OBJECTIVE: This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase (DGC) in Vibrio cholerae and how its transcription is regulated by Fur and HapR. METHODS: The roles of VCA0560 was investigated by utilizing various phenotypic assays, including colony morphological characterization, crystal violet staining, Cyclic di-GMP (c-di-GMP) quantification, and swimming motility assay. The regulation of the VCA0560 gene by Fur and HapR was analyzed by luminescence assay, electrophoretic mobility shift assay, and DNase I footprinting. RESULTS: VCA0560 gene mutation did not affect biofilm formation, motility, and c-di-GMP synthesis in V. cholerae, and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity. The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator HapR. CONCLUSION Overexpressed VCA0560 functions as an active DGC in V. cholerae, and its transcription is repressed by Fur and HapR.
Vibrio cholerae/genetics* ; Biofilms ; Quorum Sensing ; Mutation ; Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics*

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the β-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.
Vibrio parahaemolyticus/metabolism* ; Escherichia coli/metabolism* ; Bacterial Proteins/metabolism* ; Transcription Factors/genetics* ; Gene Expression Regulation, Bacterial

Animals ; Bacterial Proteins/metabolism* ; Gene Expression Regulation, Bacterial ; Plague ; Transcription Factors/genetics* ; Yersinia pestis/genetics*

OBJECTIVE: This study aimed to investigate the regulation of histone-like nucleoid structuring protein (H-NS) on biofilm formation and cyclic diguanylate (c-di-GMP) synthesis in Vibrio parahaemolyticus RIMD2210633. METHODS: Regulatory mechanisms were analyzed by the combined utilization of crystal violet staining, quantification of c-di-GMP, quantitative real-time polymerase chain reaction, LacZ fusion, and electrophoretic-mobility shift assay. RESULTS: The deletion of hns enhanced the biofilm formation and intracellular c-di-GMP levels in V. parahaemolyticus RIMD2210633. H-NS can bind the upstream promoter-proximal DNA regions of scrA, scrG, VP0117, VPA0198, VPA1176, VP0699, and VP2979 to repress their transcription. These genes encode a group of proteins with GGDEF and/or EAL domains associated with c-di-GMP metabolism. CONCLUSION One of the mechanisms by which H-NS represses the biofilm formation by V. parahaemolyticus RIMD2210633 may be via repression of the production of intracellular c-di-GMP.
Bacterial Proteins/metabolism* ; Biofilms ; Cyclic GMP/analogs & derivatives* ; Gene Expression Regulation, Bacterial ; Gentian Violet ; Histones/metabolism* ; Vibrio parahaemolyticus/genetics*

Escherichia coli biofilm is a complex membrane aggregation produced by the adhesion and secretion of extracellular polymeric substances by E. coli cells aggregated on specific media. Pathogenic E. coli will evade the immune system and the impact of various harmful factors in the environment after the formation of biofilm, causing sustained and even fatal damage to the host. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger ubiquitous in bacteria and plays a crucial role in regulating biofilm formation. This paper reviewed the recent studies about the role of c-di-GMP in the movement, adhesion, and EPS production mechanism of E. coli during biofilm formation, aiming to provide a basis for inhibiting E. coli biofilm from the perspective of c-di-GMP.
Bacterial Proteins/genetics* ; Biofilms ; Cyclic GMP/analogs & derivatives* ; Escherichia coli/metabolism* ; Escherichia coli Proteins/metabolism* ; Gene Expression Regulation, Bacterial

TetR family transcriptional regulators (TFRs) are widely distributed in bacteria and archaea, and the first discovered TFR was confirmed to control the expression of tetracycline efflux pump in Escherichia coli. TFRs can bind DNAs and ligands. Small molecule ligands can induce conformational changes of TFRs, inhibiting or promoting TFRs to control target gene expression. Currently, TFRs have a wide variety of ligands, including carbohydrates, proteins, fatty acids and their derivatives, metal ions, and so on. Due to the diversity of ligands, TFRs regulate a wide range of physiological processes, from basic carbon metabolism and nitrogen metabolism to quorum sensing and antibiotic biosynthesis. On the basis of the recent studies in our laboratory and the literature, we review here the regulatory mechanism mediated by ligands of TFRs in primary and secondary metabolism, as well as the application of ligands for TFRs in the development of gene route and the activation of antibiotic biosynthesis.
Anti-Bacterial Agents ; Bacteria/metabolism* ; Bacterial Proteins/metabolism* ; Gene Expression Regulation, Bacterial ; Ligands ; Quorum Sensing

Objective: To investigate reciprocal regulation between Fur and two RyhB homologs in 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 Conclusion Fur and the two RyhB homologs exert negative reciprocal regulation, and RyhB homologs have a positive regulatory effect on biofilm formation in
Bacterial Proteins/metabolism* ; Biofilms ; Gene Expression Regulation, Bacterial/physiology* ; Yersinia pestis/physiology*

The aim of this study was to investigate the expression of MHCⅠ gene in different tissues of Rana dybowskii under the stress of Aeromonas hydrophila (Ah), and to provide evidence for revealing the anti-infective immune response mechanism of amphibians. The experimental animal model of Aeromonas hydrophila infection was first constructed, and the pathological changes were observed by HE staining. The MHCⅠ gene α1+α2 peptide binding region of Rana dybowskii was cloned by RT-PCR and analyzed by bioinformatics. Real-time PCR was used to detect the transcription level of MHCⅠ in different tissues under Ah stress. After Ah infection, the skin, liver and muscle tissues showed signs of cell structure disappearance and texture disorder. The MHCⅠ gene α1+α2 peptide binding region fragment was 494 bp, encoding 164 amino acids, and homology with amphibians. Above 77%, the homology with mammals was as low as 14.96%, indicating that the α1+α2 region of MHC gene was less conserved among different species. The results of real-time PCR show that the liver, spleen and kidney of the experimental group were under Ah stress. The transcript levels of MHCⅠ gene in skin and muscle tissues were higher than those in the control group at 72 h, but the time to peak of each tissue was different (P<0.01), indicating that the response time of MHCⅠ gene in different tissues was different under Ah stress. This study provides a reference for further exploring the immune function of MHC molecules in anti-infection.
Aeromonas hydrophila ; Animals ; Gene Expression Profiling ; Gene Expression Regulation ; immunology ; Gram-Negative Bacterial Infections ; immunology ; Liver ; metabolism ; Ranidae ; genetics ; immunology ; microbiology ; Skin ; metabolism

OBJECTIVE: To analyze the expression and clinical significance of long non-coding RNA (lncRNA) actin filament-associated protein 1-antisense RNA1 (AFAP1-AS1) in oral squamous cell carcinoma (OSCC) and its effect on the biobehavior of OSCC cells. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of lncRNA AFAP1-AS1 in the tumor tissue and matching adjacent normal tissue of OSCC patients (n=55), SCC25 cells, and normal oral keratinocyte lines (NOK) cells. The correlation between AFAP1-AS1 expression and the clinicopathological characteristics of OSCC patients was analyzed. The relationship between AFAP1-AS1 and prognosis was also studied with the Kaplan-Meier method. AFAP1-AS1 siRNA was transfected into the SCC25 cells. Cell counting kit-8 (CCK-8) and Trans-well were used to detect cell proliferation, invasion, and migration. The expression of the invasion-associated protein, AFAP1, and Rho GTPase family members, was detected by Western blot. In addition, the immunofluorescence of the cytoskeletal actin filament was observed. RESULTS: The expression of AFAP1-AS1 was higher in the OSCC tissues than in the NOK cells, and the relative expression of AFAP1-AS1 was higher in the SCC25 cells than in the NOK cells (P<0.001). AFAP1-AS1 expression was associated with the degree of diffe-rentiation, TNM stage, lymphatic metastasis, and poor prognosis of OSCC (P<0.05). Patients with a high expression of AFAP1-AS1 had lower survival rates than those with a low expression of AFAP1-AS1 (P<0.05). After transfected by AFAP1-AS1 siRNA, the expression of AFAP1-AS1 was downregulated. The inhibition of AFAP1-AS1 expression consequently suppressed the proliferation, invasion, and migration of SCC25. Moreover, AFAP1-AS1 siRNA upregulated the expression levels of AFAP1, RhoA, Rac2, Rab10, RhoGDI, and Pfn1 but downregulated the expression of RhoC. Immunofluorescence showed that AFAP1-AS1 also reduced the formation of stress filaments in the cytoskeleton and affected the integrity of the actin fila-ment. CONCLUSIONS The expression of AFAP1-AS1 was high in the OSCC tissues and SCC25 cells and is associated with the development and prognosis of OSCC. The knockdown of AFAP1-AS1 might inhibit the proliferation and invasion of OSCC cells by regulating the integrity of the actin filament.
Actin Cytoskeleton ; Carcinoma, Squamous Cell ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Mouth Neoplasms ; RNA, Bacterial ; RNA, Long Noncoding

Bacterial biofilm refers to a tunicate-like biological group composed of polysaccharide, protein and nucleic acid secreted by bacteria on the surface of the mucous membrane or biological material. The biofilm formation is a major cause of chronic infections. Bacteria could produce some secondary metabolites during the growth and reproduction. Some of them act as signaling molecules allowing bacteria to communicate and regulate many important physiological behaviors at multiple-cell level, such as bioluminescence, biofilm formation, motility and lifestyles. Usually, these signal molecules play an important role in the formation of bacterial biofilm. We review here the effects of related signal molecules of Quorum Sensing, cyclic diguanylate, Two-Component Systems and sRNA on the biofilm formation. Focusing on these regulation mechanism of signal molecules in the process of biofilm formation is necessary for the prevention and treatment of some chronic diseases.
Bacterial Proteins ; Biofilms ; Cyclic GMP ; Gene Expression Regulation, Bacterial ; Protein Binding ; Quorum Sensing