Plasmid artificial modification: a novel method for efficient DNA transfer into bacteria.

Bacterial transformation is an essential component of many molecular biological techniques, but bacterial restriction-modification (R-M) systems can preclude the efficient introduction of shuttle vector plasmids into target bacterial cells. Whole-genome DNA sequences have recently been published for a variety of bacteria. Using homology and motif analyses, putative R-M genes can be identified from genome sequences. Introducing DNA methyltransferase genes into Escherichia coli cells causes subsequently transformed plasmids to be modified by these enzymes. We propose a new method, designated Plasmid Artificial Modification (PAM). A PAM plasmid encoding the modification enzymes expressed by the target bacterial host is transformed into E. coli (PAM host). Propagation of a shuttle vector from the PAM host to the target bacterium ensures that the plasmid will be modified such that it is protected from restriction endonuclease digestion in the target bacterium. The result will be a higher transformation efficiency. Here, we describe the use of PAM and electroporation to transform Bifidobacterium adolescentis ATCC15703. By introducing two genes encoding modification enzymes, we improved transformation efficiency 10(5)-fold.

Intra-species diversity between seven Bifidobacterium adolescentis strains identified by genome-wide tiling array analysis.

Six Bifidobacterium adolescentis strains, JCM1275, JCM1251, JCM7044, JCM7045, JCM7046, and 9-124, were analyzed using a tiling array designed according to the full genome sequence of ATCC15703. The results demonstrated deletion clusters along with single-gene mutations and deletions. Most deletions concerned genes involved in polysaccharide and cell-surface biogenesis. A dendrogram illustrating the deletions and mutations is presented and the evolution of B. adolescentis is discussed.

Improvement of bacterial transformation efficiency using plasmid artificial modification.

We have developed a method to improve the transformation efficiency in genome-sequenced bacteria, using 'Plasmid Artificial Modification' (PAM), using the host's own restriction system. In this method, a shuttle vector was pre-methylated in Escherichia coli cells, which carry all the putative genes encoding the DNA modification enzymes of the target microorganism, before electroporation was performed. In the case of Bifidobacterium adolescentis ATCC15703 and pKKT427 (3.9 kb E. coli-Bifidobacterium shuttle vector), introducing two Type II DNA methyltransferase genes lead to an enhancement in the transformation efficiency by five orders of magnitude. This concept was also applicable to a Type I restriction system. In the case of Lactococcus lactis IO-1, by using PAM with a putative Type I methyltransferase system, hsdMS1, the transformation efficiency was improved by a factor of seven over that without PAM.

Lack of effect of aciclovir on metabolism of theophylline and expression of hepatic cytochrome P450 1A2 in rats.

There is an interesting clinical report indicating that aciclovir, which is mainly excreted into urine, decreases the systemic clearance of theophylline by inhibiting cytochrome P450 (CYP) 1A2-mediated metabolism. In this study, we investigated the effect of aciclovir on the metabolism of theophylline, and on the activity and expression of hepatic CYP1A2 in rats. Theophylline (10 mg/kg) was injected intravenously into rats treated with two different dosages of aciclovir. When theophylline was simultaneously administered with aciclovir (50 mg/kg), the systemic clearance of theophylline and metabolic clearance of its major metabolites, 1-methyluric acid and 1,3-dimethyluric acid, were unchanged. In place of theophylline, when 1-methyl-3-propylxanthine (2.5 mg/kg), which is almost metabolized by CYP1A2 in rats, was coadministered intravenously with aciclovir (50 mg/kg), the pharmacokinetics of 1-methyl-3-propylxanthine was also unchanged. When theophylline was administered to rats pretreated with repeated intraperitoneal injections of aciclovir (25 mg/kg twice daily for 3 d), no significant differences in the systemic clearance of theophylline and its metabolic clearance to 1-methyluric acid and 1,3-dimethyluric acid were observed between the control and aciclovir-treated rats. This dosage of aciclovir did not change the activity of 7-ethoxyresorufin O-dealkylation, which is represented as CYP1A2 activity. In Western blot analysis, no significant change in the protein levels of hepatic CYP1A2 was observed between the control and aciclovir-treated rats. The present study suggests that aciclovir has no effect on the pharmacokinetics and metabolism of theophylline and on the activity and expression of hepatic CYP1A2 in rats.

Effect of a newly developed ketolide antibiotic, telithromycin, on metabolism of theophylline and expression of cytochrome P450 in rats.

The effects of a newly-developed ketolide antibiotic, telithromycin, on the metabolism of theophylline and the expression of hepatic cytochrome P450 (CYP) 1A2 and CYP3A2 were investigated in rats. Telithromycin at a high dose (100 mg/kg of body weight) was injected intraperitoneally once a day for 3 days. Twenty-four hours (day 4) after the final administration of telithromycin, theophylline (10 mg/kg) was administered intravenously. The presence of telithromycin significantly delayed the disappearance of theophylline from plasma. Parameters related to the pharmacokinetic interaction between theophylline and telithromycin were examined by noncompartmental methods. A significant decrease in the systemic clearance of theophylline was observed in the presence of telithromycin. Pretreatment with telithromycin significantly decreased the metabolic clearance of the major metabolites, 1-methyluric acid and 1,3-dimethyluric acid, with no change in the renal clearance of theophylline, suggesting that the decreased systemic clearance of theophylline by telithromycin is due to reduction of their metabolic clearance. Pretreatment with telithromycin significantly decreased the activity of 7-ethoxyresorufin O-deethylation and testosterone 6 beta-hydroxylation, suggesting that telithromycin decreases the activity of hepatic CYP1A2 and CYP3A2. Western blot analysis revealed that telithromycin significantly decreased the protein levels of CYP1A2 and CYP3A2 in the liver, which could explain the observed decreases in the systemic clearance of theophylline and metabolic clearance of 1-methyluric acid and 1,3-dimethyluric acid. The present study suggests that telithromycin at the dose used in this study alters the pharmacokinetics and metabolism of theophylline, due to reductions in the activity and expression of hepatic CYP1A2 and CYP3A2.

Crystallization and preliminary X-ray crystallographic studies of XynX, a family 10 xylanase from Aeromonas punctata ME-1.

Xylanases catalyze the hydrolysis of beta-1,4-glycosidic linkages within the xylan backbone. XynX is a xylanase from Aeromonas punctata ME-1 and belongs to glycoside hydrolase family 10. While most xylanases show endo-type catalytic activities, XynX shows exo-like catalytic activities, selectively producing xylobiose from birchwood xylan. In this study, XynX was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 79.0, b = 88.6, c = 93.2 A, and diffracted to beyond 1.8 A resolution.