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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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PFGE of DNA of mycobacteriophages Jolie, Mine, and 41HC.DNA of mycobacteriophages Jolie, Mine, and 41HC (not heated (NH) or heated (H) at 80°C for 10 min and cooled down in ice immediately prior to loading. DNA molecular weight (kb) on the standards is indicated at the right.
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pone-0056384-g002: PFGE of DNA of mycobacteriophages Jolie, Mine, and 41HC.DNA of mycobacteriophages Jolie, Mine, and 41HC (not heated (NH) or heated (H) at 80°C for 10 min and cooled down in ice immediately prior to loading. DNA molecular weight (kb) on the standards is indicated at the right.

Mentions: Preliminary results of the analysis of phage DNA by six restriction enzyme digestion showed that the phages were clearly different although some shared bands of comparable size for some of the restriction enzymes used (data not shown). The genome size of these newly isolated mycobacteriophages was determined by Pulse Field Gel Electrophoresis (PFGE); the results obtained showed that most of them (12/18 phages) had genomes of 42–55 kbp, while six showed genome sizes in the range of 62–70 kbp. Interestingly several of the mycobacteriophages analyzed showed bands compatible with multimers of their genomes, requiring a heating step prior to loading into the gel to avoid this behaviour (Fig. 2).This result suggested the presence of cohesive ends [34] in our mycobacteriophages genomes, in good agreement with an almost 70% of mycobacteriophage genomes with such features previously described (http://phagesdb.org/).


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)

PFGE of DNA of mycobacteriophages Jolie, Mine, and 41HC.DNA of mycobacteriophages Jolie, Mine, and 41HC (not heated (NH) or heated (H) at 80°C for 10 min and cooled down in ice immediately prior to loading. DNA molecular weight (kb) on the standards is indicated at the right.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056384-g002: PFGE of DNA of mycobacteriophages Jolie, Mine, and 41HC.DNA of mycobacteriophages Jolie, Mine, and 41HC (not heated (NH) or heated (H) at 80°C for 10 min and cooled down in ice immediately prior to loading. DNA molecular weight (kb) on the standards is indicated at the right.
Mentions: Preliminary results of the analysis of phage DNA by six restriction enzyme digestion showed that the phages were clearly different although some shared bands of comparable size for some of the restriction enzymes used (data not shown). The genome size of these newly isolated mycobacteriophages was determined by Pulse Field Gel Electrophoresis (PFGE); the results obtained showed that most of them (12/18 phages) had genomes of 42–55 kbp, while six showed genome sizes in the range of 62–70 kbp. Interestingly several of the mycobacteriophages analyzed showed bands compatible with multimers of their genomes, requiring a heating step prior to loading into the gel to avoid this behaviour (Fig. 2).This result suggested the presence of cohesive ends [34] in our mycobacteriophages genomes, in good agreement with an almost 70% of mycobacteriophage genomes with such features previously described (http://phagesdb.org/).

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