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Functional Characterization of an Aminotransferase Required for Pyoverdine Siderophore Biosynthesis in Pseudomonas aeruginosa PAO1. Chris S. Vandenende, 2004.The fluorescent dihydroxyquinoline chromophore of the pyoverdine siderophore in Pseudomonas is a condensation product of D-tyrosine and L-2,4-diaminobutyrate . Both pvdH and asd (encoding aspartate ß-semialdehyde dehydrogenase) knockout mutants of Pseudomonas aeruginosa PAO1 were unable to synthesize pyoverdine under iron-limiting conditions in the absence of L-2,4-diaminobutyrate in the culture media . The pvdH gene was subcloned, and the gene product was hyperexpressed and purified from P . aeruginosa PAO1 . PvdH was found to catalyze an aminotransferase reaction, interconverting aspartate ß-semialdehyde and L-2,4-diaminobutyrate . Steady-state kinetic analysis with a novel coupled assay established that the enzyme adopts a ping-pong kinetic mechanism and has the highest specificity for  -ketoglutarate .
The specificity of the enzyme toward the smaller keto acid pyruvate
is 41-fold lower . The enzyme has negligible activity toward other
keto acids tested . Homologues of PvdH were present in the genomes
of other Pseudomonas spp . These homologues were found in the
DNA loci of the corresponding genomes that contain other pyoverdine
synthesis genes . This suggests that there is a general mechanism
of L-2,4-diaminobutyrate synthesis in
Pseudomonas strains that produce the pyoverdine siderophore .
Cross-Regulation in Vibrio parahaemolyticus: Compensatory Activation of Polar Flagellar Genes by the Lateral Flagellar Regulator LafK. Yun-Kyeong Kim, 2004.Gene organization and hierarchical regulation of the polar flagellar genes of Vibrio parahaemolyticus, Vibrio cholerae, and Pseudomonas aeruginosa appear highly similar, with one puzzling difference . Two  54-dependent
regulators are required to direct different classes of intermediate
flagellar gene expression in V . cholerae and P . aeruginosa,
whereas the V . parahaemolyticus homolog of one of these
regulators, FlaK, appears dispensable . Here we demonstrate that there
is compensatory activation of polar flagellar genes by the lateral
flagellar regulator LafK .
Cluster II che Genes from Pseudomonas aeruginosa Are Required for an Optimal Chemotactic Response. Abel Ferrández, 2002.Pseudomonas aeruginosa, a  -proteobacterium, is motile by means of a single polar flagellum and is chemotactic to a variety of organic compounds and phosphate . P . aeruginosa has multiple homologues of Escherichia coli chemotaxis genes that are organized into five gene clusters . Previously, it was demonstrated that genes in cluster I and cluster V are essential for chemotaxis . A third cluster (cluster II) contains a complete set of che genes, as well as two genes, mcpA and mcpB, encoding methyl-accepting chemotaxis proteins . Mutations were constructed in several of the cluster II che genes and in the mcp genes to examine their possible contributions to P . aeruginosa chemotaxis . A cheB2 mutant was partially impaired in chemotaxis in soft-agar swarm plate assays . Providing cheB2 in trans complemented this defect . Further, overexpression of CheB2 restored chemotaxis to a completely nonchemotactic, cluster I, cheB-deficient strain to near wild-type levels . An mcpA mutant was defective in chemotaxis in media that were low in magnesium . The defect could be relieved by the addition of magnesium to the swarm plate medium . An mcpB mutant was defective in chemotaxis when assayed in dilute rich soft-agar swarm medium or in minimal-medium swarm plates containing any 1 of 60 chemoattractants . The mutant phenotype could be complemented by the addition of mcpB in trans . Overexpression of either McpA or McpB in P . aeruginosa or Escherichia coli resulted in impairment of chemotaxis, and these cells had smooth-swimming phenotypes when observed under the microscope . Expression of P . aeruginosa cheA2, cheB2, or cheW2 in E . coli K-12 completely disrupted wild-type chemotaxis, while expression of cheY2 had no effect . These results indicate that che cluster II genes are expressed in P . aeruginosa and are required for an optimal chemotactic response .
Cytochrome aa3 in Haloferax volcanii. Mikiei Tanaka, 2002.A cytochrome in an extremely halophilic archaeon, Haloferax volcanii, was purified to homogeneity . This protein displayed a redox difference spectrum that is characteristic of a-type cytochromes and a CN- complex spectrum that indicates the presence of heme a and heme a3 . This cytochrome aa3 consisted of 44- and 35-kDa subunits . The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved . The reduced cytochrome c partially purified from the bacterial membrane fraction was oxidized by the cytochrome aa3, providing physiological evidence for electron transfer from cytochrome c to cytochrome aa3 in archaea . Role of Pseudomonas putida tol-oprL Gene Products in Uptake of Solutes through the Cytoplasmic Membrane. María A. Llamas, 2003.Proteins of the Tol-Pal (Tol-OprL) system play a key role in the maintenance of outer membrane integrity and cell morphology in gram-negative bacteria . Here we describe an additional role for this system in the transport of various carbon sources across the cytoplasmic membrane . Growth of Pseudomonas putida tol-oprL mutant strains in minimal medium with glycerol, fructose, or arginine was impaired, and the growth rate with succinate, proline, or sucrose as the carbon source was lower than the growth rate of the parental strain . Assays with radiolabeled substrates revealed that the rates of uptake of these compounds by mutant cells were lower than the rates of uptake by the wild-type strain . The pattern and amount of outer membrane protein in the P . putida tol-oprL mutants were not changed, suggesting that the transport defect was not in the outer membrane . Consistently, the uptake of radiolabeled glucose and glycerol in spheroplasts was defective in the P . putida tol-oprL mutant strains, suggesting that there was a defect at the cytoplasmic membrane level . Generation of a proton motive force appeared to be unaffected in these mutants . To rule out the possibility that the uptake defect was due to a lack of specific transporter proteins, the PutP symporter was overproduced, but this overproduction did not enhance proline uptake in the tol-oprL mutants . These results suggest that the Tol-OprL system is necessary for appropriate functioning of certain uptake systems at the level of the cytoplasmic membrane . Intergeneric Conjugation in Streptomyces peucetius and Streptomyces sp. Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the chiC Gene. Senthamaraikannan Paranthaman, 2003.Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts . The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp . strain C5 were transformed using E . coli ET12567(pUZ8002) as a conjugal donor . When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome . Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome . The DNA sequence of the chromosomal integration site was determined and shown to be conserved . However, the core sequence, where the crossover presumably occurred in C5 and S . peucetius, is TTC . These results also showed that the  C31 integrative recombination is active and the phage attP site is functional in S . peucetius as well as in C5 . The efficiency and specificity of
C31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions . The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids . Integration of a single copy of a mutant chiC into the wild-type S . peucetius chromosome led to the production of 30-fold more chitinase .
Characterization of the First Molluscicidal Lipopolysaccharide from Moraxella osloensis. Li Tan, 2003.Moraxella osloensis is a bacterium that is mutualistically associated with Phasmarhabditis hermaphrodita, a nematode that has potential for the biocontrol of mollusk pests, especially the slug Deroceras reticulatum. We discovered that purified M . osloensis lipopolysaccharide (LPS) possesses a lethal toxicity to D . reticulatum when administered by injection but no contact or oral toxicity to this slug . The toxicity of the LPS resides in the lipid A moiety . M . osloensis LPS was semiquantitated at 6 x 107 endotoxin units per mg . The LPS is a rough-type LPS with an estimated molecular weight of 5,300 . Coinjection of galactosamine with the LPS increased the LPS's toxicity to the slug two- to four-fold . The galactosamine-induced sensitization of the slug to the LPS was reversed completely by uridine . A New Bacteroides Conjugative Transposon That Carries an ermB Gene. Anamika Gupta, 2003.The erythromycin resistance gene ermB has been found in a variety of gram-positive bacteria . This gene has also been found in Bacteroides species but only in six recently isolated strains; thus, the gene seems to have entered this genus only recently . One of the six Bacteroides ermB-containing isolates, WH207, could transfer ermB to Bacteroides thetaiotaomicron strain BT4001 by conjugation . WH207 was identified as a Bacteroides uniformis strain based on the sequence of its 16S rRNA gene . Results of pulsed-field gel electrophoresis experiments demonstrated that the transferring element was normally integrated into the Bacteroides chromosome . The element was estimated from pulsed-field gel data to be about 100 kb in size . Since the element appeared to be a conjugative transposon (CTn), it was designated CTnBST . CTnBST was able to mobilize coresident plasmids and the circular form of the mobilizable transposon NBU1 to Bacteroides and Escherichia coli recipients . A 13-kb segment that contained ermB was cloned and sequenced . Most of the open reading frames in this region had little similarity at the amino acid sequence level to any proteins in the sequence databases, but a 1,723-bp DNA segment that included a 950-bp segment downstream of ermB had a DNA sequence that was virtually identical to that of a segment of DNA found previously in a Clostridium perfringens strain . This finding, together with the finding that ermB is located on a CTn, supports the hypothesis that CTnBST could have entered Bacteroides from some other genus, possibly from gram-positive bacteria . Moreover, this finding supports the hypothesis that many transmissible antibiotic resistance genes in Bacteroides are carried on CTns .
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