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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.