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Unusual Microbial Xylanases from Insect Guts. YaLi Brennan, 2004.Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms . In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths) . Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity . Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered . Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases . Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted ß-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products . From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed . An ABC Transporter from Bacillus thuringiensis Is Essential for ß-Exotoxin I Production. Sylvain Espinasse, 2002.ß-Exotoxin I is a nonspecific insecticidal metabolite secreted by some Bacillus thuringiensis strains . Several studies of B . thuringiensis strains that have lost the capacity to produce ß-exotoxin I have suggested that there is a strong correlation between high levels of ß-exotoxin I production and the ability to synthesize crystal proteins . In this study, we showed that a mutant strain, B . thuringiensis 407-1(Cry-)(Pig+), with no crystal gene, produced considerable amounts of ß-exotoxin I together with a soluble brown melanin pigment . Therefore, ß-exotoxin I production can take place after a strain has lost the plasmids bearing the cry genes, which suggests that these curable plasmids probably contain determinants involved in the regulation of ß-exotoxin I production . Using a mini-Tn10 transposon, we constructed a library of strain 407-1(Cry-)(Pig+) mutants . We screened for nonpigmented mutants with impaired ß-exotoxin I production and identified a genetic locus harboring two genes (berA and berB) essential for ß-exotoxin I production . The deduced amino acid sequence of the berA gene displayed significant similarity to the ATP-binding domains of the DRI (drug resistance and immunity) family of ATP-binding cassette (ABC) proteins involved in drug resistance and immunity to bacteriocins and lantibiotics . The berB gene encodes a protein with six putative transmembrane helices, which probably constitutes the integral membrane component of the transporter . The demonstration that berAB is required for ß-exotoxin I production and/or resistance in B . thuringiensis adds an adenine nucleotide analog to the wide range of substrates of the superfamily of ABC proteins . We suggest that berAB confers ß-exotoxin I immunity in B . thuringiensis, through active efflux of the molecule . Heavy Metal Resistance of Biofilm and Planktonic Pseudomonas aeruginosa. Gail M. Teitzel, 2003.A study was undertaken to examine the effects of the heavy metals copper, lead, and zinc on biofilm and planktonic Pseudomonas aeruginosa . A rotating-disk biofilm reactor was used to generate biofilm and free-swimming cultures to test their relative levels of resistance to heavy metals . It was determined that biofilms were anywhere from 2 to 600 times more resistant to heavy metal stress than free-swimming cells . When planktonic cells at different stages of growth were examined, it was found that logarithmically growing cells were more resistant to copper and lead stress than stationary-phase cells . However, biofilms were observed to be more resistant to heavy metals than either stationary-phase or logarithmically growing planktonic cells . Microscopy was used to evaluate the effect of copper stress on a mature P . aeruginosa biofilm . The exterior of the biofilm was preferentially killed after exposure to elevated concentrations of copper, and the majority of living cells were near the substratum . A potential explanation for this is that the extracellular polymeric substances that encase a biofilm may be responsible for protecting cells from heavy metal stress by binding the heavy metals and retarding their diffusion within the biofilm . Characterization of an Inducible Chlorophenol O-Methyltransferase from Trichoderma longibrachiatum Involved in the Formation of Chloroanisoles and Determination of Its Role in Cork Taint of Wines. Juan-José R. Coque, 2003.A novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase catalyzing the O methylation of several chlorophenols and other halogenated phenols was purified 220-fold to apparent homogeneity from mycelia of Trichoderma longibrachiatum CECT 20431 . The enzyme could be identified in partially purified protein preparations by direct photolabeling with [methyl-3H]SAM, and this reaction was prevented by previous incubation with S-adenosylhomocysteine . Gel filtration indicated that the Mr was 112,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme was composed of two subunits with molecular weights of approximately 52,500 . The enzyme had a pH optimum between 8.2 and 8.5 and an optimum temperature of 28°C, with a pI of 4.9 . The Km values for 2,4,6-trichlorophenol and SAM were 135.9 ± 12.8 and 284.1 ± 35.1 µM, respectively . S-Adenosylhomocysteine acted as a competitive inhibitor, with a Ki of 378.9 ± 45.4 µM . The methyltransferase was also strongly inhibited by low concentrations of several metal ions, such as Cu2+, Hg2+, Zn2+, and Ag+, and to a lesser extent by p-chloromercuribenzoic acid, but it was not significantly affected by several thiols or other thiol reagents . The methyltransferase was specifically induced by several chlorophenols, especially if they contained three or more chlorine atoms in their structures . Substrate specificity studies showed that the activity was also specific for halogenated phenols containing fluoro, chloro, or bromo substituents, whereas other hydroxylated compounds, such as hydroxylated benzoic acids, hydroxybenzaldehydes, phenol, 2-metoxyphenol, and dihydroxybenzene, were not methylated .
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