| dc.description.abstract | Mycobacteriophage 13 is one of the few known transducing phages of mycobacteria. For the past several years, our laboratory has been engaged in studying the interaction of this phage with its host, Mycobacterium smegmatis. The present studies form part of this continuing project. The emphasis of this work was on characterizing the interaction of phage 13 with M. smegmatis at the genome level.
To identify the genes responsible for various biochemical functions and to localize them, conditional lethal mutants of phage 13 were isolated. The wild type strain forms turbid plaques on M. smegmatis. For isolating temperature sensitive (ts) mutants, a clear plaque forming (C) mutant was used. A number of ts mutants (that fail to grow at 40°C but grow normally at 37°C) were isolated. Six complementing ts mutants were characterized with respect to:
the time during the growth cycle when the mutated function was required,
the reversibility of the defect, and
the stability of the mutant gene product at non permissive temperature.
The mutations were mapped by recombination analysis, and a linear genetic map was constructed using recombination frequencies. This map was further extended using data available in our laboratory on twelve ts mutants characterized earlier. Based on three point crosses, the position of the plaque morphology gene (C5) was also fixed relative to these ts mutations.
Phage–Host Interaction
Upon infection, phage 13 does not significantly inhibit host specific functions. Only a slight reduction in M. smegmatis DNA synthesis was observed following infection. The ^32P labelled DNA isolated from infected cells hybridized to both phage DNA and host DNA. The extent of hybridization to phage DNA was much higher when infected cells were treated with mitomycin C, suggesting:
preferential synthesis of phage specific DNA at low drug concentrations,
even though host DNA synthesis was not strongly suppressed,
or
a possible homology between phage and host chromosomes.
To investigate the second possibility, melting and reassociation studies were conducted on the two genomes.
Thermal Denaturation and Base Composition
Thermal denaturation analysis revealed that both phage 13 and M. smegmatis genomes contain 66% GC. Analysis of base compositional distribution profiles showed that approximately 80% of both genomes share the same base composition.
Because of the high GC content, reassociation studies required high temperatures. Formamide was therefore used to lower the melting temperature (Tm). Key findings include:
Tm decreases by 0.6°C for every 1% increase in formamide, independent of GC content.
Formamide also increases the width of the melting transition.
Up to 30% formamide does not significantly affect DNA reassociation rates.
Reassociation Kinetics and Nuclease Assay
Nuclease S1 is increasingly used for monitoring DNA reassociation due to its strong discrimination between single stranded and duplex DNA. Its use at high temperatures in the presence of formamide was not reported earlier. This study established that:
S1 nuclease can be used in up to 10% formamide at temperatures as high as 68°C.
When reassociation and nuclease digestion were performed at the same temperature, reaction kinetics agreed more closely with theoretical expectations.
When ^32P labelled phage 13 DNA was reassociated in the presence of excess unlabeled M. smegmatis DNA, a significant increase in reassociation rate was observed. Kinetic analysis suggests the presence of nearly one equivalent of phage genome per host chromosome, indicating considerable sequence homology.
Fine Structure Melting Analysis
Fine structure melting analysis of phage 13 DNA was conducted at 0.2°C increments. Derivative melting profiles were fitted to Gaussian distributions using computational analysis. Thirteen subtransition domains, ranging from 59.5% to 77.1% GC, were identified.
Spheroplast Formation
In addition, a simple and rapid method for preparing spheroplasts of M. smegmatis was developed. The procedure involves:
Sensitization of cells by exposure to glycine for 2 hours.
Enzymatic removal of cell wall material using a combination of lipase and lysozyme.
This treatment converted approximately 90% of the rods into spheroplasts. Although transfection of these spheroplasts with phage 13 DNA was not efficient, they may be useful for genetic studies because they can be fused using polyethylene glycol. | |
