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Functional Genomics of Gram-Positive Microorganisms. Marta Perego, 2004. Sinorhizobium meliloti Sulfotransferase That Modifies Lipopolysaccharide. Glen E. Cronan, 2004.Sinorhizobium meliloti is a gram-negative soil bacterium found either in free-living form or as a nitrogen-fixing endosymbiont of a plant structure called the nodule . Symbiosis between S . meliloti and its plant host alfalfa is dependent on bacterial transcription of nod genes, which encode the enzymes responsible for synthesis of Nod factor . S . meliloti Nod factor is a lipochitooligosaccharide that undergoes a sulfate modification essential for its biological activity . Sulfate also modifies the carbohydrate substituents of the bacterial cell surface, including lipopolysaccharide (LPS) and capsular polysaccharide (K-antigen) (R . A . Cedergren, J . Lee, K . L . Ross, and R . I . Hollingsworth, Biochemistry 34:4467-4477, 1995) . We utilized the genomic sequence of S . meliloti to identify an open reading frame, SMc04267 (which we now propose to name lpsS), which encodes an LPS sulfotransferase activity . We expressed LpsS in Escherichia coli and demonstrated that the purified protein functions as an LPS sulfotransferase . Mutants lacking LpsS displayed an 89% reduction in LPS sulfotransferase activity in vitro . However, lpsS mutants retain approximately wild-type levels of sulfated LPS when assayed in vivo, indicating the presence of an additional LPS sulfotransferase activity(ies) in S . meliloti that can compensate for the loss of LpsS . The lpsS mutant did show reduced LPS sulfation, compared to that of the wild type, under conditions that promote nod gene expression, and it elicited a greater number of nodules than did the wild type during symbiosis with alfalfa . These results suggest that sulfation of cell surface polysaccharides and Nod factor may compete for a limiting pool of intracellular sulfate and that LpsS is required for optimal LPS sulfation under these conditions . Molecular Analysis of Carbon Monoxide-Oxidizing Bacteria Associated with Recent Hawaiian Volcanic Deposits. Kari E. Dunfield, 2004.Genomic DNA extracts from four sites at Kilauea Volcano were used as templates for PCR amplification of the large subunit (coxL) of aerobic carbon monoxide dehydrogenase . The sites included a 42-year-old tephra deposit, a 108-year-old lava flow, a 212-year-old partially vegetated ash-and-tephra deposit, and an approximately 300-year-old forest . PCR primers amplified coxL sequences from the OMP clade of CO oxidizers, which includes isolates such as Oligotropha carboxidovorans, Mycobacterium tuberculosis, and Pseudomonas thermocarboxydovorans . PCR products were used to create clone libraries that provide the first insights into the diversity and phylogenetic affiliations of CO oxidizers in situ . On the basis of phylogenetic and statistical analyses, clone libraries for each site were distinct . Although some clone sequences were similar to coxL sequences from known organisms, many sequences appeared to represent phylogenetic lineages not previously known to harbor CO oxidizers . On the basis of average nucleotide diversity and average pairwise difference, a forested site supported the most diverse CO-oxidizing populations, while an 1894 lava flow supported the least diverse populations . Neither parameter correlated with previous estimates of atmospheric CO uptake rates, but both parameters correlated positively with estimates of microbial biomass and respiration . Collectively, the results indicate that the CO oxidizer functional group associated with recent volcanic deposits of the remote Hawaiian Islands contains substantial and previously unsuspected diversity . Functional Analysis and Regulation of the Divergent spuABCDEFGH-spuI Operons for Polyamine Uptake and Utilization in Pseudomonas aeruginosa PAO1. Chung-Dar Lu, 2002.A multiple-gene locus for polyamine uptake and utilization was discovered in Pseudomonas aeruginosa PAO1 . This locus contained nine genes designated spuABCDEFGHI (spu for spermidine and putrescine utilization) . The physiological functions of the spu genes in utilization of two polyamines (putrescine and spermidine) were analyzed by using Tn5 transposon-mediated spu knockout mutants . Growth and uptake experiments support that the spuDEFGH genes specify components of a major ABC-type transport system for spermidine uptake, and enzymatic measurements indicated that spuC encodes putrescine aminotransferase with pyruvate as the amino group receptor . Although spuA and spuB mutants showed an apparent defect in spermidine utilization, the biochemical functions of the gene products have yet to be elucidated . Assays of lacZ fusions demonstrated the presence of agmatine-, putrescine-, and spermidine-inducible promoters for the spuABCDEFGH operon and the divergently transcribed spuI gene of unknown function . Since the observed induction effect of agmatine was abolished in an aguA mutant where conversion of agmatine into putrescine was blocked, putrescine or spermidine, but not agmatine, serves as the inducer molecule of the spuA-spuI divergent promoters . S1 nuclease mappings confirmed further the induction effects of the polyamines on transcription of the divergent promoters and localized the transcription initiation sites . Gel retardation assays with extracts from the cells grown on putrescine or spermidine demonstrated the presence of a polyamine-responsive regulatory protein interacting with the divergent promoter region . Finally, the absence of the putrescine-inducible spuA expression and putrescine aminotransferase (spuC) formation in the cbrB mutant indicated that the spu operons are regulated by the global CbrAB two-component system perhaps via the putative polyamine-responsive transcriptional activator . A Third Transposable Element, ISPpu12, from the Toluene-Xylene Catabolic Plasmid pWW0 of Pseudomonas putida mt-2. Peter A. Williams, 2002.A 3,372-bp insertion sequence, ISPpu12, has been identified on the archetypal toluene-xylene TOL catabolic plasmid pWW0 from Pseudomonas putida mt-2 . The insertion sequence element is located on the plasmid between bases 84397 and 87768 in a region which also contains the termini and transposase genes of the catabolic transposons Tn4651 and Tn4653 (A . Greated, L . Lambertson, P . A . Williams, and C . M . Thomas, Environ . Microbiol., in press) . ISPpu12 has terminal inverted repeats of 24 bp with three mismatches and contains four open reading frames, a tnpA homologue and three open reading frames (lspA, orf1, and orf2) of undetermined function . After insertion in vitro of a Kmr cassette into ISPpu12 either in the intergenic region between orf1 and orf2 or directly into the orf1 gene and ligation into a suicide vector, the modified ISPpu12-Km transposes at high frequency, often in multiple copies, into the chromosome of a P . putida recipient . Inactivation of lspA, orf1, and orf2 by introducing a 7-bp deletion into the 5' region of each gene had no major effect upon transposition, but a similar mutation of tnpA completely eliminated transposition . Analysis of the literature and of strains derived from the chlorobenzoate-degrading Pseudomonas sp . strain B13 suggests that the promiscuity of this element has played an important role in the history of plasmid pWW0 . Database comparisons and the accompanying paper (A . J . Weightman, A . W . Topping, K . E . Hill, L . L . Lee, K . Sakai, J . H . Slater, and A . W . Thomas, J . Bacteriol . 184:6581-6591, 2002) show that ISPpu12 is a transposable element also found in other bacteria . Crystal Structure of D-Hydantoinase from Burkholderia pickettii at a Resolution of 2.7 Angstroms: Insights into the Molecular Basis of Enzyme Thermostability. Zhen Xu, 2003.D-Hydantoinase (D-HYD) is an industrial enzyme that is widely used in the production of D-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides . This report describes the crystal structure of D-hydantoinase from Burkholderia pickettii (HYDBp) at a 2.7-Å resolution . The structure of HYDBp consists of a core (  /ß)8 triose phosphate isomerase barrel fold and a ß-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues . Although HYDBp shares only moderate sequence similarity with D-HYDs from Thermus sp . (HYDTsp) and Bacillus stearothermophilus (HYDBst), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar . Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity . Structural comparison has revealed insights into the molecular basis of the differential thermostability of D-HYDs . The more thermostable HYDTsp contains more aromatic residues in the interior of the structure than HYDBp and HYDBst . Changes of large aromatic residues in HYDTsp to smaller residues in HYDBp or HYDBst decrease the hydrophobicity and create cavities inside the structure . HYDTsp has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYDBp and HYDBst . Besides, HYDTsp also contains more rigid Pro residues . These factors are likely to make major contributions to the varying thermostability of these enzymes . This information could be exploited in helping to engineer more thermostable mesophilic enzymes .
Synthesis of GDP-Mannose and Mannosylglycerate from Labeled Mannose by Genetically Engineered Escherichia coli without Loss of Specific Isotopic Enrichment. Maria-Manuel Sampaio, 2003.We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-  -D-mannosyl-D-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment . The strain carries a deletion in the manA gene, encoding phosphomannose isomerase . This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM) . The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer . Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose . A second plasmid harbors msg, a gene from Rhodothermus marinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O--D-mannosyl-D-glycerate from GDP-mannose and endogenous glycerate . The rate-limiting step in 2-O--D-mannosyl-D-glycerate formation is the transfer of GDP-mannose to glycerate . 2-O--D-mannosyl-D-glycerate can be released from cells by treatment with cold-water shock . The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography .
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