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Putative Exopolysaccharide Synthesis Genes Influence Pseudomonas aeruginosa Biofilm Development. Masanori Matsukawa, 2004.An analysis of the Pseudomonas aeruginosa genomic sequence revealed three gene clusters, PA1381-1393, PA2231-2240, and PA3552-3558, in addition to the alginate biosynthesis gene cluster, which appeared to encode functions for exopolysaccharide (EPS) biosynthesis . Recent evidence indicates that alginate is not a significant component of the extracellular matrix in biofilms of the sequenced P . aeruginosa strain PAO1 . We hypothesized that at least one of the three potential EPS gene clusters revealed by genomic sequencing is an important component of P . aeruginosa PAO1 biofilms . Thus, we constructed mutants with chromosomal insertions in PA1383, PA2231, and PA3552 . The mutant with a PA2231 defect formed thin unstructured abnormal biofilms . The PA3552 mutant formed structured biofilms that appeared different from those formed by the parent, and the PA1383 mutant formed structured biofilms that were indistinguishable from those formed by the parent . Consistent with a previous report, we found that polysaccharides were one component of the extracellular matrix, which also contained DNA . We suggest that the genes that were inactivated in our PA2231 mutant are required for the production of an EPS, which, although it may be a minor constituent of the matrix, is critical for the formation of P . aeruginosa PAO1 biofilms . Modeling the Rate of Attachment of Listeria monocytogenes, Pantoea agglomerans, and Pseudomonas fluorescens to, and the Probability of Their Detachment from, Potato Tissue at 10°C. M. J. Garrood, 2004.The rate of attachment of bacteria to, and their subsequent detachment from, the cut surface of raw potato tissue was measured and modeled by using mathematical approaches that allowed detailed objective comparisons of adhesion processes under different conditions . Attachment was rapid and reached equilibrium after contact for 60 min . A new method to measure the probability of detachment was developed and modeled, revealing that the probability of detachment for Pseudomonas fluorescens remained unchanged for contact times between less than 5 s and 60 min . Listeria monocytogenes, however, was more easily removed initially, with the probability of detachment decreasing over the first 2 min of contact but remaining constant and equivalent to that for Pseudomonas fluorescens thereafter . For all of the bacteria tested, the number of bacteria attached after 2 min of contact was proportional to the inoculum concentration raised to the power of 0.79 . A Conserved C-Terminal Region in Gp71 of the Small Isometric-Head Phage LL-H and ORF474 of the Prolate-Head Phage JCL1032 Is Implicated in Specificity of Adsorption of Phage to Its Host, Lactobacillus delbrueckii. Victor Ravin, 2002.Thirty-five phage-resistant mutants of Lactobacillus delbrueckii subsp . lactis ATCC 15808 were selected . Thirty-three of these mutants were assigned to the Bes group, while the remaining two were grouped under the Ads designation . Bes group mutants adsorbed phage LL-H but did not allow efficient phage development . Preliminary evidence suggests that these strains exhibit a mutation that changes the DNA specificity of a restriction-modification system . The Ads group mutants did not adsorb the small isometric-head phage LL-H . The results suggest that there are at least three different types of phage receptors in L . delbrueckii: two that are specific for small isometric-head phages and one that is specific for prolate-head phage JCL1032 . Five LL-H host-range mutants which could overcome the adsorption block (a-type mutants) were selected and investigated by sequencing the genes g71 and g17, which encode minor and major tail proteins, respectively . Each of the a-type mutants carried a nucleotide change at the 3' end of gene g71 . No mutations were observed in gene g17 . Comparison of the gene product of g71 of phage LL-H with its homolog in JCL1032 (ORF474) showed that these proteins had very similar C-terminal regions . No similarities were found at the N-terminal part of the proteins . We conclude that the C-terminal portion of the protein encoded by g71 of phage LL-H and its homolog in phage JCL1032 determines the adsorption specificities of these phages on L . delbrueckii . Substrate Specificity of the Nonribosomal Peptide Synthetase PvdD from Pseudomonas aeruginosa. David F. Ackerley, 2003.Pseudomonas aeruginosa PAO1 secretes a siderophore, pyoverdinePAO, which contains a short peptide attached to a dihydroxyquinoline moiety . Synthesis of this peptide is thought to be catalyzed by nonribosomal peptide synthetases, one of which is encoded by the pvdD gene . The first module of pvdD was overexpressed in Escherichia coli, and the protein product was purified . L-Threonine, one of the amino acid residues in pyoverdinePAO, was an effective substrate for the recombinant protein in ATP-PPi exchange assays, showing that PvdD has peptide synthetase activity . Other amino acids, including D-threonine, L-serine, and L-allo-threonine, were not effective substrates, indicating that PvdD has a high degree of substrate specificity . A three-dimensional modeling approach enabled us to identify amino acids that are likely to be critical in determining the substrate specificity of PvdD and to explore the likely basis of the high substrate selectivity . The approach described here may be useful for analysis of other peptide synthetases . Compatibility of Rhizobial Genotypes within Natural Populations of Rhizobium leguminosarum Biovar viciae for Nodulation of Host Legumes. Gisèle Laguerre, 2003.Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils . Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed . The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome . Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group . With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled . The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation . By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes . Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy .
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