Abstract
Colonization of the squid Euprymna scolopes by Vibrio fischeri requires biofilm formation dependent on the 18-gene symbiosis polysaccharide locus, syp. One key regulator, SypA, controls biofilm formation by an as-yet unknown mechanism; however, it is known that SypA itself is regulated by SypE. Biofilm-proficient strains form wrinkled colonies on solid media, while sypA mutants form biofilm-defective smooth colonies. To begin to understand the function of SypA, we used comparative analyses and mutagenesis approaches. sypA (and the syp locus) is conserved in other Vibrios, including two food-borne human pathogens, Vibrio vulnificus (rbdA) and Vibrio parahaemolyticus (sypAVP). We found that both homologs could complement the biofilm defect of the V. fischeri sypA mutant, but their phenotypes varied depending on the biofilm-inducing conditions used. Furthermore, while SypAVP retained an ability to be regulated by SypE, RbdA was resistant to this control. To better understand SypA function, we examined the biofilm-promoting ability of a number of mutant SypA proteins with substitutions in conserved residues, and found many that were biofilm-defective. The most severe biofilm-defective phenotypes occurred when changes were made to a conserved stretch of amino acids within a predicted α-helix of SypA; we hypothesize that this region of SypA may interact with another protein to promote biofilm formation. Finally, we identified a residue required for negative control by SypE. Together, our data provide insights into the function of this key biofilm regulator and suggest that the SypA orthologs may play similar roles in their native Vibrio species.
Highlights
Bacteria encounter a variety of environments, not all of which are favorable, and have adopted many strategies to survive unfavorable environmental conditions
The Conservation of SypA Among Select Vibrio Species To better understand the function of SypA, we used BLAST and ClustalW2 analyses (Altschul et al, 1997, 2005; Larkin et al, 2007) to compare SypA to the SypA orthologs encoded within loci similar to the syp locus in V. vulnificus (RbdA) and V. parahaemolyticus (SypAVP)
The Function of V. fischeri SypA is Conserved Among Vibrio Species To determine if the function of SypA is conserved despite the observed differences, we assessed the ability of the two non-native genes to complement a V. fischeri sypA mutant
Summary
Bacteria encounter a variety of environments, not all of which are favorable, and have adopted many strategies to survive unfavorable environmental conditions. Biofilm formation by V. fischeri depends, in part, on expression of the 18-gene symbiosis polysaccharide locus (syp), which encodes biofilm regulators as well as the proteins necessary for the production and export of the polysaccharide component of the biofilm (Supplementary Figures S1 and S2; Yip et al, 2005, 2006). Transcription of the syp locus is controlled by an unusual and complex two-component signaling cascade comprised of two hybrid sensor kinases, RscS and SypF, and a response regulator, SypG (Supplementary Figure S1A; Visick and Skoufos, 2001; Yip et al, 2006; Hussa et al, 2008; Norsworthy and Visick, 2015). Orthologs of SypA can be found encoded within a conserved syp locus in other Vibrio species, including the human pathogens Vibrio vulnificus (RbdA) and Vibrio parahaemolyticus (SypAVP; Supplementary Figure S2). These studies give insight into the requirements for SypA to function in V. fischeri and suggest that the SypA-like proteins may function in their own species
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