Cloning and characterization of the glutamate 1-semialdehyde aminomutase gene from Xanthomonas campestris pv. phaseoli

The gene from Xanthomonas campestris pv. phaseoli for glutamate 1-semialdehyde (GSA) aminomutase, which is involved in the C5 pathway for synthesis of -aminolevulinic acid (ALA), was cloned onto a multicopy plasmid, pUC18, by the complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of deletion fragments from the initial 3.5-kb chromosomal fragment allowed the isolation of a 1.7-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.7-kb DNA fragment revealed an open reading frame (ORF) that is located downstream from a potential promoter sequence and a ribosome-binding site. The ORF encodes a polypeptide of 429 amino acid residues, and the deduced molecular mass of this polypeptide is 45,043 Da. The amino acid sequence shows a high degree of homology to the HemL proteins from other organisms, and a putative binding site for pyridoxal 5-phosphate is conserved. Correspondence to: Y. Murooka     

The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, β-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20–44·5°C and at pH values 5·2–7·4 with optimal growth at 37–41·5°C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5·0, the optimum temperature was 40°C for the endoglucanase and 50°C for the xylanase. Both enzyme activities were inhibited at 70°C. Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

Comparative assessment of the intracellular survival of the Burkholderia pseudomallei bopC mutant

Burkholderia pseudomallei, the causative agent of melioidosis, is a Gram-negative saprophytic bacterium capable of surviving within phagocytic cells. To assess the role of BopC (a type III secreted effector protein) in the pathogenesis of B. pseudomallei, a B. pseudomallei bopC mutant was used to infect J774A.1 macrophage-like cells. The bopC mutant showed significantly reduced intracellular survival in infected macrophages compared to wild-type B. pseudomallei. In addition, the bopC mutant displayed delayed escape from endocytic vesicles compared with the wild-type strain. This indicates that BopC is important, and at least in part, needed for intracellular survival of B. pseudomallei.     

Effect of acidic pH on the invasion efficiency and the type III secretion system of <Emphasis Type="Italic">Burkholderia thailandensis</Emphasis>

Burkholderia thailandensis is a close relative of Burkholderia pseudomallei. These organisms are very similar, but B. thailandensis is far less virulent than B. pseudomallei. Nucleotide sequencing and analysis of 14 B. thailandensis isolates revealed variation in the regions coding for the type III secreted BipD protein. The degree of B. thailandensis BipD sequence variation was greater than that found in B. pseudomallei. Western blot analysis indicated that, unlike B. pseudomallei, B. thailandensis type III secreted proteins including BipD and BopE could not be detected in the supernatant of culture medium unless induced by acidic conditions. In addition, culturing B. thailandensis under acidic growth conditions (pH 4.5) can induce the ability of this bacterium to invade human respiratory epithelial cells A549. The identification of an environmental stimulus that increases the invasion capability of B. thailandensis invasion is of value for those who would like to use this bacterium as a model to study B. pseudomallei virulence.     

β-Galactosidase from Lactobacillus pentosus: Purification,characterization and formation of galacto-oligosaccharides

A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 U_oNPG/mg protein. The K_m, k_cat and k_cat/K_m values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) were 38 mM, 20 s^-1, 530 M^-1·s^-1 and 1.67 mM, 540 s^-1, 325 000 M^-1·s^-1, respectively. The temperature optimum of β-galactosidase activity was 60–65°C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal β-galactosidases. Mg²⁺ ions enhanced both activity and stability significantly. L. pentosus β-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of β-(1→3) and β-(1→6) linkages, and the main transgalactosylation products identified were the disaccharides β-D-Galp-(1→6)-D -Glc, β-D-Galp-(1→3)-D -Glc, β-D -Galp-(1→6)-D -Gal, β-D -Galp-(1→3)-D -Gal, and the trisaccharides β-D -Galp-(1→3)-D -Lac, β-D -Galp-(1→6)-D -Lac.     

Effect of colony morphology variation of <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro

Background  

Primary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. Variable morphology is associated with changes in the expression of a range of putative virulence factors. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Isogenic colony morphology types II and III were generated from 5 parental type I B. pseudomallei isolates using nutritional limitation. Survival of types II and III were compared with type I for all assays.