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Role of rpoS in Stress Survival and Virulence of Vibrio cholerae
Journal of Bacteriology
Abstract
Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by rpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivated rpoS by homologous recombination. V. cholerae strains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoS mutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.
... In the first publication on chitin-induced natural competence by the Schoolnik laboratory, the authors showed that a rpoS mutant of V. cholerae was nontransformable and suggested that this phenotype might be based on RpoS's effect on hapR expression (Meibom et al., 2005). Previous work had suggested that RpoS was required for hapR transcription, since an rpoS mutant showed reduced hapR transcript levels (Yildiz et al., 2004) and decreased HA/protease (HapA) activity (Yildiz and Schoolnik, 1998) (the encoding hapA gene is positively regulated by HapR). However, recent work challenged this interpretation by showing that RpoS did not affect QS-dependent competence regulation and instead exerted its effect via the production of extracellular chitinases, therefore using the chitin-induction branch of the competence network (Dalia, 2016) (Fig. 1). ...
... Moreover, both mutants remained non-transformable even upon chitin-independent TfoX production ( Fig. 3C; rpoS::cm). Given these conflicting results compared to the convincing data provided by Dalia (Dalia, 2016), we genetically reengineered the rpoS mutant in two ways: first, by transferring the inactivated rpoS allele containing a chloramphenicol-resistance cassette from the original rpoS mutant strain (rpoS::cm corresponding to FY1; Yildiz and Schoolnik, 1998;Meibom et al., 2005) into the WT strain via natural transformation (mutant rpoS::cm-new); and second, by designing a completely new in-frame rpoS deletion strain (mutant ΔrpoS) using natural transformation coupled to flip recombination (TransFLP; Marvig and Blokesch, 2010;Silva and Blokesch, 2010;Blokesch, 2012a). Surprisingly, both of these new mutants were naturally transformable on chitin at levels that were statistically insignificant from the parental WT strain (Fig. 3A). ...
... These results generated two new questions, namely (i) why the original rpoS mutant was non-transformable while the newly engineered mutants, which were fully verified for the absence of rpoS by PCR and Sanger sequencing, were fully transformable; and (ii) why the results under chitin-inducing conditions differed compared to Dalia's work (Dalia, 2016). To address the first question, and given that the original rpoS mutant phenocopied several HapR-deficient phenotypes (Yildiz and Schoolnik, 1998), we first sequenced the strain's hapR and luxO genes. While the hapR gene was identical to the gene in the parental A1552 strain, luxO had acquired a point mutation at base pair 359 (base pair 317 according to the initial luxO ORF annotation; (Heidelberg et al., 2000). ...
The human pathogen Vibrio cholerae serves as a model organism for many important processes ranging from pathogenesis to natural transformation, which has been extensively studied in this bacterium. Previous work has deciphered important regulatory circuits involved in natural competence induction as well as mechanistic details related to its DNA acquisition and uptake potential. However, since competence was first reported for V. cholerae in 2005, many researchers have struggled with reproducibility in certain strains. In this study, we therefore compare prominent 7th pandemic V. cholerae isolates, namely strains A1552, N16961, C6706, C6709, E7946, P27459, and the close relative MO10, for their natural transformability and decipher underlying defects that mask the high degree of competence conservation. Through a combination of experimental approaches and comparative genomics based on new whole‐genome sequences and de novo assemblies, we identify several strain‐specific defects, mostly in genes that encode key players in quorum sensing. Moreover, we provide evidence that most of these deficiencies might have recently occurred through laboratory domestication events or through the acquisition of mobile genetic elements. Lastly, we highlight that differing experimental approaches between research groups might explain more of the variations than strain‐specific alterations.
... At high cell density (HCD), when intracellular HapR concentration is high, CT, TCP and biofilm formation are downregulated and production of a major protease, called haemagglutinin protease (HAP), is upregulated [17][18][19][20][21]. HAP helps in detaching adhered V. cholerae cells from the intestinal epithelial cell surface and thus it is an important virulence factor of the pathogen [22][23][24]. Apart from HapR, the stationary phase sigma factor RpoS is also involved in the regulation of HAP production [25,26]. Recently, it has been shown that deletion of the dksA gene of V. cholerae (henceforth called dksA Vc ) leads to fivefold decrease of hapA transcripts compared to that of wild-type (WT) V. cholerae cells [15]. ...
... DksA Vc positively regulates the expression of the stationary phase sigma factor RpoS Apart from HapR, the stationary phase sigma factor RpoS also positively regulates HAP production in V. cholerae [25,26] and a V. cholerae DrpoS mutant of C6709 constructed by us also produced low levels of HAP (data not shown). Therefore, to find out whether DksA Vc is involved in regulation of RpoS, we wished to determine the expression of the V. cholerae rpoS gene in the DdksA Vc genetic background using WT rpoS expression as a control. ...
... Recently, Singh et al. observed that mature biofilms in flow cells disintegrated rapidly upon stopping the flow, which results in nutrient depletion as well as accumulation of QS autoinducers [179]. These changes result in altered gene expression mainly mediated via RpoS, an alternative sigma factor rising upon nutrient limitation and HapR, the master regulator of QS with increasing cellular levels upon high cell density [180,181]. Indeed, mature biofilms with a critical size beyond approximately 18 µm show high levels of RpoS and HapR, both required to initiate dispersal [179,180]. Furthermore, the extracellular nucleases Dns and Xds have been demonstrated to be essential for detachment [4]. ...
... These changes result in altered gene expression mainly mediated via RpoS, an alternative sigma factor rising upon nutrient limitation and HapR, the master regulator of QS with increasing cellular levels upon high cell density [180,181]. Indeed, mature biofilms with a critical size beyond approximately 18 µm show high levels of RpoS and HapR, both required to initiate dispersal [179,180]. Furthermore, the extracellular nucleases Dns and Xds have been demonstrated to be essential for detachment [4]. Notably, biofilm clumps of the xds/dns double mutant were impaired for in vivo colonization, while wild type (WT) biofilm clumps outcompeted their planktonic counterparts [4]. ...
Notably, bacterial biofilm formation is increas-ingly recognized as a passive virulence factor facilitating many infectious disease processes. In this review we will focus on bacterial biofilms formed by human pathogens and highlight their relevance for diverse diseases. Along biofilm composition and regulation emphasis is laid on the intensively studied biofilms of Vibrio cholerae, Pseu-domonas aeruginosa and Staphylococcus spp., which are commonly used as biofilm model organisms and therefore contribute to our general understanding of bacterial bio-film (patho-)physiology. Finally, therapeutical interven-tion strategies targeting biofilms will be discussed.
... На основании выявленных селективных преимуществ штаммов геновариантов в условиях недостатка питательных веществ и способности к быстрому росту было высказано предположение, что в данных штаммах может быть увеличена экспрессия гена rpoS, являющегося глобальным регулятором стрессового ответа в штаммах холерного вибриона. По данным литературы ген rpoS кодирует биосинтез альтернативной сигма субъединицы σ S , связывающейся с РНК-полимеразой и контролирующей экспрессию более 25 генов в штаммах холерного вибриона, в том числе необходимых для выживания в условиях стресса [13]. Специфическое накопление белка RpoS начинается в начале стационарной фазы роста бактерий, а при попадании бактерий в неблагоприятные условия (низкий рН, повышенная температура, осмотический и оксидативный стресс, недостаток питательных веществ) обеспечивает своевременную транскрипцию набора генов, необходимых для роста клеток в данных стрессовых условиях [5,13]. ...
... По данным литературы ген rpoS кодирует биосинтез альтернативной сигма субъединицы σ S , связывающейся с РНК-полимеразой и контролирующей экспрессию более 25 генов в штаммах холерного вибриона, в том числе необходимых для выживания в условиях стресса [13]. Специфическое накопление белка RpoS начинается в начале стационарной фазы роста бактерий, а при попадании бактерий в неблагоприятные условия (низкий рН, повышенная температура, осмотический и оксидативный стресс, недостаток питательных веществ) обеспечивает своевременную транскрипцию набора генов, необходимых для роста клеток в данных стрессовых условиях [5,13]. Действительно при изучении экспрессии гена rpoS методом ОТ-ПЦР было установлено, что в штаммах геновариантов его уровень экспрессии в 1,3-7,9 раза выше, чем у типичных штаммов. ...
Aim. Study of biological properties in natural strains of genovariants of V. cholerae biovar El Tor, affecting their adaptation capacities under nutrient deficiency while comparing them with typical strains. Materials and methods. Competitive sampling was carried out through plating a mixture of cells of the two strains under investigation into autoclaved river water. Growth rate was evaluated through the optic density values. Gene expression was studied applying RT-PCR with designed primers and probes. Results. It is established that during combined cultivation of V. cholerae El Tor typical strains and genovariants under the shortage of nutrient substances (autoclaved river water), at room temperature, the level of survivability in genetically altered strains is higher than in typical strains, which points to their expressed adaptation advantages over the typical ones under the stated conditions. It is demonstrated that selective benefits of genovariant strains are provided by higher cell growth rate and increased rpoS gene expression. Conclusion. Obtained have been new data on the ability of bacterial strains of V. cholerae El Tor genovariants to rapidly grow and better express global regulator of stress response, rpoS gene, which, probably, contributes to their enhanced adaptation not only under nutrient deficiency, but under the influence of other stress factors too.
... We sequenced three V. cholerae strains (A1552, N16961, and Sa5Y) using wholegenome PacBio sequencing. V. cholerae O1 El Tor (Inaba) strain A1552 (originally named 92A1552 [9]) was isolated by the California health authorities from a traveler returning from South America (10,11), which links it to the Peruvian outbreak in the 1990s (12)(13)(14). First used for research in the Schoolnik laboratory at Stanford University, A1552 was rendered rifampicin resistant (9) and now represents the wild type in most laboratories, including ours. ...
... V. cholerae O1 El Tor (Inaba) strain A1552 (originally named 92A1552 [9]) was isolated by the California health authorities from a traveler returning from South America (10,11), which links it to the Peruvian outbreak in the 1990s (12)(13)(14). First used for research in the Schoolnik laboratory at Stanford University, A1552 was rendered rifampicin resistant (9) and now represents the wild type in most laboratories, including ours. V. cholerae O1 El Tor strain N16961 was the first sequenced strain of this species (15). ...
The bacterium Vibrio cholerae exhibits two distinct lifestyles, one as an aquatic bacterium and the other as the etiological agent of the pandemic human disease cholera. Here, we report closed genome sequences of two seventh pandemic V. cholerae O1 El Tor strains, A1552 and N16961, and the environmental strain Sa5Y.
... All V. cholerae strains are derivatives of the 7 th pandemic O1 El Tor (Inaba) strain A1552 (37). E. coli strain S17-1 λpir was used for the propagation of plasmids with the conditional R6K origin of replication and for bacterial mating. ...
Seventh pandemic Vibrio cholerae strains contain two hallmark pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. Here we use cryo-EM to reveal the mechanistic basis for plasmid defense by DdmDE. A cryo-EM structure of the DdmD helicase-nuclease reveals that it adopts an auto-inhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. A structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the non-target DNA strand. Finally, in vitro studies reveal that DdmD translocates in the 5’ to 3’ direction, while partially degrading the plasmid DNA. These findings provide critical insights into the architecture and mechanism of DdmDE systems in plasmid elimination.
... RpoS was first described as being associated with the survival of V. cholerae following exposure to hydrogen peroxide or artificial seawater (Yildiz & Schoolnik, 1998). Later, RpoS was linked to a more defined role, namely the mucosal escape response (Nielsen et al., 2006). ...
The diarrheal disease cholera is caused by the versatile and responsive bacterium Vibrio cholerae, which is capable of adapting to environmental changes. Among others, the alternative sigma factor RpoS activates response pathways, including regulation of motility‐ and chemotaxis‐related genes under nutrient‐poor conditions in V. cholerae. Although RpoS has been well characterised, links between RpoS and other regulatory networks remain unclear. In this study, we identified the ArcAB two‐component system to control rpoS transcription and RpoS protein stability in V. cholerae. In a manner similar to that seen in Escherichia coli, the ArcB kinase not only activates the response regulator ArcA but also RssB, the anti‐sigma factor of RpoS. Our results demonstrated that, in V. cholerae, RssB is phosphorylated by ArcB, which subsequently activates RpoS proteolysis. Furthermore, ArcA acts as a repressor of rpoS transcription. Additionally, we determined that the cysteine residue at position 180 of ArcB is crucial for signal recognition and activity. Thus, our findings provide evidence linking RpoS response to the anoxic redox control system ArcAB in V. cholerae.
... Because sigma factor regulons consist of several genes, they contribute to the regulation of various bacterial genes as global regulators [29,30]. In general, the stress-responsive sigma factors σ s and σ B activate the expression of genes required for bacterial survival and cell viability under extreme stress conditions [30][31][32][33][34][35]. ...
Some insertion sequence (IS) elements were actively transposed using oxidative stress conditions, including gamma irradiation and hydrogen peroxide treatment, in Deinococcus geothermalis, a radiation-resistant bacterium. D. geothermalis wild-type (WT), sigma factor gene-disrupted (∆dgeo_0606), and LysR gene-disrupted (∆dgeo_1692) mutants were examined for IS induction that resulted in non-pigmented colonies after gamma irradiation (5 kGy) exposure. The loss of pigmentation occurred because dgeo_0524, which encodes a phytoene desaturase in the carotenoid pathway, was disrupted by the transposition of IS elements. The types and loci of the IS elements were identified as ISDge2 and ISDge6 in the ∆dgeo_0606 mutant and ISDge5 and ISDge7 in the ∆dgeo_1692 mutant, but were not identified in the WT strain. Furthermore, 80 and 100 mM H2O2 treatments induced different transpositions of IS elements in ∆dgeo_0606 (ISDge5, ISDge6, and ISDge7) and WT (ISDge6). However, no IS transposition was observed in the ∆dgeo_1692 mutant. The complementary strain of the ∆dgeo_0606 mutation showed recovery effects in the viability assay; however, the growth-delayed curve did not return because the neighboring gene dgeo_0607 was overexpressed, probably acting as an anti-sigma factor. The expression levels of certain transposases, recognized as pivotal contributors to IS transposition, did not precisely correlate with active transposition in varying oxidation environments. Nevertheless, these findings suggest that specific IS elements integrated into dgeo_0524 in a target-gene-deficient and oxidation-source-dependent manner.
... RpoS controls a global adaptive response that allows many Gram-negative bacteria to survive starvation and various stresses (16)(17)(18). All isolates in the rpoS gene-deletion mutant showed motility on day 60 in three of four independent experiments ( Fig. S3) and, compared with WT, maintained high motility even after days 30 and 60 (Fig. 3G). ...
Understanding the survival strategies of pandemic cholera pathogens in aquatic environments is important for preventing their dissemination. Here, we report a phenomenon wherein long-term cultivation of Vibrio cholerae under nutrient-limited condition (M9 minimal medium supplemented with 0.2% glucose) causes mutations in flagella-related genes, thus inhibiting motility. Moreover, the motility defect reduced the transition of the viable but nonculturable state of the organisms. Maintenance of proliferative capability allows for the rapid population growth and prolonged survival in environments with nutrient sources. Diverse flagella-related genes, including flrA, flrC, flrD, flhA, flhF, fliD, fliF, fliG, fliH, fliI, fliP, fliQ, flgF, flgL, flgK, motX, and pomA, underwent mutations during the cultivation. However, only one gene (site) was mutated in a single V. cholerae . Longer cultivation for up to 300 days yielded further mutations in metabolism-related genes and the loss of virulence factors (e.g., CTX phage) and large DNA regions (~35 kb). These mutations were detected in most organisms of V. cholerae , accompanying the loss of genomic integrity. Motility-defective variants with mutations in the acetate kinase gene had become predominant in culturable cells after long-term cultivation in independently replicated experiments. These findings shed light on a survival mechanism in which V. cholerae adapts to an environmental niche by accumulating mutations.
IMPORTANCE
Vibrio cholerae undergoes a transition to a viable but non-culturable (VNC) state when subjected to various environmental stresses. We showed here that flagellar motility was involved in the development of the VNC state of V. cholerae . In this study, motility-defective isolates with mutations in various flagella-related genes, but not motile isolates, were predominantly obtained under the stress of long-term batch culture. Other genomic regions were highly conserved, suggesting that the mutations were selective. During the stationary phase of long-term culture, V. cholerae isolates with mutations in the acetate kinase and flagella-related genes were predominant. This study suggests that genes involved in specific functions in V. cholerae undergo mutations under certain environmental conditions.
... The bacterial strains and plasmids used in this work are provided in S4 and S5 Tables, respectively. The primary V. cholerae isolate used throughout this study is O1 El Tor strain A1552 [39], a representative of the ongoing 7 th cholera pandemic. Genetic manipulations are based on its published genome sequence [29]. ...
Despite extensive studies on the curve-shaped bacterium Vibrio cholerae , the causative agent of the diarrheal disease cholera, its virulence-associated regulatory two-component signal transduction system VarS/VarA is not well understood. This pathway, which mainly signals through the downstream protein CsrA, is highly conserved among gamma-proteobacteria, indicating there is likely a broader function of this system beyond virulence regulation. In this study, we investigated the VarA-CsrA signaling pathway and discovered a previously unrecognized link to the shape of the bacterium. We observed that varA -deficient V . cholerae cells showed an abnormal spherical morphology during late-stage growth. Through peptidoglycan (PG) composition analyses, we discovered that these mutant bacteria contained an increased content of disaccharide dipeptides and reduced peptide crosslinks, consistent with the atypical cellular shape. The spherical shape correlated with the CsrA-dependent overproduction of aspartate ammonia lyase (AspA) in varA mutant cells, which likely depleted the cellular aspartate pool; therefore, the synthesis of the PG precursor amino acid meso-diaminopimelic acid was impaired. Importantly, this phenotype, and the overall cell rounding, could be prevented by means of cell wall recycling. Collectively, our data provide new insights into how V . cholerae use the VarA-CsrA signaling system to adjust its morphology upon unidentified external cues in its environment.
... In conditions of metabolic stress that increase the cAMP levels in the cells, the gradient of expression of the SI cassettes will be extended, possibly favoring the expression of the appropriate combination of cassettes that will allow the cell to cope with the initial stress. Other factors such as temperature and salinity were also found to modulate cassette expression, but this is out of the scope of this review since we cannot assign such general stresses to a particular host factor, especially considering the fact that the general stress response regulator gene rpoS [21] was shown not to be involved in this process. P C promoters of 3 of the 5 MI classes have been also characterized. ...
Integrons are powerful recombination systems found in bacteria, which act as platforms capable of capturing, stockpiling, excising and reordering mobile elements called cassettes. These dynamic genetic machineries confer a very high potential of adaptation to their host and have quickly found themselves at the forefront of antibiotic resistance, allowing for the quick emergence of multi-resistant phenotypes in a wide range of bacterial species. Part of the success of the integron is explained by its ability to integrate various environmental and biological signals in order to allow the host to respond to these optimally. In this review, we highlight the substantial interconnectivity that exists between integrons and their hosts and its importance to face changing environments. We list the factors influencing the expression of the cassettes, the expression of the integrase, and the various recombination reactions catalyzed by the integrase. The combination of all these host factors allows for a very tight regulation of the system at the cost of a limited ability to spread by horizontal gene transfer and function in remotely related hosts. Hence, we underline the important consequences these factors have on the evolution of integrons. Indeed, we propose that sedentary chromosomal integrons that were less connected or connected via more universal factors are those that have been more successful upon mobilization in mobile genetic structures, in contrast to those that were connected to species-specific host factors. Thus, the level of specificity of the involved host factors network may have been decisive for the transition from chromosomal integrons to the mobile integrons, which are now widespread. As such, integrons represent a perfect example of the conflicting relationship between the ability to control a biological system and its potential for transferability.
... The wild-type Vibrio cholerae O1 El Tor strain A1552 (Yildiz and Schoolnik, 1998) and A1552 ΔmakA (Dongre et al., 2018) were used in this study. Bacterial supernatants were isolated under non-cholera toxin producing conditions. ...
Autophagy plays an essential role in the defense against many microbial pathogens as a regulator of both innate and adaptive immunity. Some pathogens have evolved sophisticated mechanisms that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy modulation mediated by the recently discovered Vibrio cholerae cytotoxin, motility-associated killing factor A (MakA). pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 (herein referring to MAP1LC3B) lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex, required for LC3 lipidation at the membranous aggregate, resulted in an inhibition of both canonical autophagy and autophagy-related processes, including the unconventional secretion of interleukin-1β (IL-1β). These findings identify a novel mechanism of host autophagy modulation and immune modulation employed by V. cholerae during bacterial infection.
... 29 ). This Inaba V. cholerae O1 was isolated in 1992 from a Peruvian traveller 29,30 and harbours the WASA-1 genomic island, a genetic hallmark of the LAT-1 sub-lineage 7,9 . An alignment of 2651 non-recombinant single nucleotide variants (SNVs) was used to calculate a maximum-likelihood phylogeny of these 532 genomes (Fig. 3a). ...
In order to control and eradicate epidemic cholera, we need to understand how epidemics begin, how they spread, and how they decline and eventually end. This requires extensive sampling of epidemic disease over time, alongside the background of endemic disease that may exist concurrently with the epidemic. The unique circumstances surrounding the Argentinian cholera epidemic of 1992–1998 presented an opportunity to do this. Here, we use 490 Argentinian V. cholerae genome sequences to characterise the variation within, and between, epidemic and endemic V. cholerae. We show that, during the 1992–1998 cholera epidemic, the invariant epidemic clone co-existed alongside highly diverse members of the Vibrio cholerae species in Argentina, and we contrast the clonality of epidemic V. cholerae with the background diversity of local endemic bacteria. Our findings refine and add nuance to our genomic definitions of epidemic and endemic cholera, and are of direct relevance to controlling current and future cholera epidemics.
... To better understand the accessory genome, including the T6SS E/I modules and to identify those genes that are novel when compared with strain N16961, we first whole-genome sequenced these strains using a longread PacBio approach followed by the de novo assembly of their genomes. As a representative strain of the seventh cholera pandemic, we used strain O1 El Tor A1552 throughout this study (Yildiz and Schoolnik, 1998). This strain is connected to a cholera outbreak in Peru in the 1990s. ...
Vibrio cholerae isolates responsible for cholera pandemics represent only a small portion of the diverse strains belonging to this species. Indeed, most V. cholerae are encountered in aquatic environments. To better understand the emergence of pandemic lineages, it is crucial to discern what differentiates pandemic strains from their environmental relatives. Here, we studied the interaction of environmental V. cholerae with eukaryotic predators or competing bacteria and tested the contributions of the hemolysin and the type VI secretion system (T6SS) to those interactions. Both of these molecular weapons are constitutively active in environmental isolates but subject to tight regulation in the pandemic clade. We showed that several environmental isolates resist amoebal grazing and that this anti-grazing defense relies on the strains' T6SS and its actin-cross-linking domain (ACD)-containing tip protein. Strains lacking the ACD were unable to defend themselves against grazing amoebae but maintained high levels of T6SS-dependent interbacterial killing. We explored the latter phenotype through whole-genome sequencing of fourteen isolates, which unveiled a wide array of novel T6SS effector and (orphan) immunity proteins. By combining these in silico predictions with experimental validations, we showed that highly similar but nonidentical immunity proteins were insufficient to provide cross-immunity among those wild strains. This article is protected by copyright. All rights reserved.
... However, general stress resistance under LSMMG was shown to be independent on this major regulator o f environmental stress (Wilson et a l, 2002a;Lynch et a l, 2004). Nevertheless, resistance to environmental stress has been frequently associated with bacterial virulence potential (Yildiz and Schoolnik, 1998;Horsburgh et a l, 2002) and should thus be considered as valuable information, particularly in the study of the effect o f gravitational forces on the virulence properties o f opportunistic pathogens. ...
Microbiological monitoring of air and surfaces within the International Space Station (ISS) has indicated that bacteria of the genus Staphylococcus are found with high frequency. Staphylococcus aureus, an opportunistic pathogen with the capacity to cause severe debilitating infection, constitutes a significant proportion of these isolates. Ground-based studies of Salmonella enterica have provided evidence that bacterial virulence is increased under the influence of simulated microgravity. These studies, and others linking space flight to reduced immune competence, provide clear evidence that extended missions may be compromised by increased risk of infection. The effects of simulated microgravity on the virulence properties of S. aureus were therefore examined. The methicillin-susceptible S. aureus (MSSA) isolates RF1, RF6 and RF11 were grown in a Synthecon High Aspect Ratio Vessel (HARV) under low shear modelled microgravity (LSMMG) and compared with cells grown under normal gravity (NO). There were no significant differences in the growth rate, antibiotic susceptibility or cell morphology of MSSA grown under LSMMG compared to NG. Growth in a modelled microgravity environment had an impact on a number of factors associated with the virulence of S. aureus. Pigment production and haemolysin secretion was significantly reduced in all three isolates under LSMMG. Global gene expression was determined by DNA microarray analysis and protein secretion examined using two-dimensional gel electrophoresis. LSMMG elicited large reductions in protein secretion by the three isolates; in particular isolate RF6 displayed a fivefold reduction in protein secretion. In total, 40 proteins were found to be down-regulated under LSMMG in a highly reproducible fashion. LC-MS/MS identified these proteins to be involved mainly with cell metabolism, including protein biosynthesis, folding and transport. DNA microarray identified significant changes in gene regulation; these were in the main associated with cell metabolism, transport, stress and virulence. For isolate RF6, the expression of a major virulence gene, hla, and the virulence regulatory system saeRS was found to be reduced two- and fivefold respectively. These data provide strong evidence that growth of S. aureus under modelled microgravity leads to a reduction in expression of virulence determinants. This observation raises the possibility that pharmacological modulation of the "microgravity trigger" that produces this avirulent phenotype would "disarm" the pathogen and resolve staphylococcal infections. This work constitutes the first step in a search for inhibitors that would prevent the secretion of a family of proteins necessary for infection to take place; the attenuated phenotype generated by such pharmacological intervention would not survive systemically and invading bacteria are likely to be removed by host immune surveillance. In contrast to conventional antibiotics, such therapeutic agents would modify rather than kill the target pathogen and consequently apply less direct selective pressure on bacterial populations.
... The wild-type Vibrio cholerae O1 El Tor strain A1552 (Yildiz and Schoolnik, 1998) ...
Autophagy plays an essential role in the defence against many microbial pathogens as a regulator of both innate and adaptive immunity. Among some pathogens, sophisticated mechanisms have evolved that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy subversion mediated by the recently discovered Vibrio cholerae cytotoxin, MakA. pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex required for LC3 lipidation at the membranous aggregate resulted in an inhibition of both canonical autophagy and autophagy-related processes including the unconventional secretion of IL-1β. These findings identify a novel mechanism of host autophagy subversion and immune modulation employed by V. cholerae during bacterial infection.
... The bacterial strains and plasmids used in this study are listed in S1 Table. The V. cholerae strain used throughout this work, A1552 [75], is a fully-sequenced [76] toxigenic O1 El Tor Inaba strain representative of the on-going 7 th cholera pandemic. ...
Type IV pili are dynamic cell surface appendages found throughout the bacteria. The ability of these structures to undergo repetitive cycles of extension and retraction underpins their crucial roles in adhesion, motility and natural competence for transformation. In the best-studied systems a dedicated retraction ATPase PilT powers pilus retraction. Curiously, a second presumed retraction ATPase PilU is often encoded immediately downstream of pilT. However, despite the presence of two potential retraction ATPases, pilT deletions lead to a total loss of pilus function, raising the question of why PilU fails to take over. Here, using the DNA-uptake pilus and mannose-sensitive haemagglutinin (MSHA) pilus of Vibrio cholerae as model systems, we show that inactivated PilT variants, defective for either ATP-binding or hydrolysis, have unexpected intermediate phenotypes that are PilU-dependent. In addition to demonstrating that PilU can function as a bona fide retraction ATPase, we go on to make the surprising discovery that PilU functions exclusively in a PilT-dependent manner and identify a naturally occurring pandemic V. cholerae PilT variant that renders PilU essential for pilus function. Finally, we show that Pseudomonas aeruginosa PilU also functions as a PilT-dependent retraction ATPase, providing evidence that the functional coupling between PilT and PilU could be a widespread mechanism for optimal pilus retraction.
... . cholerae O1 El Tor (Inaba) strain A1552 (formerly known as 92A1552-Rif r ) is a 496 rifampicin-resistant derivative of strain 92A1552(Yildiz and Schoolnik, 1998), which was 497 isolated in California from a traveller returning from South America. Epidemiological 498 investigations concluded that the transmission of this strain occurred via a contaminated seafood 499 salad that was served on an airplane between Lima, Peru and Los Angeles, California (Eberhart-500 Phillips et al., 1996; Blokesch, 2012a), which links this strain to the Peruvian cholera outbreak in 501 the 1990s. ...
Natural competence for transformation is a primary mode of horizontal gene transfer. Competent bacteria are able to absorb free DNA from their surroundings and exchange this DNA against pieces of their own genome when sufficiently homologous. However, the prevalence of non-degraded DNA with sufficient coding capacity is not well understood. In this context, we previously showed that naturally competent Vibrio cholerae use their type VI secretion system (T6SS) to actively acquire DNA from non-kin neighbors. Here, we explored the conditions of the DNA released through T6SS-mediated killing versus passive cell lysis and the extent of the transfers that occur due to these conditions. We show that competent V. cholerae acquire DNA fragments with a length exceeding 150 kbp in a T6SS-dependent manner. Collectively, our data support the notion that the environmental lifestyle of V. cholerae fosters the exchange of genetic material with sufficient coding capacity to significantly accelerate bacterial evolution.
... We further examined the effect of Fis and HapR on rpoS expression using an integrated rpoS-lacZ fusion expressed from rpoS native transcription and translation signals, as well as the production of HA/protease in wild type and mutant strains. Production of HA/protease encoded by hapA is strongly dependent on RpoS and HapR (Jobling and Holmes, 1997;Yildiz and Schoolnik, 1998;Benitez et al., 2001;Silva and Benitez, 2004). Expression of β-galactosidase activity was measured as described in (Miller, 1972) and expressed in Miller units. ...
We describe a proteomic approach to identify transcription factors binding to a target promoter. The method's usefulness was tested by identifying proteins binding to the Vibrio cholerae rpoS promoter in response to cell density. Proteins identified in this screen included the nucleoid-associated protein Fis and the quorum sensing regulator HapR.
... Ira proteins (Battesti et al., 2013) respond to specific physiological stress conditions (Hryckowian et al., 2014), as well as to the accumulation of intracellular metabolites (Battesti et al., 2015). In V. cholerae the role of RpoS is less clear as a rpoS-mutant only exhibits minor defects in intestinal colonization (Yildiz and Schoolnik, 1998;Merrell et al., 2000). However, the "mucosal escape" of V. cholerae at late stages of the infection depends on RpoS regulated gene expression (Nielsen et al., 2006). ...
The lifecycle of the causative agent of the severe secretory diarrheal disease cholera, Vibrio cholerae, is characterized by the transition between two dissimilar habitats, i.e., as a natural inhabitant of aquatic ecosystems and as a pathogen in the human gastrointestinal tract. Vibrio cholerae faces diverse stressors along its lifecycle, which require effective adaptation mechanisms to facilitate the survival fitness. Not surprisingly, the pathogen's transcriptome undergoes global changes during the different stages of the lifecycle. Moreover, recent evidence indicates that several of the transcription factors (i.e., ToxR, TcpP, and ToxT) and alternative sigma factors (i.e., FliA, RpoS, and RpoE) involved in transcriptional regulations along the lifecycle are controlled by regulated proteolysis. This post-translational control ensures a fast strategy by the pathogen to control cellular checkpoints and thereby rapidly respond to changing conditions. In this review, we discuss selected targets for regulated proteolysis activated by various stressors, which represent a key feature for fast adaptation of V. cholerae.
... We recently showed that the two regulatory proteins TfoX and TfoY foster T6SS production and, accordingly, interbacterial competition in pandemic V. cholerae (Borgeaud et al., 2015;. The previous study that addressed TfoY, however, was based on a single strain of V. cholerae, namely strain A1552 (Yildiz and Schoolnik, 1998). This human isolate originated from foodborne transmission on an airplane returning to the US from Peru/South America (Blokesch, 2012a) and belongs to the West-African South American (WASA) lineage of the currently ongoing seventh cholera pandemic (Domman et al., 2017;Matthey et al., 2018). ...
Bacteria of the genus Vibrio are common members of aquatic environments where they compete with other prokaryotes and defend themselves against grazing predators. A macromolecular protein complex called the type VI secretion system (T6SS) is used for both purposes. Previous research showed that the sole T6SS of the human pathogen V. cholerae is induced by extracellular (chitin) or intracellular (low c‐di‐GMP levels) cues and that these cues lead to distinctive signalling pathways for which the proteins TfoX and TfoY serve as master regulators. In this study, we tested whether the TfoX‐ and TfoY‐mediated regulation of T6SS, concomitantly with natural competence or motility, was conserved in non‐cholera Vibrio species, and if so, how these regulators affected the production of individual T6SSs in double‐armed vibrios. We show that, alongside representative competence genes, TfoX regulates at least one T6SS in all tested Vibrio species. TfoY, on the other hand, fostered motility in all vibrios but had a more versatile T6SS response in that it did not foster T6SS‐mediated killing in all tested vibrios. Collectively, our data provide evidence that the TfoX‐ and TfoY‐mediated signalling pathways are mostly conserved in diverse Vibrio species and important for signal‐specific T6SS induction.
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... Also, RpoS in V. cholerae enhances the production of IHF, with knock-on effects for IHF-dependent processes (Wang et al., 2012). Early work with V. cholerae showed that loss of RpoS production makes the bacterium sensitive to a variety of environmental stresses in vitro (Yildiz and Schoolnik, 1998) and interferes with its ability to colonize the mouse small intestine . The sigma factor is also involved in the latter stages of infection when the bacterium detaches from the epithelial surface. ...
Gram-negative enteropathogenic bacteria use a variety of strategies to cause disease in the human host and gene regulation in some form is typically a part of the strategy. This article will compare the toxin-based infection strategy used by the non-invasive pathogen Vibrio cholerae, the aetiological agent in human cholera, with the invasive approach used by Shigella flexneri, the cause of bacillary dysentery. Despite the differences in the mechanisms by which the two pathogens cause disease, they use environmentally-responsive regulatory hierarchies to control the expression of gene expression that have some features, and even some components, in common. The involvement of AraC-like transcription factors, the Integration Host Factor (IHF), the Factor for Inversion Stimulation (Fis), small regulatory RNAs, the RNA chaperone Hfq, horizontal gene transfer, variable DNA topology and the need to overcome the pervasive silencing of transcription by H-NS of horizontally acquired genes are all shared features. A comparison of the regulatory hierarchies in these two pathogens illustrates some striking cross-species similarities and differences among mechanisms coordinating virulence gene expression. S. flexneri, with its low infectious dose, appears to use a strategy that is centred on the individual bacterial cell, whereas V. cholerae, with a community-based, quorum-dependent approach and an infectious dose that is several orders of magnitude higher, seems to rely more on the actions of a bacterial collective
... To test whether these HapA-related and HlyA-related phenotypes are conserved in V. cholerae strains, we repeated the above-described experiments with diverse pandemic and nonpandemic V. cholerae isolates. First, we compared the wellstudied pandemic O1 El Tor isolate A1552 35 , which we primarily use in our laboratory, with two other O1 El Tor strains, namely C6706 and N16961 (Supplementary Table 1). Strain A1552 was isolated in California from a traveller returning from South America 36 , which links this strain to the Peruvian cholera outbreak in 1990s. ...
Vibrio cholerae, which causes the diarrheal disease cholera, is a species of bacteria commonly found in aquatic habitats. Within such environments, the bacterium must defend itself against predatory protozoan grazers. Amoebae are prominent grazers, with Acanthamoeba castellanii being one of the best-studied aquatic amoebae. We previously showed that V. cholerae resists digestion by A. castellanii and establishes a replication niche within the host's osmoregulatory organelle. In this study, we decipher the molecular mechanisms involved in the maintenance of V. cholerae's intra-amoebal replication niche and its ultimate escape from the succumbed host. We demonstrate that minor virulence features important for disease in mammals, such as extracellular enzymes and flagellum-based motility, have a key role in the replication and transmission of V. cholerae in its aqueous environment. This work, therefore, describes new mechanisms that provide the pathogen with a fitness advantage in its primary habitat, which may have contributed to the emergence of these minor virulence factors in the species V. cholerae.
Seventh pandemic Vibrio cholerae strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. Here we use cryogenic electron microscopy to reveal the mechanistic basis for plasmid defense by DdmDE. A structure of the DdmD helicase-nuclease reveals it adopts an auto-inhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. A structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the non-target DNA strand. In vitro studies reveal that DdmD translocates in the 5′-to-3′ direction, while partially degrading the plasmid DNA. These findings provide critical insights into the mechanism of DdmDE systems in plasmid elimination.
In response to predation by bacteriophages and invasion by other mobile genetic elements such as plasmids, bacteria have evolved specialised defence systems that are often clustered together on genomic islands. The O1 El Tor strains of Vibrio cholerae responsible for the ongoing seventh cholera pandemic (7PET) contain a characteristic set of genomic islands involved in host colonisation and disease, many of which contain defence systems. Notably, Vibrio pathogenicity island 2 contains several characterised defence systems as well as a putative Type I restriction-modification system (T1RM), which, interestingly, is interrupted by two genes of unknown function. Here, we demonstrate that the T1RM system is active, methylates the host genomes of a representative set of 7PET strains, and identify a specific recognition sequence that targets non-methylated plasmids for restriction. We go on to show that the two genes embedded within the T1RM system encode a novel two-protein modification-dependent restriction system related to the GmrSD family of Type IV restriction enzymes. Indeed, we show that this system has potent anti-phage activity against diverse members of the Tevenvirinae , a subfamily of bacteriophages with hypermodified genomes. Taken together these results expand our understanding of how this highly conserved genomic island contributes to the defence of pandemic V. cholerae against foreign DNA.
Vibrio cholerae is a pathogen that causes disease in millions of people every year by colonizing the small intestine and then secreting the potent cholera toxin. How the pathogen overcomes the colonization barrier created by the host's natural microbiota is, however, still not well understood. In this context, the type VI secretion system (T6SS) has gained considerable attention given its ability to mediate interbacterial killing. Interestingly, and in contrast to non-pandemic or environmental V. cholerae isolates, strains that are causing the ongoing cholera pandemic (7PET clade) are considered T6SS-silent under laboratory conditions. Since this idea was recently challenged, we performed a comparative in vitro study on T6SS activity using diverse strains or regulatory mutants. We show that modest T6SS activity is detectable in most of the tested strains under interbacterial competition conditions. The system's activity was also observed through immunodetection of the T6SS tube protein Hcp in culture supernatants, a phenotype that can be masked by the strains' haemagglutinin/protease. We further investigated the low T6SS activity within the bacterial populations by imaging 7PET V. cholerae at the single-cell level. The micrographs showed the production of the machinery in only a small fraction of cells within the population. This sporadic T6SS production was higher at 30 °C than at 37 °C and occurred independently of the known regulators TfoX and TfoY but was dependent on the VxrAB two-component system. Overall, our work provides new insight into the heterogeneity of T6SS production in populations of 7PET V. cholerae strains in vitro and provides a possible explanation of the system's low activity in bulk measurements.
Vibrio fluvialis is a halophilic Gram-negative bacterium regarded as an emerging unusual enteric pathogen of increasing public health concern. Our previous work has identified two type VI secretion systems (T6SSs) in V. fluvialis, VflT6SS1, and VflT6SS2, and the latter is functional in mediating interbacterial competitiveness. However, its antibacterial effectors remain to be clarified. In this work, we focused on a new potential effector/immunity pair TssI2/TsiI2. Bioinformatics analysis revealed that the C-terminal domain of TssI2 belongs to a widespread family of pesticin, and its antibacterial toxicity and corresponding protection by TsiI2 were proved via bacterial killing assays, and their action sites were localized to the periplasm of bacterial cells. The interaction of TssI2 and TsiI2 was demonstrated by the bacterial adenylate cyclase two-hybrid, protein pull-down and isothermal titration calorimetry assays. Site-directed mutagenesis demonstrated that, in addition to Glu-844, Thr-863, and Asp-869, which correspond to three reported residues in pesticin of Yersinia pestis, additional residues including Phe-837, Gly-845, Tyr-851, Gly-867, Gln-963, Trp-975, and Arg-1000 were also proved to be crucial to the bactericidal activity of TssI2. Muramidase/lysozyme-related peptidoglycan (PG) hydrolase activities of TssI2 and its variants were validated with permeabilized Escherichia coli cells and purified PG substrate. Based on sequence homologies at C-terminals in various V. fluvialis isolates, TssI2 was subdivided into five clusters (12–22% identity among them), and the antibacterial activities of representative effectors from other four Clusters were also confirmed through periplasmic over-expression in E. coli host. Two selected cognate immunities were proved to confer protection against the toxicities of their effectors. Additionally, TsiI2, which belongs to Cluster I, exhibited cross-protection to effector from Cluster V. Together, current findings expand our knowledge of the diversity and consistency of evolved VgrG effectors in V. fluvialis and on how VflT6SS2 mediates a competitive advantage to gain a better survival.
This work demonstrates that the Vibrio cholerae type six secretion system (T6SS) can actively kill prey strains within the interior of biofilm populations with substantial impact on population dynamics. We additionally show that the response regulator VxrB contributes to both T6SS killing and protection from T6SS killing within biofilms.
Cholera is a life-threatening diarrheal disease caused by the human pathogenic bacterium Vibrio cholerae. Regulatory elements are essential for bacterial transition between the natural aquatic environment and the human host. One of them is the alternative sigma factor RpoS and its anti-sigma factor RssB. Regulation principles seem to be conserved among RpoS/RssB interaction modes between V. cholerae and Enterobacteriaceae species, however the associated input and output pathways seem different. In Escherichia coli, RpoS/RssB is important for the activation of an emergency program to increase persistence and survival. Whereas, it activates motility and chemotaxis in V. cholerae, used strategically to escape from starvation conditions. We characterised a starvation-induced interaction model showing a negative feedback loop between RpoS and RssB expression. We showed by genotypic and phenotypic analysis that rssB influences motility, growth behaviour, colonization fitness, and post-infectious survival. Furthermore, we found that RssB itself is a substrate for proteolysis and a critical Asp mutation was identified and characterised to influence rssB phenotypes and their interaction with RpoS. In summary, we present novel information about the regulatory interaction between RpoS and RssB being active under in vivo colonization conditions and mark an extension to the feedback regulation circuit, showing that RssB is a substrate for proteolysis.
Inadequate access to clean water is detrimental to human health and aquatic industries. Waterborne pathogens can survive prolonged periods in aquatic bodies, infect commercially important seafood, and resist water disinfection, resulting in human infections. Environmental agencies and research laboratories require a relevant, portable, and cost-effective platform to monitor microbial pathogens and assess their risk of infection on a large scale. Advances in microfluidics enable better control and higher precision than traditional culture-based pathogen monitoring approaches. We demonstrated a rapid, high-throughput fish-based teleost (fish)-microbe (TelM) microfluidic-based device that simultaneously monitors waterborne pathogens in contaminated waters and assesses their infection potential under well-defined settings. A chamber-associated port allows direct access to the animal, while the transparency of the TelM platform enables clear observation of sensor readouts. As proof-of-concept, we established a wound infection model using Pseudomonas aeruginosa-contaminated water in the TelM platform, where bacteria formed biofilms on the wound and secreted a biofilm metabolite, pyoverdine. Pyoverdine was used as fluorescent sensor to correlate P. aeruginosa contamination to infection. The TelM platform was validated with environmental waterborne microbes from marine samples. Overall, the TelM platform can be readily applied to assess microbial and chemical risk in aquatic bodies in resource-constrained settings.
Aquatic organisms have to produce proteins or factors that help maintain a stable relationship with microbiota and prevent colonization by pathogenic microorganisms. In crustaceans and other aquatic invertebrates, relatively few of these host factors have been characterized. In this study, we show that the respiratory glycoprotein hemocyanin is a crucial host factor that modulates microbial composition and diversity in the hepatopancreas of penaeid shrimp. Diseased penaeid shrimp (Penaeus vannamei), had an empty gastrointestinal tract with atrophied hepatopancreas, expressed low hemocyanin, and high total bacterial abundance, with Vibrio as the dominant bacteria. Similarly, shrimp depleted of hemocyanin had mitochondrial depolarization, increased reactive oxygen species (ROS) levels, and dysregulation of several energy metabolism-related genes. Hemocyanin silencing together with ROS scavenger (N-acetylcysteine) treatment improved microbial diversity and decreased Vibrio dominance in the hepatopancreas. However, fecal microbiota transplantation after hemocyanin knockdown could not restore the microbial composition in the hepatopancreas. Collectively, our data provide, to our knowledge, new insight into the pivotal role of hemocyanin in modulating microbial composition in penaeid shrimp hepatopancreas via its effect on mitochondrial integrity, energy metabolism, and ROS production.
Despite extensive studies on the curve-shaped bacterium Vibrio cholerae , the causative agent of the diarrheal disease cholera, its virulence-associated regulatory two-component signal transduction system VarS/VarA is not well understood. This pathway, which mainly signals through the downstream protein CsrA, is highly conserved among gamma-proteobacteria, indicating there is likely a broader function of this system beyond virulence regulation. In this study, we investigated the VarA-CsrA signaling pathway and discovered a previously unrecognized link to the shape of the bacterium. We observed that varA -deficient V. cholerae cells showed an abnormal spherical morphology during late-stage growth. Through peptidoglycan (PG) composition analyses, we discovered that these mutant bacteria contained an increased content of disaccharide dipeptides and reduced peptide crosslinks, consistent with the atypical cellular shape. The spherical shape correlated with the CsrA-dependent overproduction of aspartate ammonia lyase (AspA) in varA mutant cells, which likely depleted the cellular aspartate pool; therefore, the synthesis of the PG precursor amino acid meso-diaminopimelic acid was impaired. Importantly, this phenotype, and the overall cell rounding, could be prevented by means of cell wall recycling. Collectively, our data provide new insights into how V. cholerae use the VarA-CsrA signaling system to adjust its morphology upon unidentified external cues in its environment.
Significance Statement
Responsible for the diarrheal disease cholera, the bacterium Vibrio cholerae tightly regulates its virulence program according to external stimuli. Here, we discovered that a sensing-response mechanism involved in the regulation of virulence also controls bacterial shape. We show that V. cholerae lacking this system lose their normal comma shape and become spherical due to an abnormal cell wall composition caused by metabolic changes that reduce available cell wall building blocks. Our study therefore sheds new light on how V. cholerae modulates its morphology based on environmental changes.
Pseudomonas aeruginosa is an opportunistic pathogen causing serious infections in immune compromised persons. These infections are difficult to erase with antibiotics, due to the formation of biofilms. The biofilm lifecycle is regulated by the second messenger molecule c-di-GMP (bis-3,5-cyclic di-guanosine monophosphate). P. aeruginosa encodes 40 genes for enzymes presumably involved in the biosynthesis and degradation of c-di-GMP. A tight regulation of expression, subcellular localized function and protein interactions control the activity of these enzymes. In this work we elucidated the transcriptional regulation of the gene encoding the membrane-bound phosphodiesterase NbdA. We previously reported a transcriptional and posttranslational role of nitric oxide (NO) on nbdA and its involvement in biofilm dispersal. NO is released from macrophages during infections but can also be produced by P. aeruginosa itself during anaerobic denitrification. Recently however, contradictory results about the role of NbdA within NO-induced biofilm dispersal were published. Therefore, the transcriptional regulation of nbdA was reevaluated to obtain insights into this discrepancy. Determination of the transcriptional start site of nbdA by 5’-RACE and subsequent identification of the promoter region revealed a shortened open reading frame (ORF) in contrast to the annotated one. In addition, putative binding sites for RpoS and AmrZ were discovered in the newly defined promoter region. Employing chromosomally integrated transcriptional lacZ reporter gene fusions demonstrated a RpoS-dependent activation and AmrZ repression of nbdA transcription. In order to investigate the impact of NO on nbdA transcription, conditions mimicking exogenous and endogenous NO were applied. While neither exogenous nor endogenous NO had an influence on nbdA promoter activity, deletion of the nitrite reductase gene nirS strongly increased nbdA transcription independently of its enzymatic activity during denitrification. The latter supports a role of NirS in P. aeruginosa apart from its enzymatic function.
IMPORTANCE
The opportunistic pathogen Pseudomonas aeruginosa possesses a network of genes encoding proteins for the turnover of the second messenger c-di-GMP involved in regulating-among others-the lifestyle switch between planktonic, motile cells and sessile biofilms. Insight into the transcriptional regulation of these genes is important for the understanding of the protein function within the cell. Determination of the transcriptional start site of the phosphodiesterase gene nbdA revealed a new promoter region and consequently a shortened open reading frame for the corresponding protein. Binding sites for RpoS and AmrZ were identified in silico and confirmed experimentally. Previously reported regulation by nitric oxide was reevaluated and a strong influence of the moonlighting protein NirS identified.
Vibrio cholerae , which causes the diarrheal disease cholera, is a species of bacteria commonly found in aquatic habitats. Within such environments, the bacterium must defend itself against predatory protozoan grazers. Amoebae are prominent grazers, with Acanthamoeba castellanii being one of the best-studied aquatic amoebae. We previously showed that V. cholerae resists digestion by A. castellanii and establishes a replication niche within the host’s osmoregulatory organelle. In this study, we deciphered the molecular mechanisms involved in the maintenance of V. cholerae ’s intra-amoebal replication niche and its ultimate escape from the succumbed host. We demonstrated that minor virulence features important for disease in mammals, such as extracellular enzymes and flagellum-based motility, play a key role role in the replication and transmission of V. cholerae in its aqueous environment. This work, therefore, describes new mechanisms that provide the pathogen with a fitness advantage in its primary habitat, which may have contributed to the emergence of these minor virulence factors in the species V. cholerae.
Preservation technologies subject bacterial cells to different levels of stress, which in the most effective cases lead to their inactivation and death. The term “stress‐ can refer to any extracellular influence that threatens the ability of microorganisms to perform their living functions. The food preservation technologies designed to rapidly inactivate microbial cells include thermal processes, irradiation, high‐pressure processing, and the use of strong oxidant compounds. Other technologies accomplish the preservation of foods by inhibiting growth; the most extensively used are low‐temperature storage (refrigeration and freezing), reduction of moisture content (concentration and drying), control of ox‐redox potential (use of controlled atmospheres and vacuum packaging), and acidification (fermentation and addition of organic acids). In nature, microorganisms are constantly exposed to similar changes in temperature, oxygen, moisture, light, pH, and chemical composition. Bacteria are able to survive thanks to a wide array of molecular responses that provide cellular protection against stresses. Bacteria are protected from changes in pH, temperature, oxidative conditions, solute concentrations, and pressure by a network of sophisticated global genetic regulatory systems and molecular stress responses specific to individual chemical or physical threats. The most important general regulators and specific genetic systems reported in representative foodborne pathogenic bacteria are highlighted in this chapter.
Lactic acid bacteria (LAB) caused many microbiological incidents in brewery industry resulting in severe loss. Meanwhile, traditional culturing method to detect LAB is time-consuming for brewers. The present review introduces the LAB as spoilage microbes in daily life, especially for lactic acid bacteria in the brewing industry targeting at the spoilage mechanism of LAB in brewing industry including the special metabolisms, the exist of the viable but nonculturable (VBNC) state and the hop resistance. At the same time, this review compares the traditional and novel rapid detection methods of this microorganism which may provide the innovative control and detection strategy of alcoholic beverage spoilage, such as improvement on microbiological quality control by using the advanced culture medium or different isothermal amplification methods.
The sigma factor RpoS (ς S ) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS in Pseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa , including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1 rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50°C, increased osmolarity, and prolonged exposure to high concentrations of H 2 O 2 . In accordance with the hypersensitivity to H 2 O 2 , catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa . The rpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoS mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101 :: aacCI mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium. On a solid medium, the FRD1 rpoS mutant produced approximately 70% less alginate than did the wild-type strain. Thus, our data indicate that although some of the functions of RpoS in P. aeruginosa physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.
How bacteria colonize surfaces and how they distinguish the individuals around them are fundamental biological questions. Type IV pili are a widespread and multipurpose class of cell surface polymers. Here we directly visualize the DNA-uptake pilus of Vibrio cholerae, which is produced specifically during growth on its natural habitat—chitinous surfaces. As predicted, these pili are highly dynamic and retract before DNA uptake during competence for natural transformation. Interestingly, DNA-uptake pili can also self-interact to mediate auto-aggregation. This capability is conserved in disease-causing pandemic strains, which typically encode the same major pilin subunit, PilA. Unexpectedly, however, we discovered that extensive strain-to-strain variability in PilA (present in environmental isolates) creates a set of highly specific interactions, enabling cells producing pili composed of different PilA subunits to distinguish between one another. We go on to show that DNA-uptake pili bind to chitinous surfaces and are required for chitin colonization under flow, and that pili capable of self-interaction connect cells on chitin within dense pili networks. Our results suggest a model whereby DNA-uptake pili function to promote inter-bacterial interactions during surface colonization. Moreover, they provide evidence that type IV pili could offer a simple and potentially widespread mechanism for bacterial kin recognition.
The conserved CsrB sRNAs are an example of sibling sRNAs, i.e., sRNAs which are present in multiple copies in genomes. This report illustrates how new copies arise through gene duplication events and highlights two evolutionary advantages of having such multiple copies: differential regulation of the multiple copies allows integration of different input signals into the regulatory network of which they are parts, and the high redundancy that they provide confers a strong robustness to the system.
Correction for ‘Engineering microbial physiology with synthetic polymers: cationic polymers induce biofilm formation in Vibrio cholerae and downregulate the expression of virulence genes’ by Nicolas Perez-Soto et al. , Chem. Sci. , 2017, 8 , 5291–5298.
Since its emergence, cholera caused by the bacterium Vibrio cholerae remains as a significant threat to human health. The continued persistence of this pathogen against many unfavourable conditions made challenging to eradicate cholera, especially in the developing countries. In the hostile conditions, the bacterium is known to form a self-enclosed polymeric structure called ‘biofilm’ which serves as a major factor responsible for its persistence and transmission. Hence, the authors aimed to understand the effectiveness of relevant physical, chemical and antibiotic treatments against the biofilm of this bacterium. For the study, the biofilm of V. cholerae O139 wild type and its isogenic Tn5-mutants that differ in their biofilm phenotype (biofilm proficient and biofilm deficient) were exposed to different levels of pH, salinity, temperature, UV radiation, H2O2, chlorination, and antibiotics. It was observed that biofilm culture of both wild type and the biofilm-proficient mutant exhibited detectable survival rate up to pH 2.0, salinity 3.5 M, temperature 50 °C, H2O2 80 mM, NaOCl 12.5 mg/L and at several folds increased antibiotic concentration (Ciprofloxacin 2 mg/L, and Doxycycline 256 mg/L) as compared to the biofilm defective mutant and the planktonic cultures of respective strains. As the biofilm of the cholera pathogen or the cholera biofilm resists several lethal challenges, it is essential to target the biofilm and its residents for the complete destruction of the infection source and thereby to prevent transmission of cholera.
Vibrio cholerae is a Gram-negative bacterium found in aquatic environments and a human pathogen of global significance. Its transition between host-associated and environmental life styles involves the tight regulation of niche-specific phenotypes such as motility, biofilm formation and virulence. V. cholerae’s transition from the host to environmental dispersal usually involves suppression of virulence and dispersion of biofilm communities. In contrast to this naturally occurring transition, bacterial aggregation by cationic polymers triggers a unique response, which is to suppress virulence gene expression while also triggering biofilm formation by V. cholerae, an artificial combination of traits that is potentially very useful to bind and neutralize the pathogen from contaminated water. Here, we set out to uncover the mechanistic basis of this polymer-triggered bacterial behavior. We found that bacteria-polymer aggregates undergo rapid autoinduction and achieve quorum sensing at bacterial densities far below those required for autoinduction in the absence of polymers. We demonstrate this induction of quorum sensing is due both to a rapid formation of autoinducer gradients and local enhancement of autoinducer concentrations within bacterial clusters, as well as the stimulation of CAI-1 and AI-2 production by aggregated bacteria. We further found that polymers cause an induction of the biofilm specific regulator VpsR and the biofilm structural protein RbmA, bypassing the usual suppression of biofilm during autoinduction. Overall, this study highlights that synthetic materials can be used to cross-wire natural bacterial responses to achieve a combination of phenotypes with potentially useful applications.
A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources. Proteins are separated according to isoelectric point by isoelectric focusing in the first dimension, and according to molecular weight by sodium dodecyl sulfate electrophoresis in the second dimension. Since these two parameters are unrelated, it is possible to obtain an almost uniform distribution of protein spots across a two-diminsional gel. This technique has resolved 1100 different components from Escherichia coli and should be capable of resolving a maximum of 5000 proteins. A protein containing as little as one disintegration per min of either 14C or 35S can be detected by autoradiography. A protein which constitutes 10 minus 4 to 10 minus 5% of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation is sufficient to permit each spot on one separation to be matched with a spot on a different separation. This technique provides a method for estimation (at the described sensitivities) of the number of proteins made by any biological system. This system can resolve proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge. Proteins whose charge is changed by missense mutations can be identified. A detailed description of the methods as well as the characteristics of this system are presented.
A study was made of the mechanisms by which visible light produces cell dormancy in Escherichia coli, resulting in loss of culturability. Visible light may act directly on the cells or generate photoproducts with a negative effect on the cells. In nonilluminated microcosms the addition of increasing concentrations of hydrogen peroxide, one of the photoproducts formed in natural aquatic systems, gave rise to the formation of nonculturable cells and injured culturable cells, and this negative effect depended on the concentration of peroxide. On the other hand, in illuminated microcosms the addition of compounds which eliminate hydrogen peroxide (i.e., catalase, sodium pyruvate, and thioglycolate) had a protective effect on the E. coli cells, as the CFU counts on minimal medium and on recuperation medium were significantly higher (P < 0.05) than those detected in the absence of these compounds. Furthermore, when hydrogen peroxide was eliminated, the CFU counts on recuperation medium did not fall significantly, indicating that nonculturable cells did not form. These results rule out the direct effect of visible light on the cells and show that hydrogen peroxide, generated photochemically, may be the cause of the loss of culturability of E. coli in illuminated systems.
The novel sigma factor (sigma S) encoded by rpoS (katF) is required for induction of many growth phase-regulated genes and expression of a variety of stationary-phase phenotypes in Escherichia coli. Here we demonstrate that wild-type cells exhibit spherical morphology in stationary phase, whereas rpoS mutant cells remain rod shaped and are generally larger. Size reduction of E. coli cells along the growth curve is a continuous and at least biphasic process, the second phase of which is absent in rpoS-deficient cells and correlates with induction of the morphogene bolA in wild-type cells. Stationary-phase induction of bolA is dependent on sigma S. The "gearbox" a characteristic sequence motif present in the sigma S-dependent growth phase- and growth rate-regulated bolAp1 promoter, is not recognized by sigma S, since stationary-phase induction of the mcbA promoter, which also contains a gearbox, does not require sigma S, and other sigma S-controlled promoters do not contain gearboxes. However, good homology to the potential -35 and -10 consensus sequences for sigma S regulation is found in the bolAp1 promoter.
The structural gene, hap, for the secreted hemagglutinin/protease (HA/protease), a putative virulence factor of Vibrio cholerae, has recently been cloned and sequenced (C. C. Häse and R. A. Finkelstein, J. Bacteriol. 173:3311-3317, 1991). The availability of the null mutant, HAP-1, and HAP-1 complemented with pCH2 (which expresses HA/protease), enabled an examination of the role of HA/protease in the virulence of V. cholerae in an animal model. However, the mutants exhibited reversible colonial variation similar but not identical to that which was previously associated with dramatic changes in virulence of parental strain 3083. Regardless of colonial morphology, the mutants were found to be fully virulent in infant rabbits. Thus, the HA/protease is not a primary virulence factor (for infant rabbits). Observations using cultured human intestinal cells indicated, instead, that the HA/protease is responsible for detachment of the vibrios from the cultured cells by digestion of several putative receptors for V. cholerae adhesins.
Many of the changes in gene expression observed when Escherichia coli cells enter stationary phase are regulated at the level of transcription initiation. A group of stationary-phase-inducible promoters, known as "gearbox" promoter, display a characteristic sequence in the -10 region which differs greatly from the consensus sequence for sigma 70-dependent promoters. Here we describe our studies on the gearbox promoters bolAp1 and mcbAp, responsible for the temporally regulated transcription of bolA and the genes involved in the synthesis of the peptide antibiotic microcin B17, respectively. Deletion analysis of mcbAp demonstrated that the stationary-phase-inducible properties of this promoter are found in a DNA fragment extending from -54 to +11 bp, surrounding the transcriptional start site, and are separable from DNA sequences responsible for the OmpR-dependent stimulation of transcription of mcbAp. In vitro transcription studies indicate that the RNA polymerase holoenzyme involved in the transcription of mcbAp contains sigma 70. In this and an accompanying paper (R. Lange and R. Hengge-Aronis, J. Bacteriol. 173: 4474-4481, 1991), experiments are described which show that the product of katF, a global regulator of stationary-phase gene expression and a putative sigma factor, is required for the expression of bolAp1 fused to the reporter gene lacZ. In contrast, mcbAp appears to be negatively regulated by katF. We discuss the implications of these results for postexponential gene expression and the role of gearbox sequences in the regulation of promoter activity.
The structural gene hap for the extracellular hemagglutinin/protease (HA/protease) of Vibrio cholerae was cloned and sequenced. The cloned DNA fragment contained a 1,827-bp open reading frame potentially encoding a 609-amino-acid polypeptide. The deduced protein contains a putative signal sequence followed by a large propeptide. The extracellular HA/protease consists of 414 amino acids with a computed molecular weight of 46,700. In the absence of protease inhibitors, this is processed to the 32-kDa form which is usually isolated. The deduced amino acid sequence of the mature HA/protease showed 61.5% identity with the Pseudomonas aeruginosa elastase. The cloned hap gene was inactivated and introduced into the chromosome of V. cholerae by recombination to construct the HA/protease-negative strain HAP-1. The cloned fragment containing the hap gene was then shown to complement the mutant strain.
Expression of more than 17 virulence genes in Vibrio cholerae is under the coordinate control of the ToxR protein. ToxR is a transmembrane protein that binds to and activates the promoter of the operon encoding cholera toxin. As yet, the ability of ToxR to activate directly other genes in this regulon has not been demonstrated. We have cloned a gene called toxT from V. cholerae 569B; the toxT gene product, like ToxR, can activate the ctx promoter in Escherichia coli. In addition, expression of other genes identified as members of the ToxR regulon (tcpA, tcpI, aldA, and tagA) can be activated in E. coli by the toxT gene product but not by ToxR. When expressed from a constitutive promoter, the toxT gene product partially suppresses the ToxR- phenotype of a toxR deletion mutant of V. cholerae. The level of toxT mRNA is greatly reduced in a toxR mutant of V. cholerae. In addition, growth conditions under which the ToxR regulon is not expressed also repress the synthesis of toxT mRNA. These results suggest that ToxR controls transcription of toxT, whose product in turn is directly responsible for activation of several virulence genes under ToxR control.
KatF is required for the expression of some 32 carbon starvation proteins in Escherichia coli including 6 previously identified as Pex. Mutants with the katF gene survive carbon and nitrogen starvation poorly. Many of the KatF-regulated starvation proteins are common to those induced by other stresses, and the mutant failed to develop starvation-mediated cross protection to osmotic, oxidative, and heat stresses. Furthermore, thermal resistance was not induced in the mutant by heat preadaptation, and it exhibited an altered pattern of protein synthesis at elevated temperature. Thus, KatF is a major switch that controls the starvation-mediated resistant state in E. coli.
The soluble hemagglutinin/protease (HA/protease) produced by Vibrio cholerae and the elastase of Pseudomonas aeruginosa are both zinc/calcium-dependent proteases. In the present study the two enzymes are compared immunologically and functionally. The N-terminal amino acid sequences of the proteins had 65% identity within the first 20 amino acids. Polyclonal antisera against each purified protein recognized the enzyme of the other species in enzyme-linked immunosorbent assay, checkerboard immunoblot, and Western blot analyses and inhibited the protease activity of both enzymes in milk and elastin agars. Like the HA/protease, the elastase hemagglutinated "responder" but not "nonresponder" chicken erythrocytes, degraded ovomucin, lactoferrin, and fibronectin, and nicked the A subunit of the cholera toxin-related heat-labile enterotoxin from Escherichia coli. Whereas none of the three proteases tested (elastase, HA/protease, or pronase E) had any obvious effect in ileal loop tests in rabbits at doses up to 50 micrograms, all three produced some detectable skin reactions at a dose of 0.1 micrograms and necrosis at a higher dose (i.e., 5 micrograms). We conclude that the V. cholerae HA/protease and the P. aeruginosa elastase are structurally, functionally, and immunologically related.
Glucose- or nitrogen-starved cultures of Escherichia coli exhibited enhanced resistance to heat (57 degrees C) or H2O2 (15 mM) challenge, compared with their exponentially growing counterparts. The degree of resistance increased with the time for which the cells were starved prior to the challenge, with 4 h of starvation providing the maximal protection. Protein synthesis during starvation was essential for these cross protections, since chloramphenicol addition at the onset of starvation prevented the development of thermal or oxidative resistance. Starved cultures also demonstrated stronger thermal and oxidative resistance than did growing cultures adapted to heat, H2O2, or ethanol prior to the heat or H2O2 challenge. Two-dimensional gel electrophoresis of 35S-pulse-labeled proteins showed that subsets of the 30 glucose starvation proteins were also synthesized during heat or H2O2 adaptation; three proteins were common to all three stresses. Most of the common proteins were among the previously identified Pex proteins (J.E. Schultz, G. I. Latter, and A. Matin, J. Bacteriol. 170:3903-3909, 1988), which are independent of cyclic AMP positive control for their induction during starvation. Induction of starvation proteins dependent on cyclic AMP was not important in these cross protections, since a delta cya strain of E. coli K-12 exhibited the same degree of resistance to heat or H2O2 as the wild-type parent did during both growth and starvation.
Some effects of visible light on the survival of Escherichia coli in waters of the Butrón river were studied by comparing illuminated and nonilluminated systems. The following count methods were used: CFU on a selective medium (eosin-methylene blue agar), CFU on a medium of recuperation (Trypticase soy agar with yeast extract and glucose), number of metabolically active cells by reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, and total number of E. coli cells as determined by the acridine orange direct-count method. In the illuminated systems, decreases in CFU of E. coli and in the number of metabolically active cells were observed. However, no decline of the total number of E. coli cells was observed. By count methods, different stages of progressive dormancy of E. coli cells were determined to exist in illuminated systems. Culturable and recoverable cells were defined as viable cells, and metabolically active cells and morphologically intact cells were defined as somnicells. Indirect activity measurements were also done by using [14C]glucose. In illuminated systems, a decrease of glucose uptake by E. coli cells was observed throughout the experiments. The assimilated fraction of [14C]glucose decreased faster than the respired fraction in illuminated systems. The percentage of respired [14C]glucose (14CO2 production) with respect to the total glucose uptake increased throughout the experiments, and the percentage of assimilated glucose decreased. Therefore, the visible light was also responsible for an additional inhibition of biosynthetic processes.
The katF gene of Escherichia coli has been sequenced revealing a 1086 base pair open reading frame from which the sequence of a 362 amino acid protein has
been deduced. The direction of transcription of katF was confirmed by expression of the gene cloned in both directions behind a T7 promoter. The KatF protein expressed in vitro migrates with an apparent size of 42 kDa. Comparison of the katF sequence to the sequence of rpoD, which encodes the sigma subunit of RNA polymerase, revealed a 181 bp region with 65% homology and a 38 bp segment that was
87% homologous. A 62 amino acid region of the predicted KatF protein sequence was found to be 85% homologous to the corresponding
sequence of a including σ70, including segment implicated in core polymerase binding. Homology was also observed with the heat shock regulatory protein
encoded by htpR.
Glucose- or nitrogen-starved cultures of Escherichia coli exhibited enhanced resistance to heat (57 degrees C) or H2O2 (15 mM) challenge, compared with their exponentially growing counterparts. The degree of resistance increased with the time for which the cells were starved prior to the challenge, with 4 h of starvation providing the maximal protection. Protein synthesis during starvation was essential for these cross protections, since chloramphenicol addition at the onset of starvation prevented the development of thermal or oxidative resistance. Starved cultures also demonstrated stronger thermal and oxidative resistance than did growing cultures adapted to heat, H2O2, or ethanol prior to the heat or H2O2 challenge. Two-dimensional gel electrophoresis of 35S-pulse-labeled proteins showed that subsets of the 30 glucose starvation proteins were also synthesized during heat or H2O2 adaptation; three proteins were common to all three stresses. Most of the common proteins were among the previously identified Pex proteins (J.E. Schultz, G. I. Latter, and A. Matin, J. Bacteriol. 170:3903-3909, 1988), which are independent of cyclic AMP positive control for their induction during starvation. Induction of starvation proteins dependent on cyclic AMP was not important in these cross protections, since a delta cya strain of E. coli K-12 exhibited the same degree of resistance to heat or H2O2 as the wild-type parent did during both growth and starvation.
The effect of NaCl on the growth rates and yields of 31 gram-negative, heterotrophic, marine bacteria was determined. The strains used were representative of aerobic genera (Alteromonas, Pseudomonas, Alcaligenes, Bdellovibrio) as well as genera comprised of facultative anaerobes (Beneckea, Photobacterium). Two media were used-the first, a medium designed for the cultivation of marine bacteria and, the second, a medium used for the cultivation of terrestrial strains. These two media differed in the concentrations of divalent cations; the terrestrial medium (TM) contained 2 mM Mg++ and 0.55 mM Ca++ while the marine medium (MM) contained 50 mM Mg++ and 10 mM Ca++. The amount of NaCl necessary for optimal growth varied in different strains and was usually considerably higher in TM (100 to 460 mM) than in MM (70 to 300 mM). Many strains which grew in MM and TM had a shorter generation time in the former than in the latter medium. In addition, four strains which grew well in MM usually failed to grow in TM. These results show that higher levels of divalent cations are either essential for growth or stimulate growth rate, indicating that for many marine strains a terrestrial medium modified by the addition of NaCl cannot support optimal growth. Fourteen terrestrial strains of the genera Pseudomonas, Alcaligenes, Acinetobacter, Salmonella, Aeromonas, and Vibrio did not have ionic requirements comparable to those of the marine strains. All of the terrestrial organisms grew in TM without added NaCl (0.068 mM Na+ was present as a contaminant). In some terrestrial organisms, growth was stimulated by the addition of NaCl, the highest stimulation being found in Vibrio cholerae. The optimal growth rates and yields for four strains of this species were observed at 2.5 to 5.0 mM NaCl while the growth rates and yields in TM with no added NaCl were 40 to 50% of the optimum.
A class of catalase-deficient mutants that was unlinked to katE was localized between mutS and cys at 59.0 min on the Escherichia coli genome. This locus was named katF. Transposon Tn10 insertions were isolated that mapped in both katE and katF loci. The catalase species present in katE+ and katF+ recombinants was found to be different from the main catalase activities, HPI and HPII, in several respects. It did not have an associated peroxidase activity; it was electrophoretically slower on native polyacrylamide gels; it eluted from DEAE-Sephadex A50 at a higher salt concentration; its Km for H2O2 was 30.9 mM as compared with 3.7 mM for HPI and HPII; its synthesis was not induced by ascorbate; and it did not cross react with HPI-HPII antisera. This new catalase was labeled HPIII.
Laboratory microecosystems (microcosms) prepared with a chemically defined sea salt solution were used to study effects of selected environmental parameters on growth and activity of Vibrio cholerae. Growth responses under simulated estuarine conditions of 10 strains of V. cholerae, including clinical and environmental isolates as well as serovars O1 and non-O1, were compared, and all strains yielded populations of approximately the same final size. Effects of salinity and temperature on extended survival of V. cholerae demonstrated that, at an estuarine salinity (25%) and a temperature of 10 degrees C, V. cholerae survived (i.e., was culturable) for less than 4 days. Salinity was also found to influence activity, as measured by uptake of 14C-amino acids. Studies on the effect of selected ions on growth and activity of V. cholerae demonstrated that Na+ was required for growth. The results of this study further support the status of V. cholerae as an estuarine bacterium.
Cholera vibrios produce a single polymeric protein that (i) causes hemagglutination; (ii) appears to participate in their attachment to gut epithelium; (iii) may mediate their detachment from gut epithelium; and (iv) is a protease that hydrolyzes fibronectin and mucin, cleaves lactoferrin, and nicks the A subunit of the choleragen-related heat-labile enterotoxin of Escherichia coli.
The influence of water temperature, salinity, and pH on the multiplication of toxigenic Vibrio cholerae serovar O1 cells and their attachment to live planktonic crustaceans, i.e., copepods, was investigated by using laboratory microcosms. By increasing water temperatures up to 30 degrees C, a pronounced effect on the multiplication of V. cholerae was demonstrated, as was attachment of the cells to live copepods. These were measured by culturable counts on agar plates and direct observation by scanning electron microscopy, respectively. Of the three salinities examined (5, 10, and 15%), maximum growth of V. cholerae and attachment to copepods occurred at 15%. An alkaline pH (8.5) was optimal both for attachment and multiplication of V. cholerae, as compared with pH 6.5 and 7.5. It is concluded that conditions affecting attachment of V. cholerae serovar O1 to live copepods observed under laboratory conditions may also occur in the natural estuarine environment and, thereby, are significant in the epidemiology of cholera.
Environmental and clinical strains of Vibrio cholerae were exposed to nutrient-free artificial seawater and filtered natural seawater microcosms for selected time intervals and examined for changes in cell morphology and number. Cells observed by transmission electron and epifluorescence microscopy were found to undergo gross alterations in cell morphology with time of exposure. The vibroid cells decreased in volume by 85% and developed into small coccoid forms surrounded by remnant cell walls. The initial number of cells inoculated into nutrient-free microcosms (culturable count and direct viable count) increased 2.5 log10 within 3 days, and even after 75 days the number of viable cells was still 1 to 2 log10 higher than the initial inoculum size. Nutrient-depleted coccoid-shaped cells were restored to normal size and assumed a bacillary shape within 3 h and began to divide within 5 h after nutrient supplementation. The increase in cell number and decrease in cell volume under nutrient-depleted conditions, as well as the rapid growth response after nutrient supplementation, may describe some of the survival mechanisms of V. cholerae in the aquatic environment.
Laboratory microcosms were employed to evaluate the influence of selected environmental parameters, organic nutrient concentration, and salinity on the growth and survival of a toxigenic strain of Vibrio cholerae LA4808. Over the range conditions tested, this strain of V. cholerae showed maximum response as determined by increased plate counts and direct microscopic counts in microcosms prepared with a chemically defined sea salts solution at a salinity of 25%, but with lower or higher salinity levels, the maximum population size declined. When added organic concentrations of less than 1,000 micrograms/liter were present, a marked salinity effect on the growth of V. cholerae was detected. However, at or above an organic nutrient concentration of 1,000 micrograms/liter, the need for an optimum salinity level was spared. From the results of this study, it is concluded that V. cholerae can grow under conditions of organic nutrient concentration and salinity typical of estuaries. Results obtained support the hypothesis that V. cholerae is an autochthonous member of the estuarine microbial community.
rpoS is the structural gene for the sigma S subunit of RNA polymerase which controls the expression of a large number of genes in Escherichia coli that are induced during entry into stationary phase or in response to increased medium osmolarity. Using a combination of primer extension experiments and a 5' deletion analysis of the region upstream of rpoS, we show that rpoS transcription is mainly driven by a single promoter (rpoSp1) located within the nlpD gene upstream of rpoS (the two relatively weak nlpD promoters contribute to the low level of rpoS expression during early exponential phase). In addition, we demonstrate that the expression of both transcriptional and translational rpoS::lacZ fusions as well as the level of rpoS mRNA originating at rpoSp1 is strongly reduced in ppGpp-deficient relA spoT mutants. However, experiments with the 5' deletion constructs indicate that a lack of ppGpp does affect transcriptional elongation rather than initiation.
rpoS, a gene that encodes an alternative sigma factor (also known as katF), is critical for the ability of Yersinia enterocolitica grown at 37 degrees C, but not at 26 degrees C, to survive diverse environmental insults such as high temperature, hydrogen peroxide, osmolarity, and low pH. However, a Y. enterocolitica rpoS mutant was not affected in expression of inv or ail, invasion of tissue culture cells, or virulence in mice.
The chromosome of Yersinia enterocolitica encodes a heat-stable enterotoxin called Yst and a surface antigen called Myf, which closely resembles enterotoxin-associated fimbriae. Both factors could act in conjunction to produce diarrhea. Production of the enterotoxin is regulated by temperature, osmolarity, and pH and occurs only when bacteria reach the stationary phase. Myf production is regulated by temperature and pH and, as we show in this work, also occurs after the exponential growth phase. In an attempt to understand the late-phase expression of yst and myf, we cloned, sequenced, and mutagenized the gene encoding RpoS, an alternative sigma factor of the RNA polymerase involved in expression of stationary-phase genes in other enterobacteria. An intact rpoS gene was necessary for full expression of yst in the stationary phase but not for the expression of myf and of pYV-encoded virulence determinants.
We report a 1.432-kb DNA sequence at 59 min on the Escherichia coli chromosome that connects the published sequences of the pcm gene for the isoaspartyl protein methyltransferase and that of the katF or rpoS (katF/rpoS) gene for a sigma factor involved in stationary-phase gene expression. Analysis of the DNA sequence reveals an open reading frame potentially encoding a polypeptide of 379 amino acids. The polypeptide sequence includes a consensus bacterial lipidation sequence present at residues 23 to 26 (Leu-Ala-Gly-Cys), four octapeptide proline- and glutamine-rich repeats of consensus sequence QQPQIQPV, and four heptapeptide threonine- and serine-rich repeats of consensus sequence PTA(S,T)TTE. The deduced amino acid sequence, especially in the C-terminal region, is similar to that of the Haemophilus somnus LppB lipoprotein outer membrane antigen (40% overall sequence identity; 77% identity in last 95 residues). The LppB lipoprotein binds Congo red dye and has been proposed to be a virulence determinant in H. somnus. Utilizing a plasmid construct with the E. coli gene under the control of a phage T7 promoter, we demonstrate the lipidation of this gene product by the incorporation of [3H]palmitic acid into a 42-kDa polypeptide. We also show that treatment of E. coli cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 46-kDa precursor. We thus designate the protein NlpD (new lipoprotein D). E. coli cells overexpressing NlpD bind Congo red dye, suggesting a common function with the H. somnus LppB protein. Disruption of the chromosomal E. coli nlpD gene by insertional mutagenesis results in decreased stationary-phase survival after 7 days.
Extracellular zinc-containing metalloproteases are widely distributed in the bacterial world. The most extensively studied are those which are associated with pathogenic bacteria or bacteria which have industrial significance. They are found practically wherever they are sought in both gram-negative and gram-positive microorganisms, be they aerobic or anaerobic. This ubiquity in itself implies that these enzymes serve important functions for the organisms which produce them. Because of the importance of zinc to enzymatic activity, it is not surprising that there is a pervasive amino acid sequence homology in the primary structure of this family of enzymes regardless of their source. The evidence suggests that both convergent and divergent evolutionary forces are at work. Within the large family of bacterial zinc-containing metalloendopeptidases, smaller family units are observed, such as thermolysin-like, elastase-like, and Serratia protease-like metalloproteases from various bacterial species. While this review was in the process of construction, a new function for zinc-containing metalloproteases was discovered: the neurotoxins of Clostridium tetani and Clostridium botulinum type B have been shown to be zinc metalloproteases with specificity for synaptobrevin, an integral membrane protein of small synaptic vesicles which is involved in neurotransmission. Additional understanding of the mode of action of proteases which contribute to pathogenicity could lead to the development of inhibitors, such as chelators, surrogate substrates, or antibodies, which could prevent or interrupt the disease process. Further studies of this broad family of metalloproteases will provide important additional insights into the pathogenesis and structure-function relationships of enzymes and will lead to the development of products, including "designer proteins," which might be industrially and/or therapeutically useful.
Shigella flexneri grown to stationary phase has the ability to survive for several hours at pH 2.5. This acid resistance, which may contribute to the low infective dose associated with shigellosis, is dependent upon the expression of the stationary-phase-specific sigma factor σs. Using random TnphoA and TnlacZ mutagenesis we isolated five acid-sensitive mutants of S. flexneri, which had lost their ability to survive at pH 2.5 for 2 h in vitro. Each transposon insertion with flanking S. flexneri DNA was cloned and sequenced. Database searches indicated that two TnlacZ mutants had an insertion within the hdeA gene, which is the first gene in the hdeAB operon. Acid resistance was restored in one of these mutants by a plasmid carrying the entire hdeAB operon. Further sequence analysis from the remaining TnlacZ and two TnphoA mutants demonstrated that they all had insertions within a previously unidentified open reading frame (ORF), which is directly downstream from the gadB gene. This putative ORF encodes a protein that has homology to a number of inner membrane amino acid antiporters. A 1.8 kb polymerase chain reaction (PCR) product containing this gene was cloned, which was able to restore acid resistance in each mutant. These fusions were induced during entry into late exponential phase and were positively regulated by RpoS. We confirmed that the expression of the acid-resistance phenotype in acidified minimal media was dependent upon the supplementation of glutamic acid and that this glutamate-dependent system was RpoS regulated. Southern hybridization revealed that both the gadC and hdeAB loci are absent in Salmonella. An rpoS deletion mutant of S. flexneri was also constructed to confirm the important role played by this gene in acid resistance. This rpoS − derivative was extremely acid sensitive. Two-dimensional gel electrophoresis of this mutant revealed that it no longer expressed 27 proteins in late log phase that were present in its isogenic parent. These data indicate that the expression of acid resistance in S. flexneri may be multifactorial and involve proteins located at different subcellular locations.
It is now well established that the σS subunit of RNA polymerase is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-inducible genes in Escherichiacoli. In this review, more recent findings will be summarized that demonstrate that σS also acts as a global regulator for the osmotic control of gene expression, and actually does so in exponentially growing cells. Thus, many σS-dependent genes are induced during entry into stationary phase as well as in response to osmotic upshift. K+ glutamate, which accumulates in hyperosmotically stressed cells, seems to specifically stimulate the activity of σS-containing RNA polymerase at σS-dependent promoters. Moreover, osmotic upshift results in an elevated cellular σS level similar to that observed in stationary-phase cells. This increase is the result of a stimulation of rpoS translation as well as an inhibition of the turnover of σS, which in exponentially growing non-stressed cells is a highly unstable protein. Whereas the RNA-binding protein HF-I, previously known as a host factor for the replication of phage Qβ RNA, is essential for rpoS translation, the recently discovered response regulator RssB, and ClpXP protease, have been shown to be required for σS degradation. The finding that the histone-like protein H-NS is also involved in the control of rpoS translation and σS turnover, sheds new light on the function of this protein in osmoregulation. Finally, preliminary evidence suggests that additional stresses, such as heat shock and acid shock, also result in increased cellular σS levels in exponentially growing cells. Taken together, σS function is clearly not confined to stationary phase. Rather, σS may be regarded as a sigma factor associated with general stress conditions.
rpoS is the structural gene for σs, which is a second vegetative sigma subunit of RNA polymerase in Escherichia coli and is involved in the expression of many stationary phase-Induced genes. Upstream of rpoS is an open reading frame (ORF) whose function and regulation have not been studied. Strong overproduction of its gene product using the IPTG-inducible tec promoter leads to the formation of bulges at the cell septum and the cell poles, and in rapidly growing cells brings about cell lysis, indicating that the gene product has a hydrolytic function in cell wall formation or maintenance. This is corroborated by sequence homology to lysostaphin, a cell wait lytic exoenzyme synthesized by two Staphylococcus strains. Using globomycin, a specific Inhibitor of signal peptidase II, we demonstrate that the product of the ORF is a novel lipoprotein (NIpD). Two transcriptional start sites for nIpD have been localized. In contrast to rpoS, nIpD is not induced during entry into stationary phase. Growth-phase-regulated transcription of rpoS is initiated at additional sites within the nIpD ORF, but the nIpD promoters contribute substantially to the basal level of rpoS expression in exponentially growing cells, indicating that nIpD and rpoS form an operon.
Starvation of a marine Vibrio sp. S14 for carbon, nitrogen and phosphorus resulted in a fourfold increase in cell number during the first 6 h in the starvation regime. This initial cell division of non-growing cells was dependent on both DNA and peptidoglycan synthesis as deduced from inhibition experiments using nalidixic acid and ampicillin. Inhibition of protein synthesis by the addition of chloramphenicol led to the cessation of both cell division and DNA synthesis after 40-60 min in the starvation regime. Starvation also induced resistance against autolytic cell wall degradation. Resistance to ampicillin-induced murein degradation was most extensive in the portion of the cell wall that was synthesized after the onset of starvation and was dependent on de novo protein synthesis. The amount of D-alanine per unit dry weight increased twofold during 24 h of starvation and an increased resistance to lysis induced by sonication was observed during this period. It is suggested that the fourfold increase in cell number during the first six hours of starvation requires proteins synthesized de novo and that new rounds of DNA replication may be initiated during non-growth subsequent to 40-60 min of starvation. While the rate of DNA synthesis during the initial 40-60 min was unaffected by the blockage of protein synthesis, the mechanism conferring autolysis resistance was effectively inhibited.
Evolution of complex regulatory pathways that control virulence factor expression in pathogenic bacteria indicates the importance to these organisms of being able to distinguish time and place. In the human intestinal pathogen Vibrio cholerae, control over many virulence genes identified to date is the responsibility of the ToxR protein. ToxR, in conjunction with a second regulatory protein called ToxS, directly activates the genes encoding the cholera toxin; other ToxR regulated genes are not activated directly by ToxR. For some of these genes, ToxR manifests its control through another activator called ToxT. Expression of toxT, which encodes a member of the AraC family of bacterial transcriptional activators, is ToxR dependent and is modulated by in vitro growth conditions that modulate expression of the ToxR virulence regulon. Thus, as in other regulatory circuits, co-ordinate expression of several genes in V. cholerae results from the activity of a cascading system of regulatory factors.
A homologue of the rpoS gene of Escherichia coli was cloned from Pseudomonas aeruginosa PAO1 by hybridization with an oligodeoxyribonucleotide probe designed from an amino-acid stretch conserved among the principal σ factors of eubacteria. Two open reading frames, the pcm gene and the orf-297 of unknown function, were found in the upstream region of rpoS, and in the same order as in E. coli. The rpoS gene of P. aeruginosa was expressed in E. coli and complemented the catalase deficiency of the rpoS mutant of E. coli. The RpoS protein of P. aeruginosa was identified by Western blot analysis in both P. aeruginosa (Pa) and the transformed E. coli. Levels of RpoS of Pa increased drastically at the onset of the stationary growth phase.
We have constructed a series of derivatives of the Ω interposon [Prentki and Krisch, Gene 29 (1984) 303–313 ] that can be used for in vitro insertional mutagenesis. Each of these DNA fragments carries a different antibiotic or Hg2+ resistance gene (ApR, CmR, TcR, KmR or HgR) which is flanked, in inverted orientation, by transcription and translation termination signals and by synthetic polylinkers. The DNA of these interposons can be easily purified and then inserted, by in vitro ligation, into a plasmid linearized either at random by DNase I or at specific sites by restriction enzymes. Plasmid molecules which contain an interposon insertion can be identified by expression of its drug resistance. The position of the interposon can be precisely mapped by the restriction sites in the flanking polylinker. To verify their properties we have used these Ω derivatives to mutagenize a broad host range plasmid which contains the entire meta-cleavage pathway of the toluene degradation plasmid pWW0 of Pseudomonas putida. Insertion of these interposons in the plasmid between the promoter and the catechol 2,3-dioxygenase (C23O) gene dramatically reduced the expression of this enzyme in Escherichia coli. We also show that when a plasmid containing an ω interposon is transferred by conjugal mobilization from E. coli to P. putida, Agrobacterium tumefaciens, Erwinia chrysanthemi, Paracoccus denitrificans or Rhizobium leguminosarum, the appropriate interposon drug resistance is usually expressed and, compared to the non-mutated plasmid, much reduced levels of C23O activity are detected. Thus, the selection and/or characterization of Ω insertional mutations can be carried out in these bacterial species.
Nutrient limitation is a critical signal in Salmonella virulence gene regulation. The katF (rpoS) gene mediates the expression of the Salmonella spv plasmid virulence genes during bacterial starvation. A katF Salmonella mutant has increased susceptibility to nutrient deprivation, oxidative stress, acid stress, and DNA damage, conditions which are relevant to the intraphagosomal environment of host macrophages. Moreover, the katF mutant has significantly reduced virulence in mice. katF encodes an alternative sigma factor of RNA polymerase which coordinately regulates Salmonella virulence.
Evolution of complex regulatory pathways that control virulence factor expression in pathogenic bacteria indicates the importance to these organisms of being able to distinguish time and place. In the human intestinal pathogen Vibrio cholerae, control over many virulence genes identified to date is the responsibility of the ToxR protein. ToxR, in conjunction with a second regulatory protein called ToxS, directly activates the genes encoding the cholera toxin; other ToxR regulated genes are not activated directly by ToxR. For some of these genes, ToxR manifests its control through another activator called ToxT. Expression of toxT, which encodes a member of the AraC family of bacterial transcriptional activators, is ToxR dependent and is modulated by in vitro growth conditions that modulate expression of the ToxR virulence regulon. Thus, as in other regulatory circuits, co-ordinate expression of several genes in V. cholerae results from the activity of a cascading system of regulatory factors.
The response of marine Vibrio sp. strain S14 (CCUG 15956) to long-term (48-h) multiple-nutrient starvation (i.e., starvation for glucose, amino acids, ammonium, and phosphate simultaneously) can be described as a three-phase process. The first phase, defined as the stringent control phase, encompasses an accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and decreases in RNA and protein synthesis during the first 40 min. In the second phase, there is a temporary increase in the rates of RNA and protein synthesis between 1 and 3 h paralleling a decrease in the ppGpp pool. The third phase includes gradual decline in macromolecular synthesis after 3 h. Using two-dimensional gel electrophoresis of pulse-labeled proteins, a total of 66 proteins were identified as starvation inducible (Sti), temporally expressed throughout the three phases of starvation. The inhibition of protein synthesis during the first phase of starvation partly disrupted the subsequent temporally ordered synthesis of starvation proteins and prevented the expression of some late starvation proteins. It was also found that the early temporal class of starvation proteins, which included the majority of the Sti proteins, was the most essential for long-term survival. Vibrio sp. strain S14 cultures prestarved (1 h) for glucose, amino acids, ammonium, or phosphate as well as cultures exposed (1 h) to CdCl2 exhibited enhanced survival during the subsequent multiple-nutrient starvation in the presence of chloramphenicol or rifampin, while heat or the addition of cyclic AMP or nalidixic acid prior to starvation had no effect. It was demonstrated that amino acid starvation and CdCl2 exposure, which induced the stringent response, were the most effective in conferring enhanced survival. A few Sti proteins were common to all starvation conditions. In addition, the total number of proteins induced by multiple-nutrient starvation significantly exceeded the sum of those induced by starvation for each of the individual nutrients.
A series of controlled expression vectors was constructed based on the wide-host-range plasmid pMMB66EH. Some of these new vectors code for the alpha-peptide of beta-galactosidase and allow the direct screening of recombinant clones by inactivation of alpha-complementation. The bla gene was replaced in some plasmids by the cat gene of Tn9 coding for chloramphenicol resistance, extending the use into beta-lactam-resistant strains. They all feature either the tac or taclac (tac-lac UV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point. These vectors were tested by subcloning the xylE gene coding for the Pseudomonas putida catechol 2,3-oxygenase and the Escherichia coli lamB gene coding for the lambda receptor. The expression of these genes in E. coli indicated that the tac promoter is five times stronger than the taclac promoter and that both were tightly regulated. The tac promoter in Pseudomonas syringae pv glycinea and Xanthomonas campestris pv vesicatoria had a strength similar to that in E. coli, while the taclac promoter was much weaker, reaching only 6.5 and 3% of the level of expression of the tac promoter, respectively. The taclac promoter, however, proved to be useful for the cloning in E. coli of DNA fragments that were unstable in vectors with stronger promoters and higher copy number. Expression of the lamB gene in Vibrio cholerae strain TRH7000 was not sufficient to permit cosmid transduction. Two subunits of the E. coli mannose permease, coded by the ptsP and ptsM genes, are also required for cosmid DNA penetration into the recipient cells.
During carbon-starvation-induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30-50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C-starvation-regulated genes we isolated and characterized chromosomal C-starvation-induced csi::lacZ fusions using the lambda placMu system. One operon fusion (csi2::lacZ) has been studied in detail. csi2::lacZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two-dimensional gel electrophoresis, cells carrying csi2::lacZ did not synthesize at least 16 proteins present in an isogenic csi2+ strain. Cells containing csi2::lacZ or csi2::Tn10 did not produce glycogen, did not develop thermotolerance and H2O2 resistance, and did not induce a stationary-phase-specific acidic phosphatase (AppA) as well as another csi fusion (csi5::lacZ). Moreover, they died off much more rapidly than wild-type cells during prolonged starvation. We conclude that csi2::lacZ defines a regulatory gene of central importanc e for stationary phase E. coli cells. These results and the cloning of the wild-type gene corresponding to csi2 demonstrated that the csi2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi2/katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose 'rpoS' ('sigma S') as appropriate designations.
At the onset of starvation Escherichia coli undergoes a temporally ordered program of starvation gene expression involving 40-80 genes which some four hours later yields cells possessing an enhanced general resistance. Two classes of genes are induced upon carbon starvation: the cst genes, requiring cyclic AMP, and the pex genes, not requiring this nucleotide for induction. The cst genes are not involved in the development of the resistant state and are concerned with escape from starvation, while the pex gene induction appears to be associated with resistance. Many of the latter are induced in response to a variety of starvation conditions. They include heat shock and oxidation resistance genes, and some utilize minor, stationary-phase-specific sigma factors for induction during starvation. The protective role of stress proteins may be due to their ability to rescue misfolded macromolecules. The starvation promoters can be potentially useful for selective expression of desired genes in metabolically sluggish populations, e.g. in high-density industrial fermentations and in situ bioremediation.
The response of Vibrio cholerae to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 30-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Total lipids and carbohydrates declined rapidly within the first 7 days, while DNA and protein exhibited a more constant decline over the 30 days of starvation. In contrast, RNA showed little decrease upon starvation. Although neutral lipids were lost, the percentage of neutral lipids did not decline as rapidly as the phospholipids. Detectable levels of poly-beta-hydroxybutyrate disappeared completely by 7 days. Carbohydrate profiles revealed the relative loss of the five-carbon sugar ribose and N-acetylglucosamine and a relative increase in the total six-carbon sugars, especially glucose. Morphologically, ribosomes appeared to exhibit no structural change, while inclusion bodies and mesosomelike structures disappeared completely, and cell wall and membrane integrity was lost. The data suggest that V. cholerae differs somewhat from other marine vibrios in its response to low nutrients but shares some characteristics in common with them. The data also suggest that certain lipids and carbohydrates may provide the endogenous energy sources needed for dormancy preparation and cell maintenance under nutrient starvation.
The transposon TnphoA was used to generate fusions between phoA, the gene for alkaline phosphatase (PhoA), and genes encoding proteins that are secreted by Vibrio cholerae. One of the PhoA+ mutants isolated showed a dramatic reduction in its ability to colonize the intestines of suckling mice. This mutant no longer produced a 20.5-kDa protein (TcpA) that we show is the major subunit of a V. cholerae pilus. Amino-terminal sequence analysis of the TcpA pilus subunit showed that it shares amino acid homology with the pilins produced by several other pathogenic bacteria. The TcpA pilus was coordinately expressed with cholera toxin under various culture conditions, and this effect appeared to be dependent on the transcriptional activator encoded by the toxR gene. We conclude that the toxR gene plays a central role in the transcriptional regulation of multiple virulence genes of V. cholerae.
Isoelectric focusing of culture supernatants from Vibrio cholerae El Tor 1621 and high protease-producing mutant strain 1621 hip revealed the presence of three different types of extracellular protease. Type I protease was the major activity in the wild-type strain and was inhibited by phenylmethylsulfonyl fluoride and by the lima bean trypsin inhibitor. Type II protease was present in the wild type and was the major activity in the high protease-producing mutant. It was resistant to inhibitors of metalloproteases and serine proteases. Two peaks of type II protease differed by 1.2 pI units in isoelectric point and by 1,500 in molecular weight. Type II protease had broad specificity, acted as a mucinase, and caused degradation of some other V. cholerae extracellular proteins, including DNase and cholera toxin. Type III protease was EDTA inhibitable and was detected only in the high protease producer. Possible roles of extracellular proteases as virulence factors in cholera pathogenesis are discussed.
Contrary to earlier findings with all other in vivo and in vitro models of cholera studied, nonchemotactic vibrio mutants showed a relatively greater fitness in 5-day-old infant mice as compared with chemotactic parent or chemotactic revertant strains. This trend was manifest in the relatively greater number of nonchemotactic mutants recovered from the upper small intestine at 4 and 18 h after intragastric infection. The same trend was also revealed in the significantly greater virulence (in terms of time to death) of nonchemotactic mutants as compared with the chemotactic parent or revertant strains. Histological studies in infant mice of the penetration of chemotactic and nonchemotactic vibrios into the mucus gel of the small intestine yielded the same findings as in all other models studied, i.e., significantly greater penetration by chemotactic vibrios. There was no correlation between the relative fitness of nonchemotactic vibrios in the small intestine of infant mice and the rate of recovery of viable nonchemotactic vibrios from that site. In contrast, excellent correlation was found between the relative fitness of nonchemotactic vibrios and a decrease in the recovery of viable cells of the chemotactic strain from the small intestine. This indicates that the relatively greater fitness of the nonchemotactic vibrios in infant mice was only apparent and that the observed phenomenon was actually due to an antibacterial mechanism which prevented the accumulation of the chemotactic strains in the small intestine rather than to any stimulating effect on the nonchemotactic mutant itself. To study the in vivo fate of the inoculum in infant mice, vibrios were labeled with either 32P, 35S, or [3H]thymidine. Specific activity determinations of the 32P label were compatible with the assumption of an accelerated rate of death of the chemotactic parent strain in the small intestine. Results with the other isotopes, however, were significantly different. Indeed, the amount of radioactivity retained in the small intestine after feeding labeled bacteria correlated more closely with the isotope used than with the strain of vibrio under study. Consequently, considerable doubt must be cast on the general validity of this not uncommon technique for determining the in vivo location and the death or survival of radioactively labeled bacteria.
The protein encoded by katF (also known as nur, appR, csi-2, abrD, and rpoS in various alleles) has been biochemically confirmed to be an alternate sigma transcription factor and renamed sigma S. Its synthesis is controlled transcriptionally and posttranscriptionally by as yet undefined mechanisms that are active well into stationary phase. sigma S controls a regulon of 30 or more genes expressed in response to starvation and during the transition to stationary phase. Proteins in the regulon, many of which have not been characterized, enhance long-term survival in nutrient-deficient medium and have a diverse group of functions including protection against DNA damage, the determination of morphological changes, the mediation of virulence, osmoprotection, and thermotolerance. Differential expression of subfamilies of genes within the regulon is effected by supplementary regulatory factors, working both individually and in combination to modulate activity of different sigma S-dependent promoters.
The gene encoding the alternative sigma factor RpoS in Salmonella typhimurium was cloned by its ability to complement acid susceptibility in rpoS mutant Escherichia coli. Sequence determination and comparison with rpoS from E. coli demonstrates a high degree of conservation, although significant differences are found within the extragenic regulatory regions.