New Gram-positive and Gram-negative bacteria were isolated from Poeni oily sludge, using enrichment procedures. The six Gram-positive strains belong to Bacillus, Lysinibacillus and Rhodococcus genera. The eight Gram-negative strains belong to Shewanella, Aeromonas, Pseudomonas and Klebsiella genera. Isolated bacterial strains were tolerant to saturated (i.e., n-hexane, n-heptane, n-decane, n-pentadecane, n-hexadecane, cyclohexane), monoaromatic (i.e., benzene, toluene, styrene, xylene isomers, ethylbenzene, propylbenzene) and polyaromatic (i.e., naphthalene, 2-methylnaphthalene, fluorene) hydrocarbons, and also resistant to different antimicrobial agents (i.e., ampicillin, kanamycin, rhodamine 6G, crystal violet, malachite green, sodium dodecyl sulfate). The presence of hydrophilic antibiotics like ampicillin or kanamycin in liquid LB-Mg medium has no effects on Gram-positive and Gram-negative bacteria resistance to toxic compounds. The results indicated that Gram-negative bacteria are less sensitive to toxic compounds than Gram-positive bacteria, except one bacteria belonging to Lysinibacillus genus. There were observed cellular and molecular modifications induced by ampicillin or kanamycin to isolated bacterial strains. Gram-negative bacteria possessed between two and four catabolic genes (alkB, alkM, alkB/alkB1, todC1, xylM, PAH dioxygenase, catechol 2,3-dioxygenase), compared with Gram-positive bacteria (except one bacteria belonging to Bacillus genus) which possessed one catabolic gene (alkB/alkB1). Transporter genes (HAE1, acrAB) were detected only in Gram-negative bacteria.
Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.
A gram-positive bacterium, strain K-540BT, which was separated from a mixed culture isolated from compost, was subjected to a polyphasic taxonomic study. Strain K-540BT was found to have 95.5%-98.7% sequence similarity with the genus Leucobacter based on 16S rRNA gene sequence analyis. Phenotypic and chemical properties of strain K-540BT were consistent with its classification in the genus Leucobacter. The major quinone system compound was MK-11 and major fatty acids were anteiso-C15:0 and iso-C16:0. Polar lipids included phosphatidylglycerol and diphosphatidylglycerol. The G+C content of the genomic DNA was 64.9 mol%. The cell wall peptidoglycan contained diaminobutyric acid and γ-aminobutyric acid. DNA-DNA hybridization results and physiological tests genotypically and phenotypically differentiated strain K-540BT from other Leucobacter species. Based on these results, strain K-540BT (=NBRC 106062T=DSM 22850T), for which the name Leucobacter exalbidus sp. nov. is proposed, represents a novel species of the genus Leucobacter.
Poly-γ-glutamic acid (γPGA), a polymer of glutamic acid, is a component of the viscosity substance of natto, a traditional Japanese food made from soybeans fermented with Bacillus subtilis (natto). Here we investigate the effects of the cell wall lytic enzymes belonging to the D,L-endopeptidases (LytE, LytF, CwlO and CwlS) on γPGA production by B. subtilis (natto). γPGA levels in a cwlO disruptant were about twofold higher than that of the wild-type strain, whereas disruption of the lytE, lytF and cwlS genes had little effect on γPGA production. The molecular size of γPGA in the cwlO disruptant was larger than that of the wild-type strain. A complementary strain was constructed by insertion of the entire cwlO gene into the amyE locus of the CwlO mutant genome, and γPGA production was restored to wild-type levels in this complementary strain. These results indicated that the peptidoglycan degradation enzyme, CwlO, plays an important role in γPGA production and affects the molecular size of γPGA.
The site-specific integrase of actinophage R4 belongs to the serine recombinase family. During the lysogenization process, it catalyzes site-specific recombination between the phage genome and the chromosome of Streptomyces parvulus 2297. An in vivo assay using Escherichia coli cells revealed that the minimum lengths of the recombination sites attB and attP are 50-bp and 49-bp, respectively, for efficient intramolecular recombination. The in vitro assay using overproduced R4 integrases as a hexahistidine (His6)-glutathione-S-transferase (GST)-R4 integrase fusion protein, showed that the purified protein preparation retains the site-specific recombination activity which catalyzes the site-specific recombination between attP and attB in the intermolecular reaction. It also revealed that the inverted repeat within attP is essential for efficient in vitro intermolecular recombination. In addition, a gel shift assay showed that His6-GST-R4 integrase bound to the 50-bp attB and 49-bp attP specifically. Moreover, based on a detailed comparison analysis of amino acid sequences of serine integrases, we found the DNA binding region that is conserved in the serine recombinase containing the large C-terminal domain. Based on the results presented on this report, attachment sites needed in vitro and in vivo for site-specific recombination by the R4 integrase have been defined more precisely. This knowledge is useful for developing new genetic manipulation tools in the future.