Biosynthesis of diaminopimelate, the precursor of lysine and a component of peptidoglycan, is an essential function of Mycobacterium smegmatis

Abstract

Diaminopimelate (DAP) is a unique metabolite used for both the biosynthesis of lysine in bacteria and the construction of the peptidoglycan of many species of bacteria, including mycobacteria. DAP is synthesized by bacteria as part of the aspartate amino acid family, which includes methionine, threonine, isoleucine, and lysine. Aspartokinase, the first enzyme in this pathway, is encoded by the ask gene in mycobacteria. Previous attempts to disrupt this gene in Mycobacterium smegmatis were unsuccessful, even when the cells were supplied with all the members of the aspartate family, suggesting that unlike other bacteria, mycobacteria may have an absolute requirement for this pathway even when growing in rich medium containing DAP. The purpose of this study was to determine if the ask gene and the aspartate pathway are essential to M. smegmatis. This study describes a test for gene essentiality in mycobacteria, utilizing a counterselectable marker (streptomycin resistance) in conjunction with a specially constructed merodiploid strain. We have used this system to show that the ask gene could not be disrupted in wild-type M. smegmatis, using selective rich medium supplemented with DAP unless there was an extra copy of ask provided elsewhere in the chromosome. Disruption of ask was also possible in a lysine auxotroph incapable of converting DAP to lysine. The ask mutant, mc 2 1278 (ask1::aph), exhibits multiple auxotrophy (Met ؊ , Thr, and Lys ؊ ) and is complemented by the ask gene. This is the first description of DAP auxotrophy in mycobacteria. The ask mutant lyses when deprived of DAP in culture, a characteristic which can be exploited for the reproducible preparation of protoplasts and mycobacterial extracts. The evidence presented here indicates that the aspartate pathway is essential to M. smegmatis and that DAP is the essential product of this pathway.Globally, tuberculosis is the leading cause of death in adults due to an infectious organism (20). It is estimated that 90 million new tuberculosis cases resulting in 30 million deaths can be expected during the last decade of this century (51). The resurgence of tuberculosis in developing nations (62), the appearance of multidrug-resistant strains of Mycobacterium tuberculosis (62), and the problem of tuberculosis in the immunocompromised (28) call for further study of mycobacteria. More knowledge about the basic biology of mycobacteria is needed in order to develop a deeper understanding of the pathogenesis of mycobacterial diseases. Furthermore, identification of biological processes specifically essential for the growth and development of mycobacteria will allow the rational design of drugs to inhibit those processes.The complex cell envelope of the mycobacteria is an outstanding feature of these organisms (13). The envelope is composed of a variety of complex lipids including the long-chain mycolic acids and unique polysaccharides such as arabinogalactan and arabinomannan (7). These components contribute to the hydrophobic nature of the mycobacterial cell s...

Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry

“…Mesodiaminopimelate (DAP) is an essential component of the peptidoglycan layer of bacterial cell walls, and disruption of its biosynthesis results in cell death, probably due to instability of the peptidoglycan (Pavelka & Jacobs, 1996). L-Lysine is produced by the enzymatic decarboxylation of meso-diaminopimelate and is essential for protein synthesis.…”

galK-based suicide vector mediated allelic exchange in Mycobacterium abscessus

“…The key to this is a suitable counterselectable marker, such as sacB (conferring sucrose sensitivity), rpsL (conferring streptomycin susceptibility in a streptomycin-resistant background) or galK (conferring 2-deoxygalactose susceptibility) [14,[27][28][29]. Others have shown that sacB does not work in M. abscessus, and the rpsL system requires a pre-existing streptomycin resistance mutation in the choromosomal copy of rpsL, which complicates strain construction [16,23,29]. Additionally, in preliminary experiments (not shown), we found it difficult to isolate streptomycin-resistant mutants that could be used with our previously developed rpsL suicide vectors.…”

“…Plasmids used for recombination were prepared using Qiagen columns (Qiagen, Valencia, CA) and, when required, the DNA was UV treated by irradiating 15 µl of a~0.5 mg ml À1 DNA preparation on a sterile, lidless polystyrene Petri dish in a UV Stratalinker 2400 (Agilent Technologies, Santa Clara, CA) on the default setting for timed exposure. One microlitre of each irradiated suicide vector was used for each electroporation, under conditions previously described for M. smegmatis [23]. Southern blotting was done with genomic DNA using the Amersham ECL DIRECT Nucleic acid [24], using iProof High-Fidelity DNA Polymerase from Bio Rad (Hercules, CA).…”

“…Genomic DNA from E. coli was prepared using a guanidinium thiocyanate method [14], while that from M. abscessus was prepared using a cetyltrimethylammonium bromide protocol [23]. Restriction and DNA modification enzymes were obtained from Fermentas (Hanover, MD) or New England Biolabs (Beverly, MA).…”

Molecular Interaction of Novel Compound 2-Methylheptyl Isonicotinate Produced by Streptomyces sp. 201 with Dihydrodipicolinate Synthase (DHDPS) Enzyme of Mycobacterium tuberculosis for its Antibacterial Activity

“…It has been observed that Mycobacterium cell walls are characterized by an unusual high DAP content. Moreover gene-knock out experiments with Mycobacterium smegmatis has demonstrated the essentiality of DAP pathway for the bacteria, where the absence of DAP results in cell lysis and death [12].…”