Combined Drug Resistance Mutations Substantially Enhance Enzyme Production in Paenibacillus agaridevorans.

This study shows that sequential introduction of drug resistance mutations substantially increased enzyme production in The triple mutant YT478 ( Gln225→stop codon, K56R, and R485H), generated by screening for resistance to streptomycin and rifampin, expressed a 1,100-fold-larger amount of the extracellular enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) than the wild-type strain. These mutants were characterized by higher intracellular -adenosylmethionine concentrations during exponential phase and enhanced protein synthesis activity during stationary phase. Surprisingly, the maximal expression of CITase mRNA was similar in the wild-type and triple mutant strains, but the mutant showed greater CITase mRNA expression throughout the growth curve, resulting in enzyme overproduction.

Activation and products of the cryptic secondary metabolite biosynthetic gene clusters by rifampin resistance (rpoB) mutations in actinomycetes.

A subset of rifampin resistance (rpoB) mutations result in the overproduction of antibiotics in various actinomycetes, including Streptomyces, Saccharopolyspora, and Amycolatopsis, with H437Y and H437R rpoB mutations effective most frequently. Moreover, the rpoB mutations markedly activate (up to 70-fold at the transcriptional level) the cryptic/silent secondary metabolite biosynthetic gene clusters of these actinomycetes, which are not activated under general stressful conditions, with the exception of treatment with rare earth elements. Analysis of the metabolite profile demonstrated that the rpoB mutants produced many metabolites, which were not detected in the wild-type strains.

Generation of Bradyrhizobium japonicum mutants with increased N2O reductase activity by selection after introduction of a mutated dnaQ gene.

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N(2)O) reductase (N(2)OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N(2)O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli.

PCR-mediated one-step deletion of targeted chromosomal regions in haploid Saccharomyces cerevisiae.

Chromosome rearrangements, especially chromosomal deletions, have been exploited as important resources for functional analysis of genomes. To facilitate this analysis, we applied a previously developed method for chromosome splitting for the direct deletion of a designed internal or terminal chromosomal region carrying many nonessential genes in haploid Saccharomyces cerevisiae. The method, polymerase chain reaction (PCR)-mediated chromosomal deletion (PCD), consists of a two-step PCR and one transformation per deletion event.

Large scale deletions in the Saccharomyces cerevisiae genome create strains with altered regulation of carbon metabolism.

Saccharomyces cerevisiae, for centuries the yeast that has been the workhorse for the fermentative production of ethanol, is now also a model system for biological research. The recent development of chromosome-splitting techniques has enabled the manipulation of the yeast genome on a large scale, and this has allowed us to explore questions with both biological and industrial relevance, the number of genes required for growth and the genome organization responsible for the ethanol production. To approach these questions, we successively deleted portions of the yeast genome and constructed a mutant that had lost about 5% of the genome and that gave an increased yield of ethanol and glycerol while showing levels of resistance to various stresses nearly equivalent to those of the parental strain.

A novel microperoxidase activity: methyl viologen-linked nitrite reducing activity of microperoxidase.

To investigate the nitrite reducing activity of microperoxidases (mps) in the presence of methyl viologen and dithionite, the fragments C14-K22 (mp9), V11-L32 (mp22), and G1-M65 (mp65) containing heme were prepared by enzymatic hydrolysis of commercially equine heart cytochrome c (Cyt c), in which His is axially coordinated to heme iron, and acts as its fifth ligand. The nitrite reducing activity of mps was measured under anaerobic condition, and the nitrite reducing activity of mps increased with the cutting of the peptide chain. The activity of the shortest nonapeptide mp9 was approximately 120-fold that of Cyt c (104 amino acid residues) and 3.