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.

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.

The CRISPR-Cas System Is Involved in OmpR Genetic Regulation for Outer Membrane Protein Synthesis in Salmonella Typhi.

The CRISPR-Cas cluster is found in many prokaryotic genomes including those of the Enterobacteriaceae family. Salmonella enterica serovar Typhi (S. Typhi) harbors a Type I-E CRISPR-Cas locus composed of cas3, cse1, cse2, cas7, cas5, cas6e, cas1, cas2, and a CRISPR1 array. In this work, it was determined that, in the absence of cas5 or cas2, the amount of the OmpC porin decreased substantially, whereas in individual cse2, cas6e, cas1, or cas3 null mutants, the OmpF porin was not observed in an electrophoretic profile of outer membrane proteins. Furthermore, the LysR-type transcriptional regulator LeuO was unable to positively regulate the expression of the quiescent OmpS2 porin, in individual S. Typhi cse2, cas5, cas6e, cas1, cas2, and cas3 mutants. Remarkably, the expression of the master porin regulator OmpR was dependent on the Cse2, Cas5, Cas6e, Cas1, Cas2, and Cas3 proteins. Therefore, the data suggest that the CRISPR-Cas system acts hierarchically on OmpR to control the synthesis of outer membrane proteins in S. Typhi.

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.

Salmonella virulence plasmid: pathogenesis and ecology.

A current view on the role of the Salmonella virulence plasmid in the pathogenesis of animal and human hosts is discussed; including the possible relevance in secondary ecological niches. Various strategies towards further studies in this respect are proposed within the One Health Concept.

CRISPR-Cas system presents multiple transcriptional units including antisense RNAs that are expressed in minimal medium and upregulated by pH in Salmonella enterica serovar Typhi.

The CRISPR-Cas system is involved in bacterial immunity, virulence, gene regulation, biofilm formation and sporulation. In Salmonella enterica serovar Typhi, this system consists of five transcriptional units including antisense RNAs. It was determined that these genetic elements are expressed in minimal medium and are up-regulated by pH. In addition, a transcriptional characterization of cas3 and ascse2-1 is included herein.

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Strain SO3 (Sequence Type 302) Isolated from a Baby with Meningitis in Mexico.

The complete genome of ITALIC! Salmonella entericaserovar Typhimurium strain SO3 (sequence type 302), isolated from a fatal meningitis infection in Mexico, was determined using PacBio technology. The chromosome hosts six complete prophages and is predicted to harbor 51 genomic islands, including 13 pathogenicity islands (SPIs). It carries the ITALIC! Salmonellavirulence plasmid (pSTV).

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Strain SO2 (Sequence Type 302) Isolated from an Asymptomatic Child in Mexico.

The complete genome sequence ofSalmonella entericaserovar Typhimurium strain SO2, isolated from an asymptomatic child in Mexico, was determined using PacBio single-molecule real-time technology. Strain SO2 has six complete chromosomal prophages, namely, ST104, Gifsy-2, ST64B, Gifsy-1, ELPhiS, and FSL SP-004, and carries aSalmonellavirulence plasmid.

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.

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.

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.

Deletion analysis of RcsC reveals a novel signalling pathway controlling poly-N-acetylglucosamine synthesis and biofilm formation in Escherichia coli.

RcsC is a hybrid histidine kinase that forms part of a phospho-relay signal transduction pathway with RcsD and RcsB. Besides the typical domains of a sensor kinase, i.e. the periplasmic (P), linker (L), dimerization and H-containing (A), and ATP-binding (B) domains, RcsC possesses a receiver domain (D) at the carboxy-terminal domain. To study the role played by each of the RcsC domains, four plasmids containing several of these domains were constructed (PLAB, LAB, AB and ABD) and transformed into Escherichia coli K-12 strain BW25113. Different amounts of biofilm were produced, depending on the RcsC domains expressed: the plasmid expressing the ABD subdomains produced the highest amount of biofilm. This phenotype was also observed when the plasmids were transformed in a ΔrcsCDB strain. Biofilm formation was abolished in the pgaABCD and nhaR backgrounds. The results indicate the existence of a novel signalling pathway that depends on RcsC, yet independent of RcsD and RcsB, that activates the pgaABCD operon and, as a consequence, biofilm formation. This signalling pathway involves the secondary metabolite acetyl phosphate and the response regulator OmpR.

Participation of the Salmonella OmpD porin in the infection of RAW264.7 macrophages and BALB/c mice.

Salmonella Typhimurium is the etiological agent of gastroenteritis in humans and enteric fever in mice. Inside these hosts, Salmonella must overcome hostile conditions to develop a successful infection, a process in which the levels of porins may be critical. Herein, the role of the Salmonella Typhimurium porin OmpD in the infection process was assessed for adherence, invasion and proliferation in RAW264.7 mouse macrophages and in BALB/c mice. In cultured macrophages, a ΔompD strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain. In contrast, overexpression of ompD caused a reduction in bacterial proliferation but did not affect adherence or invasion. In the murine model, the ΔompD strain showed increased ability to survive and replicate in target organs of infection. The ompD transcript levels showed a down-regulation when Salmonella resided within cultured macrophages and when it colonized target organs in infected mice. Additionally, cultured macrophages infected with the ΔompD strain produced lower levels of reactive oxygen species, suggesting that down-regulation of ompD could favor replication of Salmonella inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host.

The Salmonella enterica serovar Typhi LeuO global regulator forms tetramers: residues involved in oligomerization, DNA binding, and transcriptional regulation.

LeuO is a LysR-type transcriptional regulator (LTTR) that has been described to be a global regulator in Escherichia coli and Salmonella enterica, since it positively and negatively regulates the expression of genes involved in multiple biological processes. LeuO is comprised of an N-terminal DNA-binding domain (DBD) with a winged helix-turn-helix (wHTH) motif and of a long linker helix (LH) involved in dimerization that connects the DBD with the C-terminal effector-binding domain (EBD) or regulatory domain (RD; which comprises subdomains RD-I and RD-II). Here we show that the oligomeric structure of LeuO is a tetramer that binds with high affinity to DNA. A collection of single amino acid substitutions in the LeuO DBD indicated that this region is involved in oligomerization, in positive and negative regulation, as well as in DNA binding. Mutants with point mutations in the central and C-terminal regions of RD-I were affected in transcriptional activation. Deletion of the RD-II and RD-I C-terminal subdomains affected not only oligomerization but also DNA interaction, showing that they are involved in positive and negative regulation. Together, these data demonstrate that not only the C terminus but also the DBD of LeuO is involved in oligomer formation; therefore, each LeuO domain appears to act synergistically to maintain its regulatory functions in Salmonella enterica serovar Typhi.

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.

LeuO, a dormant sentinel for SPI-1?

A new mechanism for the turning-off of gene expression in Salmonella Pathogenicity Island 1 (SPI-1) is proposed by Espinosa and Casadesús, which involves the action of the LeuO quiescent regulator, by two different pathways. A major one through the activation of the hilE gene, where the HilE protein would in turn inactivate HilD, a major positive transcriptional regulator of SPI-1; and a minor HilE-HilD-independent pathway. This could constitute a back-up or an aid for the turn-off of SPI-1 genes mediated by the nucleoid protein H-NS.

The Subtleties and Contrasts of the LeuO Regulator in Salmonella Typhi: Implications in the Immune Response.

Salmonella are facultative intracellular pathogens. Salmonella infection occurs mainly by expression of two Salmonella pathogenicity Islands (SPI-1 and SPI-2). SPI-1 encodes transcriptional factors that participate in the expression of virulence factors encoded in the island. However, there are transcriptional factors encoded outside the island that also participate in the expression of SPI-1-encoded genes. Upon infection, bacteria are capable of avoiding the host immune response with several strategies that involve several virulence factors under the control of transcriptional regulators. Interestingly, LeuO a transcriptional global regulator which is encoded outside of any SPI, is proposed to be part of a complex regulatory network that involves expression of several genes that help bacteria to survive stress conditions and, also, induces the expression of porins that have been shown to be immunogens and can thus be considered as antigenic candidates for acellular vaccines. Hence, the understanding of the LeuO regulon implies a role of bacterial genetic regulation in determining the host immune response.

One Health and Food-Borne Disease: Salmonella Transmission between Humans, Animals, and Plants.

There are >2,600 recognized serovars of Salmonella enterica. Many of these Salmonella serovars have a broad host range and can infect a wide variety of animals, including mammals, birds, reptiles, amphibians, fish, and insects. In addition, Salmonella can grow in plants and can survive in protozoa, soil, and water. Hence, broad-host-range Salmonella can be transmitted via feces from wild animals, farm animals, and pets or by consumption of a wide variety of common foods: poultry, beef, pork, eggs, milk, fruit, vegetables, spices, and nuts. Broad-host-range Salmonella pathogens typically cause gastroenteritis in humans. Some Salmonella serovars have a more restricted host range that is associated with changes in the virulence plasmid pSV, accumulation of pseudogenes, and chromosome rearrangements. These changes in host-restricted Salmonella alter pathogen-host interactions such that host-restricted Salmonella organisms commonly cause systemic infections and are transmitted between host populations by asymptomatic carriers. The secondary consequences of efforts to eliminate host-restricted Salmonella serovars demonstrate that basic ecological principles govern the environmental niches occupied by these pathogens, making it impossible to thwart Salmonella infections without a clear understanding of the human, animal, and environmental reservoirs of these pathogens. Thus, transmission of S. enterica provides a compelling example of the One Health paradigm because reducing human infections will require the reduction of Salmonella in animals and limitation of transmission from the environment.

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.