Increased levels of dihydrofolate reductase in rifampin-resistant mutants of Bacillus subtilis

Abstract

Several independent, spontaneous rifampin-resistant mautants of Bacillus subtilis were isolated and found to have an increased resistance to trimethoprim, an inhibitor of dihydrofolate reductase. This increased resistance in the rif mutants was the result of a specific threefold increase in the activity of dihydrofolate reductase, since six other enzymes examined remained unchanged. This increased level of dihydrofolate reductase and the trimethoprim resistance were cotransformed (100%') with the i-if marker. These results suggest that the RNA polymerase is altered in its recognition of the gene that specifies dihydrofolate reductase.Rifampin and its derivatives are antibiotics that inhibit the activity of the DNA-dependent RNA polymerase by binding to the /3-subunit of the enzvme and preventing the initation of transcription (2, 7). Mutations to r-ifampin resistance have been reported to have pleiotropic effects on complex metabolic processes, such as: a reduced abilitv of Bacillus ssubtilis to sporulate (6,12,13); suppression of dnaA mutations (1); and inhibition of the growth of bacteriophages (11). In addition, alterations in specific enzymes have been reported in some rif nmutants: specifically, an increased level of enzymes in the arginine pathway (17), an increased level of dihvdrofolate (DHF) reductase (M. M. Aldridge, V. J. Wainscott, and J. F. Kane, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, K47, p. 144), and the absence of the enzyme glutamate svnthase (10). In this report we present results that show an increased level of DHF reductase in rif mutants of B. subtilis.All of the mutants used in this study were derivatives of the competent strain 168. Spontaneous rifampin-resistant mutants were obtained by surface spreading the gat-7 mutant (previouslI referred to as the trpX7 mutant [4,5]) NP102 on Trypticase soy agar containing (0.6%' yeast extract and 5 ,ig of rifampin (Sigma Chemical Co., St. Louis, Mo.) per ml. The plates were incubated at :370C until resistant colonies appeared. Single colonies were transferred to the rifampin-containing medium anc streaked for isolation. Cultures for enzyme assavs were routinelyr grown in 200 ml of minimal glutcose me-Present address: t)e)artiment of Biolog. (ilemison t Tiversitv, (lenison, NC 29(331. P Present address: Departm-lernt of tell Biology, Baylor College of Medicine, Houston, TIX,770:10.

Alterations in the Spectrum of Spontaneous Rifampicin-Resistance Mutations in the Bacillus subtilis rpoB Gene after Cultivation in the Human Spaceflight Environment

“… Koch et al, 2014 ). Such effects include: activation of cryptic metabolic capabilities ( Perkins and Nicholson, 2008 ); alterations in the susceptibility to other classes of antibiotics ( Kane et al, 1979 ; Kristich and Little, 2012 ); or production of various secondary metabolites ( Hu et al, 2002 ). Furthermore, it has been shown that certain mutant alleles of rpoB actually enhance the fitness of the resulting Rif R mutants under specific environmental stresses, such as growth under nutrient-limiting conditions ( Maharjan and Ferenci, 2017a ).…”