Control of biofilm formation and colonization in Vibrio fischeri: a role for partner switching?

Abstract

Bacteria employ a variety of mechanisms to promote and control colonization of their respective hosts, including restricting the expression of genes necessary for colonization to distinct situations (i.e. encounter with a prospective host). In the symbiosis between the marine bacterium Vibrio fischeri and its host squid, Euprymna scolopes, colonization proceeds via a transient biofilm formed by the bacterium. The production of this bacterial biofilm depends on a complex regulatory network that controls transcription of the symbiosis polysaccharide (syp) gene locus. In addition to this transcriptional control, biofilm formation is regulated by two proteins, SypA and SypE, which may function in an unusual regulatory mechanism known as partner switching. Best characterized in Bacillus subtilis and other Gram-positive bacteria, partner switching is a signalling mechanism that provides dynamic regulatory control over bacterial gene expression. The involvement of putative partner-switching components within V. fischeri suggests that tight regulatory control over biofilm formation may be important for the lifestyle of this organism.

Figure 1

Model of biofilm formation in V. fischeri. The symbiosis polysaccharide (sypA-R) locus is regulated at the transcriptional level via a 2-component regulatory cascade consisting of the sensor kinase, RscS, and the downstream response regulator, SypG. The regulatory proteins, SypA and SypE, exhibit antagonistic regulatory roles, promoting and inhibiting syp-dependent biofilms, respectively. These regulators control biofilm formation via an unknown mechanism that appears to function downstream of syp transcription. Biofilms are represented by the formation of a wrinkled bacterial colony (Yildiz and Visick, 2009).

Figure 2

SypA domain structure and multiple sequence alignment. (A) Domain structure of SypA. SypA contains the conserved anti-sigma factor antagonist and sulphate transporter (STAS) domain present in anti-anti-sigma factors. The conserved regulatory serine residue (S56) is indicated. (B) BLAST multiple sequence alignment (Altschul et al., 1997) and identification of conserved residues within V. fischeri SypA and the anti-anti-sigma factors RsbV and SpoIIAA of B. subtilis and BtrV of B. bronchiseptica. Serine 56 of RsbV, serine 58 of SpoIIAA, and serine 59 of BtrU are the phosphorylation targets of the respective serine kinases (Najafi et al., 1995; Yang et al., 1996). This serine residue, indicated by an S, is conserved in SypA (S56). Highlighting of the conserved identical residues (black boxes) and conserved substitutions (grey boxes) was generated using BOXSHADE Server.

Figure 3

SypE domain structure and multiple sequence alignment. (A) Domain structure of SypE. SypE is a multi-domain protein that contains a central response regulator (REC) domain flanked by an N-terminal serine kinase (RsbW) domain and a C-terminal serine phosphatase (PP2C) domain. The N-terminal RsbW domain of SypE contains the conserved N-, G1-, and G2- boxes important in anti-sigma factor activity, which are indicated by black, gray, and striped boxes, respectively. The conserved residues within the N-terminal RsbW and C-terminal PP2C domains, predicted to be important for serine kinase or serine phosphatase activity, are shown. (B) BLAST multiple sequence alignment (Altschul et al., 1997) of the N-terminal serine kinase domain of SypE with the anti-sigma factors RsbW and SpoIIAB of B. subtilis and BtrW of B. bronchiseptica. The conserved N-, G1-, and G2- boxes are outlined, and the conserved residues required for serine kinase activity are indicated in bold letters above the alignments (Dutta et al., 2000). The SypE serine kinase domain contains the conserved N-box asparagine (N52) and the G1 box aspartate and glycine residues (D81 and G83). (C) BLAST multiple sequence alignment of the C-terminal serine phosphatase domain of SypE with the serine phosphatases RsbU and SpoIIE of B. subtilis and BtrU of B. bronchiseptica. The labeled amino acids indicate the conserved residues required for serine phosphatase activity (Adler et al., 1997) SypE contains the invariant aspartate residues (D443 and D495) predicted to be important in divalent cation binding. For parts B and C, highlighting of the conserved residues (black boxes) and conserved substitutions (grey boxes) was generated using BOXSHADE server.

Figure 4

Model of partner-switching pathways. (A) A model of the B. subtilis partner-switching regulatory pathway controlling the activity of the general stress response sigma factor, sigma B. See text for detailed description of the model. (B) A model of the predicted SypA-SypE partner-switching module controlling biofilm formation. SypA and SypE possess the core components of a putative partner-switching signal pathway. The proposed model is constructed from information of the conserved protein domains and current data of biofilm regulation.