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PaeX, a Second Pectin Acetylesterase of Erwinia chrysanthemi 3937. Vladimir E. Shevchik, 2003.Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls . Pectin is a complex polysaccharide . The main chain is constituted of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification . Esterases are necessary to remove these modifications and, thus, to facilitate the further degradation of the polysaccharidic chain . In addition to PaeY, the first pectin acetylesterase identified in the E . chrysanthemi strain 3937, we showed that this bacterium produces a second pectin acetylesterase encoded by the gene paeX . The paeX open reading frame encodes a 322-residue precursor protein of 34,940 Da, including a 21-amino-acid signal peptide . Analysis of paeX transcription, by using gene fusions, revealed that it is induced by pectic catabolic products and affected by catabolite repression . The expression of paeX is regulated by the repressor KdgR, which controls all the steps of pectin catabolism; by the repressor PecS, which controls most of the pectinase genes; and by catabolite regulatory protein, the global activator of sugar catabolism . The paeX gene is situated in a cluster of genes involved in the catabolism and transport of pectic oligomers . In induced conditions, the two contiguous genes kdgM, encoding an oligogalacturonate-specific porin, and paeX are both transcribed as an operon from a promoter proximal to kdgM, but transcription of paeX can also be uncoupled from that of kdgM in noninduced conditions . PaeX is homologous to the C-terminal domain of the Butyrivibrio fibriosolvens xylanase XynB and to a few bacterial esterases . PaeX contains the typical box (GxSxG) corresponding to the active site of the large family of serine hydrolases . Purified PaeX releases acetate from various synthetic substrates and from sugar beet pectin . The PaeX activity increased after previous depolymerization and demethylation of pectin, indicating that its preferred substrates are nonmethylated oligogalacturonides . PaeX is mostly found in the periplasmic space of E . chrysanthemi . These data suggest that PaeX is mainly involved in the deacetylation of esterified oligogalacturonides that enter the periplasm by the KdgM porin . Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities. Massimiliano Cardinale, 2004.ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M . M . Fisher and E . W . Triplett, Appl . Environ . Microbiol . 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L . Ranjard et al., Appl . Environ . Microbiol . 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities . An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the façade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets . The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets . The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments . The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species . ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng µl–1, while the other primer sets failed to detect the spacers of one or more bacterial strains . Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri . mRNA Decay in Escherichia coli Comes of Age. Sidney R. Kushner, 2002. In Vitro Identification of Rns-Regulated Genes. George P. Munson, 2002.To identify Rns-regulated genes, a maltose binding protein (MBP)-Rns fusion protein was used to isolate DNA fragments from enterotoxigenic Escherichia coli genomic DNA that carry Rns binding sites . In vivo transcription fusion analysis shows that Rns positively regulates the expression of the open reading frame of yiiS, which lies immediately downstream of one MBP-Rns-bound fragment . Modulation of pPS10 Host Range by Plasmid-Encoded RepA Initiator Protein. Beatriz Maestro, 2003.We report here the isolation and analysis of novel repA host range mutants of pPS10, a plasmid originally found in Pseudomonas savastanoi . Upon hydroxylamine treatment, five plasmid mutants were selected for their establishment in Escherichia coli at 37°C, a temperature at which the wild-type form cannot be established . The mutations were located in different functional regions of the plasmid RepA initiation protein, and the mutants differ in their stable maintenance, copy number, and ability to interact with sequences of the basic replicon . Four of them have broadened their host range, and one of them, unable to replicate in Pseudomonas, has therefore changed its host range . Moreover, the mutants also have increased their replication efficiency in strains other than E . coli such as Pseudomonas putida and Alcaligenes faecalis . None of these mutations drastically changed the structure or thermal stability of the wild-type RepA protein, but in all cases an enhanced interaction with host-encoded DnaA protein was detected by gel filtration chromatography . The effects of the mutations on the functionality of RepA protein are discussed in the framework of a three-dimensional model of the protein . We propose possible explanations for the host range effect of the different repA mutants, including the enhancement of limiting interactions of RepA with specific host replication factors such as DnaA .
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