Uncovering new metabolic capabilities of Bacillus subtilis using phenotype profiling of rifampin-resistant rpoB mutants

Abstract

RNA polymerase is a central macromolecular machine controlling the flow of information from genotype to phenotype, and insights into global transcriptional regulation can be gained by studying mutational perturbations in the enzyme. Mutations in the RNA polymerase beta subunit gene rpoB causing resistance to rifampin (Rif(r)) in Bacillus subtilis were previously shown to lead to alterations in the expression of a number of global phenotypes known to be under transcriptional control, such as growth, competence for transformation, sporulation, and germination (H. Maughan, B. Galeano, and W. L. Nicholson, J. Bacteriol. 186:2481-2486, 2004). To better understand the global effects of rpoB mutations on metabolism, wild-type and 11 distinct congenic Rif(r) mutant strains of B. subtilis were tested for utilization of 95 substrates by use of Biolog GP2 MicroPlates. A number of alterations of substrate utilization patterns were observed in the Rif(r) mutants, including the utilization of novel substrates previously unknown in B. subtilis, such as gentiobiose, beta-methyl-D-glucoside, and D-psicose. The results indicate that combining global metabolic profiling with mutations in RNA polymerase provides a system-wide approach for uncovering previously unknown metabolic capabilities and further understanding global transcriptional control circuitry in B. subtilis.

FIG. 1.

Substrate utilization patterns in wt and Rif^r mutant B. subtilis strains for substrates utilized strongly (>100 U) by the wt. The horizontal line at 50 U denotes the background level (see text for details). Asterisks denote significant differences (P < 0.05) from the wt. (A) Arabinose and xylose. (B) Salicin and trehalose. (C) Pyruvic acid and uridine.

FIG. 2.

Ribose utilization in wt and Rif^r mutant B. subtilis strains. (A) Ribose utilization patterns. The horizontal line at 50 U denotes the background level. Asterisks denote significant differences from the wt (P < 0.05). (B) Kinetics of ribose utilization in the wt (circles) and RpoB Q469K (squares), Q469L (diamonds), and Q469R (triangles) mutants. Only the first 12 h are shown for clarity. (C) Growth rates (doubling times) of wt and Q469 mutant strains in SMM containing d-ribose at 55 mM or 0.55 mM. Data are averages and standard deviations of triplicate determinations. The asterisk denotes significant difference (P < 0.05).

FIG. 3.

Substrate utilization patterns in wt and Rif^r mutant B. subtilis strains for substrates utilized weakly (50 to 100 U) by the wt. Asterisks denote significant differences (P < 0.05) from the wt. (A) Gentiobiose, β-d-methyl glucoside, and psicose. (B) Cellobiose and arbutin. (C) Amino acids l-alanine, l-asparagine, l-glutamic acid, and l-serine.

FIG. 4.

Summary of substrate utilization by Rif^r mutants. (A) Substrates utilized strongly (>100 U) by the wt. (B) Substrates utilized weakly (50 to 100 U) by the wt. Numbers of substrates whose utilization was significantly increased (black bars) or decreased (hatched bars) relative to the wt (P < 0.05 by ANOVA) are shown above the corresponding mutation.