Compensatory increase in<Emphasis Type="Italic"> ahpC</Emphasis> gene expression and its role in protecting<Emphasis Type="Italic"> Burkholderia pseudomallei</Emphasis> against reactive nitrogen intermediates

In the human pathogen Burkholderia pseudomallei, katG encodes the antioxidant defense enzyme catalase-peroxidase. Interestingly, a B. pseudomallei mutant, disrupted in katG, is hyperresistant to organic hydroperoxide. This hyperresistance is due to the compensatory expression of the alkyl hydroperoxide reductase gene (ahpC) and depends on a global regulator OxyR. The KatG-deficient mutant is also highly resistant to reactive nitrogen intermediates (RNI). When overproduced, the B. pseudomallei AhpC protein, protected cells against killing by RNI. The levels of resistance to both organic peroxide and RNI returned to those of the wild-type when the katG mutant was complemented with katG. These studies establish the partially overlapping defensive activities of KatG and AhpC.     

Agrobacterium tumefaciens membrane-bound ferritin plays a role in protection against hydrogen peroxide toxicity and is negatively regulated by the iron response regulator

An Agrobacterium tumefaciens membrane-bound ferritin (mbfA) mutant was generated to assess the physiological functions of mbfA in response to iron and hydrogen peroxide (H(2) O(2) ) stresses. Wild-type and the mbfA mutant strains showed similar growth under high- and low-iron conditions. The mbfA mutant was more sensitive to H(2) O(2) than wild-type strain. Expression of a functional mbfA gene could complement the H(2) O(2) -hypersensitive phenotype of the mbfA mutant and a rhizobial iron regulator (rirA) mutant, suggesting that MbfA protects cells from H(2) O(2) toxicity by sequestering intracellular free iron, thus preventing the Fenton reaction. The expression of mbfA could be induced in response to iron and to H(2) O(2) treatment. The iron response regulator (irr) also acted as a repressor of mbfA expression. An irr mutant had high constitutive expression of mbfA, which partly contributed to the H(2) O(2) -hyperresistant phenotype of the irr mutant. The data reported here demonstrate an important role of A.?tumefaciens MbfA in the cellular defence against iron and H(2) O(2) stresses.     

Mini-Tn7 vectors as genetic tools for gene cloning at a single copy number in an industrially important and phytopathogenic bacteria, Xanthomonas spp.

Transposon mini-Tn 7 vectors insert into the chromosome of several Gram-negative bacteria in a site-specific manner. Here, we showed the application of mini-Tn 7 as single copy site-specific integration vector system for Xanthomonas campestris pv. campestris . The transposition of the mini-Tn 7 into the bacterial genome was detected at a Tn 7 attachment ( att Tn 7 ) site located downstream of glmS1 . Furthermore, using a newly constructed vector pBBR1FLP2 containing the flipase (FLP) recombinase for site-specific excision of the sequence between the FLP recombinase target (FRT) sites, and a sacB counter selection marker, an unmarked mini-Tn 7 insertion mutant was created. Mini-Tn 7 insertion did not affect bacterial virulence on the tested plant. The mini-Tn 7 and FLP–FRT systems also work well in Xanthomonas oryzae pv. oryzae .     

Gene Cloning and Characterization of a Novel Highly Organic Solvent Tolerant Lipase from Proteus sp. SW1 and its Application for Biodiesel Production

Proteus sp. SW1 was found to produce an extracellular solvent tolerant lipase. The gene, lipA, encoding a bacterial lipase, was cloned from total Proteus sp. SW1 DNA. lipA was predicted to encode a 287 amino acid protein of 31.2?kDa belonging to the Group I proteobacterial lipases. Purified His-tagged LipA exhibited optimal activity at pH 10.0 and 55??C. It was highly stable in organic solvents retaining 112% of its activity in 100% isopropanol after 24?h, and exhibited more than 200% of its initial activity upon exposure to 60% acetone, ethanol, and hexane for 18?h. Biodiesel synthesis reactions, using a single step addition of 13% an acyl acceptor ethanol, showed that LipA was highly effective at converting palm oil into biodiesel.     

Characterization of the Opisthorchis viverrini genome

The methylations of trematode genomic DNA were analyzed using restriction enzymes and Southern blot hybridization. Restriction enzymes MspI, HpaII, HhaI were used to probe CpG methylation while MboI, Sau3A, DpnI were used for A methylation. The results revealed that Opisthorchis viverrini, Fasciola gigantica and Gigantocotyle siamensis had neither CpG nor A methylations. The presence of highly repeated DNA elements was also demonstrated in O. viverrini genomic DNA.