The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii

To investigate regulatory networks in Legionella pneumophila, the gene encoding the homolog of the Escherichia coli stress and stationary-phase sigma factor RpoS was identified by complementation of an E. coli rpoS mutation. An open reading frame that is approximately 60% identical to the E. coli rpoS gene was identified. Western blot analysis showed that the level of L. pneumophila RpoS increased in stationary phase. An insertion mutation was constructed in the rpoS gene on the chromosome of L. pneumophila, and the ability of this mutant strain to survive various stress conditions was assayed and compared with results for the wild-type strain. Both the mutant and wild-type strains were more resistant to stress when in stationary phase than when in the logarithmic phase of growth. This finding indicates that L. pneumophila RpoS is not required for a stationary-phase-dependent resistance to stress. Although the mutant strain was able to kill HL-60- and THP-1-derived macrophages, it could not replicate within a protozoan host, Acanthamoeba castellanii. These data suggest that L. pneumophila possesses a growth phase-dependent resistance to stress that is independent of RpoS control and that RpoS likely regulates genes that enable it to survive in the environment within protozoa. Our data indicate that the role of rpoS in L. pneumophila is very different from what has previously been reported for E. coli rpoS.     

Amino acid residue Ala-62 in the FimH fimbrial adhesin is critical for the adhesiveness of meningitis-associated Escherichia coli to collagens

Adhesion of meningitis-associated Escherichia coli O18acK1H7 to collagens was characterized. The E. coli strain IHE 3034 adhered to type IV and type I collagens but not to type III collagen immobilized on glass. Collagens lack terminal mannosyl units, yet the bacterial adhesion was completely abolished in the presence of alpha-methyl-D-mannoside. A cat cassette was introduced into the filmA gene of IHE 3034, and the resulting mutant strain IHE 3034-2 failed to adhere to collagens. In contrast, insertion of a Gm cassette into the sfaA gene of IHE 3034, encoding the S-fimbrillin, had no significant effect on the adhesiveness. The fim cluster from IHE 3034 was cloned and expressed in trans in the fimA::cat mutant strain IHE 3034-2. The complemented strain IHE 3034-2(pRPO-1) exhibited adhesiveness to type IV and type I collagens, confirming the function of the type 1 fimbria in the adhesion. We have previously shown that the type 1 fimbria from E. coli K-12 strain PC31 does not confer bacterial adhesiveness to collagens. The fimH genes from E. coli IHE 3034 as well as from PC31 were expressed in the fimH-null strain MS4. The FimH from IHE 3034 potentiated collagen adherence, whereas the FimH from PC31 was inactive. Sequence comparison of fimH from IHE 3034 and PC31 revealed five amino-acid differences in the predicted mature FimH proteins: at residues 27, 62, 70, 78 and 201. Each of these residues in the IHE 3034-FimH were individually substituted to the corresponding amino acid in the PC31-FimH. The substitution S62-->A completely abolished collagen adhesiveness. The reverse substitution A62-->S in the PC31-FimH as well as in the FimH from another E. coli strain induced collagen adhesiveness to the level seen with IHE 3034-FimH. Out of nine fimH genes analysed from isolates of E. coli, collagen adhesiveness as well as alanine at position 62 in FimH were found only in two O18acK1H7 isolates with the isoenzyme profile ET type 1. Our results demonstrate that the amino-acid residue Ala-62 in the FimH lectin is critical for the adhesion to collagens by a highly virulent clonal group of E. coli.     

rpoS mutants in archival cultures of Salmonella enterica serovar typhimurium

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.     

Further studies on RpoS in enterobacteria: identification of rpoS in Enterobacter cloacae and Kluyvera cryocrescens

RpoS, the alternative sigma factor sigma(s), is important for bacterial survival under extreme conditions. Many enterobacteria are opportunistic human pathogens and their ability to survive in a changing environment could be an essential step for their virulence. To determine the presence of this gene in enteric bacteria, an Escherichia coli rpoS probe was constructed and used to detect the presence of this gene in different species. A gene homologous to rpoS was found in Citrobacter amalonaticus, Enterobacter cloacae, Klebsiella planticola, Kluyvera cryocrescens, Serratia rubidaea, Shigella sonnei, and Yersinia ruckeri. Providencia stuartii and Proteus vulgaris were the only tested enterobacteria that did not show any signal with the E. coli rpoS probe or that did not lead to amplification of an rpoS fragment using specific primers. The rpoS gene from E. cloacae and from K. cryocrescens was cloned and sequenced and a mutant allele was constructed in E. cloacae. Survival rates under different harsh conditions were followed in order to determine the effect of rpoS inactivation in exponential- and stationary-phase cells of both strains. E. cloacae rpoS mutants were more sensitive to extreme pH, high osmolarity, and high temperature than the wild-type.     

Phenotypic diversity caused by differential RpoS activity among environmental Escherichia coli isolates

Enteric bacteria deposited into the environment by animal hosts are subject to diverse selective pressures. These pressures may act on phenotypic differences in bacterial populations and select adaptive mutations for survival in stress. As a model to study phenotypic diversity in environmental bacteria, we examined mutations of the stress response sigma factor, RpoS, in environmental Escherichia coli isolates. A total of 2,040 isolates from urban beaches and nearby fecal pollution sources on Lake Ontario (Canada) were screened for RpoS function by examining growth on succinate and catalase activity, two RpoS-dependent phenotypes. The rpoS sequence was determined for 45 isolates, including all candidate RpoS mutants, and of these, six isolates were confirmed as mutants with the complete loss of RpoS function. Similarly to laboratory strains, the RpoS expression of these environmental isolates was stationary phase dependent. However, the expression of RpoS regulon members KatE and AppA had differing levels of expression in several environmental isolates compared to those in laboratory strains. Furthermore, after plating rpoS+ isolates on succinate, RpoS mutants could be readily selected from environmental E. coli. Naturally isolated and succinate-selected RpoS mutants had lower generation times on poor carbon sources and lower stress resistance than their rpoS+ isogenic parental strains. These results show that RpoS mutants are present in the environment (with a frequency of 0.003 among isolates) and that, similarly to laboratory and pathogenic strains, growth on poor carbon sources selects for rpoS mutations in environmental E. coli. RpoS selection may be an important determinant of phenotypic diversification and, hence, the survival of E. coli in the environment.     

Functional heterogeneity of RpoS in stress tolerance of enterohemorrhagic Escherichia coli strains

The stationary-phase sigma factor (RpoS) regulates many cellular responses to environmental stress conditions such as heat, acid, and alkali shocks. On the other hand, mutations at the rpoS locus have frequently been detected among pathogenic as well as commensal strains of Escherichia coli. The objective of this study was to perform a functional analysis of the RpoS-mediated stress responses of enterohemorrhagic E. coli strains from food-borne outbreaks. E. coli strains belonging to serotypes O157:H7, O111:H11, and O26:H11 exhibited polymorphisms for two phenotypes widely used to monitor rpoS mutations, heat tolerance and glycogen synthesis, as well as for two others, alkali tolerance and adherence to Caco-2 cells. However, these strains synthesized the oxidative acid resistance system through an rpoS-dependent pathway. During the transition from mildly acidic growth conditions (pH 5.5) to alkaline stress (pH 10.2), cell survival was dependent on rpoS functionality. Some strains were able to overcome negative regulation by RpoS and induced higher beta-galactosidase activity without compromising their acid resistance. There were no major differences in the DNA sequences in the rpoS coding regions among the tested strains. The heterogeneity of rpoS-dependent phenotypes observed for stress-related phenotypes was also evident in the Caco-2 cell adherence assay. Wild-type O157:H7 strains with native rpoS were less adherent than rpoS-complemented counterpart strains, suggesting that rpoS functionality is needed. These results show that some pathogenic E. coli strains can maintain their acid tolerance capability while compromising other RpoS-dependent stress responses. Such adaptation processes may have significant impact on a pathogen's survival in food processing environments, as well in the host's stomach and intestine.     

Altered stationary-phase response in a Borrelia burgdorferi rpoS mutant

The homolog of the chromosomally encoded stationary-phase sigma factor RpoS in Borrelia burgdorferi was inactivated using gyrB(r) as a selectable marker. Two-dimensional nonequilibrium pH gradient electrophoresis of stationary-phase cell lysates identified at least 11 differences between the protein profiles of the rpoS mutant and wild-type organisms. Wild-type B. burgdorferi had a growth phase-dependent resistance to 1 N NaCl, similar to the stationary-phase response reported for other bacteria. The B. burgdorferi rpoS mutant strain was less resistant to osmotic stress in stationary phase than the isogenic rpoS wild-type organism. The results indicate that the B. burgdorferi rpoS homolog influences protein composition and participates in stationary-phase-dependent osmotic resistance. This rpoS mutant will be useful for studying regulation of gene expression in response to changing environmental conditions.     

Expression of the Fis protein is sustained in late-exponential- and stationary-phase cultures of Salmonella enterica serovar Typhimurium grown in the absence of aeration

The classic expression pattern of the Fis global regulatory protein during batch culture consists of a high peak in the early logarithmic phase of growth, followed by a sharp decrease through mid-exponential growth phase until Fis is almost undetectable at the end of the exponential phase. We discovered that this pattern is contingent on the growth regime. In Salmonella enterica serovar Typhimurium cultures grown in non-aerated SPI1-inducing conditions, Fis can be detected readily in stationary phase. On the other hand, cultures grown with standard aeration showed the classic Fis expression pattern. Sustained Fis expression in non-aerated cultures was also detected in some Escherichia coli strains, but not in others. This novel pattern of Fis expression was independent of sequence differences in the fis promoter regions of Salmonella and E. coli. Instead, a clear negative correlation between the expression of the Fis protein and of the stress-and-stationary-phase sigma factor RpoS was observed in a variety of strains. An rpoS mutant displayed elevated levels of Fis and had a higher frequency of epithelial cell invasion under these growth conditions. We discuss a model whereby Fis and RpoS levels vary in response to environmental signals allowing the expression and repression of SPI1 invasion genes.     

Impact of rpoS deletion on the proteome of Escherichia coli grown planktonically and as biofilm

To investigate the role of rpoS in gene expression of Escherichia coli cells grown as biofilms, we compared the proteomes of a rpoS mutant and the wild-type strain. Experiments were performed on planktonic cells (in exponential or stationary growth phase) and biofilms developed on glass wool. Spot-by-spot comparison of gels obtained from biofilm and planktonic wild-type organisms showed that the intensity of between 22 and 30% of detected spots was affected by the growth mode, depending of the control used. Principal component analysis, used to interpret the variations in protein spot densities, discriminated exponential-phase cells (wild-type and mutant) from the other incubation conditions and secondarily 72-old cultures. The statistical analysis demonstrated that the rpoS mutation did not significantly modify the proteome of exponential-growth phase cells, the differences involving only 3% of the proteome. However, increasing the incubation time from 8 to 72 h noticeably increased the number of changed proteins. A cluster analysis showed that RpoS plays a role in the special nature of the gene expression of biofilm cells but lower than in stationary-phase bacteria. We identified 35 rpoS-regulated proteins that were already or not described as controlled by this sigma factor. For some of them, the mode of regulation by RpoS was obviously dependent on the culture condition (planktonic vs biofilm).     

The effects of low-shear stress on Adherent-invasive Escherichia coli

The impact of low-shear stress (LSS) was evaluated on an Adherent-invasive Escherichia coli clinical isolate (AIEC strain O83:H1) from a Crohn's disease patient. High-aspect ratio vessels (HARVs) were used to model LSS conditions to characterize changes in environmental stress resistance and adhesion/invasive properties. Low-shear stress-grown cultures exhibited enhanced thermal and oxidative stress resistance as well as increased adherence to Caco-2 cells, but no changes in invasion were observed. An AIEC rpoS mutant was constructed to examine the impact of this global stress regulator. The absence of RpoS under LSS conditions resulted in increased sensitivity to oxidative stress while adherence levels were elevated in comparison with the wild-type strain. TnphoA mutagenesis and rpoS complementation were carried out on the rpoS mutant to identify those factors involved in the LSS-induced adherence phenotype. Mutagenesis results revealed that one insertion disrupted the tnaB gene (encoding tryptophan permease) and the rpoS tnaB double mutant exhibited decreased adherence under LSS. Complementation of the tnaB gene, or medium supplemented with exogenous indole, restored adhesion of the rpoS tnaB mutant under LSS conditions. Overall, our study demonstrated how mechanical stresses such as LSS altered AIEC phenotypic characteristics and identified novel functions for some RpoS-regulated proteins.     

Variation in resistance to high hydrostatic pressure and rpoS heterogeneity in natural isolates of Escherichia coli O157:H7

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.     

GASP phenotype: presence in enterobacteria and independence of sigmaS in its acquisition

The appearance of growth advantage in stationary phase or GASP was originally detected in Escherichia coli. The presence of this phenotype in other enterobacteria such as Enterobacter cloacae, Salmonella typhimurium, Providencia stuartii and Shigella dysenteriae is described in this work. E. cloacae GASP strains presented lower levels of RpoS than the parental strain, although no mutation in the gene or its promoter was detected. This work offers evidence of GASP rpoS-independent pathways as GASP was also acquired in knock-out rpoS E. cloacae and E. coli strains.     

Involvement of rpoS in the survival of Escherichia coli in the viable but non-culturable state

When exposed to stress-provoking environmental conditions such as those of ground waters, many medically important bacteria have been shown to be capable of activating a survival strategy known as the viable but non-culturable (VBNC) state. In this state bacteria are no longer culturable on conventional growth media, but the cells maintain their viability and pathogenicity genes/factors and can start dividing again, in a part of the cell population, upon restoration of favourable environmental conditions. Little is known about the genetic mechanisms underlying the VBNC state. In this study we show evidence of involvement of the rpoS gene in persistence of Escherichia coli in the VBNC state. The kinetics of entry into the non-culturable state and duration of cell viability were measured in two E. coli mutants carrying an inactivated rpoS gene and compared with those of the parents. For these experiments, laboratory microcosms consisting of an artificial oligotrophic medium incubated at 4 degrees C were used. The E. coli parental strains reached the non-culturable state in 33 days when the plate counts were evaluated on Luria-Bertani agar containing sodium pyruvate, whereas cells of the rpoS mutants lost their culturability in only 21 days. Upon reaching unculturability the parents yielded respiring cells and cells with intact membranes for at least the next three weeks and resuscitation was allowed during this time. In contrast, the RpoS- mutant cells demonstrated intact membranes for only two weeks and a very restricted (<7 days) resuscitation capability. Guanosine 3',5'-bispyrophosphate (ppGpp) acts as a positive regulator during the production and functioning of RpoS. A mutant deficient in ppGpp production behaved like the rpoS mutants, while overproducers of ppGpp displayed a vitality at least comparable to that of RpoS+ strains. These results suggest that the E. coli parental strains enter the VBNC state which lasts for, at least, three weeks, after which apparently all the cells die. The rpoS mutants do not activate this survival strategy and early die. This implies involvement of the rpoS gene in E. coli persistence in the VBNC state.     

rpoS基因在肠杆菌CGMCC 5087响应环境胁迫中的功能

[背景]苯乙醇(2-Phenylethanol,2-PE)是一种具有玫瑰香气味的高级香料添加剂,被广泛应用于香水、化妆品、食品和医药等领域.目前,利用工程菌合成苯乙醇有很好的应用前景.我们分离到一株肠杆菌(Enterobactersp.)CGMCC 5087,其可以通过苯丙酮酸途径合成2-PE.然而该菌的生长受到不同环...     

Positive selection for loss of RpoS function in Escherichia coli

Though RpoS, an alternative sigma factor, is required for survival and adaptation of Escherichia coli under stress conditions, many strains have acquired independent mutations in the rpoS gene. The reasons for this apparent selective loss and the nature of the selective agent are not well understood. In this study, we found that some wild type strains grow poorly in succinate minimal media compared with isogenic strains carrying defined RpoS null mutations. Using an rpoS+ strain harboring an operon lacZ fusion to the highly-RpoS dependent osmY promoter as an indicator strain, we tested if this differential growth characteristic could be used to selectively isolate mutants that have lost RpoS function. All isolated (Suc+) mutants exhibited attenuated beta-galactosidase expression on indicator media suggesting a loss in either RpoS or osmY promoter function. Because all Suc+ mutants were also defective in catalase activity, an OsmY-independent, RpoS-regulated function, it was likely that RpoS activity was affected. To confirm this, we sequenced PCR-amplified products containing the rpoS gene from 20 independent mutants using chromosomal DNA as a template. Sequencing and alignment analyses confirmed that all isolated mutants possessed mutated alleles of the rpoS gene. Types of mutations detected included single or multiple base deletions, insertions, and transversions. No transition mutations were identified. All identified point mutations could, under selection for restoration of beta-galactosidase, revert to rpoS+. Revertible mutation of the rpoS gene can thus function as a genetic switch that controls expression of the regulon at the population level. These results may also help to explain why independent laboratory strains have acquired mutations in this important regulatory gene.