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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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Plaque morphology changes by temperature or addition of antibiotics.A. Aliquots containing ≈100 PFU of phages Jolie1 and Bahia1 were adsorbed to aliquots of stationary M. smegmatis cultures grown at 30°C or 37°C, mixed with 4 ml of top agar and poured on 7H9-Gly-agar plates. Incubation proceeded for 48 h a30°C, 37°C or 42°C. For the sake of simplicity only the results at 30°C are displayed as results with cells grown at 37°C were identical. B. Bacteriophage 39HC was plated on lawns of M. smegmatis mc2155 grown on 7H9-Gly medium or in the same medium containing either Isoniazid (INH) or a commercial combination of Ampicillin-Sulbactam (Ap-Sb). Antibiotics were added to the bottom agar to final concentrations of 1.0 µg/ml (INH), or 5.0 µg/ml (Ap-Sb), respectively. For plate preparation, 4 ml of the top agar and 100 ml of an overnight bacterial culture (1×109 CFU) were used. Magnification is shown at 2.5X of the original size.
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pone-0056384-g001: Plaque morphology changes by temperature or addition of antibiotics.A. Aliquots containing ≈100 PFU of phages Jolie1 and Bahia1 were adsorbed to aliquots of stationary M. smegmatis cultures grown at 30°C or 37°C, mixed with 4 ml of top agar and poured on 7H9-Gly-agar plates. Incubation proceeded for 48 h a30°C, 37°C or 42°C. For the sake of simplicity only the results at 30°C are displayed as results with cells grown at 37°C were identical. B. Bacteriophage 39HC was plated on lawns of M. smegmatis mc2155 grown on 7H9-Gly medium or in the same medium containing either Isoniazid (INH) or a commercial combination of Ampicillin-Sulbactam (Ap-Sb). Antibiotics were added to the bottom agar to final concentrations of 1.0 µg/ml (INH), or 5.0 µg/ml (Ap-Sb), respectively. For plate preparation, 4 ml of the top agar and 100 ml of an overnight bacterial culture (1×109 CFU) were used. Magnification is shown at 2.5X of the original size.

Mentions: Surprisingly, while all the mycobacteriophages gave lysis plaques at 30°C, ten of them (First, 20ES, 21AM, 21AS, 32HC, Bahia1, 40AC, 41HC, 19ES and CRB1) were able to form plaques (comparable to or smaller than the ones obtained at 30°C) at 37°C but none propagated at 42°C (Fig. 1A).


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)

Plaque morphology changes by temperature or addition of antibiotics.A. Aliquots containing ≈100 PFU of phages Jolie1 and Bahia1 were adsorbed to aliquots of stationary M. smegmatis cultures grown at 30°C or 37°C, mixed with 4 ml of top agar and poured on 7H9-Gly-agar plates. Incubation proceeded for 48 h a30°C, 37°C or 42°C. For the sake of simplicity only the results at 30°C are displayed as results with cells grown at 37°C were identical. B. Bacteriophage 39HC was plated on lawns of M. smegmatis mc2155 grown on 7H9-Gly medium or in the same medium containing either Isoniazid (INH) or a commercial combination of Ampicillin-Sulbactam (Ap-Sb). Antibiotics were added to the bottom agar to final concentrations of 1.0 µg/ml (INH), or 5.0 µg/ml (Ap-Sb), respectively. For plate preparation, 4 ml of the top agar and 100 ml of an overnight bacterial culture (1×109 CFU) were used. Magnification is shown at 2.5X of the original size.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585329&req=5

pone-0056384-g001: Plaque morphology changes by temperature or addition of antibiotics.A. Aliquots containing ≈100 PFU of phages Jolie1 and Bahia1 were adsorbed to aliquots of stationary M. smegmatis cultures grown at 30°C or 37°C, mixed with 4 ml of top agar and poured on 7H9-Gly-agar plates. Incubation proceeded for 48 h a30°C, 37°C or 42°C. For the sake of simplicity only the results at 30°C are displayed as results with cells grown at 37°C were identical. B. Bacteriophage 39HC was plated on lawns of M. smegmatis mc2155 grown on 7H9-Gly medium or in the same medium containing either Isoniazid (INH) or a commercial combination of Ampicillin-Sulbactam (Ap-Sb). Antibiotics were added to the bottom agar to final concentrations of 1.0 µg/ml (INH), or 5.0 µg/ml (Ap-Sb), respectively. For plate preparation, 4 ml of the top agar and 100 ml of an overnight bacterial culture (1×109 CFU) were used. Magnification is shown at 2.5X of the original size.
Mentions: Surprisingly, while all the mycobacteriophages gave lysis plaques at 30°C, ten of them (First, 20ES, 21AM, 21AS, 32HC, Bahia1, 40AC, 41HC, 19ES and CRB1) were able to form plaques (comparable to or smaller than the ones obtained at 30°C) at 37°C but none propagated at 42°C (Fig. 1A).

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