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

Abstract

The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a genetic exchange element that combines central aspects of bacteriophage-mediated transduction and natural transformation. RcGTA particles resemble a small double-stranded DNA bacteriophage, package random ϳ4-kb fragments of the producing cell genome, and are released from a subpopulation (<1%) of cells in a stationary-phase culture. RcGTA particles deliver this DNA to surrounding R. capsulatus cells, and the DNA is integrated into the recipient genome though a process that requires homologs of natural transformation genes and RecA-mediated homologous recombination. Here, we report the identification of the LexA repressor, the master regulator of the SOS response in many bacteria, as a regulator of RcGTA activity. Deletion of the lexA gene resulted in the abolition of detectable RcGTA production and an ϳ10-fold reduction in recipient capability. A search for SOS box sequences in the R. capsulatus genome sequence identified a number of putative binding sites located 5= of typical SOS response coding sequences and also 5= of the RcGTA regulatory gene cckA, which encodes a hybrid histidine kinase homolog. Expression of cckA was increased >5-fold in the lexA mutant, and a lexA cckA double mutant was found to have the same phenotype as a ⌬cckA single mutant in terms of RcGTA production. The data indicate that LexA is required for RcGTA production and maximal recipient capability and that the RcGTA-deficient phenotype of the lexA mutant is largely due to the overexpression of cckA. IMPORTANCEThis work describes an unusual phenotype of a lexA mutant of the alphaproteobacterium Rhodobacter capsulatus in respect to the phage transduction-like genetic exchange carried out by the R. capsulatus gene transfer agent (RcGTA). Instead of the expected SOS response characteristic of prophage induction, this lexA mutation not only abolishes the production of RcGTA particles but also impairs the ability of cells to receive RcGTA-borne genes. The data show that, despite an apparent evolutionary relationship to lambdoid phages, the regulation of RcGTA gene expression differs radically. The gene transfer agent of Rhodobacter capsulatus (RcGTA) combines features of bacteriophage-mediated transduction with natural transformation. The genes that encode the structural proteins of RcGTA particles are endogenous to the R. capsulatus chromosome and are located in at least two loci (1). In the stationary phase of R. capsulatus cultures, a subset (Ͻ1%) of the cell population produces RcGTA particles that resemble a tailed bacteriophage. Essentially random ϳ4-kb fragments of genomic DNA are packaged into the RcGTA particles, and particles are released by cell lysis mediated by a canonical endolysin/holin system, as for bacteriophage release (2-4). RcGTA particles recognize a capsular polysaccharide receptor on R. capsulatus cells using spikes located on the RcGTA capsid (5). The current understanding is that RcGTA appears to inject DNA into the periplasmic space, and homologs...

Insights into Origin and Evolution of α-proteobacterial Gene Transfer Agents

“…RcGTA homologs that are scattered across the genome (Supplementary Figures S2b and 4 S2c). Given that in the RcGTA genome, the structural genes are in one locus ( Figure 1A) and 5 are presumed to be co-transcribed (Kuchinski et al 2016), our observations suggest that 6 functional GTAs might be restricted mainly to α-proteobacteria. (Figure 4).…”

The Protease ClpXP and the PAS Domain Protein DivL Regulate CtrA and Gene Transfer Agent Production in Rhodobacter capsulatus

“…Impairment of CckA autophosphorylation greatly increases RcGTA protein production. The role of CckA and CtrA in RcGTA production is not fully understood: overexpression of cckA decreases RcGTA production by an unknown mechanism (37). Although a nonphosphorylatable CtrA(D51A) mutant had increased intracellular levels of capsid protein (20), no apparent change in total (intracellular plus extracellular) capsid protein was observed for the cckA deletion mutant (20,21,37).…”

Lateral gene transfer, bacterial genome evolution, and the Anthropocene

“…This suggests that GTAs have evolved from prophages, and are now specifically adapted agents of LGT encoded within the host genome . A proportion of host cells lyse to release GTA particles, whose DNA is taken up by recipient cells, using machinery similar to that involved in transformation . The DNA transfer to recipient cells is efficient enough to suggest that GTAs drive high frequencies of LGT in the world's oceans …”

“…It is consequently not surprising that induction of the SOS response leads to a burst of activity in mobile DNA elements . SOS is associated with induction of prophages, expression and production of GTAs, and excision of IS elements, ICEs, and transposons . SOS promotes excision of pathogenicity islands that can subsequently be packaged into phage‐like particles .…”

“…Bactericidal antibiotics trigger the SOS response, thus simultaneously generating mobile DNA elements and releasing them into the environment. Induction of bacteriophages and GTAs can lead to cell lysis as part of the natural life cycle of these agents . The successful lateral transfer of naked or phage‐encapsulated DNA can occur well after initial release and does not require host and recipient cells to be present at the same time or place.…”