Quorum‐sensing regulation of a capsular polysaccharide receptor for the Rhodobacter capsulatus gene transfer agent (RcGTA)

Abstract

The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) resembles a small tailed bacteri-ophage that packages almost random genomic DNA segments that may be transferred to other R. capsula-tus cells. Gene transfer agents are produced by a number of prokaryotes; however, no receptors have been identified. We investigated the RcGTA recipient capability of wild-type R. capsulatus cells at different culture growth phases, and found that the frequency of RcGTA-dependent acquisition of an allele increases as cultures enter the stationary phase. We also found that RcGTA adsorption to cells follows a similar trend. RcGTA recipient capability and adsorption were found to be dependent on the GtaR/I quorum-sensing (QS) system. Production of an extracellular polysaccharide was found to be regulated by GtaR/I QS, as was production of the cell capsule. A number of QS-regulated putative polysaccharide biosynthesis genes were identified, and mutagenesis of two of these genes, rcc01081 and rcc01932, yielded strains that lack a capsule. Furthermore, these mutants were impaired in RcGTA recipient capability and adsorption, as was a non-encapsulated wild-type isolate of R. capsulatus. Overall, our results indicate that capsular polysaccha-ride is a receptor for the gene transfer agent of R. cap-sulatus, RcGTA.

Phosphate Concentration and the Putative Sensor Kinase Protein CckA Modulate Cell Lysis and Release of the Rhodobacter capsulatus Gene Transfer Agent

“…Two recent reports indicated that RcGTA is produced by a subset of the bacterial population and that the fraction of RcGTA gene-expressing cells in the total cell population is increased in an RcGTA overproducer strain, DE442 (14,20). Additionally, maximal gene transduction is dependent on the growth phase of recipient cultures, largely due to quorum-sensing regulation of the production of a capsule receptor needed for binding of RcGTA (21). The production of RcGTA is induced as cultures enter the stationary phase and particles are released through cell lysis (19,20), which requires the endolysin encoded by rcc00555 (14).…”

The SOS Response Master Regulator LexA Regulates the Gene Transfer Agent of Rhodobacter capsulatus and Represses Transcription of the Signal Transduction Protein CckA

“…S3 and Table S1 in the supplemental material). This PCR product was digested with SphI and cloned into suicide plasmid pZDJ (27). The resultant plasmid was used as the template for a PCR with primers del_lexA_F and del_l-exA_R (see Fig.…”

“…The ⌬280pG::lacZ mutant strain used for transposon Tn5 mutagenesis (31) was created by PCR amplifying the RcGTA promoter region as described previously (32) and subcloning it into SacI/BamHI double-digested suicide plasmid pZDJ (27), resulting in pZDJ::pGTA. The lacZ gene was PCR amplified from plasmid pXCA601 (33) with primers lacZ_F_BamHI and lacZ_R_HindIII (see Table S1 in the supplemental material), and the resulting fragment was digested with BamHI and HindIII and subcloned into BamHI/HindIII double-digested plasmid pBluescript, resulting in pBS-lacZ.…”

“…The ⌬lexA and ⌬lexA ⌬cckA mutants were constructed in a WT SB1003-derived strain called SBpG that has an mCherry reporter gene fused to the promoter for the main RcGTA structural gene cluster (pGTA) on the chromosome. The integration of the fusion sequence was performed by introducing it into R. capsulatus for allelic exchange on suicide plasmid pZDJ (27).…”

Involvement of the response regulator CtrA in the extracellular DNA production of the marine bacterium <i>Rhodovulum sulfidophilum</i>

“…However, GTAs are not inducible by mitomycin C (Marrs, 1974) and do not form plaques (Solioz and Marrs, 1977). The expression of GTA genes studied to date is controlled by cellular regulatory systems (Brimacombe et al, 2013;Schaefer et al, 2002) and functions as truly gene transfer agents for the host cells. GTAs play a role in lateral gene transfer in nature and, thus, affect the evolution of prokaryotic genomes.…”