Chemotactic responses of Pseudomonas strains to naphthalene in modified capillary assays. Naphthalene chemotaxis was restored when pHG59 was introduced into P. putida G7.C1(pHG100). Cells were grown on naphthalene. The results are shown in both photographic (top) and schematic (bottom) form. Naphthalene crystals are visible inside the mouths of the capillary tubes. The accumulation of a cloud of cells around the mouth of a capillary tube over time indicates a chemotactic response.

NahY is necessary for naphthalene chemotaxis, as determined by capillary assay. The mutant P. putida G7 Y1 is not chemotactic to naphthalene, but the mutation is complemented by the introduction of pHG125, a clone containing nahY. The wild-type strain also exhibited a positive response to naphthalene by the agarose-in-plug method, whereas the nahY mutant strain G7 Y1 did not (data not shown). Cells were grown on naphthalene.

The naphthalene catabolic plasmid and degradation pathway in P. putida G7. (A) NAH7. The region of NAH7 involved in naphthalene degradation is indicated by arrows. Two subclones made from NAH7, pHG100 and pHG59, are shown. Only EcoRI sites in or around the naphthalene degradation genes are shown (E, EcoRI). (B) Naphthalene catabolic pathway. The meta pathway, which converts catechol to acetyl-coenzyme A (acetyl-CoA) and pyruvate, is encoded on the NAH7 plasmid.

Plasmid pHG59. (A) Restriction and gene map of pHG59. Sufficient sequencing was done to determine the locations of the starts and stops of all the genes illustrated. B, BsiWI; E, EcoRI; N, NsiI; P, PstI; Sa, SalI; Sm, SmaI. Not all SalI or NsiI sites are shown. (B) Subclones of pHG59. Plasmid pHG97 (see Table 1 for construction details) was derived from a 4-kb SmaI-EcoRI fragment of pHG59. A kanamycin resistance cassette was introduced into the NsiI sites of this 4-kb fragment. The second clone, pHG125, contains nahY and was used for complementation analysis. (C) Transcript analysis of nahJ, nahX, and nahY. The intergenic regions between nahJ and nahX and between nahX and nahY that were amplified by RT-PCR are shown.

Phylogenetic tree of the predicted N-terminal periplasmic domains of selected bacterial transducer proteins constructed by the AllAll program of the Computational Biochemistry Research Group (1). The proteins included, sources, and National Center for Biotechnology Information Entrez protein accession numbers are as follows: Tap, MCP IV (dipeptide chemoreceptor) from E. coli, 2506839; Tcp, methyl-accepting chemotaxis citrate transducer from S. typhimurium, 400235; Tsr, MCP I (serine chemoreceptor) from E. coli, 400233; Tar, MCP II (aspartate chemoreceptor protein) from E. coli, 2506837; Trg, MCP III (ribose and galactose chemoreceptor protein) from E. coli, 2506838; PctC, chemotaxis transducer protein (amino acid chemoreceptor) from P. aeruginosa, 2626833; PctB, chemotaxis transducer protein (amino acid chemoreceptor) from P. aeruginosa, 2626836; McpA, methyl-accepting chemoreceptor (presumed) from Rhodobacter capsulatus, 2126470; HtrII, sensory rhodopsin II transducer (methyl-accepting phototaxis protein II) from Halobacterium salinarium, 3023997; McpA, MCP (amino acid chemoreceptor) from Bacillus subtilis, 730002; McpB, MCP (amino acid chemoreceptor) from B. subtilis, 730003; TlpB, MCP (amino acid chemoreceptor) from B. subtilis, 730959; YvaQ, methyl-accepting chemotaxis-like protein from B. subtilis, 2635882; McpB, methyl-accepting chemoreceptor (presumed) from R. capsulatus, 2126471; DcrH, methyl-accepting chemoreceptor (presumed) from Desulfovibrio vulgaris, 887858; HlyB, hemolysin secretion protein precursor from Vibrio cholerae, 123206; NahY, chemotaxis transducer protein for naphthalene from P. putida, AF100302.