Homologues of Genetic Transformation DNA Import Genes Are Required for Rhodobacter capsulatus Gene Transfer Agent Recipient Capability Regulated by the Response Regulator CtrA

Abstract

Gene transfer agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that homologues of three genes involved in natural transformation in other bacteria, comEC, comF, and comM, are essential for RcGTA-mediated gene acquisition. This paper gives genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in <1% of a cell population, CtrA, which is also required for gene acquisition in recipient cells, is expressed in the vast majority of the population. Our results indicate that RcGTA-mediated gene transfer combines key aspects of two bacterial horizontal gene transfer mechanisms, where donor DNA is packaged in transducing phage-like particles and recipient cells take up DNA using natural transformation-related machinery. Both of these differentiated subsets of a culture population, donors and recipients, are dependent on the same response regulator, CtrA. IMPORTANCEHorizontal gene transfer (HGT) is a major driver of bacterial evolution and adaptation to environmental stresses. Traits such as antibiotic resistance or metabolic properties can be transferred between bacteria via HGT; thus, HGT can have a tremendous effect on the fitness of a bacterial population. The three classically described HGT mechanisms are conjugation, transformation, and phage-mediated transduction. More recently, the HGT factor GTA was described, where random pieces of producing cell genome are packaged into phage-like particles that deliver DNA to recipient cells. In this report, we show that transport of DNA borne by the R. capsulatus RcGTA into recipient cells requires key genes previously thought to be specific to natural transformation pathways. These findings indicate that RcGTA combines central aspects of phage-mediated transduction and natural transformation in an efficient, regulated mode of HGT.T he first evidence of prokaryotic genetic exchange was transformation, a term coined in 1928 by Griffith (1). Subsequently, conjugation was observed in 1946 (2), followed by transduction, which was discovered in 1952 (3). More recently, another prokaryotic mode of horizontal gene transfer, dependent on an extracellular particle called a gene transfer agent (GTA), was described (4), and GTAs subsequently have been discovered in diverse prokaryotes (5).GTAs varying in morphology have been reported, although most resemble small, tailed double-stranded DNA (dsDNA) bacteriophages. The general criteria that define a GTA are (i) the DNA packaged within the head is insufficient to carry the GTA structural genes; (ii) all GTA particles package only random parts o...

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

“…This is especially complicated when evaluating the RcGTA production and recipient capability systems that are regulated by the CckAChpT-CtrA phosphorelay, because CckA may have opposing activities, depending on the magnitude of expression. Furthermore, our results showed that dprA is not differentially expressed in the ⌬lexA mutant, and because competence genes are coregulated with dprA, they are likely not differentially expressed (6,7).…”

“…LexA appears to directly repress the expression of the histidine kinase homolog CckA, which is involved in the CckA-ChpT-CtrA phosphorelay that regulates several aspects of RcGTA biology, including production, recipient capability, particle maturation, and particle release (4,6,7,12). Because an SOS box is present in the promoter sequence of cckA in environmental isolates B6, YW1, and YW2 (data not shown), whose genomes have been sequenced, in addition to strain SB1003, repression of cckA by LexA appears to be conserved in all strains of R. capsulatus.…”

“…An RcGTA stock was prepared from a DE442 overproducer mutant harboring a plasmid containing the rcc01080 gene (pd1080Gm) interrupted by a gentamicin resistance (Gm r ) cassette (6). This RcGTA stock was used because the Gm r gene is not present in the WT and mutant R. capsulatus strains studied in our work.…”

“…It appears that nonphosphorylated CtrA is needed for expression of RcGTA structural genes early in the induction process and that phosphorylated CtrA (CtrAϳP) is needed late in the induction process for cell lysis and release of RcGTA particles (4,11,12). Furthermore, cells lacking the ctrA gene are unable to act as recipients in RcGTA-mediated gene transfer, because the CtrA protein is also required to induce the expression of comEC, comF, comM, and dprA (6,7).…”

“…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 of the natural-transformation genes comEC and comF are required for the transfer of the DNA fragment into the cytoplasm (6). Integration of the DNA into the recipient's chromosome requires comM, recA, and dprA and may result in allele exchange, again as in natural transformation (7).…”