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Biochemical and biophysical studies of Bacillus subtilis envelops under hyper osmotic stress
Abstract
The behaviour and state of the envelopes from B. subtilis cultures grown in Luria Bertani (LB) medium with and without 1.5 M NaCl are compared. Under hypertonic conditions, the hydrophobicity of the cultures increases. The phospholipid and fatty acid (FA) compositions show important differences: a higher cardiolipin (CL) content [at the expense of phosphatidylglycerol (PG)], and a higher unsaturated and straight chain FA content. The fluidity of the membranes, determined with fluorescent probes, indicates an increase in viscosity of the cytoplasmic membrane. The consequences of these variations in membrane permeability and osmotolerance are discussed.
... Our results show that similar levels of branched-chain fatty acids were observed for L. monocytogenes Scott A at 37 ‡C with or without salt in the culture medium (7% of NaCl). However, an increase of the percentage of branched-chain fatty acids was observed for the CNL strain, in contrast to what has been reported for a bacterium closely related to L. monocytogenes : Bacillus subtilis which showed a decrease of the percentage of branched-chain fatty acids in hyperosmotic medium [25]. Moreover, when compared with the control condition (37 ‡C^0% NaCl) the C 15 to C 17 ratio was two-and ¢vefold lower in both Scott A and CNL cells, respectively at 37 ‡C^7% of NaCl. ...
... The increase of ai17:0 amount in both strains in hyperosmotic medium suggests that these membranes may be less £uid when compared to membranes from control conditions grown cells. These results are consistent with the suggestion that adaptive changes in both strains have occurred in the presence of salt to counter the disturbance of the cellular homeostasis under the hyperosmotic treatment [25]. ...
... Moreover, i15:0 levels declined in hyperosmotic medium over the entire temperature range studied, and remained with lower values than those of control conditions in L. monocytogenes Scott A cells. A decrease in isobranched-chain fatty acids was reported for B. subtilis when cultivated in hyperosmotic medium [25]. The lowering of the level of i15:0, the increase in ai17:0 amount, and the maintenance of the ai15:0 at a steady state or its low decrease over the entire temperature range in a hyperosmotic medium lead probably to a more rigid membrane, in order to retain intracellular osmocompatible compounds during the osmoadaptation process as reported for B. subtilis [25]. ...
Cells of two strains of Listeria monocytogenes CNL 895807 and Scott A were grown to late exponential phase at different growth temperatures (37, 20 and 4 ‡C) with or without NaCl (7%), and their fatty acid compositions were analysed. The results showed that low thermal adaptation response of L. monocytogenes CNL was different than that of the Scott A strain, and it was based on both an increase of anteiso-branched-chain fatty acids and a significant decrease of straight-chain fatty acids. However, the main modifications observed in the Scott A strain when grown at a low temperature were a decrease of the proportion of ai17:0 and an increase of ai15:0. In hyperosmotic medium and over the entire temperature range (4 ‡C, 20 ‡C and 37 ‡C) the two L. monocytogenes strains showed a cellular fatty acid profile dominated by ai15:0. In addition, a decrease of the two major straight-chain fatty acids (14:0 and 16:0) was observed in the CNL strain. These results demonstrated that the CNL strain showed different behaviours of low thermal and salt adaptation to maintain membrane fluidity, which are based both on an increase of anteiso-branched-chain fatty acids, and a significant decrease of straight-chain fatty acids.
... Additionally, these studies also saw the upregulation of enzymes involved in altering the membrane composition to produce a more rigid, less fluid membrane. Such changes, including increased levels of saturated FAs and cardiolipin, have also been reported in B. subtilis (62,63), Escherichia coli (64), and S. aureus (65) subjected to salt stress, which produced thicker cell walls, possibly preventing NaCl entrance and the escape of compatible solutes (62). Increased levels of saturated FAs also increase the melting point of the membrane, making cells more resistant to higher temperatures (66). ...
... Additionally, these studies also saw the upregulation of enzymes involved in altering the membrane composition to produce a more rigid, less fluid membrane. Such changes, including increased levels of saturated FAs and cardiolipin, have also been reported in B. subtilis (62,63), Escherichia coli (64), and S. aureus (65) subjected to salt stress, which produced thicker cell walls, possibly preventing NaCl entrance and the escape of compatible solutes (62). Increased levels of saturated FAs also increase the melting point of the membrane, making cells more resistant to higher temperatures (66). ...
... As mentioned previously, cardiolipin levels positively correlate with increased osmotolerance, cold tolerance, and acid tolerance in both L. monocytogenes and other bacteria alike (62,65,70). This diphosphatidylglycerol lipid is a multifunctional molecule that participates in cell division, energy metabolism, and membrane transport (71). ...
Listeria monocytogenes is a food-borne pathogen, whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened using a microplate assay, for strains displaying increased or decreased desiccation (43% RH, 15°C) survival in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food grade stainless steel (SS) coupons in TSB+1% glucose. Single transposon insertions in mutants exhibiting >0.5 log CFU/cm(2) change in desiccation survival relative to the wildtype were determined by sequencing arbitrary PCR products. Strain morphology, motility and osmotic stress survival (TSB-glu+20% NaCl) was also analyzed. The initial screen selected 129 desiccation sensitive (DS) and 61 desiccation tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagella-less strains contained transposons in flagella biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY) genes, while others harboured transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. Interrupted genes in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (p<0.05) different survival relative to the wildtype when exposed to osmotic stress, demonstrating some genes likely have similar roles in surviving the two water stresses.
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... Salt stress also exerts pleiotropic effects on microbial physiology, including changes in membrane composition [11,12], cell wall properties [13], exopolysaccharide structural content [14], cell swarming [15,16], and iron homeostasis [17][18][19]. ...
... The adaptation of B. subtilis to high salt conditions is commonly accompanied by the changes in the composition of the cell membrane, especially in its fatty acid and lipid components [13,15]. Thus, it has been reported that the increase in the content of cardiolipin, and saturated straight-chain and unsaturated fatty acids may correlate with higher salt resistance of B. subtilis [12], suggesting that fatty acid metabolism may be involved in Bacillus sp. N16-5 response to salt stress. ...
The alkaliphilic halotolerant bacterium Bacillus sp. N16-5 is often exposed to salt stress in its natural habitats. In this study, we used one-colour microarrays to investigate adaptive responses of Bacillus sp. N16-5 transcriptome to long-term growth at different salinity levels (0%, 2%, 8%, and 15% NaCl) and to a sudden salt increase from 0% to 8% NaCl. The common strategies used by bacteria to survive and grow at high salt conditions, such as K+ uptake, Na+ efflux, and the accumulation of organic compatible solutes (glycine betaine and ectoine), were observed in Bacillus sp. N16-5. The genes of SigB regulon involved in general stress responses and chaperone-encoding genes were also induced by high salt concentration. Moreover, the genes regulating swarming ability and the composition of the cytoplasmic membrane and cell wall were also differentially expressed. The genes involved in iron uptake were down-regulated, whereas the iron homeostasis regulator Fur was up-regulated, suggesting that Fur may play a role in the salt adaption of Bacillus sp. N16-5. In summary, we present a comprehensive gene expression profiling of alkaliphilic Bacillus sp. N16-5 cells exposed to high salt stress, which would help elucidate the mechanisms underlying alkaliphilic Bacillus spp. survival in and adaptation to salt stress.
... Estos ácidos grasos, si bien no son los más abundantes, son característicos de este género y aumentan desde menos del 1% hasta valores cercanos al 10%. Varios autores (2,18,20) han comunicado variaciones de ácidos grasos ramificados, iso y anteiso como respuesta al estrés ambiental, pero en ningún caso las bacterias que estudiaron contenían n:0 iso OH, ni n:0 10 metil. ...
... Otros autores han encontrado cambios significativos en los ácidos grasos de las membranas celulares, Guillot et al (13) encontraron que en Lactococcus lactis se incrementa la proporción de ácidos grasos ciclopropano y que no se producen cambios en la relación nosaturados/saturados, López et al (20), trabajando con Bacillus subtilis encontraron un aumento significativo de los ácidos grasos saturados de cadena lineal con una disminución de los ácidos grasos saturados, en particular los iso ramificados. Valderrama et al (31), estudiaron la acción de la concentración salina sobre Halomonas salina y comunicaron que la concentración salina tiene una gran influencia en la composición de ácidos grasos y que activaría la ciclopropano sintetasa con disminución de los ácidos grasos monoinsaturados. ...
Variation in the composition of Rhodococcus rodochrous GNP-OHP-38r cell membrane fatty acids in response to temperature and salinity. The members of the genus Rhodococcus are frequent and abundant inhabitants of polluted areas with hydrocarbons and they resist the salinity present in the central Patagonia. This genus has good capacity to eliminate pollution produced by hydrocarbons that constitutes the biggest pollutant agent in the region. The present work studies the answer in the composition of its fatty acids under the combined action of the temperature and saline concentration of an isolated stump of a landfarming system. The strategy of Rhodococcus rodochrous strain GNP-OHP-38r in front of the thermal-osmotic stress is the increase of the percentage of the total saturated fatty acids (n:0); fatty acids branched in the terminal carbon with hidroxyl group in position 2 (n:0 iso 2 OH) and saturated with group methyl in carbon 10 (n:0 10 metil) when the temperature is increased. These acids increase w hile the percentage of n:1 cis decrease.
... In Bacillus subtilis, the composition of the cytoplasmic membrane and cell wall are adjusted, and the aggregation capability of the cells is largely damaged (Steil et al., 2003). Moreover, salt stress alters the cell envelope composition of Rhizobacteria, causing changes in proteins, periplasmic glucans, capsular lipopolysaccharides (Altabe et al., 1994), and the composition of the cell envelopes, which play an important role in adaptation of osmotic stress (López et al., 2000). Under stressful conditions, certain bacteria like Pseudomonas produce exopolysaccharides (EPS), which enhance water retention and regulate the diffusion of carbon sources in the microbial environment to protect microbial from hydric stress and fluctuations in water potential (Sandhya et al., 2009). ...
Brucella can inhabit hostile environments, including osmotic stress. How Brucella responds collectively to osmotic stress is largely unexplored, particularly in spatially structured communities such as a biofilm. To gain insight into this growth mode, we set out to characterize the Brucella melitensis 16M biofilm, describe its phenotype, and carry out a comparative transcriptomic analysis between biofilms under osmotic stress and control conditions. We determined that the bacteria challenged with 1.5 M NaCl had a reduced ability to aggregate and form clumps and develop a biofilm; however, the salt stress promoted the release of the outer membrane vesicles from the biofilm. Together with the genotypical response to osmotic stress, we identified 279 differentially expressed genes in B. melitensis 16M grown under osmotic conditions compared with control conditions; 69 genes were upregulated and 210 downregulated. Under osmotic stress, the main changed genes of biofilm were predicted to be involved in flagellar assembly, cell envelope, translation, small RNA regulation, transport and binding proteins, and energy metabolism. In addition, the ABC transporter was enriched in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. We highlight 12 essential ABC transporter genes associated with a bacterial response to osmotic stress at the biofilm stage, including one specific locus, BME_RS12880, mediating betaine accumulation in biofilms to eliminate osmotic stress. The current study results can help researchers gain insights into B. melitensis 16M biofilm adaptation to osmotic stress and provide information for developing intervention strategies to control Brucella .
... In a similar manner to decreased temperatures, certain cyanobacteria (Huflejt et al., 1990) and other microorganisms (López et al., 2000) have been reported to respond to osmotic stress by increasing the desaturation of membrane lipids. Although the molecular interactions are not comprehensively understood, in cyanobacteria this is appears to protect the photosynthetic machinery against damage upon exposure to high salinity [see review (Yang et al., 2020)]. ...
The ability to produce medium-chain length aliphatic hydrocarbons is strictly conserved in all photosynthetic cyanobacteria, but the molecular function and biological significance of these compounds still remain poorly understood. This study gives a detailed view to the changes in intracellular hydrocarbon chain saturation in response to different growth temperatures and osmotic stress, and the associated physiological effects in the model cyanobacterium Synechocystis sp. PCC 6803. We show that the ratio between the representative hydrocarbons, saturated heptadecane and desaturated heptadecene, is reduced upon transition from 38°C toward 15°C, while the total content is not much altered. In parallel, it appears that in the hydrocarbon-deficient ∆ado (aldehyde deformylating oxygenase) mutant, phenotypic and metabolic changes become more evident under suboptimal temperatures. These include hindered growth, accumulation of polyhydroxybutyrate, altered pigment profile, restricted phycobilisome movement, and ultimately reduced CO2 uptake and oxygen evolution in the ∆ado strain as compared to Synechocystis wild type. The hydrocarbons are present in relatively low amounts and expected to interact with other nonpolar cellular components, including the hydrophobic part of the membrane lipids. We hypothesize that the function of the aliphatic chains is specifically associated with local fluidity effects of the thylakoid membrane, which may be required for the optimal movement of the integral components of the photosynthetic machinery. The findings support earlier studies and expand our understanding of the biological role of aliphatic hydrocarbons in acclimation to low temperature in cyanobacteria and link the proposed role in the thylakoid membrane to changes in photosynthetic performance, central carbon metabolism, and cell growth, which need to be effectively fine-tuned under alternating conditions in nature.
... Obtained results were similar to those observed by Soumitra et al, (2007) who reported a reduction in the growth of A. brasilense bacteria by addition of NaCl, by accumulation of organic osmolytes as flexible modes of adaptation and osmoadaptation strategies, which become the crucial factor for the stability of enzymes and other cellular components under salt stress. Bacteria adapt to salinity by cell envelope (Lopez et al, 2000). Also, potassium ions, glycine betaine, proline, proline betaine, trehalose and ectoines could accumulating as osmolytes. ...
... The biosynthetic pathways and expression of genes involved are studied in detail under varying concentrations of salt. Salt stress exerts pleiotropic effects on multiple parameters dealing with physiology, membrane composition, cell wall properties (Lopez et al. 1998(Lopez et al. , 2000, EPS structural content (Lloret et al. 1995), cell swarming (Steil et al. 2003;Xu et al. 2005), and iron homeostasis (Hoffmann et al. 2002;Argandona et al. 2010;Gancz and Merrell 2011). Synthesis of organic compatible solutes, such as glycine, betaine, ectoine, proline, and trehalose, is activated or upregulated due to salt stress (Sleator and Hill 2002;Hahne et al. 2010). ...
Microorganisms are omnipresent, irrespective of environmental conditions, microbes have a huge diversity that adapt to most absurd physiology, stringent growth parameters and utilize bizarre nutrients as sole energy source. Environment poses many different ecological niches that rarely favor life of higher organisms, but these conditions provide impetus to a large microbial diversity that harbors tremendous biotechnological potential. Extreme conditions with acute pH, daunting salinity, lethal temperature, critical pressures present with physiological extremities while heavy radiations, heavy metals and organic solvents etc give chemically challenging environments to microbes. A substantial percentage of microbes are strictly extremophiles, a huge micro flora tolerant to extremities and many tolerant to multiple extremities exists simultaneously in nature. Halophiles possess inherent properties to survive under these extreme conditions and yet produce certain novel biomolecules of immense industrial potential. For survival halophilic bacteria adopt various strategies including certain physiological changes, definite structural features and higher degree of molecular individuality. In present chapter we attempt a summation of different adaptations of halophilic bacteria and possible significant and attractive proposition towards commercialization.
... The composition of the cell envelope plays an important role in osmo-adaptation [94]. Cell envelope-related changes triggered by salt stress include alterations in the structure and composition of the peptidoglycan layer [95], and changes in membrane and/or periplasmic protein composition, lipid composition, periplasmic glucan levels, and capsular polysaccharide biosynthesis [96]. ...
Background
Soil salinization is a worldwide problem that is intensifying because of the effects of climate change. An effective method for the reclamation of salt-affected soils involves initiating plant succession using fast growing, nitrogen fixing actinorhizal trees such as the Casuarina. The salt tolerance of Casuarina is enhanced by the nitrogen-fixing symbiosis that they form with the actinobacterium Frankia. Identification and molecular characterization of salt-tolerant Casuarina species and associated Frankia is imperative for the successful utilization of Casuarina trees in saline soil reclamation efforts. In this study, salt-tolerant and salt-sensitive Casuarina associated Frankia strains were identified and comparative genomics, transcriptome profiling, and proteomics were employed to elucidate the molecular mechanisms of salt and osmotic stress tolerance. ResultsSalt-tolerant Frankia strains (CcI6 and Allo2) that could withstand up to 1000 mM NaCl and a salt-sensitive Frankia strain (CcI3) which could withstand only up to 475 mM NaCl were identified. The remaining isolates had intermediate levels of salt tolerance with MIC values ranging from 650 mM to 750 mM. Comparative genomic analysis showed that all of the Frankia isolates from Casuarina belonged to the same species (Frankia casuarinae). Pangenome analysis revealed a high abundance of singletons among all Casuarina isolates. The two salt-tolerant strains contained 153 shared single copy genes (most of which code for hypothetical proteins) that were not found in the salt-sensitive(CcI3) and moderately salt-tolerant (CeD) strains. RNA-seq analysis of one of the two salt-tolerant strains (Frankia sp. strain CcI6) revealed hundreds of genes differentially expressed under salt and/or osmotic stress. Among the 153 genes, 7 and 7 were responsive to salt and osmotic stress, respectively. Proteomic profiling confirmed the transcriptome results and identified 19 and 8 salt and/or osmotic stress-responsive proteins in the salt-tolerant (CcI6) and the salt-sensitive (CcI3) strains, respectively. Conclusion
Genetic differences between salt-tolerant and salt-sensitive Frankia strains isolated from Casuarina were identified. Transcriptome and proteome profiling of a salt-tolerant strain was used to determine molecular differences correlated with differential salt-tolerance and several candidate genes were identified. Mechanisms involving transcriptional and translational regulation, cell envelop remodeling, and previously uncharacterized proteins appear to be important for salt tolerance. Physiological and mutational analyses will further shed light on the molecular mechanism of salt tolerance in Casuarina associated Frankia isolates.
... Cells treated with 18% NaCl exhibited a signicant increase in the levels of unsaturated fatty acids and a decrease in the proportion of saturated fatty acids ( Fig. 4 and 5). These results are similar to those reported in other bacteria, 31,32 suggesting that modulation of the fatty acids composition is necessary for survival during salt stress. In addition, increase of the ratio of UFA/SFA can protect the cell from other environmental stresses in LAB has also been observed. ...
Tetragenococcus halophilus, a moderately halophilic Gram-positive lactic acid bacteria, was widely existed in many food fermentation systems, where salt stress is an environmental condition commonly encountered. In this study, a partial proteome reference map of T. halophilus was established by using the proteomics approach. A total of 120 proteins were identified and the cluster of orthologous groups (COG), codon adaptation index (CAI), and GRAVY value analyses of the identified proteins were performed. To better understand the salt tolerance mechanisms and stress responses, we carried out a comparative proteomic and physiological study of T. halophilus during salt stress. Comparative proteomic analysis revealed 26 differentially expressed proteins that are mainly involved in cellular metabolism, energy production, stress response, and others. Analysis of the cytoplasmic membrane fatty acids showed that higher proportions of unsaturated fatty acid and higher mean chain length were observed in the cells challenged by salt stress. In addition, the salt shocked cells exhibited higher amounts of intracellular proline, glycine betaine, and trehalose compared with those without salt stress. Results presented in this study may further elucidate the mechanisms of T. halophilus underlying salt stress.
... In other bacteria, the proportion of PG rises dramatically ( B. subtilis, Oceanomonas baumannii, and Lactococcus lactis) (Table 1). The references are 1 (López et al. 2000), 2 (López et al. 2006), 3 (Dare et al. 2014), 4 (Romantsov et al. 2007, 5 (Romantsov et al. 2009b), 6 (Sevin and Sauer 2014), 7 (Oliver et al. 2014), 8 (Guillot et al. 2000), 9 (Brown et al. 2000, 10 (Brown et al. 2003), 11 (Danevcic et al. 2005), and 12 (Lopalco et al. 2013) Bacterial phospholipid composition also depends on the growth phase and the growth medium. For example, the proportion of PG increased dramatically in stationary phase when B. subtilis and Listeria monocytogenes were cultivated on rich media (LB and BHI, respectively), whereas the proportion of PG in E. coli cultivated on MOPS minimal medium decreased in stationary phase (Table 1). ...
Changing environmental osmotic pressure causes transmembrane water fluxes that may impair cellular functions. Bacteria mitigate water fluxes by controlling the solute content of their cytoplasm. Increasing osmotic pressure triggers solute synthesis or uptake via osmosensing transporters, whereas osmotic downshock triggers solute release via mechanosensitive channels. Membrane lipids are implicated in the subcellular localization and function of membrane-based osmoregulatory systems. Zwitterionic phosphatidylethanolamine (PE) and anionic phosphatidylglycerol (PG) and cardiolipin (CL) are the predominant phospholipids in most bacteria, but their proportions vary widely. For many species, anionic lipids increase in proportion during cultivation in high salinity media. Evidence suggests that interactions among anionic lipid headgroups and cytoplasm-exposed areas of osmosensory transporters ProP, BetP, and OpuA are fundamental to their osmosensory response. CL-dependent targeting of transporter ProP to the CL-rich environment at the poles of Escherichia coli cells further modulates the osmolality response. Protein-lipid interactions are also fundamental to the gating of mechanosensitive channels MscL and MscS by membrane tension. Future work should encompass further characterization of the impacts of lipid composition on key physical properties of the membrane, as well as the regulation of lipid composition and membrane properties in response to environmental cues. The roles of lipids in the structural mechanisms of osmosensing and mechanosensitive channel gating are not fully understood. Osmosensory systems provide useful paradigms for the study of both protein-lipid interactions and the role of subcellular localization in bacterial lipid and protein function.
... salivarius incubated in mandarin juices was lower than that obtained in MRS Broth. The change in cell wall hydrophobicity could be a result of bacterial stress to certain culture conditions, such as low pH, high temperature and hyperosmotic stress (Lopez et al., 2000;Remeta et al., 2002). It seems that in those samples with 300 g/kg of trehalose addition, slightly higher pH and created interactions between trehalose, probiotic microorganisms and juices cloud, protected L. salivarius spp. ...
... A substantial number of genes involved in cell wall synthesis or modification are also represented among the salt-induced genes, indicating that B. subtilis modifies its cell wall when it is continuously exposed to high salinity (Steil et al., 2003). Genes falling into this functional group have also been identified as osmotically controlled in other studies (Fischer and Bremer, 2012;Lopez et al., 1998Lopez et al., , 2000Palomino et al., 2009). ...
As a result of the biophysical properties of the cytoplasm and the semipermeable nature of the cytoplasmic membrane, fluctuations in the environmental osmolarity trigger water fluxes along the osmotic gradient in or out of the cell under hypo- and hyperosmotic conditions. These water fluxes impinge on the magnitude of turgor and cellular hydration, and thereby either threaten the integrity of the cell (low-osmolarity environments) or cause dehydration of the cytoplasm (high-osmolarity environments). No microbial cell can actively transport water, and hence it can only regulate indirectly water fluxes across the cellular membrane by actively modulating the osmotic potential of the cytoplasm. It does so by importing ions (primarily K+) and compatible solutes, organic osmolytes that are highly compliant with cellular physiology, to trigger water influx. Conversely, it transiently opens mechanosensitive channels to rapidly jettison water-attracting ions and organic compounds to curb water influx. Here we use the model system for Gram-positive bacteria, the soil-dwelling Bacillus subtilis, to review the cellular and genetic osmostress responses that allow it to cope effectively with changes in the external osmolarity. This is a frequently occurring challenge in the upper layers of the soil that results from flooding and desiccation processes. We provide an overview of possible mechanisms that allow the B. subtilis cell to sense osmotic changes in its environment, process this information, and change its gene expression profile accordingly in a way to ultimately enhance its competitiveness under osmotically stressful growth conditions.
... For instance, some genes added to the SigB regulon in clade A organisms may enhance the synthesis of cardiolipin and that of the ABC transporter involved in its release [56]. This molecule increases the hydrophobicity and viscosity of the cell membrane by changing its composition [57]. These changes had been related to an increase in survival in the high osmotic environment of B. subtilis [58], as well as to nonspecific resistance to antibiotics and antimicrobial peptides, by decreasing the permeability of the cell envelope [59,60]. ...
Bacteria of the Bacillus cereus group are environmental Gram-positive spore-forming bacteria ubiquitously distributed. Despite the high degree of genetic similarity among the different strains, they show strong phenotypic variability, from mammal or entomopathogen strains to soil-dwelling saprophytes, and from psychrophylic to thermotolerant strains. Most of the phenotypes are linked to the presence of large plasmids that encode for diverse toxins. However, other processes, like mutation or recombination, also participate in shaping the evolution and population structure of these bacteria. Here we review different aspects of the evolution of this group.
... In our laboratory, we have studied the response to osmotic stress of several gram-positive strains and species and observed several cell envelope modifications when Bacillus or Lactobacillus strains were exposed to high-salt conditions (Lopez et al. 2000;Piuri et al. 2003Piuri et al. , 2005Palomino et al. 2009Palomino et al. , 2010Palomino et al. , 2013. In particular, we have shown that growth in high-salt conditions of Lactobacillus casei BL23, a species that lacks S-layers, causes several pleiotropic effects, including differences in the susceptibility to enzymatic lysis, increased sensitivity to cationic antimicrobials such as nisin, and increased capacity to form biofilm on artificial surface and transformation ability. ...
In this work, we studied the role of surface layer (S-layer) proteins in the adaptation of Lactobacillus acidophilus ATCC 4356 to the osmotic stress generated by high salt. The amounts of the predominant and the auxiliary S-layer proteins SlpA and SlpX were strongly influenced by the growth phase and high-salt conditions (0.6 M NaCl). Changes in gene expression were also observed as the mRNAs of the slpA and slpX genes increased related to the growth phase and presence of high salt. A growth stage-dependent modification on the S-layer protein profile in response to NaCl was observed: while in control conditions, the auxiliary SlpX protein represented less than 10 % of the total S-layer protein, in high-salt conditions, it increased to almost 40 % in the stationary phase. The increase in S-layer protein synthesis in the stress condition could be a consequence of or a way to counteract the fragility of the cell wall, since a decrease in the cell wall thickness and envelope components (peptidoglycan layer and lipoteichoic acid content) was observed in L. acidophilus when compared to a non-S-layer-producing species such as Lactobacillus casei. Also, the stationary phase and growth in high-salt medium resulted in increased release of S-layer proteins to the supernatant medium. Overall, these findings suggest that pre-growth in high-salt conditions would result in an advantage for the probiotic nature of L. acidophilus ATCC 4356 as the increased amount and release of the S-layer might be appropriate for its antimicrobial capacity.
... responses . How - ever , caution must be exercised in making such predictions . In the related B . subtilis , growth in NaCl leads to changes in fatty acid composition that one would predict as being fluidizing ( Ló pez et al . , 1998 ) , but direct measurements of membrane fluidity using fluorescent probes showed that the opposite was the case ( Ló pez et al . , 2000 ) . ...
The phenotypic adaptation of membrane lipids in seven strains of the food-poisoning bacterium Bacillus cereus, isolated from Bangladeshi rice, is reported in relation to their ability to grow under conditions of low water activity (a w ), reduced temperature and the presence of soluble rice starch. The strains have different membrane phospholipid head-group and fatty acyl compositions, and they display individual differences in their responses to both low a w and reduced temperature. The extent of the increase in anionic membrane lipids in response to low a w varies from strain to strain, is solute specific and in one strain does not occur. Growth is stimulated by the presence of soluble rice starch and results in a large rise in the proportion of diphosphatidylglycerol (DPG) at the expense of phosphatidylglycerol (PG), without any change in the proportion of total anionic phospholipids. Growth at 15 °C compared with 37 °C increases the proportions of DPG and phosphatidylethanolamine at the expense of PG. At the lower temperature there are changes in phospholipid fatty acyl composition characteristic of those expected to maintain membrane fluidity, including increases in the amount of total branched fatty acids and the anteiso-/iso-branched ratio, and a decrease in the equivalent chain-length, but there are strain differences in how those changes were achieved. In contrast to some other bacilli, there are persistent large increases in the proportions of unsaturated fatty acyl chains in phospholipids during growth at 15 °C.
... Machado et al. (2004) reported increased saturated and cyclic forms of fatty acids in Lactobacillus casei at high osmolality. A similar type of observation was also reported in the case of L. monocytogenes strain CNL and Bacillus subtilis (Chihib et al. 2003;Lopez et al. 2000;Zhu et al. 2005). Unlike previous findings, our data show that iso fatty acid content increased with osmolality, while the content of anteiso forms decreased in both the organisms (Fig. 2). ...
The response of cellular fatty acids to various environmental stresses was studied using two endophytic species of Micrococcus. A total of 18 samples with three biological replicates from low, moderate and high stress conditions of salt (0.5, 5 and 10 % NaCl), pH (5, 7 and 10) and temperatures (15, 25 and 41 °C) were analysed. Branched chain fatty acids dominated in both the organisms, while saturated and unsatu-rated fatty acids were detected less frequently. The mole percentage of isoforms of branched chain fatty acids gradually increased with increasing salinity and showed more than a twofold increase at higher concentration of salt (10 %). Unlike Micrococcus yunnanensis DSM 21948 T , Micrococcus aloeverae MCC 2184 T showed more agreement with previous findings related to stress tolerance in other bacteria. Data indicate that iso fatty acids are responsible for the growth of Micrococcus at high salt concentration. In addition, instead of individual fatty acids, the ratio of the total content of iso/ anteiso forms modulates membrane fluidity and functions during environmental stress in Micrococcus. For a comparative study of salinity stress in Gram-positive and Gram-negative bacteria, the strain of Halomonas was alsoincluded.
... Bacteria in general, however, are able to adapt to changing environmental conditions by physicochemical modification of their surface properties, which may result in an increase in cell surface hydrophobicity. This surface modification is a well characterized stress response of bacteria (Lopez et al. 2000;Wick et al. 2003). Changes in the structure of lipopolysaccharides and the release of membrane vesicles were described as underlying mechanisms (Al-Tahhan et al. 2000;Baumgarten et al. 2012). ...
Soil–water repellency (SWR) is a widely observed phenomenon with severe impacts, but a physicochemical framework to explain the process of SWR development is still a major field of research. Recent studies have shown that microbial biomass residues, in particular cell fragments, contribute significantly to the formation of soil organic matter (SOM) and can decrease wettability. It was also shown that osmotic stress increases the hydrophobicity of bacterial cell surfaces. If microorganisms are an important source of SOM, the attachment of cells and their residues on mineral grains should decrease wettability of minerals, and the effect should be more pronounced in case of osmotic stress. Cultures of Pseudomonas putida, either unstressed or exposed to osmotic stress, and cell fragments were mixed with minerals and the impact on surface wetting properties was investigated by determining the solid-water contact angle (CA). Attachment of bacteria to quartz surfaces resulted in a significant increase in hydrophobicity of the surfaces (CA increase by up to 90°), in particular for stressed cells. Cell fragments and cytosol were also found to decrease wettability significantly (CAs of up to 100°). These findings may explain various phenomena related to SWR, like critical soil–water content, and may be one important explanation for the formation of SWR after irrigation with treated sewage effluents. The results also support the hypothesis of a microbial origin of SWR, in which macromolecular biological structures may have a greater impact than specific classes of organic compounds.
... Однако анализ экспрессии генов с помощью ДНКмикрочипов не выявил индукции генов десатураз при гиперосмотическом стрессе [41]. Однако на клетках Bacillus subtilis показано, что гиперосмотический стресс вызывает снижение текучести мембран с последующим возрастанием количества ненасыщенных жирных кислот в мембранных липидах [42]. Этот процесс до конца не изучен и может быть связан как с индукцией генов десатураз, так и с увеличением активности предсуществующих десатураз. ...
35, 127276 Москва Тел./факс: 095-977-93-72, эл. почта: losda@ippras.ru Холоднокровные организмы часто испытывают изменения парамет-ров окружающей среды, и их способность к выживанию напрямую зависит от адаптационных способностей. Перепады температур и осмолярности внешней среды вызывают изменения в текучести клеточных мембран. Эти изменения являются необходимыми для запуска ответа на стрессовые воз-действия, что в конечном итоге и обеспечивает адаптацию. Молекулярные механизмы, отвечающие за восприятие изменения текучести мембран, пока полностью не охарактеризованы. Тем не менее применение новых подхо-дов – метода мутагенеза, направленного на изменение текучести мембран, и анализ экспрессии целого генома с помощью ДНК-микрочипов – позво-лили значительно продвинуться в понимании механизмов регуляции теку-чести мембран и идентификации сенсоров, воспринимающих сигналы абиотических стрессов. В этой статье рассматриваются механизмы, регули-рующие текучесть мембран, возможные сенсоры, воспринимающие сигна-лы об изменении текучести, а также экспрессия генов, отвечающих за адап-тацию к меняющимся условиям окружающей среды. Ключевые слова: ДНК-микрочипы, липиды мембран, низкотемпературные сенсоры, осмосенсоры, текучесть мембран, температурный стресс. Введение Температурный и осмотический стрессы вызывают изменения физи-ческих свойств клеточных мембран живых организмов. Вероятно, клетки воспринимают эти изменения с помощью сенсорных белков, локализо-ванных в мембранах. Эти белки передают сигналы от окружающей среды по сетям передачи и в результате регулируют экспрессию генов стрессо-вого ответа [1, 2]. Химические и генетические изменения физических свойств мембран могут оказывать сходный эффект на экспрессию генов, принимающих участие в акклиматизации [3–5]. Физическое состояние мембранных липидов также может регулировать активность мембраносвя-занных белков – ионных каналов [6], рецепторных протеин-киназ [7, 8] и сенсорных белков [9, 10].
... Further, bacterial EPS mitigate saline stress by reducing the content of Na + available for plant uptake (Upadhyay et al. 2012). Salinity stress also has been reported to alter the cell envelope composition of the rhizobacteria resulting in changes in proteins, periplasmic glucans, and capsular, EPS and LPS, fatty acid composition, and cross-linkage of peptidoglycan (Jofré et al. 1998;Piuri et al. 2005) and is suggested that composition of the cell envelops play important role in osmoadaptation (López et al. 2000). ...
... A significant increase in fluorescent anisotropy of DPH was observed when the cells were exposed to hypertonic conditions, for example, when a high KCl concentration (0Á6 mol l À1 ) was added at the beginning of the fermentation (T0.6i). Similar observations have been reported in Bacillus subtilis cells cultured under hypersaline conditions (Lopez et al. 2000), in Escherichia coli (Beney et al. 2004) and Saccharomyces cerevisiae (Laroche et al. 2001) cells submitted to high osmotic pressure, but never in lactic acid bacteria. ...
AimsIn order to help cells to better resist the stressful conditions associated with the freeze-drying process during starter production, we investigated the effect of various osmotic conditions on growth, survival and acidification activity of Lactobacillus buchneri R1102, after freeze-drying and during storage for three months at 25°C.Methods and resultsHigh survival rates during freeze-drying, but not during storage, were obtained when 0.1 M KCl was added at the beginning of fermentation, without any change in membrane properties and betaine accumulation. This condition made it possible to maintain a high acidification rate throughout the process. In contrast, the addition of 0.6 M KCl concentrations at the beginning of fermentation led to a high survival rate during storage that was related to high intracellular betaine levels, low membrane fluidity and high cycC19:0 concentrations. However, these modifications induced the degradation of acidification activity during storage. When a moderate stress was applied by combining 0.1 M KCl at the beginning and 0.6 M KCl at the end of fermentation, betaine accumulated in the cells without any membrane alteration, allowing them to maintain high acidification activity and survival rate during storage.Conclusion
Specific osmotic conditions during fermentation induced intracellular betaine accumulation and modifications of membrane characteristics, thus affecting stress resistance of Lact. buchneri R1102. A slight osmotic stress made it possible to maintain a high acidification activity, whereas a high osmotic stress at the end of fermentation led to the preservation of cell survival during freeze-dried storage.Significance and impact of the studyThis study revealed that the survival and preservation of acidification activity of freeze-dried Lact. buchneri R1102 during starter production can be improved by using appropriate osmotic conditions.This article is protected by copyright. All rights reserved.
... Le changement du profil d'acides gras dans des conditions hyperosmotiques conduit probablement à la rigidité membranaire permettant la rétention d'osmoprotecteurs intracellulaires durant le processus d'osmoadaptation [26]. Ce processus a été identifié chez Bacillus subtilis [60]. ...
Listeria monocytogenes est un pathogène psychrotrophe d’origine alimentaire,
responsable d’infections humaines invasives. C’est un microorganisme
ubiquitaire et capable de survivre dans des conditions de stress
froid et salin rencontrés dans l’industrie de la transformation et le traitement
des aliments. Une stratégie utilisée par plusieurs bactéries et en particulier
par L. monocytogenes pour l’adaptation au stress osmotique est l’accumulation
des solutés compatibles. Ce pathogène utilise deux types de réponses
adaptatives aux basses températures : l’ajustement de la fluidité membranaire
par un changement de la composition des acides gras membranaires et
l’accumulation d’osmolytes de l’environnement. Nous décrivons dans cette
revue les mécanismes mis en jeu par L. monocytogenes pour répondre aux
stress froid et osmotique liés aux procédés de fabrication alimentaire, et à
montrer l’implication de cette adaptation en sécurité des aliments.
... Other NaCl tolerance mechanisms include changes of membrane lipid composition to compensate for the decrease in membrane fluidity. Levels of unsaturated fatty acids in membrane lipids increased in B. subtilis under osmotic stress (Ló pez et al., 2000; Los and Murata, 2004). An increase of branched fatty acids in Gram-positive halophilic and halotolerant bacteria or unsaturated fatty acids in Gram-negative and Gram-positive halophilic bacteria increases the membrane fluidity when grown in media with increasing salt concentrations (Kates, 1986; Russell, 1989). ...
Desulfovibrio vulgaris Hildenborough strains with significantly increased tolerance to NaCl were obtained via experimental evolution. A NaCl-evolved strain, ES9-11, isolated from a population cultured for 1200 generations in medium amended with 100 mM NaCl, showed better tolerance to NaCl than a control strain, EC3-10, cultured for 1200 generations in parallel but without NaCl amendment in medium. To understand the NaCl adaptation mechanism in ES9-11, we analyzed the transcriptional, metabolite and phospholipid fatty acid (PLFA) profiles of strain ES9-11 with 0, 100- or 250 mM-added NaCl in medium compared with the ancestral strain and EC3-10 as controls. In all the culture conditions, increased expressions of genes involved in amino-acid synthesis and transport, energy production, cation efflux and decreased expression of flagellar assembly genes were detected in ES9-11. Consistently, increased abundances of organic solutes and decreased cell motility were observed in ES9-11. Glutamate appears to be the most important osmoprotectant in D. vulgaris under NaCl stress, whereas, other organic solutes such as glutamine, glycine and glycine betaine might contribute to NaCl tolerance under low NaCl concentration only. Unsaturation indices of PLFA significantly increased in ES9-11. Branched unsaturated PLFAs i17:1 ω9c, a17:1 ω9c and branched saturated i15:0 might have important roles in maintaining proper membrane fluidity under NaCl stress. Taken together, these data suggest that the accumulation of osmolytes, increased membrane fluidity, decreased cell motility and possibly an increased exclusion of Na(+) contribute to increased NaCl tolerance in NaCl-evolved D. vulgaris.
... Gene cluster 5 also appears to coordinate the enhanced synthesis of cardiolipin (CL), stimulated by at least three genes in this cluster, including CL synthase (TU #1), and phosphatidylglycerophosphate synthase and 3-ketoacyl(acyl-carrier-protein) reductase (TU #2). Increased CL synthesis would change the composition of the cell membrane, increasing the hydrophobicity and viscosity of this membrane [54], which could have varied phenotypic effects. Increased CL levels stimulate protein translocation across the cell membrane in B. subtilis [55] and E. coli [56], and are important for high osmolarity survival in B. subtilis [57] and S. aureus [58]. ...
Background
The Bacillus cereus sensu lato group currently includes seven species (B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, B. thuringiensis, B. weihenstephanensis and B. cytotoxicus) that recent phylogenetic and phylogenomic analyses suggest are likely a single species, despite their varied phenotypes. Although horizontal gene transfer and insertion-deletion events are clearly important for promoting divergence among these genomes, recent studies have demonstrated that a major basis for phenotypic diversity in these organisms may be differential regulation of the highly similar gene content shared by these organisms. To explore this hypothesis, we used an in silico approach to evaluate the relationship of pathogenic potential and the divergence of the SigB-dependent general stress response within the B. cereus sensu lato group, since SigB has been demonstrated to support pathogenesis in Bacillus, Listeria and Staphylococcus species.
Results
During the divergence of these organisms from a common “SigB-less” ancestor, the placement of SigB promoters at varied locations in the B. cereus sensu lato genomes predict alternative structures for the SigB regulon in different organisms. Predicted promoter changes suggesting differential transcriptional control of a common gene pool predominate over evidence of indels or horizontal gene transfer for explaining SigB regulon divergence.
Conclusions
Four lineages of the SigB regulon have arisen that encompass different gene contents and suggest different strategies for supporting pathogenesis. This is consistent with the hypothesis that divergence within the B. cereus sensu lato group rests in part on alternative strategies for regulation of a common gene pool.
... Severe salt stress has been shown to have pleiotropic effects on the physiology of the soil bacterium, B. subtilis as the composition of the cytoplasmic membrane is altered [52], cell wall properties are adjusted, and swarming capability of the cells is largely impaired [53]. Salinity stress also has been reported to alter the cell envelope composition of the rhizobacteria resulting in changes in proteins, periplasmic glucans, and capsular, exo and lipopolysaccharides [3,54] and is suggested that composition of the cell envelops play important role in osmoadaptation [55]. Certain bacteria like Pseudomonas survive under stress conditions due to the production of exopolysaccharides (EPS), which protects microorganisms from hydric stress and fluctuations in water potential by enhancing water retention and regulating the diffusion of carbon sources in microbial environment [56]. ...
Pseudomonas pseudoalcaligenes, MSP-538 an efficient biocontrol agent against Xanthomonas oryzae pv. oryzae, the bac-terial blight pathogen of rice, was obtained from the salty soils of the coastal agricultural belt of southern coast of India. Greenhouse studies revealed that the root colonization potential of the strain was not hampered with higher salinity in soil. As a means of salt tol-erance, the strain de novo-synthesized, the osmolytes, Ala, Gly, Glu, Ser, Thr, and Asp in the cytosol, as revealed by HPLC-Amino acid analyser profile. The PAGE protein profiling of total cell proteins and membrane proteins revealed many induced/repressed proteins as Salt Stress Proteins (SSPs). Differential Display (DD) of salt stress related genes was carried out using random primers, which revealed an array of up-regulated and down regulated genes.
... However, genome-wide analysis of transcription in Synechocystis, using DNA microarrays, indicated that hyperosmotic stress does not activate the transcription of genes for desaturases Shoumskaya et al., 2005). Nevertheless, in Bacillus subtilis, hyperosmotic stress caused a decrease in the fluidity of cell membranes and subsequently increased the levels of unsaturated fatty acids in membrane lipids (Lopez et al., 2000). This phenomenon might be due to the enhanced expression of the genes for desaturases, or to the activation of preexisting desaturases. ...
Plants and other photosynthetic organisms experience frequent changes in environment. Their ability to survive depends on
their capacity to acclimate to such changes. In particular, fluctuations in temperature and/or osmolarity affect the fluidity
of cytoplasmic and thylakoid membranes. The molecular mechanisms responsible for the perception of changes in membrane fluidity
have not been fully characterized. However, the analysis of genome-wide gene expression with DNA microarrays has provided
a powerful new approach to studies of the contribution of membrane fluidity to gene expression and to the identification of
environmental sensors. In this chapter, we summarize the knowledge on the mechanisms that regulate membrane fluidity, on putative
sensors that perceive changes in membrane fluidity, and on the subsequent expression of genes that ensures acclimation to
a new set of environmental conditions.
... Severe salt stress has been shown to have pleiotropic effects on the physiology of the soil bacterium, B. subtilis as the composition of the cytoplasmic membrane is altered [52], cell wall properties are adjusted, and swarming capability of the cells is largely impaired [53]. Salinity stress also has been reported to alter the cell envelope composition of the rhizobacteria resulting in changes in proteins, periplasmic glucans, and capsular, exo and lipopolysaccharides [3,54] and is suggested that composition of the cell envelops play important role in osmoadaptation [55]. Certain bacteria like Pseudomonas survive under stress conditions due to the production of exopolysaccharides (EPS), which protects microorganisms from hydric stress and fluctuations in water potential by enhancing water retention and regulating the diffusion of carbon sources in microbial environment [56]. ...
Rhizobacteria have been reported to be beneficial to the plants in many different ways. Increasing salinity in the coastal agricultural zones has been shown to be a threat to the plant and microbial life in the area. Exposure of microorganisms to high-osmolality environments triggers rapid fluxes of cell water along the osmotic gradient out of the cell, thus causing a reduction in turgor and dehydration of the cytoplasm. The microorganisms have developed various adaptations to counteract the outflow of water. The first response to osmotic up shifts and the resulting efflux of cellular water is uptake of K(+) and cells start to accumulate compatible solutes. Yet another mechanism is by altering the cell envelope composition resulting in changes in proteins, periplasmic glucans, and capsular, exo and lipopolysaccharides. Bacteria also initiate a program of gene expression in response to osmotic stress by high NaCl concentrations, which are manifested as a set of proteins produced in increased amounts in response to the stress. Genomics, transcriptomics and proteomics approaches have revealed the key components in molecular basis of bacteria salt adaptation. Understanding the mechanisms of osmo-adaptation in rhizobacteria would also be relevant from an ecological and an applicative point of view. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
... Van Loosdrecht et al. (1987b) found that, for a certain species, bacterial cells became more hydrophobic at high growth rates. It has been shown that the change in cell wall hydrophobicity can result from bacterial stress response to certain culture conditions, such as low pH, high temperature, and hyperosmotic stress (Bossier and Verstraete, 1996; Mattarelli et al., 1999; Lopez et al., 2000; Remeta et al., 2002). In the present work, however, there was no significant difference in cell wall hydrophobicity between YB-030518 and YB- 034325 at pH 5.5, 7.0 and 8.5, at 5 °C and 20 °C (Table 1). ...
The cell wall hydrophobicity of Bacillus sp. YB-030518 and YB-034325 isolated from carp (Cyprinus carpio) ponds and its changes under different culture conditions were studied on the basis of the amount of bacteria in a hydrocarbon/water two-phase system. We investigated the effect of cell wall hydrophobicity on the role of these bacteria as probiotics in bioremediation based on shrimp feed. Culture conditions such as growth phase, pH and temperature influence the hydrophobic properties of the Bacillus sp. cell surface. In the exponential growth phase (18 h), the hydrophobicity of both YB-030518 and YB-034325 was significantly higher (P < 0.05) than that at 96 h. The hydrophobicity of YB-034325 was significantly higher (P < 0.05) than that of YB-030518 (5 °C at pH 5.5 and pH 8.5; 20 °C at pH 5.5 and pH 7.0). However, for any single probiotic, there was no statistical difference (P > 0.05) in cell wall hydrophobicity at different pH values (5.5, 7.0 and 8.5) or at different temperatures (5 °C and 20 °C). The relative growth rate of YB-034325 with a high level of hydrophobicity was significantly higher (P < 0.05) than that of YB-030518. The bioremediation capability at 48 h and 96 h was significantly higher in YB-034325 (P < 0.05) compared to YB-030518 and the Control. Moreover, the mean bioremediation capability of treatment with probiotics Bacillus sp. YB-030518 and YB-034325 was significantly higher (P < 0.05) than that of the Control.
... Very similar spectra and fluorescence lifetimes were obtained on liposomes consisting of POPE, DOPG and TOCL, which are considered models of the cytoplasmic membranes of bacteria [12,13]. This is in line with prior observations [6]. ...
Daptomycin is a lipopeptide antibiotic that binds to and depolarizes bacterial cell membranes. Its antibacterial activity requires calcium and correlates with the content of phosphatidylglycerol in the target membrane. Daptomycin has been shown to form oligomers on liposome membranes. We here use perylene excimer fluorescence to further characterize the membrane-associated oligomer. To this end, the N-terminal fatty acyl chain was replaced with perylene-butanoic acid. The perylene derivative retains one third of the antibacterial activity of native daptomycin. On liposomes containing phosphatidylcholine and phosphatidylglycerol, as well as on Bacillus subtilis cells, the perylene-labeled daptomycin forms excimers, which shows that the N-terminal acyl chains of neighboring oligomer subunits are in immediate contact with one another. In a lipid bicelle system, oligomer formation can be titrated with stoichiometric amounts of phosphatidylglycerol. Therefore, the interaction of daptomycin with a single molecule of phosphatidylglycerol is sufficient to trigger daptomycin oligomerization.
... This is indeed the case with PC alone (Fig. 5B), which confirms that oligomerization does not occur on these membranes. The composition of Bacillus cereus and Bacillus subtilis cell membranes has been modeled with liposomes consisting of cardiolipin (TOCL, 17%), phosphatidylglycerol (DOPG, 40%) and phosphatidylethanolamine (POPE, 43%) [20,21]. On such liposome membranes, oligomerization is again observed (Fig. 5C). ...
Daptomycin is a lipopeptide antibiotic that kills Gram-positive bacteria by membrane depolarization. While it has long been assumed that the mode of action of daptomycin involves the formation of membrane-associated oligomers, this has so far not been experimentally demonstrated. We here use FRET between native daptomycin and an NBD-labeled daptomycin derivative to show that such oligomerization indeed occurs. The oligomers are observed in the presence of calcium ions on membrane vesicles isolated from Bacillus subtilis, as well as on model membranes containing the negatively charged phospholipid phosphatidylglycerol. In contrast, oligomerization does not occur on membranes containing phosphatidylcholine only, nor in solution at micromolar daptomycin concentrations. The requirements for oligomerization of daptomycin resemble those previously reported for antibacterial activity, suggesting that oligomerization is necessary for the activity.
... In principle, changing lipid structure could influence membrane protein function by altering chemical and/or physical properties of the membrane (e.g. via chemical phenomena like hydrophobic interactions, hydrogen bonding and electrostatic interactions, or by affecting physical properties like membrane fluidity, membrane tension or interfacial curvature). For example, osmotically induced changes of the phospholipid composition of B. subtilis have been shown to alter physical properties of its cytoplasmic membrane [45,46]. Some effects of lipids on protein function require direct lipidprotein interactions and others do not [43,90]. ...
Cells control their own hydration by accumulating solutes when they are exposed to high osmolality media and releasing solutes in response to osmotic down-shocks. Osmosensory transporters mediate solute accumulation and mechanosensitive channels mediate solute release. Escherichia coli serves as a paradigm for studies of cellular osmoregulation. Growth in media of high salinity alters the phospholipid headgroup and fatty acid compositions of bacterial cytoplasmic membranes, in many cases increasing the ratio of anionic to zwitterionic lipid. In E. coli, the proportion of cardiolipin (CL) increases as the proportion of phosphatidylethanolamine (PE) decreases when osmotic stress is imposed with an electrolyte or a non-electrolyte. Osmotic induction of the gene encoding CL synthase (cls) contributes to these changes. The proportion of phosphatidylglycerol (PG) increases at the expense of PE in cls(-) bacteria and, in Bacillus subtilis, the genes encoding CL and PG synthases (clsA and pgsA) are both osmotically regulated. CL is concentrated at the poles of diverse bacterial cells. A FlAsH-tagged variant of osmosensory transporter ProP is also concentrated at E. coli cell poles. Polar concentration of ProP is CL-dependent whereas polar concentration of its paralogue LacY, a H(+)-lactose symporter, is not. The proportion of anionic lipids (CL and PG) modulates the function of ProP in vivo and in vitro. These effects suggest that the osmotic induction of CL synthesis and co-localization of ProP with CL at the cell poles adjust the osmolality range over which ProP activity is controlled by placing it in a CL-rich membrane environment. In contrast, a GFP-tagged variant of mechanosensitive channel MscL is not concentrated at the cell poles but anionic lipids bind to a specific site on each subunit of MscL and influence its function in vitro. The sub-cellular locations and lipid dependencies of other osmosensory systems are not known. Varying CL content is a key element of osmotic adaptation by bacteria but much remains to be learned about its roles in the localization and function of osmoregulatory proteins.
The Gram‐positive bacterium Bacillus subtilis is frequently exposed to hyperosmotic conditions. In addition to the induction of genes involved in the accumulation of compatible solutes, high salinity exerts widespread effects on B. subtilis physiology, including changes in cell wall metabolism, induction of an iron limitation response, reduced motility, and suppression of sporulation. We performed a combined whole‐transcriptome and proteome analysis of B. subtilis 168 cells continuously cultivated at low or high (1.2 M NaCl) salinity. Our study revealed significant changes in the expression of more than one‐fourth of the protein‐coding genes and of numerous non‐coding RNAs. New aspects in understanding the impact of high salinity on B. subtilis include a sustained low‐level induction of the SigB‐dependent general stress response and strong repression of biofilm formation under high‐salinity conditions. The accumulation of compatible solutes such as glycine betaine aids the cells to cope with water stress by maintaining physiologically adequate levels of turgor and also affects multiple cellular processes through interactions with cellular components. Therefore, we additionally analyzed the global effects of glycine betaine on the transcriptome and proteome of B. subtilis and revealed that it influences gene expression not only under high‐salinity, but also under standard growth conditions. This article is protected by copyright. All rights reserved.
Changing environmental osmotic pressure causes transmembrane water fluxes that may impair cellular functions. Bacteria mitigate water fluxes by controlling the solute content of their cytoplasm. Increasing osmotic pressure triggers solute synthesis or uptake via osmosensing transporters, whereas osmotic downshock triggers solute release via mechanosensitive channels. Membrane lipids are implicated in the subcellular localization and function of membrane-based osmoregulatory systems. Zwitterionic phosphatidylethanolamine (PE) and anionic phosphatidylglycerol (PG) and cardiolipin (CL) are the predominant phospholipids in most bacteria, but their proportions vary widely. For many species, anionic lipids increase in proportion during cultivation in high salinity media. Evidence suggests that interactions among anionic lipid headgroups and cytoplasm-exposed areas of osmosensory transporters ProP, BetP, and OpuA are fundamental to their osmosensory response. CL-dependent targeting of transporter ProP to the CL-rich environment at the poles of Escherichia coli cells further modulates the osmolality response. Protein-lipid interactions are also fundamental to the gating of mechanosensitive channels MscL and MscS by membrane tension. Future work should encompass further characterization of the impacts of lipid composition on key physical properties of the membrane, as well as the regulation of lipid composition and membrane properties in response to environmental cues. The roles of lipids in the structural mechanisms of osmosensing and mechanosensitive channel gating are not fully understood. Osmosensory systems provide useful paradigms for the study of both protein-lipid interactions and the role of subcellular localization in bacterial lipid and protein function.
Spore-forming bacteria are major players in life on Earth, and they are fascinating, as they adapt to many ecological niches. The spores are able to resist high temperatures, to dry out and to recover and germinate when outgrowth conditions become available. Spore-formers are mainly Gram-positive firmicute bacteria with low GC content; the Bacillus and Clostridia genera include prominent species. Bacillus subtilis is one of the most thoroughly studied bacteria, serving as a model to study sporulation and the regulatory networks controlling this differentiation system. B. subtilis is also a bacterium widely used for producing enzymes and metabolites of industrial importance. Among the spore-forming firmicutes, the Bacillus cereus group presents the most remarkable diversity. Frequently designated as “the Good, the Bad and the Ugly”, this bacterial group covers eight closely related species. They include the mammal pathogen Bacillus anthracis, responsible for anthrax disease, the insect pathogen Bacillus thuringiensis, used worldwide as a bioinsecticide to control pests of agricultural, forest and medical importance, and B. cereus senso stricto, an important food contaminant responsible for gastrointestinal disorders and, occasionally, more serious systemic diseases in immunecompromised individuals. The specific mammal toxicity of B. anthracis, the insecticidal activity of B. thuringiensis and the emetic activity of some B. cereus strains are due to plasmid genes. However, in addition to these specific traits, all these bacteria share chromosomal genes conferring the ability to colonize and to grow in abiotic and biotic environments. Interestingly, and in sharp contrast to these pathogens, a few B. cereus strains are used as probiotics in livestock animals and even, formerly, for humans, indicating the versatility of this group of bacteria. Among the pathogenic Clostridia, Clostridium botulinum and Clostridium perfringens are important sources of foodborne poisonings and infections, and the emergent opportunistic human pathogen Clostridium difficile is responsible for severe nosocomial intestinal infections. Due to the high resistance of the spores, to their adhesion properties and to their capacity to form biofilms, these anaerobic Clostridia and facultative anaerobic Bacilli are difficult to eliminate and have a direct economic impact upon the food industry and human health.This special issue of Research in Microbiology contains eight articles covering three main topics highlighted during the first BSPIT (Bactéries sporulantes pathogènes ou d'intérêt technologique) meeting which was held in France (Paris, July 6th and 7th, 2015) and hosted by the SFM (Société Française de Microbiologie), and which was mainly attended by French-speaking scientists.
Variations in the supply of water and the concomitant changes in salinity and osmolality are among the most significant environmental parameters affecting the survival and growth of microorganisms (Galinski and Trüper 1994; Csonka and Epstein 1996; Wood 1999; Bremer and Krämer 2000). Microorganisms can colonize a wide variety of ecological niches with a considerable spectrum of salinitiFs and osmotic conditions that range from concentrated salt brines to fresh water sources (Ventosa et al.1998). Furthermore, within a single bacterial habitat, there also can be drastic fluctuations from the prevalent osmotic milieu.
In the present scenario increased agricultural productivity is achieved by indiscriminate and excessive application of agrochemicals which lead to health and environmental hazards. Extensive research in developing eco-friendly strategy to reduce or replace the application of agrochemicals revealed plant growth-promoting rhizobacteria (PGPR) as potential candidate. PGPR are an important group of bacterial communities residing in rhizosphere and exert beneficial effects on host plant through various mechanisms. The mode of action through which PGPR enhance plant fitness under different biotic and abiotic stress have been a target of research from several years. Hence, in this chapter, mechanism involved in PGPR-mediated growth promotion and suppression of biotic and abiotic stress in plants is reviewed. PGPR is known to exert its beneficial effect on plant through several mechanisms. Plant growth-promoting mechanisms include root colonization, nitrogen fixation, phosphate solubilization, production of IAA, and other phytohormones, siderophore, and volatiles. Suppression of biotic stress is through competition for space, food and nutrition with phytopathogens, production of antibiotic, siderophores, volatiles, and by inducing systemic resistance (ISR). Abiotic stress (especially drought and salt) management by means of PGPR involves modification of physiological and biochemical activities of host plant by producing abscisic acid and other phytohormones, lowering the ethylene by activity of ACC deaminase, and producing antioxidants, which are collectively termed as induced systemic tolerance (IST). Hence expression of these PGPR traits and factors affecting on it should be studied thoroughly in order to use it to its best. Under experimental or field conditions, these traits may express singly or simultaneously which is affected by various biotic and abiotic factors which alter the performance of introduced PGPR.
The effects of NaCl reduction (10.0, 7.5, 5.0, 2.5, and 0% NaCl) and its substitution with KCl (50% substitution at each given concentration) on morphology of Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium longum was investigated using transmission electron microscopy. Changes in membrane composition, including fatty acids and phospholipids, were investigated using gas chromatography and thin layer chromatography. Adhesion ability of these bacteria to human intestinal epithelial-like Caco-2 cells, as affected by NaCl and its substitution with KCl, was also evaluated. Bacteria appeared elongated and the intracellular content appeared contracted when subjected to salt stress, as observed by transmission electron microscopy. Fatty acid content was altered with an increase in the ratio of unsaturated to saturated fatty acid content on increasing the NaCl-induced stress. Among the phospholipids, phosphatidylglycerol was reduced, whereas phosphatidylinositol and cardioplipin were increased when the bacteria were subjected to salt stress. There was a significant reduction in adhesion ability of the bacteria to Caco-2 cells when cultured in media supplemented with NaCl; however, the adhesion ability was improved on substitution with KCl at a given total salt concentration. The findings provide insights into bacterial membrane damage caused by NaCl.
Carried out Two experiments laboratory in the Department of Horticulture -College of Agriculture Diyala University in 2011 to study the effect of salinity on the growth Azospirillum brasilense bacteria, as it included the first experiment salinisation of soil salt levels different (1.8,4 ,6 ,8 and 10) ds.m-1 for periods to incubation different (4, 8, 12 and 16) days, the results showed increasing levels of salinity caused a reduction significantly the numbers of bacteria. affected the periods of incubation affected significantly in the numbers of cells of bacteria. Gave period incubation (16) days on highest values for the numbers of bacterial cells. Was a significant effect of interaction in the numbers of bacterial cells, as it reached the highest numbers at the level of salinity (1.8) ds.m-1 and at period incubation (16) days and gave the level (10) ds.m-1 at period incubation (8) days lower the values Second experiment studied the effect of five salts (NaCl , Na 2 So 4 , MgCl 2 .6H 2 o , MgSo 4 .7H 2 o and CaCl 2 .2H 2 o), with six concentrations (0, 0.04, 0.08, 0.12, 0.16 and 0.20) Mole.L -1 on the numbers bacterial cells Azospirillum brasilense, the results showed that increasing the level of salt reduced the numbers of bacteria cells a significant decrease irrespective of the type of salt. Mgcl 2 salt gave the highest numbers irrespective of the level of salt and not significant differences for salt (MgSo 4 , CaCl 2), while the salt gave (NaCl 2) lower the values followed by salt (Na 2 So 4). That increasing levels of salinity affected significantly reduce the numbers of bacteria and all types of salts studied, as the salt (NaCl 2) and at (0.16) Mole.L -1 did not record any growth of bacteria, and salt (Na 2 So 4) did not record any growth at (0.20) ds.m -1 , recorded salt (MgSo 4) at (0.04) Mole.L -1 higher values for the numbers of bacteria.
Microbacterium esteraromaticum is a microorganism that is frequently obtained of cultivations coming from landfarming or hydrocarbons biorremediation processes in the central Patagonia (Argentina) and it is subjected to variations of temperature and changes of salinity that take place naturally. Its adaptability to these climatic conditions took us to study the changes of adaptation that take place in the fatty acids of its membranes. This work studied the combined effect of temperature and salt concentration on the membrane fatty acids composition of Microbacterium esteraromaticum GNP5b strain. M. esteraromaticum, as a result of temperature increases, elongated the fatty acids chains (17 atoms of carbon), decreased 15:0 anteiso, and modified the percentage of fatty acids 15:0 iso starting from the respective anteiso. With salinity increases it modifies its composition of fatty acids according to incubation temperature. At 14 and 28 °C it increases the 15:0 iso and 17:0 iso in detriment of their homologous anteiso. At 37 °C this group of fatty acids does not follow the same previous patterns. The chain length, expressed as the index C15/C17, is erratic with salinity increases.
The probiotic Gram-positive bacterium Lactobacillus casei BL23 is naturally confronted with salt stress habitats. It has been previously reported that growth in high salt medium containing 0.8M NaCl leads to modifications in the cell envelope of this bacterium. In this study, we report that L. casei BL23 has an increased ability to form biofilms and to bind cations in high salt conditions. This behavior correlated with modifications of surface properties involving teichoic acids, which are important cell wall components. Also in this high salt condition we showed that L. casei BL23 produces lower level of the cell wall polymer lipoteichoic acid (LTA) and that this anionic polymer has a shorter average chain length and a lower level of D-alanyl-substitution. Moreover analysis of the transcript levels of the dltABCD operon, encoding the enzymes required for the incorporation of D-alanine into anionic polymers, showed a 16-fold reduction in mRNA levels, which is consistent with a decrease in D-alanine substitutions on LTA. Furthermore, a 13-fold reduction in the transcript levels was observed for the gene LCABL_09330 coding for a putative LTA synthase. To provide further experimental evidence that LCABL_09330 is a true LTA synthase (LtaS) in L. casei BL23, the enzymatic domain was cloned and expressed in E. coli. The purified protein was able to hydrolyze the membrane lipid phosphatidylglycerol as expected for an LTA synthase enzyme, and hence LCABL_09330 was renamed LtaS. The purified enzyme showed Mn2+-ion dependent activity, and its activity was modulated by differences in NaCl concentration. The decrease in both ltaS transcript levels and enzyme activity observed in high salt condition might influence the length of the LTA backbone chain. A putative function of the modified LTA structure is discussed that is compatible with the growth under salt stress conditions and with the overall envelope modifications taking place during this stress condition.
Biogranulation that includes anaerobic and aerobic granular sludge processes is a promising biotechnology for wastewater treatment. The formation and structure of biogranules are associated very closely with cell hydrophobicity. This paper therefore attempts to review the essential role of cell hydrophobicity in the formation of granular sludge. Cell hydophobicity could be induced by culture conditions, and in turn initiates cell-to-cell aggregation that is a crucial step towards biogranulation. In this paper, the factors that may influence cell hydrophobicity were discussed, while a thermodynamic interpretation of cell hydrophobicity was also presented. Meanwhile, a selection pressure-based inducing strategy for cell hydrophobicity was further proposed. It appears that cell hydrophobicity is a triggering force of biogranulation, and high cell hydrophobicity seems to be a prerequisite of biogranulation. However, the knowledge regarding the role of cell hydrophobicity in biogranulation process is far from complete. Some future research niches are then outlined.
The yqiHIK gene cluster from Bacillus subtilis is predicted to encode an extracellular lipoprotein (YqiH), a secreted N-acetylmuramoyl-l-alanine amidase (YqiI), and a cytoplasmic glycerophosphodiester phosphodiesterase (YqiK). Reverse transcriptase PCR (RT-PCR)
analysis showed that the yqiHIK genes are transcribed as an operon. Consistent with the in silico prediction, we found that the purified YqiI protein exhibited hydrolytic activity toward peptidoglycan sacculi. Transcription
studies with yqiH-treA reporter fusion strains revealed that the expression of yqiHIK is subjected to finely tuned osmotic control, but enhanced expression occurs only in severely osmotically stressed cells.
Primer extension analysis pinpointed the osmotically responsive yqiHIK promoter, and site-directed mutagenesis was employed to assess functionally important sequences required for promoter activity
and osmotic control. Promoter variants with constitutive activity were isolated. A deletion analysis of the yqiHIK regulatory region showed that a 53-bp AT-rich DNA segment positioned 180 bp upstream of the −35 sequence is critical for
the activity and osmotic regulation of the yqiHIK promoter. Hence, the expression of yqiHIK is subjected to genetic control at a distance. Upon the onset of growth of cells of the B. subtilis wild-type strain in high-salinity medium (1.2 M NaCl), we observed gross morphological deformations of cells that were then
reversed to a rod-shaped morphology again when the cells had adjusted to the high-salinity environment. The products of the
yqiHIK gene cluster were not critical for reestablishing rod-shaped morphology, but the deletion of this operon yielded a B. subtilis mutant impaired in growth in a defined minimal medium and at high salinity.
Conjugation experiments with Bacillus thuringiensis and transfer kinetics demonstrated that salt stress has a positive impact on plasmid transfer efficiency. Compared to standard
osmotic conditions (0.5% NaCl), plasmid transfer occurred more rapidly, and at higher frequencies (>100-fold), when bacteria
were exposed to a high-salt stress (5% NaCl) in liquid brain heart infusion (BHI). Under milder salt conditions (2.5% NaCl),
only a 10-fold effect was observed in Luria-Bertani broth and no difference was detected in BHI. These observations are particularly
relevant in the scope of potential gene exchanges among members of the Bacillus cereus group, which includes food-borne contaminants and pathogens.
The application of physical stress to microorganisms is the most widely used method to induce cell inactivation and promote food stability. To survive, microorganisms have evolved both physiological and genetic mechanisms to tolerate some extreme physical conditions. This is clearly of significance to the food industry in relation to survival of pathogens or spoilage organisms in food. In some microorganisms, the "cold shock response" has been observed in response to abrupt changes to lower temperatures. This results in the production of specific sets of proteins ( cold shock proteins), the continued synthesis of proteins involved in transcription and translation, and the repression of heat shock proteins. The addition of weak acid preservatives ( for example, sorbates, benzoates) also induces a specific pattern of gene expression ( for example, 'Acid Tolerance Response'), which is likely to be required for optimal adaptation of bacteria to weak acid preservatives and low pH. The primary mode of the antimicrobial action of low pH is to reduce the internal cell pH (pHi) below the normal physiological range tolerated by the cell, leading to growth inhibition. Survival mechanisms involve maintaining pH homeostasis, and this is achieved by a combination of passive and active mechanisms. Microorganisms adapt to osmotic stress by accumulating non-ionic or compatible solutes such as trehalose, glycerol, sucrose, and mannitol. These compatible solutes help balance the osmotic pressure and help preserve protein function inside the cells. By understanding and controlling such mechanisms of adaptation, it may be possible to prevent growth of key microorganisms in food products.
In its natural habitats, Bacillus subtilis is exposed to changing osmolarity, necessitating adaptive stress responses. Transcriptomic and proteomic approaches can provide
a picture of the dynamic changes occurring in salt-stressed B. subtilis cultures because these studies provide an unbiased view of cells coping with high salinity. We applied whole-genome microarray
technology and metabolic labeling, combined with state-of-the-art proteomic techniques, to provide a global and time-resolved
picture of the physiological response of B. subtilis cells exposed to a severe and sudden osmotic upshift. This combined experimental approach provided quantitative data for
3,961 mRNA transcription profiles, 590 expression profiles of proteins detected in the cytosol, and 383 expression profiles
of proteins detected in the membrane fraction. Our study uncovered a well-coordinated induction of gene expression subsequent
to an osmotic upshift that involves large parts of the SigB, SigW, SigM, and SigX regulons. Additionally osmotic upregulation
of a large number of genes that do not belong to these regulons was observed. In total, osmotic upregulation of about 500
B. subtilis genes was detected. Our data provide an unprecedented rich basis for further in-depth investigation of the physiological
and genetic responses of B. subtilis to hyperosmotic stress.
The plant growth-promoting rhizobacterium, Ochrobactrum sp. 11a displays a high intrinsic salinity tolerance and has been used in this work to study the molecular basis of bacterial responses to high concentrations of NaCl. A collection of Ochrobactrum sp. 11a mutants was generated by Tn5-B21 mutagenesis and screened for sensitivity to salinity. One clone, designated PBP and unable to grow on glutamate mannitol salt agar medium supplemented with 300 mM NaCl was selected and further characterized. The PBP mutant carries a single transposon insertion in a gene showing a high degree of identity to the serine-type d-alanyl-d-alanine carboxypeptidase gene of Ochrobactrum anthropi. Interestingly, the expression of this gene was shown to be upregulated by salt in the PBP mutant. Moreover, evidence is presented for the requirement of the gene product for adaptation to high-salt conditions as well as to overcome the toxicity of LiCl, KCl, sucrose, polyethylene glycol (PEG), AlCl(3), CuSO(4), and ZnSO(4). In addition to the altered tolerance to both ionic and osmotic stresses, the PBP mutant exhibited changes in colony and cell morphology, exopolysaccharide production, and an increased sensitivity to detergents.
The study was focused on the role of the penicillin binding protein PBP4* of Bacillus subtilis during growth in high salinity rich media. Using pbpE-lacZ fusion, we found that transcription of the pbpE gene is induced in stationary phase and by increased salinity. This increase was also corroborated at the translation level for PBP4* by western blot. Furthermore, we showed that a strain harboring gene disruption in the structural gene (pbpE) for the PBP4* endopeptidase resulted in a salt-sensitive phenotype and increased sensitivity to cell envelope active antibiotics (vancomycin, penicillin and bacitracin). Since the pbpE gene seems to be part of a two-gene operon with racX, a racX::pRV300 mutant was obtained. This mutant behaved like the wild-type strain with respect to high salt. Electron microscopy showed that high salt and mutation of pbpE resulted in cell wall defects. Whole cells or purified peptidoglycan from WT cultures grown in high salt medium showed increased autolysis and susceptibility to mutanolysin. We demonstrate through zymogram analysis that PBP4* has murein hydrolyze activity. All these results support the hypothesis that peptidoglycan is modified in response to high salt and that PBP4* contributes to this modification.
A mutant of Escherichia coli has been isolated, the growth of which is inhibited by low concentrations (1 mm) of NaCl. High levels of magnesium, calcium, or strontium in the medium permit growth in the presence of sodium. The metal content of the inhibited mutant is normal, but the strain is unable to tolerate levels of sodium to which the wild type is indifferent. Immediately after the addition of sodium to cultures of the mutant, rates of synthesis of protein, ribonucleic acid, deoxyribonucleic acid, and total lipid are unchanged, but more cardiolipin and less phosphatidylethanolamine are produced. The direct enzymatic cause of this change, which affects membrane function, is not known. Studies of the metabolism of phosphatidylglycerol in vivo after pulse-labeling with [2-(3)H]glycerol reveal that a major pathway both in wild-type and mutant strains involves the cleavage of labeled glycerol from phosphatidylglycerol.
The contribution of lipoteichoic acid (LTA) to the hydrophobic surface properties of group A streptococci was investigated in aqueous dextran-polyethylene glycol two-phase systems. Enzymatic digestions were performed to characterize the hydrophobic surface structure. The results obtained indicated that LTA is a major factor responsible for the hydrophobic character of the cell surface of group A streptococci. This was further supported by the similarity of partition in polymer two-phase systems between whole group A streptococci and tritiated LTA extracted from a group A streptococcal strain. Surface LTA was also determined on intact organisms by a new method measuring the adsorption of antibodies to LTA to the bacterial surface. A correlation was found between the content of surface LTA and the hydrophobicity of the group A streptococci. We conclude that surface-associated LTA is the major factor determining surface hydrophobicity of group A streptococci.
The adherence of Streptococcus pyogenes cells to hexadecane droplets was measured by vortexing water suspensions of streptococci with hexadecane. It was found that adherence of the organisms to hexadecane droplets was abolished by pretreating the organisms with trypsin, pepsin at pH 4.5, or HCl solutions at 95 degrees C. Streptococcal adherence was best expressed in organisms harvested during the stationary phase of growth and was inhibited by fatty acid-free albumin because of the interaction of the protein with the streptococcal surfaces. The data suggest that adherence to hexadecane droplets measures the availability on the surface of S. pyogenes cells of lipophilic residues that are either hydrophobic regions of surface protein structures or, more likely, glycolipids complexed with and oriented by surface proteins.
All microorganisms possess a positive turgor, and maintenance of this outward-directed pressure is essential since it is generally considered as the driving force for cell expansion. Exposure of microorganisms to high-osmolality environments triggers rapid fluxes of cell water along the osmotic gradient out of the cell, thus causing a reduction in turgor and dehydration of the cytoplasm. To counteract the outflow of water, microorganisms increase their intracellular solute pool by amassing large amounts of organic osmolytes, the so-called compatible solutes. These osmoprotectants are highly congruous with the physiology of the cell and comprise a limited number of substances including the disaccharide trehalose, the amino acid proline, and the trimethylammonium compound glycine betaine. The intracellular amassing of compatible solutes as an adaptive strategy to high-osmolality environments is evolutionarily well-conserved in Bacteria, Archaea, and Eukarya. Furthermore, the nature of the osmolytes that are accumulated during water stress is maintained across the kingdoms, reflecting fundamental constraints on the kind of solutes that are compatible with macromolecular and cellular functions. Generally, compatible solutes can be amassed by microorganisms through uptake and synthesis. Here we summarise the molecular mechanisms of compatible solute accumulation in Escherichia coli and Bacillus subtilis, model organisms for the gram-negative and gram-positive branches of bacteria.
The role of unsaturated fatty acids in membrane lipids in the tolerance of the photosynthetic machinery to salt stress was studied by comparing the desA-/desD- mutant of Synechocystis sp. PCC 6803, which contained monounsaturated fatty acids, with the wild-type strain, which contained a full complement of polyunsaturated fatty acids. In darkness, the loss of oxygen-evolving photosystem II activity in the presence of 0.5 M NaCl or 0.5 M LiCl was much more rapid in desA-/desD- cells than in wild-type cells. Oxygen-evolving activity that had been lost during incubation with 0.5 M NaCl in darkness returned when cells were transferred to conditions that allowed photosynthesis or respiration. Recovery was much greater in wild-type than in desA-/desD- cells, and it was prevented by lincomycin. Thus, the unsaturation of fatty acids is important in the tolerance of the photosynthetic machinery to salt stress. It appears also that the activity and synthesis of the Na+/H+ antiporter system might be suppressed under high-salt conditions and that this effect can be reversed, in part, by the unsaturation of fatty acids in membrane lipids.
Changes in the cell envelopes in response to acidity were studied in two strains of Mesorhizobium loti differing in their tolerance to pH. When the less acid-tolerant strain LL22 was grown at pH 5.5, membrane phosphatidylglycerol decreased and phosphatidylcholine increased, compared with cells grown at pH 7.0. On the other hand, when the more acid-tolerant strain LL56 was grown at pH 5.5, phosphatidylglycerol, phosphatidylethanolamine, and lysophospholipid decreased 25%, 39%, and 51% respectively, while phosphatidyl-N-methylethanolamine and cardiolipin increased 26% and 65% respectively compared with cells grown at pH 7.0. The longest-chain fatty acids (19:0 cy and 20:0) increased in both strains at pH 5.5, while in LL56 these fatty acids increased still further at pH 4.0. Variations in other wall and membrane properties such as cell hydrophobicity, lypopolysaccharides, and protein composition of the outer membrane in relation to acid pH are also discussed.
The lipid-water phase diagrams of polar- and galacto-lipids extracted from maize chloroplasts are described and the structures of the various phases are analysed. The general conformation of the paraffin chains is found to be liquid-like. One phase is lamellar, formed by identical planar lipid leaflets, filled by the paraffin chains and separated by layers of water. In the case of the polar lipids the thickness of the water layer increases more than 150 Å with increasing water concentration, whereas in the case of the galacto-lipids only a small amount of water can be incorporated between the lipid leaflets (the maximum thickness of the water layer is 20 Å) and the water in excess remains as a separate phase. This property, previously observed with other lipids, is related to the presence of net electrical charges in polar lipids. In the “dry” region of the phase diagram other phases are observed: one is a hexagonal array of infinite stiff rods, filled by the polar elements of the system, the other is formed by rods of similar structure but of finite length, joined three by three to form two interwoven three-dimensional networks, organized in a body-centred cubic lattice. The hexagonal phase is observed for both the polar- and galacto-lipids and the cubic phase only in galacto-lipids. The structures of the lamellar and of the cubic phases are consistent with the analysis of the intensity of the X-ray reflections. The observations are compared to those previously made with other lipid systems, and some conclusions of general interest are drawn.
Pulse-chase experiments with [2-3H]glycerol and [14C]acetate revealed that in Staphylococcus aureus lipoteichoic acid biosynthesis plays a dominant role in membrane lipid metabolism. In the chase, 90% of the glycerophosphate moiety of phosphatidylglycerol was incorporated into the polymer: 25 phosphatidylglycerol + diglucosyldiacylglycerol → (glycerophospho)25-diglucosyldiacylglycerol + 25 diacylglycerol.
Glycerophosphodiglucosyldiacylglycerol was shown to be an intermediate, confirming that the hydrophilic chain is polymerized on the final lipid anchor.
Total phosphatidylglycerol served as the precursor pool and was estimated to turn over more than twice for lipoteichoic acid synthesis in one bacterial doubling. Of the resulting diacylglycerol approximately 10% was used for the synthesis of glycolipids and the lipid anchor of lipoteichoic acid. The majority of diacylglycerol recycled via phosphatidic acid to phosphatidylglycerol. Synthesis of bisphosphatidylglycerol was negligible and only a minor fraction of phosphatidylglycerol passed through the metabolically labile lysyl derivative.
In contrast to normal growth, energy deprivation caused an immediate switch-over from the synthesis of lipoteichoic acid to the synthesis of bisphosphatidylglycerol.
The effect of salt stress on lipid composition and its relationship with ethanol tolerance in Saccharomyces cerevisiae was studied. Amounts of phospholipids as well as that of sterols decreased, whereas that of protein and glycolipids increased with increasing salt concentration. Relative proportion of amino phospholipids (phosphatidylethanolamine and phosphatidylserine) decreased, whereas that of phosphatidylcholine showed a reverse trend. Cells grown under increasing salt concentration were more resistant to ethanol-induced leakage of UV-absorbing substances, an index of ethanol endurance. Results showed an overlap between osmotolerance and ethanol tolerance in this strain.
Proefschrift Nijmegen. Met lit. opg. - Met samenvatting in het Nederlands. Titel op omslag: Lipoteichoic acids of bifidobacterium bifidum subsp. pennsylvanicum.
Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/30145/1/0000522.pdf
Data from several membrane systems are presented to confirm an empirical means of correcting diphenylhexatriene fluorescence for depolarization caused by sample turbidity. The depolarization proportionally constants obtained are not equal, but are shown to vary with (a) the physical state of the membrane, (b) the cholesterol content of the membrane, (c) the protein content of the membrane, and (d) the method of membrane preparation or isolation. It is concluded that depolarization corrections must always be considered when diphenylhexatriene fluorescence anisotropy is used to compare the fluidities within different membrane bilayers.
Branched-chain fatty acids were first found by the author in certain bacteria some years ago. In this extensive review the following chapters are included: Fatty acid composition of Bacillus sp., biosynthesis of fatty acids, factors affecting fatty acid pattern and content, function of branched-chain fatty acids, grouping of Bacillus sp. according to fatty acid patterns and its significance on taxonomy, evolution of fatty acid systems. The occurrence of iso and anteiso fatty acids in microbes is not as rare as first thought. They are the major component of the fatty acids found within certain genera. This can be used in taxonomy and in the hypothesis of evolution in microorganisms. Fatty acids are mostly found on cell membranes. Branched-chain fatty acids are rare in animals but certain recent data suggest that their importance in animals has not been fully appraised. (Csonka - Radlett)
A lipoteichoic acid and a membrane glycolipid were isolated from Bacillus licheniformis 6346 MH-1. The fatty acid composition of the two preparations were similar. Most of the fatty acids were of the branched chain type. The glycolipid was shown to be a diacyl derivative of O-beta-D-glucopyranosyl-(1 leads to 6)-O-beta-D-glucopyranosyl-(1 leads to 3)-glycerol. The lipoteichoic acid contained lipid, polyglycerol phosphate, and glucosamine. The lipid was released by treatment with hydrofluoric acid and by hydrolysis in dilute acid and was shown to have a structure identical with that of the membrane glycolipid.
The initiation of sporulation in Bacillus subtilis is controlled by the Spo0A transcription factor which is activated by phosphorylation through a phosphorelay mechanism that is dependent upon the activity of one or more protein kinases. The enzymatic activity of one of these protein kinases, KinA, was found to be inhibited in vitro by certain fatty acids. The most potent inhibitors have at least one unsaturated double bond in the cis configuration and a chain length of 16-20 carbon atoms. Homologous isomers with a trans double bond are not inhibitory. Saturated straight- or branched-chain fatty acids are either much weaker inhibitors or have no effect. The inhibitors prevent autophosphorylation of KinA and are non-competitive with ATP. B. subtilis phospholipids were found to contain at least one as yet unidentified type of fatty acid that, when present in an unesterified form, inhibited KinA. The results suggest that the concentration of a specific unsaturated fatty acid may act as a signal linking the initiation of sporulation to the status of membrane synthesis and septation or some other specific membrane-associated activity.
The cytoplasmic membrane of Bacillus subtilis 168, prepared from cells in the stationary phase, has been found to contain protein (62%), RNA (22%) and lipid (16%).
Analyses of the membrane lipid revealed the presence of phospholipids (75%), neutral lipid (10%) and a compound identified as a diglucosyl diglyceride. The major phospholipids were diphosphatidyl glycerol and phosphatidyl ethanolamine, with small amounts of phosphatidyl glycerol and lipoamino acids.
Branched chain fatty acids comprised over 75% of the total fatty acids of both whole cells and membranes. Iso and anteiso acids with 15 and 17 carbon atoms were the major components, together with small amounts of iso acids containing 14 and 16 carbon atoms and n-acids. No unsaturated acids were present.
A procedure has been devised to isolate mutants of Bacillus subtilis with structurally defective membranes. The procedure used to screen for the mutants involved comparison of the stability of protoplasts of the mutant with those of the wild type in a medium of sufficient osmotic strength to stabilize wild-type protoplasts. Mutagenized cells were grown as clones on agar plates, and then replicated onto plates containing 0.5 m lactose, which is sufficient to stabilize wild-type protoplasts. The colonies on the lactose-containing plates were then treated with lysozyme to convert the cells to protoplasts. Colonies of wild-type protoplasts remained opaque; however, colonies of mutant protoplasts lysed and became clear. Twenty-nine osmotically fragile mutants were isolated in this manner; the membranes of several mutants were found to contain alterations in the composition of their proteins or lipids.
1.1. Cardiolipin of Staphylococcus aureus 209 P markedly increases and phosphatidylglycerol and lysylphosphatidylglycerol diminish as the NaCl concentration of the culture medium increases.2.2. The major change in fatty acid composition lies in the proportion of branched acids, resulting in an increase of the branched fatty acids in cardiolipin when cultured in high NaCl medium.
Bacillus subtilis contained at least five phospholipids, four of which have been isolated and identified as a polyglycerol phospholipid, probably cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, and lysylphosphatidylglycerol. Further purification of the latter phosphoglyceride was obtained by high-voltage electrophoresis, and it was shown that this treatment removed amino acid-containing, nonlipidic material from the phosphoglyceride. This associated material, which is not covalently linked to the lipid, gave rise to minor amounts of a number of amino acids, other than lysine, in acid hydrolysates of the lysylphosphatidylglycerol. The phospholipid composition of B. subtilis appeared to depend on the growth conditions. Addition of glucose to the medium lowered the pH during growth; this was accompanied by an increase in the amount of lysylphosphatidylglycerol and a decrease in the phosphatidylglycerol content, when compared with growth at neutral pH. The amount of the other phospholipids and the total amount of phospholipid remained constant under the different conditions. The shape and the osmotic susceptibility of the protoplasts of this organism appeared to depend on the growth conditions. Cells harvested from a neutral growth medium gave spherical protoplasts which lysed rapidly, whereas cells grown in an acidic medium maintained their rod-shaped form to a great extent after the cell wall had been removed, even after being suspended in a hypotonic medium. The latter observation suggests the presence of a more rigid membranous structure in cells which have been exposed to a low environmental pH during growth.
Bacillus subtilis cultures submitted to an osmotic upshock (1.5 M NaCl) lysed unless stationary phase had been reached. Several physiological variations were observed, such as delayed growth (adaptation), a filamentous bacterial appearance, RecA-dependent osmoresistance (SOS), and cross-induction by a previous stress (heat shock). Osmoresistance and sporulation seem to share pathways of regulation such as inhibition in the presence of glucose and glutamine and derepression in a catabolite-resistant mutant such as degUh. However, spores were not obtained on hypertonic media. Mutants of later sporulation stages (spoII, spoIII) presented a response similar to that of the wild-type parent, indicating that both processes probably shared early controls. Null mutations in any of the known key modulators of sporulation (spoOA or degU) resulted in similar levels of osmosensitivity. Sensor mutations in kinA and degS also led to strains with altered responses, the kinA mutant being even more osmosensitive than the degS mutant. Several spoOA mutant phenotypes are due to this gene's control of abrB, a regulator of stationary-phase events, and an abrB mutation relieved the osmosensitivity of the spoOA-containing mutant but had no effect on a wild-type strain.
Coexisting gel and liquid-crystalline phospholipid phase domains can be observed in synthetic phospholipid vesicles during the transition from one phase to the other and, in vesicles of mixed phospholipids, at intermediate temperatures between the transitions of the different phospholipids. The presence of cholesterol perturbs the dynamic properties of both phases to such an extent as to prevent the detection of coexisting phases. 6-Lauroyl-2-dimethylaminopahthalene (Laurdan) fluorescence offers the unique advantage of well resolvable spectral parameters in the two phospholipid phases that can be used for the detection and quantitation of coexisting gel and liquid-crystalline domains. From Laurdan fluorescence excitation and emission spectra, the generalized polarization spectra and values were calculated. By the generalized polarization phospholipid phase domain coexistence can be detected, and each phase can be quantitated. In the same phospholipid vesicles where without cholesterol domain coexistence can be detected, above 15 mol% and, remarkably, at physiological cholesterol concentrations, > or = 30 mol%, no separate Laurdan fluorescence signals characteristic of distinct domains can be observed. Consequences of our results on the possible size and dynamics of phospholipid phase domains and their biological relevance are discussed.
The envelope properties of B. subtilis cultures grown in LB and LBN hyperosmotic media (LB + 1.5 M NaCl) were compared. Since hypertonic cultures showed a Spo-phenotype, a Spo-mutant grown in LB was also analyzed. LBN cultures showed extensive filamentation and presented different sensitivities toward phage infection (phi29 and phi105), or antibiotics whose targets are at wall (lysozyme, penicillin G) or membrane level (polymyxin B, phosphonomycin). Results of the biochemical composition revealed that during hyperosmotic growth, the cell wall increased in thickness, and among the membrane lipids, glycolipid and cardiolipin increased in parallel with a decrease in phosphatidylglycerol. The fatty acid composition was also modified, and an increase in saturated straight chain with a decrease of saturated iso-branched fatty acids was observed. The increase of monounsaturated 18-1 (omega-9) fatty acid was probably related to the absence of sporulation observed in hypertonic media, since its increase has been shown to inhibit the KinA sensor of sporulation. The significance of the other wall and membrane composition variations (and hydrophobic surface properties) in relation to the osmotic adaptation are discussed.
In Bacillus subtilis, osmotolerance is a stationary phase-dependent, adaptive response inhibiting sporulation and sharing common regulators with this process. The extent of this inhibition was determined by measuring transcription activity of promoter lacZ fusions to early sigma genes (spoIIG and spoIIA coding for precursors of sigmaE and sigmaF) and to reporters of them (spoIID, spoIIQ and spoIIIG), in the absence and presence of 0.6 M or 1 M NaCl. The transcription activity of these sigma precursors, normally occurring at the onset of the stationary phase, was reduced to 30-50% of their maximal expression in hyperosmotic conditions; expression of genes under their control was, however, more inhibited (<10%). Therefore, sporulation was blocked at the sigma sigmaE and sigmaF activation steps. This assumption was confirmed by electron microscopic examinations of hyperosmotic cultures, which presented asymmetric septa characteristic of stage II mutants. Discussion was focused on the particular composition and/or structure of membranes during hyperosmotic growth and their involvement in the arrest of sporulation.
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