The nahR gene of the 83-kilobase naphthalene degradation plasmid NAH7 of Pseudomonas putida encodes a 34-kilodalton polypeptide which binds to the nah and sal promoters to activate transcription of the degradation genes in response to the inducer salicylate. The DNA sequence of the nahR gene was determined, and a derived amino acid sequence of the NahR protein was obtained. A computer search for homologous proteins showed that within the first 124 amino-terminal residues, NahR has approximately 35% identity with the transcriptional activator proteins encoded by the nodD genes of Rhizobium species. Allowing for ultraconservative amino acid substitutions, greater than 47% overall similarity was found between NahR and NodD, while 32% similarity was found between NahR and another transcription activator, LysR of Escherichia coli. The region of greatest similarity among all three proteins contained a probable helix-turn-helix DNA-binding motif as suggested by homology with the proposed consensus sequence for Cro-like DNA-binding domains. The high level of amino acid identity between NahR and NodD, in conjunction with the observations that nahR and nodD are 45% homologous in DNA sequence, are divergently transcribed from homologous promoters near the structural genes they control, and have similar DNA-binding sites, strongly suggests that these two genes evolved from a common ancestor.The 83-kilobase (kb) NAH7 plasmid from the soil bacterium Pseudomonas putida encodes enzymes for the metabolism of naphthalene or salicylate as the sole carbon and energy source (8). The 14 genes encoding the enzymes for this metabolism are organized in two operons: nah (nahA-F), encoding six enzymes required for metabolism of naphthalene to salicylate and pyruvate, and sal (nahG-M), encoding eight enzymes which metabolize salicylate to pyruvate and acetaldehyde (43). Expression of these enzymes is increased over 20-fold by growth in the presence of the inducer salicylate or the nonmetabolizable inducer 2-aminobenzoate (2). Induction requires the product of only one regulatory gene, nahR, encoding a 36-kilodalton (kDa) polypeptide (33,36,44); it mediates induction by activating transcription from both the nah and sal promoters (33). NahR activates transcription from the sal promoter in trans, only in the presence of salicylate. Deletion experiments have shown that the sal promoter sequences between -83 and -45 are required for both DNA binding and transcription activation by NahR (35,36). Other experiments have shown that the NahR protein protects a highly conserved region (-82 to -47) of both the nah and sal promoters from DNase I digestion in the presence or absence of salicylate and that this binding is necessary for transcription activation (35). It has been suggested that the promoter-bound NahR protein, upon binding salicylate, undergoes a conformational change which results in increased transcription from that promoter (35).An analogous system is found in another genus of soil bacteria, Rhizobium. Members of this genus harbor larg...
Pseudomonas solanacearum is an important phytopathogen which excretes a variety of extracellular enzymes. Pulse-chase experiments showed that one of these enzymes, a 0-1,4-endoglucanase (EGL) encoded by the egl gene, is synthesized as a higher-molecular-weight precursor polypeptide (pEGL) which is subsequently excreted into the extracellular medium as a 43-kilodalton mature protein. Si nuclease transcript mapping and DNA sequence analysis were used to identify the transcription start site and the possible translation start site of egl. Pulse-chase experiments and comparison of the putative NH2-terminal amino acid sequence of pEGL with the actual NH2-terminal amino acid sequence of mature excreted EGL suggested that pEGL has a 45-residue leader sequence preceding the N terminus of EGL which is proteolytically cleaved during export to the extracellular environment. The first 20 residues of the leader sequence resembled a typical lipoprotein signal peptide. The excretion of EGL by P. solanacearum apparently requires a membrane potential since it was blocked by carbonyl cyanide m-chlorophenyl hydrazone.
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