Salicylate is a precursor of pyochelin in Pseudomonas aeruginosa and both compounds display siderophore activity. To elucidate the salicylate biosynthetic pathway, we have cloned and sequenced a chromosomal region of P. aeruginosa PAO1 containing two adjacent genes, designated pchB and pchA, which are necessary for salicylate formation. The pchA gene encodes a protein of 52 kDa with extensive similarity to the chorismate-utilizing enzymes isochorismate synthase, anthranilate synthase (component I) and p-aminobenzoate synthase (component I), whereas the 11 kDa protein encoded by pchB does not show significant similarity with other proteins. The pchB stop codon overlaps the presumed pchA start codon. Expression of the pchA gene in P. aeruginosa appears to depend on the transcription and translation of the upstream pchB gene. The pchBA genes are the first salicylate biosynthetic genes to be reported. Salicylate formation was demonstrated in an Escherichia coli entC mutant lacking isochorismate synthase when this strain expressed both the pchBA genes, but not when it expressed pchB alone. By contrast, an entB mutant of E. coli blocked in the conversion of isochorismate to 2,3-dihydro-2,3-dihydroxybenzoate formed salicylate when transformed with a pchB expression construct. Salicylate formation could also be demonstrated in vitro when chorismate was incubated with a crude extract of P. aeruginosa containing overproduced PchA and PchB proteins; salicylate and pyruvate were formed in equimolar amounts. Furthermore, salicylate-forming activity could be detected in extracts from a P. aeruginosa pyoverdin-negative mutant when grown under iron limitation, but not with iron excess. Our results are consistent with a pathway leading from chorismate to isochorismate and then to salicylate plus pyruvate, catalyzed consecutively by the iron-repressible PchA and PchB proteins in P. aeruginosa.

The arcABC operon of Pseudomonas aeruginosu encodes arginine deiminase, catabolic ornithine carbamoyltransferase and carbamate kinase, respectively. We have determined the nucleotide sequences of the arcA and urcC genes. The arcA open reading frame specifies a polypeptide of 46.3 kDa. The same molecular mass was obtained for the subunit of purified arginine deiminase after electrophoresis under denaturing conditions. The N-terminal amino acid sequence of arginine deiminase was in agreement with the corresponding nucleotide sequence. The native arginine deiminase had an estimated molecular mass of 175 -180 kDa, suggesting a tetrametric structure. The enzyme was activated by Mgz+ or Mn2+ and strongly inhibited by Zn". The apparent K,,, for L-arginine was 0.04 mM in the presence of MgZf and 0.47 mM without Mgz+. The arcC open reading frame codes for a 33-kDa protein, confirming the molecular mass previously reported for the subunit of carbamate kinase. The translation-initiation site of arcC was determined by deletion mapping. Two regions of dyad symmetry found between arcA and arcC might stabilize the putative arcABC transcript in the upstream (arcA) region; this might contribute to the high level of arcA expression as compared to the moderate level of arcC expression.Carbamate kinase had 37% sequence similarity (and 13.5% identity) with the C-terminal part of carbamoylphosphate synthetase (large subunit) from Escherichiu coli. Arginine deiminase had no apparent similarity with argininosuccinate lyase. Thus, the arcA and arcC genes do not appear to be closely related to arginine biosynthetic genes, whereas it had previously been shown that the arcB gene has a high degree of identity with the arginine biosynthetic argF genes of P. aeruginosa and E. c o kThe latter part of arginine biosynthesis and the catabolic arginine-deiminase pathway have a number of metabolites in common (Fig. 1). In Pseudomonas ueruginosa the enzymes which catalyze these reactions are all distinct [I, 21. A futile cycle is avoided by several control mechanisms. Carbamoylphosphate synthetase is derepressed by limitation of arginine or pyrimidines, activated by ornithine and inhibited by UMP [3]. The anabolic ornithine carbamoyltransferase is repressed by arginine and forms, with citrulline, a dead-end complex, which prevents the back reaction [4 -61. The three enzymes of the arginine deiminase pathway are coordinately induced by oxygen limitation and by arginine [7]. The catabolic ornithine carbamoyltransferase shows highly cooperative carbamoyl-phosphate binding and hence does not catalyze the anabolic reaction in vivo. AMP, CMP and inorganic phosphate are activators of the enzyme [8, 91 (V.S., unpublished results). Carbamate kinase is inhibited by ATP [lo]. Thus, conditions of energy depletion favour the functioning of the arginine deiminase pathway. Correspondence to D. Haas, Mikrobiologischcs Institut, ETHAhbreviution. X-gal, 5-bromo-4-chloro-3-indolyl-~-~-galactoside. Enzymes. Arginase (EC 3.5.3.1); arginine deiminase (EC 3.5.3.6 The gene...

We examine the expression of constitutive or repressible, monocistronic genes from Pseudomonas aeruginosa and Escherichia coli after their transfer to the heterologous host. To this end, chromosomal DNA from P. aeruginosa was cloned into the mobilizable broad-host-range vector pKT240; recombinant plasmids carrying the argA, argF, or proC genes were identified by complementation of the corresponding auxotrophic mutations. The isofunctional E. coli genes and the E. coli proB gene were subcloned into pKT240 from existing recombinant plasmids. The enzyme expression specified by the Pseudomonas genes in E. coli, calculated per gene copy, ranged from 0.3%-5% of the levels observed in Pseudomonas. Fusion of the P. aeruginosa proC gene to the E. coli consensus tac promoter resulted in very high proC enzyme production in E. coli, indicating that, at least in this case, the expression barrier is essentially at the level of transcriptional initiation. The E. coli argA and argF enzymes, which are controlled by repression in their native host, were synthesized constitutively in P. aeruginosa at 5% of the levels measured in E. coli under derepressed conditions. The constitutive E. coli proB and proC genes were expressed at high levels (ca. 50%) in the heterologous host. These results support the idea that P. aeruginosa may be a more permissive host than E. coli for the heterologous expression of genes from gram-negative bacteria.