Format

Send to

Choose Destination
J Bacteriol. 2016 Jul 28;198(16):2263-74. doi: 10.1128/JB.00322-16. Print 2016 Aug 15.

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions.

Author information

1
Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Diseases Research, McMaster University, Hamilton, Ontario, Canada.
2
Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Diseases Research, McMaster University, Hamilton, Ontario, Canada Department of Biochemistry, Western University, London, Ontario, Canada.
3
Computer Science and Mathematics Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA, and Department of Microbiology, University of Tennessee, Knoxville, Tennessee, USA.
4
Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada howell@sickkids.ca burrowl@mcmaster.ca.
5
Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Diseases Research, McMaster University, Hamilton, Ontario, Canada howell@sickkids.ca burrowl@mcmaster.ca.

Abstract

FimV 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.

IMPORTANCE:

FimV 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.

PMID:
27297880
PMCID:
PMC4966435
DOI:
10.1128/JB.00322-16
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center