The Proline Variant of the W[F/L/M][T/S]R Cyclic Di-GMP Binding Motif Suppresses Dependence on Signal Association for Regulator Function.

Vibrio vulnificus is an estuarine bacterium and potent opportunistic human pathogen. It enters the food chain by asymptomatically colonizing a variety of marine organisms, most notably oysters. Expression of the brp-encoded extracellular polysaccharide, which enhances cell-surface adherence, is regulated by cyclic di-GMP (c-di-GMP) and the activator BrpT. The Vibrio cholerae and Vibrio parahaemolyticus homologs VpsT and CpsQ, directly bind c-di-GMP via a novel W[F/L/M][T/S]R motif, and c-di-GMP binding is absolutely required for activity. Notably, BrpT belongs to a distinct subclass of VpsT-like regulators that harbor a proline in the third position of the c-di-GMP binding motif (WLPR), and the impact of this change on activity is unknown. We show that the brp locus is organized as two linked operons with BrpT specifically binding to promoters upstream of brpA and brpH Expression data and structural modeling suggested that BrpT might be less dependent on c-di-GMP binding for activity than VpsT or CpsQ. We show that the affinity of BrpT for c-di-GMP is low and that signal binding is not a requisite for BrpT function. Furthermore, a BrpT mutant engineered to carry a canonical WLTR motif (BrpTP124T) bound c-di-GMP with high affinity and its activity was now c-di-GMP dependent. Conversely, introduction of the WLPR motif into VpsT suppressed its dependence on c-di-GMP for activity. This is the first demonstration of reduced dependence on signal association for regulator function within this motif family. Thus, BrpT defines a new class of VpsT-like transcriptional regulators, and the WLPR motif variant may similarly liberate the activity of other subclass members.IMPORTANCE A Vibrio genome may encode nearly 100 proteins that make, break, and bind c-di-GMP, underscoring its central role in the physiology of these bacteria. The activity of the biofilm regulators VpsT of V. cholerae and CpsQ of V. parahaemolyticus is regulated by the direct binding of c-di-GMP via a novel W[F/L/M][T/S]R motif. The V. vulnificus homolog, BrpT, bears an unusual WLPR variant and remains active at low intracellular c-di-GMP levels. This suggests that the WLPR motif may also liberate the activity of other members of this subclass. A single point mutation at the 3rd position of the motif was sufficient to moderate dependence on c-di-GMP binding for activator function, highlighting the simplicity with which complex bacterial signaling networks can be rewired.

The Agrobacterium tumefaciens CheY-like protein ClaR regulates biofilm formation.

The switch from a motile, planktonic existence to an attached biofilm is a major bacterial lifestyle transition that is often mediated by complex regulatory pathways. In this report, we describe a CheY-like protein required for control of the motile-to-sessile switch in the plant pathogen Agrobacterium tumefaciens. This regulator, which we have designated ClaR, possesses two distinct CheY-like receiver (REC) domains and is involved in the negative regulation of biofilm formation, through production of the unipolar polysaccharide (UPP) adhesin and cellulose. The ClaR REC domains share predicted structural homology with characterized REC domains and contain the majority of active site residues known to be essential for protein phosphorylation. REC1 is missing the conserved aspartate (N72) residue and although present in REC 2 (D193), it is not required for ClaR-dependent regulation suggesting that phosphorylation, which modulates the activity of many CheY-like proteins, appears not to be essential for ClaR activity. We also show that ClaR-dependent negative regulation of attachment is diminished significantly in mutants for PruA and PruR, proteins known to be involved in a pterin-mediated attachment regulation pathway. In A. tumefaciens, pterins are required for control of the intracellular signal cyclic diguanylate monophosphate through the DcpA regulator, but our findings suggest that pterin-dependent ClaR control of attachment can function independently from DcpA, including dampening of c-di-GMP levels. This report of a novel CheY-type biofilm regulator in A. tumefaciens thus also adds significant details to the role of pterin-mediated signalling.

Environmental Calcium Initiates a Feed-Forward Signaling Circuit That Regulates Biofilm Formation and Rugosity in Vibrio vulnificus.

Poor clinical outcomes (disfigurement, amputation, and death) and significant economic losses in the aquaculture industry can be attributed to the potent opportunistic human pathogen Vibrio vulnificusV. vulnificus, as well as the bivalves (oysters) it naturally colonizes, is indigenous to estuaries and human-inhabited coastal regions and must endure constantly changing environmental conditions as freshwater and seawater enter, mix, and exit the water column. Elevated cellular c-di-GMP levels trigger biofilm formation, but relatively little is known regarding the environmental signals that initiate this response. Here, we show that calcium is a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers expression of the brp extracellular polysaccharide that enhances biofilm formation. A transposon screen for the loss of calcium-induced PbrpA expression revealed CysD, an enzyme in the sulfate assimilation pathway. Targeted disruption of the pathway indicated that the production of a specific metabolic intermediate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), was required for calcium-induced PbrpA expression and that PAPS was separately required for development of the physiologically distinct rugose phenotype. Thus, PAPS behaves as a second messenger in V. vulnificus Moreover, c-di-GMP and BrpT (the activator of brp expression) acted in concert to bias expression of the sulfate assimilation pathway toward PAPS and c-di-GMP accumulation, establishing a feed-forward regulatory loop to boost brp expression. Thus, this signaling network links extracellular calcium and sulfur availability to the intracellular second messengers PAPS and c-di-GMP in the regulation of V. vulnificus biofilm formation and rugosity, survival phenotypes underpinning its evolution as a resilient environmental organism.IMPORTANCE The second messenger c-di-GMP is a key regulator of bacterial physiology. The V. vulnificus genome encodes nearly 100 proteins predicted to make, break, and bind c-di-GMP. However, relatively little is known regarding the environmental signals that regulate c-di-GMP levels and biofilm formation in V. vulnificus Here, we identify calcium as a primary environmental signal that specifically increases intracellular c-di-GMP concentrations, which in turn triggers brp-mediated biofilm formation. We show that PAPS, a metabolic intermediate of the sulfate assimilation pathway, acts as a second messenger linking environmental calcium and sulfur source availability to the production of another intracellular second messenger (c-di-GMP) to regulate biofilm and rugose colony formation, developmental pathways that are associated with environmental persistence and efficient bivalve colonization by this potent human pathogen.