mec-12 is one of a dozen genes required for touch receptor neuron function in Caenorhabditis elegans. Some mec-12 mutants (mechanosensory-defective) lack the large-diameter microtubules that are characteristic of these neurons (15 protofilaments, as opposed to 11). Mutants of mec-7, a alpha-tubulin encoding gene, have a similar phenotype. We have identified the nature of mec-12 by germline transformation rescue and characterization of a point mutation. Sequence analysis of the mec-12 encoded product (MEC-12) indicates that it corresponds to a novel C. elegans alpha-tubulin. MEC-12 is the only identified C. elegans alpha-tubulin that contains a lysine at position 40, a known site of post-translational acetylation. Some mec-12 mutations eliminate microtubule acetylation as assayed immunocyto-chemically; phenotypic rescue using a MEC-12 variant lacking the lysine-40 showed that acetylation is not required for MEC-12 activity. Although functionally needed only in the touch neurons, mec-12 is expressed in several other neuron types. These results support the notion that tubulin isotype diversity contributes to the formation of distinct classes of microtubules; 15-protofilament microtubule assembly requires MEC-12 alpha-tubulin and MEC-7 beta-tubulin, which are both highly expressed in the touch receptor neurons. MEC-12 is the first reported alpha-tubulin isotype that appears to be required in a single class of neuronal microtubules.

Two genes important in DNA repair, recA and lexA, were recently identified in Xanthomonas campestris pathovar citri (X.c. pv. citri). An open reading frame located immediately downstream of lexA and recA has now been isolated from this pathovar and characterized. This 486-bp open reading frame encodes a protein of 162 amino acids and shares substantial sequence similarity with recX of other bacterial species. The X.c. pv. citri RecX protein was overexpressed in Escherichia coli and purified; SDS-polyacrylamide gel electrophoresis revealed a molecular size of 18 kDa for the purified protein. Whereas Northern blot analysis failed to detect recX mRNA in X.c. pv. citri, recX transcripts were detected in this pathovar by reverse transcription and polymerase chain reaction analysis. The increased abundance of recX transcript in X.c. pv. citri revealed that the recX promoter was activated by exposure of cells to DNA-damaging agents. Southern blot and polymerase chain reaction analyses revealed the presence of a recX-related gene in all nine additional X. campestris pathovars tested. The genetic arrangement of lexA-recA-recX was apparent in X. campestris and each of the three genes transcribed from their own promoters.

A cluster of genes encoding polyhydroxybutyrate (PHB) depolymerase (phaZ), PHB synthase (phaC), phasin (phaP), and the regulator protein (phaR) was previously identified in Rhodobacter sphaeroides FJ1 (R. sphaeroides FJ1). In this study, we investigated the role of the PhaR protein on the expression of the pha genes. Immunoblot analysis revealed that the expressions of phaP, phaZ and phaR genes in wild-type cells of R. sphaeroides FJ1 are repressed during the active growth phase, with the exception of phaC. A phaR deletion mutant of R. sphaeroides FJ1 was constructed, and the basal level of phaP and phaZ expression in this mutant was markedly increased. Electrophoretic mobility shift assays demonstrated that PhaR binds to the promoter region of phaP as well as those of phaR and phaZ. These results suggest that the PhaR protein is a repressor of phaP, phaR, and phaZ genes in R. sphaeroides FJ1.

The phaC, phaP, phaR, and phaZ genes are involved in the synthesis, accumulation, and degradation of poly-beta-hydroxybutyrate (PHB). These genes encode the PHB synthase, phasin, regulatory protein, and PHB depolymerase, respectively, and are located in the same locus in the genome of Rhodobacter sphaeroides FJ1, a purple nonsulfur bacterium capable of producing PHB. We have previously found that the PhaR protein binds to the promoter regions of phaP, phaR, and phaZ and represses their expression. In this study, we determined that PhaR binds to an 11-bp palindromic sequence, 5'-CTGCN(3)GCAG-3', located at nucleotides -69 to -59 and -97 to -87 relative to the translation start site of phaP. Substitution of the three spacer nucleotides with any three or four nucleotides in this sequence had no effect on PhaR binding, but all other base deletions or substitutions in this sequence abolished its ability to bind PhaR both in vitro and in vivo. These results suggest that PhaR regulates the expression of phaP in R. sphaeroides FJ1.

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