Alphaproteobacteria commonly produce an adhesin that is anchored to the exterior of the envelope at one cell pole. In Caulobacter crescentus this adhesin, known as the holdfast, facilitates attachment to solid surfaces and cell partitioning to air-liquid interfaces. An ensemble of two-component signal transduction (TCS) proteins controls C. crescentus holdfast biogenesis by indirectly regulating expression of HfiA, a potent inhibitor of holdfast synthesis. We performed a genetic selection to discover direct hfiA regulators that function downstream of the adhesion TCS system and identified rtrC, a hypothetical gene. rtrC transcription is directly activated by the adhesion TCS regulator, SpdR. Though its primary structure bears no resemblance to any defined protein family, RtrC binds and regulates dozens of sites on the C. crescentus chromosome via a pseudo-palindromic sequence. Among these binding sites is the hfiA promoter, where RtrC functions to directly repress transcription and thereby activate holdfast development. Either RtrC or SpdR can directly activate transcription of a second hfiA repressor, rtrB. Thus, environmental regulation of hfiA transcription by the adhesion TCS system is subject to control by an OR-gated type I coherent feedforward loop; these regulatory motifs are known to buffer gene expression against fluctuations in regulating signals. We have further assessed the functional role of rtrC in holdfast-dependent processes, including surface adherence to a cellulosic substrate and formation of pellicle biofilms at air-liquid interfaces. Strains harboring insertional mutations in rtrC have a diminished adhesion profile in a competitive cheesecloth binding assay and a reduced capacity to colonize pellicle biofilms in select media conditions. Our results add to an emerging understanding of the regulatory topology and molecular components of a complex bacterial cell adhesion control system.
Alphaproteobacteria commonly produce an adhesin that is anchored to the exterior of the envelope at one cell pole. In Caulobacter crescentus, this adhesin enables permanent attachment to solid surfaces and is known as the holdfast. An ensemble of two-component signal transduction (TCS) proteins control C. crescentus holdfast biogenesis by indirectly regulating expression of HfiA, a potent inhibitor of holdfast synthesis. A genetic selection to discover direct hfiA regulators that function downstream of this adhesion TCS system identified a hypothetical gene that we have named rtrC. Though the primary structure of RtrC bears no resemblance to any defined protein family, RtrC directly binds and regulates dozens of sites on the C. crescentus chromosome via a pseudo-palindromic motif. Among these binding sites is the hfiA promoter, where RtrC functions to directly repress transcription and thereby activate holdfast development. RtrC, the DNA-binding response regulator SpdR, and the transcription factor RtrB together form an OR-gated type I coherent feedforward loop (C1-FFL) that regulates hfiA transcription. C1-FFL motifs are known to buffer gene expression against transient loss of regulating signals, which often occurs in fluctuating natural environments. We conclude that the formerly hypothetical gene, rtrC, encodes a transcription factor that functions downstream of the C. crescentus TCS adhesion control system to regulate development of the holdfast adhesin.
A suite of molecular sensory systems enablesCaulobacterto control growth, development, and reproduction in response to levels of essential elements. The enhancer binding protein NtrC and its cognate sensor histidine kinase NtrB are well-established regulators of nitrogen assimilation in bacteria, but their precise functions inCaulobactermetabolism and cell development remain largely undefined. Deletion ofC. crescentus ntrCslowed cell growth in complex medium, whilentrBandntrCwere essential for growth when ammonium was the sole nitrogen source due to their requirement for glutamine synthase (glnA) expression. Random transposition of a conserved IS3-family mobile genetic element frequently rescued the growth defect ofntrCmutant strains by restoring transcription of theglnBAoperon, revealing a possible role for IS3 transposition in shaping the evolution ofCaulobacterpopulations during nitrogen limitation. The chromosome ofC. crescentusharbors dozens of NtrC binding sites, with a large fraction located near genes involved in polysaccharide biosynthesis. The majority of NtrC binding sites align with those of the nucleoid associated protein, GapR, an essential protein involved in chromosome organization, or MucR1, a cell cycle regulator. Thus, NtrC is predicted to directly impact the regulation of cell cycle and cell development. Indeed, loss of NtrC function led to elongated polar stalks and elevated synthesis of cell envelope polysaccharides. These phenotypes were rescued by supplementing media with glutamine or by ectopic expression of theglnBAoperon. This study establishes regulatory connections between NtrC, nitrogen metabolism, polar morphogenesis, and envelope polysaccharide synthesis inCaulobacter.
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