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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.

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Hydrocarbons (HCs) are environmentally friendly natural refrigerants and are expected to be promising alternative candidates to replace some currently used halogenated hydrocarbon refrigerants. Some available data sets for HCs used to formulate the equations of state (EoS) for them are relatively old, so we point out that new data with less uncertainty are expected to play an essential role in updating the EoS for HCs. Therefore, a set of PVT property measurements for hydrocarbon refrigerants including propane, n-butane, and isobutane was conducted in the present study. A newly developed vibrating-tube densimeter was employed for the measurements, and then a total of 430 liquid PVT properties were obtained, including those at the saturation boundaries. The measurement range is (240 to 380) K for temperature and up to 7 MPa for pressure. The measurement uncertainty is about 3 mK for temperature, 0.26 kPa + 0.022% for pressure, and 0.1 kg‚m -3 + 0.024% for density. The present data were compared with available thermodynamic models that are currently considered to be the most reliable. A set of modified Tait equations of state for the liquid phase are also discussed.

Bifidobacterium longum is a nonpathogenic anaerobic bacterium among normal bacterial flora. Recently, it was reported that B. longum accumulated in hypoxic solid tumors. The gene of interest was expressed in transfected B. longum by the shuttle vector pBLES100 in solid tumors. In this report, we constructed pBLES100-S-eCD, which included the cytosine deaminase gene. We confirmed by western blotting that transfected B. longum produced cytosine deaminase. In addition, transfected B. longum produced cytosine deaminase that converted 5-fluorocytosine into 5-fluorouracil. B. longum could be useful for enzyme/pro-drug therapy of hypoxic solid tumors.

ABSTRACTFunctional analysis ofBifidobacteriumgenes is essential for understanding host-Bifidobacteriuminteractions with beneficial effects on human health; however, the lack of an effective targeted gene inactivation system in bifidobacteria has prevented the development of functional genomics in this bacterium. Here, we report the development of a markerless gene deletion system involving a double crossover inBifidobacterium longum. Incompatible plasmid vectors were used to facilitate a second crossover step. The conditional replication vector pBS423-ΔrepA, which lacks the plasmid replication generepA, was integrated into the target gene by a first crossover event. Subsequently, the replicative plasmid pTBR101-CM, which harborsrepA, was introduced into this integrant to facilitate the second crossover step and subsequent elimination of the excised conditional replication vector from the cells by plasmid incompatibility. The proposed system was confirmed to work as expected inB. longum105-A using the chromosomal full-length β-galactosidase gene as a target. Markerless gene deletion was tested using theagagene, which encodes α-galactosidase, whose substrates include raffinose. Almost all the pTBR101-CM-transformed strains became double-crossover recombinants after subculture, and 4 out of the 270 double-crossover recombinants had lost the ability to assimilate raffinose. Genotype analysis of these strains revealed markerless gene deletion ofaga. Carbohydrate assimilation analysis and α-galactosidase activity measurement were conducted using both the representative mutant and a plasmid-basedaga-complemented strain. These functional analyses revealed thatagais the only gene encoding a functional α-galactosidase enzyme inB. longum105-A.

Bifidobacteria are the main component of the human microflora. We constructed a temperature-sensitive (Ts) plasmid by random mutagenesis of the Bifidobacterium-Escherichia coli shuttle vector pKKT427 using error-prone PCR. Mutant plasmids were introduced into Bifidobacterium longum 105-A and, after screening approximately 3,000 colonies, candidate clones that grew at 30 °C but not at 42 °C were selected. According to DNA sequence analysis of the Ts plasmid, five silent and one missense mutations were found in the repB region. The site-directed mutagenesis showed only the missense mutation to be relevant to the Ts phenotype. We designated this plasmid pKO403. The Ts phenotype was also observed in B. longum NCC2705 and Bifidobacterium adolescentis ATCC15703. Single-crossover homologous-recombination experiments were carried out to determine the relationship between the length of homologous sequences encoded on the plasmid and recombination frequency: fragments greater than 1 kb gave an efficiency of more than 10(3) integrations per cell. We performed gene knockout experiments using this Ts plasmid. We obtained gene knockout mutants of the pyrE region of B. longum 105-A, and determined that double-crossover homologous recombination occurred at an efficiency of 1.8 %. This knockout method also worked for the BL0033 gene in B. longum NCC2705.

The interactions of three forms of HU dimer from Escherichia coli with DNA were compared. The complexes formed between HU and short DNA fragments (35 to 132 bp), uncurved oligodeoxyribonucleotide (oligo) with and without 5-bp deoxyriboadenosine (dA) stretches and curved oligo with 5-bp dA stretches, were analyzed by the gel retardation technique. The binding of HU homodimers to the DNA was less efficient than that of HU heterodimer, and the HU-1 homodimer had lower DNA-binding capacity than the HU-2 homodimer. The equilibrium dissociation constant (KD) for DNA of the HU-1 homodimer was 3 times that of the HU heterodimer. The HU dimers had two- to fourfold higher affinity for DNA duplexes containing 5-bp dA stretches, particularly for curved DNA. The binding of HU heterodimer to the DNA was inhibited more by the curved DNA competitor than the uncurved DNA competitor without dA stretches.