Although phages able to infect mycobacteria were reported several years ago, they were mainly used for typification purposes (13, 23, 61) -except for the very early work by Y. Mizuguchi on lysogeny by mycobacteriophages, mostly only available in Japanese language- until Dr. Bill Jacobs and Dr. Graham Hatfull joined forces to tackle the genetic analysis of M. tuberculosis. By the early 90s, although the chemical composition of the lipid-rich cell envelope of this pathogen had been analyzed in detail, and there were several studies on the immunological and cell biology aspects of the infection, up to that moment there had been no means to genetically study the tubercle bacilli or any other mycobacteria. The lack of natural or chemically induced competence for transformation was one of the reasons for this road block. Jacobs and Hatfull assumed that -as has happened in other bacterial systems- phages may provide regulatory signals and genetic elements that could be used for vector design. Thus, they started to look for novel mycobacteriophages in the environment and, while embarking in a very fruitful "phage-hunting" program (81), they constructed cloning vectors containing promoters of mycobacteriophages L5 and D29 (80, 98). Those cloning vectors, which were rapidly adopted by the scientific community, constituted the first generation of vectors for genetic manipulation of mycobacteria, although suitable conditions for transformation through electroporation had to be developed for their widespread utilization (45, 97). Temperature-sensitive derivatives of phage TM4, deficients in replication at high temperature, were also obtained, allowing the regulated delivery of transposons -carrying genes encoding for resistance to antibiotics- into the mycobacterial chromosome (12). In that way, random insertions which may cause auxotrophies could easily be obtained. Since the genetic sequence of the transposon is known, junction DNA sequences could be obtained with little difficulty leading to the identification of chromosomal position of the insertion site. Therefore, insertional mutants of M. tuberculosis H37Rv (a virulent lab strain) could be used for infection of susceptible animal models such as mice or even in vitro infection of macrophages, which is the usual niche for this pathogen. Thus, the place -and therefore the gene- in the genome where the transposon was inserted could be identified, allowing for the first studies focused on the impact of the loss of specific genes in the virulence of M. tuberculosis (58, 59, 92-94). Through a highly defined PCR-based cloning system in a cosmid, Hatfull and Jacobs were also able to make precise deletions in any gene of the mycobacterial genome, provided that the sequence of adjacent DNA was known (11). That was the first time in which "custom-made" mutants were obtained in mycobacteria by using mycobacteriophages (although it was also accomplished at the same time by the utilization of suicide plasmids by Gicquels group at the Institut Pasteur), thus opening the door for an exhaustive functional and structural protein analysis. Moreover, as mentioned above, mutants carrying specific gene deletions could be tested for their virulence attenuation, helping to pin down the genes involved in pathogenesis. Thus, phages were not only a successful and handy tool-box for development of cloning vectors but also a scaffolding used for strain construction by means of transduction of chromosomal fragments. Unfortunately, that could only be achieved in the non-pathogenic saprophytic M. smegmatis, since so far no generalized transducing phage has been described for M. tuberculosis. From that point on, the knowledge gathered by the application of these novel phage-based technologies has been breathtaking, leading -among several other examples- to the understanding of the differences during the onset of the infection between the pathogenic M. tuberculosis and the vaccine strain M. bovis var. BCG, the identification of most of the targets for currently used anti-tubercular drugs and epidemiological studies on the distribution of strains with different level of virulence (14, 39, 59, 109, 114). As mentioned before, the quest for novel tools for genetic analysis led these researchers to a phage-hunting program that was used for undergraduate student mentorship in biological sciences (47). During that exciting period, 50 new phages were isolated, sequenced and characterized, giving copious amounts of information on gene organization and horizontal gene transfer in mycobacteria (37, 38, 46, 66, 67, 74, 82, 83). The genetic analysis of those newly isolated phages showed high diversity, but also a remarkable degree of mosaicism, product of illegitimate recombination (81). Hatfull et al. (46) recently reported a summary of the information obtained in the analysis of 37 of those 50 new mycobacteriophages, pointing out that all of them were double-stranded DNA phages, most of them belonged to the group of Sipho-viridae phages with long flexible non-contractile tails, while a few belonged to the group of the Myoviridae phages that contain contractile tails. Most of the mycobacteriophages showed isometric heads at the electron microscope, although some of them (e.g. Che9c, Corndog) exhibited large elongated heads. While lytic genetic cassettes (encoding for enzymes involved in "poking" holes in the bacterial cell membrane to help virion release) have been detected in the majority of the sequenced phages, integration components (attP sites and recombinases) are roughly present in half of them (46). A very interesting outcome of those studies is the detection of proteins involved in recombination, some of which are homologous to the well-known RecA bacterial protein. In a strikingly important and elegant approach, the Hatfulls group has recently developed a method based on recombinogenic enzymes from phage Che9, which might soon replace the specialized transduction method (104-107). The "recombineering" method avoids the costly in vitro cosmid packaging, thus it is much cheaper and will soon become the preferred method for creating specific deletions in the mycobacterial chromosome. Although more studies are needed, it is clear that phages are a treasure chest, which will be providing a large number of genetic elements for mycobacteria manipulation for a long time.
Mossberg Model 183 Manual
15. Bonington A, Strang JI, Klapper PE, Hood SV, Parish A, Swift PJ, et al. TB PCR in the early diagnosis of tuberculous meningitis: evaluation of the Roche semi-automated COBAS Amplicor MTB test with reference to the manual Amplicor MTB PCR test. Tuber Lung Dis 2000; 80: 191-6. 2ff7e9595c
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