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Two chemotactic transducer genes (termed pctB and pctc) and an open reading frame (orfl) were found in the -flanking region which was previously identified as a chemotactic transducer gene in Ps0udomonas eeruginosa. The pctB and pcK: genes encode predicted polypeptides of 629 and 632 amino acids, respectively. Overall, PctB and Pctc had 81 and 75% amino acid identities with PctA, respectively. A null mutant strain PCT2, which contained a deletion in the entire pctC, orfl, pctA and pctB genes, did not show chemotaxis towards all 20 commonly occurring Loamino acids. This mutant strain also failed to respond to amino acid catabolites (cadaverine, baminobutyrate and putrescine) that are strong attractants for the wild-type strain PAOl. To study the role of each gene product in L-amino acid taxis, plasmids harbouring the pctC, orfl, pctA, or pets genes were constructed and introduced into strain PO2 by transformation. The orfl gene did not complement the defect in chemotaxis of strain -2.The pctA gene restored the ability of strain PCT2 to respond to 18 L-amino acids, suggesting that PctA plays a major role in detecting L-amino acids in P. a0ruglnosa. The pctB and pctC genes complemented the defect in chemotaxis to only seven (Ala, Arg, Glu, Lys, Met, Tyr, Gln) and two (His, Pro) Lamino acids, respectively.

Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phb and pha) of Pseudomonas sp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer {poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]} consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In thephb locus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbCPs), β-ketothiolase (PhbAPs), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbBPs) were found. The genetic organization showed a putative promoter region, followed byphbB Ps-phbA Ps-phbC Ps. Upstream from phbB Ps was found thephbR Ps gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. The phbR Ps gene was found to be transcribed in the opposite direction from the three structural genes. Cloning of phbR Ps in a relatively high-copy vector in Pseudomonas sp. strain 61-3 elevated the levels of β-galactosidase activity from a transcriptional phbpromoter-lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbRPs is a positive regulatory protein controlling the transcription of phbBAC Ps in this bacterium. In the pha locus, two genes encoding PHA synthases (PhaC1Ps and PhaC2Ps) were flanked by a PHA depolymerase gene (phaZ Ps), and two adjacent open reading frames (ORF1 and phaD Ps), and the gene order was ORF1, phaC1 Ps,phaZ Ps, phaC2 Ps, andphaD Ps. Heterologous expression of the cloned fragments in PHA-negative mutants of Pseudomonas putida andRalstonia eutropha revealed that PHB synthase and two PHA synthases of Pseudomonas sp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.

Male gametogenesis in plants can be impaired by an incompatibility between nuclear and mitochondrial genomes, termed cytoplasmic male sterility (CMS). A sterilizing factor resides in mitochondria, whereas a nuclear factor, Restorer-of-fertility (Rf), restores male fertility. Although a majority of plant Rf genes are thought to encode a family of RNA-binding proteins called pentatrico-peptide repeat (PPR) proteins, we isolated a novel type of Rf from sugar beet. Two BACs and one cosmid clone that constituted a 383-kbp contig covering the sugar beet Rf1 locus were sequenced. Of 41 genes borne by the contig, quadruplicated genes were found to be associated with specific transcripts in Rf1 flower buds. The quadruplicated genes encoded a protein resembling OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. Construction of transgenic plants revealed that one of the four genes (bvORF20) was capable of restoring partial pollen fertility to CMS sugar beet; the level of restoration was comparable to that evaluated by a crossing experiment. However, the other genes lacked such a capability. A GFP-fusion experiment showed that bvORF20 encoded a mitochondrial protein. The corresponding gene was cloned from rf1rf1 sugar beet and sequenced, and a solitary gene that was similar but not identical to bvORF20 was found. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product.

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A Pseudomonas aeruginosa mutant, defective in taxis toward L-serine but responsive to peptone, was selected by the swarm plate method after N-methyl-N-nitrosoguanidine mutagenesis. The mutant, designated PCT1, was fully motile but failed to show chemotactic responses to glycine, L-serine, L-threonine, and L-valine. PCT1 also showed weaker responses to some other commonly occurring L-amino acids than did the wild-type strain PAO1. A chemotactic transducer gene, denoted pctA (Pseudomonas chemotactic transducer A), was cloned by phenotypic complementation of PCT1. Nucleotide sequence analysis showed that the pctA gene encodes a putative polypeptide of 629 amino acids with a calculated mass of 68,042. A hydropathy plot of the predicted polypeptide suggested that PctA may be an integral membrane protein with two potential membrane-spanning regions. The C-terminal domain of PctA showed high homology with the enteric methyl-accepting chemotaxis proteins (MCPs). The most significant amino acid sequence similarity was found in the region of MCPs referred to as the highly conserved domain. Pseudomonas aeruginosa is an obligately aerobic bacterium and is capable of swimming by rotating a single polar flagellum. This organism inhabits a wide range of environmental niches, from soil and water to the human host; it is an opportunistic pathogen that is among the most frequently isolated bacteria in nosocomial infections (8). P. aeruginosa, like most other motile bacteria, exhibits chemotactic responses to a wide range of chemical stimuli, including L-amino acids (5), sugars (22), organic acids (21), thiocyanic and isothiocyanic esters (24), and P i (16). Several lines of evidence suggest the presence of regulatory mechanisms underlying the chemotaxis by P. aeruginosa. The taxis toward L-amino acids is subject to control by nitrogen availability in a manner similar to the control of various enzymes of nitrogen metabolism (5). The strength of the chemotactic responses to glucose and citrate is also dependent on prior growth of the bacterial cells on those carbon sources (22). P i taxis in P. aeruginosa is induced by P i limitation and requires the phoU gene for its negative regulation (17). However, these regulatory mechanisms are still poorly understood at the molecular level.Biochemical and genetic information concerning chemotactic transducers has been accumulated with the enteric bacteria Escherichia coli (3) and Salmonella typhimurium (26), the gram-positive bacterium Bacillus subtilis (12), and the soil bacterium Rhizobium meliloti (11). The methyl-accepting chemotaxis proteins (MCPs) are the best-characterized signal transducers of the chemosensory apparatus (3, 26). These transducers recognize ligand-occupied forms of various periplasmic binding proteins or small molecules directly and are known to be covalently modified by dynamic methylation and demethylation reactions in response to chemical stimuli (6). P. aeruginosa possesses approximately 73-kDa proteins that are labeled with L-[methyl-3 H]methionine in response to...

Polyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C 3 to C 5 , mediumchain-length (MCL) PHAs consist of monomer units of C 6 to C 14 , and SCL-MCL PHAs consist of monomers ranging in size from C 4 to C 14 . Although previous studies using recombinant Escherichia coli have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant E. coli. The 3-ketoacyl-acyl carrier protein synthase III gene (fabH) from E. coli was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the Aeromonas caviae PHA synthase gene (phaC), or the pPPAC plasmid, which harbors the Pseudomonas sp. strain 61-3 PHA synthase gene (phaC1), and the abilities of these strains to accumulate PHA from glucose were assessed. It was found that overexpression of several of the mutant fabH genes enabled recombinant E. coli to induce the production of monomers of C 4 to C 10 and subsequently to produce unusual PHA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PHA production.

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