A mycobacterial extracytoplasmic sigma factor involved in survival following heat shock and oxidative stress - PubMed (original) (raw)

A mycobacterial extracytoplasmic sigma factor involved in survival following heat shock and oxidative stress

N D Fernandes et al. J Bacteriol. 1999 Jul.

Erratum in

Abstract

Extracytoplasmic function (ECF) sigma factors are a heterogeneous group of alternative sigma factors that regulate gene expression in response to a variety of conditions, including stress. We previously characterized a mycobacterial ECF sigma factor, SigE, that contributes to survival following several distinct stresses. A gene encoding a closely related sigma factor, sigH, was cloned from Mycobacterium tuberculosis and Mycobacterium smegmatis. A single copy of this gene is present in these and other fast- and slow-growing mycobacteria, including M. fortuitum and M. avium. While the M. tuberculosis and M. smegmatis sigH genes encode highly similar proteins, there are multiple differences in adjacent genes. The single in vivo transcriptional start site identified in M. smegmatis and one of two identified in M. bovis BCG were found to have -35 promoter sequences that match the ECF-dependent -35 promoter consensus. Expression from these promoters was strongly induced by 50 degrees C heat shock. In comparison to the wild type, an M. smegmatis sigH mutant was found to be more susceptible to cumene hydroperoxide stress but to be similar in logarithmic growth, stationary-phase survival, and survival following several other stresses. Survival of an M. smegmatis sigH sigE double mutant was found to be markedly decreased following 53 degrees C heat shock and following exposure to cumene hydroperoxide. Expression of the second gene in the sigH operon is required for complementation of the sigH stress phenotypes. SigH is an alternative sigma factor that plays a role in the mycobacterial stress response.

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Figures

FIG. 1

FIG. 1

The sigH loci of M. smegmatis and M. tuberculosis. The sigH genes are highly conserved, but the genes immediately downstream from sigH in these species show no similarity. There are several other differences in the organization of the flanking regions of the chromosome in these species (see text).

FIG. 2

FIG. 2

Alignment of the inferred amino acid sequences of M. tuberculosis SigH (mtsigH.pep), P. aeruginosa AlgU (psealgu.pep), and M. tuberculosis SigE (mtsigE.pep). Identical residues are shaded. M. tuberculosis SigH is 40% identical to M. tuberculosis SigE and 26% identical to P. aeruginosa AlgU, with the greatest similarity in regions 2 and 4.

FIG. 3

FIG. 3

Primer extension mapping of the mycobacterial sigH transcription start sites in vivo. (A) M. smegmatis. (B) M. bovis BCG. Primer extension reactions were performed with RNA isolated after growth at 37°C (lane 1) or after 50°C heat shock (lane 2). (C) Sequence 5′ of the M. smegmatis P1 (MsP1) and the M. bovis BCG P2 (MtP2) transcription start sites compared to the ECF-dependent promoter −35 consensus sequence. (D) Sequence 5′ of the M. bovis BCG P1 transcription start site (MTP1) compared to the M. smegmatis rpsL promoter. Identical residues are shaded. The M. tuberculosis sequence 5′ of MtP1 and MtP2 is identical to that of M. bovis BCG.

FIG. 4

FIG. 4

Autoregulation of sigH transcription in M. smegmatis. Primer extension reactions were performed with RNA isolated from mc2-155 (lane 1) and RH280 (lane 2) after growth at 37°C and from mc2-155 (lane 3) and RH280 (lane 4) after 50°C heat shock.

FIG. 5

FIG. 5

RT-PCR of M. smegmatis RNA. Primers flanking sigH and the second gene in the operon were used to perform RT-PCR on 2 μg of M. smegmatis mc2-155 total RNA. The PCR product was electrophoresed in 1% agarose containing ethidium bromide. Lane 1, 100-bp ladder (bright bands are at 500 and 1,000 bp); lane 2, RT-PCR of RNA isolated following growth at 37°C; lane 3, RT-PCR of RNA isolated following 50°C heat shock; lane 4, PCR of RNA isolated following 50°C heat shock without an initial RT reaction. A single band of the expected size (979 bp) is seen only in lane 3, indicating that both genes are expressed from a single RNA transcript.

FIG. 6

FIG. 6

(A) Survival of M. smegmatis mc2-155 (sigH+), RH244 (sigE), RH280 (sigH), and RH315 (sigH sigE) following exposure to 0.25 mM cumene hydroperoxide. Strains were grown at 37°C to log phase (OD600 of 0.1 to 0.5) and diluted in medium to approximately equal densities, cumene hydroperoxide was added, and dilutions were removed for plating at the indicated times. (B) Inhibition of growth of M. smegmatis strains around paper discs impregnated with 10 μl of 40 mM cumene hydroperoxide. The ability of a single copy of sigH (pRH1335) and a single copy of sigH plus the second gene in the operon (pRH1341) to complement the increased susceptibility of RH280 and RH315 was determined. The diameter of the zone of no growth surrounding the disc was measured on day 3. Each error bar represents ±1 standard deviation.

FIG. 7

FIG. 7

Survival of M. smegmatis mc2-155 (sigH+), RH244 (sigE), RH280 (sigH), and RH315 (sigH sigE) following 53°C heat shock. Strains were grown at 37°C to log phase (OD600 of 0.1 to 0.5), diluted in medium to approximately equal densities, and then transferred to a 53°C water bath. The ability of a single copy of sigH (pRH1335) and a single copy of sigH plus the second gene in the operon (pRH1341) to complement the increased susceptibility of RH315 was determined. Colonies were counted following 4 days of incubation. Each error bar represents ±1 standard deviation. □, mc2-155/pMH94A; ◊, RH244/pMH94A; ○, RH280/pMH94A; ▵, RH315/pMH94A; ⊞, RH315/pRH1335; ⧫, RH315/pRH1341.

FIG. 8

FIG. 8

Western blot analysis of DnaK expression in wild-type and mutant strains of M. smegmatis. Western blotting of M. smegmatis lysates was performed with the anti-mycobacterial DnaK monoclonal antibody IT-41 (HAT3). Lanes 1 and 2, RH244 (sigE); lanes 3 and 4: RH280 (sigH); lanes 5 and 6, RH315 (sigE sigH); lanes 7 and 8, mc2-155. Lanes 1, 3, 5, and 7, lysates made following growth at 37°C; lanes 2, 4, 6, and 8, lysates made following 50°C heat shock.

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