The LeuO regulator and quiescence: About transcriptional roadblocks, multiple promoters, and crispr-cas.

LeuO is a LysR-type transcriptional regulator in bacteria. It determines the regulation of numerous genes related to stress response and virulence. Thus, four exciting areas of research are discussed herein. One pertains the leuO gene, which in S. Typhi and in E. coli contains multiple forward promoters as well as reverse promoters, even though it is expressed at very low levels, that is, it is quiescent. Such multiplicity might allow for a greater plasticity in regulation, or even aid in maintaining the quiescence, in processes that appear to involve many nucleoid-associated proteins in a second area of opportunity. A third one relates to the effector-binding domain of the LeuO regulator, which is highly conserved in S. enterica and in E. coli and determines its activity as a regulator of transcription. A fourth area regards the role of the CRISPR-Cas system in gene regulation in S. Typhi; a system that is regulated by LeuO.

A multi-drug resistant Salmonella Typhimurium ST213 human-invasive strain (33676) containing the bla CMY-2 gene on an IncF plasmid is attenuated for virulence in BALB/c mice.

BACKGROUND: Classical strains of Salmonella enterica serovar Typhimurium (Typhimurium) predominantly cause a self-limiting diarrheal illness in humans and a systemic disease in mice. In this study, we report the characterization of a strain isolated from a blood-culture taken from a 15-year old woman suffering from invasive severe salmonellosis, refractory to conventional therapy with extended-spectrum cephalosporin (ESC). RESULTS: The strain, named 33676, was characterized as multidrug-resistant Salmonella serogroup A by biochemical, antimicrobial and serological tests. Multilocus sequence typing (MLST) and XbaI macrorestrictions (PFGE) showed that strain 33676 belonged to the Typhimurium ST213 genotype, previously described for other Mexican Typhimurium strains. PCR analyses revealed the presence of IncA/C, IncFIIA and ColE1-like plasmids and the absence of the Salmonella virulence plasmid (pSTV). Conjugation assays showed that the ESC-resistance gene bla CMY-2 was carried on the conjugative IncF plasmid, instead of the IncA/C plasmid, as found in previously studied ST213 strains. Although the IncA/C plasmid conferred most of the observed antimicrobial resistances it was not self-conjugative; it was rather able to conjugate by co-integrating with the IncF plasmid. Strain 33676 was fully attenuated for virulence in BALB/c mice infections. Both type-three secretion system (T3SS), encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), were functional in the 33676 strain and, interestingly, this strain produced the H2 FljB flagellin instead of the H1 FliC flagellin commonly expressed by S. enterica strains. CONCLUSIONS: Strain 33676 showed two main features that differentiate it from the originally described ST213 strains: 1) the bla CMY-2 gene was not carried on the IncA/C plasmid, but on a conjugative IncF plasmid, which may open a new route of dissemination for this ESC-resistance gene, and 2) it expresses the H2 FljB flagella, in contrast with the other ST213 and most Typhimurium reference strains. To our knowledge this is the first report of an IncF bla CMY-2-carrying plasmid in Salmonella.

HilD induces expression of Salmonella pathogenicity island 2 genes by displacing the global negative regulator H-NS from ssrAB.

Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) have essential roles in the pathogenesis of Salmonella enterica. Previously, we reported transcriptional cross talk between SPI-1 and SPI-2 when the SPI-1 regulator HilD induces expression of the SsrA/B two-component system, the central positive regulator of SPI-2, during the growth of Salmonella to late stationary phase in LB rich medium. Here, we further define the mechanism of the HilD-mediated expression of ssrAB. Expression analysis of cat transcriptional fusions containing different regions of ssrAB revealed the presence of negative regulatory sequences located downstream of the ssrAB promoter. In the absence of these negative cis elements, ssrAB was expressed in a HilD-independent manner and was no longer repressed by the global regulator H-NS. Consistently, when the activity of H-NS was inactivated, the expression of ssrAB also became independent of HilD. Furthermore, electrophoretic mobility shift assays showed that both HilD and H-NS bind to the ssrAB region containing the repressing sequences. Moreover, HilD was able to displace H-NS bound to this region, whereas H-NS did not displace HilD. Our results support a model indicating that HilD displaces H-NS from a region downstream of the promoter of ssrAB by binding to sites overlapping or close to those sites bound by H-NS, which leads to the expression of ssrAB. Although the role of HilD as an antagonist of H-NS has been reported before for other genes, this is the first study showing that HilD is able to effectively displace H-NS from the promoter of one of its target genes.

In silico identification and experimental characterization of regulatory elements controlling the expression of the Salmonella csrB and csrC genes.

The small RNAs CsrB and CsrC of Salmonella indirectly control the expression of numerous genes encoding widespread cellular functions, including virulence. The expression of csrB and csrC genes, which are located in different chromosomal regions, is coordinated by positive transcriptional control mediated by the two-component regulatory system BarA/SirA. Here, we identified by computational analysis an 18-bp inverted repeat (IR) sequence located far upstream from the promoter of Salmonella enterica serovar Typhimurium csrB and csrC genes. Deletion analysis and site-directed mutagenesis of the csrB and csrC regulatory regions revealed that this IR sequence is required for transcriptional activation of both genes. Protein-DNA and protein-protein interaction assays showed that the response regulator SirA specifically binds to the IR sequence and provide evidence that SirA acts as a dimer. Interestingly, whereas the IR sequence was essential for the SirA-mediated expression of csrB, our results revealed that SirA controls the expression of csrC not only by binding to the IR sequence but also by an indirect mode involving the Csr system. Additional computational, biochemical, and genetic analyses demonstrated that the integration host factor (IHF) global regulator positively controls the expression of csrB, but not of csrC, by interacting with a sequence located between the promoter and the SirA-binding site. These findings contribute to the better understanding of the regulatory mechanism controlling the expression of CsrB and CsrC.

OmpR phosphorylation regulates ompS1 expression by differentially controlling the use of promoters.

The Salmonella enterica ompS1 gene encodes a quiescent porin that belongs to the OmpC/OmpF family. In the present work we analysed the regulatory effects of OmpR phosphorylation on ompS1 expression. We found that in vivo, OmpR in its phosphorylated form (OmpR-P) was important in the regulation of the two ompS1 promoters: OmpR-P activated the P1 promoter and repressed the P2 promoter in an EnvZ-dependent manner; expression occurs from the P2 promoter in an ompR mutant. In vitro, OmpR-P had a higher DNA-binding-affinity to the ompS1 promoter region than OmpR and OmpRD55A, showing an affinity even higher than that of equivalent DNA regions in the 5'-upstream regulatory sequence of the major porin-encoding genes ompC and ompF. By analysing different environmental conditions, we found that glucose and glycerol enhanced ompS1 expression in the wild-type strain. Interestingly the stimulation by glycerol was OmpR-dependent while the effect of glucose was still observed in the absence of OmpR. Acetyl phosphate produced by the AckA-Pta pathway did not influence ompS1 regulation. These data indicate the important role of the phosphorylation in the activity of OmpR on the differential regulation of both ompS1 promoters and its impact on the pathogenesis.

Salmonella Typhi OmpS1 and OmpS2 porins are potent protective immunogens with adjuvant properties.

Salmonella enterica serovar Typhi (S. Typhi) is the causal agent of typhoid fever, a disease that primarily affects developing countries. Various antigens from this bacterium have been reported to be targets of the immune response. Recently, the S. Typhi genome has been shown to encode two porins--OmpS1 and OmpS2--which are expressed at low levels under in vitro culture conditions. In this study, we demonstrate that immunizing mice with either OmpS1 or OmpS2 induced production of specific, long-term antibody titres and conferred protection against S. Typhi challenge; in particular, OmpS1 was more immunogenic and conferred greater protective effects than OmpS2. We also found that OmpS1 is a Toll-like receptor 4 (TLR4) agonist, whereas OmpS2 is a TLR2 and TLR4 agonist. Both porins induced the production of tumour necrosis factor and interleukin-6, and OmpS2 was also able to induce interleukin-10 production. Furthermore, OmpS1 induced the over-expression of MHC II molecules in dendritic cells and OmpS2 induced the over-expression of CD40 molecules in macrophages and dendritic cells. Co-immunization of OmpS1 or OmpS2 with ovalbumin (OVA) increased anti-OVA antibody titres, the duration and isotype diversity of the OVA-specific antibody response, and the proliferation of T lymphocytes. These porins also had adjuvant effects on the antibody response when co-immunized with either the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus [A(H1N1)pdm09]. Taken together, the data indicate that OmpS1 and OmpS2, despite being expressed at low levels under in vitro culture conditions, are potent protective immunogens with intrinsic adjuvant properties.

Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid.

BACKGROUND: Our observation that in the Mexican Salmonella Typhimurium population none of the ST19 and ST213 strains harbored both the Salmonella virulence plasmid (pSTV) and the prevalent IncA/C plasmid (pA/C) led us to hypothesize that restriction to horizontal transfer of these plasmids existed. We designed a conjugation scheme using ST213 strain YU39 as donor of the blaCMY-2 gene (conferring resistance to ceftriaxone; CRO) carried by pA/C, and two E. coli lab strains (DH5α and HB101) and two Typhimurium ST19 strains (SO1 and LT2) carrying pSTV as recipients. The aim of this study was to determine if the genetic background of the different recipient strains affected the transfer frequencies of pA/C. RESULTS: YU39 was able to transfer CRO resistance, via a novel conjugative mechanism, to all the recipient strains although at low frequencies (10-7 to 10-10). The presence of pSTV in the recipients had little effect on the conjugation frequency. The analysis of the transconjugants showed that three different phenomena were occurring associated to the transfer of blaCMY-2: 1) the co-integration of pA/C and pX1; 2) the transposition of the CMY region from pA/C to pX1; or 3) the rearrangement of pA/C. In addition, the co-lateral mobilization of a small (5 kb) ColE1-like plasmid was observed. The transconjugant plasmids involving pX1 re-arrangements (either via co-integration or ISEcp1-mediated transposition) obtained the capacity to conjugate at very high levels, similar to those found for pX1 (10-1). Two versions of the region containing blaCMY-2 were found to transpose to pX1: the large version was inserted into an intergenic region located where the "genetic load" operons are frequently inserted into pX1, while the short version was inserted into the stbDE operon involved in plasmid addiction system. This is the first study to report the acquisition of an extended spectrum cephalosporin (ESC)-resistance gene by an IncX1 plasmid. CONCLUSIONS: We showed that the transfer of the YU39 blaCMY-2 gene harbored on a non- conjugative pA/C requires the machinery of a highly conjugative pX1 plasmid. Our experiments demonstrate the complex interactions a single strain can exploit to contend with the challenge of horizontal transfer and antibiotic selective pressure.

Salmonella Typhimurium ST213 is associated with two types of IncA/C plasmids carrying multiple resistance determinants.

BACKGROUND: Salmonella Typhimurium ST213 was first detected in the Mexican Typhimurium population in 2001. It is associated with a multi-drug resistance phenotype and a plasmid-borne blaCMY-2 gene conferring resistance to extended-spectrum cephalosporins. The objective of the current study was to examine the association between the ST213 genotype and blaCMY-2 plasmids. RESULTS: The blaCMY-2 gene was carried by an IncA/C plasmid. ST213 strains lacking the blaCMY-2 gene carried a different IncA/C plasmid. PCR analysis of seven DNA regions distributed throughout the plasmids showed that these IncA/C plasmids were related, but the presence and absence of DNA stretches produced two divergent types I and II. A class 1 integron (dfrA12, orfF and aadA2) was detected in most of the type I plasmids. Type I contained all the plasmids carrying the blaCMY-2 gene and a subset of plasmids lacking blaCMY-2. Type II included all of the remaining blaCMY-2-negative plasmids. A sequence comparison of the seven DNA regions showed that both types were closely related to IncA/C plasmids found in Escherichia, Salmonella, Yersinia, Photobacterium, Vibrio and Aeromonas. Analysis of our Typhimurium strains showed that the region containing the blaCMY-2 gene is inserted between traA and traC as a single copy, like in the E. coli plasmid pAR060302. The floR allele was identical to that of Newport pSN254, suggesting a mosaic pattern of ancestry with plasmids from other Salmonella serovars and E. coli. Only one of the tested strains was able to conjugate the IncA/C plasmid at very low frequencies (10-7 to 10-9). The lack of conjugation ability of our IncA/C plasmids agrees with the clonal dissemination trend suggested by the chromosomal backgrounds and plasmid pattern associations. CONCLUSIONS: The ecological success of the newly emerging Typhimurium ST213 genotype in Mexico may be related to the carriage of IncA/C plasmids. We conclude that types I and II of IncA/C plasmids originated from a common ancestor and that the insertion and deletion of DNA stretches have shaped their evolutionary histories.

The S. Typhi leuO gene contains multiple functional promoters.

The S. Typhi leuO gene, which codes for the LysR-type transcriptional regulator LeuO, contains five forward promoters named P3, P1, P2, P5 and P4, and two reverse promoters, P6 and P7. The activity of the forward promoters was revealed by primer extension using gene reporter fusions in an S. Typhi hns lrp mutant strain. Likewise, the activity of the reverse promoters was revealed in an hns background. Derepression of the transcription of the chromosomal gene was confirmed by RT-PCR in the hns lrp mutant. The leuOP1 transcriptional reporter fusion, which contained only the major P1 promoter, had a lower expression in a relA spoT mutant strain, indicating that the steady-state levels of the (p)ppGpp alarmone positively regulate it. In contrast, the leuOP3, leuOP5P4, leuOP6 and leuOP7 transcriptional fusions were derepressed in the relA spoT background, indicating that the alarmone has a negative effect on their expression. Thus, the search for genetic regulators and environmental cues that would differentially derepress leuO gene expression by antagonizing the action of the H-NS and Lrp nucleoid-associated proteins, or that would fine-tune the expression of the various promoters, will further our understanding of the significance that multiple promoters have in the control of LeuO expression.

Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains.

BACKGROUND: Bacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome. RESULTS: We found a low genetic diversity for both housekeeping and accessory genes. Sequence type 19 (ST19) was supported as the founder genotype of STs 213, 302 and 429. We found a temporal pattern in which the derived ST213 is replacing the founder ST19 in the four geographic regions analyzed and a geographic trend in the number of resistance determinants. The distribution of the accessory genes was not random among chromosomal genotypes. We detected strong associations among the different accessory genes and the multilocus chromosomal genotypes (STs). First, the Salmonella virulence plasmid (pSTV) was found mostly in ST19 isolates. Second, the plasmid-borne betalactamase cmy-2 was found only in ST213 isolates. Third, the most abundant integron, IP-1 (dfrA12, orfF and aadA2), was found only in ST213 isolates. Fourth, the Salmonella genomic island (SGI1) was found mainly in a subgroup of ST19 isolates carrying pSTV. The mapping of accessory genes and multilocus genotypes on the dendrogram derived from macrorestiction fingerprints allowed the establishment of genetic subgroups within the population. CONCLUSION: Despite the low levels of genetic diversity of core and accessory genes, the non-random distribution of the accessory genes across chromosomal backgrounds allowed us to discover genetic subgroups within the population. This study provides information about the importance of the accessory genome in generating genetic variability within a bacterial population.

Population analysis of D6-like plasmid prophage variants associated with specific IncC plasmid types in the emerging Salmonella Typhimurium ST213 genotype.

The Salmonella enterica serovar Typhimurium sequence type 213 (ST213) emerged as a predominant genotype in Mexico. It is characterized by harboring multidrug resistance (MDR) IncC plasmids (previously IncA/C) and the lack of the Salmonella virulence plasmid (pSTV). Here we show that the D6-like plasmid prophage is present in most of the ST213 strains. We used the reported nucleotide sequence of YU39 plasmid (pYU39_89) to design a PCR typing scheme for the D6-like plasmid prophages, and determined the complete nucleotide sequences for the D6-like prophages of three additional ST213 strains (YU07-18, SL26 and SO21). Two prophage variants were described: i) a complete prophage, containing homologous sequences for most of the genetic modules described in P1 and D6 phages, which most likely allow for the lytic and lysogenic lifestyles; and ii) an incomplete prophage, lacking a 15 kb region containing morphogenesis genes, suggesting that it is defective. The tail fiber gene inversion region was the most divergent one between D6 and pYU39_89 genomes, suggesting the production of a distinct set of tail fibers, which could be involved in host range preferences. A glutaminyl-tRNA synthetase gene (glnS), which could be involved in providing host cell increased fitness or plasmid maintenance functions, was found in all D6-like genomes. Population level analysis revealed a biogeographic pattern of distribution of these plasmid-phages and specific associations with variants of MDR IncC plasmids. Statistically significant associations were found between the two prophage variants (p75 or p89), the type of IncC plasmids (I or II) and geographic isolation regions (Sonora, San Luis Potosí, Michoacán and Yucatán). This work integrates results from molecular typing, genomics and epidemiology to provide a broad overview for the evolution of an emergent Salmonella genotype.

IS200 and multilocus sequence typing for the identification of Salmonella enterica serovar Typhi strains from Indonesia.

In this work, IS200 and multi-locus sequence typing (MLST) were used to analyze 19 strains previously serotyped as Salmonella enterica serovar Typhi and isolated in Indonesia (16 strains), Mexico (2 strains), and Switzerland (1 strain). Most of the strains showed the most common Typhi sequence types, ST1 and ST2, and a new Typhi genotype (ST1856) was described. However, one isolate from Mexico and another from Indonesia were of the ST365 and ST426 sequence types, indicating that they belonged to serovars Weltevreden and Aberdeen, respectively. These results were supported by the amplification of IS200 fragments, which rapidly distinguish Typhi from other serovars. Our results demonstrate the utility of IS200 and MLST in the classification of Salmonella strains into serovars. These methods provide information on the clonal relatedness of strains isolated worldwide.

Complete Genome Sequence of a Human-Invasive Salmonella enterica Serovar Typhimurium Strain of the Emerging Sequence Type 213 Harboring a Multidrug Resistance IncA/C Plasmid and a blaCMY-2-Carrying IncF Plasmid.

Salmonella enterica subsp. enterica serovar Typhimurium strain 33676 was isolated in Mexico City, Mexico, from a patient with a systemic infection, and its complete genome sequence was determined using PacBio single-molecule real-time technology. Strain 33676 harbors an IncF plasmid carrying the extended-spectrum cephalosporin gene blaCMY-2 and a multidrug resistance IncA/C plasmid.

The two-component system CpxR/A represses the expression of Salmonella virulence genes by affecting the stability of the transcriptional regulator HilD.

Salmonella enterica can cause intestinal or systemic infections in humans and animals mainly by the presence of pathogenicity islands SPI-1 and SPI-2, containing 39 and 44 genes, respectively. The AraC-like regulator HilD positively controls the expression of the SPI-1 genes, as well as many other Salmonella virulence genes including those located in SPI-2. A previous report indicates that the two-component system CpxR/A regulates the SPI-1 genes: the absence of the sensor kinase CpxA, but not the absence of its cognate response regulator CpxR, reduces their expression. The presence and absence of cell envelope stress activates kinase and phosphatase activities of CpxA, respectively, which in turn controls the level of phosphorylated CpxR (CpxR-P). In this work, we further define the mechanism for the CpxR/A-mediated regulation of SPI-1 genes. The negative effect exerted by the absence of CpxA on the expression of SPI-1 genes was counteracted by the absence of CpxR or by the absence of the two enzymes, AckA and Pta, which render acetyl-phosphate that phosphorylates CpxR. Furthermore, overexpression of the lipoprotein NlpE, which activates CpxA kinase activity on CpxR, or overexpression of CpxR, repressed the expression of SPI-1 genes. Thus, our results provide several lines of evidence strongly supporting that the absence of CpxA leads to the phosphorylation of CpxR via the AckA/Pta enzymes, which represses both the SPI-1 and SPI-2 genes. Additionally, we show that in the absence of the Lon protease, which degrades HilD, the CpxR-P-mediated repression of the SPI-1 genes is mostly lost; moreover, we demonstrate that CpxR-P negatively affects the stability of HilD and thus decreases the expression of HilD-target genes, such as hilD itself and hilA, located in SPI-1. Our data further expand the insight on the different regulatory pathways for gene expression involving CpxR/A and on the complex regulatory network governing virulence in Salmonella.

Complete Genome Sequencing of a Multidrug-Resistant and Human-Invasive Salmonella enterica Serovar Typhimurium Strain of the Emerging Sequence Type 213 Genotype.

Salmonella enterica subsp. enterica serovar Typhimurium strain YU39 was isolated in 2005 in the state of Yucatán, Mexico, from a human systemic infection. The YU39 strain is representative of the multidrug-resistant emergent sequence type 213 (ST213) genotype. The YU39 complete genome is composed of a chromosome and seven plasmids.

IS200 and multilocus sequence typing for the identification of Salmonella enterica serovar Typhi strains from Indonesia

In this work, IS200 and multi-locus sequence typing (MLST) were used to analyze 19 strains previously serotyped as Salmonella enterica serovar Typhi and isolated in Indonesia (16 strains), Mexico (2 strains), and Switzerland (1 strain). Most of the strains showed the most common Typhi sequence types, ST1 and ST2, and a new Typhi genotype (ST1856) was described. However, one isolate from Mexico and another from Indonesia were of the ST365 and ST426 sequence types, indicating that they belonged to serovars Weltevreden and Aberdeen, respectively. These results were supported by the amplification of IS200 fragments, which rapidly distinguish Typhi from other serovars. Our results demonstrate the utility of IS200 and MLST in the classification of Salmonella strains into serovars. These methods provide information on the clonal relatedness of strains isolated worldwide (AU)

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LeuO antagonizes H-NS and StpA-dependent repression in Salmonella enterica ompS1.

The ompS1 gene encodes a quiescent porin in Salmonella enterica. We analysed the effects of H-NS and StpA, a paralogue of H-NS, on ompS1 expression. In an hns single mutant expression was derepressed but did not reach the maximum level. Expression in an stpA single mutant showed the same low repressed level as the wild type. In contrast, in an hns stpA background, OmpS1 became abundant in the outer membrane. The expression of ompS1 was positively regulated by LeuO, a LysR-type quiescent regulator that has been involved in pathogenesis. Upon induction of the cloned leuO gene into the wild type, ompS1 was completely derepressed and the OmpS1 porin was detected in the outer membrane. LeuO activated the P1 promoter in an OmpR-dependent manner and P2 in the absence of OmpR. LeuO bound upstream of the regulatory region of ompS1 overlapping with one nucleation site of H-NS and StpA. Our results are thus consistent with a model where H-NS binds at a nucleation site and LeuO displaces H-NS and StpA.

Salmonella enterica serovar Typhimurium ompS1 and ompS2 mutants are attenuated for virulence in mice.

Salmonella enterica serovar Typhimurium mutants with mutations in the ompS1 and ompS2 genes, which code for quiescent porins, were nevertheless highly attenuated for virulence in a mouse model, indicating a role in pathogenesis. Similarly, a strain with a mutation in the gene coding for LeuO, a positive regulator of ompS2, was also attenuated.

OmpR and LeuO positively regulate the Salmonella enterica serovar Typhi ompS2 porin gene.

The Salmonella enterica serovar Typhi ompS2 gene codes for a 362-amino-acid outer membrane protein that contains motifs common to the porin superfamily. It is expressed at very low levels compared to the major OmpC and OmpF porins, as observed for S. enterica serovar Typhi OmpS1, Escherichia coli OmpN, and Klebsiella pneumoniae OmpK37 quiescent porins. A region of 316 bp, between nucleotides -413 and -97 upstream of the transcriptional start point, is involved in negative regulation, as its removal resulted in a 10-fold increase in ompS2 expression in an S. enterica serovar Typhi wild-type strain. This enhancement in expression was not observed in isogenic mutant strains, which had specific deletions of the regulatory ompB (ompR envZ) operon. Furthermore, ompS2 expression was substantially reduced in the presence of the OmpR D55A mutant, altered in the major phosphorylation site. Upon random mutagenesis, a mutant where the transposon had inserted into the upstream regulatory region of the gene coding for the LeuO regulator, showed an increased level of ompS2 expression. Augmented expression of ompS2 was also obtained upon addition of cloned leuO to the wild-type strain, but not in an ompR isogenic derivative, consistent with the notion that the transposon insertion had increased the cellular levels of LeuO and with the observed dependence on OmpR. Moreover, LeuO and OmpR bound in close proximity, but independently, to the 5' upstream regulatory region. Thus, the OmpR and LeuO regulators positively regulate ompS2.