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Analysis of the role of the Listeria monocytogenes F0F1-ATPase operon in the acid tolerance response
Abstract
As little is known about the genes involved in the induction of an acid tolerance response in Listeria monocytogenes, the role of the F0F1-ATPase was analyzed as a consequence of its role in the acid tolerance of a number of other bacteria and its conserved nature. It was found that acid adapted cells treated with N,N'-dicyclohexylcarbodiimide (DCCD) exhibited greatly enhanced sensitivity to low pH stress. Degenerate primers were designed to amplify and sequence a portion of the atpD gene. Subsequently, a PCR product from atpA to atpD was identified. While we were unable to create a deletion in the atpA gene, the plasmid pORI19 was inserted in a region between atpA and atpG to reduce, rather than eliminate, expression of the downstream genes. As expected this mutant displayed enhanced resistance to neomycin and exhibited slower growth than the wild type strain. This mutant could still induce an acid tolerance response and remained susceptible to DCCD treatment, but its relative acid sensitivity was difficult to assess as a consequence of its slow growth.
... The carbamoyl-phosphate is further metabolized by ArcC into ammonium and carbon dioxide with the production of ATP. This ATP may contribute to the extrusion of cytoplasmic protons by the F o F 1 -ATPase, another mechanism that has been shown to play a role in the maintenance of pH homeostasis [34]. A lesser-known mechanism of acid stress resistance in L. monocytogenes is the agmatine deiminase (AgDI) pathway [17,33,35,36] ( Figure 1B). ...
... Mechanisms other than decarboxylation also play an important role in acid stress resistance. The F o F 1 -ATPase enzyme complex plays a major role in the regulation of intracellular pH in a number of bacteria [9], and the contribution of this proton translocating ATPase to acid resistance has also been studied in E. coli and L. monocytogenes [34,43,44]. A study by Cotter et al. [34] suggests that the F o F 1 -ATPase system plays a role in the acid tolerance response (ATR) of L. monocytogenes. ...
... The F o F 1 -ATPase enzyme complex plays a major role in the regulation of intracellular pH in a number of bacteria [9], and the contribution of this proton translocating ATPase to acid resistance has also been studied in E. coli and L. monocytogenes [34,43,44]. A study by Cotter et al. [34] suggests that the F o F 1 -ATPase system plays a role in the acid tolerance response (ATR) of L. monocytogenes. Inhibition of the F o F 1 -ATPase by N,N'-dicyclohexylcarbodiimide (DCCD) prevents proton translocation out of the cell and severely affects the ability of bacteria to respond to acid stress. ...
Acidity is one of the principal physicochemical factors that influence the behavior of microorganisms in any environment, and their response to it often determines their ability to grow and survive. Preventing the growth and survival of pathogenic bacteria or, conversely, promoting the growth of bacteria that are useful (in biotechnology and food production, for example), might be improved considerably by a deeper understanding of the protective responses that these microorganisms deploy in the face of acid stress. In this review, we survey the molecular mechanisms used by two unrelated bacterial species in their response to low pH stress. We chose to focus on two well-studied bacteria, Escherichia coli (phylum Proteobacteria) and Listeria monocytogenes (phylum Firmicutes), that have both evolved to be able to survive in the mammalian gastrointestinal tract. We review the mechanisms that these species use to maintain a functional intracellular pH as well as the protective mechanisms that they deploy to prevent acid damage to macromolecules in the cells. We discuss the mechanisms used to sense acid in the environment and the regulatory processes that are activated when acid is encountered. We also highlight the specific challenges presented by organic acids. Common themes emerge from this comparison as well as unique strategies that each species uses to cope with acid stress. We highlight some of the important research questions that still need to be addressed in this fascinating field.
... Pourtant l'examination de la distribution de gadAB chez des bactéries entériques montre que bien que toutes les souches de E. coli (173), pathogènes ou non, possèdent ces gènes, la majorité des autres genres testés ne les possèdent pas (Grant et al., 2001). (Cotter et al., 2000). ...
... Ability to resist to low pH and bile, two stresses encountered in stomach and intestine, could be important factors for survival and asymptomatic carriage of a L. monocytogenes strain. In reponse to low pH in gastric fluids, intracellular homeostasis of L. monocytogenes is partially dependent on the ability of the F 0 F 1 -ATPase to pump excess protons from cytoplasm (Cotter et al., 2000). In addition, L. monocytogenes apparently utilizes a glutamate decarboxylase system (GAD) homologous to that used by E. coli (Cotter et al., 2001). ...
... De plus, le glutamate libre est parfois ajouté de manière délibérée dans les aliments pour augmenter leur saveur umami. En conséquence, la glutamate décarboxylase pourrait donc être un des mécanismes majeurs impliqués dans la survie de L. monocytogenes dans l'aliment et par voie de conséquence dans notre système digestif.Par ailleurs, il a été observé que le niveau d'activité GAD pouvait être variable en fonction de la souche considérée(Cotter et al., 2001a).L'opéron codant pour l'ATPase F 0 F 1 a été identifié chez L. monocytogenes grâce à l'utilisation d'oligonucléotides dégénérés provenant de la séquence de l' H + ATPase de Salmonella(Cotter et al., 2000). La F 0 F 1 -ATPase est une enzyme multimérique composée d'une partie catalytique F 1 et d'une partie intégrée dans la membrane F 0. F 1 comprend Introduction bibliographique les sous-unités α, β, γ, δ et ε et se charge d'hydrolyser ou de synthétiser de l'ATP.F 0 comprend les sous-unités a, b et c qui, par leur organisation, fonctionnent comme un canal permettant la translocation des protons. ...
Alors que l’intestin est le dernier réservoir qui sépare le milieu extérieur des tissus de l’hôte, il peut héberger des souches de Listeria monocytogenes suite à l’ingestion d’aliments contaminés. Les critères requis pour parvenir dans l’intestin sont liés aux stress rencontrés chez l’hôte. Le premier critère analysé est la capacité de la souche à activer sa glutamate décarboxylase (GAD) à pH acide car ce système protégerait la souche de l’acidité de l’estomac. Les souches de portage fécal résistent particulièrement bien à l’acidité du milieu simulant les fluides gastriques en présence de glutamate. Peu de souches d’origine alimentaire possédent des niveaux d’activité GAD comparables à ceux observés pour les souches de portage asymptomatique et les souches responsables de cas de listériose chez l’Homme. La résistance de L. monocytogenes aux sels biliaires est par contre un caractère commun à l’ensemble des souches étudiées quelle que soit leur origine. L’étude des capacités invasives des 14 souches de portage asymptomatique dans la cellule hôte a montré que 10 souches de portage fécal avaient un niveau de virulence atténué sur l’œuf embryonné de poulet de 14 jours contrairement aux souches liées à des épisodes épidémiques ou sporadiques. Cinq de ces souches de portage asymptomatique ont des capacités réduites d’entrée dans la cellule épithéliale Caco-2 en culture. L’Internaline A, une protéine de virulence bactérienne impliquée dans l’entrée de la bactérie dans la cellule épithéliale, apparaît être le facteur déficient pour ces 5 souches. L’analyse de séquences partielles du gène inlA a permis de déceler des mutations ponctuelles responsables de l’apparition de codons stop prématurés et de la production des formes tronquées d’Internaline A observées par Western-blot. L’utilisation de la PCR-RFLP sur les régions variables du gène inlA de souches d’origines diverses nous a permis de détecter la présence de cette déficience chez des souches d’origine alimentaire. La fréquence de ce type de souches potentiellement non invasives dans l’aliment pourrait donc expliquer au moins en partie, l’existence du portage asymptomatique et l’incidence relativement faible de cas de listérioses en proportion de la fréquence estimée de L. monocytogenes ingérées.
... Recently, it has been shown that B. cereus ATCC14579 cells can employ complex survival strategies involving decarboxylase and deiminase systems which are implicated in intracellular pH (pH i ) homeostasis (Senouci-Rezkallah et al. 2011). In response to low pH, Proton pumps play a major role in pH i homeostasis in Listeria monocytogenes (Cotter et al. 2000). ATPases from different sources have very similar structures (Santana et al. 1994). ...
... On the other hand, its role is to create a proton gradient (used for a variety of transport processes) with the energy provided by ATP hydrolysis and to maintain the intracellular pH via proton extrusion (Kakinuma 1998), this is the case for the oral streptococci Streptococcus mutans and Streptococcus sanguis (Bender et al. 1986), Lactobacillus acidophilus (Kullen and Klaenhammer 1999) and Lactococcus lactis (Koebmann et al. 2000). The proton translocating F 1 F 0 -ATPase enzyme complex plays a significant role in the regulation of intracellular pH in a number of bacteria (Cotter et al. 2000). In Enterococcus faecalis, Lactobacillus brevis and Enterococcus hirae, a high increase in F 1 F 0 -ATPase activity was observed when cells were grown at low pH (Kobayashi et al. 1984(Kobayashi et al. , 1986Arikado et al. 1999). ...
... This reduction was more marked in acid-adapted cells than unadapted cells. Comparable results were observed in L. monocytogenes and Salmonella Typhimurium, acid-adapted cells grown in batch culture and treated with DCCD were much more sensitive than treated unadapted cells to exposure to pH 3.5 and 3.3, respectively (Foster and Hall 1991;Cotter et al. 2000). These data show the role of F 1 F 0 -ATPase in B. cereus acid adaptation. ...
This study examined the involvement of ATPase activity in the acid tolerance response (ATR) of Bacillus cereus ATCC14579 strain. In the current work, B. cereus cells were grown in anaerobic chemostat culture at external pH (pHe ) 7.0 or 5.5 and at a growth rate of 0.2 h(-1) . Population reduction and internal pH (pHi ) after acid shock at pH 4.0 was examined either with or without ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) and ionophores valinomycin and nigericin. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted cells) compared with cells grown at pH 7.0 (unadapted cells), indicating that B. cereus cells grown at low pHe were able to induce a significant ATR and Exercise-induced increase in ATPase activity. However, DCCD and ionophores had a negative effect on the ability of B. cereus cells to survive and maintain their pHi during acid shock. When acid shock was achieved after DCCD treatment, pHi was markedly dropped in unadapted and acid-adapted cells. The ATPase activity was also significantly inhibited by DCCD and ionophores in acid-adapted cells. Furthermore, transcriptional analysis revealed that atpB (ATP beta chain) transcripts was increased in acid-adapted cells compared to unadapted cells before and after acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. These adaptations depend on the ATPase activity induction and pHi homeostasis. Our data demonstrate that the ATPase enzyme can be implicated in the cytoplasmic pH regulation and in acid tolerance of B. cereus acid-adapted cells.
© 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
... No significant difference was observed about the survival rate of strains ΔaguA1(Δlmo0038) and ΔAgDI (Δlmo0036-lmo0042) in acidic broth (data not shown). F 0 F 1 -ATPase system is essential for L. monocytogenes, in which mutantion will cause a lethal effect to the bacteria [25] . we didn't evaluate its contribution to acid resistance of L. monocytogenes. ...
... we didn't evaluate its contribution to acid resistance of L. monocytogenes. Datta et al. [25] and Cotter et al. [26] treated L. monocytogenes LS2 and LO28 at the exponential phase with N, N'-Dicyclohexylcarbodiimide (DCCD), an F 0 F 1 -ATPase inhibitor, which resulted in significantly reducing survival rate of the bacteria under the lethally acidic condition [25,26] . ...
... we didn't evaluate its contribution to acid resistance of L. monocytogenes. Datta et al. [25] and Cotter et al. [26] treated L. monocytogenes LS2 and LO28 at the exponential phase with N, N'-Dicyclohexylcarbodiimide (DCCD), an F 0 F 1 -ATPase inhibitor, which resulted in significantly reducing survival rate of the bacteria under the lethally acidic condition [25,26] . ...
Listeria monocytogenes is an important zoonotic foodborne pathogen, which can cause a severe invasive illness to susceptible humans and animals with high mortality. As L. monocytogenes is widely distributed in natural environments, the bacterium is easy to contaminate food processing facilities and the products to be ingested by host. But during the transition from a saprophyte to intracellular pathogen, one of the biggest challenge L. monocytogenes encounters is the acid stress. To combat the acidic environments, the bacterium developed several acid resistance systems, including acid tolerance response (ATR), F0F1-ATPase, glutamate decarboxylase (GAD), arginine deiminase (ADI) and agmatine deiminase (AgDI). In this study, we comprehensively evaluated the contributions of different acid resistance systems and explored the different roles of the three GAD components under acidic conditions. We found that the GadD2 of GAD system made the largest contribution to the survival of L. monocytogenes in artificial gastric juice (AGJ) and acidic brain heart infusion (BHI), which was followed by the global stress regulator SigB, GadD3 of GAD system, AguA1 of AgDI system and ArcA of ADI system. Transcription analysis showed that the mRNA level of the three GADs were consistent with their contribution to acid resistance. Similar results were observed in the other three representative strains EGDe, Lm850658 and M7. We further obtained the purified GADs and their poly-antibodies to demonstrate that the contribution of the three GADs were determined by the protein levels in L. monocytogenes. Further studies are needed to focus on the regulation of different expression of the GAD system.
... Weak acids under their unprotonated form can diffuse into the cell and dissociate, releasing a proton and leading to acidification of the cytoplasm. Strong acids are not able to permeate through the cell membrane but by lowering the external pH and increasing the pH gradient, they increase the proton permeability and thereby lead to the reduction of pH int Beales, 2004;Cotter et al., 2000;Krebs et al., 1983). Bacteria can survive thanks to their ability to regulate their pH int , a process primarily driven by controlled movement of cations across the membrane (Beales, 2004). ...
... monocytogenes . In L. monocytogenes, the increase of acid tolerance due to glutamate decarboxylase system is only provided when glutamate decarboxylase acts in concert with glutamate/GABA antiporter (Cotter et al., 2000). ...
Predictive microbiology is a tool to assess food microbiological risks and optimise food processes. However predictive microbiology which predicts the bacterial behaviour does not take bacterial physiological state into consideration. The aim ofthis work is to identify molecular biomarkers to assess the impact of bacterial adaptation on the subsequent acid resistance. In this study, the acid resistance of Bacillus weihenstephanensis wasinvestigated and mRNAs were identified as acid resistance biomarkers.(i) The bacterial acid resistance of different mildstress adapted cells was quantified using cultural methods. Mild salt-adapted cells were less resistant than cells grown in optimal conditions;and the latter less resistant than acid-adaptedcells. However, the bacterial resistance of B.weihenstephanensis followed the same patternwhen facing acidic changes of the environment and that, whatever the environmental condition previously encountered.(ii) For RT-qPCR gene expression quantifications a specific rotative PCR device based on the PallGeneDisc® Technology was developed.(iii) Omic data and bacterial acid resistances correlation allows the selection of biomarkers to track the more resistant and the more sensitive cells present within the bacterial population.(iv) Both linear and non linear correlations allowed to define two types of biomarkers: ‘Directbiomarker’ for which expression patterns uponmild stress treatment were linearly correlated to the subsequent acid resistance and ‘long-actingbiomarkers’ which were transiently up-regulatedduring mild stress exposure and correlated to increased acid resistance over time.(v) A multivariate analysis was performed to correlate the acid bacterial resistance and the gene expression of vegetative cells. This mathematical method provides the advantage to take gene expressions and their interactions into account and allowed the selection of 9 genes as acid resistance biomarkers of B. weihenstephanensis. Finally, some promising results were also obtained. There by, it would be feasible to use gene expression at a given time to predict the bacterialsurvival behaviour in lethal acid conditions.
... Proteins such as GroEL, ATP synthase, and various transcriptional regulators showed increased synthesis under acid stress growth [32]. F 0 F 1 -ATPase is a multi-subunit enzyme involved in the acid adaptation of L. monocytogenes, serving as an ATP driven proton channel across the cell membrane, helping maintain cellular pH homeostasis [33]. An inadequately maintained intracellular homeostasis can be the cause of loss of cell viability [34]. ...
Background: Listeria monocytogenes represents high risk for consumers, since it can grow under refrigeration and acidic environments by developing acid tolerance response (ATR). Objective: The aim of this work was to study the growth and survival of ATR L. monocytogenes strain Scott A in media acidified with malic acid, lactic acid, or blueberry extract. Methods: Bacterial growth was evaluated using tryptic soy broth with yeast extract (TSBYE) combined with the different acid solutions and incubated at 25°C for 24 h. An optical density system measured growth every 15 minutes for 24 h. RESULTS: Complete inhibition of L. monocytogenes occurred in presence of treatments including malic acid pH 2.0 and 3.0; lactic acid pH 2.0, 3.0, and 4.0; and with blueberry extract pH 2.0 in the mixture. No growth was observed in treatments under pH 4.5. Turbidity values of media mixed with blueberry extract at pH 3.0, 4.0, and 5.0 showed no statistical difference at 18 h and growth media had pH of 6.13, 6.53, and 6.78, respectively. CONCLUSIONS: Lactic acid was more effective inhibiting bacterial growth compared to malic acid. Blueberry extract was not effective acidifying the final pH of the TSBYE solutions, therefore L. monocytogenes survived in media acidified with low pH blueberry extract. Treatments with blueberry extract had the least antibacterial effect in this study.
... Two important physiological functions have been assigned to the F 0 F 1 -ATP synthase in bacteria including oxidative phosphorylation by the synthesis of ATP aerobically from ADP and inorganic phosphate using the energy of an electrochemical ion gradient and the reverse reaction whereby a transmembrane ion gradient or proton motive force (PMF) is generated anaerobically at the expense of ATP hydrolysis when the driving force is low 33 . The generation of this PMF could be important to overcome an acidic environment by the increase of the intracellular pH 34 . It is known that pathogenic elicitors can induce a cytosolic acidification due to H + influx in plants [35][36][37] . ...
Fire blight, caused by the enterobacterium Erwinia amylovora, is a destructive disease, which can affect most members of the Rosaceae family. Since no significant genomic differences have been found by others to explain differences in virulence, we used here a gel-based proteomic approach to elucidate mechanisms and key players that allow the pathogen to survive, grow and multiply inside its host. Therefore, two strains with proven difference in virulence were grown under controlled conditions in vitro as well as in planta (infected apple rootstocks). Proteomic analysis including 2DE and mass spectrometry revealed that proteins involved in transcription regulation were more abundant in the in planta condition for both strains. In addition, genes involved in RNA processing were upregulated in planta for the highly virulent strain PFB5. Moreover, the upregulation of structural components of the F0F1-ATP synthase are major findings, giving important information on the infection strategy of this devastating pathogen. Overall, this research provides the first proteomic profile of E. amylovora during infection of apple rootstocks and insights into the response of the pathogen in interaction with its host.
... In food systems, it has been demonstrated that in glutamate-rich products, the survival rate of L. monocytogenes is significantly improved [107]. In addition to glutamate:GABA antiporter, other proton pumps such as F 0 F 1 -ATPase have also been proposed as active mechanisms to maintain homeostasis in acidified environments [108]. ...
Although many efforts have been made to control Listeria monocytogenes in the food industry, growing pervasiveness amongst the population over the last decades has made this bacterium considered to be one of the most hazardous foodborne pathogens. Its outstanding biocide tolerance capacity and ability to promiscuously associate with other bacterial species forming multispecies communities have permitted this microorganism to survive and persist within the industrial environment. This review is designed to give the reader an overall picture of the current state-of-the-art in L. monocytogenes sessile communities in terms of food safety and legislation, ecological aspects and biocontrol strategies.
... Little is known about the S. aureus ATP synthase; however, it was shown to be important for growth and survival (38,51), reinforcing the hypothesis that mutations in atpE need to allow some level of functionality. For other Bacillales like L. monocytogenes, it was reported that ATP synthase also plays a role in pH homeostasis (52) and that deletion of ATP synthase in B. subtilis greatly affects growth (53). Our previous work showed that TO and its analog FC04-100 possess a narrow yet specific spectrum of activity against the SCVs of Bacillales (13). ...
Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of deadly hospital-acquired infections. The discovery of anti- Staphylococcus antibiotics and new classes of drugs not susceptible to the mechanisms of resistance shared among bacteria is imperative. We recently showed that tomatidine (TO), a steroidal alkaloid from solanaceous plants, possesses potent antibacterial activity against S. aureus small colony variants (SCVs), the notoriously persistent form of this bacterium that has been associated with recurrence of infections. Here, using genomic analysis of in vitro -generated TO-resistant S. aureus strains to identify mutations in genes involved in resistance, we identified the bacterial ATP synthase as the cellular target. Sequence alignments were performed to highlight the modified sequences and the structural consequences of the mutations were evaluated in structural models. Overexpression of the atpE gene in S. aureus SCVs or introducing the mutation found in the atpE gene of one of the high-level TO-resistant S. aureus mutant into the Bacillus subtilis atpE gene provided resistance to TO and further validated the identity of the cellular target. FC04-100, a TO derivative which also possesses activity against non-SCV strains, prevents high-level resistance development in prototypic strains and limits the level of resistance observed in SCVs. An ATP synthesis assay allowed the observation of a correlation between antibiotic potency and ATP synthase inhibition. The selectivity index (inhibition of ATP production by mitochondria vs . bacterial ATP synthase) is estimated to be >10 ⁵ -fold for FC04-100.
... Ferritine se liant à l'ADN + + + (Dussurget et al., 2005) ATPase-F 0 F 1 ATPase membranaire d'efflux de proton + (Cotter et al., 2000) RecA Régulateur de la réponse SOS + + (van der Veen et al., 2010) ...
Les fluctuations hydriques influencent l'activité et la viabilité des microorganismes. L’humidité relative de l’air est ainsi un paramètre potentiellement efficace pour maîtriser le développement et la persistance de microorganismes pathogènes. Cependant, l’efficacité de ce paramètre reste peu connue par rapport à d’autres facteurs environnementaux comme le pH ou la température. Le séchage est l'étape finale des procédures de nettoyage et de désinfection en industrie agroalimentaire. Cependant, il est aujourd'hui utilisé de façon empirique alors qu'il pourrait permettre d'améliorer la décontamination des surfaces dans les ateliers et diminuer la persistance des microorganismes. Malgré les procédures de nettoyage et de désinfection, la bactérie Listeria monocytogenes est fréquemment retrouvée dans l'industrie agroalimentaire et y persiste pendant de longues périodes. Evaluer et comprendre l'impact des fluctuations hydriques sur la survie de L. monocytogenes afin d'en optimiser la destruction sur une surface, constituent ainsi les principaux objectifs de cette thèse.Dans un premier temps, 30 souches de L. monocytogenes isolées de différents environnements de transformation des aliments et présentant différents sérotypes et niveaux de virulence ont été exposées à un stress hyperosmotique et à un séchage. Cette approche a permis d'analyser les différences de résistance entre les souches et de sélectionner quatre souches présentant différents profils de résistance pour poursuivre les travaux.Afin de définir les conditions de fluctuations hydriques les plus létales pour les souches de L. monocytogenes, différents paramètres ont été étudiés tels que la vitesse de déshydratation, le niveau d’humidité relative, la vitesse de réhydratation, l’application de cycles successifs de déshydratation-réhydratation et le milieu de séchage.Enfin, les mécanismes cellulaires induisant la mort de L. monocytogenes lors des fluctuations hydriques ont été explorés par spectroscopie infrarouge à transformée de Fourier, microscopie à force atomique, cytométrie en flux et par séchage en anaérobiose. Ces différentes méthodes ont permis de révéler qu'un stress mécanique et structural sont majoritairement responsables de la mort de L. monocytogenes.L’ensemble de ce travail démontre que la maîtrise du niveau et des variations de l’humidité relative de l’air est un moyen efficace pour détruire L. monocytogenes et offre de réelles perspectives d’application pour améliorer l’hygiène des ateliers de production alimentaire.
... The transcriptomic responses of L. monocytogenes to a variety of environmental stimuli such as heat (Ripio, Vazquez-Boland, Vega, Nair, & Berche, 1998;Hanawa et al., 1999;Hanawa, Yamanishi, Murayama, Yamamoto, & Kamiya, 2002;Gaillot, Pellegrini, Bregenholt, Nair, & Berche, 2000;Van der Veen, Hain, Wouters, Hossain, de Vos, Abee et al., 2007), cold (Phan-Thanh & Gormon, 1995;Nelson et al., 2004;Schmid et al., 2009;Durack, Ross, & Bowman, 2013), acid (Cotter, Gahan, & Hill, 2000Milecka, Samluk, Wasiak, & Krawczyk-Balska, 2015) and osmotic shock (Sleator, Gahan, Abee, & Hill, 1999;Sleator, Gahan, & Hill, 2001a;Duche, Tremoulet, Glaser, & Labadie, 2002;Brondsted, Kallipolitis, Ingmer, & Knochel, 2003;Sleator & Hill, 2005) as well as the presence of antimicrobials (Dutta, Elhanafi, & Kathariou, 2013;Elhanafi, Dutta, & Kathariou, 2010;Van der Veen & Abee, 2010;Laursen, Bahl, Licht, Gram, & Knudsen, 2015;Liu, Basu, Miller, & McMullen, 2014;Pleitner, Trinetta, Morgan, Linton, & Oliver, 2014;Romanova, Wolffs, Brovko, & Griffiths, 2006) in vitro and in an actual food matrix (Liu & Ream, 2008;Olesen, Thorsen, & Jespersen, 2010;Bae, Crowley, & Wang, 2011;Alessandria, Rantsiou, Dolci, Zeppa, & Cocolin, 2013;Mataragas et al., 2015;Hadjilouka et al., 2016) have been extensively studied improving our understanding of the pathogen's physiology (Hadjilouka, Paramithiotis, & Drosinos, 2015b). Genes associated with virulence potential as well as ones that contribute to the activation of response mechanisms have been in the epicenter of these studies. ...
The aim of the present study was to assess the transcriptomic response of L. monocytogenes isolates on the exposure to lemongrass essential oil. Overnight culture of six strains previously isolated from a strawberry sample was spread over the surface of BHI agar and exposed to 5 and 10 μL of lemongrass essential oil that were applied on a Whatman paper placed on the lid. Three of them survived in the former case and one in the latter; in all cases biomass was collected and RNA was isolated. The expression of the key virulence genes prfA, sigB, plcA, plcB, hlyA, inlA, inlB, inlC, inlJ, lmo2470 and lmo2672, as well as accA, acpP and fapR involved in fatty acid biosynthesis/metabolism and murE and pbpB involved in peptidoglycan biosynthesis was assessed by RT-qPCR. Transcription of murE, pbpB, accA, fapR, prfA, sigB, inlA and lmo2672 was not affected. On the contrary, downregulation of hly and inlJ was observed for all strains. Moreover, upregulation of acpP and downregulation of plcA, plcB, inlB, inlC and lmo2470 were observed according to the strain. The increased amount of lemongrass essential oil affected significantly the expression of acpP, hly, and inlJ.
... In addition to GAD and ADI systems, other proton pumps such as F 0 F 1 -ATPase have also been suggested as active mechanisms to maintain L. monocytogenes homeostasis in low (mild) pH environments (Cotter et al., 2000). ATP produced during arginine conversion under ADI mechanisms is used by F 0 F 1 -ATPase to generate a proton gradient, enabling H + expulsion and homeostasis restoration (Smith et al., 2013). ...
The foodborne pathogen Listeria monocytogenes is the causative agent of human listeriosis, a severe disease, especially dangerous for the elderly, pregnant women, and newborns. Although this infection is comparatively rare, it is often associated with a significant mortality rate of 20–30% worldwide. Therefore, this microorganism has an important impact on food safety. L. monocytogenes can adapt, survive and even grow over a wide range of food production environmental stress conditions such as temperatures, low and high pH, high salt concentration, ultraviolet lights, presence of biocides and heavy metals. Furthermore, this bacterium is also able to form biofilm structures on a variety of surfaces in food production environments which makes it difficult to remove and allows it to persist for a long time. This increases the risk of contamination of food production facilities and finally foods. The present review focuses on the key issues related to the molecular mechanisms of the pathogen survival and adaptation to adverse environmental conditions. Knowledge and understanding of the L. monocytogenes adaptation approaches to environmental stress factors will have a significant influence on the development of new, efficient, and cost-effective methods of the pathogen control in the food industry, which is critical to ensure food production safety.
... For example, one proposed strategy is that the production of ATP in the process of ADI pathway enables bacteria to provide energy for F 1 F O -ATPase to pump cytoplasmic protons and so maintain cytosolic pH homeostasis under acidic environments. Similar strategies have been employed by many pathogenic bacteria to survive within acidified phagosomes [80][81][82][83]. ...
Antibacterial resistance to infectious diseases is a significant global concern for health care organizations; along with aging populations and increasing cancer rates, it represents a great burden for government healthcare systems. Therefore, the development of therapies against bacterial infection and cancer is an important strategy for healthcare research. Pathogenic bacteria and cancer have developed a broad range of sophisticated strategies to survive or propagate inside a host and cause infection or spread disease. Bacteria can employ their own metabolism pathways to obtain nutrients from the host cells in order to survive. Similarly, cancer cells can dysregulate normal human cell metabolic pathways so that they can grow and spread. One common feature of the adaption and disruption of metabolic pathways observed in bacterial and cancer cell growth is amino acid pathways; these have recently been targeted as a novel approach to manage bacterial infections and cancer therapy. In particular, arginine metabolism has been illustrated to be important not only for bacterial pathogenesis but also for cancer therapy. Therefore, greater insights into arginine metabolism of pathogenic bacteria and cancer cells would provide possible targets for controlling of bacterial infection and cancer treatment. This review will summarize the recent progress on the relationship of arginine metabolism with bacterial pathogenesis and cancer therapy, with a particular focus on arginase and arginine deiminase pathways of arginine catabolism.
... (Shabala et al., 2002a,b;Cheng et al., 2015) by efficient mechanisms for pH-homeostasis (Gray et al., 2006). Together with the electrical membrane potential , the pH gradient across the membrane constitutes the proton motive force, which is essential for generation of ATP by the F 1 F 0 ATPase (Booth, 1985;Cotter et al., 2000). Failure to maintain pH homeostasis, e.g., following exposure to bacteriocins such as pediocin and nisin, leads to loss of cell viability and, therefore, may be used as a sensitive indicator of bacterial death at the single cell level (Budde and Jakobsen, 2000;Kastbjerg et al., 2009). ...
Bacteriocins are antimicrobial peptides naturally produced by many bacteria and were shown to be effective against various pathogens including Listeria monocytogenes. L. monocytogenes is a food-borne pathogen that frequently causes disease outbreaks around the world with fatal outcomes in at-risk individuals. Thus, bacteriocins are a promising solution to prevent contaminations with L. monocytogenes and other microorganisms during food production and preservation. In the present study, we constructed L. monocytogenes EGD-e/pNZ-Phelp-pHluorin, a strain that constitutively expresses the pH-sensitive fluorescent protein pHluorin, as a sensor strain to detect disruption of the pH gradient by the membrane-damaging activity of bacteriocins. The ratiometric fluorescence properties of pHluorin were validated both in crude extracts and permeabilized cells of this sensor strain. L. monocytogenes EGD-e/pNZ-Phelp-pHluorin was used to assess membrane damaging activity of the bacteriocins nisin A and pediocin PA-1 and to determine the minimal concentrations required for full disruption of the pH gradient across the membrane. Moreover, the sensor strain proved useful to analyze the presence of compounds affecting membrane integrity in supernatants of a nisin Z-producing Lactococcus lactis strain at different timepoints during growth. Supernatants of this strain that were active in disrupting the pH gradient across the membrane were also shown to inhibit growth of L. monocytogenes. In summary, the presented results suggest that the generated sensor strain is a convenient, fast and reliable tool to identify and characterize novel bacteriocins and other compounds that target membrane integrity.
... Exposure of L. monocytogenes to adverse conditions can result in cross-protection against additional stresses such as its entry and colonization in the host (Hill et al., 2002;). The ability of L. monocytogenes to respond to low pH conditions plays an integral role in its survival and resistance to acidic foods (Cotter et al., 2000), thus impacting on food processing and preservation protocols. The organism can become highly resistant to even extremely acidic conditions due to stress hardening (Lou and Yousef, 1997). ...
Listeria monocytogenes is the causative agent of listeriosis which can survive under a wide range of adverse conditions. This gram positive organism is of major concern to the food industry due to the regulatory environment and concerns for consumer food safety. Due to its ubiquitous presence in the environment and psychrotolerant nature, its presence is anticipated even under refrigerated conditions. Adaptation to environmental stresses such as heat, acid, salt, cold provides protection to Listeria cells against wide range of other stresses. Biofilm formation by this organism presents a major risk specifically in the food industry, further introducing the possibility of post-process contamination. Thus, this pathogen is of great concern to manufacturers due to its resistance to a number of food preservation practices. In this chapter we describe the survival strategies employed by L. monocytogenes and how the novel non-thermal approaches of ozone and atmospheric cold plasma (ACP) may prove useful for control and inactivation of this pathogen. Studies presenting the effects of system and process control parameters associated with Listeria inactivation efficacy and the resulting control measures that may be considered for effective processing applications are highlighted. Further exploration of the potential to optimise both ozone and ACP for control of Listeria spp is given in the context of the wider research in the area.
... coli, S. aureus and Salmonella). This response will protect the cells from normally lethal acid stress after exposure to mild acid stress (Kroll and Patchett, 1992;Gahan et al., 1996;Datta and Benjamin, 1997;Cotter et al., 2000). The physiological state of the cells will change, which will markedly alter their chances of surviving the hosts' defence system (Buchanan et al., 2000). ...
... The former may react with protons resulting in pH value increase whereas the latter may be used for biosynthetic processes or to remove protons via the F 0 F 1 ATPase enzyme complex (Stack et al. 2008). The operon of the latter consists of nine genes and was characterized by Cotter et al. (2000). The contribution of fri, a gene encoding a ferritin-like protein, in acid stress tolerance was recently reported by Milecka et al. (2015). ...
Listeria monocytogenes is a facultative intracellular bacterium and hence, this bacterium is able to survive and propagate in macrophages (M. Yet, in contrast to this notion, a majority of L. monocytogenes is killed by M indicating that M play a critical role in protection against L. monocytogenes infection. Because the number of L. monocytogenes engulfed and killed by M depends on whether or not M are activated, experimental listeriosis has been employed for analyzing M activities by many investigators. There are several methods for analyzing phagocytic and listericidal activities of M Yet, in most cases expensive materials and complicated methods are required. In this review, in addition to methods recently employed for determination of phagocytic and listericidal activities of M, simple and excellent, though old-fashioned, method is also introduced.
... GadB is induced under acid stress condition via the SigB stress regulon (Cotter et al., 2001). In addition an H + ATPase has been shown to have a role in acid survival and in the induction of an acid-tolerance response (Cotter et al., 2000). The genes encoding the ATPase (atpC and atpD) appear to be regulated by PrfA (Glaser et al., 2001). ...
The genus Listeria contains ten species of Gram-positive bacteria, L. monocytogenes, L. fleischmannii, L. grayi, L. innocua, L. ivanovii, L. marthii, L. rocourtiae, L. seeligeri, L. weihenstephanensis, and L. welshimeri, and has been classified (along with members of the genus Brochothrix: B. thermosphacta and B. campestris) within the family Listeriaceae. Members of this family produce short rods that may form filaments. Cells stain Gram-positive, and the cell walls contain meso-diaminopimelic acid. The major lipid components include saturated straight-chain and methyl-branched fatty acids. Endospores are not produced; menaquinones are the sole respiratory quinones. Growth is aerobic and facultatively anaerobic; glucose is fermented to lactate and other products. L. monocytogenes (and to a lesser extent L. ivanovii) which are pathogenic to humans and a range of other animals, and the disease is primarily transmitted by consumption of contaminated food or feed. Human listeriosis is an opportunistic infection which most often affects those with severe underlying illness, the elderly, pregnant women, and both unborn and newly delivered infants. The reported incidence of human listeriosis varies between countries from 10 cases per million of the total population. Because of the severity of infection, listeriosis is one of the major causes of death from a preventable foodborne illness. Studies of the molecular biology of L. monocytogenes have identified a number of virulence factors that promote uptake into nonprofessional phagocytic cells and the process of movement from cell-to-cell by recruiting host cell proteins and remodeling the host cell cytoskeleton. This has made L. monocytogenes also of interest both as a tool to help understand eukaryotic cell biology and as a potential therapeutic agent for intracellular delivery of drugs and as a cancer vaccine. The presence of L. monocytogenes remains a major challenge for the food industry. Its psychrotrophic nature means that it can grow at or below refrigeration temperatures and it is also relatively tolerant of high solute concentrations, resists desiccation, and therefore can overcome mild food preservation techniques. L. monocytogenes is able to form biofilms and can colonize food processing equipment and environments, leading to cross-contamination of processed foods. Hence it is of particular concern in ready-to-eat foods.
... Owing to its pathogenic lifestyle, this organism must develop a variety of mechanisms to rapidly adjust to the adverse environments. The glutamate decarboxylase (Gad) system , operon encoding F 0 -F 1 -ATPase (Cotter et al., 2000), arginine deiminase (Adi) system (Ryan et al., 2009), DNA repair system, activator of the SOS response (RecA) (Van der Veen and Abee, 2011), and the gene encoding for the nucleotide excision repair pathway (uvrA) (Kim et al., 2006) have been reported to be important for pH homeostasis in L. monocytogenes. ...
... Stationary and exponential DCCD-treated cells achieved different survival rates, suggesting a growth phase-dependent phenomenon; stationary phase cells attained 1% survival, in contrast to exponential phase that had only 0.001% survival. Later, Cotter, Gahan, and Hill (2000) demonstrated the involvement of the F 1 F 0 -ATPase complex in acid tolerance response. Acid-adapted L. monocytogenes LO28 cells (pH 5.5 adjusted with 3 M lactic acid) survived to low pH (pH 3.5) 3 logs higher than DCCDtreated cells after 2 h of acid exposure. ...
Dairy products, in particular soft cheeses, pose a major concern to the dairy industry and public health authorities as they are the leading source of listeriosis outbreaks, a severe foodborne infection affecting pregnant women, children, elderly and immunocompromised people, with a high (20–30%) mortality rate. Cheeses offer a suitable environment for the survival and growth of Listeria monocytogenes, allowing this pathogen to display tolerance responses that can favour its presence in cheese and persistence in dairy processing plants. Extensive food safety regulations in the EU towards prevention of contamination of dairy products with L. monocytogenes have been implemented. However due to the specific abilities of this pathogen to overcome the processing hurdles, its control remains a challenge. Compliance with the Good Manufacturing Practices, observation of Hazard Analysis Critical Control Points (HACCP) and the surveillance of the pathogen in the cheese processing environment are crucial to provide consumers with a safe product. This review aims to provide an overview on the current knowledge about the potential for the transmission of L. monocytogenes in cheese and its abilities to overcome the challenging processing conditions and implications for the behaviour of the pathogen in the host.
... L. monocytogenes has several mechanisms to maintain its internal pH (pH i ) under acid stress (Table 1) including the F 0 F 1 -ATPase (Cotter et al., 2000), the glutamatic acid decarboxylase (GAD; Feehily et al., 2014), and the arginine and agmatine deiminases (ADI and AgDI; Lund et al., 2014). The F 0 F 1 -ATPase is involved in ATR initiation during mild pH stress (Mclaughlin and Rees, 2009). ...
Listeria monocytogenes is a human food-borne facultative intracellular pathogen that is resistant to a wide range of stress conditions. As a consequence, L. monocytogenes is extremely difficult to control along the entire food chain from production to storage and consumption. Frequent and recent outbreaks of L. monocytogenes infections illustrate that current measures of decontamination and preservation are suboptimal to control L. monocytogenes in food. In order to develop efficient measures to prevent contamination during processing and control growth during storage of food it is crucial to understand the mechanisms utilized by L. monocytogenes to tolerate the stress conditions in food matrices and food processing environments. Food-related stress conditions encountered by L. monocytogenes along the food chain are acidity, oxidative and osmotic stress, low or high temperatures, presence of bacteriocins and other preserving additives, and stresses as a consequence of applying alternative decontamination and preservation technologies such high hydrostatic pressure, pulsed and continuous UV light, pulsed electric fields (PEF). This review is aimed at providing a summary of the current knowledge on the response of L. monocytogenes toward these stresses and the mechanisms of stress resistance employed by this important food-borne bacterium. Circumstances when L. monocytogenes cells become more sensitive or more resistant are mentioned and existence of a cross-resistance when multiple stresses are present is pointed out.
... L. monocytogenes is a successful pathogen in terms of its ability to withstand acidic environments. Acid tolerance, though the mechanism is still not fully understood, makes L. monocytogenes survival possible in low pH foods and beverages (Cotter, Gahan, & Hill, 2000). Recently, one of the least expected cases of listeriosis outbreak was linked to the consumption of caramel-coated apples in USA in 2014, a previously unreported vehicle for L. monocytogenes. ...
When testing conditions that prevail in fruit juice industry it was found that Listeria monocytogenes ATCC 7644 and a wild strain of Listeria innocua were highly hydrophobic, expressed swimming and twitching, co‐aggregated with yeast cells and produced exopolysaccharide, all characteristics that would favor the adhesive process. In mono‐species biofilms, L. innocua adhered on stainless steel at significantly higher counts than L. monocytogenes achieving values of 6.64 ± 0.01 and 5.80 ± 0.21 log CFU/cm². In dual species biofilms with resident yeasts, L. innocua cells counts increase significantly in the presence of Candida tropicalis and Candida krusei and decrease significantly in the presence of Rhodotorula mucilaginosa and Candida kefyr. When cocultured with Candida tropicalis, the cell numbers of L. monocytogenes had a significant increase. These results revealed synergic and antagonistic interactions among species.
Practical applications
The study supports the plausibility that interactions between L. monocytogenes and members of resident microbiota, such as C. tropicalis, C. krusei, C. kefyr, and R. mucilaginosa, might play an important role for the survival and dissemination of L. monocytogenes. Apple juice processing conditions was used as simulation of the real condition in fruit juice processing environment and these results will alert to fruit juice industry to adopt the best cleaning and disinfecting practices against Listeria.
... In order for L. monocytogenes to survive low pH environments, the bacterium has evolved several mechanisms that allow it to maintain pH homeostasis. These include the arginine deiminase system [9], an F 0 F 1 ATPase [10], the adaptive acid tolerance response (ATR) [11] and the glutamate decarboxylase (GAD) system [12]. The GAD system has been shown in L. monocytogenes to be important for survival in synthetic gastric fluid [12] but not in the presence of organic acids [8] commonly found in foods. ...
The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains.
... So, there is a great probability that the unknown and uncultivable bacteria have the potential to become warehouse of novel industrial enzymes, natural compounds to produce novel bioprocesses and technology for diagnosis and medicine and agriculturally important resources (Kimura 2018). To explore the taxonomic diversity as well as functional characteristics of bioresources, culture-independent technologies such as phospholipid and fatty acid analysis (PLFA) (Cotter et al. 2000;Buyer and Sasser 2012;Mrozik et al. 2014), DNA microarrays (Wagner et al. 2007), fluorescence in situ hybridization (FISH) (Ivanov et al. 2003), quantitative real-time polymerase chain reaction (qRT-PCR) (Fierer et al. 2005), different genetic fingerprinting techniques, viz., denaturing-gradient gel electrophoresis or temperature-gradient gel electrophoresis (DGGE/TGGE) (Muyzer et al. 1993), single-strand conformation polymorphism (SSCP) (Schwieger and Tebbe 1998), amplified ribosomal DNA restriction analysis (ARDRA) (Uchiyama et al. 2002;Lagacé et al. 2004), terminal restriction fragment length polymorphism (T-RFLP) (Liu et al. 1997), and ribosomal intergenic spacer analysis (RISA) (Fisher and Triplett 1999;Fechner et al. 2010). However these techniques are not described as these are not in the scope of this chapter. ...
Freshwater ecosystem encompasses varied and rich diversity of habitat conditions and is home to diverse microbial community. Each of these diverse microbial community plays a specific function that contributes to the importance of such an ecosystem in the global carbon cycle through consumption and emission of carbon dioxide and thereby in the regulation of the global climate. However, unlike their counterpart of other ecosystem, viz., terrestrial, the microbial diversity studies of freshwater ecosystem have been comparatively scanty mainly because many of the methods suitable for their identification were only developed recently. The conventional methods of studying microbes that relied on cultivation of the organism for identification in laboratories left out many that were uncultivable under laboratory conditions. Recently, several tools and techniques including both traditional and high-throughput state-of-the-art technologies have been explored to decipher the microbial diversities of freshwater ecosystem. Advancement in tools and techniques related to microbial diversity studies has provided new insights into microbial diversity and their functioning in freshwater ecosystem. In this chapter we discuss the different traditional methods followed by molecular biology techniques that are used to decipher microbial diversity of both cultivable and non-cultivable microbes of freshwater ecosystem. We discuss in detail about the cutting-edge high-throughput technologies, viz., metagenomics, metatranscriptomics, and metaproteomics that are aiding in increasing our understanding of the freshwater microbial diversity as well as their functioning.
... Similarly, a five-gene stress survival islet (Islet-1) enhances growth in suboptimal conditions (pH and salt) in acidified media with HCl as well as lactic acid (Ryan et al., 2010). Furthermore, the insertion of Tn917 plasmid in a thiamine (thiT) gene exhibited significantly more acid-sensitivity than WT at low pH in HCl pH-adjusted media (Madeo et al., 2012) and a modified F 1 F 0 -ATPase operon was also investigated (Cotter et al. 2000). Finally, comparison of pH and a w stress tolerance in 138 L. monocytogenes strains revealed that the presence of gene ORF2110 enhance growth in sodium lactate supplemented low pH BHI broth (Van Der Veen et al., 2008). ...
A collection of Listeria monocytogenes isolates from various food products, food processing environments and clinical sources (n = 153) were evaluated for their tolerance to acetic, lactic and propionic acids. A large variation in tolerance was observed amongst isolates under mildly acidic conditions (pH 5.3) for acetic (5–20 mM undissociated acid) and propionic acid (2–10 mM undissociated acid) but there was less variation for lactic acid (3–6 mM undissociated acid). Analysis of the isolate genome sequences for a complement of genes previously shown to have a role in acid tolerance revealed that thiT, gadT2, gadD2 and gadD3 genes were linked to higher acetic acid tolerance (P < 0.05) while lisRK was linked to higher tolerance to propionic acid (P = 1 × 10⁻¹¹). An absence of plasmid genes was also linked with isolates showing higher tolerance for all acids. Scoary GWAS analysis revealed that a total of 333, 207, and 333 genes were associated with acid tolerance for acetic, lactic, and propionic acid, respectively (P < 0.05). However, the p-value adjusted with Bonferroni's method for multiple comparisons did not reveal any significant associations. Isolates were grouped into clonal complexes (CC) using Multi Locus Sequence Typing (MLST) and MIC values for the three acids were determined for representative strains. One complex, CC18, showed significantly higher (P ≤ 0.05) acetic and propionic acid MIC values than other groups, whereas only CC7 type isolates revealed significantly higher (P ≤ 0.001) lactic acid MIC values. The results demonstrate that MLST typing could be linked to acid tolerance phenotypic traits which is important in predicting the behaviour of L. monocytogenes in food products.
... Several mechanisms, schematically depicted in Figure 2, are known to maintain intracellular pH (pH i ) to values compatible with L. monocytogenes vitality. These include the F 0 F 1 -ATPase, the glutamate decarboxylase (GAD) system, and the arginine and agmatine deiminases (ADI and AgDI, respectively) (Cotter et al., 2000;Feehily et al., 2014;Cheng et al., 2017). The F 0 F 1 -ATPase, a multi-subunit enzyme system, is involved in the acid tolerance response initiation upon mild acidic pH stress. ...
The pathogenic microorganism Listeria monocytogenes is ubiquitous and responsible for listeriosis, a disease with a high mortality rate in susceptible people. It can persist in different habitats, including the farm environment, the food production environments, and in foods. This pathogen can grow under challenging conditions, such as low pH, low temperatures, and high salt concentrations. However, L. monocytogenes has a high degree of strain divergence regarding virulence potential, environmental adaption, and stress response. This review seeks to provide the reader with an up-to-date overview of clonal and serotype-specific differences among L. monocytogenes strains. Emphasis on the genes and genomic islands responsible for virulence and resistance to environmental stresses is given to explain the complex adaptation among L. monocytogenes strains. Moreover, we highlight the use of advanced diagnostic technologies, such as whole-genome sequencing, to fine-tune quantitative microbiological risk assessment for better control of listeriosis.
... The first group comprised genes encoding the following factors involved in energy production: F 1 F 0 -ATP synthase, cytochrome oxidases and NADH-quinone oxidoreductase (Supplementary Table 3). The activation of the F 1 F 0 -ATPase/ATP-synthase has been reported under acid conditions in bacterial species such as E. coli (Sun et al., 2012) and Listeria monocytogenes (Cotter et al., 2000). In our study, the induction of three of the F 1 F 0 -ATPase/ATP-synthase genes was observed: atpG, atpD, and atpC, the latter encoding the subunit participating in proton translocation. ...
Brucellae are facultative intracellular coccobacilli causing brucellosis, one of the most widespread bacterial zoonosis affecting wildlife animals, livestock and humans. The genus Brucella comprises classical and atypical species, such as Brucella suis and Brucella microti , respectively. The latter is characterized by increased metabolic activity, fast growth rates, and extreme acid resistance at pH 2.5, suggesting an advantage for environmental survival. In addition, B. microti is more acid-tolerant than B. suis at the intermediate pH of 4.5. This acid-resistant phenotype of B. microti may have major implications for fitness in soil, food products and macrophages. Our study focused on the identification and characterization of acid resistance determinants of B. suis and B. microti in Gerhardt’s minimal medium at pH 4.5 and 7.0 for 20 min and 2 h by comparative RNA-Seq-based transcriptome analysis, validated by RT-qPCR. Results yielded a common core response in both species with a total of 150 differentially expressed genes, and acidic pH-dependent genes regulated specifically in each species. The identified core response mechanisms comprise proton neutralization or extrusion from the cytosol, participating in maintaining physiological intracellular pH values. Differential expression of 441 genes revealed species-specific mechanisms in B. microti with rapid physiological adaptation to acid stress, anticipating potential damage to cellular components and critical energy conditions. Acid stress-induced genes encoding cold shock protein CspA, pseudogene in B. suis , and stress protein Dps were associated with survival of B. microti at pH 4.5. B. suis response with 284 specifically regulated genes suggested increased acid stress-mediated protein misfolding or damaging, triggering the set-up of repair strategies countering the consequences rather than the origin of acid stress and leading to subsequent loss of viability. In conclusion, our work supports the hypothesis that increased acid stress resistance of B. microti is based on selective pressure for the maintenance of functionality of critical genes, and on specific differential gene expression, resulting in rapid adaptation.
... L. monocytogenes have been shown to have lisR (response regulator) and lisK (histidine kinase). The study by Cotter, Gahan, and Hill (2000) has demonstrated the role of LisRK in survival at low pH and regulation of virulence. Further studies indicated the tolerance to increased salt stress is due to the potassium dependent expression of kdpE and orfX gene products. ...
Listeria monocytogenes, during its transmission from the non-host to host environment, encounters an array of stress and a range of polymicrobial community. It has enhanced regulatory mechanisms to survive in non-host environments and adhere to both the abiotic and biotic surfaces. The biofilm markers and the virulence factors that assist L. monocytogenes in encountering the stress are tightly regulated. To effectively regulate the gene expression, L. monocytogenes employs quorum sensing as one of the mechanisms of stress response systems. The quorum-sensing system of L. monocytogenes shares homologs across genus and domain; thus, potentially capable of responding to communication signals released from other bacteria and host. The stress responses in L. monocytogenes could be modulated by interaction with the surrounding microflora, food components, and the host. Unrevealing molecular interactions that associate bacterial communication with stress response will assist in developing food processing technologies and novel therapeutics. In the current review, we discuss the stress response system in L. monocytogenes as modulated by inter-species and cross-domain bacterial communication. Also, the effect of host-released metabolites affecting the virulence properties of L. monocytogenes has been highlighted.
... long-term survival) in FPEs (Carpentier and Cerf 2011). The remarkable persistence of L. monocytogenes is due in part to a complex array of molecular mechanisms that provide increased protection against low pH (Cotter, Gahan and Hill 2000;Feehily et al. 2014;Lund, Tramonti and De Biase 2014), high osmotic pressure (Bucur et al. 2018), oxidative stress (Harter et al. 2017), disinfectants (Elhanafi, Dutta and Kathariou 2010;Muller et al. 2013Muller et al. , 2014, UVC (Kim et al. 2006) and temperatures as low as -0.4 • C (Chan and Wiedmann 2009). In addition to chromosomally encoded stress response systems, it has been revealed that L. monocytogenes plasmids contribute to stress tolerance. ...
Listeria monocytogenes is the causative agent of the highly fatal foodborne disease listeriosis and can persist in food production environments. Recent research highlights the involvement of L. monocytogenes plasmids in different stress response mechanisms, which contribute to its survival in food production facilities. Ultraviolet (UV) light in the UVC spectrum (200 to 280 nm) is used in food production to control microbial contamination. Although plasmid-encoded UV resistance mechanisms have been described in other bacteria, no research indicates that L. monocytogenes plasmids contribute to the UV stress response. The plasmids of L. monocytogenes strains 6179, 4KSM, and R479a are genetically distinct and were utilized to study the roles of plasmids in the UV response. Wildtype and plasmid-cured variant cells were grown to logarithmic or late-stationary phase, plated on agar plates, and exposed to UVC for 60 or 90 s, and colony-forming units (CFUs) were determined. CFUs of 6179 and 4KSM, bearing pLM6179 and p4KSM, respectively, were significantly (p-value < 0.05) higher than the plasmid-cured strains in both logarithmic and stationary phases. No difference in survival was observed for the R479a strain. Our data show for the first time that certain L. monocytogenes plasmids contribute to the survival of UVC light stress.
... Another mechanism for acid tolerance is the F0F1-ATPase complex. The F0F1-ATPase enzyme has two different domains: a cytoplasmic catalytic portion (F 1 ) responsible for ATP synthesis or hydrolysis and the integral membrane domain (F 0 ), functioning as a proton channel [98]. The enzyme synthesizes ATP aerobically using protons passing into the cell cytoplasm (oxidative phosphorylation) or hydrolyzes ATP as protons exit the cell, generating a proton motive force (PMF) [99]. ...
Listeria monocytogenes is a ubiquitous opportunistic pathogen responsible for the well-known listeriosis disease. This bacterium has become a common contaminant of food, threatening the food processing industry. Once consumed, the pathogen is capable of traversing epithelial barriers, cellular invasion, and intracellular replication through the modulation of virulence factors such as internalins and haemolysins. Mobile genetic elements (plasmids and transposons) and other sophisticated mechanisms are thought to contribute to the increasing antimicrobial resistance of L. monocytogenes. The environmental persistence of the pathogen is aided by its ability to withstand environmental stresses such as acidity, cold stress, osmotic stress, and oxidative stress. This review seeks to give an insight into L. monocytogenes biology, with emphasis on its virulence factors, antimicrobial resistance, and adaptations to environmental stresses.
... The two pathways can reduce the proton concentrations in the cytoplasm by incorporating intracellular protons with glutamate and ammonia, respectively (Cotter Ryan et al., 2008). Meanwhile, F 0 F 1 -ATPase can also work in a reverse direction by hydrolyzing ATP and pumping protons out of the cell (Cotter et al., 2000). Moreover, the changes of acid anions could be a probable cause to influence the ATR of bacteria (Hirshfield et al., 2003). ...
This study evaluated the acid tolerance response (ATR) of two strains of Listeria monocytogenes (serotype 1/2a and 4b) and one strain of Listeria innocua under different mildly acid conditions. Cells were incubated in combinations of three concentrations of lactic acid medium (3, 4.75, and 15 mM) and three external pH's (pHex 5.0, 6.0, and 6.5), plus, a HCl control, and a blank control (pH 7.4). Results showed that lactic acid induced lower log reduction of all three strains when challenged in severe acid conditions (pH 3.0) after being habituated at a pHex of 5.5 or 6.0 until the log phase, compared with a pHex of 6.5 or the two controls. This indicates that when the pHex was either 5.5 or 6.0 this induced a higher ATR of the strains, which may be caused by the ability of the strains to retain intracellular pH (pHi) homeostasis with pHi maintained in the range of 7.4-7.9. It was also found that a pHex of 5.5 resulted in the highest pHi of the strains across all incubated conditions, which indicates that the pHi may play an important role in the induction of ATR when Listeria cells are habituated in lactic acid, and if the higher pHi can be maintained, the ATR would be stronger. The concentration of lactic acid per se has no significant effect on ATR, which it is proposed was due to the pHi homeostasis maintained within the cells. However, the difference in ATR among three strains was also significant, which cannot be explained by the stable pHi of all tested strains. Therefore, other underlying mechanisms to mediate ATR under different conditions need to be explored in further studies.
... The effect of various environmental stimuli on the in vitro transcriptomic responses of several L. monocytogenes strains has been assessed. Among them, the effect of carbohydrates (Milenbachs et al. 1997, Milenbachs Lukowiak et al. 1997, Renzoni et al. 1997, high hydrostatic pressure processing (Bowman et al. 2008), extracellular pH value (Behari and Youngman 1998), thermal, acidic and osmotic shock (Phan-Thanh and Gormon 1995, Ripio et al. 1998, Sleator et al. 1999, 2001a,b, Hanawa et al. 1999, Cotter et al. 2000,Gaillot et al. 2000, Duche et al. 2002, Brondsted et al. 2003, Nelson et al. 2004, van der Veen et al. 2007, Schmid et al. 2009, Durack et al. 2013, Milecka et al. 2015, essential oils (Hadjilouka et al. 2017) and various antimicrobials (Romanova et al. 2006, Elhanafi et al. 2010, Kastbjerg et al. 2010, van der Veen and Abee 2010, Stasiewicz et al. 2011, Dutta et al. 2013, Shi et al. 2013, Pleitner et al. 2014, Laursen et al. 2015, Hadjilouka et al. 2016a) has been thoroughly studied (NicAogain et al. 2016). On the other hand, the transcription of virulence genes during growth on food matrices has only been marginally studied. ...
... Its ability to adapt to relatively harsh environmental conditions is one of the important features enabling its persistence in food processing environments and for establishing infection in the host. L. monocytogenes can maintain its intracellular pH (pH i ) homeostasis when exposed to an environmental pH (pH ex ) of 4.5, and survives well at pH ex 3.5 (Cotter et al., 2000). Pre-exposure of L. monocytogenes cells to mild acid stress (pH ex 4.5) may induce an acid tolerance response (ATR) that can render them more resistant to fatal acidic stress (O'Byrne and Karatzas, 2008). ...
Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. RsbR, an upstream regulator of the sigma B (SigB) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion (ΔrsbR), complementation (C-ΔrsbR), and phosphorylation site mutations in the rsbR (RsbR-T175A, RsbR-T209A, and RsbR-T175A-T209A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsbR reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175A disabled RsbR complementation, while RsbR-T209A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of RsbR are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.
Some Listeria monocytogenes strains are persistent in food processing environments, where this pathogen may be subjected to various stresses. This study aimed to elucidate the response of persistent strains of L. monocytogenes to low pH and H2O2 exposure. Almost all of the persistent strains examined were highly susceptible to low pH, whereas H2O2 susceptibility was comparable to that of control strains. Two persistent strains isolated from the same sample, however, exhibited lower susceptibility to low pH. These findings suggest an acid-susceptible phenotype predominates in the habitat, indicating that environmental conditions contribute to the establishment of persistence. Representative strains exhibiting acid-susceptible and less acid-susceptible phenotypes were further investigated regarding acid response characteristics. Less acid-susceptible strains exhibited increased survival in acidified brain heart infusion (BHI) broth compared with acidified phosphate-buffered saline (PBS). These strains also exhibited increased survival in acidified PBS containing glucose and glutamate, which are involved in acid response mechanisms, compared with acidified PBS alone. However, neither acidified BHI broth nor exogenous glucose and glutamate increased survival of acid-susceptible strains. An adaptive acid tolerance response of the acid-susceptible phenotype was observed, but this was limited compared with that of the less acid-susceptible phenotype.
Acids have been widely used in food processing to enhance flavour, prevent the deterioration of food and eliminate or retard the growth of foodborne pathogens. The inhibition of foodborne pathogens can be due to disruption of outer structure of the microbial cell, inhibition of metabolic processes, or damage to the macro-cellular components such as nucleic acids and enzymes. However, some foodborne pathogens might survive, adapt, and develop tolerance to acid stress via a wide range of molecular mechanisms. Various intrinsic and extrinsic factors can affect the development of acid tolerance responses. In this chapter, we reviewed the acids commonly used in the food industry. We also discussed different modes of action of acids, the tolerance response induced in foodborne pathogens after being exposed to acid, and the various factors affecting the development of acid tolerance response.
Bacteriocins from lactic acid bacteria are natural preservatives that inhibit foodborne pathogenic microorganisms and have recently attracted much attention. Acetic acid metabolism is part of the core of central metabolism. To elucidate the acetic acid stress response of L. paracasei HD1.7, we performed high-throughput RNA-seq to compare gene expression under different concentrations of acetic acid stress. The results showed that with increasing acetic acid stress, the concentration of bacteriocin was increased, its production rate was accelerated, and the number of differentially expressed genes also increased. The continuously upregulated and downregulated KEGG pathways adjusted to the increase in acetic acid stress. The late-stage regulatory system of bacteriocin synthesis was related to ABC transporters (OppABCDF system). Purine and pyrimidine metabolism may provide ATP for ABC transporters. Acetic acid stress reduced glutamate biosynthesis, but arginine biosynthesis increased to attenuate acid stress. In addition, the KEGG enrichment analysis results of different environmental stresses (acetic acid, HCl, citric acid and ethanol stress) indicated that ABC transporters and fatty acid metabolism were the keys to bacteria adapting to environmental stress. This experiment lays the foundation for further research on bacteriocins.
Our understanding of the tetrahydrofuran (THF) degradation in complex environment is limited. The majority of THF degrading genes reported are group V soluble diiron monooxygenases and share greater than 95% homology with one another. In this study, we used sole-carbon-source incubation combined with high-throughput metagenomic sequencing to investigate this contaminant's degradation in environmental samples. We identified as-yet-uncultivated microbe from the genera Pseudonocardia and fungi Scedosporium sp. (Scedosporium sp. was successfully isolated) as THF degraders as containing THF degradation genes, while microbes from the genera Bordetella, Pandoraea and Rhodanobacter functioned as main cooperators by utilizing acidic intermediates and providing anti-acid mechanisms. Furthermore, a 9387-bp THF degradation cluster designated thmX from the as-yet-uncultivated Pseudonocardia (with 6 main ORFs and with 79–93% amino acid sequence identity with previously reported clusters) was discovered. We also found a THF-degrading related cytochrome P450 monooxygenase from the genus Scedosporium and predicted its cognate reductase for the first time. All the genes and clusters mentioned above were successfully amplified from samples and cloned into the suitable expression vectors. This study will provide novel insights for understanding of THF degradation mechanisms under acid stress conditions and mining new THF degradation genes.
As part of their lifecycle neutralophilic bacteria are often exposed to varying environmental stresses, amongst which fluctuations in pH are the most frequent. In particular, acid environments can be encountered in many situations from fermented food to the gastric compartment of the animal host. Herein we review the current knowledge of the molecular mechanisms adopted by a range of Gram-positive and Gram-negative bacteria, mostly those affecting human health, for coping with acid stress. Because organic and inorganic acids have deleterious effects on the activity of the biological macromolecules to the point of significantly reducing growth and even threatening their viability, it is not unexpected that neutralophilic bacteria have evolved a number of different protective mechanisms, that provide them with an advantage in otherwise life-threatening conditions. The overall logic of these is to protect the cell from the deleterious effects of a harmful level of protons. Among the most favoured mechanisms are the pumping out of protons, production of ammonia, and proton-consuming decarboxylation reactions, as well as modifications of the lipid content in the membrane. Several examples are provided to describe mechanisms adopted to sense the external acidic pH. Particular attention is paid to Escherichia coli extreme acid resistance mechanisms, the activity of which ensure survival and may be directly linked to virulence.This article is protected by copyright. All rights reserved.
Preservation technologies subject bacterial cells to different levels of stress, which in the most effective cases lead to their inactivation and death. The term “stress‐ can refer to any extracellular influence that threatens the ability of microorganisms to perform their living functions. The food preservation technologies designed to rapidly inactivate microbial cells include thermal processes, irradiation, high‐pressure processing, and the use of strong oxidant compounds. Other technologies accomplish the preservation of foods by inhibiting growth; the most extensively used are low‐temperature storage (refrigeration and freezing), reduction of moisture content (concentration and drying), control of ox‐redox potential (use of controlled atmospheres and vacuum packaging), and acidification (fermentation and addition of organic acids). In nature, microorganisms are constantly exposed to similar changes in temperature, oxygen, moisture, light, pH, and chemical composition. Bacteria are able to survive thanks to a wide array of molecular responses that provide cellular protection against stresses. Bacteria are protected from changes in pH, temperature, oxidative conditions, solute concentrations, and pressure by a network of sophisticated global genetic regulatory systems and molecular stress responses specific to individual chemical or physical threats. The most important general regulators and specific genetic systems reported in representative foodborne pathogenic bacteria are highlighted in this chapter.
Staphylococcus aureus exhibits many defenses against host innate immunity, including the ability to replicate in the presence of nitric oxide (NO·). S. aureus NO· resistance is a complex trait and hinges on the ability of this pathogen to metabolically adapt to the presence of NO·. Here, we employed deep sequencing of transposon junctions (Tn-Seq) in a library generated in USA300 LAC to define the complete set of genes required for S. aureus NO· resistance. We compared the list of NO·-resistance genes to the set of genes required for LAC to persist within murine skin infections (SSTIs). In total, we identified 168 genes that were essential for full NO· resistance, of which 49 were also required for S. aureus to persist within SSTIs. Many of these NO·-resistance genes were previously demonstrated to be required for growth in the presence of this immune radical. However, newly defined genes, including those encoding SodA, MntABC, RpoZ, proteins involved with Fe-S-cluster repair/homeostasis, UvrABC, thioredoxin-like proteins and the F1F0 ATPase, have not been previously reported to contribute to S. aureus NO· resistance. The most striking finding was that loss of any genes encoding components of the F1F0 ATPase resulted in mutants unable to grow in the presence of NO· or any other condition that inhibits cellular respiration. In addition, these mutants were highly attenuated in murine SSTIs. We show that in S. aureus, the F1F0 ATPase operates in the ATP-hydrolysis mode to extrude protons and contribute to proton-motive force. Loss of efficient proton extrusion in the ΔatpG mutant results in an acidified cytosol. While this acidity is tolerated by respiring cells, enzymes required for fermentation cannot operate efficiently at pH ≤ 7.0 and the ΔatpG mutant cannot thrive. Thus, S. aureus NO· resistance requires a mildly alkaline cytosol, a condition that cannot be achieved without an active F1F0 ATPase enzyme complex.
Bacteria capable of causing foodborne infections must negotiate a long and tortuous passage from the environment to the site of infection in the susceptible host. Foodborne pathogens may encounter stressful environments during the production, preparation and storage of food. Following consumption they are exposed to the low pH of the stomach and survivors subsequently encounter volatile fatty acids, bile and low oxygen in the small intestine. Bacteria that survive to this point must compete with established gut flora for niches and nutrients and must overcome, among other insults, antimicrobial peptides produced by their competitors (Dunne et al., 1999). Those organisms capable of invasion subsequently penetrate the gut epithelium and are internalized within phagosomes, specialized organelles which prevent bacterial multiplication by means of acidic pH, and through the production of defensins (oxygen-independent mechanisms), hydrogen peroxide and superoxide radicals (oxygen-dependent mechanisms). To survive and grow in these inhospitable environments, foodborne pathogens possess mechanisms to overcome these stresses, and thus are capable of colonization resulting in either clinical or sub-clinical infection. In this chapter, the mechanisms employed by foodborne pathogens to adapt to the host environment and cause disease have been considered. Our primary focus is the pathogenesis of Listeria monocytogenes and Salmonella enterica serovar Typhimurium as examples of Gram-positive and Gram-negative foodborne pathogens capable of causing invasive disease in a mouse model of infection.
Stress Responses, Adaptation, and Virulence of Bacterial Pathogens During Host Gastrointestinal Colonization, Page 1 of 2
Abstract
Bacterial pathogens are exposed to a variety of stressful conditions while spreading to and colonizing new hosts to cause infection. Gastrointestinal pathogens such as Campylobacter, Escherichia, Helicobacter, Listeria, Salmonella, and Shigella species encounter numerous stresses during host colonization and infection. During transit through the gastrointestinal tract these pathogens are exposed to physical stresses (pH and osmotic stresses) as well as noxious substances (reactive oxygen and nitrosative species). Bacteria respond to these stresses by altering their transcriptome/proteome in an adaptive manner to either overcome the stress or resist the stress long enough to transition to more favorable conditions. The following sections will present the current state of knowledge for each stress response mentioned above and how these defenses contribute to bacterial virulence. Virulence Mechanisms of Bacterial Pathogens, Fifth Edition
Numerous commensal and pathogenic Gram-negative and Gram-positive bacteria are referred to as neutralophiles because they grow best at pH levels close to neutrality. Thus, exposure to harsh-to-mild acidic environments, such as those encountered in the digestive tract of animal hosts, in the phagosome of macrophages, in fermented foods, but also in the soil or in acid mine drainage, is a rather common encounter for neutralophiles during their life cycle. As a result, it is not surprising that most of them have evolved sophisticated molecular mechanisms to cope with low pH. These protective mechanisms provide neutralophiles with the ability to sense acid pH and keep under control the intracellular acidification of the cytoplasm, thus avoiding protons from reaching such harmful levels as to compromise cellular vitality, which relies on the proper functioning of many biological macromolecules at pH levels near neutrality. The aim of this chapter is to provide an overview of the most commonly employed, and best characterized, molecular systems in a number of Gram-positive and Gram-negative bacteria. How they work inside the cell and how their activity can be linked to virulence are highlighted. The biochemistry and distribution of the glutamate-dependent acid resistance system among orally acquired bacteria are described in some detail.
Optochin, a cinchona alkaloid derivative discovered over 100 years ago, possesses highly selective antibacterial activity towards Streptococcus pneumoniae. Pneumococcal disease remains the leading source of bacterial pneumonia and meningitis worldwide. The structure activity relationships of optochin were examined through modification to both the quinoline and quinuclidine subunits, which led to the identification of analogue 48 with substantially improved activity. Resistance and molecular modeling studies indicate 48 likely binds to the c-ring of ATP synthase near the conserved glutamate 52 ion binding site while mechanistic studies demonstrated 48 causes cytoplasmic acidification. Initial pharmacokinetic and drug metabolism analysis of optochin and 48 revealed limitations of these quinine analogues, which were rapidly cleared resulting in poor in vivo exposure through hydroxylation pendant to the quinuclidine and O-dealklyation of the quinoline. Collectively, the results provide a foundation to advance 48 and highlight ATP synthase as a promising target for antibiotic development.
Lis.te'ri.a. N.L. fem. n. Listeria named after Lord Lister, English surgeon and pioneer of antisepsis. Firmicutes / “Bacilli” / Bacillales / “Listeriaceae” / Listeria Regular, short rods, 0.4–0.5 × 1–2 µm with parallel sides and blunt ends. Usually occur singly or in short chains. In older or rough cultures, filaments of ≥6 µm in length may develop. Gram-positive with even staining, but some cells, especially in older cultures, lose their ability to retain the Gram strain. Not acid-fast. Capsules not formed. Do not form spores. All species motile with peritrichous flagella when cultured <30°C. Aerobic and facultative anaerobic. Colonies (24–48 h) are 0.5–1.5 mm in diameter, round, translucent, low convex with a smooth surface and entire margin, non-pigmented with a crystalline central appearance. May be sticky when removed from agar surfaces, usually emulsify easily, and may leave a slight impression on the agar surface after removal. Older cultures (3–7 d) are larger, 3–5 mm in diameter, have a more opaque appearance, sometimes with a sunken center: rough colonial forms may develop. In 0.25% (w/v) agar, 8% (w/v) gelatin and 1.0% (w/v) glucose semi-solid medium, growth along the stab after 24 h at 37°C is followed by irregular, cloudy extensions into the medium. Growth spreads slowly through the entire medium. An umbrella-like zone of maximal growth occurs 3–5 mm below the surface. Temperature limits of growth <0 to 45°C; optimal growth at 30–37°C. Do not survive heating at 60°C for 30 min. Growth occurs between pH 6 and pH 9. Growth occurs in nutrient broth supplemented with up to 10% (w/v) NaCl. Catalase-positive, oxidase-negative. Cytochromes produced. Homofermentative anaerobic catabolism of glucose results in production of l(+)-lactic acid, acetic acid plus other end products. Acid but no gas produced from other sugars. Methyl-red-positive, Voges–Proskauer-positive. Exogenous citrate not utilized. Organic growth factors are required. Indole is not produced. Esculin and sodium hippurate are hydrolyzed. Urea is not hydrolyzed. Gelatin, casein, and milk are not hydrolyzed. DNA G+C content (mol%): 36–42.5 (Tm). Type species: Listeria monocytogenes Pirie 1940a.
As with most foodborne pathogens, Listeria monocytogenes, during its life cycle, encounters many environmental stresses, beginning with its residence in soil, through the food production chain until it encounters host challenges to accomplish a successful infection process. Exposure to a single stress or a combination of stresses can compromise the integrity of the bacterial cell, and to circumvent those injuries the pathogen is equipped with mechanisms that can sense sublethal stress and trigger its gene expressing arsenal to reprogram its mode of survival by expressing stress tolerance factors. Such tolerance factors were initially identified using laboratory media. However, these approaches did not reliably translate those tolerance responses in real food matrices that the pathogen requires, and the molecular elements associated with those responses. Using different omics approaches, recent studies are revealing the tolerance responses associated with different food matrices and their possible impact in enabling the pathogen to better survive in vivo stress challenges. This knowledge is useful for developing new and more efficient control strategies and improving food safety, especially for minimally processed foods.
Accurate identification of the infection source and the transmission route are necessary for the effective implementation of preventive measures against microbial food-borne pathogens. Advances in the field of molecular biology has allowed the development of sophisticated techniques able to detect differences at genomic level and through which studies of epidemiological nature may be conducted. Techniques, such as pulsed-field gel electrophoresis (PFGE), multilocus variable number of tandem repeats analysis (MLVA), and multilocus sequence typing (MLST) have been thoroughly studied and extensively applied. These techniques are characterized by specific strengths and weaknesses that need to be taken into consideration before any conclusion is drawn. In this chapter all information related to typing approaches of Listeria monocytogenes, Salmonella serovars, Escherichia coli O157:H7, and Campylobacter spp. are integrated and critically discussed.
The product of the spollM gene of Bacillus subtilis is required for complete septum migration and forespore engulfment during sporulation. To investigate whether expression of spollM is required in the forespore compartment of the sporangium, we have constructed a new integrational vector, pKSV7, which contains temperature-sensitive replication functions derived from pE194ts. The presence of the conditionally defective replication origin greatly stimulates plasmid excision when sporulation occurs at the permissive temperature. This facilities the use of a genetic technique employed by Illing et al [1] to distinguish genes whose expression must occur in the forespore from genes that may be expressed exclusively in the mother cell compartment. The results of the integration/excision experiments using pKSV7 support the conclusion that spollM must be expressed in the mother cell. The conditional integration analysis of porespore and mother cell fractions suggests that spollM is also expressed in the mother cell. The conditional integration vector pKSV7 replicates at high copy number in E coli and allows the identification of inserts in the polylinker cluster by disruption of α-complementation and thus should be useful other kinds of genetic manipulations in B subtilis.
L-malate transport was studied in membrane vesicles from Leuconostoc oenos MLE(-) (mutant lacking malolactic enzyme) which were fused with liposomes containing beef heart cytochrome c oxidase as a proton-motive-force-generating system. In these hybrid membranes, accumulation of L-malate was observed in response to a pH gradient (ΔpH), with the inside alkaline, but was strongly inhibited by a membrane potential (ΔΨ) of normal polarity (inside negative). Imposition of a ΔΨ, with the inside positive, by means of valinomycin-mediated potassium influx, resulted in a rapid accumulation of L-malate, indicating that L-malate was taken up in an anionic form. The results are consistent with a uniport mechanism facilitating the uptake of monoanionic L-malate, the dominant species at the low pH of the experiments. Kinetic analysis of ΔpH-driven L-malate uptake in the pH range 3.0-5.8, yielded apparent affinity constants that varied less than twofold when calculated on the basis of the concentrations of monoanionic L-malate, whereas the values differed 2-3 orders of magnitude for the other species. At L-malate concentrations above 1 mM, a non-saturable transport component became apparent which may reflect passive influx of L-malic acid. Substrate specificity studies indicated that citrate and L-malate (and possibly D-lactate and L-lactate) compete for a single general carboxylate transport system. The carboxylate transport system catalysed homologous L-malate and heterologous L-malate/citrate exchange with rates similar to the rate of L-malate efflux. Since metabolic energy is conserved during malolactic fermentation in L. oenos, the underlying mechanism most likely involves electrogenic monoanionic L-malate uptake, in combination with H+ consumption in the cytoplasm, followed by diffusion outwards of lactic acid plus carbon dioxide.
Lactobaciflus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (Δψ), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate that histidine uptake, histamine efflux, and histidine/histamine exchange are electrogenic processes. Histidine/histamine exchange is much faster than the unidirectional fluxes of these substrates, is inhibited by an inside-negative Δψ and is stimulated by an inside positive Δψ. These data suggest that the generation of metabolic energy from histidine decarboxylation results from an electrogenic histidine/histamine exchange and indirect proton extrusion due to the combined action of the decarboxylase and carrier-mediated exchange. The abundance of amino acid decarboxylation reactions among bacteria suggests that this mechanism of metabolic energy generation and/or pH regulation is widespread.
We discuss our recent results on the Escherichia coli F-ATPase, in particular its catalytic site in the beta subunit and regulation of H+ transport by the gamma subunit. Affinity labelling experiments suggest that beta Lys-155 in the glycine-rich sequence is near the gamma-phosphate moiety of ATP bound at the catalytic site. The enzyme loses activity upon introduction of missense mutations in beta Lys-155 or beta Thr-156 and changes catalytic properties upon introduction of other mutations. By analysis of mutations and their pseudo revertants, residues beta Ser-174, beta Glu-192 and beta Val-198 were found to be located near the glycine-rich sequence. The combined approaches of chemical labelling and genetics have been fruitful in visualizing the structure of the catalytic site. Analysis of mutations in the gamma subunit suggests that this subunit has an essential role in coupling catalysis with proton translocation.
The acid tolerance response (ATR) is an adaptive system triggered at external pH (pHo) values of 5.5 to 6.0 that will protect cells from more severe acid stress (J. Foster and H. Hall, J. Bacteriol. 172:771-778, 1990). Correlations between the internal pH (pHi) of adapted versus unadapted cells at pHo of 3.3 indicate that the ATR system produces an inducible pH-homeostatic function. This function serves to maintain the pHi above 5 to 5.5. Below this range, cells rapidly lose viability. Development of this pH homeostasis mechanism was sensitive to protein synthesis inhibitors and operated only to augment the pHi at pHo values below 4. In contrast, classical constitutive pH homeostasis was insensitive to protein synthesis inhibitors and was efficient only at pHo values above 4. Physiological studies indicated an important role for the Mg(2+)-dependent proton-translocating ATPase in affording ATR-associated survival during exposure to severe acid challenges. Along with being acid intolerant, cells deficient in this ATPase did not exhibit inducible pH homeostasis. We speculate that adaptive acid tolerance is important to Salmonella species in surviving acid encounters in both the environment and the infected host.
L-Malate transport in Lactobacillus plantarum was inducible, and the pH optimum was 4.5. Malate uptake could be driven by an artificial proton gradient (delta pH) or an electroneutral lactate efflux. Because L-lactate efflux was unable to drive L-malate transport in the absence of a delta pH, it did not appear that the carrier was a malate-lactate exchanger. The kinetics of malate transport were, however, biphasic, suggesting that the external malate concentration was also serving as a driving force for low-affinity malate uptake. Because the electrical potential (delta psi, inside negative) inhibited malate transport, it appeared that the malate transport-lactate efflux couple was electrogenic (net negative) at high concentrations of malate. De-energized cells that were provided with malate only generated a large proton motive force (greater than 100 mV) when the malate concentration was greater than 5 mM, and malate only caused an increase in cell yield (glucose-limited chemostats) when malate accumulated in the culture vessel. The use of the malate gradient to drive malate transport (facilitated diffusion) explains how L. plantarum derives energy from malolactic fermentation, a process which does not involve substrate-level phosphorylation.
The gram-positive bacterium Listeria monocytogenes is an ubiquitous, intracellular pathogen which has been implicated within the past decade as the causative organism in several outbreaks of foodborne disease. Listeriosis, with a mortality rate of about 24%, is found mainly among pregnant women, their fetuses, and immunocompromised persons, with symptoms of abortion, neonatal death, septicemia, and meningitis. Epidemiological investigations can make use of strain-typing procedures such as DNA restriction enzyme analysis or electrophoretic enzyme typing. The organism has a multifactorial virulence system, with the thiol-activated hemolysin, listeriolysin O, being identified as playing a crucial role in the organism's ability to multiply within host phagocytic cells and to spread from cell to cell. The organism occurs widely in food, with the highest incidences being found in meat, poultry, and seafood products. Improved methods for detecting and enumerating the organism in foodstuffs are now available, including those based on the use of monoclonal antibodies, DNA probes, or the polymerase chain reaction. As knowledge of the molecular and applied biology of L. monocytogenes increases, progress can be made in the prevention and control of human infection.
In the Gram-negative anaerobe, Oxalobacter formigenes, the generation of metabolic energy depends on the transport and decarboxylation of oxalate. We have now used assays of reconstitution to study the movements of oxalate and to characterize the exchange of oxalate with formate, its immediate metabolic derivative. Membranes of O. formigenes were solubilized with octyl-beta-D-glucopyranoside in the presence of 20% glycerol and Escherichia coli phospholipid, and detergent extracts were reconstituted by detergent dilution. [14C]Oxalate was taken up by proteoliposomes loaded with unlabeled oxalate, but not by similarly loaded liposomes or by proteoliposomes containing sulfate in place of oxalate. Oxalate transport did not depend on the presence of sodium or potassium, nor was it affected by valinomycin (1 microM), nigericin (1 microM), or a proton conductor, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (5 microM) when potassium was at equal concentration on either side of the membrane. Such data suggest the presence of an overall neutral oxalate self-exchange, independent of common cations or anions. Kinetic analysis of the reaction in proteoliposomes gave a Michaelis constant (Kt) for oxalate transport of 0.24 mM and a maximal velocity (Vmax) of 99 mumol/min/mg of protein. A direct exchange of oxalate and formate was indicated by the observations that formate inhibited oxalate transport and that delayed addition of formate released [14C]oxalate accumulated during oxalate exchange. Moreover, [14C]formate was taken up by oxalate-loaded proteoliposomes (but not liposomes), and this heterologous reaction could be blocked by external oxalate. Further studies, using formate-loaded proteoliposomes, suggested that the heterologous exchange was electrogenic. Thus, for assays in which N-methylglucamine served as both internal and external cation, formate-loaded particles took up oxalate at a rate of 2.4 mumol/min/mg of protein. When external or internal N-methylglucamine was replaced by potassium in the presence of valinomycin, there was, respectively, a 7-fold stimulation or an 8-fold inhibition of oxalate accumulation, demonstrating that net negative charge moved in parallel with oxalate during the heterologous exchange. The work summarized here suggests the presence of an unusually rapid and electrogenic oxalate2-:formate1- antiport in membranes of O. formigenes. Since a proton is consumed during the intracellular decarboxylation that converts oxalate into formate plus CO2, antiport of oxalate and formate would play a central role in a biochemical cycle consisting of (a) oxalate influx, (b) oxalate decarboxylation, and (c) formate efflux.(ABSTRACT TRUNCATED AT 400 WORDS)
We cloned and sequenced an operon of nine genes coding for the subunits of the Bacillus subtilis F0F1 ATP synthase. The arrangement of these genes in the operon is identical to that of the atp operon from Escherichia coli and from three other Bacillus species. The deduced amino acid sequences of the nine subunits are very similar to their counterparts from other organisms. We constructed two B. subtilis strains from which different parts of the atp operon were deleted. These B. subtilis atp mutants were unable to grow with succinate as the sole carbon and energy source. ATP was synthesized in these strains only by substrate-level phosphorylation. The two mutants had a decreased growth yield (43 and 56% of the wild-type level) and a decreased growth rate (61 and 66% of the wild-type level), correlating with a twofold decrease of the intracellular ATP/ADP ratio. In the absence of oxidative phosphorylation, B. subtilis increased ATP synthesis through substrate-level phosphorylation, as shown by the twofold increase of by-product formation (mainly acetate). The increased turnover of glycolysis in the mutant strain presumably led to increased synthesis of NADH, which would account for the observed stimulation of the respiration rate associated with an increase in the expression of genes coding for respiratory enzymes. It therefore appears that B. subtilis and E. coli respond in similar ways to the absence of oxidative phosphorylation.
The atp and fur genes are involved in the acid tolerance response of Salmonella typhimurium. An atp::Tn10 mutant was avirulent in the mouse typhoid model when assayed by oral and intraperitoneal routes. However, a fur mutant was completely virulent by the intraperitoneal route. No relevant differences in intracellular survival or invasion rates were observed for the two mutants in macrophages and epithelial cells. These data indicate that separate acid tolerance response genes may have different roles in S. typhimurium virulence.
A system for generating chromosomal insertions in lactococci is described. It is based on the conditional replication of lactococcal pWV01-derived Ori+ RepA- vector pORI19, containing lacZ alpha and the multiple cloning site of pUC19. Chromosomal AluI fragments of Lactococcus lactis were cloned in pORI19 in RepA+ helper strain Escherichia coli EC101. The frequency of Campbell-type recombinants, following introduction of this plasmid bank into L. lactis (RepA-), was increased by combining the system with temperature-sensitive pWV01 derivative pVE6007. Transformation of L. lactis MG1363 (pVE6007) with the pORI19 bank of lactococcal chromosomal fragments at the permissive temperature allowed replication of several copies of a recombinant plasmid from the bank within a cell because of the provision in trans of RepA-Ts from pVE6007. A temperature shift to 37 degrees C resulted in loss of pVE6007 and integration of the pORI19 derivatives at high frequencies. A bank of lactococcal mutants was made in this way and successfully screened for the presence of two mutations: one in the monocistronic 1.3-kb peptidoglycan hydrolase gene (acmA) and one in the hitherto uncharacterized maltose fermentation pathway. Reintroduction of pVE6007 into the Mal- mutant at 30 degrees C resulted in excision of the integrated plasmid and restoration of the ability of ferment maltose. The integration plasmid (pMAL) was rescued by using the isolated plasmid content of a restored Mal+ colony to transform E. coli EC101. Nucleotide sequencing of the 564-bp chromosomal fragment in pMAL revealed an internal part of an open reading frame of which the translated product showed significant homology with ATP-binding proteins MalK of E. coli, Salmonella typhimurium, and Enterobacter aerogenes and MsmK of Streptococcus mutans. This combined use of two types of conditional replicating pWV01-derived vectors represents a novel, powerful tool for chromosomal gene inactivation, targeting, cloning, and sequencing of the labelled gene.
During its life cycle, Salmonella typhimurium is exposed to a variety of acidic conditions. Survival in the acidic environments within the host may require the adaptive acid tolerance response (ATR), which is characterized by the induction of several Salmonella proteins upon exposure to mildly acidic conditions. These induced proteins protect the bacterium from death under severe acid challenge. The goal of this study was to examine the role of ATR in Salmonella pathogenesis. Initially, we observed that differences exist between the virulent S. typhimurium strains and the laboratory S. typhimurium strain LT2 with respect to their ATR. Mutations affecting the ATR of S. typhimurium LT2, including atrB, atrC (polA), atrD, atbR, and fur, were crossed into virulent Salmonella strains, and the resultant transductants were screened for virulence in mice and acid sensitivity. Surprisingly, with the exception of the fur mutation, none of the muatations had a major effect on acid resistance or virulence in the pathogenic strains. The fur mutants showed a 1-to 3-log increase in the 50% lethal dose; however, the magnitude of its effect was dependent on the strain background. Strains containing two or three different atr mutations were constructed, and these were also examined for acid sensitivity and virulence. The double and triple mutants that contained an atrC mutation no longer displayed an ATR. Those mutants which were more acid sensitive were also highly attenuated, suggesting a strong correlation between the ability to mount and ATR and virulence in S. typhimurium. Comparison of the ability of the various atr single, double, and triple mutants to survive within macrophages showed that strains containing an atrC mutation survived much less than the wild type in bone marrow-derived macrophages. No difference in survival within J774 macrophage like cells were detected.
The ability of Listeria monocytogenes to tolerate low-pH environments is of particular importance because the pathogen encounters such environments in vivo, both during passage through the stomach and within the macrophage phagosome. In our study, L. monocytogenes was shown to exhibit a significant adaptive acid tolerance response following a 1-h exposure to mild acid (pH 5.5), which is capable of protecting cells from severe acid stress (pH 3.5). Susceptibility to pH 3.5 acid is growth phase dependent. Stationary-phase Listeria cultures are naturally resistant to the challenge pH (pH 3.5), while exponential-phase cultures require adaptation at pH 5.5 to induce acid tolerance. Adaptation requires protein synthesis, since treatment with chloramphenicol prevents the development of acid tolerance. Induction of the acid tolerance response also protects L. monocytogenes against the effect of other environmental stresses. Acid-adapted cells demonstrate increased tolerance toward thermal stress, osmotic stress, crystal violet, and ethanol. Following prolonged exposure of L. monocytogenes to pH 3.5, we isolated mutants which constitutively demonstrate increased acid tolerance at all stages of the growth cycle. These mutants do not display full acid tolerance, but their resistance to low pH can be further increased following adaptation to mild-acid conditions. The mutants demonstrated increased lethality for mice relative to that of the wild type when inoculated by the intraperitoneal route. When administered as lower inocula, the mutants reached higher levels in the spleens of infected mice than did the wild type. The data suggest that low-pH conditions may have the potential to select for L. monocytogenes mutants with increased natural acid tolerance and increased virulence.
We have previously shown that tolerance to severe acid stress (pH 3.5) can be induced in Listeria monocytogenes following a 1-h adaptation to mild acid (pH 5.5), a phenomenon termed the acid tolerance response (ATR) (B. O'Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ. Microbiol. 62:1693-1698, 1966). In an attempt to determine the industrial significance of the ATR, we have examined the survival of adapted and nonadapted cells in a variety of acidic foods. Acid adaptation enhanced the survival of L. monocytogenes in acidified dairy products, including cottage cheese, yogurt, and whole-fat cheddar cheese. Acid-adapted L. monocytogenes cultures also demonstrated increased survival during active milk fermentation by a lactic acid culture. Similarly, acid-adapted cells showed greatly improved survival in low-pH foods (orange juice and salad dressing) containing acids other than lactic acid. However, in foods with a marginally higher pH, such as mozzarella cheese, a commercial cottage cheese, or low-fat cheddar cheese, acid adaptation did not appear to enhance survival. We have previously isolated mutants of L. monocytogenes that are constitutively acid tolerant in the absence of an induction step (O'Driscoll et al., Appl. Environ. Microbiol. 62:1693-1698, 1996). In the present study, one such mutant, ATM56, demonstrated an increased ability to survive in low-pH foods and during milk fermentation when compared with the wild-type strain. Significant numbers of ATM56 could be recovered even after 70 days in both whole-fat and low-fat cheddar cheese. Collectively, the data suggest that ATR mechanisms, whether constitutive or induced, can greatly influence the survival of L. monocytogenes in low-pH food environments.
The acid tolerance of a Listeria monocytogenes serotype 4b strain was studied by measuring its ability to survive at an acidic pH at 37 degrees C. The acid tolerance of L. monocytogenes was much lower than those of Escherichia coli O157:H7 and Shigella flexneri strains. This observation suggested a higher infective dose for L. monocytogenes than E. coli O157:H7 and Shigella. The susceptibility of L. monocytogenes to acidic pH was dependent upon growth medium pH and growth phase of the culture. Nisin and some other ionophores reduced the acid tolerance of both stationary-phase and log-phase cultures of L. monocytogenes. These studies indicated that nisin might be a useful candidate for controlling acid tolerance of L. monocytogenes.
Exposing Listeria monocytogenes LO28 to sublethal pH induces protection against normally lethal pH conditions, a phenomenon known as the acid tolerance response. We identified a mutant, L. monocytogenes ATR1, which is incapable of inducing such tolerance, either against low pH or against any other stress tested. The virulence of this mutant was considerably decreased, suggesting that the acid tolerance response contributes to in vivo survival of L. monocytogenes.
lisRK encodes a two-component regulatory system in the food pathogen Listeria monocytogenes LO28. Following identification of the operon in an acid-tolerant Tn917 mutant, a deletion in the histidine kinase component was shown to result in a growth phase variation in acid tolerance, an ability to grow in high ethanol concentrations, and a significant reduction in virulence.
Listeria monocytogenes is capable of withstanding low pH after initial exposure to sublethal acidic conditions, a phenomenon termed the acid tolerance response (B. O'Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ. Microbiol. 62:1693-1698, 1996). Treatment of L. monocytogenes LO28 with chloramphenicol during acid adaptation abrogated the protective effect, suggesting that de novo protein synthesis is required for the acid tolerance response. Analysis of protein expression during acid adaptation by two-dimensional gel electrophoresis revealed changes in the levels of 53 proteins. Significant protein differences were also evident between nonadapted L. monocytogenes LO28 and a constitutively acid-tolerant mutant, ATM56. In addition, the analysis[S_TABC] revealed differences in protein expression between cells induced with a weak acid (lactic acid) and those induced with a strong acid (HCl). Comparison of both acid-adapted LO28 and ATM56 revealed that both are capable of maintaining their internal pH (pH(infi)) at higher levels than nonadapted control cells during severe acid stress. Collectively, the data demonstrate the profound alterations in protein synthesis which take place during acid adaptation in L. monocytogenes and ultimately lead to an increased ability to survive severe stress conditions.
The gram-positive bacterium Listeria monocytogenes is an ubiquitous, intracellular pathogen which has been implicated within the past decade as the causative organism in several outbreaks of foodborne disease. Listeriosis, with a mortality rate of about 24%, is found mainly among pregnant women, their fetuses, and immunocompromised persons, with symptoms of abortion, neonatal death, septicemia, and meningitis. Epidemiological investigations can make use of strain-typing procedures such as DNA restriction enzyme analysis or electrophoretic enzyme typing. The organism has a multifactorial virulence system, with the thiol-activated hemolysin, listeriolysin O, being identified as playing a crucial role in the organism's ability to multiply within host phagocytic cells and to spread from cell to cell. The organism occurs widely in food, with the highest incidences being found in meat, poultry, and seafood products. Improved methods for detecting and enumerating the organism in foodstuffs are now available, including those based on the use of monoclonal antibodies, DNA probes, or the polymerase chain reaction. As knowledge of the molecular and applied biology of L. monocytogenes increases, progress can be made in the prevention and control of human infection.
Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (delta psi), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate that histidine uptake, histamine efflux, and histidine/histamine exchange are electrogenic processes. Histidine/histamine exchange is much faster than the unidirectional fluxes of these substrates, is inhibited by an inside-negative delta psi and is stimulated by an inside positive delta psi. These data suggest that the generation of metabolic energy from histidine decarboxylation results from an electrogenic histidine/histamine exchange and indirect proton extrusion due to the combined action of the decarboxylase and carrier-mediated exchange. The abundance of amino acid decarboxylation reactions among bacteria suggests that this mechanism of metabolic energy generation and/or pH regulation is widespread.
This chapter discusses concepts and techniques originating outside the bacterial world, and especially upon Peter Mitchell's chemiosmotie hypothesis. It aims to construct a framework to hang the present and future experimental data on. The chapter describes the balance of the charge account and with the physical state of the cytoplasmic ions. It discusses the use of ionophores. It also discusses the theoretical concepts, surveys methods for measuring membrane potentials and then turns to the emerging evidence for electrogenic proton transport as the metabolic basis of electrical potentials across bacterial membranes. The emerging evidence for electrogenic transport of protons by vectorial ATPase and a respiratory chain, with the generation of an electrical potential across the plasma membrane are discussed. The chapter discusses the transport of the alkali metal ions and the interrelationship of the cation fluxes. It also discusses the significance of the cations transport process.
The intracellular pH (pHi) optimum for glycolysis in Streptococcus mutans Ingbritt was determined to be 7.0 by use of the ionophore gramicidin for manipulation of pHi. Glycolytic activity decreased to zero as the pHi was lowered from 7.0 to 5.0. In contrast, glycolysis had an extracellular pH (pHo) optimum of 6.0 with a much broader profile. The relative insensitivity of glycolysis to the lowering of pHo was attributed to the ability of S. mutans to maintain a transmembrane pH gradient (delta pH, inside more alkaline) at low pHo. At a pHo of 5.0, glycolyzing cells of S. mutans maintained a delta pH of 1.37 +/- 0.09 units. The maintenance of this delta pH was dependent on the concentration of potassium ions in the extracellular medium. Potassium was rapidly taken up by glycolyzing cells of S. mutans at a rate of 70 nmol/mg dry weight/min. This uptake was dependent on the presence of both ATP and a proton motive-force (delta p). The addition of N-N'-dicyclohexylcarbodiimide (DCCD) to glycolyzing cells of S. mutans caused a partial collapse of the delta pH. Growth of S. mutants at pHo 5.5 in continuous culture resulted in the maintenance of a delta pH larger than that produced by cells grown at pH 7.0. These results suggest the presence of a proton-translocating F1Fo-ATPase in S. mutans whose activity is regulated by the intracellular pH and transmembrane electrical potential (delta psi). The production of an artificial delta p of 124 mV across the cell membrane of S. mutans did not result in proton movement through the F1Fo-ATPase coupled to ATP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Salmonella typhimurium was adapted to acid by exposure to hydrochloric acid at pH 5.8 for one to two doublings. Acid-adapted cells had increased resistance to inactivation by organic acids commonly present in cheese, including lactic, propionic, and acetic acids. Recovery of cells during the treatment with organic acids was increased 1,000-fold by inclusion of 0.1% sodium pyruvate in the recovery medium. Acid-adapted S. typhimurium cells survived better than nonadapted cells during a milk fermentation by a lactic acid culture. Acid-adapted cells also showed enhanced survival over a period of two months in cheddar, Swiss, and mozzarella cheeses kept at 5 degrees C. Acid adaptation was found in Salmonella spp., including Salmonella enteritidis, Salmonella choleraesuis subsp. choleraesuis serotype heidelberg, and Salmonella choleraesuis subsp. choleraesuis serotype javiana, associated with food poisoning. These observations support the theory that acid adaptation is an important survival mechanism enabling Salmonella spp. to persist in fermented dairy products and possibly other acidic food products.
The highly conserved portion of the catalytic subunit (beta-subunit) of the membrane-bound, proton-translocating ATPase from three strains of oral streptococci has been amplified via the polymerase chain reaction. Hybridization studies demonstrated the existence of homology between Escherichia coli and Bacillus megaterium beta-subunit probes at the streptococcal DNA level. Highly degenerate primers, based on the E. coli and B. megaterium amino acid (aa) sequences, were used to amplify the homologues in Streptococcus mutans, S. sanguis and S. sobrinus. The 600 bp fragment from S. sobrinus has been cloned and its nucleotide (nt) sequence determined. Comparison of its nt and deduced aa sequence to that of E. coli and B. megaterium reveals a high degree of homology at the aa level.
Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5'-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.
Experiments, relevant to growth in milk, were done to delineate the aerobic and anaerobic growth of Listeria species on selected sugars in several media. All species grew on glucose aerobically, forming lactic acid and (or) acetic acid. Anaerobically, only lactic acid was formed; cell yields were 80% of those obtained aerobically. When incubated aerobically, small amounts (1.5 microns/mL) of isovaleric acid, 2-hydroxyisovaleric acid, and trace amounts of isobutyric acid were formed. These products were characteristically formed by 26 strains representing all the species of Listeria. Added leucine stimulated isovaleric acid formation. Anaerobic fermentations of glucose could be followed by 60 to 80% cell lysis; less lysis occurred in air. Anaerobically, only hexoses and pentoses supported growth; aerobically, maltose and lactose supported growth of some strains, but sucrose did not support growth of any strain tested. Listeria grayi and Listeria murrayi utilized the galactose and glucose moieties of lactose for growth; Listeria monocytogenes and Listeria innocua used only the glucose moiety. Glucosamine and N-acetylglucosamine supported aerobic and anaerobic growth as well as glucose, and their presence stimulated the utilization of lactose by "lactose-negative" strains. Analyses of cultures grown at 5 degrees C in sterile milk treated with glucose oxidase supported the conclusion that the glucose of the milk was the major, if not the limiting, substrate that supported growth.
Four Escherichia coli mutants with defects in the alpha subunit of H+-ATPase (F0F1) (strain KF154, Pro-281----Leu; KF101 and KF131, Ala-285----Val; KF114, Arg-376----Cys) were isolated, and the kinetic properties of their F1-ATPases were studied. All the mutations so far identified are clustered in the two defined regions of the alpha subunit. With F1 of strain KF114, as with F1 of uncA401 (Ser-373----Phe; T. Noumi, M. Futai, and H. Kanazawa (1984) J. Biol. Chem. 259, 10076-10079), the rate of multisite hydrolysis of ATP was 4 X 10(-3)-fold lower than that with wild-type F1, suggesting that residues Ser-373 and Arg-376 or the regions in their vicinities are essential for positive catalytic cooperativity. With F1 from strain KF101, multisite hydrolysis was higher (about 40% of that of the wild type), but the F1 was unstable and showed defective interaction with the membrane sector (F0). The F1 from KF154 had lower multisite hydrolysis (about 10% of that of the wild type) but could support slow growth by oxidative phosphorylation.
The distribution of [14C]dimethyloxazolidinedione was employed to measure the internal pH of Streptococcus faecalis. Glycolyzing cells maintained an internal pH more alkaline than that of the medium by 0.5–1 unit. The pH difference disappeared upon exhaustion of glucose. Arginine metabolism did not establish a pH gradient.The pH gradient was abolished by metabolic inhibitors of two kinds: inhibitors of the membrane-bound ATPase including N,N′-dicyclohexylcarbodiimide and chlorhexidine; and proton conductors including tetrachlorosalicylanilide, carbonylcyanide m-chlorophenylhydrazone and the antibiotics nigericin and monensin. The antibiotics valinomycin and monactin which facilitate K+ diffusion did not affect the pH gradient so long as the external K+ concentration was relatively high; at low K+ concentrations they lowered the internal pH.To study the relationship of internal pH to K+ accumulation we employed cells in which K+ was completely replaced by Na+. Such Na+-loaded cells also maintained a pH gradient during glycolysis, albeit a small one (0.3 unit), which was again abolished by dicyclohexylcarbodiimide and by proton conductors. Addition of K+ raised the internal pH to the level characteristic of K+-loaded cells. The results suggest that the pH gradient is formed by energy-dependent extrusion of protons from the cell; this renders the interion alkaline and generates a membrane potential (interior negative). K+ accumulation apparently results from movement of K+ down its electrochemical gradient.
A neomycin-resistant mutant of Escherichia coli, NR70, lacking membrane-bound Mg(2+)-adenosine triphosphatase (EC 3.6.1.3) activity has been isolated. Both whole cells and membrane vesicles exhibit a reduced ability to accumulate amino acids and sugars. Other membrane-related functions such as oxygen consumption, the in vivo hydrolysis of o-nitrophenyl-beta-d-galactoside, and the phosphoenolpyruvate-dependent phosphotransferase system did not exhibit reduced activities in NR70. Amino acid transport could be partially restored by the addition of N,N'-dicyclohexylcarbodiimide. The results suggest that a role of the Mg(2+)-adenosine triphosphatase may be to participate in the coupling of energy derived from the electron transport chain to other processes such as transport.
F1ATPase from the Escherichia coli mutant of H+-ATPase, AN120 (uncA401), has less than 1% of the wild type activity and has been shown to be defective in the alpha subunit by in vitro reconstitution experiments. In the present study, the mutation site was located within a domain of the subunit by recombinant DNA technology. For this, a series of recombinant plasmids carrying various portions of the alpha subunit gene were constructed and used for genetic recombination with AN120. Analysis of the recombinants indicated that the mutation site could be located between amino acid residues 370 and 387. The biochemical properties of the mutant F1 were analyzed further using the fluorescent ATP analog DNS-ATP (2'-(5-dimethylaminonaphthalene-1-sulfonyl)-amino-2'-deoxy ATP). The single turnover process of E. coli F1ATPase proposed by Matsuoka et al. [(1982) J. Biochem. 92, 1383-1398.] was compared in the mutant and wild type F1's. Mutant F1 bound DNS-ATP and hydrolyzed it as efficiently as wild type F1. Results showed that binding of ATP to a low affinity site, possibly in the beta subunit, caused decrease of fluorescence of DNS-ATP in the wild type F1 and that this effect of ATP binding was inhibited by DCCD (dicyclohexyl carbodiimide). However, this effect was not inhibited by DCCD in the mutant F1, suggesting that in the proposed process some step(s) after ATP binding to the low affinity site differed in the mutant and wild F1's. When Pi was added to F1 bound to DNS-ATP or to aurovertin, a fluorescent probe capable of binding to the beta subunit, the opposite changes of fluorescence of these probes in the mutant and wild type F1's were observed, suggesting that the conformational change induced by phosphate binding was altered in the mutant F1. On the basis of the estimated mutation site and the biochemical properties of the mutant F1, the correlation of the domain of this site in the alpha subunit with the function of F1 ATPase is discussed.
The ATP synthase occurs in remarkably conserved form in procaryotic and eucaryotic cells. Thus, present knowledge of ATP synthase is derived from studies of the enzyme from different organisms, each offering specific experimental possibilities. In recent times, research on the H+-conducting F0 part of the ATP synthase has been greatly stimulated by two developments in the Escherichia coli system. Firstly, the purification and reconstitution of the whole ATP synthase as well as the proton conductor F0 from E. coli have been achieved. These functionally active preparations are well defined in terms of subunit composition, similar to the thermophilic enzyme from PS-3 studied by Kagawa's group. Secondly, the genetics and the molecular cloning of the genes of all the F0 subunits from E. coli yielded information on the function of subunit polypeptides and essential amino acid residues. Furthermore, the amino acid sequence of hydrophobic F0 subunits, which are difficult to analyze by protein-chemical techniques, could be derived from the nucleotide sequence of the genes. These achievements provide the framework to study specific aspects of the structure and function of the F0 subunits.