[Show abstract][Hide abstract] ABSTRACT: An aerobic endospore-forming bacilli (NVH 391-98T) was isolated during a severe food poisoning outbreak in France in 1998, and four other similar strains have since been isolated, also mostly from food poisoning cases. Based on 16S rRNA gene sequence similarity, these strains were shown to belong to the B. cereus Group (over 97% similarity with the current Group species), and phylogenetic distance from other validly described species of the genus Bacillus was less than 95%. Based on 16S rRNA gene sequence similarity and MLST data, these new strains were shown to form a robust and well-separated cluster in the B. cereus Group, and constituted the most distant cluster from species of this Group. Major fatty acids (iso-C15:0, C16:0, iso-C17:0, anteiso-C15:0, iso-C16:0, iso-C13:0) supported the affiliation of these strains to the Bacillus genus, and more specifically to the B. cereus Group. NVH 391-98T taxon was more specifically characterized by abundance of iso-C15:0 and low amounts of iso-C13:0 compared to other members of the B. cereus Group. Genome similarity together with DNA-DNA hybridization values and physiological and biochemical tests made it possible to genotypically and phenotypically differentiate NVH 391-98T taxon from the six current B. cereus Group species. NVH 391-98T represents therefore a new species, for which the name Bacillus cytotoxicus sp. nov. is proposed, with the type strain NVH 391-98T (=DSM 22905T =CIP 110041T).
Full-text · Article · Feb 2012 · International Journal of Systematic and Evolutionary Microbiology
[Show abstract][Hide abstract] ABSTRACT: Lactic acid bacteria (LAB) are a phylogenetically diverse group named for their main attribute in food fermentations, that is, production of lactic acid. However, several LAB are genetically equipped for aerobic respiration metabolism when provided with exogenous sources of heme (and menaquinones for some species). Respiration metabolism is energetically favorable and leads to less oxidative and acid stress during growth. As a consequence, the growth and survival of several LAB can be dramatically improved under respiration-permissive conditions. Respiration metabolism already has industrial applications for the production of dairy starter cultures. In view of the growth and survival advantages conferred by respiration, and the availability of heme and menaquinones in natural environments, we recommend that respiration be accepted as a part of the natural lifestyle of numerous LAB.
Full-text · Article · Apr 2011 · Current Opinion in Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Lactic acid bacteria (LAB) include those designated as generally recognized as safe (LABGRAS), used in dairy industries, and opportunistic pathogens like most of the streptococceae. They are usually classified as strict
fermentative bacteria producing mainly lactic acid as the end product of carbohydrate catabolism and they are oxygen-sensitive.
Oxygen, in conjunction with the reducing environment, can generate highly toxic byproducts: superoxide (O2.−), hydrogen peroxide (H2O2), and hydroxyl radical (HO.). These species damage macromolecules like enzymes, leading to growth arrest or mortality in LAB. However, in the last decade,
a basic functional oxygen-dependent respiratory chain has been identified in several LAB, suggesting that they might be better
adapted to an oxygen environment than we thought previously. Interestingly, LAB are defective in their capacity to synthesize
heme (and quinone in some LAB), both essential cofactors in respiratory chains. This chapter focuses on recent studies of
oxygen toxicity, the respiratory metabolism in LAB, exemplified by Lactococcus lactis, and the signaling pathway associated with oxidative stress responses.
[Show abstract][Hide abstract] ABSTRACT: Heme is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular heme partners were identified to date and are not well conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (group B Streptococcus) is a heme auxotroph, which acquires exogenous heme to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a heme-binding protein. AhpC binds hemin with a K(d) of 0.5 microm and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo. A group B Streptococcus ahpC mutant displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in heme intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a heme-binding protein.
Full-text · Article · Mar 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Lactic acid bacteria (LAB) species like L. lactis are traditionally considered as obligate fermentative bacteria because even in aerobiosis they use sugar degradation for substrate-level phosphorylation, i.e. ATP production. However, recent studies revealed that this bacterium and some other LAB are capable of activating a heme-dependent cytochrome oxidase (CydAB) and thus undergo a respiration metabolism. Nevertheless, respiratory chain activation is allowed only when cells have access to heme (and additionally menaquinone for some LAB) in the environment because they cannot synthesize these compounds. Respiration increases the biomass yield and extends the long term survival of stored cells. These benefits of respiration are explained in different ways: i) Respiratory chain activity consumes oxygen, limiting the formation of toxic reactive oxygen species. ii) Respiratory chain generates a pH gradient, which potentially increases ATP production via H+-ATPase activity. iii) Respiration metabolism decreases lactic acid production, limiting acid stress. However, LAB have to cope with heme toxicity. Although heme has clear metabolic benefits the intracellular pool of free heme must be stringently controlled to prevent damage to macromolecules like DNA. In L. lactis, a potential efflux pump system, consisting of an ATPase (YgfA) and a permease (YgfB), is specifically highly induced in response to exogenous heme. Interestingly, the ygfA and ygfB genes are in an operon with ygfC, a potential regulator of the TetR family. Our studies implicate the ygfCBA operon is involved in modulating the free heme level and is regulated by YgfC.
Full-text · Article · Jan 2010 · Japanese Journal of Lactic Acid Bacteria
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial drugs targeting the reportedly essential type II fatty acid synthesis (FASII) pathway have been recently acclaimed for their efficacy against infections caused by multiresistant Gram-positive bacteria. Our findings show that the strategy for antibiotic development based on FASII pathway targets is fundamentally flawed by the fact that exogenous fatty acids fully bypass inhibition of this pathway in both in vitro and in vivo conditions. We demonstrate that major Gram-positive pathogens-such as streptococci, pneumococci, enterococci and staphylococci-overcome drug-induced FASII pathway inhibition when supplied with exogenous fatty acids, and human serum proves to be a highly effective source of fatty acids. For opportunist pathogen Streptococcus agalactiae, growth in serum leads to an overall decrease of FASII gene expression. No antibiotic inhibitor could have a stronger effect than the inactivation of the target gene, so we challenged the role of FASII using deletion mutants. Our results unequivocally show that the FASII target enzymes are dispensable in vivo during S. agalactiae infection. The results of this study largely compromise the use of FASII-based antimicrobials for treating sepsis caused by Gram-positive pathogens.
[Show abstract][Hide abstract] ABSTRACT: Quinones are essential components of the respiration chain that shuttle electrons between oxidoreductases. We characterized the quinones synthesized by Lactococcus lactis, a fermenting bacterium that activates aerobic respiration when a haem source is provided. Two distinct subgroups were characterized: Menaquinones (MK) MK-8 to MK-10, considered as hallmarks of L. lactis, are produced throughout growth. MK-3 and demethylMK-3 [(D)MK-3] are newly identified and are present only late in growth. Production of (D)MK-3 was conditional on the carbon sugar and on the presence of carbon catabolite regulator gene ccpA. Electron flux driven by both (D)MK fractions was shared between the quinol oxidase and extracellular acceptors O(2), iron and, with remarkable efficiency, copper. Purified (D)MK-3, but not MK-8-10, complemented a menB defect in L. lactis. We previously showed that a respiratory metabolism is activated in Group B Streptococcus (GBS) by exogenous haem and MK, and that this activity is implicated in virulence. Here we show that growing lactococci donate (D)MK to GBS to activate respiration and stimulate growth of this opportunist pathogen. We propose that conditions favouring (D)MK production in dense microbial ecosystems, as present in the intestinal tract, could favour implantation of (D)MK-scavengers like GBS within the complex.
[Show abstract][Hide abstract] ABSTRACT: Numerous Streptococcaceae produce an H2O-forming NADH oxidase, Nox-2, which has been generally implicated in aerobic survival. We examined the roles of Nox-2 in Group B Streptococcus (GBS), a leading agent of neonatal infections. While nox2 inactivation caused an aerobic growth arrest, no improvement was seen by addition of antioxidants to cultures, suggesting that this defect was not due to accumulation of toxic oxygen species. Using several approaches, we show that the observed inability of the nox2 mutant to grow aerobically is mainly due to an underlying defect in fatty acid (FA) biosynthesis: (i) the nox2 aerobic growth defect is fully and rapidly complemented by adding oleic acid to culture medium, and (ii) direct assimilation of this unsaturated FA in both wild type (WT) and nox2 GBS membranes is demonstrated and correlated with mutant growth rescue. We propose that NAD+ depletion in the nox2 mutant results in reduced acetyl-CoA production, which perturbs FA biosynthesis and hence blocks growth in aerobiosis. The nox2 aerobic growth defect was also complemented when GBS respiration metabolism was activated by exogenous haem and menaquinone. The membrane NADH oxidase activity generated by the functional respiratory chain thus compensates the cytoplasmic NADH oxidase deficiency. The nox2 mutant was attenuated for virulence, as assessed in lung, intraperitoneal and intravenous murine infection models. As the nox2 defect seems only to affect aerobic growth of GBS, its reduced virulence supports the suggestion that aerobic conditions and NADH oxidase activities are relevant to the GBS infection process.
Full-text · Article · Dec 2006 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Most bacteria require iron for growth. However, as it may not be directly available under aerobic conditions, bacteria may use iron-sequestering molecules, such as bacterially encoded siderophores, or heme, which is the major iron source in the animal host. Bacteria may also assimilate heme for purposes other than as an iron source. Once internalised, heme can activate, for example, a heme-dependent catalase and a cytochrome oxidase. In bacterial pathogen Streptococcus agalactiae, heme, in association with exogenous menaquinone, activates a respiratory chain. Respiration has radical effects on carbon metabolism. GBS respiration-grown cells display improved survival in an aerobic environment and greater virulence in a murine septicemia model. GBS might benefit from its ecological niches to capture heme and menaquinone, i.e., from other bacteria when it colonizes host mucosa, or from blood-containing organs during septicemia.
[Show abstract][Hide abstract] ABSTRACT: Group B Streptococcus (GBS) is a common constituent of the vaginal microflora, but its transmission to newborns can cause life-threatening sepsis, pneumonia and meningitis. Energy metabolism of this opportunist pathogen has been deduced to be strictly fermentative. We discovered that GBS undergoes respiration metabolism if its environment supplies two essential respiratory components: quinone and haem. Respiration metabolism led to significant changes in growth characteristics, including a doubling of biomass and an altered metabolite profile under the tested conditions. The GBS respiratory chain is inactivated by: (i) withdrawing haem and/or quinone, (ii) treating cultures with a respiration inhibitor or (iii) inactivating the cydA gene product, a subunit of cytochrome bd quinol oxidase, in all cases resulting in exclusively fermentative growth. cydA inactivation reduced GBS growth in human blood and strongly attenuated virulence in a neonatal rat sepsis model, suggesting that the animal host may supply the components that activate GBS respiration. These results suggest a role of respiration metabolism in GBS dissemination. Our findings show that environmental factors can increase the flexibility of GBS metabolism by activating a newly identified respiration chain. The need for two environmental factors may explain why GBS respiration metabolism was not found in previous studies.
Full-text · Article · May 2005 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: The catabolic control protein CcpA is the highly conserved regulator of carbon metabolism in Gram-positive bacteria. We recently showed that Lactococcus lactis, a fermenting bacterium in the family of Streptococcaceae, is capable of respiration late in growth when haem is added to aerated cultures. As the start of respiration coincides with glucose depletion from the medium, we hypothesized that CcpA is involved in this metabolic switch and investigated its role in lactococcal growth under aeration and respiration conditions. Compared with modest changes observed in fermentation growth, inactivation of ccpA shifts metabolism to mixed acid fermentation under aeration conditions. This shift is due to a modification of the redox balance via derepression of NADH oxidase, which eliminates oxygen and decreases the NADH pool. CcpA also plays a decisive role in respiration metabolism. Haem addition to lag phase ccpA cells results in growth arrest and cell mortality. Toxicity is due to oxidative stress provoked by precocious haem uptake. We identify the repressor of the haem transport system and show that it is a target of CcpA activation. We propose that CcpA-mediated repression of haem uptake is a means of preventing oxidative damage at the start of exponential growth. CcpA thus appears to govern a regulatory network that coordinates oxygen, iron and carbon metabolism.
Full-text · Article · Nov 2003 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Oxygen is a major determinant of both survival and mortality of aerobic organisms. For the facultative anaerobe Lactococcus lactis, oxygen has negative effects on both growth and survival. We show here that oxygen can be beneficial to L. lactis if heme is present during aerated growth. The growth period is extended and long-term survival is markedly improved compared
to results obtained under the usual fermentation conditions. We considered that improved growth and survival could be due
to the capacity of L. lactis to undergo respiration. To test this idea, we confirmed that the metabolic behavior of lactococci in the presence of oxygen
and hemin is consistent with respiration and is most pronounced late in growth. We then used a genetic approach to show the
following. (i) The cydA gene, encoding cytochromed oxidase, is required for respiration and plays a direct role in oxygen utilization. cydA expression is induced late in growth under respiration conditions. (ii) ThehemZ gene, encoding ferrochelatase, which converts protoporphyrin IX to heme, is needed for respiration if the precursor, rather
than the final heme product, is present in the medium. Surprisingly, survival improved by respiration is observed in a superoxide
dismutase-deficient strain, a result which emphasizes the physiological differences between fermenting and respiring lactococci.
These studies confirm respiratory metabolism in L. lactis and suggest that this organism may be better adapted to respiration than to traditional fermentative metabolism.
Full-text · Article · Sep 2001 · Journal of Bacteriology