Extracellular Polysaccharide Receptor and Receptor-Binding Proteins of the Bacteriophage-like Gene Transfer Agent RcGTA.

A variety of prokaryotes produce a bacteriophage-like gene transfer agent (GTA), and the alphaproteobacterial RcGTA is a model GTA. Some environmental isolates of lack the ability to acquire genes transferred by the RcGTA (recipient capability). In this work, we investigated the reason why strain 37b4 lacks recipient capability. The RcGTA head spike fiber and tail fiber proteins have been proposed to bind extracellular oligosaccharide receptors, and strain 37b4 lacks a capsular polysaccharide (CPS). The reason why strain 37b4 lacks a CPS was unknown, as was whether the provision of a CPS to 37b4 would result in recipient capability. To address these questions, we sequenced and annotated the strain 37b4 genome and used BLAST interrogations of this genome sequence to search for homologs of genes known to be needed for recipient capability. We also created a cosmid-borne genome library from a wild-type strain, mobilized the library into 37b4, and used the cosmid-complemented strain 37b4 to identify genes needed for a gain of function, allowing for the acquisition of RcGTA-borne genes. The relative presence of CPS around a wild-type strain, 37b4, and cosmid-complemented 37b4 cells was visualized using light microscopy of stained cells. Fluorescently tagged head spike fiber and tail fiber proteins of the RcGTA particle were created and used to measure the relative binding to wild-type and 37b4 cells. We found that strain 37b4 lacks recipient capability because of an inability to bind RcGTA; the reason it is incapable of binding is that it lacks CPS, and the absence of CPS is due to the absence of genes previously shown to be needed for CPS production in another strain. In addition to the head spike fiber, we found that the tail fiber protein also binds to the CPS.