Characterization of the replicator region of megaplasmid pTAV3 of Paracoccus versutus and search for plasmid-encoded traits The GenBank accession number for the sequence reported in this paper is AF390867.

ABSTRACT⌽LM21 is a temperate phage isolated from Sinorhizobium sp. strain LM21 (Alphaproteobacteria). Genomic analysis and electron microscopy suggested that ⌽LM21 is a member of the family Siphoviridae. The phage has an isometric head and a long noncontractile tail. The genome of ⌽LM21 has 50,827 bp of linear double-stranded DNA encoding 72 putative proteins, including proteins responsible for the assembly of the phage particles, DNA packaging, transcription, replication, and lysis. Virion proteins were characterized using mass spectrometry, leading to the identification of the major capsid and tail components, tape measure, and a putative portal protein. We have confirmed the activity of two gene products, a lytic enzyme (a putative chitinase) and a DNA methyltransferase, sharing sequence specificity with the cell cycle-regulating methyltransferase (CcrM) of the bacterial host. Interestingly, the genome of Sinorhizobium phage ⌽LM21 shows very limited similarity to other known phage genome sequences and is thus considered unique. IMPORTANCEProphages are known to play an important role in the genomic diversification of bacteria via horizontal gene transfer. The influence of prophages on pathogenic bacteria is very well documented. However, our knowledge of the overall impact of prophages on the survival of their lysogenic, nonpathogenic bacterial hosts is still limited. In particular, information on prophages of the agronomically important Sinorhizobium species is scarce. In this study, we describe the isolation and molecular characterization of a novel temperate bacteriophage, ⌽LM21, of Sinorhizobium sp. LM21. Since we have not found any similar sequences, we propose that this bacteriophage is a novel species. We conducted a functional analysis of selected proteins. We have demonstrated that the phage DNA methyltransferase has the same sequence specificity as the cell cycle-regulating methyltransferase CcrM of its host. We point out that this phenomenon of mimicking the host regulatory mechanisms by viruses is quite common in bacteriophages.

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“…LM21 using a genomic DNA purification kit (Thermo Scientific). Triparental mating was performed as previously described (24,25).…”

Section: Methodsmentioning

confidence: 99%

Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity

Dziewit

Oscik

Bartosik

et al. 2014

J Virol

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“…The stability of plasmids during growth under nonselective conditions was tested as described previously (10). Briefly, stationaryphase cultures were diluted in fresh medium without antibiotic selection and cultivated for approximately 10, 20, and 30 generations.…”

Section: Methodsmentioning

confidence: 99%

The SXT Conjugative Element and Linear Prophage N15 Encode Toxin-Antitoxin-Stabilizing Systems Homologous to the tad-ata Module of the Paracoccus aminophilus Plasmid pAMI2

et al. 2007

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A group of proteic toxin-antitoxin (TA) cassettes whose representatives are widely distributed among bacterial genomes has been identified. These cassettes occur in chromosomes, plasmids, bacteriophages, and noncomposite transposons, as well as in the SXT conjugative element of Vibrio cholerae. The following four homologous loci were subjected to detailed comparative studies: (i) tad-ata from plasmid pAMI2 of Paracoccus aminophilus (the prototype of this group), (ii) gp49-gp48 from the linear bacteriophage N15 of Escherichia coli, (iii) s045-s044 from SXT, and (iv) Z3230-Z3231 from the genomic island of enterohemorrhagic Escherichia coli O157:H7 strain EDL933. Functional analysis revealed that all but one of these loci (Z3230-Z3231) are able to stabilize heterologous replicons, although the host ranges varied. The TA cassettes analyzed have the following common features: (i) the toxins are encoded by the first gene of each operon; (ii) the antitoxins contain a predicted helix-turn-helix motif of the XRE family; and (iii) the cassettes have two promoters that are different strengths, one which is located upstream of the toxin gene and one which is located upstream of the antitoxin gene. All four toxins tested are functional in E. coli; overexpression of the toxins (in the absence of antitoxin) results in a bacteriostatic effect manifested by elongation of bacterial cells and growth arrest. The toxins have various effects on cell viability, which suggests that they may recognize different intracellular targets. Preliminary data suggest that different cellular proteases are involved in degradation of antitoxins encoded by the loci analyzed.Bacterial plasmids have modular structures, since it is possible to dissect them into several functional cassettes. The core region of the plasmid backbone is composed of a set of conserved modules, coding for replication and stability functions, which are crucial for plasmid maintenance. Comparative analyses of plasmid genomes have revealed that the same modules or closely related modules can be found in various combinations in different plasmids hosted by phylogenetically distinct bacteria. The plasticity of plasmid genomes is the result of horizontal gene transfer, as well as numerous recombinational events, which take place in diverse bacterial hosts. Recent advances in bacterial genomics have revealed that shuffling of modules is not limited to plasmids but is a much more common phenomenon, occurring in all bacterial mobile genetic elements. These elements may thus be considered combinations of exchangeable functional cassettes (78).Shuffling of modules, initially defined in plasmids, bacteriophages, and transposable elements, has resulted in (i) the generation of novel mobile elements, such as conjugative transposons (containing phage-related recombination modules plus plasmid-borne modules for conjugational transfer) (64), and (ii) the appearance of chimeric elements exhibiting new, unusual properties, including transposable bacteriophage Mu (containing a transposition mod...

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“…The construction of the shuttle entrapment vector was performed in E. coli TG1 in two steps. The E. coli-specific mobilizable vector pABW1 (Bartosik et al, 1997) was digested with PstI and ligated with the 2?9 kb entrapment cartridge from pGBG1 (Schneider et al, 2000). The resulting plasmid (pMMB1) was digested with XbaI and KpnI and ligated with a 5?6 kb linear form of the repABC-containing mini-replicon pTAV320 (Bartosik et al, 1998), recovered from shuttle plasmid pABW22A (Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…1; see Methods for details). This plasmid was a fusion of: (i) an E. coli-specific vector, pABW1 (Bartosik et al, 1997); (ii) mini-replicon pTAV320, carrying a repABC-type replicon originating from low-copy-number plasmid pTAV1 (107 kb) of Paracoccus versutus UW1 (Bartosik et al, 1998); and (iii) a cI-tetA selective cartridge (Schneider et al, 2000).…”

Section: Identification Of Tnppa1mentioning

confidence: 99%

Identification of a transposable genomic island of Paracoccus pantotrophus DSM 11072 by its transposition to a novel entrapment vector pMMB2

et al. 2006

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A novel shuttle entrapment vector, pMMB2, was used to identify a large transposable element, TnPpa1 (44?3 kb), of Paracoccus pantotrophus DSM 11072. TnPpa1 has a composite structure with divergently oriented copies of a cryptic transposon, Tn3434 (Tn3 family), located at both termini. The core region of the element contains a large set of putative genes, whose products show similarity to enzymes involved in central intermediary metabolism (e.g. tricarboxylic acid cycle or 2-methylcitrate cycle), transporters, transcriptional regulators and conserved proteins of unknown function. A 4?2 kb DNA segment of TnPpa1 is homologous to a region of chromosome cII of Rhodobacter sphaeroides 2.4.1, which exemplifies the mosaic structure of this element. TnPpa1 is bordered by 5 bp long directly repeated sequences and is located within a mega-sized replicon, pWKS5, in the DSM 11072 genome. Spontaneous inversion of the core region of TnPpa1 was detected in the host genome. Analysis of the distribution of TnPpa1 in three other strains of P. pantotrophus revealed that this element was present exclusively within DSM 11072, which suggests its relatively recent acquisition by lateral transfer. The identification of TnPpa1 (which may be considered a transposable genomic island) provides evidence for the transposition and lateral transfer of large DNA segments of chromosomal origin (carrying various housekeeping genes), which may have a great impact on the evolution of bacterial genomes.

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Characterization of the replicator region of megaplasmid pTAV3 of Paracoccus versutus and search for plasmid-encoded traits The GenBank accession number for the sequence reported in this paper is AF390867.

Cited by 23 publications

References 43 publications

Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity

Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity

The SXT Conjugative Element and Linear Prophage N15 Encode Toxin-Antitoxin-Stabilizing Systems Homologous to the tad-ata Module of the Paracoccus aminophilus Plasmid pAMI2

Identification of a transposable genomic island of Paracoccus pantotrophus DSM 11072 by its transposition to a novel entrapment vector pMMB2

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