Two Trk/Ktr/HKT-type potassium transporters, TrkG and TrkH, perform distinct functions in Escherichia coli K-12.

Escherichia coli K-12 possesses two versions of Trk/Ktr/HKT-type potassium ion (K+) transporters, TrkG and TrkH. The current paradigm is that TrkG and TrkH have largely identical characteristics, and little information is available regarding their functional differences. Here, we show using cation uptake experiments with K+ transporter knockout mutants that TrkG and TrkH have distinct ion transport activities and physiological roles. K+-transport by TrkG required Na+, whereas TrkH-mediated K+ uptake was not affected by Na+. An aspartic acid located five residues away from a critical glycine in the third pore-forming region might be involved in regulation of Na+-dependent activation of TrkG. In addition, we found that TrkG but not TrkH had Na+ uptake activity. Our analysis of K+ transport mutants revealed that TrkH supported cell growth more than TrkG; however, TrkG was able to complement loss of TrkH-mediated K+ uptake in E. coli. Furthermore, we determined that transcription of trkG in E. coli was downregulated but not completely silenced by the xenogeneic silencing factor H-NS (histone-like nucleoid structuring protein or heat-stable nucleoid-structuring protein). Taken together, the transport function of TrkG is clearly distinct from that of TrkH, and TrkG seems to have been accepted by E. coli during evolution as a K+ uptake system that coexists with TrkH.

Regulation of constant cell elongation and Sfm pili synthesis in Escherichia coli via two active forms of FimZ orphan response regulator.

Escherichia coli (E. coli) has multiple copies of the chaperone-usher (CU) pili operon in five fimbria groups: CU pili, curli, type IV pili, type III secretion pili, and type IV secretion pili. Commensal E. coli K-12 contains 12 CU pili operons. Among these operons, Sfm is expressed by the sfmACDHF operon. Transcriptome analyses, reporter assays, and chromatin immunoprecipitation PCR analyses reported that FimZ directly binds to and activates the sfmA promoter, transcribing sfmACDHF. In addition, FimZ regularly induces constant cell elongation in E. coli, which is required for F-type ATPase function. The bacterial two-hybrid system showed a specific interaction between FimZ and the α subunit of the cytoplasmic F1 domain of F-type ATPase. Studies performed using mutated FimZs have revealed two active forms, I and II. Active form I is required for constant cell elongation involving amino acid residues K106 and D109. Active form II additionally required D56, a putative phosphorylation site, to activate the sfmA promoter. The chromosomal fimZ was hardly expressed in parent strain but functioned in phoB and phoP double-gene knockout strains. These insights may help to understand bacterial invasion restricted host environments by the sfm γ-type pili.

The hdeD Gene Represses the Expression of Flagellum Biosynthesis via LrhA in Escherichia coli K-12.

Escherichia coli survives under acid stress conditions by the glutamic acid-dependent acid resistance (GAD) system, which enzymatically decreases intracellular protons. We found a linkage between GAD and flagellar systems in E. coli. The hdeD gene, one of the GAD cluster genes, encodes an uncharacterized membrane protein. A reporter assay showed that the hdeD promoter was induced in a GadE-dependent manner when grown in the M9 glycerol medium. Transcriptome analysis revealed that most of the transcripts were from genes involved in flagellum synthesis, and cell motility increased not only in the hdeD-deficient mutant but also in the gadE-deficient mutant. Defects in both the hdeD and gadE increased the intracellular level of FliA, an alternative sigma factor for flagellum synthesis, activated by the master regulator FlhDC. The promoter activity of the lrhA gene, which encodes repressor for the flhDC operon, was found to decrease in both the hdeD- and gadE-deficient mutants. Transmission electron microscopy showed that the number of flagellar filaments on the hdeD-, gadE-, and lrhA-deficient cells increased, and all three mutants showed higher motility than the parent strain. Thus, HdeD in the GAD system activates the lrhA promoter, resulting in a decrease in flagellar filaments in E. coli cells. We speculated that the synthesis of HdeD, stimulated in E. coli exposed to acid stress, could control the flagellum biosynthesis by sensing slight changes in pH at the cytoplasmic membrane. This could help in saving energy through termination of flagellum biosynthesis and improve bacterial survival efficiency within the animal digestive system. IMPORTANCE E. coli cells encounter various environments from the mouth down to the intestines within the host animals. The pH of gastric juice is lower than 2.0, and the bacterial must quickly respond and adapt to the following environmental changes before reaching the intestines. The quick response plays a role in cellular survival in the population, whereas adaptation may contribute to species survival. The GAD and flagellar systems are important for response to low pH in E. coli. Here, we identified the novel inner membrane regulator HdeD, encoding in the GAD cluster, to repress the synthesis of flagella. These insights provide a deeper understanding of how the bacteria enter the animal digestive system, survive, and form colonies in the intestines.

Cytotoxic Mechanism of Excess Polyamines Functions through Translational Repression of Specific Proteins Encoded by Polyamine Modulon.

Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an Escherichia coli strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.] encoded by members of polyamine modulon, which are essential for cell growth and viability. In particular, synthesis of proteins that have unusual locations of the Shine-Dalgarno (SD) sequence in their mRNAs was inhibited. In order to elucidate the molecular mechanism of cytotoxicity by the excessive accumulation of spermidine, the spermidine-dependent structural change of the bulged-out region in the mRNA at the initiation site of the rmf mRNA was examined using NMR analysis. It was suggested that the structure of the mRNA bulged-out region is affected by excess spermidine, so the SD sequence of the rmf mRNA cannot approach initiation codon AUG.

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing.

Regulatory role of CsqR (YihW) in transcription of the genes for catabolism of the anionic sugar sulfoquinovose (SQ) in Escherichia coli K-12.

The binding sites of YihW, an uncharacterized DeoR-family transcription factor (TF) of Escherichia coli K-12, were identified using Genomic SELEX screening at two closely located sites, one inside the spacer between the bidirectional transcription units comprising the yihUTS operon and the yihV gene, and another one upstream of the yihW gene itself. Recently the YihUTS and YihV proteins were identified as catalysing the catabolism of sulfoquinovose (SQ), a hydrolysis product of sulfoquinovosyl diacylglycerol (SQDG) derived from plants and other photosynthetic organisms. Gel shift assay in vitro and reporter assay in vivo indicated that YihW functions as a repressor for all three transcription units. De-repression of the yih operons was found to be under the control of SQ as inducer, but not of lactose, glucose or galactose. Furthermore, a mode of its cooperative DNA binding was suggested for YihW by atomic force microscopy. Hence, as a regulator of the catabolism of SQ, we renamed YihW as CsqR.

Cross-regulation between two common ancestral response regulators, HprR and CusR, in Escherichia coli.

The uncharacterized two-component system YedVW of Escherichia coli is involved in stress response to hydrogen peroxide. To identify the H2O2-sensing role of YedV, a set of single Cys-to-Ala substitution mutants were constructed. One particular mutant with C165A substitution in the membrane domain rendered YedV inactive in H2O2-dependent transcription of its regulatory target hiuH. We then proposed to rename YedVW to HprSR (hydrogen peroxide response sensor/regulator). One unique characteristic of HprR is the overlapping of its recognition sequence with that of the Cu(II)-response two-component system regulator CusR. Towards understanding this unique regulation system, in this study we analysed the interplay between HprR and CusR with respect to transcription of hiuH, a regulatory target of HprR, and cusC, a target of CusR. Under low protein concentrations in vitro and in vivo, two regulators recognize and transcribe both hiuH and cusC promoters, albeit at different efficiency, apparently in a collaborative fashion. This is a new type of transcription regulation of the common target genes in response to different external signals. Upon increase in protein concentrations, however, HprR and CusR compete with each other in transcription of the common targets, thereby exhibiting a competitive interplay.

Transcription factor CecR (YbiH) regulates a set of genes affecting the sensitivity of Escherichia coli against cefoperazone and chloramphenicol.

Genomic SELEX (systematic evolution of ligands by exponential enrichment) screening was performed for identification of the binding site of YbiH, an as yet uncharacterized TetR-family transcription factor, on the Escherichia coli genome. YbiH was found to be a unique single-target regulator that binds in vitro within the intergenic spacer located between the divergently transcribed ybiH-ybhGFSR and rhlE operons. YbhG is an inner membrane protein and YbhFSR forms a membrane-associated ATP-binding cassette (ABC) transporter while RhlE is a ribosome-associated RNA helicase. Gel shift assay and DNase footprinting analyses indicated one clear binding site of YbiH, including a complete palindromic sequence of AATTAGTT-AACTAATT. An in vivo reporter assay indicated repression of the ybiH operon and activation of the rhlE operon by YbiH. After phenotype microarray screening, YbiH was indicated to confer resistance to chloramphenicol and cefazoline (a first-generation cephalosporin). A systematic survey of the participation of each of the predicted YbiH-regulated genes in the antibiotic sensitivity indicated involvement of the YbhFSR ABC-type transporter in the sensitivity to cefoperazone (a third-generation cephalosporin) and of the membrane protein YbhG in the control of sensitivity to chloramphenicol. Taken together with the growth test in the presence of these two antibiotics and in vitro transcription assay, it was concluded that the hitherto uncharacterized YbiH regulates transcription of both the bidirectional transcription units, the ybiH-ybhGFSR operon and the rhlE gene, which altogether are involved in the control of sensitivity to cefoperazone and chloramphenicol. We thus propose to rename YbiH as CecR (regulator of cefoperazone and chloramphenicol sensitivity).

Cross talk in promoter recognition between six NarL-family response regulators of Escherichia coli two-component system.

Bacterial two-component system (TCS) is composed of the sensor kinase (SK) and the response regulator (RR). After monitoring an environmental signal or condition, SK activates RR through phosphorylation, ultimately leading to the signal-dependent regulation of genome transcription. In Escherichia coli, a total of more than 30 SK-RR pairs exist, each forming a cognate signal transduction system. Cross talk of the signal transduction takes place at three stages: signal recognition by SK (stage 1); RR phosphorylation by SK (stage 2); and target recognition by RR (stage 3). Previously, we analyzed the stage 2 cross talk between the whole set of E. coli SK-RR pairs and found that the cross talk takes place for certain combinations. As an initial attempt to identify the stage 3 cross talk at the step of target promoter recognition by RR, we analyzed in this study the cross-recognition of target promoters by six NarL-family RRs, EvgA, NarL, NarP, RcsB, UhpA, and UvrY. Results of both in vivo and in vitro studies indicated that the stage 3 cross talk takes place for limited combinations, in particular, including a multifactor-regulated ydeP promoter.

Expanded roles of two-component response regulator OmpR in Escherichia coli: genomic SELEX search for novel regulation targets.

The two-component system (TCS) is a sophisticated bacterial signal transduction system for regulation of genome transcription in response to environmental conditions. The EnvZ-OmpR system is one of the well-characterized TCS of Escherichia coli, responding to changes in environmental osmolality. Regulation has largely focused on the differential expression of two porins, OmpF and OmpC, which transport small molecules across the outer membrane. Recently, it has become apparent that OmpR serves a more global regulatory role and regulates additional targets. To identify the entire set of regulatory targets of OmpR, we performed the genomic SELEX screening of OmpR-binding sites along the E. coli genome. As a result, more than 30 novel genes have been identified to be under the direct control of OmpR. One abundant group includes the genes encoding a variety of membrane-associated transporters that mediate uptake or efflux of small molecules, while another group encodes a set of transcription regulators, raising a concept that OmpR is poised to control a diverse set of responses by altering downstream transcriptional regulators.

Role for σ38 in prolonged survival of Escherichia coli in Drosophila melanogaster.

Bacteria adapt themselves to host environments by altering the pattern of gene expression. The promoter-recognizing subunit σ of bacterial RNA polymerase plays a major role in the selection of genes to be transcribed. Among seven σ factors of Escherichia coli, σ(38) is responsible for the transcription of genes in the stationary phase and under stressful conditions. We found a transient increase of σ(38) when E. coli was injected into the hemocoel of Drosophila melanogaster. The loss of σ(38) made E. coli rapidly eliminated in flies, and flies infected with σ(38)-lacking E. coli stayed alive longer than those infected with the parental strain. This was also observed in fly lines defective in humoral immune responses, but not in flies in which phagocytosis was impaired. The lack of σ(38) did not influence the susceptibility of E. coli to phagocytosis, but made them vulnerable to killing after engulfment. The changes caused by the loss of σ(38) were recovered by the forced expression of σ(38)-encoding rpoS as well as σ(38)-regulated katE and katG coding for enzymes that detoxify reactive oxygen species. These results collectively suggested that σ(38) contributes to the prolonged survival of E. coli in Drosophila by inducing the production of enzymes that protect bacteria from killing in phagocytes. Considering the similarity in the mechanism of innate immunity against invading bacteria between fruit flies and humans, the products of these genes could be new targets for the development of more effective antibacterial remedies.

Regulatory role of transcription factor SutR (YdcN) in sulfur utilization in Escherichia coli.

Sulfur makes up 1 % of the dry mass of bacteria, and it is an abundant element (0.1 %) on earth. Sulfur in the environment is, however, mostly in oxidized forms and inaccessible to living organisms. At present, the entire assimilation pathway of external sulfur to sulfur-containing biomolecules and its regulation in Escherichia coli remain poorly understood, except for the metabolic pathway of cysteine synthesis, the first-step metabolite of sulfur assembly. During the search for regulation targets of uncharacterized transcription factors by Genomic SELEX screening, we found that the hitherto uncharacterized YdcN regulates a set of genes involved in the utilization of sulfur, including the generation of sulfate and its reduction, the synthesis of cysteine, the synthesis of enzymes containing Fe-S as cofactors, and the modification of tRNA with use of sulfur-containing substrates. Taking these findings together, we propose renaming YdcN as SutR (regulator of sulfur utilization).

Cooperative regulation of the common target genes between H2O2-sensing YedVW and Cu²âº-sensing CusSR in Escherichia coli.

YedVW is one of the uncharacterized two-component systems (TCSs) of Escherichia coli. In order to identify the regulation targets of YedVW, we performed genomic SELEX (systematic evolution of ligands by exponential enrichment) screening using phosphorylated YedW and an E. coli DNA library, and identified YedW-binding sites within three intergenic spacers, yedW-hiuH, cyoA-ampG and cusR-cusC, along the E. coli genome. Using a reporter assay system, we found that transcription of hiuH, encoding 5-hydroxyisourate hydrolase, was induced at high concentrations of either Cu(2+) or H2O2. Cu(2+)-dependent expression of hiuH was observed in the yedWV knockout mutant, but was reduced markedly in the cusRS-null mutant. However, H2O2-induced hiuH expression was observed in the cusRS-null mutant, but not in the yedWV-null mutant. Gel mobility shift and DNase I footprinting analyses showed binding of both YedW and CusR to essentially the same sequence within the hiuH promoter region. Taken together, we concluded that YedVW and CusSR formed a unique cooperative TCS pair by recognizing and regulating the same targets, but under different environmental conditions - YedVW played a role in H2O2 response regulation, whilst CusSR played a role in Cu(2+) response regulation.

Induction of the Escherichia coli yijE gene expression by cystine.

Cystine is formed from two molecules of the cysteine under oxidized conditions, but is reversibly converted to cysteine by reduction. Growth of Escherichia coli is retarded in the presence of excess cystine. Transcriptome analysis showed 11 up-regulated and 26 down-regulated genes upon exposure to excess cystine. The reporter assay confirmed regulation by cystine of the expression of one up-regulated membrane gene, yijE, and two down-regulated membrane genes, yhdT and yihN. In order to identify the as yet unidentified gene encoding cystine efflux transporter, the putative cystine efflux candidate, yijE gene, was over-expressed. Expression of the yijE gene suppressed the slow growth of E. coli in the presence of high concentration of extracellular cystine. In good agreement, the knock-out of yijE gene increased the sensibility to cystine. These observations altogether imply that the yijE gene is involved in response to cystine in E. coli.

Profiling of protein thiophosphorylation by Phos-tag affinity electrophoresis: evaluation of adenosine 5'-O-(3-thiotriphosphate) as a phosphoryl donor in protein kinase reactions.

Adenosine 5'-O-(3-thiotriphosphate) (ATPγS) has been widely used as a phosphoryl donor to trace protein kinase activities. However, the question remains whether particular kinases accept ATPγS as readily as they accept natural ATP. We investigated the characteristics of several kinase reactions in the presence of ATPγS by using Phos-tag affinity electrophoresis. The Phos-tag gel permitted quantitative analysis of thiophosphorylated proteins produced by kinase reactions in vitro and it identified differences in the efficiencies of utilization of ATPγS and ATP in these reactions. Using the method, we evaluated the utility of ATPγS as a phosphoryl donor in studies on bacterial two-component systems. Histidine kinases accepted ATPγS as readily as they accepted ATP in autophosphorylation reactions. However, downstream phosphotransfer reactions with ATPγS were markedly slower than the corresponding reactions with ATP. In an analysis of the sluggish thiophosphate transfer, we found that detergent-denatured thiophosphorylated histidine kinases gradually hydrolyzed at the P-N bond, even at neutral pH, during incubation for 24 h, whereas the native form of the thiophosphorylated enzymes were much more stable. Profiling of protein thiophosphorylation by using Phos-tag affinity electrophoresis might provide new insights into the characteristics of various types of kinase reactions with ATPγS.

Expression levels of transcription factors in Escherichia coli: growth phase- and growth condition-dependent variation of 90 regulators from six families.

The expression pattern of the genome in Escherichia coli is controlled by regulating the utilization of a limited number of RNA polymerases between a total of 4600 genes on its genome. The distribution pattern of RNA polymerase on the genome changes after two steps of protein-protein interaction with seven sigma subunits and about 300 transcription factors (TFs). Based on a systematic search for the regulation target promoters recognized by each TF, we propose two novel concepts: each TF regulates a number of target promoters; and each promoter is regulated by many TFs. In parallel, attempts have been made to determine the intracellular concentrations of all TFs using two systems: quantitative immunoblot analysis using TF-specific antibodies; and reporter assay of TF promoter activities. The direct measurement of TF protein level has so far been published for a set of 60 regulators with known functions. This study describes the determination of growth phase-dependent expression levels of 90 TFs using the reporter assay system. The translational fusion vector was constructed from the TF promoter sequence including an N-terminal proximal TF segment and the reporter GFP. At the beginning of cell growth, high-level expression was observed only for a small number of TFs. In the exponential phase, approximately 80 % TFs are expressed, but the expressed TF species change upon transfer to the stationary phase. Significant changes in the pattern of TF expression were observed between aerobic and anaerobic conditions. The list of intracellular levels of TFs provides further understanding to the transcription regulation of the E. coli genome under various stressful conditions.

The hierarchic network of metal-response transcription factors in Escherichia coli.

Enterobacteria such as Escherichia coli are able to survive under various environments within host animals by changes of the expression pattern of its genome. The selective expression of genes in its genome takes place by controlling the promoter recognition properties of RNA polymerase by protein-protein interplays with transcription factors. In this review, I describe the regulatory network formed by the metal-sensing transcription factors in E. coli. Comprehensive analyses identify the set of regulation targets for a total of 13 metal-response transcription factors, indicating that nine species of transcription factors are local regulators while four species of transcription factors are global regulators. The signal transduction pathways for these metal-response regulons show not only the complex cross-talks but also the hierarchic multi-regulatory network. This regulatory network seems to play a role for E. coli survival to colonize in a large intestine within host animals.

Identification of the set of genes, including nonannotated morA, under the direct control of ModE in Escherichia coli.

ModE is the molybdate-sensing transcription regulator that controls the expression of genes related to molybdate homeostasis in Escherichia coli. ModE is activated by binding molybdate and acts as both an activator and a repressor. By genomic systematic evolution of ligands by exponential enrichment (SELEX) screening and promoter reporter assays, we have identified a total of nine operons, including the hitherto identified modA, moaA, dmsA, and napF operons, of which six were activated by ModE and three were repressed. In addition, two promoters were newly identified and direct transcription of novel genes, referred to as morA and morB, located on antisense strands of yghW and torY, respectively. The morA gene encodes a short peptide, MorA, with an unusual initiation codon. Surprisingly, overexpression of the morA 5' untranslated region exhibited an inhibitory influence on colony formation of E. coli K-12.

Involvement of EnvZ-OmpR two-component system in virulence control of Escherichia coli in Drosophila melanogaster.

Bacteria adapt to environmental changes by altering gene expression patterns with the aid of signal transduction machinery called the two-component regulatory system (TCS), which consists of two protein components, a sensor kinase and response regulator. We examined the role of the TCS in bacterial adaptation to host environments using genetically tractable organisms, Escherichia coli as a pathogen and Drosophila melanogaster as a host. To determine the strength of the transcription promoters of TCS-encoding genes in Drosophila, adult flies were infected with a series of E. coli strains that expressed GFP driven by the promoters of genes coding for 27 sensor kinases and 32 response regulators of E. coli TCS followed by the measurement of fluorescence intensities. We further analyzed EnvZ-OmpR among the TCS encoded by genes having stronger promoters. A mutant E. coli strain lacking EnvZ-OmpR had a higher pathogenic effect on fly survival than that of the parental strain, and the forced expression of envZ and ompR in the mutant strain lowered its pathogenicity. The lack of EnvZ-OmpR did not affect the growth of E. coli in a culture medium as well as the level of colony-formable E. coli in flies. An increase in E. coli virulence with the loss of EnvZ-OmpR was observed in flies defective in an Imd-mediated humoral response, and both the mutant and parental strains were equally engulfed by hemocytes in vitro. These results suggest that EnvZ-OmpR mitigated the virulence of E. coli in Drosophila by a mechanism not accompanied by a change of bacterial burden. This behavior of E. coli is most likely a bacterial strategy to achieve persistent infection.

Screening of promoter-specific transcription factors: multiple regulators for the sdiA gene involved in cell division control and quorum sensing.

Prokaryotic DNA-binding transcription factors (TFs) bind in close vicinity of the promoter and regulate transcription through interplay with the DNA-dependent RNA polymerase. Promoters associated with the genes involved in stress response have recently been found to be under the control of multiple regulators, each monitoring one specific environmental condition or factor. In order to identify TFs involved in regulation of one specific promoter, we have developed a PS-TF (promoter-specific TF) screening system, in which the binding of purified TFs to a test promoter was analysed by gel-shift assay. This PS-TF screening system was applied for detection of TFs involved in regulation of the promoter for the Escherichia coli sdiA gene encoding the master regulator of cell division and quorum sensing. After screening of a total of 191 purified TFs (two-thirds of the predicted E. coli TFs), at least 15 TFs have been identified to bind to the sdiA promoter, including five two-component system (TCS) regulators, ArcA, CpxR, OmpR, RcsB and TorR. In this study, we focus on these five TFs for detailed analysis of their regulatory roles in vivo. Under normal growth conditions in LB medium, all these TFs repressed the sdiA promoter and the repression levels correlated with their intracellular levels. Taken together, we propose that these TCS regulators repress transcription in vivo of the sdiA gene, ultimately leading to suppression of cell division.