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Cyclic di-GMP (c-di-GMP), a ubiquitous secondary messenger in bacteria, affects multiple bacterial behaviors including motility and biofilm formation. c-di-GMP is synthesized by diguanylate cyclase harboring a GGDEF domain and degraded by phosphodiesterase harboring an either EAL or HD-GYP domain. Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis, harbors more than 60 genes involved in c-di-GMP metabolism. However, roles of most of these genes including vpa0198, which encodes a GGDEF-domain containing protein, are still completely unknown. AphA and OpaR are the master quorum sensing (QS) regulators operating at low (LCD) and high cell density (HCD), respectively. QsvR integrates into QS to control gene expression via direct regulation of AphA and OpaR. In this study, we showed that deletion of vpa0198 remarkably reduced c-di-GMP production and biofilm formation, whereas promoted the swimming motility of V. parahaemolyticus. Overexpression of VPA0198 in the vpa0198 mutant strain significantly reduced the swimming and swarming motility and enhanced the biofilm formation ability of V. parahaemolyticus. In addition, transcription of vpa0198 was under the collective regulation of AphA, OpaR and QsvR. AphA activated the transcription of vpa0198 at LCD, whereas QsvR and OpaR coordinately and directly repressed vpa0198 transcription at HCD, thereby leading to a cell density-dependent expression of vpa0198. Therefore, this work expanded the knowledge of synthetic regulatory mechanism of c-di-GMP in V. parahaemolyticus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The type VI secretion system 2 (T6SS2) gene cluster of Vibrio parahaemolyticus comprises three operons: VPA1027-1024, VPA1043-1028, and VPA1044-1046. AcsS is a LysR-like transcriptional regulator that play a role in activating flagella-driven motility in V. parahaemolyticus. However, its potential roles in other cellular pathways remain poorly understood. In this study, we conducted a series of experiments to investigate the regulatory effects of AcsS on the transcription of VPA1027 (hcp2), VPA1043, and VPA1044. The findings revealed that AcsS indirectly inhibits the transcription of these genes. Additionally, deletion of acsS resulted in enhanced adhesion of V. parahaemolyticus to HeLa cells. However, disruption of T6SS2 alone or in conjunction with AcsS significantly diminished the adhesion capacity of V. parahaemolyticus to HeLa cells. Therefore, it is suggested that AcsS suppresses cell adhesion in V. parahaemolyticus by downregulating the transcription of T6SS2 genes., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Vibrio parahaemolyticus utilizes a polar flagellum for swimming in liquids and employs multiple lateral flagella to swarm on surfaces and in viscous environments. The VPA0961 protein is an LysR family transcriptional regulator that can regulate the swimming and swarming motility of V. parahaemolyticus, but the detailed regulatory mechanisms are not yet fully understood. Herein, we designated the protein as AcsS, which stands for activator of swimming and swarming motility. Our data provided evidence that deleting the acsS gene significantly reduced both swimming and swarming motility of V. parahaemolyticus. Furthermore, AcsS was found to activate the expression of both polar (flgA, flgM, flgB, and flgK) and lateral (motY, fliM, lafA, and fliD) flagellar genes. Overexpression of AcsS in Escherichia coli induced the expression of flgA, motY, and lafA, but did not affect the expression of flgB, flgK, flgM, fliM, and fliD. Interestingly, His-tagged AcsS did not bind to the upstream DNA regions of all the tested genes, suggesting indirect regulation. In conclusion, AcsS positively regulated the swimming and swarming motility of V. parahaemolyticus by activating the transcription of polar and lateral flagellar genes. This work enriched our understanding of the gene expression regulation within the dual flagellar systems of V. parahaemolyticus., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Motile bacteria navigate toward favorable conditions and away from unfavorable environments using chemotaxis. Mechanisms of sensing attractants are well understood; however, molecular aspects of how bacteria sense repellents have not been established. Here, we identified malate as a repellent recognized by the MCP2201 chemoreceptor in a bacterium Comamonas testosteroni and showed that it binds to the same site as an attractant citrate. Binding determinants for a repellent and an attractant had only minor differences, and a single amino acid substitution in the binding site inverted the response to malate from a repellent to an attractant. We found that malate and citrate affect the oligomerization state of the ligand-binding domain in opposing way. We also observed opposing effects of repellent and attractant binding on the orientation of an alpha helix connecting the sensory domain to the transmembrane helix. We propose a model to illustrate how positive and negative signals might be generated., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Guo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: To investigate reciprocal regulation between Fur and two RyhB homologs in Yersinia pestis ( Y. pestis ), as well as the roles of RyhBs in biofilm formation., Methods: Regulatory relationships were assessed by a combination of colony morphology assay, primer extension, electrophoretic mobility shift assay and DNase I footprinting., Results: Fur bound to the promoter-proximal DNA regions of ryhB1 and ryhB2 to repress their transcription, while both RyhB1 and RyhB2 repressed the expression of Fur at the post-transcriptional level. In addition, both RyhB1 and RyhB2 positively regulated Y. pestis biofilm exopolysaccharide (EPS) production and the expression of hmsHFRS and hmsT ., Conclusion: Fur and the two RyhB homologs exert negative reciprocal regulation, and RyhB homologs have a positive regulatory effect on biofilm formation in Y. pestis ., (Copyright © 2021 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Microorganisms possess diverse mechanisms to regulate investment into individual cellular processes according to their environment. How these regulatory strategies reflect the inherent trade-off between the benefit and cost of resource investment remains largely unknown, particularly for many cellular functions that are not immediately related to growth. Here, we investigate regulation of motility and chemotaxis, one of the most complex and costly bacterial behaviors, as a function of bacterial growth rate. We show with experiment and theory that in poor nutritional conditions, Escherichia coli increases its investment in motility in proportion to the reproductive fitness advantage provided by the ability to follow nutrient gradients. Since this growth-rate dependent regulation of motility genes occurs even when nutrient gradients are absent, we hypothesize that it reflects an anticipatory preallocation of cellular resources. Notably, relative fitness benefit of chemotaxis could be observed not only in the presence of imposed gradients of secondary nutrients but also in initially homogeneous bacterial cultures, suggesting that bacteria can generate local gradients of carbon sources and excreted metabolites, and subsequently use chemotaxis to enhance the utilization of these compounds. This interplay between metabolite excretion and their chemotaxis-dependent reutilization is likely to play an important general role in microbial communities., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Vibrio parahaemolyticus is a prevalent seafoodborne enteropathogen that has become a global concern since the spread of its pandemic strain in 1996. This study investigates the responses of this pathogen to the oxidative disinfectants hydrogen peroxide, chlorine dioxide, and peracetic acid. Expression of the regulator genes oxyR and rpoS, determined by reverse transcription PCR, in V. parahaemolyticus wild-type, oxyR mutant, and rpoS mutant strains exhibited similar patterns in response to the tested oxidative disinfectants. The transcription of the rpoS gene was markedly enhanced in the oxyR mutant strain in the exponential phase. The expression of catalase KatE1 was tracked by using a LacZ fusion reporter in these strains. The experimental results revealed that KatE1 was a significant scavenger of hydrogen peroxide and peracetic acid in V. parahaemolyticus, and RpoS may partially compensate for the regulatory role of OxyR in the oxyR mutant strain. In contrast to its responses to hydrogen peroxide and paracetic acid, KatE1 was not the primary scavenger of chlorine dioxide in these V. parahaemolyticus strains. This study shows that these disinfectants activated a basic oxidative response in this pathogen with different features.

Transmembrane chemoreceptors are widely present in Bacteria and Archaea. They play a critical role in sensing various signals outside and transmitting to the cell interior. Here, we report the structure of the periplasmic ligand-binding domain (LBD) of the transmembrane chemoreceptor MCP2201, which governs chemotaxis to citrate and other organic compounds in Comamonas testosteroni. The apo-form LBD crystal revealed a typical four-helix bundle homodimer, similar to previously well-studied chemoreceptors such as Tar and Tsr of Escherichia coli. However, the citrate-bound LBD revealed a four-helix bundle homotrimer that had not been observed in bacterial chemoreceptor LBDs. This homotrimer was further confirmed with size-exclusion chromatography, analytical ultracentrifugation and cross-linking experiments. The physiological importance of the homotrimer for chemotaxis was demonstrated with site-directed mutations of key amino acid residues in C. testosteroni mutants., (© 2019 John Wiley & Sons Ltd.)

QseB is a response regulator of the QseBC two-component system (TCS) which is associated with quorum sensing and functions as a global regulator of flagella, biofilm formation, and virulence. The function of QseB and its interaction with QseC has been the subject of study in some organisms, however, little work was done in Salmonella enterica serovar Typhi (S. Typhi). The objective of this study was to investigate the effect of QseB on biofilm formation and virulence in S. Typhi. It showed that the biofilm formation ability of qseC mutant was limited as compared to the wild type strain. We also show overexpression of qseB was in a qseC mutant. Interestingly, deletion of qseB in a qseC mutant restored a wild type phenotype. These results suggested that QseB may account for the impaired biofilm formation in the absence of QseC. Furthermore, deletion of qseB in wild type cells decreased biofilm formation, whereas overexpression of qseB in wild type cells increased biofilm formation. Quantitative real-time PCR also revealed the up-regulation of some fimbria-associated genes in a qseB overexpression strain. These results indicate that QseB may enhance biofilm formation in the presence of QseC. Taken together, we hypothesize that QseB has dual regulatory functions which are dependent upon its cognate sensor. Additionally, invasion of HeLa cells was enhanced in qseB mutant but attenuated in a qseC mutant compared with wild-type. The β-galactosidase activity of invF::lacZ was increased in qseB mutant but decreased in qseC mutant which was consistent with invasion results. In conclusion, QseB may have dual regulatory functions concerning biofilm formation and plays a negative role in virulence of S. Typhi., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mycoplasma hyorhinis (M. hyorhinis) is an important pathogen of pigs. In previous studies, the variable lipoprotein (Vlp) family has been shown to play a role in mediating M. hyorhinis cytoadhesion. Herein, we performed several experiments to study the function of each Vlp family member in detail, especially examining the cytoadhesion functional domain and how the repeat unit copy number impacts on function. Recombinant proteins rVlpII, composed of region II from all seven Vlp members; rVlpIII, composed of repeat peptides from region III of all of Vlp members; as well as a series of recombinant rVlp proteins for each member containing different repeat unit copy numbers were constructed. All of the proteins were expressed in Escherichia coli and purified by affinity chromatography. The recombinant proteins, as well as seven keyhole limpet hemocyanin-conjugated Vlp peptides containing two copies of the repeat unit, were analyzed for their adherence to swine tracheal epithelial cells using a microtiter plate adherence assay. Both rVlpII and rVlpIII proteins were able to bind to cell membrane proteins. Among the repeat unit peptides, only PepVlpB and PepVlpG were able to bind to cell membrane proteins. All of the Vlp members had cytoadhesion capability. The adhesion abilities of the proteins containing 0 or 3 copies of the repeat unit were stronger than those of the proteins containing 12 copies. For rVlpA, rVlpB, rVlpD, rVlpF and rVlpG, the proteins containing no copies bound stronger than the proteins containing 3 copies. In contrast, the adherence of rVlpC3 was stronger than that of rVlpC0. There was no significant difference between the adherence of rVlpE3 and that of rVlpE0. Our results suggest that the major cytoadhesion sites of Vlps are mainly contained in region II, the function of which would be blocked by region III when region III is longer., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The linear plasmid pBSSB1 mediates the flagellar phase variation in H:z66 positive Salmonella enterica serovar Typhi ( S. Typhi). The gene named stp17 ( S . Typhi plasmid number 17 gene) is located on pBSSB1 and encodes the protein STP17. The expression pattern at the protein-level and function of STP17 remains unknown. In this study, the recombinant protein STP17 His6 was expressed, purified and used to prepare the polyclonal anti-STP17 antibody. We detected protein-level expression of stp17 in S. Typhi and further investigated the protein expression characteristics of stp17 in different growth phases by western blot analysis. The effects of STP17 on bacterial growth and motility were analyzed. In addition, the structure of STP17 was predicted and the active site of STP17 was identified by site-directed mutagenesis. The results showed that STP17 was expressed stably in the wild type strain of S . Typhi. STP17 expression at the protein level peaks when cultures reach an OD 600 value of 1.2. The growth rate and motility of the Δ stp17 strain were significantly decreased compared with the wild type strain ( P < 0.05) and this phenotype was restored in the stp17 complementary strain. Moreover, the growth rate and motility of the stp17 over-expression strain was greater than the wild type strain. STP17 contains nine Helix segments, six Stand segments and some Coil segments in the secondary structural level. The top-ranked 3-D structure of STP17 predicted by I-TASSER contains a putative ATPase domain and the amino acid residues of GLY16, GLY19, LYS20, ASN133, LYS157, and LYS158 may be the active site residues of STP17. Finally, STP17 was able to catalyze the ATP to ADP reaction, suggesting that STP17 may be an ATPase. To our knowledge, this is the first report describing the protein expression characteristics of STP17 in S. Typhi, showing that STP17 promotes bacterial growth and motility, which may be associated with its potential ATPase activity.

The type Ⅳ secretion system (T4SS) of H. pylori encoded by cag pathogenicity island mediates the injection of the toxin CagA, which is considered as a paradigm for bacterial carcinogenesis. T4SS is generally composed of a set of proteins, the majority of which still remain unclear. In this study, we have identified CagI, one of the T4SS proteins, which interacted with CagA and played an important role for CagA stability., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Vibrio parahaemolyticus is a leading cause of seafood-associated diarrhea and gastroenteritis. This bacteria expresses a major virulence determinant called T3SS1. Expression of T3SS1 is tightly regulated by the ExsA-ExsC-ExsD regulatory system. The transcription of exsA and probably exsC is repressed directly by the H-NS protein. In this study, the regulation of exsD by H-NS was investigated using quantitative RT-PCR, primer extension, LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays. The results showed that His-H-NS protected a single region from 61 bp to 174 bp upstream of exsD against DNase I digestion, and a transcription start site located at 105 bp upstream of exsD was detected and its activity was repressed by H-NS. Therefore, a single H-NS-dependent promoter was transcribed for exsD in V. parahaemolyticus. Thus, all three genes in the ExsA-ExsC-ExsD regulatory system of T3SS1 are directly repressed by H-NS in V. parahaemolyticus., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Vacuolating cytotoxin antigen (VacA) is one of the major virulence factors in Helicobacter pylori (H. pylori), which is responsible for cell vacuolar degeneration and apoptotic cell death. A candidate host factor which mediates this process is cortactin, a protein associated with the processes of colonization and adhesion of H. pylori in gastric epithelium., Aim: To investigate the role of cortactin in VacA-induced apoptosis of gastric epithelial cells., Methods: Cortactin expression and shRNA lentiviral constructs were developed and transduced into the human gastric cancer cell line, AGS. VacA protein was purified from H. pylori cultures, acid-activated, and co-incubated with the transduced cell populations. Apoptosis was detected by flow cytometry, and the levels of the pro- and anti-apoptotic proteins Bax and Bcl-2 were determined by Western blot., Results: Acid-activated purified VacA induced apoptosis in the parental AGS cells. Increased expression of cortactin (AGS/cortactin) led to a greater percentage of cells undergoing apoptosis. In contrast, knockdown of cortactin with shRNA (AGS/cortactin-shRNA) decreased the percentage of apoptotic cells. The protein levels of pro- and anti-apoptotic proteins Bax and Bcl-2 were increased and decreased in AGS/cortactin cells relative to the parental AGS cells. In the AGS/cortactin-shRNA cells, Bax protein levels were decreased, while Bcl-2 protein was increased., Conclusions: The results indicate that cortactin is involved in the regulation of apoptosis induced by VacA in gastric cells.

Lipid-associated membrane proteins (LAMPs) are important in the pathogenicity of the Mycoplasma genus of bacteria. We investigated whether Mycoplasma hyopneumoniae LAMPs have pathogenic potential by inducing apoptosis in a St. Jude porcine lung epithelial cell line (SJPL). LAMPs from a pathogenic strain of M. hyopneumoniae (strain 232) were used in the research. Our investigation made use of diamidino-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) analysis, and Annexin-V-propidium iodide staining. After LAMP treatment for 24 h, typical changes were induced, chromosomes were concentrated, apoptotic bodies were observed, the 3'-OH groups of cleaved genomes were exposed, and the percentage of apoptotic cells reached 36.5 ± 11.66%. Caspase 3 and caspase 8 were activated and cytochrome c (cyt c) was released from the mitochondria into the cytoplasm; poly ADP ribose polymerase (PARP) was digested into two fragments; p38 mitogen-activated protein kinase (MAPK) was phosphorylated; and the expression of pro-apoptosis protein Bax increased while the anti-apoptosis protein Bcl-2 decreased. LAMPs also stimulated SJPL cells to produce nitric oxide (NO) and superoxide. This study demonstrated that LAMPs from M. hyopneumoniae can induce apoptosis in SJPL cells through the activation of caspase 3, caspase 8, cyt c, Bax, and p38 MAPK, thereby contributing to our understanding of the pathogenesis of M. hyopneumoniae, which should improve the treatment of M. hyopneumoniae infections.

Comamonas testosteroni CNB-1 behaves chemotactically toward a wide range of organic compounds, and 19 methyl-accepting chemotaxis proteins (MCPs) were annotated from the genome of strain CNB-2, a plasmid-curing derivative from strain CNB-1. The MCP-free mutant CNB-1Δ20 completely lost its chemotactic responses. In this study, we found that a chemoreceptor, namely MCP2983, restored chemotactic responses toward nine carboxylic acids and ten aromatic compounds to CNB-1Δ20. Isothermal titration calorimetry analysis indicated that the ligand-binding domain (LBD) of MCP2983 specifically binds to cis-aconitate but not other tested compounds. Deletion of the LBD of MCP2983 impaired chemotactic responses toward cis-aconitate as well as other tested compounds, indicating that the LBD of MCP2983 was essential for triggering chemotactic responses. Five amino acid residues (M(81), S(156), E(157), I(158), and L(159)) that are located at a putative ligand-binding pocket were identified to be involved in binding to cis-aconitate. So far, the MCP2983 represents the sole biochemically identified chemoreceptor that specifically binds to cis-aconitate and is able to trigger chemotaxis towards diverse organic compounds.

Aim: An RNA-seq analysis recently identified a 236-nucleotide transcript upstream from malS in Salmonella enterica serovar Typhi. Here, we investigated its molecular characteristics and function., Materials & Methods: RACE and northern blotting were used to determine the molecular characteristics of the sequence, and mutagenesis, microarray, immunoblotting and an invasion assay were used to investigate the functions of the transcript., Results: The transcript was identified as the malS 5'-untranslated region (UTR), which could influence the expression of the flagellar and SPI-1 genes and the invasion of HeLa cells by S. Typhi. Deletion of bax increased the expression of the invasion genes and the invasive capacity of S. Typhi, whereas the expression of the malS 5'-UTR reduced the expression of bax., Conclusion: The malS 5'-UTR reduces the expression of bax and increases the invasive capacity of S. Typhi.

Bacterial chemotaxis towards aromatic compounds has been frequently observed; however, knowledge of how bacteria sense aromatic compounds is limited. Comamonas testosteroni CNB-1 is able to grow on a range of aromatic compounds. This study investigated the chemotactic responses of CNB-1 to 10 aromatic compounds. We constructed a chemoreceptor-free, non-chemotactic mutant, CNB-1Δ20, by disruption of all 19 putative methyl-accepting chemotaxis proteins (MCPs) and the atypical chemoreceptor in strain CNB-1. Individual complementation revealed that a putative MCP (tagged MCP2201) was involved in triggering chemotaxis towards all 10 aromatic compounds. The recombinant sensory domain of MCP2201 did not bind to 3- or 4-hydroxybenzoate, protocatechuate, catechol, benzoate, vanillate and gentisate, but bound oxaloacetate, citrate, cis-aconitate, isocitrate, α-ketoglutarate, succinate, fumarate and malate. The mutant CNB-1ΔpmdF that lost the ability to metabolize 4-hydroxybenzoate and protocatechuate also lost its chemotactic response to these compounds, suggesting that taxis towards aromatic compounds is metabolism-dependent. Based on the ligand profile, we proposed that MCP2201 triggers taxis towards aromatic compounds by sensing TCA cycle intermediates. Our hypothesis was further supported by the finding that introduction of the previously characterized pseudomonad chemoreceptor (McpS) for TCA cycle intermediates into CNB-1Δ20 likewise triggered chemotaxis towards aromatic compounds., (© 2013 John Wiley & Sons Ltd.)

Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumonia in swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the main pathogenicity factors in mycoplasma diseases. In this study, we investigated the effects of M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in LAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number of TUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. In addition, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis in PAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased production of nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic rates increase and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is suggested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophage via NO production, superoxide anion production, and caspase-3 activation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: To study the transcriptional regulation mechanism of dps by OxyR in Yersinia pestis., Methods: Total RNAs were extracted from the wide-type (WT) strain and the oxyR null mutant (deltaoxyR). Primer extension assay was used to detect the promoter activity (the amount of primer extension product) of dps in WT and that in deltaoxyR. Then, quantitative RT-PCR was done to calculate the transcriptional variation of dps between the WT and deltaoxyR. The entire promoter-proximal region of the dps gene was amplified by PCR from Y. pestis strain 201. Over-expressed His-OxyR was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Then, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-OxyR to dps promoter region in vitro. Finally, Escherichia coli OxyR consensus was deployed to predict the OxyR binding site to dps promoter region., Results: The primer extension assay detected only one transcriptional start site located at 40bp uptream of dps, whose transcript was under positive regulation by OxyR. The bioinformatics analysis indicated that His-OxyR bound to a single region from 111 bp to 78bp upstream of dps., Conclusion: The transcription of dps was directly activated by OxyR in Yersinia pestis.

Objective: To study the role of RpoE and RpoS on the influence of the metabolism and growth of bacterial under hyperosmotic stress., Methods: The rpoS/rpoE double deletion mutant of Salmonella enterica serovar typhi (S. typhi) was prepared by homologous recombination through the suicide plasmid mediated. The recombination was visualized by PCR. Growth curves were drawn by using photometric value A600 as the ordinate and cultivation time as abscissa. The survival abilities of bacterial were compared under hyperosmotic stress. Statistical differences of early logarithmic growth stage (4 h) and laters logarithmic growth stage (12 h) were analyzed by one-way ANOVA. The expression difference of metabolism related genes of wild-type and mutant strains of S. Typhi incubated under hyperosmotic stress were investigated by Salmonella genomic DNA microarray. Real-time quantitative PCR (qRT-PCR) was performed to validate the results of microarray assay in some selected genes., Results: The rpoS/rpoE double deletion mutant of S. Typhi was successfully generated. The analysis of growth curve showed that the 4-hour and 12-hour A600 values were separately 0.503 ± 0.018 and 2.060 ± 0.112 in rpoS deletion mutant strains, 0.293 ± 0.053 and 1.933 ± 0.115 in rpoE deletion mutant strains, and 0.051 ± 0.007 and 0.963 ± 0.111 in rpoS/rpoE double deletion mutant strains; all of which were lower than the values of wild-type strains, who were 0.725 ± 0.097 and 2.496 ± 0.171, respectively. The difference were statistically significant (P < 0.05). The genomic DNA microarray revealed that 42 genes relevant with bacterial metabolism were influenced by RpoE and RpoS. Results of qRT-PCR showed that the expression values of rpsE, rbsK, nusG and etuB in rpoS deletion mutant strains were (1.86 ± 0.14)×10(6), (1.37 ± 0.11)×10(6), (2.72 ± 0.58)×10(6) and (8.27 ± 1.01)×10(6) copies/µg, respectively; while those in rpoE deletion mutant strains were (2.19 ± 0.17)×10(6), (1.51 ± 0.12)×10(6), (2.73 ± 0.57)×10(6) and (9.63 ± 1.42)×10(6) copies/µg, respectively. Compared with the values in wild-type strains, which were separately (1.94 ± 0.10)×10(6), (1.52 ± 0.11)×10(6), (2.39 ± 0.52)×10(6) and (10.83 ± 1.52)×10(6) copies/µg, the differences was not statistical significance (P > 0.05). However, compared with the values in rpoS/rpoE double mutant strains, which were separately (5.64 ± 0.59)×10(6), (4.17 ± 0.40)×10(6), (9.44 ± 1.22)×10(6) and (2.95 ± 0.88)×10(6) copies/µg, the difference was significant (P < 0.05)., Conclusion: RpoE and RpoS could influence the expression of lots of metabolism genes. Together, they regulated the metabolism and growth of S. Typhi under hyperosmotic stress.

Decreased expression (twofold) of a putative yehUTS operon of which yehUT encodes a putative YehU/YehT two-component system in the ompR mutant from Salmonella enterica serovar Typhi (S. Typhi) GIFU10007 under hypotonic growth condition was observed by qRT-PCR. Purified recombinant protein OmpR(His6) of GIFU10007 was shown to bind the upstream region of the yehU gene by the gel-shift assay. In addition, the yehT deletion mutant (ΔyehT) displayed differential expression (twofold or higher) of 26 genes under the condition by the DNA microarray analysis. Altogether, OmpR might regulate the YehUT system in S. Typhi under hypotonic growth condition.

pBSSB1 is a 27 kb non-bacteriophage-related linear plasmid first found in Salmonella enterica serovar Typhi (S. Typhi), but the mechanism underlying the replication of pBSSB1 is currently unknown. Previous reports showed that the factor for inversion stimulation (Fis) encoded by fis can affect the replication, transcription and other processes through binding DNA. Here, a fis deletion mutant of S. Typhi (Δfis) was prepared through the homologous recombination mediated by suicide plasmid and the loss of pBSSB1 in Δfis was observed surprisingly by pulsed field gel electrophoresis (PFGE). Subsequently, the loss of pBSSB1 was verified by PCR and Southern blot. In addition, the motility of Δfis was deficient and the flagellin of Δfis could not be detected by 2-dimensional polyacrylamide gel electrophoresis. All these results show that Fis is essential for the stability of pBSSB1 and affects the motility of S. Typhi.

The type VI secretion system (T6SS) of Salmonella enterica serovar Typhi (S. typhi) is associated with Salmonella pathogenicity island 6 (SPI-6). Though the T6SS gene cluster is intact in S. typhi, the protein complex is believed to be non-functional due to the presence of a pseudogene form of SciI (VipB homolog), a key component. We detected the SciK-his6 in the supernatant of the wild type strain of S. typhi containing the plasmid over-expressing SciK (hcp homolog) with a his6 epitope at the C-terminus, which suggested that the T6SS in S. typhi is functional. We also identified four genes that were essential to T6SS function: sciC (vasA homolog), sciS (vasK homolog), sciG (clpV homolog), and vrgS (vgrG homolog). Further analysis revealed that S. typhi T6SS is cytotoxic to human epithelial cells, but does not influence bacterial growth and mobility. RcsB, PmrA, and Hfq were identified as regulators of S. typhi T6SS gene expression; however, PhoP appears to not be involved. Taken together, the data demonstrate the functionality of S. typhi T6SS and confirm the important role of T6SS for S. typhi's ability to invade and infect epithelial cells.

Salmonella enterica serovar Typhi (S. Typhi) is the cause of typhoid fever, a food-borne disease that is prevalent worldwide, most particularly in developing countries. RNA polymerase sigma factors RpoE (σ(E)) and RpoS (σ(S)) govern transcription initiation of two sets of genes in Escherichia and Salmonella. It was previously suggested that some genes might be coregulated by RpoE and RpoS in Salmonella under conditions of environmental stress, but experimental evidence has been lacking. We therefore constructed rpoS deletion (ΔrpoS) and double rpoE/rpoS deletion (ΔrpoE/ΔrpoS) mutants of S. Typhi and compared their growth properties with an rpoE mutant (ΔrpoE) and wild-type strains under conditions of hyperosmotic stress. We report that the ΔrpoE, ΔrpoS, and ΔrpoE/ΔrpoS strains grew more slowly under hyperosmotic stress conditions than the wild-type strain, and the ΔrpoE/ΔrpoS strain grew most slowly. The global transcriptional profiles of ΔrpoE, ΔrpoS, ΔrpoE/ΔrpoS after 30 min of hyperosmotic stress were investigated using a Salmonella genomic DNA microarray. The results of microarray indicated that the expression levels of 38 genes were markedly reduced during hyperosmotic stress in the double mutant ΔrpoE/ΔrpoS strain, but expression levels were not significantly affected by single ΔrpoE or ΔrpoS mutations. This was confirmed for several key genes by qRT-PCR. This study therefore indicated crosstalk between sigma factors RpoE and RpoS in S. Typhi under hyperosmotic conditions and provides new insights into the regulatory networks of S. Typhi.