Department of Biology, Imperial College of Science, Technology and Medicine, Imperial College Road, London SW7 2AZ, UK1
Author for correspondence: Huw D. Williams. Tel: +44 20 75945383. Fax: +44 20 75842056. e-mail: h.d.williams{at}ic.ac.uk
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ABSTRACT |
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Keywords: dormancy, Mycobacterium tuberculosis, purine, VBNC, oxygen
The GenBank accession number for the sequence reported in this paper is AJ278609.
a Present address: LGC, Queens Road, Middlesex TW11 0LY, UK.
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INTRODUCTION |
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We are studying the mechanisms by which mycobacteria survive nutrient deprivation as a model for mycobacterial persistence. We have previously described the physiological changes accompanying the adaptation of Mycobacterium smegmatis to carbon-starved stationary phase (Smeulders et al., 1999 ). Carbon-starved cultures maintain viability for at least 2 years. M. smegmatis may sense when the carbon source is approaching limiting concentrations and then initiate an adaptive response to stationary-phase survival. During early stationary phase cells undergo reductive cell division and become more resistant to environmental stress and stabilize their mRNA. However, it is apparent that continuous cell growth and cell division occurs in stationary phase. Strains with altered colony morphology appear and take over stationary-phase cultures, and competition experiments between stationary-phase-adapted and exponential-phase-adapted strains show that variants appear that have growth and survival advantages in stationary phase (Smeulders et al., 1999
).
We have recently described the isolation of mutants that are defective in stationary-phase survival in both rich medium and during carbon starvation (Keer et al., 2000 ). These were identified from a Tn611 (Guilhot et al., 1994
) mutant library by screening for mutants that had lost viability in carbon-starved stationary phase. These mutants were also defective in stationary-phase survival in rich medium. Interestingly, all the mutants were disrupted in genes with homologues in the M. tuberculosis genome (Keer et al., 2000
). The identification of one of these as a gene (ponA) encoding a penicillin-binding protein is consistent with the need for cell division in stationary phase (Smeulders et al., 1999
; Keer et al., 2000
).
Our previous work used O2-sufficient, stationary-phase cultures. However, one model for mycobacterial persistence that has been used in a number of laboratories is O2 starvation (Wayne, 1994 ; Wayne & Hayes, 1996
; Lim et al., 1999
). In this model, cultures of the bacteria are subjected to a temporal gradient of O2 depletion by incubating in sealed tubes. This model is based on the idea that bacteria may be deprived of O2 in the caseous, necrotic centres of tuberculous lesions. Two genes encoding sigma factors are upregulated during O2-limited stationary phase (Hu & Coates, 1999a
), as is the 16 kDa
-crystallin heat-shock protein (Hu & Coates, 1999b
; Yuan et al., 1996
) and the M. tuberculosis hmp gene product, which is homologous to the Escherichia coli flavohaemoglobin (Hu et al., 1999
), as well as a histone-like protein in M. smegmatis (Lee et al., 1998
).
An important question is whether the adaptation mechanisms for survival are the same under carbon- (O2-sufficient) and O2-starvation conditions. One way to address this question is to look for mutants that are defective in carbon- but not O2-starvation survival and vice versa. In this study we have looked at the O2-starvation survival of mutants previously identified as being impaired in stationary-phase survival under O2-sufficient, carbon-starved conditions, and we describe the characterization of a mutant that is defective in O2-but not carbon-starvation survival.
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METHODS |
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Isolation of transposon mutants impaired in stationary-phase survival.
M. smegmatis mutants impaired in stationary-phase survival were isolated from a mutant library constructed by transposon mutagenesis using Tn611 (Guilhot et al., 1994 ), as described by Keer et al. (2000)
.
Cloning the M. smegmatis purF gene.
A M. smegmatis gene library in the cosmid vector LAWRIST4 was kindly provided by Neil Stoker, London School of Hygiene and Tropical Medicine. Five hundred and seventy-six cosmid clones in LAWRIST4 were screened in hybridization experiments with the 640 bp PstI/HindIII fragment isolated from the Tn611 mutant 329B to identify cosmids containing putative purF genes. Two hybridizing cosmids were isolated and 1·5 kb PstI, 3·8 kb BamH1 and 4·5 kb SacI fragments were subcloned from these cosmids into pBluescript. The 1·5 kb fragment was completely sequenced and contained the majority of the M. smegmatis purF gene. Sequencing fragments from the other clones allowed us to obtain the complete purF sequence. To complement 329B, the 4·5 kb SacI fragment from pPUR3 was subcloned into the mycobacterial shuttle cosmid pNBV1 (Howard et al., 1995 ) to form pPUR10 (Table 1
).
Transduction.
Mycobacteriophage I3 lysates from Tn611 mutant strains were prepared according to Sundar Raj & Ramakrishnan (1970) . Mutations were transduced by infecting 5x109 c.f.u. exponentially growing M. smegmatis mc2155 with mutant I3 lysates at a m.o.i. of 2, and transductants were selected on Lab-lemco plates containing kanamycin.
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RESULTS |
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Identification of purF as the disrupted gene in mutant 329B: cloning and sequencing of purF
To identify the gene disrupted in the purine auxotroph 329B, chromosomal DNA isolated from the mutant strain 329B was digested with NotI, which does not cut within the transposon vector itself, producing large chromosomal fragments. The fragments were recircularized and transformed into E. coli. Selection for kanamycin- and streptomycin-resistant colonies yielded plasmids containing the transposon insertion in its entirety plus additional flanking genomic sequences. PstI digestion of the clones produced the parental pCG79 fragments, plus an extra PstI fragment containing a portion of flanking chromosomal DNA. This fragment was sequenced and a BLAST search indicated it to be highly homologous to purF genes from a range of bacteria (data not shown). The purF gene encodes phosphoribosylpyrophosphate amidotransferease, which catalyses the first committed step in purine biosynthesis. This PstI fragment was used to screen a M. smegmatis genomic cosmid library in LAWRIST4. Two cosmid clones were isolated and 1·5 kb PstI, 3·8 kb PstI and 4·5 kb SacI fragments were subcloned from these into pBluescript (Table 1). The 1·5 kb PstI fragment containing the majority of the gene was used for sequencing. The GenBank accession number for this sequence is AJ278609. Alignment with M. tuberculosis and Mycobacterium leprae sequences shows very clearly the strong homology between the PurF proteins from these three mycobacterial species (Fig. 3
).
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DISCUSSION |
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The purF mutant 329B was isolated using a screen to find mutants defective in carbon-starvation survival (Keer et al., 2000 ). 329B grew to give visible turbidity under the screen conditions of growth in 0·02% glycerol containing Hartmansde Bont medium, presumably because there was sufficient carryover of purines in the inoculum to allow some growth. Its apparent loss of viability in the initial screen, compared to the wild-type, could in part reflect a lower final cell density due to purine limitation. It could also be due to the effects of O2 limitation in the microtitre plates, which were sealed with film to prevent evaporation of the medium during extended 37 °C incubation.
Intriguingly, 329B purF loses viability during O2 starvation in HdB minimal medium that has been supplemented with purines. One explanation is that purines are required for survival but are not taken up by the bacteria during the early stages of O2 starvation. Alternatively, the O2-starvation phenotype may not be a consequence of starvation for purines directly but results from a shortage of one or more intermediates in the purine biosynthesis pathway. Important in this context is the finding that a Rhizobium etli purF mutant and certain other purine biosynthesis mutants overexpress cytochrome cbb3, a cytochrome c oxidase (Soberon et al., 1997 ; García-Horsman et al., 1994
). Analysis of the transcription of the fixNOQP operon, encoding cytochrome cbb3, in different R. etli purine biosynthetic mutants, suggests that 5 aminoimidazole-4-carboxamide ribonucleotide (AICAR) or a related metabolite is a negative regulator of this oxidase (Soberon et al., 1997
). It would be interesting to investigate the survival phenotypes of other M. smegmatis purine biosynthesis mutants. To our knowledge, nothing is known about the respiratory chain of M. smegmatis. In preliminary studies, we have found that while overall respiratory capacity, determined as NADH oxidase activity, is unchanged in a M. smegmatis purF mutant, cytochrome c oxidase activity is two- to threefold higher in the mutant compared to the wild-type, although this activity does not reduce to wild-type levels upon complementation with pPUR10 (Keer et al., unpublished results). However, the cytochrome c oxidase activities and cytochrome c levels of M. smegmatis are low compared with, for example, Pseudomonas aeruginosa, which has a cytochrome cbb3 (Keer et al., unpublished results). This requires further investigation to show directly whether or not overexpression of a specific terminal oxidase is responsible for the O2-starvation phenotype. How could loss of control of a terminal oxidase affect survival during O2 starvation? One possibility is that inappropriate levels of oxidase expression may affect the adaptation to O2 starvation, by preventing electron flux through the most favourable respiratory pathway. Alternatively, a defect in oxidase regulation may affect exit from stationary phase, which is required for viability determination of samples taken from the O2-starved culture. There is a precedent for this in the stationary-phase exit defect found in E. coli cytochrome-bd-deficient mutants (Siegele & Kolter, 1993
; Siegele et al; 1996
; Goldman et al., 1996
). However, there may be other explanations for the effect of purF mutation on O2-starvation survival. In Lactobacillus lactis there is evidence for cellular metabolic pathways being intimately related to stress responses (Duwat et al., 1999
; Rallu et al., 2000
). Changes in guanine nucleotide pools affect the ability to synthesize (p)ppGpp and related signal molecules, although one might expect (p)ppGpp also to have a role in carbon-starvation survival. Studies of acid stress of L. lactis suggest that modification of the flux through the purine nucleotide pathway or increased pppGpp concentrations are perceived as intracellular stress signals in this organism, leading to multistress resistance (Rallu et al., 2000
).
M. smegmatis loses viability more rapidly in the early stages of O2-starved stationary phase compared to carbon-starved stationary phase. This may reflect the dynamic state of the culture under carbon-starvation conditions, with new variants with improved survival capabilities appearing during incubation (Smeulders et al., 1999 ), their growth being fuelled by nutrients (carbon sources) released by dying cells and the availability of O2 as a terminal electron acceptor for respiration. During O2 starvation there is no terminal electron acceptor available and so cryptic growth would only be possible if the bacteria used an undiscovered fermentation pathway. The finding that there are mutations which are important for carbon-starved stationary phase but not for O2-starved stationary phase, and that the purF mutant is affected in O2- but not carbon-starvation survival suggests that different survival mechanisms do operate under both conditions. It is interesting that mutant 317C, which is defective in a putative penicillin-binding protein (Keer et al., 2000
), was not markedly defective in survival during O2 starvation while it is in rich medium and following carbon starvation. The need for a penicillin-binding protein in stationary-phase survival is consistent with our previous demonstration of the dynamic nature of M. smegmatis stationary-phase cultures and the appearance of new variants, which presumably requires growth and cell division (Smeulders et al., 1999
). The absence of a survival phenotype of this mutant during O2 starvation raises the issue of whether O2-starved cultures are similarly dynamic.
How do the purF mutants recover culturability from day 815 onwards under O2 starvation? Southern blotting showed that recovery did not involve movement of the transposon and the survival kinetics of a culture pre-adapted to O2 starvation were not consistent with the accumulation of suppressor mutations (Fig. 5). Therefore, we are left with the explanation that the dramatic loss in viability followed by a partial recovery is the phenotype of the purF mutant. This is supported by the complementation experiment in which pPUR10 complemented the ability of 329B to grow on unsupplemented minimal medium and partially, but not completely, its ability to survive O2 starvation to wild-type levels. The lack of full complementation may be due to the multicopy complementation leading to an imbalance in the levels of purine biosynthetic intermediates. Two possible mechanisms can explain the recovery in the culturability of the purF mutant in O2-starved stationary phase. Firstly, as conditions change in stationary phase, regrowth (cryptic growth) of the viable population (typically 103104 c.f.u. ml-1 at day 812), occurs using nutrients released by dead cells. This seems improbable during O2 starvation for the reasons discussed above. However, the possibility cannot be ruled out that an unknown fermentation pathway is available to M. smegmatis under these conditions or that a terminal electron acceptor is made available following the breakdown of dead cells. A second explanation is that the loss of culturability during the early days of O2 starvation results from a significant fraction of the population becoming dormant or active but nonculturable (ANC). We prefer the term ANC here to viable but nonculturable (VBNC) for the reasons discussed by others (Barer, 1997
; Barer & Harwood, 1999
; Kell et al., 1998
). The outcome of dormancy or the ANC state is a transient loss of culturabilty, although the physiological properties of the cells would differ markedly between these two states. If the cells become dormant they would enter a reversible state of low metabolic activity while if they became ANC they would remain metabolically active. The dormant or ANC cells then resuscitate and recover culturability as conditions change in stationary phase. Resuscitation would not necessitate regrowth. We cannot distinguish between these possibilities at present although their investigation will form the basis of future experiments. Interestingly, a recent investigation by Bogosian et al. (2000)
indicates that the apparent resuscitation of Vibrio vulnificus from a VBNC (ANC) state (Whitesides & Oliver, 1997
) may be explained by the emergence of an injured, hydrogen-peroxide-sensitive subpopulation. An intriguing possibility is that something similar may be happening in the M. smegmatis purF mutant.
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ACKNOWLEDGEMENTS |
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Received 17 July 2000;
revised 2 October 2000;
accepted 30 October 2000.