RpoS Levels Research Articles

Response Regulator RssB의 활성 조절

많은 세균들은 환경적 스트레스에 대항하기 위해 세균 생존에 유용한 특정 유전자들의 전사를 유도하는 대체시그마 인자 RpoS를 활용한다. 세포 내 RpoS 단백질의 농도는 주로 ClpXP 단백질 분해효소의 조절을 통해 결정된다. RpoS를 ClpXP로 전달하기 위해서는 adaptor 단백질 RssB가 반드시 필요하다. Two-component-type response regulator RssB는 RpoS와 지속적으로 상호작용을 하지만, 다양한 환경변화에 의해 RssB-RpoS 상호작용이 억제되어 세균에서 RpoS 양을 증가시킨다. 본 총설에서는 최근까지 연구 된 RssB-RpoS 상호작용에 관여하는 RssB의 anti-adaptor 단백질 IraD, IraM, IraP 등의 조절인자들과 RssB의 N-terminal 수용체 도메인의 인산화에 대해 설명하고 요약하였다. 이러한 RssB의 정교한 활성을 통한 RpoS 분해조절 과정은 외부환경 스트레스로부터 보다 효율적으로 세균을 보호할 수 있다. Against environmental stresses, many bacteria utilize the alternate sigma factor RpoS that induces transcription of the specific set of genes helpful in promoting bacterial survival. Intracellular levels of RpoS are determined mainly by its turnover through proteolysis of ClpXP protease. Delivery of RpoS to ClpXP strictly requires the adaptor protein RssB. The two-component-type response regulator RssB constantly interacts with RpoS, but diverse environmental changes inhibit this interaction through modification of RssB activity, which increases RpoS levels in bacteria. This review discusses and summarizes recent findings on regulatory factors in RssB-RpoS interactions, including IraD, IraM, IraP anti-adaptor proteins of RssB and phosphorylation of N-terminal receiver domain of RssB. New information shows that the coordinated regulation of RssB activity in controlling RpoS turnover confers efficient bacterial defense against stresses.

Contributions of Environmental Signals and Conserved Residues to the Functions of Carbon Storage Regulator A of Borrelia burgdorferi

Carbon storage regulator A of Borrelia burgdorferi (CsrABb) contributes to vertebrate host-specific adaptation by modulating activation of the Rrp2-RpoN-RpoS pathway and is critical for infectivity. We hypothesized that the functions of CsrABb are dependent on environmental signals and on select residues. We analyzed the phenotype of csrABb deletion and site-specific mutants to determine the conserved and pathogen-specific attributes of CsrABb. Levels of phosphate acetyltransferase (Pta) involved in conversion of acetyl phosphate to acetyl-coenzyme A (acetyl-CoA) and posttranscriptionally regulated by CsrABb in the csrABb mutant were reduced from or similar to those in the control strains under unfed- or fed-tick conditions, respectively. Increased levels of supplemental acetate restored vertebrate host-responsive determinants in the csrABb mutant to parental levels, indicating that both the levels of CsrABb and the acetyl phosphate and acetyl-CoA balance contribute to the activation of the Rrp2-RpoN-RpoS pathway. Site-specific replacement of 8 key residues of CsrABb (8S) with alanines resulted in increased levels of CsrABb and reduced levels of Pta and acetyl-CoA, while levels of RpoS, BosR, and other members of rpoS regulon were elevated. Truncation of 7 amino acids at the C terminus of CsrABb (7D) resulted in reduced csrABb transcripts and posttranscriptionally reduced levels of FliW located upstream of CsrABb. Electrophoretic mobility shift assays revealed increased binding of 8S mutant protein to the CsrA binding box upstream of pta compared to the parental and 7D truncated protein. Two CsrABb binding sites were also identified upstream of fliW within the flgK coding sequence. These observations reveal conserved and unique functions of CsrABb that regulate adaptive gene expression in B. burgdorferi.

Manganese and Zinc Regulate Virulence Determinants in Borrelia burgdorferi

Borrelia burgdorferi, the causative agent of Lyme disease, must adapt to two diverse niches, an arthropod vector and a mammalian host. RpoS, an alternative sigma factor, plays a central role in spirochetal adaptation to the mammalian host by governing expression of many genes important for mammalian infection. B. burgdorferi is known to be unique in metal utilization, and little is known of the role of biologically available metals in B. burgdorferi. Here, we identified two transition metal ions, manganese (Mn(2+)) and zinc (Zn(2+)), that influenced regulation of RpoS. The intracellular Mn(2+) level fluctuated approximately 20-fold under different conditions and inversely correlated with levels of RpoS and the major virulence factor OspC. Furthermore, an increase in intracellular Mn(2+) repressed temperature-dependent induction of RpoS and OspC; this repression was overcome by an excess of Zn(2+). Conversely, a decrease of intracellular Mn(2+) by deletion of the Mn(2+) transporter gene, bmtA, resulted in elevated levels of RpoS and OspC. Mn(2+) affected RpoS through BosR, a Fur family homolog that is required for rpoS expression: elevated intracellular Mn(2+) levels greatly reduced the level of BosR protein but not the level of bosR mRNA. Thus, Mn(2+) and Zn(2+) appeared to be important in modulation of the RpoS pathway that is essential to the life cycle of the Lyme disease spirochete. This finding supports the emerging notion that transition metals such as Mn(2+) and Zn(2+) play a critical role in regulation of virulence in bacteria.

Identification of regulatory factors of the stationary phase sigma factor RpoS in Escherichia coli

The sigma factor RpoS is the master regulator of the general stress response that helps cells survive harsh environmental conditions by associating with the RNA polymerase and regulating gene expression. While numerous stresses are known to up‐regulate RpoS, many of the molecular steps leading from stresses to changes in RpoS levels remain unknown. To screen for the novel regulators, we used an RpoS’‐‘LacZ fusion. We transformed the reporter strains with an overexpression library, screening for plasmids that resulted in colonies with either more or less RpoS as observed on Xgal indicator plates. We verified that RpoS levels were altered in the presence of the plasmid with Western blots and quantified the effect on RpoS expression beta‐galactosidase assays in both RpoS transcription and translation lacZ fusions. We identified the genes on the plasmids as gadY, rbsD, yhhI, yhjR, ykgR and part of the dsdA gene in the screen. Our results suggested all of the factors, with the exception of rbsD, regulated RpoS in a post‐transcriptional manner either at the level of translation or protein stability. The small RNA gadY caused a strong inhibitory effect on RpoS expression and yhhI, ykgR and yhjR causing a mild increase expression of RpoS. In summary, we have found multiple new factors that affect RpoS at the post‐transcriptional level.

Escherichia coli Lacking RpoS Are Rare in Natural Populations of Non-Pathogens

The alternative sigma factor RpoS controls a large regulon that allows E. coli to respond to a variety of stresses. Mutations in rpoS can increase rates of nutrient acquisition at the cost of a decrease in stress resistance. These kinds of mutations evolve rapidly under certain laboratory conditions where nutrient acquisition is especially challenging. The frequency of strains lacking RpoS in natural populations of E. coli is less clear. Such strains have been found at frequencies over 20% in some collections of wild isolates. However, laboratory handling can select for RpoS-null strains and may have affected some of these strain collections. Other studies have included an unknown diversity of strains or only used a phenotypic proxy as a measure of RpoS levels. We directly measured RpoS levels in a collection of E. coli that includes the full diversity of the species and that was handled in a manner to minimize the potential for laboratory evolution. We found that only 2% of strains produce no functional RpoS. Comparison of these strains in multiple labs shows that these rpoS mutations occurred in the laboratory. Earlier studies reporting much higher levels of RpoS polymorphism may reflect the storage history of the strains in laboratories rather than true frequency of such strains in natural populations.

Growth and survival parameter estimates and relation to RpoS levels in serotype O157:H7 and non-O157 Shiga toxin-producing Escherichia coli

This study analysed the growth and survival of 18 strains of the six serotypes of non-O157 Shiga toxin-producing Escherichia coli (STEC) (O26, O45, O103, O111, O121 and O145) most frequently implicated in human illness and compared them with Escherichia coli O157:H7 strain ATCC43895. The data from growth in Luria-Bertani broth (LB)-HCl (pH 4.0, 4.5, 4.8), LB-lactate (pH 4.5 and 4.8) and LB-NaCl (5%, 7%) were fitted to modified Gompertz equations to enable quantitative comparisons across strains and media conditions. Serogroup O45 strains had growth rates that were equal to or significantly greater than the O157:H7 control strain in all growth conditions tested. The growth rate was independent from the maximum growth achieved, but three strains (103A, 121A and 45B) had significantly faster growth and greater maximum cell densities in LB-NaCl 5% (strain 103A), LB-HCl pH 4·0 (strain 121A) and LB-NaCl 7% (strain 45B). Survival in LB-HCl pH 3.0 of four strains (103C, 111B, 26B and 26C) was significantly greater and five strains (26A, 45A, 111A, 121A and 145A) were significantly reduced in comparison with the O157:H7 control strain. None of the STEC strains had greater survival in LB-NaCl 12% than the O157:H7 control strain. A significant association was found between the exponential phase, but not stationary phase, RpoS level and survival of STEC. Some STEC strains had growth or survival properties that exceeded those of the O157:H7 control strain, but none of the non-O157 STEC had both significantly greater growth and survival properties. STEC survival was associated with the exponential-phase RpoS level. Results from this study define the variability in growth and survival of STEC strains that will be useful defining food product formulations and process interventions to control STEC. The presence of exponential phase σ(s) expands the significance of this alternative sigma factor.

Cellular Variability of RpoS Expression Underlies Subpopulation Activation of an Integrative and Conjugative Element

Conjugative transfer of the integrative and conjugative element ICEclc in the bacterium Pseudomonas knackmussii is the consequence of a bistable decision taken in some 3% of cells in a population during stationary phase. Here we study the possible control exerted by the stationary phase sigma factor RpoS on the bistability decision. The gene for RpoS in P. knackmussii B13 was characterized, and a loss-of-function mutant was produced and complemented. We found that, in absence of RpoS, ICEclc transfer rates and activation of two key ICEclc promoters (Pint and PinR) decrease significantly in cells during stationary phase. Microarray and gene reporter analysis indicated that the most direct effect of RpoS is on PinR, whereas one of the gene products from the PinR-controlled operon (InrR) transmits activation to Pint and other ICEclc core genes. Addition of a second rpoS copy under control of its native promoter resulted in an increase of the proportion of cells expressing the Pint and PinR promoters to 18%. Strains in which rpoS was replaced by an rpoS-mcherry fusion showed high mCherry fluorescence of individual cells that had activated Pint and PinR, whereas a double-copy rpoS-mcherry–containing strain displayed twice as much mCherry fluorescence. This suggested that high RpoS levels are a prerequisite for an individual cell to activate PinR and thus ICEclc transfer. Double promoter–reporter fusions confirmed that expression of PinR is dominated by extrinsic noise, such as being the result of cellular variability in RpoS. In contrast, expression from Pint is dominated by intrinsic noise, indicating it is specific to the ICEclc transmission cascade. Our results demonstrate how stochastic noise levels of global transcription factors can be transduced to a precise signaling cascade in a subpopulation of cells leading to ICE activation.

Physiological adaptation of Escherichia coli after transfer onto refrigerated ground meat and other solid matrices: A molecular approach

Bacteria on meat are subjected to specific living conditions that differ drastically from typical laboratory procedures in synthetic media. This study was undertaken to determine the behavior of bacteria when transferred from a rich-liquid medium to solid matrices, as is the case during microbial process validation. Escherichia coli cultured in Brain–Heart Infusion (BHI) broth to different growth phases were inoculated in ground beef (GB) and stored at 5°C for 12 days or spread onto BHI agar and cooked meat medium (CMM), and incubated at 37°C for several hours. We monitored cell densities and the expression of σ factors and genes under their control over time. The initial growth phase of the inoculum influenced growth resumption after transfer onto BHI agar and CMM. Whatever the solid matrix, bacteria adapted to their new environment and did not perceive stress immediately after inoculation. During this period, the σE and σH regulons were not activated and rpoD mRNA levels adjusted quickly. The rpoS and gadA mRNA levels did not increase after inoculation on solid surfaces and displayed normal growth-dependent modifications. After transfer onto GB, dnaK and groEL gene expression was affected more by the low temperature than by the composition of a meat environment.

General stress sigma factor RpoS influences time required to enter the viable but non‐culturable state in Salmonella enterica

In stressful conditions, bacteria enter into the viable but non-culturable (VBNC) state; in this state, they are alive but fail to grow on conventional media on which they normally grow and develop into colonies. The molecular basis underlying this state is unknown. We investigated the role of the alternative sigma factor RpoS (σ(38)) in the VBNC induction using Salmonella Dublin, Salmonella Oranienburg and Salmonella Typhimurium LT2. VBNC was induced by osmotic stress in LT2 and Oranienburg. Dublin also entered the VBNC state, but more slowly than LT2 and Oranienburg did. The LT2 rpoS gene was initiated from an alternative initiation codon, TTG; therefore, LT2 had smaller amounts of RpoS than Dublin and Oranienburg. Oranienburg had a single amino acid substitution (D118N) in RpoS (RpoS(SO)). Disruption of rpoS caused rapid VBNC induction. VBNC induction was significantly delayed by Dublin-type RpoS (RpoS(SD)), but only slightly by RpoS(SO). These results indicate that RpoS delays VBNC induction and that the rapid induction of VBNC in LT2 and Oranienburg may be due to lower levels of RpoS and to the D118N amino acid substitution, respectively. Reduced RpoS intracellular level was observed during VBNC induction. During the VBNC induction, Salmonella might regulate RpoS which is important for maintenance of culturablity under stresses.

The stationary phase sigma factor, RpoS, regulates the production of a carbapenem antibiotic, a bioactive prodigiosin and virulence in the enterobacterial pathogen Serratia sp. ATCC 39006

Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium that is virulent in plant (potato) and invertebrate animal (Caenorhabditis elegans) models. It produces two secondary metabolite antibiotics, a prodigiosin and a carbapenem, and the exoenzymes pectate lyase and cellulase. We showed previously that deletion of the RNA chaperone Hfq abolished antibiotic production and attenuated virulence in both animal and plant hosts. Hfq and dependent small RNAs (sRNAs) are known to regulate the post-transcriptional expression of rpoS, which encodes σ(S), the stationary phase sigma factor subunit of RNA polymerase. An S39006 hfq deletion mutant showed decreased transcript levels of rpoS. Therefore, in this study we investigated whether the phenotypes regulated by Hfq were mediated through its control of rpoS. Whereas loss of Hfq abolished prodigiosin and carbapenem production and attenuated virulence in both C. elegans and potato, characterization of an S39006 rpoS mutant showed unexpectedly elevated prodigiosin and carbapenem production. Furthermore, the rpoS mutant exhibited attenuated animal pathogenesis, but not plant pathogenesis. Additionally, a homologue of the Hfq-dependent sRNA, RprA, was identified and shown to regulate prodigiosin production in a manner consistent with its role in positively regulating translation of rpoS mRNA. Combined, these results demonstrate that Hfq regulation of secondary metabolism and plant pathogenesis is independent of RpoS and establishes RpoS and RprA as regulators of antibiotic production.

Characterization of Edwardsiella tarda rpoN: roles in σ70 family regulation, growth, stress adaption and virulence toward fish

Edwardsiella tarda EIB202, a Gram-negative pathogen with strong virulence, is an opportunistic pathogen capable of causing edwardsiellosis with high mortality to fish. Alternative sigma factor 54 (RpoN) is an important regulator of virulence and stress resistance genes in many bacterial species and mainly responsible for transcription of genes in nitrogen utilization. In this study, the in-frame rpoN deletion mutant was constructed to analyze the function of RpoN in Edwardsiella tarda firstly. Compared to the wild-type and complemented strain rpoN (+), the ΔrpoN was impaired in terms of the ability to survive under oxidative stress, osmotic stress and acid resistance, as well as the growth in Luria-Bertani medium, demonstrating essential roles of RpoN in stress resistance and nitrogen utilization. In addition, the ΔrpoN displayed markedly decreased biofilm formation and chondroitinase activity and was attenuated in virulence reflected in the increased median lethal dose value and extended infection cycle. Real-time polymerase chain reaction revealed that the expression levels of σ(70) class changed in varying degrees in the rpoN mutant. Especially, the expression levels of rpoS and fliA were down-regulated 4.1-fold and 7.9-fold in stationary phase in comparison with the wild type, respectively. Furthermore, two differential expression genes, znuA and flhC, were detected in the wild type and ΔrpoN using the method of differential display reverse transcription PCR.

Antitoxin DinJ influences the general stress response through transcript stabilizer CspE

Antitoxins are becoming recognized as proteins that regulate more than their own synthesis; for example, we found previously that antitoxin MqsA of the Escherichia coli toxin/antitoxin (TA) pair MqsR/MqsA directly represses the gene encoding the stationary-phase sigma factor RpoS. Here, we investigated the physiological role of antitoxin DinJ of the YafQ/DinJ TA pair and found DinJ also affects the general stress response by decreasing RpoS levels. Corroborating the reduced RpoS levels upon producing DinJ, the RpoS-regulated phenotypes of catalase activity, cell adhesins and cyclic diguanylate decreased while swimming increased. Using a transcriptome search and DNA-binding assays, we determined that the mechanism by which DinJ reduces RpoS is by repressing cspE at the LexA palindrome; cold-shock protein CspE enhances translation of rpoS mRNA. Inactivation of CspE abolishes the ability of DinJ to influence RpoS. Hence, DinJ influences the general stress response indirectly by regulating cspE.

Oligopeptide Permease A5 Modulates Vertebrate Host-Specific Adaptation of Borrelia burgdorferi

Borrelia burgdorferi, the agent of Lyme disease, undergoes rapid adaptive gene expression in response to signals unique to its arthropod vector or vertebrate hosts. Among the upregulated genes under vertebrate host conditions is one of the five annotated homologs of oligopeptide permease A (OppA5, BBA34). A mutant lacking oppA5 was constructed in an lp25-deficient isolate of B. burgdorferi strain B31, and the minimal regions of infectivity were restored via a shuttle vector pBBE22 with or without an intact copy of bba34. Immunoblot analysis of the bba34 mutant revealed a reduction in the levels of RpoS, BosR, and CsrA(Bb) with a concomitant reduction in the levels of OspC, DbpA, BBK32, and BBA64. There were no changes in the levels of OspA, NapA, P66, and three other OppA orthologs. Quantitative transcriptional analysis correlated with the changes in the protein levels. However, the bba34 mutant displayed comparable infectivities in the C3H/HeN mice and the wild-type strain, despite the reduction in several pathogenesis-related proteins. Supplementation of the growth medium with increased levels of select components, notably sodium acetate and sodium bicarbonate, restored the levels of several proteins in the bba34 mutant to wild-type levels. We speculate that the transport of acetate appears to contribute to the accumulation of key metabolites, like acetyl phosphate, that facilitate the adaptation of B. burgdorferi to the vertebrate host by the activation of the Rrp2-RpoN-RpoS pathway. These studies underscore the importance of solute transport to host-specific adaptation of B. burgdorferi.

The constancy of global regulation across a species: the concentrations of ppGpp and RpoS are strain-specific in Escherichia coli

BackgroundSigma factors and the alarmone ppGpp control the allocation of RNA polymerase to promoters under stressful conditions. Both ppGpp and the sigma factor σS (RpoS) are potentially subject to variability across the species Escherichia coli. To find out the extent of strain variation we measured the level of RpoS and ppGpp using 31 E. coli strains from the ECOR collection and one reference K-12 strain.ResultsNine ECORs had highly deleterious mutations in rpoS, 12 had RpoS protein up to 7-fold above that of the reference strain MG1655 and the remainder had comparable or lower levels. Strain variation was also evident in ppGpp accumulation under carbon starvation and spoT mutations were present in several low-ppGpp strains. Three relationships between RpoS and ppGpp levels were found: isolates with zero RpoS but various ppGpp levels, strains where RpoS levels were proportional to ppGpp and a third unexpected class in which RpoS was present but not proportional to ppGpp concentration. High-RpoS and high-ppGpp strains accumulated rpoS mutations under nutrient limitation, providing a source of polymorphisms.ConclusionsThe ppGpp and σS variance means that the expression of genes involved in translation, stress and other traits affected by ppGpp and/or RpoS are likely to be strain-specific and suggest that influential components of regulatory networks are frequently reset by microevolution. Different strains of E. coli have different relationships between ppGpp and RpoS levels and only some exhibit a proportionality between increasing ppGpp and RpoS levels as demonstrated for E. coli K-12.

ProQ Is an RNA Chaperone that Controls ProP Levels in Escherichia coli

Transporter ProP mediates osmolyte accumulation in Escherichia coli cells exposed to high osmolality media. The cytoplasmic ProQ protein amplifies ProP activity by an unknown mechanism. The N- and C-terminal domains of ProQ are predicted to be structurally similar to known RNA chaperone proteins FinO and Hfq from E. coli. Here we demonstrate that ProQ is an RNA chaperone, binding RNA and facilitating both RNA strand exchange and RNA duplexing. Experiments performed with the isolated ProQ domains showed that the FinO-like domain serves as a high-affinity RNA-binding domain, whereas the Hfq-like domain is largely responsible for RNA strand exchange and duplexing. These data suggest that ProQ may regulate ProP production. Transcription of proP proceeds from RpoD- and RpoS-dependent promoters. Lesions at proQ affected ProP levels in an osmolality- and growth phase-dependent manner, decreasing ProP levels when proP was expressed from its own chromosomal promoters or from a heterologous plasmid-based promoter. Small RNA molecules are known to regulate cellular levels of sigma factor RpoS. ProQ did not act by changing RpoS levels since proQ lesions did not influence RpoS-dependent stationary phase thermotolerance and they affected ProP production and activity similarly in bacteria without and with an rpoS defect. Taken together, these results suggest that ProQ does not regulate proP transcription. It may act as an RNA-binding protein to regulate proP translation.

The uncertain consequences of transferring bacterial strains between laboratories - rpoS instability as an example

BackgroundMicrobiological studies frequently involve exchanges of strains between laboratories and/or stock centers. The integrity of exchanged strains is vital for archival reasons and to ensure reproducible experimental results. For at least 50 years, one of the most common means of shipping bacteria was by inoculating bacterial samples in agar stabs. Long-term cultures in stabs exhibit genetic instabilities and one common instability is in rpoS. The sigma factor RpoS accumulates in response to several stresses and in the stationary phase. One consequence of RpoS accumulation is the competition with the vegetative sigma factor σ70. Under nutrient limiting conditions mutations in rpoS or in genes that regulate its expression tend to accumulate. Here, we investigate whether short-term storage and mailing of cultures in stabs results in genetic heterogeneity.ResultsWe found that samples of the E. coli K-12 strain MC4100TF exchanged on three separate occasions by mail between our laboratories became heterogeneous. Reconstruction studies indicated that LB-stabs exhibited mutations previously found in GASP studies in stationary phase LB broth. At least 40% of reconstructed stocks and an equivalent proportion of actually mailed stock contained these mutations. Mutants with low RpoS levels emerged within 7 days of incubation in the stabs. Sequence analysis of ten of these segregants revealed that they harboured each of three different rpoS mutations. These mutants displayed the classical phenotypes of bacteria lacking rpoS. The genetic stability of MC4100TF was also tested in filter disks embedded in glycerol. Under these conditions, GASP mutants emerge only after a 3-week period. We also confirm that the intrinsic high RpoS level in MC4100TF is mainly due to the presence of an IS1 insertion in rssB.ConclusionsGiven that many E. coli strains contain high RpoS levels similar to MC4100TF, the integrity of such strains during transfers and storage is questionable. Variations in important collections may be due to storage-transfer related issues. These results raise important questions on the integrity of bacterial archives and transferred strains, explain variation like in the ECOR collection between laboratories and indicate a need for the development of better methods of strain transfer.

Inactivation of bb0184 , Which Encodes Carbon Storage Regulator A, Represses the Infectivity of Borrelia burgdorferi

The genome of Borrelia burgdorferi, the Lyme disease spirochete, encodes a homolog (the bb0184 gene product) of the carbon storage regulator A protein (CsrA(Bb)); recent studies reported that CsrA(Bb) is involved in the regulation of several infectivity factors of B. burgdorferi. However, the mechanism involved remains unknown. In this report, a csrA(Bb) mutant was constructed and complemented in an infectious B31A3 strain. Subsequent animal studies showed that the mutant failed to establish an infection in mice, highlighting that CsrA(Bb) is required for the infectivity of B. burgdorferi. Western blot analyses revealed that the virulence-associated factors OspC, DbpB, and DbpA were attenuated in the csrA(Bb) mutant. The Rrp2-RpoN-RpoS pathway (σ(54)-σ(S) sigma factor cascade) is a central regulon that governs the expression of ospC, dbpB, and dbpA. Further analyses found that the level of RpoS was significantly decreased in the mutant, while the level of Rrp2 remained unchanged. A recent study reported that the overexpression of BB0589, a phosphate acetyl-transferase (Pta) that converts acetyl-phosphate to acetyl-coenzyme A (CoA), led to the inhibition of RpoS and OspC expression, suggesting that acetyl-phosphate is an activator of Rrp2. Along with this report, we found that CsrA(Bb) binds to the leader sequence of the bb0589 transcript and that the intracellular level of acetyl-CoA in the csrA(Bb) mutant was significantly increased compared to that of the wild type, suggesting that more acetyl-phosphate was being converted to acetyl-CoA in the mutant. Collectively, these results suggest that CsrA(Bb) may influence the infectivity of B. burgdorferi via regulation of acetate metabolism and subsequent activation of the Rrp2-RpoN-RpoS pathway.

Antagonistic regulation of motility and transcriptome expression by RpoN and RpoS in Escherichia coli

Bacteria generally possess multiple σ factors that, based on structural and functional similarity, divide into two families: σ(70) and σ(N) . Many studies have revealed σ factor competition within the σ(70) family, while the competition between σ(N) and σ(70) families has yet to be fully explored. Here we report a global antagonistic effect on gene expression between two alternative σ factors, σ(N) (RpoN) and a σ(70) family protein σ(S) (RpoS). Mutations in rpoS and rpoN were found to inversely affect a number of cellular traits, such as the expression of flagellar genes, σ(N) -controlled growth on poor nitrogen sources, and σ(S) -directed expression of acid phosphatase AppA. Transcriptome analysis reveals that about 60% of genes in the RpoN regulon are under reciprocal RpoS control. Furthermore, loss of RpoN led to increased levels of RpoS, while RpoN levels were unaffected by the rpoS mutation. Expression of the flagellar σ(F) factor (FliA), another σ(70) family protein, is controlled positively by RpoN but negatively by RpoS. This positive control by RpoN is likely mediated through the flagellar regulator FlhDC, whose expression is RpoN-dependent. These findings unveil a complex regulatory interaction among σ(N) , σ(S) and σ(F) , which modulates motility, nitrogen utilization, stress response and many other cellular functions.

Molecular and Evolutionary Bases of Within-Patient Genotypic and Phenotypic Diversity in Escherichia coli Extraintestinal Infections

Although polymicrobial infections, caused by combinations of viruses, bacteria, fungi and parasites, are being recognised with increasing frequency, little is known about the occurrence of within-species diversity in bacterial infections and the molecular and evolutionary bases of this diversity. We used multiple approaches to study the genomic and phenotypic diversity among 226 Escherichia coli isolates from deep and closed visceral infections occurring in 19 patients. We observed genomic variability among isolates from the same site within 11 patients. This diversity was of two types, as patients were infected either by several distinct E. coli clones (4 patients) or by members of a single clone that exhibit micro-heterogeneity (11 patients); both types of diversity were present in 4 patients. A surprisingly wide continuum of antibiotic resistance, outer membrane permeability, growth rate, stress resistance, red dry and rough morphotype characteristics and virulence properties were present within the isolates of single clones in 8 of the 11 patients showing genomic micro-heterogeneity. Many of the observed phenotypic differences within clones affected the trade-off between self-preservation and nutritional competence (SPANC). We showed in 3 patients that this phenotypic variability was associated with distinct levels of RpoS in co-existing isolates. Genome mutational analysis and global proteomic comparisons in isolates from a patient revealed a star-like relationship of changes amongst clonally diverging isolates. A mathematical model demonstrated that multiple genotypes with distinct RpoS levels can co-exist as a result of the SPANC trade-off. In the cases involving infection by a single clone, we present several lines of evidence to suggest diversification during the infectious process rather than an infection by multiple isolates exhibiting a micro-heterogeneity. Our results suggest that bacteria are subject to trade-offs during an infectious process and that the observed diversity resembled results obtained in experimental evolution studies. Whatever the mechanisms leading to diversity, our results have strong medical implications in terms of the need for more extensive isolate testing before deciding on antibiotic therapies.

Influence of rpoS mutations on the response of Salmonella enterica serovar Typhimurium to solar radiation

Salmonella enterica serovar Typhimurium is an important pathogen, and exhibits considerable resistance to the lethal effects of solar radiation. To evaluate the involvement of the RpoS transcription factor in the defense mechanisms of this organism, the sunlight response of a wild type strain (ATCC14028) was compared with that of an rpoS mutant, which exhibited increased sensitivity. Kinetics of cell death was complex in both strains, probably due to the presence of a variety of targets for the radiation. When ultraviolet radiation was excluded from the incident sunlight, lethal effects were abolished independently of the allelic state of rpoS. Reduction of oxygen concentration in the irradiation medium provided moderate protection to ATCC14028, but notably improved survival of the mutant. Similar assays were developed with another S. enterica strain (DA1468), which is a derivative of strain LT2 and produces low levels of RpoS. In this strain the loss of viability reveals the dependence on solar ultraviolet and oxygen concentration found for ATCC14028, but radiation resistance was slightly reduced. Increased sensitivity was observed in an rpoS mutant derived from DA1468, indicating that RpoS functions related to photoprotection are conserved in this strain. In addition, notable differences in the shape of the survival curves obtained for mutants derived from ATCC14028 and DA1468 were found, suggesting that genes beyond RpoS control are relevant in the sunlight response of these mutants.