Molecular techniques in mycobacteriology

The bacterial genus Mycobacterium belongs to the group of Grain-positive bacteria and holds many species, displaying a broad spectrum of properties. About one third is pathogenic to humans or animals, the rest of the mycobacterial species being relatively harmless soil- or water dwellers. Among the pathogenics are notorious species such as Mycobacterium tuberculosis and Mycobacterium leprae. On an annual basis, 3 million people die of tuberculosis, and 15 million suffer from leprosy. Therefore the pathogenic mycobacteria are subject of intensive study, with as main goals the understanding of the processes of infection and the development of medication and vaccination. But also the non-pathogenic mycobacteria are subject of intensive study. Some species are able to degrade recalcitrant organic compounds, which makes them interesting for environmental applications. The biotechnological production of valuable intermediates in the synthesis of pharmaceuticals might benefit as well from the application of mycobacteria. Unfortunately, the genus Mycobacterium is not very cooperative. On the one hand there are obvious intrinsic problems in the working with pathogenic microorganisms, on the other hand, mycobacteria are relatively inaccessible to the standard microbiological methods. This is for a great part caused by the presence of a thick cell wall, which impedes the standard methods for the introduction of modified DNA-molecules (transformation) from being applied. Furthermore, the fact that Mycobacterium is evolutionarily distant from bacterial species as Escherichia coli and Bacillus sp. , for which most molecular biological techniques are developed, causes the incompatibility of DNA-vectors between Mycobacterium and E. coli Chapter 2 of this thesis gives an overview of recent developments in these areas of research. The development of transformation techniques and DNA-vectors is described in detail. The development of DNA-vectors exhibits a clear tendency from species-specific towards so-called broad-host-range vectors, that can maintain themselves in other bacterial species as well. Chapter 3 reports on the development of a transformation technique for Mycobacterium aurum, based on electroporation of mycobacteria with intact cell walls. Employment of this technique rendered the solution to the problems encountered when trying to transform M. aurum in the 'classical' way, even after partial removal of the cell wall. With these 'spheroplasts' the regeneration of the cell wall appeared to be the barrier to successful transformation. Transformation of bacteria using the technique of electroporation is based on the submission of a suspension of bacteria to a strong electrical field for a short period of time. In this way the cell membranes become transiently permeable to macromolecules. The plasmid employed, pAL8, originated from a closely related Mycobacterium species, enhancing the prospect of successful expression in M. aurum. Indeed, pAL8 could maintain and express itself in M. aurum, thus enabling the optimization of the transformation technique for M. aurum. By treating the cells with wall degrading agents prior to electroporation, transformation yields could still be enhanced. Unfortunately, pAL8 is not very suitable as a vector for the transport of manipulated genes (cloning). Therefore, further investigations were aimed at finding suitable vectors for the cloning of mycobacterial genes. In a first approach a series of plasmids from related Gram-positive microorganisms were tested for transformation capability of M. aurum. Surprisingly, the vector plasmid that was least related to Mycobacterium was the only one to be expressed in M. aurum. This plasmid, pJRD215, is a construct based on a so-called broad-host-range plasmid, which is found in many Gram-negative bacterial species, none of which is related to Mycobacterium. In fact, this was the first report of the transformation of a Gram-positive bacterium with a Grainnegative broad-host-range plasmid. Many bacterial species involved in biotechnological processes are Gram-positive, and might be transformed with pJRD215 as well. Chapter 5 reports the transformation of five more Grainpositive bacterial species that could be transformed with pJRD215, which widens the range of application of this plasmid.