Functional and genetic study of M. tuberculosis glutamine synthetase (GS) and other factors possibly involved in GS metabolism

[摘要] ENGLISH ABSTRACT: Sequence analysis showed that the essential glnA1 gene of Mycobacterium tuberculosis mightbe closely related to an actinomycetes progenitor sequence and that this sequence may haveundergone duplication into other non-essential GS encoding genes in some Actinobateria,notably the mycobacteria. Also, the M. tuberculosis glnA1 sequence remains conservedthroughout the evolution of M. tuberculosis. It was also shown that glnA1 is widely expressed inM. tuberculosis infected human pulmonary tissue, where M. tuberculosis might be present inaltered phenotypes. These results imply that glnA1 is under selective pressure againstevolutionary change.At transcriptional level it was shown that M. tuberculosis glnA1 might be expressed from twoalternate promoter sites, but that these promoter sites may not be controlled by environmentalnitrogen (in the form of ammonium) variation. We also showed that M. tuberculosis GS iseffectively exported by M. smegmatis to the cell wall, but that GS secretion into the exogenousenvironment does not occur. Also, evidence has been presented which suggested that M.tuberculosis GS might be modified at the C-terminus, probably as part of a mechanism thatretains GS in the cytosol. This hypothesis was further substantiated where it was demonstratedthat two GS isoforms of different size (short isoform in cytosol, longer isoform in cell wall) arepresent in M. bovis BCG. It is unknown what causes this modification, since it couldn't beobserved in M. smegmatis, but it was suggested that it might be through the action of a cis- ortrans-acting element present in proximity of the M. tuberculosis glnA1 gene. It was also shownthat a cluster of genes found immediately downstream of the M. tuberculosis glnA1 sequencemight be regulated in an operonic fashion under conditions of elevated environmental nitrogenconcentrations. Two of the genes (glnE and glnA2) in this operon arrangement have beenpreviously shown to be involved in nitrogen and glutamine metabolism. The function of the othergene, Rv2223c, is unknown. It was shown that Rv2223c homologs are mostly found in themycobacteria and that it may encode an exported protease. It was hypothesised that thissequence and its adjacently located progenitor sequence, Rv2224c, might be involved in M.tuberculosis GS mediated metabolism. It was showed that over-expression of Rv2223c andRv2224c may be toxic to E. coli and mycobacterial hosts, such as M. smegmatis, but thatinhibition of transcription of these genes may be fatal to M. bovis BCG and M. tuberculosisH37Rv. It was also shown that Rv2223c is widely expressed in M. tuberculosis infected humantissue, which was comparable to that of glnA1. The results presented in this study shed more light on the distribution and transcriptionalregulation of GS in mycobacteria and has identified new genes that might be involved in GSregulation. These results may present new approaches to tuberculosis control and therebycontribute to alleviate the burden of the disease.

[发布机构] Stellenbosch University