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. 1999 Jun;181(11):3486-93.
doi: 10.1128/JB.181.11.3486-3493.1999.

Regulation of hmp gene transcription in Mycobacterium tuberculosis: effects of oxygen limitation and nitrosative and oxidative stress

Y Hu  1 , P D Butcher, J A Mangan, M A Rajandream, A R Coates
Affiliations

Regulation of hmp gene transcription in Mycobacterium tuberculosis: effects of oxygen limitation and nitrosative and oxidative stress

Y Hu et al. J Bacteriol. 1999 Jun.

Abstract

The Mycobacterium tuberculosis hmp gene encodes a protein which is homologous to flavohemoglobin in Escherichia coli. Northern blotting analysis demonstrated that hmp transcription increased when a microaerophilic culture became oxygen limited as it entered stationary phase at 20 days. There was a fivefold increase of the hmp transcripts during early stationary phase compared with the value which was observed in the exponential growth phase. This induction of hmp transcription was not due to changes in the mRNA stability since the half-life of hmp mRNA was very short in a 20-day microaerophilic culture. No induction of hmp mRNA was observed during entry into stationary phase when the culture was continuously aerated. hmp transcription was induced after a short exposure of a late-exponential-phase culture to anaerobic conditions. These data indicate that oxygen limitation is the trigger for hmp gene transcription. In addition, when a microaerophilic culture entered into the stationary phase at 20 days, transcription of hmp increased to a small extent after exposure to S-nitrosoglutathione (a nitric oxide [NO] releaser) and sodium nitroprusside (an NO+ donor) and decreased after exposure to paraquat (a superoxide generator) and H2O2. In log phase (4 days) and late stationary phase (40 days), the transcription of hmp was unaffected by nitrosative and oxidative stress. Three primer extension products were observed. The -10 region is 100% identical to that of promoter T3 in mycobacteria and shows a strong similarity to the -10 sequence of hmp and rpoS promoters in E. coli. These observations of hmp mRNA induction in response to O2 limitation and nitrosative stress suggest that the hmp gene of M. tuberculosis may have a role in protection of the organism from NO killing under microaerophilic conditions.

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Figures

FIG. 1
FIG. 1
Growth curves of M. tuberculosis H37Rv. The bacilli were grown in 7H9 medium containing 0.05% Tween 80 supplemented with 10% albumin dextrose complex without shaking and with shaking as described in Materials and Methods. Viability was estimated as CFU per milliliter at 0, 4, 10, 20, 30, 40, 50, 55, and 60 days of unagitated incubation (black squares; curve a) and at 0, 4, 10, and 30 days of agitated incubation (white squares; curve b). The values shown are the averages of three independent experiments.
FIG. 2
FIG. 2
Northern blotting analysis of steady-state levels of the hmp mRNA in M. tuberculosis. (A) RNA was extracted at 4, 10, 20, 30, and 55 days of microaerophilic incubation across growth curve a (Fig. 1) and analyzed by formaldehyde-agarose gel electrophoresis and Northern blotting as described in Materials and Methods. The filter was hybridized with an hmp gene-specific probe. The blot was stripped and reprobed with cspA-, ftsZ mRNA-, and 16S rRNA-specific probes. (B) Densitometric analysis of the autoradiographs and two other independent experiments showing the steady-state levels of the hmp, cspA, and ftsZ transcripts. The quantification was based on several exposures of different periods. The signal obtained from each band for each mRNA was divided by the corresponding signal of 16S rRNA. The corrected data of the bands for each mRNA were plotted against the days of incubation and expressed relative to maximal value.
FIG. 3
FIG. 3
Northern blot analysis of decay of hmp mRNA in 20-day-old stationary-phase bacilli. (A) Total RNA was extracted from the bacilli at 0, 1, 3, 5, and 10 min after addition of 100 μg of rifampin per ml. (B) Densitometric analysis of decay of hmp mRNA. The quantification is based on several exposures of the autoradiographs (A) and two others from independent experiments for different periods. The signals of the bands were plotted against the times of the RNA isolation and expressed as the percentages of the initial values. The half-life calculated from the blots was 0.8 ± 0.05 min.
FIG. 4
FIG. 4
Effects of aerobic and anaerobic conditions on transcription of the hmp gene. (A) RNA was extracted at 4, 10, and 20 days of aerobic incubation (Fig. 1, growth curve b) and analyzed by Northern blotting. The filter was hybridized to detect hmp mRNA (upper panel) and then reprobed to detect cspA mRNA (middle panel) and 16S rRNA (lower panel) after being stripped. (B) RNA was extracted from a mid-log-phase culture (7 days) (Fig. 1, growth curve b) after exposure to anaerobic conditions for 0, 1, and 4 h and subjected to Northern blotting analysis. Hybridization of hmp mRNA (upper panel) and rehybridization of cspA mRNA (middle panel) and 16S rRNA (lower panel) were performed as described for panel A. These results have been independently confirmed in two additional experiments.
FIG. 5
FIG. 5
Northern blot analysis of the hmp mRNA level in response to nitrosative and oxidative stresses. RNA was extracted from a 20-day microaerophilic culture after exposure to different stress conditions described in Materials and Methods and subjected to Northern blot analysis. (A) Northern blot hybridization of hmp mRNA. The same blots were stripped and hybridized with the 16S rRNA-specific probe. (B) Densitometric analysis of hmp mRNA level in response to stresses. The values shown are the averages of two independent experiments. Ctrl, control; PQ, paraquat.
FIG. 6
FIG. 6
Determination of the hmp transcription initiation site. Primer extension analysis was performed as described in Materials and Methods with total RNA prepared from cultures at different growth phases. Lanes G, A, T, and C contained sequence reactions generated with the same primer. Lanes 2, 3, 4, 5, and 6 indicate the primer extension products with the RNA isolated from the bacilli after 4, 15, 20, 25, and 30 days of unagitated incubation (Fig. 1, growth curve a), respectively. A reaction without RNA is shown in lane 1. Primer extension points are marked by asterisks in the DNA sequence shown on the right, which is the nontranscribed strand and is the complement of the sequence that is readable from the sequencing ladder.
FIG. 7
FIG. 7
DNA sequence of the hmp gene regulatory region. Shown are the partial region encoding hmp and a 146-bp upstream sequence. The three primer extension points which correlated with nucleotides G, A, and C are indicated by asterisks. The putative promoter regions are highlighted by boxes and labelled −10 and −35. The putative ribosome-binding site is marked with a double underline and is labelled SD. The start codon is in boldface. The primers used for generating the Northern blotting probe are underlined.
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