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Analysis of novel mycobacteriophages indicates the existence of different strategies for phage inheritance in mycobacteria.

Stella EJ, Franceschelli JJ, Tasselli SE, Morbidoni HR - PLoS ONE (2013)

Bottom Line: Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100-400 nm, genome size in the 50-70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed.Although all the mycobacteriophages propagated at 30°C, eight of them failed to propagate at 37°C.In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.

View Article: PubMed Central - PubMed

Affiliation: Cátedra de Microbiología, Facultad de Ciencias Médicas, Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT
Mycobacteriophages have been essential in the development of mycobacterial genetics through their use in the construction of tools for genetic manipulation. Due to the simplicity of their isolation and variety of exploitable molecular features, we searched for and isolated 18 novel mycobacteriophages from environmental samples collected from several geographic locations. Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100-400 nm, genome size in the 50-70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed. Although all the mycobacteriophages propagated at 30°C, eight of them failed to propagate at 37°C. Since some of our phages yielded pinpoint plaques, we improved plaque detection by including sub-inhibitory concentrations of isoniazid or ampicillin-sulbactam in the culture medium. Thus, searches for novel mycobacteriophages at low temperature and in the presence of these drugs would allow for the isolation of novel members that would otherwise not be detected. Importantly, while eight phages lysogenized Mycobacterium smegmatis, four of them were also capable of lysogenizing Mycobacterium tuberculosis. Analysis of the complete genome sequence obtained for twelve mycobacteriophages (the remaining six rendered partial genomic sequences) allowed for the identification of a new singleton. Surprisingly, sequence analysis revealed the presence of parA or parA/parB genes in 7/18 phages including four that behaved as temperate in M. tuberculosis. In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.

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Related in: MedlinePlus

Genome organization of mycobacteriophage First.The scheme shows the gene annotation of mycobacteriophage First, the dashed arrow at gp25 represents the (−1) frameshift that generates gp 24 and gp25.
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pone-0056384-g003: Genome organization of mycobacteriophage First.The scheme shows the gene annotation of mycobacteriophage First, the dashed arrow at gp25 represents the (−1) frameshift that generates gp 24 and gp25.

Mentions: The fact that First was capable of infecting both M. smegmatis and M. tuberculosis, and its initial classification as temperate led us to analyze its genome to detect the presence of putative attP, int and xis sequences. Bioinformatic analysis allowed for the identification of open reading frames and possible functions to be assigned (Table S4). The sequence of the ends of this 53028 bp mycobacteriophage, determined by PCR and sequencing of the product, showed a 10 bp 3′ defined overhang with the sequence 5′-CGGTCGGTTA- 3′. The genomic organization of First (represented in Fig. 3) is highly similar to the one displayed by members of Group A, having the gene order terminase, portal, protease, scaffold, capsid, head-tail joining genes, major tail subunits, tapemeasure protein and minor tail protein subunits. First also shares another feature of Cluster A mycobacteriophages in having additional non-structural genes between the terminase and the left end of its genome, identified as the lysis cassette genes. We also detected the presence of a translational (−1) frameshift located between First ORF24 and ORF25, encoding for the tail assembly chaperones; other common feature of Cluster A members. As many other mycobacteriophages, First encodes proteins hypothetically involved in metabolism of nucleotides, such as thymidylate synthase and ribonucleotide reductase. The sequence and annotation of First has been deposited in GenBank under accession number JX899358.


Analysis of novel mycobacteriophages indicates the existence of different strategies for phage inheritance in mycobacteria.

Stella EJ, Franceschelli JJ, Tasselli SE, Morbidoni HR - PLoS ONE (2013)

Genome organization of mycobacteriophage First.The scheme shows the gene annotation of mycobacteriophage First, the dashed arrow at gp25 represents the (−1) frameshift that generates gp 24 and gp25.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585329&req=5

pone-0056384-g003: Genome organization of mycobacteriophage First.The scheme shows the gene annotation of mycobacteriophage First, the dashed arrow at gp25 represents the (−1) frameshift that generates gp 24 and gp25.
Mentions: The fact that First was capable of infecting both M. smegmatis and M. tuberculosis, and its initial classification as temperate led us to analyze its genome to detect the presence of putative attP, int and xis sequences. Bioinformatic analysis allowed for the identification of open reading frames and possible functions to be assigned (Table S4). The sequence of the ends of this 53028 bp mycobacteriophage, determined by PCR and sequencing of the product, showed a 10 bp 3′ defined overhang with the sequence 5′-CGGTCGGTTA- 3′. The genomic organization of First (represented in Fig. 3) is highly similar to the one displayed by members of Group A, having the gene order terminase, portal, protease, scaffold, capsid, head-tail joining genes, major tail subunits, tapemeasure protein and minor tail protein subunits. First also shares another feature of Cluster A mycobacteriophages in having additional non-structural genes between the terminase and the left end of its genome, identified as the lysis cassette genes. We also detected the presence of a translational (−1) frameshift located between First ORF24 and ORF25, encoding for the tail assembly chaperones; other common feature of Cluster A members. As many other mycobacteriophages, First encodes proteins hypothetically involved in metabolism of nucleotides, such as thymidylate synthase and ribonucleotide reductase. The sequence and annotation of First has been deposited in GenBank under accession number JX899358.

Bottom Line: Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100-400 nm, genome size in the 50-70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed.Although all the mycobacteriophages propagated at 30°C, eight of them failed to propagate at 37°C.In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.

View Article: PubMed Central - PubMed

Affiliation: Cátedra de Microbiología, Facultad de Ciencias Médicas, Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT
Mycobacteriophages have been essential in the development of mycobacterial genetics through their use in the construction of tools for genetic manipulation. Due to the simplicity of their isolation and variety of exploitable molecular features, we searched for and isolated 18 novel mycobacteriophages from environmental samples collected from several geographic locations. Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100-400 nm, genome size in the 50-70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed. Although all the mycobacteriophages propagated at 30°C, eight of them failed to propagate at 37°C. Since some of our phages yielded pinpoint plaques, we improved plaque detection by including sub-inhibitory concentrations of isoniazid or ampicillin-sulbactam in the culture medium. Thus, searches for novel mycobacteriophages at low temperature and in the presence of these drugs would allow for the isolation of novel members that would otherwise not be detected. Importantly, while eight phages lysogenized Mycobacterium smegmatis, four of them were also capable of lysogenizing Mycobacterium tuberculosis. Analysis of the complete genome sequence obtained for twelve mycobacteriophages (the remaining six rendered partial genomic sequences) allowed for the identification of a new singleton. Surprisingly, sequence analysis revealed the presence of parA or parA/parB genes in 7/18 phages including four that behaved as temperate in M. tuberculosis. In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.

Show MeSH
Related in: MedlinePlus