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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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Partitioning loci location in mycobacteriophages.Schematic representations of the mycobacteriophage genomes showing the location of putative parA and parB genes and sequences of tandem repeats (TR) functioning as putative centromers. Genes transcribed leftward are indicated. RedRock is included as a representative of parA/parB containing mycobacteriophages as published by Hatfull [31].
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pone-0056384-g004: Partitioning loci location in mycobacteriophages.Schematic representations of the mycobacteriophage genomes showing the location of putative parA and parB genes and sequences of tandem repeats (TR) functioning as putative centromers. Genes transcribed leftward are indicated. RedRock is included as a representative of parA/parB containing mycobacteriophages as published by Hatfull [31].

Mentions: Due to the detection of parA and parB genes in First, and taking in consideration previous reports showing the presence of such genes in Leptospira[39], [40] and Escherichia coli[41], [42] bacteriophages and in some mycobacteriophages [31], we analyzed the presence of the mentioned genes in all our isolates as well as their relatedness to others available in public databases. The results showed that parA could be identified in mycobacteriophages 20ES, 40AC, 41HC, CRB1 in addition to First. Also, analysis of the partial genome sequences of mycobacteriophages 21AM and 21AS, revealed the presence of hypotetical ParA proteins. Interestingly, parB genes were found in all the above mentioned mycobacteriophages excepted for phage 40AC, that behaves as a lytic phage. Thus, from our reported eight “lysogenic” mycobacteriophages, six contain parA/parB genes ((Table 2). Moreover, a search in a non-redundant database showed the presence of centrally located parA and parB genes in the genomes of Cluster A mycobacteriophages RedRock, PackMan, Alma, EricB, Hammer, Gladiator, Jeffabunny, DaVinci and Blue7 and in mycobacteriophage TA17A, a Cluster B member [35], [43]. Our own phages also display a central position for parA and parB genes except in the case of phage 41HC, in which the partitioning locus is located in a terminal position (Fig. 4).


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)

Partitioning loci location in mycobacteriophages.Schematic representations of the mycobacteriophage genomes showing the location of putative parA and parB genes and sequences of tandem repeats (TR) functioning as putative centromers. Genes transcribed leftward are indicated. RedRock is included as a representative of parA/parB containing mycobacteriophages as published by Hatfull [31].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056384-g004: Partitioning loci location in mycobacteriophages.Schematic representations of the mycobacteriophage genomes showing the location of putative parA and parB genes and sequences of tandem repeats (TR) functioning as putative centromers. Genes transcribed leftward are indicated. RedRock is included as a representative of parA/parB containing mycobacteriophages as published by Hatfull [31].
Mentions: Due to the detection of parA and parB genes in First, and taking in consideration previous reports showing the presence of such genes in Leptospira[39], [40] and Escherichia coli[41], [42] bacteriophages and in some mycobacteriophages [31], we analyzed the presence of the mentioned genes in all our isolates as well as their relatedness to others available in public databases. The results showed that parA could be identified in mycobacteriophages 20ES, 40AC, 41HC, CRB1 in addition to First. Also, analysis of the partial genome sequences of mycobacteriophages 21AM and 21AS, revealed the presence of hypotetical ParA proteins. Interestingly, parB genes were found in all the above mentioned mycobacteriophages excepted for phage 40AC, that behaves as a lytic phage. Thus, from our reported eight “lysogenic” mycobacteriophages, six contain parA/parB genes ((Table 2). Moreover, a search in a non-redundant database showed the presence of centrally located parA and parB genes in the genomes of Cluster A mycobacteriophages RedRock, PackMan, Alma, EricB, Hammer, Gladiator, Jeffabunny, DaVinci and Blue7 and in mycobacteriophage TA17A, a Cluster B member [35], [43]. Our own phages also display a central position for parA and parB genes except in the case of phage 41HC, in which the partitioning locus is located in a terminal position (Fig. 4).

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