Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H(2)S) and carbon disulphide (CS(2)). The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS(2) into H(2)S and carbon dioxide (CO(2)), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS(2) hydrolase from Acidianus A1-3. The enzyme monomer displays a typical β-carbonic anhydrase fold and active site, yet CO(2) is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical β-carbonic anhydrases and the formation of a single 15-Å-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme's substrate specificity for CS(2), which is hydrophobic. The transposon sequences that surround the gene encoding this CS(2) hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient β-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS(2) metabolism.

Mycobacterium tuberculosis can persist for many years within host lung tissue without causing clinical disease. Little is known about the state in which the bacilli survive, although it is frequently referred to as dormancy. Some evidence suggests that cells survive in nutrient-deprived stationary phase. Therefore, we are studying stationary-phase survival of Mycobacterium smegmatis as a model for mycobacterial persistence. M. smegmatis cultures could survive 650 days of either carbon, nitrogen, or phosphorus starvation. In carbonlimited medium, cells entered stationary phase before the carbon source (glycerol) had been completely depleted and glycerol uptake from the medium continued during the early stages of stationary phase. These results suggest that the cells are able to sense when the glycerol is approaching limiting concentrations and initiate a shutdown into stationary phase, which involves the uptake of the remaining glycerol from the medium. During early stationary phase, cells underwent reductive cell division and became more resistant to osmotic and acid stress and pool mRNA stabilized. Stationary-phase cells were also more resistant to oxidative stress, but this resistance was induced during late exponential phase in a cell-density-dependent manner. Upon recovery in fresh medium, stationary-phase cultures showed an immediate increase in protein synthesis irrespective of culture age. Colony morphology variants accumulated in stationary-phase cultures. A flat colony variant was seen in 75% of all long-term-stationary-phase cultures and frequently took over the whole population. Cryo scanning electron microscopy showed that the colony organization was different in flat colony strains, flat colonies appearing less well organized than wild-type colonies. Competition experiments with an exponential-phase-adapted wild-type strain showed that the flat strain had a competitive advantage in stationary phase, as well a providing evidence that growth and cell division occur in stationary-phase cultures of M. smegmatis. These results argue against stationary-phase M. smegmatis cultures entering a quiescent state akin to dormancy but support the idea that they are a dynamic population of cells.

We identified a response regulator in Mycobacterium smegmatis which plays an important role in adaptation to oxygen-starved stationary phase. The regulator exhibits strong sequence similarity to DevR/Rv3133c of M. tuberculosis. The structural gene is present on a multigene locus, which also encodes a sensor kinase. A devR mutant of M. smegmatis was adept at surviving growth arrest initiated by either carbon or nitrogen starvation. However, its culturability decreased several orders of magnitude below that of the wild type under oxygenstarved stationary-phase conditions. Two-dimensional gel analysis revealed that a number of oxygen starvation-inducible proteins were not expressed in the devR mutant. Three of these proteins are universal stress proteins, one of which is encoded directly upstream of devR. Another protein closely resembles a proposed nitroreductase, while a fifth protein corresponds to the ␣-crystallin (HspX) orthologue of M. smegmatis. None of the three universal stress proteins or nitroreductase, and a considerably lower amount of HspX was detected in carbon-starved wild-type cultures. A fusion of the hspX promoter to gfp demonstrated that DevR directs gene expression when M. smegmatis enters stationary phase brought about, in particular, by oxygen starvation. To our knowledge, this is the first time a role for a two-component response regulator in the control of universal stress protein expression has been shown. Notably, the devR mutant was 10 4 -fold more sensitive than wild type to heat stress. We conclude that DevR is a stationary-phase regulator required for adaptation to oxygen starvation and resistance to heat stress in M. smegmatis.

A bank of 600 insertional mutants of Mycobacterium smegmatis was screened for mutants defective in stationary-phase survival. Of 74 mutants picked by the initial screen, 21 had stationary-phase survival defects and 7 of these were studied in more detail. In general, mutants survived stationary phase significantly less well in rich medium than under carbon-starvation conditions. In all cases the loss of viability in stationary phase was not complete even after prolonged incubation. All mutants showed an initial decrease in viability, during the first 40 d in stationary phase, followed by an increase in viable counts that returned viability close to the levels of the wild-type. Southern hybridization experiments showed that recovery of viability was not a consequence of precise excision or movement of the transposon. Two of the survival mutants differed from the wild-type in their colony morphology, and recovery of their viability in stationary phase was coincident with the return of wild-type colony morphology. It is possible that second-site suppressor mutations accumulate that alleviate the effects of the original mutation. For five of the mutants the DNA flanking the site of transposition was amplified by ligation-mediated PCR and sequenced to identify the disrupted locus. In each case, homologous genes were identified in the Mycobacterium tuberculosis genome, three of which have clearly predicted functions in M. tuberculosis as a penicillin-binding protein, in biotin biosynthesis and as a polyketide synthase. This is the first identification of genes implicated in the stationary-phase survival of mycobacteria.

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c Carbon disulfide (CS 2 ) and carbonyl sulfide (COS) are important in the global sulfur cycle, and CS 2 is used as a solvent in the viscose industry. These compounds can be converted by sulfur-oxidizing bacteria, such as Acidithiobacillus thiooxidans species, to carbon dioxide (CO 2 ) and hydrogen sulfide (H 2 S), a property used in industrial biofiltration of CS 2 -polluted airstreams. We report on the mechanism of bacterial CS 2 conversion in the extremely acidophilic A. thiooxidans strains S1p and G8. The bacterial CS 2 hydrolases were highly abundant. They were purified and found to be homologous to the only other described (archaeal) CS 2 hydrolase from Acidianus strain A1-3, which forms a catenane of two interlocked rings. The enzymes cluster in a group of ␤-carbonic anhydrase (␤-CA) homologues that may comprise a subclass of CS 2 hydrolases within the ␤-CA family. Unlike CAs, the CS 2 hydrolases did not hydrate CO 2 but converted CS 2 and COS with H 2 O to H 2 S and CO 2 . The CS 2 hydrolases of A. thiooxidans strains G8, 2Bp, Sts 4-3, and BBW1, like the CS 2 hydrolase of Acidianus strain A1-3, exist as both octamers and hexadecamers in solution. The CS 2 hydrolase of A. thiooxidans strain S1p forms only octamers. Structure models of the A. thiooxidans CS 2 hydrolases based on the structure of Acidianus strain A1-3 CS 2 hydrolase suggest that the A. thiooxidans strain G8 CS 2 hydrolase may also form a catenane. In the A. thiooxidans strain S1p enzyme, two insertions (positions 26 and 27 [PD] and positions 56 to 61 [TPAGGG]) and a nine-amino-acid-longer C-terminal tail may prevent catenane formation.

In this study it was demonstrated that a range of transposon mutants of Mycobacterium smegmatis, previously described as having impaired survival in carbon-starved stationary phase, were not markedly affected in O 2 -starved stationary-phase survival. One exception was 329B, a purine auxotroph, which showed a precipitous reduction in viability from 10 8 to 10 3 c.f.u. ml N1 during the first 5-10 d in O 2 -starved stationary phase. This was followed by an equally rapid recovery in culturability to a level within 10-100-fold of wildtype levels by 10-20 d into stationary phase. Transduction of the mutation into a clean genetic background demonstrated that the phenotype was due to the transposon insertion, which was shown to be in the purF gene. purF encodes phosphoribosylpyrophosphate amidotransferase, which catalyses the first committed step in purine biosynthesis. The M. smegmatis purF gene, which encodes a protein with a very high degree of similarity to the PurF homologues of Mycobacterium tuberculosis and Mycobacterium leprae, was cloned and shown to substantially complement the O 2 -starvation phenotype. The recovery in culturabilty of the purF mutant in O 2 -starved stationary phase did not involve movement of the transposon. In addition, when cells that had recovered culturability were retested, their survival kinetics in stationary phase were identical to the original culture, indicating that their recovery was not explained by the accumulation of suppressor mutations. It is concluded that the survival curve in O 2 -starved stationary phase for the purF mutant represents its true phenotype and is not a result of subsequent genetic changes in the culture. It is argued that the purF cells lose culturability for a finite period of time in stationary phase. Whether this is due to a fraction of the population dying and then regrowing using a previously undiscovered fermentation pathway, or becoming transiently dormant, or entering an active nonculturable state and subsequently undergoing resuscitation cannot be distinguished at this stage.

We have characterized the induction kinetics of ϳ1,700 proteins during entry into and survival in carbonstarved stationary phase by Mycobacterium smegmatis. Strikingly, among the patterns of expression observed were a group of proteins that were expressed in exponential-phase cultures and severely repressed in 48-h stationary-phase cultures (Spr or stationary-phase-repressed proteins) but were synthesized again at high levels in >128-day stationary-phase cultures (Spr 1281 proteins). A number of Spr 1281 proteins were identified, and they included the heat shock protein DnaK, the tricarboxylic acid cycle enzyme succinyl coenzyme A synthase, a FixA-like flavoprotein, a single-stranded DNA binding protein, and elongation factor Tu (EF-Tu). The identification of EF-Tu as an Spr 1281 protein is significant, as ribosomal components are known to be expressed in a growth rate-dependent way. We interpreted these data in terms of a model whereby stationaryphase mycobacteria comprise populations of cells that differ in both their growth status and gene expression patterns. To investigate this further, we constructed gene fusions between the rpsL gene promoter (which heads the Mycobacterium smegmatis operon encoding the tuf gene encoding EF-Tu) or the rrnA promoter gene and an unstable variant of green fluorescent protein. While the majority of cells in old stationary-phase cultures had low levels of fluorescence and so rpsL expression, a small but consistently observed population of approximately 1 in 1,000 cells was highly fluorescent. This indicates that a small fraction of the cells was expressing rpsL at high levels, and we argue that this represents the growing subpopulation of cells in stationary-phase cultures.The ability of Mycobacterium tuberculosis to persist in host tissues significantly contributes to its success as a pathogen. This leads to latent infections being the most frequent outcome of the interaction between this bacterium and its host (22). Very little is understood about the physiological state of persistent M. tuberculosis, and the study of in vitro models that may mimic the persistent state is important to obtain leads to understanding the survival of mycobacteria under nongrowing conditions and their persistence in vivo. It is commonly thought that viable bacteria inhabit granulomatous lesions within the lung and are maintained in a persistent state by low oxygen availability, and this has been studied extensively in the in vitro Wayne model (37,38). However, it is also plausible that bacilli survive in lung lesions under stationary-phase conditions resulting from a specific nutrient starvation. Nyka showed that starved cells of M. tuberculosis become chromophobic, like cells isolated from lung lesions, but they can survive for at least 2 years without the presence of nutrients and then recover rapidly when fresh nutrients are encountered (25). Betts and coworkers studied cultures of M. tuberculosis starved in buffer using proteomic and transcriptomic approaches. The transcriptional profiling dat...