Gary J. Sarkis

DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics

Genetic studies of Mycobacterium tuberculosis and other mycobacterial pathogens have suffered from the lack of a sophisticated genetic system. To address this issue we have developed a viral system through a detailed characterization of mycobacteriophage L5, a temperate phage that infects both fast‐ and slow‐growing mycobacteria. We describe here the complete DNA sequence of the L5 genome and initial characterization of L5 virion structure and gene expression. In addition to providing a genetic‘toolbox’for the mycobacteria we find that L5 offers a new paradigm for dsDNA phages, being phenotypically temperate but employing genetic strategies for phage growth usually associated with lytic bacteriophages.

L5 LUCIFERASE REPORTER MYCOBACTERIOPHAGES : A SENSITIVE TOOL FOR THE DETECTION AND ASSAY OF LIVE MYCOBACTERIA

Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug‐sensitive and drug‐resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.

CONSTRUCTION OF D29 SHUTTLE PHASMIDS AND LUCIFERASE REPORTER PHAGES FOR DETECTION OF MYCOBACTERIA

Diseases caused by Mycobacterium tuberculosis, M. leprae and M. avium, cause significant morbidity and mortality worldwide. Effective treatments require that the organisms be speciated and that drug susceptibilities for the causative organisms be characterized. Reporter phage technology has been developed as a rapid and convenient method for identifying mycobacterial species and evaluating drug resistance. In this report we describe the construction of luciferase reporter phages from mycobacteriophage D29 DNA. Shuttle phasmids were first constructed with D29 in order to identify non-essential regions of the D29 genomes and to introduce unique cloning sites within that region. Using this approach, we observed that all of the D29 shuttle phasmids had the cosmid vector localized to one area of the phage genome near one cohesive end. These shuttle phasmids had been constructed with a cosmid that could be readily excised from the D29 genome with different sets of restriction enzymes. Luciferase reporter phages were made by substituting the luciferase cassette for the cosmid vector. Recombinant phages with the luciferase cassette fall into two groups. One group produced light and had the expression cassette oriented with the promoter directing transcription away from the cohesive end. In contrast, the other group had the expression cassette in the opposite orientation and failed to produce light during lytic infection, but did produce light in L5 lysogens which are known to repress D29 promoters. These results suggest that a phage promoter of the D29 phage can occlude the expression of a promoter introduced into this region. D29 luciferase reporter phages are capable of detecting low numbers of L5 lysogens like L5 luciferase phages. However, unlike L5 luciferase phages, D29 luciferase phages can readily infect M. tuberculosis and M. bovis BCG, demonstrating that these phages can be used to evaluate drug susceptibilities of many types of mycobacteria.

Structure of a synaptic gamma delta resolvase tetramer covalently linked to two cleaved DNAs.

The structure of a synaptic intermediate of the site-specific recombinase gammadelta resolvase covalently linked through Ser10 to two cleaved duplex DNAs has been determined at 3.4 angstrom resolution. This resolvase, activated for recombination by mutations, forms a tetramer whose structure is substantially changed from that of a presynaptic complex between dimeric resolvase and the cleavage site DNA. Because the two cleaved DNA duplexes that are to be recombined lie on opposite sides of the core tetramer, large movements of both protein and DNA are required to achieve strand exchange. The two dimers linked to the DNAs that are to be recombined are held together by a flat interface. This may allow a 180 degrees rotation of one dimer relative to the other in order to reposition the DNA duplexes for strand exchange.

Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.

Decline in protease activities with age in the nematode Caenorhabditis elegans

The activities of 3 lysosomal proteases in the nematode Caenorhabditis elegans are markedly lower in older animals. The aspartyl protease cathepsin D declines about 10-fold from day 3 (early adulthood) to day 11 (near the mean lifespan); this reflects a net decline in the amount of cathepsin D antigen. The specific activity of the thiol protease cathepsin Ce1 declines about 2.5-fold over the same period, and the specific activity of the thiol protease cathepsin Ce2 declines about 8-fold. The activity of a new non-lysosomal protease, designated cathepsin CeX, is invariant with age. The data are consistent with the hypothesis that reduced protease activity in older animals may cause a decline in the rate of protein turnover with age, but do not prove this hypothesis.