Transcriptional Regulation of opaR, qrr2–4 and aphA by the Master Quorum-Sensing Regulator OpaR in Vibrio parahaemolyticus

Background: Vibrio parahaemolyticus is a leading cause of infectious diarrhea and enterogastritis via the fecal-oral route. V. harveyi is a pathogen of fishes and invertebrates, and has been used as a model for quorum sensing (QS) studies. LuxR is the master QS regulator (MQSR) of V. harveyi, and LuxR-dependent expression of its own gene, qrr2–4 and aphA have been established in V. harveyi. Molecular regulation of target genes by the V. parahaemolyticus MQSR OpaR is still poorly understood. Methodology/Principal Findings: The bioinformatics analysis indicated that V. parahaemolyticus OpaR, V. harveyi LuxR, V. vulnificu SmcR, and V. alginolyticus ValR were extremely conserved, and that these four MQSRs appeared to recognize the same conserved cis-acting signals, which was represented by the consensus constructs manifesting as a position frequency matrix and as a 20 bp box, within their target promoters. The MQSR box-like sequences were found within the upstream DNA regions of opaR, qrr2–4 and aphA in V. parahaemolyticus, and the direct transcriptional regulation of these target genes by OpaR were further confirmed by multiple biochemical experiments including primer extension assay, gel mobility shift assay, and DNase I footprinting analysis. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and OpaR-binding sites were determined for the five target genes of OpaR, which gave a structural map of the OpaR-dependent promoters. Further computational promote

Isolation and Characterization of 89K Pathogenicity Island-Positive ST-7 Strains of Streptococcus suis Serotype 2 from Healthy Pigs, Northeast China

Streptococcus suis is a swine pathogen which can also cause severe infection, such as meningitis, and streptococcal-like toxic shock syndrome (STSS), in humans. In China, most of the S. suis infections in humans were reported in the southern areas with warm and humid climates, but little attention had been paid to the northern areas. Data presented here showed that the virulent serotypes 1, 2, 7, and 9 of S. suis could be steadily isolated from the healthy pigs in the pig farms in all the three provinces of Northeast China. Notably, a majority of the serotype 2 isolates belonged to the 89K pathogenicity island-positive ST-7 clone that had historically caused the human STSS outbreaks in the Sichuan and Jiangsu provinces of China, although the human STSS case caused by S. suis had never been reported in northern areas of China. Data presented here indicated that the survey of S. suis should be expanded to or reinforced in the northern areas of China

Phenotypic and transcriptional analysis of the osmotic regulator OmpR in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>The osmotic regulator OmpR in <it>Escherichia coli </it>regulates differentially the expression of major porin proteins OmpF and OmpC. In <it>Yersinia enterocolitica </it>and <it>Y. pseudotuberculosis</it>, OmpR is required for both virulence and survival within macrophages. However, the phenotypic and regulatory roles of OmpR in <it>Y. pestis </it>are not yet fully understood.</p> <p>Results</p> <p><it>Y. pestis </it>OmpR is involved in building resistance against phagocytosis and controls the adaptation to various stressful conditions met in macrophages. The <it>ompR </it>mutation likely did not affect the virulence of <it>Y. pestis </it>strain 201 that was a human-avirulent enzootic strain. The microarray-based comparative transcriptome analysis disclosed a set of 224 genes whose expressions were affected by the <it>ompR </it>mutation, indicating the global regulatory role of OmpR in <it>Y. pestis</it>. Real-time RT-PCR or <it>lacZ </it>fusion reporter assay further validated 16 OmpR-dependent genes, for which OmpR consensus-like sequences were found within their upstream DNA regions. <it>ompC</it>, <it>F</it>, <it>X</it>, and <it>R </it>were up-regulated dramatically with the increase of medium osmolarity, which was mediated by OmpR occupying the target promoter regions in a tandem manner.</p> <p>Conclusion</p> <p>OmpR contributes to the resistance against phagocytosis or survival within macrophages, which is conserved in the pathogenic yersiniae. <it>Y. pestis </it>OmpR regulates <it>ompC</it>, <it>F</it>, <it>X</it>, and <it>R </it>directly through OmpR-promoter DNA association. There is an inducible expressions of the pore-forming proteins OmpF, C, and × at high osmolarity in <it>Y. pestis</it>, in contrast to the reciprocal regulation of them in <it>E. coli</it>. The main difference is that <it>ompF </it>expression is not repressed at high osmolarity in <it>Y. pestis</it>, which is likely due to the absence of a promoter-distal OmpR-binding site for <it>ompF</it>.</p

Regulatory effects of cAMP receptor protein (CRP) on porin genes and its own gene in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>The cAMP receptor protein (CRP) is a global bacterial regulator that controls many target genes. The CRP-cAMP complex regulates the <it>ompR-envZ </it>operon in <it>E. coli </it>directly, involving both positive and negative regulations of multiple target promoters; further, it controls the production of porins indirectly through its direct action on <it>ompR-envZ</it>. Auto-regulation of CRP has also been established in <it>E. coli</it>. However, the regulation of porin genes and its own gene by CRP remains unclear in <it>Y. pestis</it>.</p> <p>Results</p> <p><it>Y. pestis </it>employs a distinct mechanism indicating that CRP has no regulatory effect on the <it>ompR-envZ </it>operon; however, it stimulates <it>ompC </it>and <it>ompF </it>directly, while repressing <it>ompX</it>. No transcriptional regulatory association between CRP and its own gene can be detected in <it>Y. pestis</it>, which is also in contrast to the fact that CRP acts as both repressor and activator for its own gene in <it>E. coli</it>. It is likely that <it>Y. pestis </it>OmpR and CRP respectively sense different signals (medium osmolarity, and cellular cAMP levels) to regulate porin genes independently.</p> <p>Conclusion</p> <p>Although the CRP of <it>Y. pestis </it>shows a very high homology to that of <it>E. coli</it>, and the consensus DNA sequence recognized by CRP is shared by the two bacteria, the <it>Y. pestis </it>CRP can recognize the promoters of <it>ompC</it>, <it>F</it>, and <it>X </it>directly rather than that of its own gene, which is different from the relevant regulatory circuit of <it>E. coli</it>. Data presented here indicate a remarkable remodeling of the CRP-mediated regulation of porin genes and of its own one between these two bacteria.</p

Mixing state and particle hygroscopicity of organic-dominated aerosols over the Pearl River Delta region in China

Simultaneous measurements of aerosol hygroscopicity and particle-phase chemical composition were performed at a suburban site over the Pearl River Delta region in the late summer of 2016 using a self-assembled hygroscopic tandem differential mobility analyzer (HTDMA) and an Aerodyne quadruple aerosol chemical speciation monitor (ACSM), respectively. The hygroscopic growth factor (HGF) of the Aitken mode (30 nm, 60 nm) and accumulation mode (100 nm, 145 nm) particles were obtained under 90% relative humidity (RH). An external mixture was observed for particles of every size during this study, with a dominant mode of more-hygroscopic (MH) particles, as aged aerosols dominated due to the anthropogenic influence. The HGF of lesshygroscopic (LH) mode particles increased, while their number fractions decreased during the daytime due to a reduced degree of external mixing that probably resulted from the condensation of gaseous species. These LH mode particles in the early morning or late afternoon could be possibly dominated by carbonaceous material emitted from local automobile exhaust during rush hours. During polluted days with air masses flowing mainly from the coastal areas, the chemical composition of aerosols had a clear diurnal variation and a strong correlation with the mean HGF. Closure analysis was carried out between the HTDMA-measured HGF and the ACSM-derived hygroscopicity using various approximations for the hygroscopic growth factor of organic compounds (HGF(org)). Considering the assumptions regarding the differences in the mass fraction of each component between PM1 and 145 nm particles, the hygroscopicity-composition closure was achieved using an HGF(org) of 1.26 for the organic material in the 145 nm particles and a simple linear relationship between the HGForg and the oxidation level inferred from the O : C ratio of the organic material was suggested. Compared with the results from other environments, HGF(org) obtained from our measurements appeared to be less sensitive to the variation of its oxidation level, which is, however, similar to the observations in the urban atmosphere of other megacities in China. This finding suggests that the anthropogenic precursors or the photooxidation mechanisms might differ significantly between the suburban and urban atmosphere in China and those in other background environments. This may lead to different characteristics of the oxidation products in secondary organic aerosols (SOA) and therefore to a different relationship between the HGF(org) and its O : C ratio.Peer reviewe

Cell Membrane Is Impaired, Accompanied by Enhanced Type III Secretion System Expression in Yersinia pestis Deficient in RovA Regulator

BACKGROUND: In the enteropathogenic Yersinia species, RovA regulates the expression of invasin, which is important for enteropathogenic pathogenesis but is inactivated in Yersinia pestis. Investigation of the RovA regulon in Y. pestis at 26 °C has revealed that RovA is a global regulator that contributes to virulence in part by the direct regulation of psaEFABC. However, the regulatory roles of RovA in Y. pestis at 37 °C, which allows most virulence factors in mammalian hosts to be expressed, are still poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: The transcriptional profile of an in-frame rovA mutant of Y. pestis biovar Microtus strain 201 was analyzed under type III secretion system (T3SS) induction conditions using microarray techniques, and it was revealed that many cell-envelope and transport/binding proteins were differentially expressed in the ΔrovA mutant. Most noticeably, many of the T3SS genes, including operons encoding the translocon, needle and Yop (Yersinia outer protein) effectors, were significantly up-regulated. Analysis of Yop proteins confirmed that YopE and YopJ were also expressed in greater amounts in the mutant. However, electrophoresis mobility shift assay results demonstrated that the His-RovA protein could not bind to the promoter sequences of the T3SS genes, suggesting that an indirect regulatory mechanism is involved. Transmission electron microscopy analysis indicated that there are small loose electron dense particle-like structures that surround the outer membrane of the mutant cells. The bacterial membrane permeability to CFSE (carboxyfluorescein diacetate succinimidyl ester) was significantly decreased in the ΔrovA mutant compared to the wild-type strain. Taken together, these results revealed the improper construction and dysfunction of the membrane in the ΔrovA mutant. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the RovA regulator plays critical roles in the construction and functioning of the bacterial membrane, which sheds considerable light on the regulatory functions of RovA in antibiotic resistance and environmental adaptation. The expression of T3SS was upregulated in the ΔrovA mutant through an indirect regulatory mechanism, which is possibly related to the altered membrane construction in the mutant

Molecular Characterization of Transcriptional Regulation of rovA by PhoP and RovA in Yersinia pestis

BACKGROUND: Yersinia pestis is the causative agent of plague. The two transcriptional regulators, PhoP and RovA, are required for the virulence of Y. pestis through the regulation of various virulence-associated loci. They are the global regulators controlling two distinct large complexes of cellular pathways. METHODOLOGY/PRINCIPAL FINDINGS: Based on the LacZ fusion, primer extension, gel mobility shift, and DNase I footprinting assays, RovA is shown to recognize both of the two promoters of its gene in Y. pestis. The autoregulation of RovA appears to be a conserved mechanism shared by Y. pestis and its closely related progenitor, Y. pseudotuberculosis. In Y. pestis, the PhoP regulator responds to low magnesium signals and then negatively controls only one of the two promoters of rovA through PhoP-promoter DNA association. CONCLUSIONS/SIGNIFICANCE: RovA is a direct transcriptional activator for its own gene in Y. pestis, while PhoP recognizes the promoter region of rovA to repress its transcription. The direct regulatory association between PhoP and RovA bridges the PhoP and RovA regulons in Y. pestis

Characterization of an aspartate aminotransferase encoded by YPO0623 with frequent nonsense mutations in Yersinia pestis

Yersinia pestis, the causative agent of plague, is a genetically monomorphic bacterial pathogen that evolved from Yersinia pseudotuberculosis approximately 7,400 years ago. We observed unusually frequent mutations in Y. pestis YPO0623, mostly resulting in protein translation termination, which implies a strong natural selection. These mutations were found in all phylogenetic lineages of Y. pestis, and there was no apparent pattern in the spatial distribution of the mutant strains. Based on these findings, we aimed to investigate the biological function of YPO0623 and the reasons for its frequent mutation in Y. pestis. Our in vitro and in vivo assays revealed that the deletion of YPO0623 enhanced the growth of Y. pestis in nutrient-rich environments and led to increased tolerance to heat and cold shocks. With RNA-seq analysis, we also discovered that the deletion of YPO0623 resulted in the upregulation of genes associated with the type VI secretion system (T6SS) at 26°C, which probably plays a crucial role in the response of Y. pestis to environment fluctuations. Furthermore, bioinformatic analysis showed that YPO0623 has high homology with a PLP-dependent aspartate aminotransferase in Salmonella enterica, and the enzyme activity assays confirmed its aspartate aminotransferase activity. However, the enzyme activity of YPO0623 was significantly lower than that in other bacteria. These observations provide some insights into the underlying reasons for the high-frequency nonsense mutations in YPO0623, and further investigations are needed to determine the exact mechanism