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Molecular and Functional Characterization of a Unique Sucrose Hydrolase from Xanthomonas axonopodis pv . glycines. Hong-Suk Kim, 2004.A novel sucrose hydrolase (SUH) from Xanthomonas axonopodis pv . glycines, a causative agent of bacterial pustule disease on soybeans, was studied at the functional and molecular levels . SUH was shown to act rather specifically on sucrose (Km = 2.5 mM) but not on sucrose-6-phosphate . Protein analysis of purified SUH revealed that, in this monomeric enzyme with an estimated molecular mass of 70,223 ± 12 Da, amino acid sequences determined for several segments have corresponding nucleotide sequences in XAC3490, a protein-coding gene found in the genome of X . axonopodis pv . citri . Based on this information, the SUH gene, consisting of an open reading frame of 1,935 bp, was cloned by screening a genomic library of X . axonopodis pv . glycines 8ra . Database searches and sequence comparison revealed that SUH has significant homology to some family 13 enzymes, with all of the crucial invariant residues involved in the catalytic mechanism conserved, but it shows no similarity to known invertases belonging to family 32 . suh expression in X . axonopodis pv . glycines requires sucrose induction, and insertional mutagenesis resulted in an absence of sucrose-inducible sucrose hydrolase activity in crude protein extracts and a sucrose-negative phenotype . Recombinant SUH, overproduced in Escherichia coli and purified, was shown to have the same enzymatic characteristics in terms of kinetic parameters . Chromosomal Amplification of the Escherichia coli lipB Region Confers High-Level Resistance to Selenolipoic Acid. Sean W. Jordan, 2002.One of the mutants (slr7 mutant) of a wild-type Escherichia coli strain resistant to selenolipoic acid reported previously (K . E . Reed, T . W . Morris, and J . E . Cronan, Jr., Proc . Natl . Acad . Sci . USA 91:3720-3724, 1994) unexpectedly grew on minimal medium following transductional introduction of a lipA null mutation . We report that the slr7 strain carries a duplication of the lip chromosomal region that causes the phenotype of the mutant strain . The IntI-Like Tyrosine Recombinase of Shewanella oneidensis Is Active as an Integron Integrase. François Drouin, 2002.We have found an integron-like integrase gene and an attI site in Shewanella oneidensis as part of a small chromosomal integron . We have cloned this gene and tested the ability of the integrase to excise cassettes from various integrons . Most cassettes flanked by two attC sites are readily excised, while cassettes in the "first" position, with an attI1 or attI3 site on one end, are not excised . An exception is a cassette with attI2 on one end . The attI2 site, from Tn7, has greater similarity to the attI site adjacent to the integrase of S . oneidensis than do attI1 or attI3 . We cloned the attI site of S . oneidensis and observed the integration of two different cassettes . We have, therefore, demonstrated the function of this integron-like integrase . CelI, a Noncellulosomal Family 9 Enzyme from Clostridium thermocellum, Is a Processive Endoglucanase That Degrades Crystalline Cellulose. Rachel Gilad, 2003.The family 9 cellulase gene celI of Clostridium thermocellum, was previously cloned, expressed, and characterized (G . P . Hazlewood, K . Davidson, J . I . Laurie, N . S . Huskisson, and H . J . Gilbert, J . Gen . Microbiol . 139:307-316, 1993) . We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein . The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b . The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70°C, respectively, using carboxymethylcellulose as a substrate . Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase . A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates . A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined . This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates . Synthetic lac Operator Substitutions for Studying the Nitrate- and Nitrite-Responsive NarX-NarL and NarQ-NarP Two-Component Regulatory Systems of Escherichia coli K-12. Valley Stewart, 2003.The NarX and NarQ sensor-histidine kinases control phosphorylation of the NarL and NarP response regulators in response to the respiratory oxidants nitrate and nitrite . Target operon transcription is activated by the Fnr protein in response to anaerobiosis, and it is further activated and/or repressed by the phospho-NarL and phospho-NarP proteins, which bind to heptamer DNA sequences . The location and arrangement of heptamers vary widely among different target operon control regions . We have constructed a series of monocopy lac operon control region constructs in which the primary operator O1-lac has been replaced by 7-2-7 heptamer pairs from the nrfA, nirB, napF, and fdnG operon control regions . These constructs provide tools for dissecting various aspects of ligand interactions with sensor-kinases, sensor interactions with response regulators, and phospho-response regulator interactions with DNA targets . Expression of the lacZ gene from these constructs was repressed to various degrees by nitrate and nitrite . In response to nitrate, the nrfA and nirB operon 7-2-7 heptamer pairs at operator O1 each mediated greater than 100-fold repression of lacZ gene expression, whereas the napF operon 7-2-7 heptamer pair mediated approximately tenfold repression . Introduction of narL, narP, narX, and narQ null alleles in various combinations allowed the in vivo interactions between different sensor-regulator pairs to be evaluated and compared . PCR Assay of the groEL Gene for Detection and Differentiation of Bacillus cereus Group Cells. Yu-Hsiu Chang, 2003.Strains of species in the Bacillus cereus group are potentially enterotoxic . Thus, the detection of all B . cereus group strains is important . As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the B . cereus group, we explored the potential of the groEL gene as a phylogenetic marker . A phylogenetic analysis of the groEL sequences of 78 B . cereus group strains revealed that the B . cereus group strains were split into two major clusters, one including six B . mycoides and one B . pseudomycoides (cluster II) and the other including two B . mycoides and the rest of the B . cereus group strains (cluster I) . Cluster I was further differentiated into two subclusters, Ia and Ib . The sodA gene sequences of representative strains from different clusters were also compared . The phylogenetic tree constructed from the sodA sequences showed substantial similarity to the tree constructed from the groEL sequences . Based on the groEL sequences, a PCR assay for detection and identification of B . cereus group strains was developed . Subsequent restriction fragment length polymorphism (RFLP) analysis verified the PCR amplicons and the differentiation of the B . cereus group strains . RFLP with MboI was identical for all the B . cereus group strains analyzed, while RFLP with MfeI or PstI classified all B . cereus and B . thuringiensis strains into two groups . All cluster II B . mycoides and B . pseudomycoides strains could be discriminated from other B . cereus group bacteria by restriction analysis with TspRI .
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