Abstract
σE is one of the 13 sigma factors encoded by the Mycobacterium tuberculosis chromosome, and its involvement in stress response and virulence has been extensively characterized. Several sigma factors are post-translationally regulated by proteins named anti-sigma factors, which prevent their binding to RNA polymerase. Rv1222 (RseA), whose gene lays immediately downstream sigE, has been proposed in the past as the σE-specific anti sigma factor. However, its role as anti-sigma factor was recently challenged and a new mechanism of action was hypothesized predicting RseA binding to RNA polymerase and DNA to slow down RNA transcription in a not specific way. In this manuscript, using specific M. tuberculosis mutants, we showed that by changing the levels of RseA expression, M. tuberculosis growth rate does not change (as hypothesized in case of non-specific decrease of RNA transcription) and has an impact only on the transcription level of genes whose transcriptional control is under σE, supporting a direct role of RseA as a specific anti-σE factor.
Highlights
ΣE is one of the 13 sigma factors encoded by the Mycobacterium tuberculosis chromosome, and its involvement in stress response and virulence has been extensively characterized
Rv1222 leads to a general decrease of RNA transcription and a consequent reduced growth rate, hypothesizing that deletion of this gene would result in an increase of M. tuberculosis growth rate
We analyzed whether rv1222 overexpression would decrease M. tuberculosis growth rate, as already observed for M. smegmatis and E. coli by Rudra and coll.[15]
Summary
Was able to inhibit transcription regardless the source of RNAP, for inhibition occurred by using holo RNAP or core RNAP coming either from M. tuberculosis, Escherichia coli or Bacillus subtilis. Recombinants were isolated as white colonies on plates containing sucrose and X-gal and the occurrence of a double crossover leading to sigE and rv1222 deletion was confirmed by PCR screening with the couple of primers RP1520/RP1521 (couple A) and RP1522/RP1523 (couple B) (Fig. 1B). These primer couples amplify two DNA regions whose length is 2552 bp and 2562 bp respectively in the wt strain, and 1247 bp and 1257 bp respectively in the correct mutant. The resulting plasmid, named pLCM1 was electroporated into H37Rv and TB340 to obtain TB515 and TB516 respectively
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