Exponential-phase glycogen recycling is essential for growth of Mycobacterium smegmatis

Abstract

Bacterial glycogen is a polyglucose storage compound that is thought to prolong viability during stationary phase. However, a specific role for glycogen has not been determined. We have characterized SMEG53, a temperature-sensitive mutant of Mycobacterium smegmatis that contains a mutation in glgE, encoding a putative glucanase. This mutation causes exponentially growing SMEG53 cells to stop growing at 42 degrees C in response to high levels of glycogen accumulation. The mutation in glgE is also associated with an altered growth rate and colony morphology at permissive temperatures; the severity of these phenotypes correlates with the amount of glycogen accumulated by the mutant. Suppression of the temperature-sensitive phenotype, via a decrease in glycogen accumulation, is mediated by growth in certain media or multicopy expression of garA. The function of GarA is unknown, but the presence of a forkhead-associated domain suggests that this protein is a member of a serine-threonine kinase signal transduction pathway. Our results suggest that in M. smegmatis glycogen is continuously synthesized and then degraded by GlgE throughout exponential growth. In turn, this constant recycling of glycogen controls the downstream availability of carbon and energy. Thus, in addition to its conventional storage role, glycogen may also serve as a carbon capacitor for glycolysis during the exponential growth of M. smegmatis.

FIG. 1

Growth curves of SMEG53 and mc²155 cultures at 30°C following a temperature shift from 30 to 42°C. Cells grown exponentially at 30°C were diluted to an OD₆₀₀ of 0.3 in fresh medium; and then one half was incubated at 30°C, and the other half was incubated at 42°C. Cell densities were determined at OD₆₀₀ over the time period shown. Samples: SMEG53, 30°C (□); SMEG53, 42°C (■); mc²155, 30°C (○); mc²155, 42°C (●). Shown is a representative example of three independent experiments. The time point where the morphological change is first noted in SMEG53 at 42°C is indicated by the symbol containing the asterisk.

FIG. 2

Amino acid similarities between the M. smegmatis glgE gene product (SMEG), the M. tuberculosis homolog Rv1327c (TB), and one of the S. coelicolor pep1 gene products (PEP1). Alignments were performed with CLUSTAL W, and the amino acid shading was performed with McBoxshade version 2.11. Black shading depicts amino acid residues conserved in all three of the proteins; grey shading depicts residues conserved in two. The histidine mutated in the SMEG53 glgE gene product is indicated by the asterisk.

FIG. 3

Genetic organization of the SMEG53 complementing genes glgE (A) and garA (B). The region shown depicts the smallest amount of DNA required for the restoration of high-temperature growth to SMEG53 based on subclone insert size or overlapping clone analysis. Shadings denote complete (black) and partial (shaded) open reading frames. Arrows indicate direction of transcription, and lines depict intervening DNA. Also shown is the putative gene product of garA. The FHA domain predicted by PROSITE is boxed, and the three invariant amino acid residues associated with this domain are shown in bold.

FIG. 4

Glycogen content of SMEG53 grown under conditions suppressing the temperature-sensitive phenotype. Exponentially growing cultures were diluted to an OD₆₀₀ of 0.3 in fresh medium and then shifted to 42°C. Cells were grown to mid-log phase (OD₆₀₀ = 0.75 to 0.85), and then glycogen assays performed as before. Each value represents the mean of three assays ± standard deviation. (A) Mutant cells containing pMD30, pAEB234 (garA), or pAEB235 (glgE), grown in 7H9 containing 10 μg of kanamycin per ml. (B) SMEG53 cells grown in 7H9, 7H9 containing 0.2 M NaCl (NaCl), or M9.

FIG. 5

(A and B) Colonial morphology of wild-type (A) and SMEG53 (B) colonies grown at 30°C on 7H10. (C and D) Colonies of SMEG53 complemented with pAEB235 (glgE; C) or pAEB234 (garA; D) and grown at 42°C on 7H10. The colonies shown in panel A are representative of the morphology found for the wild-type under all conditions tested.