Journal of Bacteriology, December 2002, p . 6760-6767, Vol . 184, No . 24

Obviously, this monocausal model of persistence oversimplifies the situation in vivo . However, if it does reflect some aspects of the life of this parasite in its host, the development of drugs that target hypoxic dormant bacilli could have a profound impact on tuberculosis therapy . A combination therapy consisting of conventional antimycobacterials (targeting growing bacilli) and antidormancy drugs (hitting the dormant subpopulation) might considerably shorten the time necessary for treatment (6, 41) .

Mycobacterium tuberculosis is an aerosol-transmitted biosafety level 3 pathogen . To overcome the associated experimental limitations, we established the attenuated BCG strain of M . bovis as a model organism to study the hypoxic life of the tubercle bacillus . We demonstrated that BCG shows a dormancy response that is strikingly similar to the behavior shown by M . tuberculosis when grown in the oxygen-limited Wayne culture system (11, 12, 16, 20, 22, 29) . To identify dormancy-induced proteins, we previously subjected BCG grown in the Wayne system to a proteome analysis (1) . In this work, the response regulator Rv3133c, a putative phosphorylation-dependent transcription factor identified in the M . tuberculosis H37Rv genome project (2), was found to be upregulated immediately upon entry into dormancy . In contrast, this protein is not detectable in either aerated growing or aerated stationary-phase cultures . The temporal expression profile in the Wayne system and the apparent dormancy specificity, together with the probable biochemical function of the protein as a transcription factor, suggest that the molecule could play a key regulatory role in the adaptation of bacilli to survival under hypoxic conditions (1) .

Together with the response regulator, three other proteins, -crystallin (5, 14, 17, 26, 31, 36, 43, 44) and two "conserved hypothetical" proteins, universal stress protein domain-containing Rv2623 (7, 14, 19, 26, 31) and cystathionine ß-synthase domain-containing Rv2626c (14, 26, 31) are upregulated upon entry into dormancy (1) . The observed temporal coinduction of these three proteins along with the response regulator upon entry into dormancy could indicate that they are under the control of this transcription factor . To determine whether Rv3133c plays a role in adaptation to hypoxic survival and in the upregulation of the three coinduced proteins, we deleted its coding sequence from the BCG genome . We report the phenotypic consequences of this loss-of-function mutation on growth and viability and the proteome of the bacillus grown in the Wayne dormancy culture system .

Oxygen depletion was monitored via decolorization of the redox indicator methylene blue as described previously (16) . Growth and survival were determined by turbidity measurements in a Bacharach Coleman model 35 photometer (Thomas Scientific) and by determination of CFU on agar after plating of appropriate dilutions as described previously (16) . In contrast to the hypoxic stationary-phase cells grown in the Wayne system, clumping of bacilli occurred throughout aerated stationary-phase growth in roller bottles . To reduce clumping for CFU determinations, the roller bottle cultures were sonicated five times for 15 s each with 1-min ice breaks at level 2 of an Ultrasonic processor XC sonicator (Misonix) before plating .

DNA manipulations. DNA manipulations were done according to standard protocols (28) . DNA purifications were carried out with Qiagen kits as recommended by the supplier . DNA was introduced into BCG by electroporation as described before (29) with minor modifications . A lambda ZAPII library of genomic DNA from BCG strain ATTC 35734 was constructed with Stratagene reagents (B . Murugasu-Oei and T . Dick, unpublished data) . A 429-bp probe specific to dosR was generated by PCR with the primers TCGTAGGTGAGGCGGGTTC and CGGCGATCTGCTTGTTGGT, designed according to the sequence of the gene in M . tuberculosis H37Rv (Rv3133c [2]) . The library was screened, and positive clones were excised as pBluescriptSK plasmids with helper phages according to the protocol provided by the supplier .

One clone (pCB1, Fig . 1A) carrying a 5.9-kb insert containing the dosR gene plus flanking regions was subjected to DNA sequencing on a Perkin-Elmer ABI Prism 377 sequencer . To facilitate subsequent manipulations of the dosR operon, a 4.7-kb BamHI-XbaI fragment (BamHI site in the polylinker of pBluescriptSK, XbaI site downstream of Rv3132c within the genomic fragment) was excised from pCB1 and ligated with BamHI- and XbaI-digested pNEB193 (New England Biolabs) . The resulting plasmid was digested with DraIII, resulting in a 607-bp deletion within the 654-bp dosR coding sequence . The product was blunt ended with T4 DNA polymerase and ligated with the blunt-ended, 1.2-kb PstI fragment carrying the kanamycin resistance gene from pUCK4 (Pharmacia) . The 5.4-kb genomic DNA fragment carrying the kanamycin resistance cassette replacing the dosR coding sequence was released via BamHI-PstI digestion (PstI site in the polylinker of pNEB193) and ligated into BamHI- and PstI-digested pJQ200SK (25), resulting in the gene replacement plasmid pCB2 (Fig . 1A) .

 
pNEB193 carrying the 4.7-kb BamHI-XbaI fragment was also used to generate the replacement construct for the Rv3132c coding sequence . The plasmid was digested with BstXI and XhoI, resulting in an 895-bp deletion within the 1,737-bp Rv3132c coding sequence . The product was blunt ended and ligated with the kanamycin resistance cassette as before . The 5.1-kb genomic DNA fragment carrying the kanamycin resistance cassette replacing the Rv3132c coding sequence was transferred to pJQ200SK as before, resulting in the Rv3132c gene replacement plasmid pCB3 (Fig . 1A) . Sequencing the deletion sites showed that the kanamycin resistance cassette was inserted in the same transcriptional orientation as dosR and Rv3132c . Gene replacement was carried out according to the two-step protocol, taking advantage of the properties of the suicide vector pJQ200SK to confer sensitivity to sucrose and resistance to gentamicin (23) .

Briefly, BCG was transformed with 1 µg of the gene replacement constructs and selected on agar containing kanamycin (20 µg ml^-1) . Replica plating of kanamycin-resistant colonies on agar containing sucrose (2%) and gentamicin (5 µg ml^-1) identified strains that were sucrose sensitive and gentamicin resistant, indicating genomic recombination of the whole-gene replacement constructs . These recombinants were grown in broth to allow a second, intrachromosomal recombination step to occur . For selection of second-step recombinants that had lost the pJQ200SK sequence and the flanking copy of the native, wild-type genes, cultures were plated on agar containing sucrose and kanamycin . Sucrose- and kanamycin-resistant colonies were replica plated on agar containing gentamicin to confirm gentamicin sensitivity and thus loss of the pJQ200SK sequence . For rescue of the phenotypes caused by disruption of dosR, the 4.7-kb BamHI-XbaI genomic fragment containing the dosR operon was released from pNEB193 (see above) via BamHI and XbaI digestion and ligated into BamHI- and XbaI-digested pNBV1, which confers resistance to hygromycin (50 µg ml^-1) (10), resulting in pCB4 (Fig . 1A) .

Two-dimensional gel electrophoretic analysis. Bacilli were broken up by disrupting the cells with glass beads in a Mini Beadbeater cell disruptor as described previously (1) . Then 100 µg of total protein was subjected to isoelectric focusing with pH 4 to 7 Immobiline dry strips and an IPGphor isoelectric focusing unit (Amersham Pharmacia) and separated in the second dimension by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis (Protean IIxi system; Bio-Rad) as described before (1) .

Nucleotide sequence accession number. The sequence of the dosR locus as shown in pCB1 (Fig . 1A) has been deposited in GenBank under accession number AF522461 .

To determine the function of DosR, its coding sequence was replaced with a kanamycin resistance cassette (Fig . 1A, pCB2; see Materials and Methods for details) . The replacement of the dosR gene by its inactivated allele (dosR::km) was confirmed by Southern blotting, as shown in Fig . 1B .

Hypoxia-induced death of BCG dosR::km. To determine the role of the dosR gene in the dormancy response, BCG dosR::km was grown in the oxygen-limited Wayne culture system . This system is based on growth of the bacilli in sealed tubes with stirring (16, 39) . Initially the cultures grow exponentially and consume oxygen . A temporal oxygen gradient is self-generated, and upon encountering a microaerobic threshold level after about 4 to 5 days, the wild type is deflected from exponential growth and enters a transition phase in which the turbidity of the culture increases slowly up to day 10, after which the A₆₀₀ stays constant (Fig . 2A) (1) . Wild-type bacilli in the hypoxic stationary phase are in a state of dormancy, i.e., they are nonreplicating and maintain viability (16, 39) .

 
In the initial aerobic exponential growth phase observed in this system, BCG dosR::km was indistinguishable from the wild type (Fig . 2A) . This shows that the loss of dosR did not affect the growth of the bacillus . BCG dosR::km terminated aerobic exponential growth at the same time (i.e., after 4 to 5 days) and at the same A₆₀₀ as the wild type . However, in contrast to the wild type, BCG dosR::km underwent the transition from exponential growth to constant turbidity by day 6, i.e., the characteristic extended transition phase of the wild type has been lost in the dosR::km strain . This result could indicate that dosR is involved in the transition (adaptation) from aerobic exponential growth to hypoxic nonreplicating survival .

To determine the effect of the dosR loss-of-function mutation on the actual viability of the hypoxic nongrowing culture, CFU were determined . Figure 2B shows that the CFU of BCG dosR::km and the wild type were identical up to day 5 in the Wayne system, when both strains terminated aerobic exponential growth . Consistent with the slight increase in A₆₀₀ over the next 5 days during the extended transition phase, a moderate increase in CFU was observed for the wild type at day 10 . After that, the CFU of the wild type stayed constant for 40 days of total cultivation time, after which the experiments were terminated . In contrast to the survival of hypoxia shown by the wild type, Fig . 2B shows that the CFU of BCG dosR::km started to decline at day 10 . After 40 days of total incubation time, the viable-cell count of the BCG dosR::km culture was 1,500-fold lower than that at day 5, when exponential growth terminated (Fig . 2B) .

The growth and survival analyses were carried out with an independently isolated dosR::km strain (Fig . 1B, lane 3), yielding identical results (data not shown) . Figure 2 also shows that both phenotypes exhibited by BCG dosR::km, loss of the extended transition phase and hypoxic death, were rescued by a wild-type copy of the dosR operon carried by plasmid pCB4 (Fig . 1A) . Taken together, these results show that the dosR disruption affected the transition phase and caused massive death of the oxygen-starved culture . Hence, dosR is essential for the adaptation of BCG to survival of hypoxia .

Loss of dormancy proteins in BCG dosR::km. Previously we demonstrated that DosR is upregulated immediately upon oxygen starvation-induced termination of growth and that -crystallin and the two conserved hypothetical proteins Rv2623 and Rv2626c are temporally coinduced with the response regulator (Fig . 3A) (1) . To reveal a possible regulatory role of the dosR gene in the induction of these dormancy proteins, BCG dosR::km was grown in the Wayne culture system, and a proteome analysis was carried out . Protein extracts were prepared from cultures that were terminating growth (Fig . 2A, arrow) and were subjected to two-dimensional gel electrophoresis . BCG dosR::Km had lost the DosR protein, as expected (compare protein spot numbered 1 in Fig . 3A with empty circle numbered 1 in Fig . 3B) .

 
Strikingly, Fig . 3B also shows the loss of induction of all three dormancy proteins that were coinduced with DosR in wild-type bacilli . The proteome analysis was carried out with the independently isolated dosR::km strain (Fig . 1B, lane 3), yielding identical results (data not shown) . Figure 3C shows that the regulatory phenotype shown by BCG dosR::km could be rescued by a wild-type copy of the dosR operon carried by plasmid pCB4 (Fig . 1A) . These results demonstrate that the induction of -crystallin, Rv2623, and Rv2626c by dormancy depends on DosR and thus that DosR plays a regulatory role in the dormancy response .

Minor phenotypic consequences in BCG Rv3132c::km. The apparent cotranscription of the histidine kinase Rv3132c (27) with the dosR response regulator (see Fig . 1A, pCB1) could indicate that the two proteins form a two-component system in which the kinase is involved in phosphorylation of DosR . Thus, Rv3132c could play the role of a dormancy sensor kinase, regulating DosR activity . To determine whether the kinase is indeed essential for the dormancy response, the gene was replaced with a kanamycin resistance cassette (Fig . 1A, pCB3; Fig . 1C), and the resulting Rv3132c::km strain was grown in the Wayne culture system .

Figure 2A shows that the Rv3132c::km strain appeared to maintain a wild-type-like extended transition phase, although the turbidity of the culture at day 10 was somewhat lower . Viability of the hypoxic, nongrowing BCG Rv3132c::km culture showed a moderate 15-fold reduction after 40 days of growth in the Wayne system, compared to the 1,500-fold hypoxic kill observed for the dosR::km mutant under the same conditions . Consistent with the moderate effect of the loss of kinase function on growth and survival in the Wayne culture system, Fig . 3D shows that BCG Rv3132c::km maintained inducible expression of the dormancy proteins; although the induced level of the dormancy proteins appeared to be somewhat reduced compared to the induction of the proteins in the wild type . The growth, survival, and proteome analyses were carried out with an independently isolated Rv3132c::km strain (Fig . 1C, lane 3), yielding identical results (data not shown) .

BCG Rv3132c::km thus showed a moderate phenotype compared to the loss of extended transition phase, loss of hypoxic viability, and loss of expression of dormancy proteins observed for BCG dosR::Km when grown in the Wayne culture system . These results indicate that, in comparison to the key role observed for dosR, the Rv3132c kinase plays a minor role in the dormancy response . Furthermore, these results confirm that the phenotypic effects of the dosR inactivation were not due to polar effects on the downstream-located Rv3132c kinase gene .

Wild-type-like survival of BCG dosR::km in aerated stationary phase. Previously we demonstrated that upregulation of DosR is specific to oxygen starvation-induced termination of growth, i.e., to the dormancy response . Termination of growth by nutrient starvation under non-oxygen-limited conditions in aerated roller bottles is not accompanied by an increase in the steady-state level of DosR (1) . Based on the lack of DosR induction in aerated stationary-phase cultures, one would predict that the protein does not play a role in the survival of aerated nongrowing culture . To test the dormancy specificity of dosR function, BCG dosR::km was grown in aerated roller bottles, and growth and survival were determined . Both strains terminated exponential growth after 5 days of cultivation, at an A₆₀₀ of 0.8 . They showed an identical transition phase and the same viable-cell counts in the aerated stationary phase after a total cultivation time of 20 days (8 x 10⁷ and 6 x 10⁷ CFU ml^-1 for the wild-type and BCG dosR::km strains, respectively) and 40 days (3 x 10⁶ and 2 x 10⁶ CFU ml^-1 for the wild-type and BCG dosR::km strains, respectively) . These results show that growth and survival of BCG dosR::km under non-oxygen-limited conditions in roller bottles were indistinguishable from those of the wild type . Hence, the function of dosR in maintaining the viability of nongrowing cultures is specific for hypoxic dormancy, and dormancy-specific induction of DosR correlates with the dormancy specificity of its function .

The deletion of the coding sequence of the histidine kinase Rv3132c located downstream of dosR had moderate consequences on hypoxic survival and induction of dormancy proteins . This suggests that the Rv3132c kinase plays only a minor role in the dormancy response and that other histidine kinases could be involved in dormancy signaling . The situation in the signal transduction pathway in Mycobacterium dormancy could thus be similar to sporulation signaling in Bacillus subtilis, in which multiple histidine kinases control (via a complex phosphorelay system) the phosphorylation status of the master regulator of sporulation, the response regulator Spo0A (13) .

Our finding that -crystallin induction depends on dosR but not on the Rv3132c histidine kinase is consistent with data from Sherman et al . (31) . Those authors were unable to generate a dosR gene replacement mutant (as a "result of technical difficulties?" [31]) . Hence, they interfered with the expression of dosR by interrupting the conserved hypothetical protein gene Rv3134c, which is located upstream of dosR (see Fig . 1A) . By employing an anti--crystallin antibody, it was shown that this mutation resulted in loss of induction of -crystallin under low-oxygen culture conditions . Complementation experiments suggested that this was due to reduced dosR expression and not to the inactivation of Rv3134c (31) . In contrast, an Rv3132c histidine kinase-disrupted strain maintained inducible -crystallin expression . Consistent with our data, the Rv3134c-disrupted strain did not show significant loss of viability in stationary-phase cultures grown in aerated roller bottles . Growth and survival of the Rv3134c and Rv3132c mutant strains in an oxygen-limited, Wayne-type culture system were not characterized by the authors (31) .

The identification of DosR as an essential dormancy response regulator in our present study promises rapid progress in understanding the biochemistry underlying the quiescent hypoxic life of these obligate aerobes . The dosR::km strain allows definition of the complete regulon (and thus the dormancy-essential enzymatic functions) under control of this transcription factor by comparative DNA microarray analyses . Even more important, perhaps, BCG dosR::km represents the first Mycobacterium mutant that shows not only a strong but also an apparently specific survival phenotype in hypoxic dormant bacilli generated in the Wayne culture system . We demonstrated previously that DosR is not detectable in aerated stationary-phase culture (1), and we show here that, consistent with that observation, survival of the dosR loss-of-function mutant was indistinguishable from that of the wild type under these conditions . Thus, the dosR::km mutant provides us with a probe for testing the relevance of hypoxic dormant bacilli for the life of the parasite in vivo .

If hypoxic dormant bacilli do play a role in the persistence of the bacilli during drug treatment, one would predict that chemotherapy of active disease and/or latency caused by the dosR::km strain is more effective than the treatment of wild-type infection . DosR loss-of-function bacilli cannot weather the onslaught of conventional drugs by "hiding" in their dormant form, and hence, the time required to eradicate infection should be reduced . Furthermore, hypoxic dormancy of the bacillus might play a role in the progression of active disease and/or in the establishment and maintenance of latency of infection . If that is the case, the dosR::km mutant is expected to show attenuated virulence . Animal models are in place to test these predictions (8) . Thus, DosR and its dormancy regulon could provide a rich source of targets for a fundamentally new level of chemotherapeutic intervention against tuberculosis . Not hitting growth-related targets in proliferating bacilli but knocking out dormancy-related targets in sleepers may turn out to be the way to go in the development of future, more effective antimycobacterials .

T.D . is an adjunct staff member of the Department of Microbiology, National University of Singapore . This work was supported by the Agency for Science, Technology and Research (A*STAR), Singapore .

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