The Structural Basis for Promoter −35 Element Recognition by the Group IV σ Factors

The control of bacterial transcription initiation depends on a primary σ factor for housekeeping functions, as well as alternative σ factors that control regulons in response to environmental stresses. The largest and most diverse subgroup of alternative σ factors, the group IV extracytoplasmic function σ factors, directs the transcription of genes that regulate a wide variety of responses, including envelope stress and pathogenesis. We determined the 2.3-Å resolution crystal structure of the −35 element recognition domain of a group IV σ factor, Escherichia coli σ(E) (4), bound to its consensus −35 element, GGAACTT. Despite similar function and secondary structure, the primary and group IV σ factors recognize their −35 elements using distinct mechanisms. Conserved sequence elements of the σ(E) −35 element induce a DNA geometry characteristic of AA/TT-tract DNA, including a rigid, straight double-helical axis and a narrow minor groove. For this reason, the highly conserved AA in the middle of the GGAACTT motif is essential for −35 element recognition by σ(E) (4), despite the absence of direct protein–DNA interactions with these DNA bases. These principles of σ(E) (4)/−35 element recognition can be applied to a wide range of other group IV σ factors

Identification of nucleotides critical for activity of the sigma(E)-dependent rpoEp3 promoter in Salmonella enterica serovar Typhimurium

We previously described a two-plasmid system for the identification of promoters recognized by <i>Salmonella enteric</i> serovar Typhimurium (<i>S</i> . Typhimurium) σ<sup>E</sup>. The <i>S</i> . Typhimurium σ<sup>E</sup>-dependent <i>rpoEp3</i> promoter was active in the <i>E. coli</i> two-plasmid system only after arabinose-induced expression of <i>S</i> . Typhimurium <i>rpoE</i> . In the present study, we have exploited this two-plasmid system for the identification of nucleotides critical for activity of the <i>rpoEp3</i> promoter. A library of randomly mutated DNA fragments containing the <i>rpoEp3</i> promoter was cloned upstream of a <i>lacZα</i> reporter gene and screened for activity in the presence of <i>S</i> . Typhimurium σ<sup>E</sup>. The clones exhibiting reduced LacZ activity were sequenced to identify the mutations. The activity of the mutated <i>rpoEp3</i> promoters were studied further using a luciferase-based promoter-probe plasmid. All of the important nucleotides of the <i>rpoEp3</i> promoter (in capital) were located in the −35 (ggAActt) and −10 (TctaA) regions. The critical nucleotides were also the most conserved in known σ<sup>E</sup>-dependent promoters. The study also revealed the importance of the 16-bp spacing between −10 and −35 region, as reducing the spacing to 15-bp greatly reduced activity of the promoter. This method should be generally applicable for the identification of important nucleotides in the cognate promoters of other σ factors

New members of the Escherichia coli sigma(E) regulon identified by a two-plasmid system

A previously established method, based on a two-plasmid system, was used to identify promoters recognized by RNA polymerase containing the extracytoplasmic stress response sigma factor σ<sup>E</sup> in <i>Escherichia coli</i>. In addition to previously identified <i>rpoE</i>-dependent promoters, 11 new promoters potentially directing the expression of 15 genes were identified that were active only after over-expression of <i>rpoE</i>. The promoters were confirmed and transcriptional start points of the promoters were determined by primer extension analysis and S1-nuclease mapping. All the promoters contained sequences similar to the consensus sequence of <i>rpoE</i>-dependent promoters. The new <i>rpoE</i>-dependent promoters governed expression of genes encoding proteins involved in primary metabolism (<i>fusA, tufA, recR</i>), phospholipid and lipopolysaccharide biosynthesis (<i>psd, lpxP</i>), signal transduction (<i>sixA</i>), proposed inner or outer membrane proteins (<i>bacA, sbmA, smpA, yeaY</i>), and proteins with unknown function (<i>ybaB, yaiW, yiiS, yiiT, yfeY</i>)

Transcriptional analysis of the rpoE gene encoding extracytoplasmic stress response sigma factor sigmaE in Salmonella enterica serovar Typhimurium

The <i>rpoE</i> gene of <i>Salmonella enterica</i> serovar Typhimurium (<i>S</i>. Typhimurium), which encodes the extracytoplasmic stress response sigma factor σ<sup>E</sup>, is critically important for the virulence of <i>S</i>. Typhimurium. We analysed expression of <i>rpoE</i> by wild-type and mutant bacteria grown in different conditions by S1-nuclease mapping using RNA, and using in vivo reporter gene fusions. Three promoters, <i>rpoEp1, rpoEp2</i> and <i>rpoEp3</i>, were located upstream of the <i>S</i>. Typhimurium <i>rpoE</i> gene. The promoters were differentially expressed during growth and under several stress conditions including cold shock. Expression from the <i>rpoEp3</i> promoter was absent in an <i>S</i>. Typhimurium <i>rpoE</i> mutant, demonstrating its dependence upon σE. The level of mRNA corresponding to <i>rpoEp3</i> was also higher in a <i>cpxR</i> mutant, indicating a negative regulation of the promoter by the Cpx system. Using this <i>rpoE</i>-dependent promoter, we optimised a two-plasmid system for identification of promoters recognised by <i>S</i>. Typhimurium σE. The <i>rpoEp3</i> promoter was active in the <i>Escherichia coli</i> two-plasmid system and has an identical transcription start point as in <i>S</i>. Typhimurium but only after induction of <i>S</i>. Typhimurium <i>rpoE</i> expression

Small outer-membrane lipoprotein, SmpA, is regulated by oE and has a role in cell envelope integrity and virulence of Salmonella enterica serovar Typhimurium

SmpA is a small outer-membrane lipoprotein that is a component of the essential YaeT outer-membrane protein assembly complex. In Salmonella enterica serovar Typhimurium (S. Typhimurium), expression of the smpA gene was shown to be directed by two promoters, smpAp1 and smpAp2. The more distal promoter, smpAp1, is dependent upon the extracytoplasmic stress response sigma factor sigma(E). An smpA null mutant was constructed in S. Typhimurium SL1344 and was shown to be more sensitive than its wild-type parent to growth at high temperature and in the presence of sodium cholate, SDS plus EDTA, and the hydrophobic antibiotic rifampicin. The lack of SmpA in S. Typhimurium elicits a sigma(E)-dependent stress response. These findings are indicative of altered outer-membrane integrity in the smpA mutant, probably due to a defect in outer-membrane protein biogenesis. SmpA was not important for entry or survival within murine macrophages; however, the S. Typhimurium smpA mutant was attenuated in mice by both the oral and parenteral routes of infection, and SmpA appeared to be most important for the growth of S. Typhimurium at systemic sites

Role of the alternative sigma factors sigmaE and sigmaS in survival of Salmonella enterica serovar Typhimurium during starvation, refrigeration and osmotic shock

The ability of Salmonella enterica serovar Typhimurium to survive environmental stress requires specific, coordinated, responses, which induce resistance to the stress condition. This study investigated the relative contribution of sigmaE and sigmaS, the sigma factors regulating extracytoplasmic and general stress response functions, respectively, to survival at low temperature and also in media of differing osmotic strength, conditions relevant to food preservation. To determine if low-temperature storage is a signal for sigmaE- and sigmaS-mediated survival, the ability of S. Typhimurium rpoE, rpoS and rpoE/rpoS mutants to survive in a saline starvation-survival model at a refrigeration temperature (4.5 degrees C) was examined. Under these conditions, the rpoE mutant was significantly (P<0.05) compromised compared to the parent and to an rpoS mutant. The double mutant in rpoE and rpoS displayed a cumulative defect in survival. In hyperosmotic environments (low aw) containing 6 % NaCl and at refrigeration temperature, both sigma factors were important for maximum survival but sigmaS played the dominant role. Analysis of the metabolic activity of starved populations at 4.5 and 37 degrees C revealed significantly (P<0.001) elevated electron-transport system activity in mutants in rpoE and rpoS, indicating a role for sigmaE- and sigmaS-regulated genes in maintaining energy homeostasis. Together these data demonstrate that sigmaE and sigmaS are important for survival of S. Typhimurium in conditions encountered during food processing and that the relative contribution of sigmaE and sigmaS is critically dependent on the precise nature of the stress

Bacterial community assembly based on functional genes rather than species

The principles underlying the assembly and structure of complex microbial communities are an issue of long-standing concern to the field of microbial ecology. We previously analyzed the community membership of bacterial communities associated with the green macroalga Ulva australis, and proposed a competitive lottery model for colonization of the algal surface in an attempt to explain the surprising lack of similarity in species composition across different algal samples. Here we extend the previous study by investigating the link between community structure and function in these communities, using metagenomic sequence analysis. Despite the high phylogenetic variability in microbial species composition on different U. australis (only 15% similarity between samples), similarity in functional composition was high (70%), and a core of functional genes present across all algal-associated communities was identified that were consistent with the ecology of surface- and host-associated bacteria. These functions were distributed widely across a variety of taxa or phylogenetic groups. This observation of similarity in habitat (niche) use with respect to functional genes, but not species, together with the relative ease with which bacteria share genetic material, suggests that the key level at which to address the assembly and structure of bacterial communities may not be “species” (by means of rRNA taxonomy), but rather the more functional level of genes

Salmonella enterican Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection

HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on sigma(E) (RpoE). S. Typhimurium htrA also possesses a sigma(E)-dependent promoter; however, we found that the absence of sigma(E) had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S. Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was sigma(E) dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor sigma(70). These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E. coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 degrees C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 in vitro or in vivo

Genome-Wide Scan of the Gene Expression Kinetics of Salmonella enterica Serovar Typhi during Hyperosmotic Stress

Salmonella enterica serovar Typhi is a human enteroinvasive pathogen that canovercome the stress caused by the high osmolarity of the human small intestine and causesystemic infection. To investigate the global transcriptional regulations of S. entericaserovar Typhi exposed to a hyperosmotic environment, a genomic oligo-DNA microarraycontaining 4474 Salmonella genes was prepared. A wild strain of S. enterica serovar TyphiGIFU10007 was grown in LB medium containing 50 mM NaCl to simulate a low osmoticenvironment. The hyperosmotic stress was simulated by an osmotic up-shift, whichincreased the concentration of NaCl in the LB from 50 mM to 300 mM. Genome-wide geneexpressions of S. enterica serovar Typhi at 15 min, 30 min, 60 min, and 120 min after theosmotic up-shift were investigated by the microarray analysis. Gene expression profiles insomewhat later stage (60 ~120 min) of the stress were quite different from those in the earlystage (0 ~ 30 min) of the stress. At 120 min after the osmotic stress, the expression levels of889 genes were obviously changed. However, expression levels of only 382 genes weresignificantly changed at 15 min after the osmotic stress. The expression levels of most SPI-1genes associated with invasion of the pathogen were increased at 120 min after the osmoticup-shift, but were not obviously changed at 15 min or 30 min after the osmotic stress.Expressions of a central regulatory gene, phoP, and sigma factor genes rpoE, rpoD, andrpoS were also changed with different profiles during the osmotic stress. These resultsindicated that the invasive ability of the pathogen is significantly increased after 2 h of hyperosmotic stress, and regulator PhoP and sigma factors RpoE, RpoD appear to participate in the network regulatory mechanisms that benefit the pathogen to adapt hyperosmotic environmental conditions. The later increased invasive ability of S. enterica serovar Typhi after hyperosmotic stress may be one reason why the pathogen performs invading in the distal ileum of human and not in areas of the upper small intestine