Identification and Characterization of a New Organic Hydroperoxide Resistance (ohr) Gene with a Novel Pattern of Oxidative Stress Regulation from Xanthomonas campestris pv. phaseoli

We have isolated a new organic hydroperoxide resistance (ohr) gene from Xanthomonas campestris pv. phaseoli. This was done by complementation of an Escherichia coli alkyl hydroperoxide reductase mutant with an organic hydroperoxide-hypersensitive phenotype. ohr encodes a 14.5-kDa protein. Its amino acid sequence shows high homology with several proteins of unknown function. An ohr mutant was subsequently constructed, and it showed increased sensitivity to both growth-inhibitory and killing concentrations of organic hydroperoxides but not to either H₂O₂ or superoxide generators. No alterations in sensitivity to other oxidants or stresses were observed in the mutant. ohr had interesting expression patterns in response to low concentrations of oxidants. It was highly induced by organic hydroperoxides, weakly induced by H₂O₂, and not induced at all by a superoxide generator. The novel regulation pattern of ohr suggests the existence of a second organic hydroperoxide-inducible system that differs from the global peroxide regulator system, OxyR. Expression of ohr in various bacteria tested conferred increased resistance to tert-butyl hydroperoxide killing, but this was not so for wild-type Xanthomonas strains. The organic hydroperoxide hypersensitivity of ohr mutants could be fully complemented by expression of ohr or a combination of ahpC and ahpF and could be partially complemented by expression ahpC alone. The data suggested that Ohr was a new type of organic hydroperoxide detoxification protein.     

Bacillus subtilis SSE4 produces subtulene A, a new lipopeptide antibiotic possessing an unusual C15 unsaturated β-amino acid

Subtulene A, a new cyclic lipopeptide, was isolated from the culture broth of Bacillus subtilis SSE4. This antibiotic compound contained the seven common α-amino acids, l-Asn-1, d-Tyr-2, d-Asn-3, l-Gln-4, l-Pro-5, d-Asn-6, l-Ser-7 and the unique β-amino acid-8 present in the iturin family. 1D and 2D NMR, as well as MS analyses, identified the β-amino acid as 3-amino-13-methyltetradec-8-enoic acid, an Iso C15 long chain β-amino acid. B. subtilis SSE4 was also found to produce iturin A. B. subtilis SSE4 culture filtrate exhibited both antifungal and antibacterial activities.     

Discovery of indazoles as inhibitors of Tpl2 kinase

Synthesis, modeling and structure-activity relationship of indazoles as inhibitors of Tpl2 kinase are described. From a high throughput screening effort, we identified an indazole hit compound 5 that has a single digit micromolar Tpl2 activity. Through SAR modifications at the C3 and C5 positions of the indazole, we discovered compound 31 with good potency in LANCE assay and cell-based p-Erk assay.     

Mutations in oxyR resulting in peroxide resistance in Xanthomonas campestris

A spontaneous Xanthomonas campestris pv. phaseoli H(2)O(2)-resistant mutant emerged upon selection with 1 mM H(2)O(2). In this report, we show that growth of this mutant under noninducing conditions gave high levels of catalase, alkyl hydroperoxide reductase (AhpC and AhpF), and OxyR. The H(2)O(2) resistance phenotype was abolished in oxyR-minus derivatives of the mutant, suggesting that elevated levels and mutations in oxyR were responsible for the phenotype. Nucleotide sequence analysis of the oxyR mutant showed three nucleotide changes. These changes resulted in one silent mutation and two amino acid changes, one at a highly conserved location (G197 to D197) and the other at a nonconserved location (L301 to R301) in OxyR. Furthermore, these mutations in oxyR affected expression of genes in the oxyR regulon. Expression of an oxyR-regulated gene, ahpC, was used to monitor the redox state of OxyR. In the parental strain, a high level of wild-type OxyR repressed ahpC expression. By contrast, expression of oxyR5 from the X. campestris pv. phaseoli H(2)O(2)-resistant mutant and its derivative oxyR5G197D with a single-amino-acid change on expression vectors activated ahpC expression in the absence of inducer. The other single-amino-acid mutant derivative of oxyR5L301R had effects on ahpC expression similar to those of the wild-type oxyR. However, when the two single mutations were combined, as in oxyR5, these mutations had an additive effect on activation of ahpC expression.     

The unique glutathione reductase from Xanthomonas campestris: gene expression and enzyme characterization

The glutathione reductase gene, gor, was cloned from the plant pathogen Xanthomonas campestris pv. phaseoli. Its gene expression and enzyme characteristics were found to be different from those of previously studied homologues. Northern blot hybridization, promoter-lacZ fusion, and enzyme assay experiments revealed that its expression, unlike in Escherichia coli, is OxyR-independent and constitutive upon oxidative stress conditions. The deduced amino acid sequence shows a unique NADPH binding motif where the most highly conserved arginine residue, which is critical for NADPH binding, is replaced by glutamine. Interestingly, a search of the available Gor amino acid sequences from various sources, including other Xanthomonas species, revealed that this replacement is specific to the genus Xanthomonas. Recombinant Gor enzyme was purified and characterized, and was found to have a novel ability to use both, NADPH and NADH, as electron donor. A gor knockout mutant was constructed and shown to have increased expression of the organic peroxide-inducible regulator gene, ohrR.     

Separation and purification of phycocyanin from Spirulina sp. using a membrane process

The highest purity ratio of phycocyanin extract was obtained when fresh biomass was used as raw material. The crude extract was purified by membrane process using microfiltration and ultrafiltration. Membrane of pore sizes 5 μm, at feed flow rate of 150 mL min⁻¹, permeate flux of 58.5 L h⁻¹ m⁻² was selected for coarse filtration and membrane with pore size 0.8/0.2 μm at the flow rate of 100 mL min⁻¹, permeate flux of 336 L h⁻¹ m⁻² was selected for fine filtration, giving phycocyanin recovery of 88.6% and 82.9%, respectively. For ultrafiltration, membrane with MWCO at 50 kDa, 69 kPa and 75 mL min⁻¹ of flow rate with a mean permeate flux 26.8 L h⁻¹ m⁻² and a retention rate of 99% was found to be optimal. Under these filtration conditions, food grade phycocyanin with the purity around 1.0 containing c-phycocyanin as the major component was obtained.     

Regulation of the katG-dpsA operon and the importance of KatG in survival of Burkholderia pseudomallei exposed to oxidative stress

Homologues of the catalase-peroxidase gene katG and the gene for the non-specific DNA binding protein dpsA were identified downstream of oxyR in Burkholderia pseudomallei. Northern experiments revealed that both katG and dpsA are co-transcribed during oxidative stress. Under conditions where the katG promoter is not highly induced, dpsA is transcribed from a second promoter located within the katG-dpsA intergenic region. A katG insertion mutant was found to be hypersensitive to various oxidants. Analysis of katG expression in the oxyR mutant indicates that OxyR is a dual function regulator that represses the expression of katG during normal growth and activates katG during exposure to oxidative stress. Both reduced and oxidized OxyR were shown to bind to the katG promoter.