Mutants of Mycobacterium smegmatis impaired in stationary-phase survival The GenBank accession numbers for the sequences determined in this work are: AJ277088 (mutant 272A), AJ277089 (mutant 272E), AJ27790 (mutant 317C), AJ277152 (mutant 492A) and AJ276883 (mutant 3910D).

Abstract

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.

Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase

“…Detector outputs were acquired using Astra software (Wyatt Technology). 8 The identities of the purified wildtype proteins were confirmed by enzyme assay and liquid chromatography-electrospray ionization tandem mass spectrometry, whilst the molecular weight of the mutants were confirmed using MALDI mass spectrometry, as described in Supporting Methods. All mass spectrometry services were performed at the Adelaide Proteomics Center, The University of Adelaide.…”

“…As the cell membrane provides a defensive barrier against environmental toxins, host immune factors and antibiotic agents [6] and [7], the metabolic pathways related to the synthesis of membrane lipids have been suggested as promising targets for the development of new antibiotics. Genetic studies have revealed important roles for biotin biosynthesis in Mycobacterium tuberculosis during growth, infection and the latency phase, thus establishing this pathway as a potential drug target for new anti-tuberculosis therapies [3], [8], [9], [10], [11] and [12]. In addition, biotin biosynthesis has been intensively studied in other bacteria and the enzymes involved in the pathway have been implicated in bacterial virulence during infection and intracellular replication [13].…”

Formation and resuscitation of ‘non-culturable’ cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase

“…Recently, Keer et al (2000) have found that several mutants of Mycobacterium smegmatis, defective in stationary phase survival under aerobic or anaerobic conditions, revealed a transient decrease in viability followed by an increase in culturability. The authors suggest at least two possible explanations for this effect : (a) re-growth (cryptic growth) of viable cells, and (b) formation of ' non-culturable ' (and possibly dormant) bacteria in stationary phase and their subsequent resuscitation (Keer et al, 2000(Keer et al, , 2001. Since the authors did not measure culturability by MPN counts, direct evidence was neither sought nor obtained for resuscitation of non-culturable cells.…”

Biotin biosynthesis in Mycobacterium tuberculosis: physiology, biochemistry and molecular intervention

“…Access to genome-wide, insertional mutagenic libraries of Mycobacteria has facilitated phenotypic screening to identify key metabolic pathways required under a variety of conditions (Sassetti et al, 2001. For example disruption of bioA attenuated the growth of M. smegmatis on carbon-depleted media, imitating the nutrient deprived state experienced by stationary phase bacteria during latency (Keer et al, 2000). As the ability of Mycobacteria to grow inside macrophages is related to its virulence and pathogenicity (Russell, 2001), manipulation of in vitro growth conditions can also imitate conditions experienced in vivo.…”