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Essential role of calcium in extending RTX adhesins to their target.

ABSTRACT: RTX adhesins are long, multi-domain proteins present on the outer membrane of many Gram-negative bacteria. From this vantage point, adhesins use their distal ligand-binding domains for surface attachment leading to biofilm formation. To expand the reach of the ligand-binding domains, RTX adhesins maintain a central extender region of multiple tandem repeats, which makes up most of the proteins' large molecular weight. Alignments of the 10-15-kDa extender domains show low sequence identity between adhesins. Here we have produced and structurally characterized protein constructs of four tandem repeats (tetra-tandemers) from two different RTX adhesins. In comparing the tetra-tandemers to each other and already solved structures from Marinomonas primoryensis and Salmonella enterica, the extender domains fold as diverse beta-sandwich structures with widely differing calcium contents. However, all the tetra-tandemers have at least one calcium ion coordinated in the linker region between beta-sandwich domains whose role appears to be the rigidification of the extender region to help the adhesin extend its reach.

SUBMITTER: Vance TDR

PROVIDER: S-EPMC7493085 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Essential role of calcium in extending RTX adhesins to their target.

Vance Tyler D R TDR Ye Qilu Q Conroy Brigid B Davies Peter L PL

Journal of structural biology: X 20200908

RTX adhesins are long, multi-domain proteins present on the outer membrane of many Gram-negative bacteria. From this vantage point, adhesins use their distal ligand-binding domains for surface attachment leading to biofilm formation. To expand the reach of the ligand-binding domains, RTX adhesins maintain a central extender region of multiple tandem repeats, which makes up most of the proteins' large molecular weight. Alignments of the 10-15-kDa extender domains show low sequence identity betwee ...[more]

PMID: 32984811

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Project description:Biofilm formation by the Gram-negative, Gammaproteobacteria Pseudomonas fluorescens relies on the repeats-in-toxin adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through their respective type 1 secretion systems, and retention at the cell surface. Published work has shown that retention of the adhesins occurs via a post-translational mechanism involving the cyclic-di-GMP receptor LapD and the protease LapG. However, little is known about the underlying mechanisms that regulate the level of these adhesins. Here, we demonstrate that the master regulator FleQ modulates biofilm formation by both transcriptionally and post-transcriptionally regulating LapA and MapA. We find that a ΔfleQ mutant has a biofilm formation defect compared to the wild-type (WT) strain, which is attributed in part to a decrease in LapA and MapA abundance in the cell, despite the ΔfleQ mutant having increased levels of lapA and mapA transcripts compared to the WT strain. Through transposon mutagenesis and subsequent genetic analysis, we found that overstimulation of the Gac/Rsm pathway partially rescues biofilm formation in the ΔfleQ mutant background. Collectively, these findings provide evidence that FleQ regulates biofilm formation by both transcriptionally regulating the expression of the lapA and mapA genes and post-transcriptionally regulating the abundance of LapA and MapA, and that activation of the Gac/Rsm pathway can post-transcriptionally enhance biofilm formation by P. fluorescens. IMPORTANCE Biofilm formation is a highly coordinated process that bacteria undergo to colonize a variety of surfaces. For Pseudomonas fluorescens, biofilm formation requires the production and localization of repeats-in-toxin adhesins to the cell surface. To date, little is known about the underlying mechanisms that regulate biofilm formation by P. fluorescens. Here, we identify FleQ as a key regulator of biofilm formation that modulates both gene expression and abundance of LapA and MapA through both a transcriptional and post-transcriptional mechanism. We provide further evidence implicating activation of the Gac/Rsm system in FleQ-dependent regulation of biofilm formation. Together, our findings uncover evidence for a dual mechanism of transcriptional and post-transcriptional regulation of the LapA and MapA adhesins.

Dataset Information

Essential role of calcium in extending RTX adhesins to their target.

Publications

Essential role of calcium in extending RTX adhesins to their target.

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