Dataset Information

The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family.

ABSTRACT: The synthesis of exotoxin A (ETA) by Pseudomonas aeruginosa is a complex, regulated event. Several ETA putative regulatory mutants of P. aeruginosa PA103 have previously been characterized (S. E. H. West, S. A. Kaye, A. N. Hamood, and B. H. Iglewski, Infect. Immun. 62:897-903, 1994). In addition to ETA production, these mutants, PA103-15, PA103-16, and PA103-19, were also deficient in the production of protease and in regA P1 promoter activity. RegA is a positive regulator of ETA transcription. We cloned a gene, designated vfr for virulence factor regulator, that restored ETA and protease production to parental levels in these mutants. In addition, transcription from the regA P1 promoter was restored. In Escherichia coli, when vfr was overexpressed from a phage T7 promoter, a protein with an apparent molecular mass of 28.5 kDa was produced. Analysis of the deduced amino acid sequence of vfr revealed that the expected protein is 67% identical and 91% similar over a 202-amino-acid overlap to the E. coli cyclic AMP receptor protein (CAP or Crp). The cloned vfr gene complemented the beta-galactosidase- and tryptophanase-deficient phenotypes of E. coli RZ1331, a crp deletion mutant. However, the E. coli crp gene under the control of the tac promoter did not complement the ETA-deficient or protease-deficient phenotype of PA103-15 or PA103-16. The ability of vfr to restore both ETA and protease production to these mutants suggests that vfr is a global regulator of virulence factor expression in P. aeruginosa.

SUBMITTER: West SE

PROVIDER: S-EPMC197210 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family.

West S E SE Sample A K AK Runyen-Janecky L J LJ

Journal of bacteriology 19941201 24

The synthesis of exotoxin A (ETA) by Pseudomonas aeruginosa is a complex, regulated event. Several ETA putative regulatory mutants of P. aeruginosa PA103 have previously been characterized (S. E. H. West, S. A. Kaye, A. N. Hamood, and B. H. Iglewski, Infect. Immun. 62:897-903, 1994). In addition to ETA production, these mutants, PA103-15, PA103-16, and PA103-19, were also deficient in the production of protease and in regA P1 promoter activity. RegA is a positive regulator of ETA transcription. ...[more]

PMID: 8002577

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Project description:FimV is a Pseudomonas aeruginosa inner membrane hub protein that modulates levels of the second messenger, cyclic AMP (cAMP), through the activation of adenylate cyclase CyaB. Although type IVa pilus (T4aP)-dependent twitching motility is modulated by cAMP levels, mutants lacking FimV are twitching impaired, even when exogenous cAMP is provided. Here we further define FimV's cAMP-dependent and -independent regulation of twitching. We confirmed that the response regulator of the T4aP-associated Chp chemotaxis system, PilG, requires both FimV and the CyaB regulator, FimL, to activate CyaB. However, in cAMP-replete backgrounds-lacking the cAMP phosphodiesterase CpdA or the CheY-like protein PilH or expressing constitutively active CyaB-pilG and fimV mutants failed to twitch. Both cytoplasmic and periplasmic domains of FimV were important for its cAMP-dependent and -independent roles, while its septal peptidoglycan-targeting LysM motif was required only for twitching motility. Polar localization of the sensor kinase PilS, a key regulator of transcription of the major pilin, was FimV dependent. However, unlike its homologues in other species that localize flagellar system components, FimV was not required for swimming motility. These data provide further evidence to support FimV's role as a key hub protein that coordinates the polar localization and function of multiple structural and regulatory proteins involved in P. aeruginosa twitching motility.IMPORTANCEPseudomonas aeruginosa is a serious opportunistic pathogen. Type IVa pili (T4aP) are important for its virulence, because they mediate dissemination and invasion via twitching motility and are involved in surface sensing, which modulates pathogenicity via changes in cAMP levels. Here we show that the hub protein FimV and the response regulator of the Chp system, PilG, regulate twitching independently of their roles in the modulation of cAMP synthesis. These functions do not require the putative scaffold protein FimL, proposed to link PilG with FimV. PilG may regulate asymmetric functioning of the T4aP system to allow for directional movement, while FimV appears to localize both structural and regulatory elements-including the PilSR two-component system-to cell poles for optimal function.

Project description:UnlabelledFimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV861-919, containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV689, a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions.ImportanceFimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs.

Dataset Information

The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family.

Publications

The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family.

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