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

Westbye, Alexander B.; Leung, Molly M.; Florizone, Sarah Marie; Taylor, Terumi A.; Johnson, J. A.; Fogg, Paul C. M.; Beatty, J. Thomas

doi:10.1128/jb.00669-13

Cited by 45 publications

(67 citation statements)

References 53 publications

(96 reference statements)

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“…Transcription of these lysis genes was shown to be regulated by the histidine sensor kinase CckA. Evidently, a holin-endolysin system, not recognized in the early studies by Lang and Beatty, mediates GTA release from this alphaproteobacterium (60). It is likely that this mechanism generally provides the means for GTA release.…”

Section: Involvement Of Holins In Bacterial Gene Transfermentioning

confidence: 97%

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Holins in Bacteria, Eukaryotes, and Archaea: Multifunctional Xenologues with Potential Biotechnological and Biomedical Applications

2015

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bHolins form pores in the cytoplasmic membranes of bacteria for the primary purpose of releasing endolysins that hydrolyze the cell wall and induce cell death. Holins are encoded within bacteriophage genomes, where they promote cell lysis for virion release, and within bacterial genomes, where they serve a diversity of potential or established functions. These include (i) release of gene transfer agents, (ii) facilitation of programs of differentiation such as those that allow sporulation and spore germination, (iii) contribution to biofilm formation, (iv) promotion of responses to stress conditions, and (v) release of toxins and other proteins. There are currently 58 recognized families of holins and putative holins with members exhibiting between 1 and 4 transmembrane ␣-helical spanners, but many more families have yet to be discovered. Programmed cell death in animals involves holin-like proteins such as Bax and Bak that may have evolved from bacterial holins. Holin homologues have also been identified in archaea, suggesting that these proteins are ubiquitous throughout the three domains of life. Phage-mediated cell lysis of dualmembrane Gram-negative bacteria also depends on outer membrane-disrupting "spanins" that function independently of, but in conjunction with, holins and endolysins. In this minireview, we provide an overview of their modes of action and the first comprehensive summary of the many currently recognized and postulated functions and uses of these cell lysis systems. It is anticipated that future studies will result in the elucidation of many more such functions and the development of additional applications.

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Section: Involvement Of Holins In Bacterial Gene Transfermentioning

confidence: 97%

“…Westbye et al (60) identified the promoter of the first gene in this GTA gene cluster and showed that gene transfer frequencies depend on the growth medium. Surprisingly, millimolar concentrations of phosphate posttranslationally inhibited the lysis-dependent release of GTA from the cells.…”

Section: Involvement Of Holins In Bacterial Gene Transfermentioning

confidence: 99%

Holins in Bacteria, Eukaryotes, and Archaea: Multifunctional Xenologues with Potential Biotechnological and Biomedical Applications

2015

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“…R. capsulatus strains lacking the unlinked cckA, chpT, or ctrA genes are unable to act as transduction donors either because RcGTA structural gene transcripts are not induced (ctrA mutants) or because maturation and release of RcGTA are impaired (cckA and chpT mutants). 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 …”

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confidence: 99%

“…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)(3)(4). RcGTA particles recognize a capsular polysaccharide receptor on R. capsulatus cells using spikes located on the RcGTA capsid (5).…”

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

et al. 2016

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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...

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“…The RcGTA primary structural gene cluster consists of an ϳ15-kb region of the chromosome, and additional essential factors, including a holin and endolysin, are encoded in distant genome regions (5,7,9). Production of RcGTA requires the CtrA response regulator, and transcription is modulated by the GtaI/R quorumsensing system (10)(11)(12).…”

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

et al. 2015

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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...

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

Cited by 45 publications

References 53 publications

Holins in Bacteria, Eukaryotes, and Archaea: Multifunctional Xenologues with Potential Biotechnological and Biomedical Applications

Holins in Bacteria, Eukaryotes, and Archaea: Multifunctional Xenologues with Potential Biotechnological and Biomedical Applications

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

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

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