Proteins of Mycobacterium bovis BCG induced in the Wayne dormancy model

Abstract

Oxygen starvation triggers the shiftdown of the obligate aerobe Mycobacterium bovis BCG to a state of dormancy. Two-dimensional electrophoresis showed a drastic up-regulation of the alpha-crystallin homolog, the putative response regulator Rv3133c, and the two conserved hypothetical proteins Rv2623 and Rv2626c in dormant bacilli.

FIG. 1

Growth of BCG in the Wayne dormancy culture system and in aerated roller bottles. Solid circles show the growth curve of BCG under the oxygen-limited conditions of the Wayne dormancy culture system. Aerobic exponential precultures were diluted to an A₆₀₀ of 0.005 with Dubos Tween-albumin broth (Difco) and grown in sealed tubes (2 by 12.5 cm; 17-ml culture volume) under gentle stirring at 170 rpm as described previously (12, 24). Anaerobiosis in the stationary phase was monitored using the redox indicator methylene blue (24). Empty circles show the growth curve of BCG under non-oxygen-limited conditions in roller bottles. Precultures were diluted to an A₆₀₀ of 0.05 and grown in roller bottles (10 by 14 cm; 100-ml culture volume). Cultures were aerated in a roller apparatus at 1 rpm. The roller bottles were opened daily for turbidity measurements and to allow exchange of the air. Mean values and standard deviations from four independent experiments are shown. Arrows indicate the time points when samples were taken for protein and RNA analyses.

FIG. 2

Temporal profile of protein contents of BCG grown in the Wayne dormancy culture system. Protein extracts were prepared at the time points (A to D) indicated by the arrows in Fig. 1. Samples (100 μg) of total protein were prepared from ≈ 100 ml of culture at time point A and ≈ 35 to 50 ml of culture at time points B to D and were subjected to two-dimensional electrophoresis. Panels A to D show silver-stained gels corresponding to the four time points. Arrows 1 to 4 indicate dormancy-induced protein spots. The arrowhead shows the 22-kDa alkyl hydroperoxide reductase (AhpC, Rv2428; 6, 35) which was found to be down-regulated in the hypoxic stationary phase. Molecular masses (MW) are indicated in kilodaltons. The experiment was carried out four times with the same results. Growth phase-dependent protein contents were also analyzed using isoelectric focusing strips at pHs 3 to 10. No additional dormancy-induced proteins were detected.

FIG. 3

Steady-state levels of mRNAs encoding dormancy-induced proteins in growing and dormant cultures. Autoradiograms of Northern blots of total RNA from exponentially growing (lanes A, from time point A in Fig. 1) and hypoxic stationary-phase cultures (lanes D, from time point D in Fig. 1) grown in the Wayne dormancy culture system are shown. The blots were hybridized with [α-³²P]dATP-labeled probes specific to the transcripts encoding the dormancy-induced proteins: 1, α-crystallin homolog; 2, 23-kDa response regulator; 3, 32-kDa conserved hypothetical protein; 4, 16-kDa conserved hypothetical protein. X-ray films were exposed for 1 day. Sizes are indicated in bases. 16S rRNA shows the blots after rehybridization with a probe specific to 16S rRNA, demonstrating equal loading of rRNA. Five micrograms of total RNA (prepared from ≈20 ml of culture at time point A and from ≈35 ml of culture at time point D) was loaded. At the bottom, the agarose gel electrophoretic analysis of reverse transcriptase (RT) PCRs carried out with primers specific to the dormancy-induced genes and 150 ng of total RNA is shown. Carrying out the identical reactions but without the reverse transcription step did not yield any bands, demonstrating that the bands generated by reverse transcriptase PCR were derived from RNA and not from contaminating genomic DNA. A control PCR with genomic DNA yielded bands of the same size observed for the reverse transcriptase PCRs. Reverse transcriptase PCRs with primers specific to 16S rRNA yielded bands of the same intensity independent of the RNA sample; thus confirming equal loading of total RNA into the reverse transcriptase reactions (data not shown). All experiments were repeated once, yielding the same results. The values on the right are numbers of nucleotides.

FIG. 4

Steady-state levels of dormancy-induced proteins in aerated roller bottle cultures. Protein extracts were prepared at the time points (A to D) indicated by the arrows in Fig. 1. Samples of total protein (100 μg) were prepared from ≈25 ml of culture at time point A and ≈10 to 15 ml of culture at time points B to D and were subjected to two-dimensional electrophoresis. Panels A to D show silver-stained gels corresponding to the four time points. Arrows 1 to 4 indicate the migration areas of the dormancy-induced protein spots shown in Fig. 2. The arrowhead shows the 22-kDa alkyl hydroperoxide reductase found to be down-regulated in the aerated stationary phase. Molecular masses (MW) are indicated in kilodaltons. The experiment was carried out four times with the same results.