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Global Analysis of Proteins Synthesized by Mycobacterium smegmatis Provides Direct Evidence for Physiological Heterogeneity in Stationary-Phase Cultures^¶

Marian C. J. Blokpoel,^1, Marjan J. Smeulders,^1, Julia A. M. Hubbard,² Jacquie Keer,^1, and Huw D. Williams^1*

Division of Biology, Faculty of Life Sciences, Imperial College London, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, United Kingdom,¹ Computational, Analytical and Structural Sciences, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts SG1 2NY, United Kingdom²

Received 26 April 2005/ Accepted 19 July 2005

We have characterized the induction kinetics of 1,700 proteins during entry into and survival in carbon-starved 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 (Spr₁₂₈ proteins). A number of Spr₁₂₈ 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 Spr₁₂₈ 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 stationary-phase 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.

^¶ Supplemental material for this article may be found at http://jb.asm.org/.

M.C.J.B. and M. J. S. contributed equally to this work.

Present address: LGC Ltd., Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.