Search

The natural plasmid pBM19 carries the key mdh gene needed for the oxidation of methanol into formaldehyde by Bacillus methanolicus. Five more genes, glpX, fba, tkt, pfk, and rpe, with deduced roles in the cell primary metabolism, are also located on this plasmid. By using real-time PCR, we show that they are transcriptionally upregulated (6- to 40-fold) in cells utilizing methanol; a similar induction was shown for two chromosomal genes, hps and phi. These seven genes are involved in the fructose bisphosphate aldolase/sedoheptulose bisphosphatase variant of the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation. Curing of pBM19 causes higher methanol tolerance and reduced formaldehyde tolerance, and the methanol tolerance is reversed to wild-type ievels by reintroducing mdh. Thus, the RuMP pathway is needed to detoxify the formaldehyde produced by the methanol dehydrogenase-mediated conversion of methanol, and the in vivo transcription levels of mdh and the RuMP pathway genes reflect the methanol tolerance level of the cells. The transcriptional inducer of hps and phi genes is formaldehyde, and not methanol, and introduction of multiple copies of these two genes into B. methanolicus made the cells more tolerant of growth on high methanol concentrations. The recombinant strain also had a significantly higher specific growth rate on methanol than the wild type. While pBM19 is critical for growth on methanol and important for formaldehyde detoxification, the maintenance of this plasmid represents a burden for B. methanolicus when growing on mannitol. Our data contribute to a new and fundamental understanding of the regulation of B. methanolicus methylotrophy.

Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50[degrees]C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.

Research has been conducted on Bacillus methanolicus MGA3 steady-state methanol-limited cultures. The role of the growth-inhibitory effects of methanol pulses which perturb steady-state methanol concentrations has been investigated and the results are reported.

The first genetic transformation system, shuttle vectors and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus are presented. Two low-copy-number Escherichia coli-B. methanolicus shuttle plasmids, pDQ507 and pDQ508 are shown. pDQ508 contains the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 contains a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule.

The restriction endonuclease Bacillus methanolicus (B. methanolicus) T1 (BmeT1) has been isolated and characterized. BmeT1 is an isoschizomer of BclI that recognizes the DNA sequence 5' TGATCA 3'. Data suggested the existence of a subset of methylate Sau3AI (GATC) sites and a second subset of nonmethylated Sau3AI sites within the B. methanolicus chromosome. Data can be used to develop strategies for inhibiting BmeT1 restriction of DNA introduced into B. methanolicus.

A comprehensive model for the kinetic aspects of alkaline phosphatase in the Escherichia coli periplasm which considers all substrate levels is suggested. The model assumes that the phosphate released from the periplasm was four times more than intake of substrate. The model accurately represents the kinetic behavior of various cell properties which indicates that the enzyme kinetic parameters obtained in dilute solution are applicable in vivo.

Streptomyces lavendulae has a genetic locus mcr that exhibits high-level resistance to mitomycin (MMC) when cloned into S. lavendulae. This locus has two genes mcrA and mcrB which are responsible for resistance and cellular self-protection. The resistance mechanism is involved in the prevention of reductive activation of MMC and DNA alkylation. Major similarities are observed between the deduced mcrA protein product and three oxidoreductases derived from bacterial, plant and animal systems.

The gene coding the thermostable meso-diaminopimelate (DAP) decarboxylase, derived from Bacillus methanolicus MGA3, was isolated, cloned and sequenced. The data obtained proved that the B. methanolicus MGA3 DAP decarboxylase is similar to the DAP decarboxylase of B. subtilis. Amino acid sequence studies of bacterial DAP decarboxylases, eukaryotic ornithine decarboxylases and arginine decarboxylases reveal several areas of conservation. The pattern of inhibition proves that the functioning of the enzyme in lysine-over-producing strains of B. methanolicus MGA3 may restrict lysine synthesis.

A study was conducted on the effect of diffusion in the in vivo kinetics of alkaline phosphatase of the Escherichia coli periplasm. Results showed that substrate diffusion to the enzyme induced as much as a 1000-fold change in enzyme concentration per cell. This observation indicated diffusion's role as a rate-limiting step of the enzymatic reaction in the cells.

A gene coding for the lysine-sensitive aspartokinase II of the methylotrophic thermotolerant Bacillus sp. strain MGA3 was isolated and characterized. Sequence analysis showed that the MGA3 aspartokinase II is a tetramer composed of alpha and beta dimers, having a total mass of 122,000 Da. Homology comparisons showed that the MGA3 aspartokinase is 76% identical to the B. subtilis aspartokinase II. In addition, the analysis of a 300-nucleotide intervening sequence showed that the enzyme may be regulated by a transcription attenuating mechanism similar to that proposed for the B. subtilis tryptophan operon.

The gene encoding citrate synthase from Bacillus sp. strain C4 was isolated and sequenced. The compelete nucleleotide sequence for the entire 3.1-kb HindIII fragment was derived revealing one major open reading frame which coding for citrate synthase, ctsA. The citrate synthase isolated from the strain C4 was a dimer with a subunit of Mr of 42, 000. The N-terminal amino acid sequence was found to be identifical with the predicted gene sequnce. The amino acid sequence was also compared with citrate synthase secreted by other gram-negative bacteria.

The cloning and DNA sequencing of a partial putative dap operon including aspartate semialdehyde dehydrogenase (asd), dihydrodipicolinate synthase (dapA), and dapG encoding AKI and the distant gene yclM encoding AKIII is reported. The results obtained provide further insight into aspartokinase (AK) regulation and function and valuable information that could be applied to engineer L-lysine-producing Bacillus methanolicus strains.

Results indicate that Bacillus methanolicus exhibit several forms of citrae synthase and glutamate secretion can be altered to increase L-lysine secretion by regulaing citrate synthase activity. In the citrate synthase deficient-mutant , the level of of glutamate secreted is reduced 7-fold and the ratio of L-lysine to glutamate secreted is enhance 4-5-fold as compared to the wild type.