Identification and Characterization of VpsR and VpsT Binding Sites in Vibrio cholerae

Zamorano‐Sánchez, David; Fong, Jiunn C. N.; Kilic, Sefa; Erill, Ivan; Yildiz, Fitnat H.

doi:10.1128/jb.02439-14

Cited by 67 publications

(132 citation statements)

References 61 publications

Supporting

Mentioning

126

Contrasting

Order By: Relevance

“…4C), located from Ϫ138 to Ϫ124, 36 bp upstream of PepsC1. This binding site matches 11/14 bases in the recently described VpsR binding site consensus sequence (37). Promoter truncation analysis indicated the region encoding this putative binding site is essential for c-di-GMP induction of epsC (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cyclic Di-GMP and VpsR Induce the Expression of Type II Secretion in Vibrio cholerae

et al. 2017

View full text Add to dashboard Cite

Vibrio cholerae is a human pathogen that alternates between growth in environmental reservoirs and infection of human hosts, causing severe diarrhea. The second messenger cyclic di-GMP (c-di-GMP) mediates this transition by controlling a wide range of functions, such as biofilms, virulence, and motility. Here, we report that c-di-GMP induces expression of the extracellular protein secretion (eps) gene cluster, which encodes the type II secretion system (T2SS) in V. cholerae. Analysis of the eps genes confirmed the presence of two promoters located upstream of epsC, the first gene in the operon, one of which is induced by c-di-GMP. This induction is directly mediated by the c-di-GMP-binding transcriptional activator VpsR. Increased expression of the eps operon did not impact secretion of extracellular toxin or biofilm formation but did increase expression of the pseudopilin protein EpsG on the cell surface.IMPORTANCE Type II secretion systems (T2SSs) are the primary molecular machines by which Gram-negative bacteria secrete proteins and protein complexes that are folded and assembled in the periplasm. The substrates of T2SSs include extracellular factors, such as proteases and toxins. Here, we show that the widely conserved second messenger cyclic di-GMP (c-di-GMP) upregulates expression of the eps genes encoding the T2SS in the pathogen V. cholerae via the c-di-GMP-dependent transcription factor VpsR.KEYWORDS Vibrio cholerae, biofilm, VpsR, type II secretion, cyclic di-GMP V ibrio cholerae is a major bacterial pathogen responsible for the diarrheal disease cholera, causing an estimated 2.8 million infections each year resulting in approximately 91,000 deaths (1). V. cholerae is endemic to coastal waterways in tropical countries, where it persists in the environment as a biofilm on chitinous surfaces and periodically causes outbreaks in human populations. V. cholerae can rapidly spread and multiply under favorable environmental conditions, as seen in the recent 2010 Haiti outbreak (2). A fundamental property that allows V. cholerae to cause disease is its ability to transition from environmental reservoirs to human hosts. This transition is in part regulated by the second-messenger molecule cyclic di-GMP (c-di-GMP) (3-5).c-di-GMP is a nearly ubiquitous second-messenger molecule in bacteria that controls a range of physiological functions, including biofilm formation, motility, and virulence factor expression (6). c-di-GMP is synthesized by diguanylate cyclases (DGCs) (7) and degraded by phosphodiesterases (PDEs) (8,9). Together, these enzymes alter the concentration of c-di-GMP in the cell in response to environmental inputs. c-di-GMP has been shown to repress motility and virulence to promote a sessile, biofilmassociated lifestyle by stimulating the production of exopolysaccharide (EPS) matrix substances and adhesins while inhibiting flagellar activity or expression (3, 10-12). Levels of intracellular c-di-GMP have been proposed to be high in environmental

show abstract

Section: Resultsmentioning

confidence: 99%

Cyclic Di-GMP and VpsR Induce the Expression of Type II Secretion in Vibrio cholerae

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Transcription factors belonging to several other protein families bind c-di-GMP as an effector, and it will be interesting to explore their mechanisms of action in depth. These include VpsT from Vibrio cholerae, which inversely regulates biofilm matrix production and motility in response to c-di-GMP and is a member of the LuxR family of regulators (43,44). In Xanthomonas campestris, c-di-GMP acts as a negative regulator of the protein Clp, impairing the binding of Clp to DNA and the induction of genes important for virulence (45).…”

Section: Discussionmentioning

confidence: 99%

FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa

2016

View full text Add to dashboard Cite

The transcription factor FleQ from Pseudomonas aeruginosa derepresses expression of genes involved in biofilm formation when intracellular levels of the second messenger cyclic diguanosine monophosphate (c-di-GMP) are high. FleQ also activates transcription of flagellar genes, and the expression of these genes is highest at low intracellular c-di-GMP. FleQ thus plays a central role in mediating the transition between planktonic and biofilm lifestyles of P. aeruginosa. Previous work showed that FleQ controls expression of the pel operon for Pel exopolysaccharide biosynthesis by converting from a repressor to an activator upon binding c-di-GMP. To explore the activity of FleQ further, we carried out DNase I footprinting at three additional biofilm gene promoters, those of psl, cdrAB, and PA2440. The expression of cdrAB, encoding a cell surface adhesin, was sufficiently responsive to FleQ to allow us to carry out in vivo promoter assays. The results showed that, similarly to our observations with the pel operon, FleQ switches from a repressor to an activator of cdrAB gene expression in response to c-di-GMP. From the footprinting data, we identified a FleQ DNA binding consensus sequence. A search for this conserved sequence in bacterial genome sequences led to the identification of FleQ binding sites in the promoters of the siaABCD operon, important for cell aggregation, and the bdlA gene, important for biofilm dispersal, in P. aeruginosa. We also identified FleQ binding sites upstream of lapA-like adhesin genes in other Pseudomonas species. IMPORTANCEThe transcription factor FleQ is widely distributed in Pseudomonas species. In all species examined, it is a master regulator of flagellar gene expression. It also regulates diverse genes involved in biofilm formation in P. aeruginosa when intracellular levels of the second messenger c-di-GMP are high. Unlike flagellar genes, biofilm-associated genes are not always easy to recognize in genome sequences. Here, we identified a consensus DNA binding sequence for FleQ. This allowed us to survey Pseudomonas strains and find new genes that are likely regulated by FleQ and possibly involved in biofilm formation. Cyclic diguanosine monophosphate (c-di-GMP) is an intracellular second messenger that is produced by bacteria and has diverse effects on bacterial physiology. c-di-GMP binds to riboswitches and effector proteins to modulate transcription, translation, and protein activities (1, 2). In the opportunistic pathogen Pseudomonas aeruginosa, the transcription factor FleQ binds c-di-GMP to derepress expression of genes for biofilm components (3, 4). Biofilms are surface-attached communities of bacteria embedded in a matrix made of exopolysaccharides, proteins, and DNA. Biofilm infections are particularly difficult to treat because bacteria are protected by the biofilm matrix, making them resistant to antimicrobial treatment compared to planktonic cells (5). Genes controlled by FleQ include pel genes, coding for Pel exopolysaccharide; psl genes, coding for Psl exopolys...

show abstract

“…The complementation of lonA was carried out using a Tn7-based system that inserted the ORF of lonA accompanied by 178 bp of upstream genomic sequence into the conserved Tn7 site at the 3= end of the glmS gene, as previously described (28). Transcriptional fusions were constructed by cloning the upstream regulatory regions of selected genes into the pBBRlux plasmid, as previously described (13). The exact lengths of the upstream regulatory region used for the construction of these fusions are listed in Table S1 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

“…The transcription of the genes that code for these matrix components and their biosynthetic enzymes is primarily controlled by the transcriptional regulatory proteins VpsR, VpsT, and HapR (10)(11)(12). VpsR and VpsT bind directly to the upstream regulatory regions of the vps-I and vps-II operons and directly activate the expression of vps genes (13). Quorum sensing (QS) acts to repress biofilm formation in V. cholerae at high cell densities by activating the production of HapR, which then directly affects the transcription of vps structural genes and vpsT (12,13).…”

mentioning

confidence: 99%

“…VpsR and VpsT bind directly to the upstream regulatory regions of the vps-I and vps-II operons and directly activate the expression of vps genes (13). Quorum sensing (QS) acts to repress biofilm formation in V. cholerae at high cell densities by activating the production of HapR, which then directly affects the transcription of vps structural genes and vpsT (12,13).…”

mentioning

confidence: 99%

See 1 more Smart Citation

The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae

et al. 2016

Self Cite

View full text Add to dashboard Cite

The presence of the Lon protease in all three domains of life hints at its biological importance. The prokaryotic Lon protease is responsible not only for degrading abnormal proteins but also for carrying out the proteolytic regulation of specific protein targets. Posttranslational regulation by Lon is known to affect a variety of physiological traits in many bacteria, including biofilm formation, motility, and virulence. Here, we identify the regulatory roles of LonA in the human pathogen Vibrio cholerae. We determined that the absence of LonA adversely affects biofilm formation, increases swimming motility, and influences intracellular levels of cyclic diguanylate. Whole-genome expression analysis revealed that the message abundance of genes involved in biofilm formation was decreased but that the message abundances of those involved in virulence and the type VI secretion system were increased in a lonA mutant compared to the wild type. We further demonstrated that a lonA mutant displays an increase in type VI secretion system activity and is markedly defective in colonization of the infant mouse. These findings suggest that LonA plays a critical role in the environmental survival and virulence of V. cholerae. IMPORTANCEBacteria utilize intracellular proteases to degrade damaged proteins and adapt to changing environments. The Lon protease has been shown to be important for environmental adaptation and plays a crucial role in regulating the motility, biofilm formation, and virulence of numerous plant and animal pathogens. We find that LonA of the human pathogen V. cholerae is in line with this trend, as the deletion of LonA leads to hypermotility and defects in both biofilm formation and colonization of the infant mouse. In addition, we show that LonA regulates levels of cyclic diguanylate and the type VI secretion system. Our observations add to the known regulatory repertoire of the Lon protease and the current understanding of V. cholerae physiology.

show abstract

Identification and Characterization of VpsR and VpsT Binding Sites in Vibrio cholerae

Cited by 67 publications

References 61 publications

Cyclic Di-GMP and VpsR Induce the Expression of Type II Secretion in Vibrio cholerae

Cyclic Di-GMP and VpsR Induce the Expression of Type II Secretion in Vibrio cholerae

FleQ DNA Binding Consensus Sequence Revealed by Studies of FleQ-Dependent Regulation of Biofilm Gene Expression in Pseudomonas aeruginosa

The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae

Contact Info

Product

Resources

About