Regulation of iron-sulphur cluster biogenesis in Mycobacterium tuberculosis

[摘要] ENGLISH ABSTRACT: Iron-sulphur (Fe-S) clusters are protein cofactors that are important for the functioning of many proteins involved in diverse processes in Mycobacterium tuberculosis. Complex Fe-S cluster biogenesis systems are required for their synthesis, to protect the clusters from the deleterious effects of reactive oxygen species in vivo. The suf system is the primary Fe-S cluster biogenesis system in M. tuberculosis and the components are encoded in an operon consisting of seven genes (Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466). The first gene in the operon, Rv1460, is predicted to encode a transcriptional regulator based on homology with the cyanobacterial suf operon repressor, SufR. This study aimed to determine whether Rv1460 is involved in the regulation of suf operon expression and M. tuberculosis physiology.In order to address this knowledge gap, attempts were made to generate three distinct Rv1460 deletion mutants in M. tuberculosis H37Rv. Surprisingly, generation of only one of the mutants, Rv1460stop, in which Rv1460 is truncated by insertion of a premature stop codon, was successful. This suggests that Rv1460 is not essential for the in vitro growth of M. tuberculosis. Our inability to generate the ΔRv1460 and Rv1460ΔDNAbd mutants may be due to polar effects on the expression of downstream genes, which make these mutants non-viable. Analysis of the Rv1460stop mutant's growth revealed that Rv1460 is required for normal growth on solid and in liquid media, under standard culture conditions. The Rv1460stop mutant was more sensitive to ROS, indicating the importance of Rv1460 in oxidative stress response, and potentially implicating it in intracellular survival and pathogenesis. The Rv1460stop mutant's growth was, surprisingly, not impaired under iron limitation.Gene expression studies done on the wild-type, Rv1460stop mutant and complementation strain revealed that Rv1460 acts as a transcriptional repressor of itself and the rest of the suf operon, since transcript levels of both Rv1460 and Rv1461 increased in the Rv1460stop mutant. Rv1460 was shown to be co-transcribed with the rest of the operon. Transcript levels, however, also suggested that Rv1460 may be independently transcribed from the rest of the gene cluster. Electrophoretic mobility shift assays done with recombinant Rv1460 demonstrated binding of Rv1460 to the Rv1460 promoter and within Rv1461. This indicates that Rv1460 mediates transcriptional control through direct interaction with the suf operon DNA. Recombinant Rv1460 was shown to form dimers in solution and to coordinate an Fe-S cluster in vitro, which has important implications for its function as a regulator, because the affinity of Fe-S cluster containing regulators for DNA is often influenced by the presence and redox state of their cluster. The role of three conserved cysteine residues (C203, C216, C244), predicted to be involved in Fe-S cluster coordination in Rv1460, could not be confirmed, as replacing these residues with serine residues did not alter their ability to coordinate an Fe-S cluster. The regulation of the suf operon in M. tuberculosis is multi-faceted, probably because Fe-S cluster biogenesis needs to be fine-tuned to allow survival within the host. This study indicates that Rv1460 plays a key role in this regulation and in M. tuberculosis physiology.

[发布机构] Stellenbosch University