Mycobacterium bovis BCG response regulator essential for hypoxic dormancy

Abstract

Obligately aerobic tubercle bacilli are capable of adapting to survive hypoxia by developing into a nonreplicating or dormant form. Dormant bacilli maintain viability for extended periods. Furthermore, they are resistant to antimycobacterials, and hence, dormancy might play a role in the persistence of tuberculosis infection despite prolonged chemotherapy. Previously, we have grown dormant Mycobacterium bovis BCG in an oxygen-limited Wayne culture system and subjected the bacilli to proteome analysis. This work revealed the upregulation of the response regulator Rv3133c and three other polypeptides (alpha-crystallin and two "conserved hypothetical" proteins) upon entry into dormancy. Here, we replaced the coding sequence of the response regulator with a kanamycin resistance cassette and demonstrated that the loss-of-function mutant died after oxygen starvation-induced termination of growth. Thus, the disruption of this dormancy-induced transcription factor resulted in loss of the ability of BCG to adapt to survival of hypoxia. Two-dimensional gel electrophoresis of protein extracts from the gene-disrupted strain showed that the genetic loss of the response regulator caused loss of the induction of the other three dormancy proteins. Thus, the upregulation of these dormancy proteins requires the response regulator. Based on these two functions, dormancy survival and regulation, we named the Rv3133c gene dosR for dormancy survival regulator. Our results provide conclusive evidence that DosR is a key regulator in the oxygen starvation-induced mycobacterial dormancy response.

FIG. 1.

dosR locus, gene replacement constructs, rescue plasmid, and Southern blot analyses of gene replacement mutants. (A) Schematic diagrams (not to scale) of the dosR locus in BCG(pCB1) and derivatives thereof (pCB2 to -4) are shown. pCB2, dosR gene replacement plasmid; pCB3, Rv3132c gene replacement plasmid; pCB4, rescue plasmid. Horizontal arrows show open reading frames, and the numbers below the arrows in pCB1 indicate the corresponding Rv numbers of open reading frames as annotated for the M. tuberculosis H37Rv genome (2). Empty boxes labeled Km represent the kanamycin resistance cassette. Only relevant restriction sites are indicated: B, BamHI; S, SphI; D, DraIII; N, NotI; Bs, BstXI; Xh, XhoI; E, EcoRI; X, XbaI; P, PstI. Underlined restriction sites were used for deletion of coding sequences. S-probe and N-probe indicate SphI and NotI fragments used as probes for the Southern blots shown in B and C, respectively. Solid line, genomic DNA; dotted line, polylinker sequence. Vector backbones of the plasmids are indicated on the rightside. pBlueSK, pBluescriptSK (Stratagene); pJQ200SK, suicide vector (25); pNBV1, E. coli-mycobacterium shuttle vector (10). The asterisk indicates the site of a single-base-pair polymorphism that was detected between BCG and M. tuberculosis H37Rv. Codon 283 of Rv3132c in H37Rv (ATT) is ACT in BCG, resulting in an Ile-to-Thr amino acid replacement in the sensor domain of the kinase. (B) Southern blot analyses of ΔdosR::km gene replacement mutants. S-probe, DNA from the wild type (lane 1) and two independently isolated dosR gene replacement mutants (lanes 2 and 3) was digested with SphI and EcoRI, transferred to a membrane, and probed with the 1-kb SphI fragment (S-probe) shown in A. BCG wild-type genomic DNA showed the expected hybridization band, corresponding to the size of the SphI fragment used as the probe. In contrast, the ΔdosR::km mutants, which have lost an SphI site due to deletion of the dosR coding sequence, showed a band of 3.7 kb, corresponding to the expected size of the genomic SphI-EcoRI fragment containing the disrupted ΔdosR::km allele (compare pCB1 with pCB2). Km-probe, probing of the blot with a 1.2-kb PstI fragment carrying the kanamycin resistance cassette detected the same hybridization band in the ΔdosR::km strains that was detected by using the 1-kb SphI fragment as the probe, confirming that the 3.7-kb SphI-EcoRI band observed for these strains indeed contained the kanamycin resistance cassette and, hence, the disrupted dosR gene. (C) Southern blot analyses of ΔRv3132c::km gene replacement mutants. N-probe, DNA from the wild type (lane 1) and two independently isolated Rv3132c gene replacement mutants (lanes 2 and 3) was digested with NotI and XbaI, transferred to a membrane, and probed with the 0.8-kb NotI fragment (N-probe) shown in A. Wild-type genomic DNA showed the expected hybridization band, corresponding to the size of the NotI fragment used as the probe. In contrast, the ΔRv3132c::km mutants, which have lost a NotI site due to deletion of the Rv3132c coding sequence, showed a band of 3.2 kb corresponding to the expected size of the genomic NotI-XbaI fragment containing the disrupted ΔRv3132c::km allele (compare pCB1 and pCB3). Km-probe, probing of the blot with the 1.2-kb PstI fragment carrying the kanamycin resistance cassette (Km-probe) detected the same hybridization band in the ΔRv3132c::km strains that was detected with the 0.8-kb NotI fragment as a probe, confirming that the 3.2-kb NotI-XbaI band in BCG ΔRv3132c::km genomic DNA contained the kanamycin resistance cassette and hence the disrupted gene. DNA from both the ΔdosR::km and ΔRv3132c::km mutants showed no hybridization signal with the pJK200SK vector as the probe, confirming that the strains had lost all vector sequences (data not shown). Size markers are from the 1-kb ladder; 1 to 2 μg of genomic DNAs was loaded.

FIG. 2.

Growth and survival of wild-type BCG and ΔdosR::km, ΔdosR::km(pCB4), and ΔRv3132c::km strains in the Wayne dormancy culture system. Exponentially growing cultures were diluted and grown in sealed tubes with stirring. (A) Growth monitored by turbidity measurement. (B) Growth monitored by colony count determination. wt, wild type; ΔdosR::km, dosR gene replacement mutant; ΔdosR::km(pCB4), ΔdosR::km mutant transformed with the dosR rescue plasmid pCB4; ΔRv3132c::km, Rv3132c gene replacement mutant (see Fig. 1A). Growth and survival of ΔdosR::km transformed with pNBV1, i.e., the vector backbone used to carry the dosR genomic fragment in the pCB4 rescue construct (Fig. 1A), was indistinguishable from the behavior of the untransformed ΔdosR::km strain (data not shown). The experiments were carried out three times with duplicate cultures. Standard deviations are shown. The arrow pointing upwards indicates the time at which samples were taken for the protein analyses shown in Fig. 3. f and d, fading and complete decolorization of the oxygen indicator methylene blue, respectively.

FIG. 3.

Two-dimensional gel electrophoretic analyses of protein extracts from wild-type BCG and ΔdosR::km, ΔdosR::km(pCB4), and ΔRv3132c::km strains grown in the Wayne dormancy culture system. Protein extracts were prepared at the time when cultures grown in the Wayne system terminated growth at day 5, as indicated by the arrow in Fig. 2A. Then 100-μg samples of total protein were subjected to two-dimensional gel electrophoresis. Silver-stained gels are shown. (A) Wild-type BCG. (B) BCG ΔdosR::km. (C) BCG ΔdosR::km(pCB4). Two protein spots that were detected in the pCB4-transformed ΔdosR::km strain upon termination of growth are marked by arrows; their identity remains to be determined. (D) BCG ΔRv3132c::km. Circles labeled 1 to 4 indicate dormancy-induced protein spots. In B, where the dormancy proteins were not detectable, the circles indicate their expected migration positions. 1, DosR; 2, α-crystallin; 3, conserved hypothetical protein Rv2626c; 4, conserved hypothetical protein Rv2623. Sizes are indicated in kilodaltons. The experiments were repeated twice with independently prepared cultures, yielding the same results. The experiments were also carried out for all four strains with protein extracts from 20-day-old Wayne cultures, yielding the same results that were obtained for 5-day-old cultures (data not shown).