Transcription of the nfrA-ywcH operon from Bacillus subtilis is specifically induced in response to heat

Abstract

The NfrA protein, an oxidoreductase from the soil bacterium Bacillus subtilis, is synthesized during the stationary phase and in response to heat. Analysis of promoter mutants revealed that the nfrA gene belongs to the class III heat shock genes in B. subtilis. An approximate 10-fold induction at both the transcriptional and the translational levels was found after thermal upshock. This induction resulted from enhanced synthesis of mRNA. Genetic and Northern blot analyses revealed that nfrA and the gene downstream of nfrA are transcribed as a bicistronic transcriptional unit. The unstable full-length transcript is processed into two short transcripts encoding nfrA and ywcH. The nfrA-ywcH operon is not induced by salt stress or by ethanol. According to previously published data, the transcription of class III genes in general is activated in response to the addition of these stressors. However, this conclusion is based on experiments which lacked a valid control. Therefore, it seems possible that the transcription of all class III genes is specifically induced by heat shock.

FIG. 1

(A) Induction of P_nfrA by different kinds of stress. β-Galactosidase (β-gal) expression in response to different stressors was determined. β-Galactosidase activity was plotted versus time. Filled bars, control at 37°C; empty bars, heat-shocked cells; shaded bars, salt-stressed cells; hatched bars, cells grown in the presence of 5% ethanol. Samples were collected at different times after stress was applied. All experiments were done in triplicate. (B) Influence of temperature on the heat shock response. Cells were grown at 37°C before an aliquot was shifted to a new temperature. Samples were collected at different times after stress was applied. β-Galactosidase activity was plotted versus time. Filled circles, control at 37°C; empty circles, 45°C; squares, 47°C; triangles, 49°C; inverted triangles, 51°C; diamonds, 53°C; hexagons, 55°C. (C) Western blot analysis of NfrA synthesis. Equal amounts of cell extracts were subjected to Western blot analysis. Samples were taken from stressed and unstressed cells at different times after stress was applied.

FIG. 2

Northern blot analysis of nfrA transcription. Heat stress was applied. At the indicated times, cells were harvested and RNA was isolated. Equal amounts of RNA were used for the analysis. The positions of the 23S and 16S rRNAs are indicated. (A) RNA from heat-shocked cells. Time after heat shock and growth temperature are indicated. Different bands of 0.8 and 2 kb are visible. (B) RNA from puromycin-treated cells. Puromycin (20 μg/ml) (+) was added to the cell suspension; −, no puromycin. Cells were harvested at the indicated times, and RNA was isolated.

FIG. 3

Decay of nfrA mRNA before and after heat shock. RNA was isolated at different times after stress was applied. (A) Decay of the nfrA mRNA at 37°C. (B) Decay of the nfrA mRNA at 50°C. (C) Decay curves for the nfrA mRNAs at 37°C (empty circles) and 50°C (filled circles). The amount of RNA at time zero was set to 1 for both graphs. Times after the addition of rifampin are given.

FIG. 4

Sequence of the P_nfrA promoter region. The putative −10, −16, and −35 regions are indicated by gray backgrounds. The ς^D promoter (34) is indicated by boxes. Only the effects of single mutations within this region are shown. Mutations resulting in elevated promoter activity are shown above the wild-type sequence; mutations resulting in reduced transcriptional activity are shown below. M, A or C; S, G or C; K, G or T; V, A, G, or C.

FIG. 5

Regulation of P_nfrA-P_xylA hybrids. The sequences of the respective promoter variants are shown. Sequences derived from P_xylA are shown by a shaded background. The promoters were cloned into plasmid pDL; the resulting plasmids were integrated into the amyE gene of B. subtilis 168 to give the respective B. subtilis strains. BgaB activity was determined with cell extracts from unstressed and heat-shocked cells. The induction ratios are shown at the right. The predicted transcriptional start points of the respective RNAs are indicated by bold letters. The uninduced activities (in Miller units) of the respective promoter derivatives were as follows: DIPA4, 3 ± 0.5 U; B49, 1 ± 0.1 U; DXyl1035, 0.6 ± 0.1 U; DXyl10, 62 ± 6; DXyl35, 0.6 ± 0.2 U; DXyl10Δ2, 1 ± 0.1 U; DXyl35Δ2, 0.5 ± 0.1 U; and DXyl, 62 ± 7 U. The activity of the control, B. subtilis DL, was 0.2 ± 0.1 U (not shown).

FIG. 6

Comparison of nfrA and ywcH induction. (A) Induction of transcriptional fusions of lacZ to the respective gene during growth in NB supplemented with glucose and glutamate. Filled circles, growth of B. subtilis IPA43, given as OD₆₀₀ units; open circles, β-galactosidase (β-gal) activity of B. subtilis IPA43 (nfrA-lacZ fusion in nfrA); squares, β-galactosidase activity of B. subtilis IPA44 (ywcH-lacZ fusion in ywcH); inverted triangles, β-galactosidase activity of B. subtilis IPA44E (ywcH-lacZ fusion in amyE); triangles, β-galactosidase activity of B. subtilis DH32M (control). (B) Heat shock induction of nfrA and ywcH. Filled symbols, activity in cells grown at 37°C; empty symbols, activity of stressed cells; circles, B. subtilis DIPA4 (nfrA-bgaB); squares, B. subtilis DIPA44 (ywcH-bgaB).

FIG. 7

(A) Northern blot analysis of ywcH transcription. RNA was isolated from cells before (37°C) and after (50°C) heat shock or during the exponential (OD₆₀₀ = 0.2) and stationary (OD₆₀₀ = 1.4) growth phases. Equal amounts of RNA were used for the analysis. The positions of the 23S and 16S rRNAs are indicated. Two bands specifically hybridizing to a ywcH probe were obtained. (B) Mapping of the ywcH 5′ end. RNA was isolated from B. subtilis 168 grown at the indicated temperatures. The sequence ladder used as a size marker was obtained by using the same primer as that used for the primer extension reaction. The putative start codon of translation is indicated (TAC). Whereas the 5′ end of the RNA is upstream from the putative translational start codon in heat-shocked cells, it is downstream from this codon in unstressed cells (arrows at left). (C) S1 nuclease mapping of the 3′ end of nfrA. RNA was isolated from cells before (37°C) and after (50°C) heat shock or from stationary-phase cells grown in NB or NB with glucose and glutamate (GG). Equal amounts of RNA were used for the analysis. Restriction fragments of known lengths and a sequence ladder of a known sequence were used as size markers. Four different signals were obtained in the S1 nuclease reaction. The 912-base fragment was derived from an unprocessed probe. The 875-base fragment (large filled arrow) was obtained by S1 nuclease processing of a probe hybridized to an unprocessed nfrA-ywcH transcript. The 397-base fragment (hatched arrow) was processed at the 5′ end of the stem-loop structure. The 378-base fragment (empty arrow) was processed at the single mismatch within the stem-loop structure. (D) Graphic illustration of the results obtained by S1 nuclease mapping and primer extension. The labeling of the arrows is like that in panel C. The 875-base fragment is not due to RNA processing but is specific for the strategy of the experiment. It indicates that readthrough occurs.