Resistance to high concentrations of nalidixic acid in Pseudomonas aeruginosa PAO was due to mutations in one locus designated nalA, which was mapped by transduction between hex-9001 and leu-10. The nalA mutants were cross-resistant to pipemidic acid, a nalidixic acid analog, at relatively low concentrations. Replicative DNA synthesis was resistant to both drugs in permeabilized cells of nalA mutants. A locus coding for low-level resistance to nalidixic acid, nalB, was cotransducible with pyrB, proC, and met-28. The nalB mutants were also resistant to low levels of pipemidic acid, novobiocin, and P-lactam antibiotics (e.g., carbenicillin, azlocillin, and cefsulodin), but not to other drugs, such as gentamicin, rifampin, kanamycin, or tetracycline. In nalB mutants, DNA replication showed wild-type sensitivity to nalidixic acid, whereas carbenicillin-induced filamentation required higher drug levels than in the wild-type strain. Thus, nalB mutations appear to decrease cell permeability to some antibiotics. The sensitivity of replicative DNA synthesis to nalidixic acid and novobiocin was very similar in P. aeruginosa and Escherichia coli; by contrast, the concentrations of these drugs needed to inhibit growth of P. aeruginosa were higher than those reported for E. coli by one or two orders of magnitude.
Pseudomonas aeruginosa PAO was able to grow in the absence of exogenous terminal electron acceptors, provided that the medium contained 30 to 40 mM L-arginine and 0.4% yeast extract. Under strictly anaerobic conditions (02 at <1 ppm), growth could be measured as an increase in protein and proceeded in a nonexponential way; arginine was largely converted to ornithine but not entirely consumed at the end of growth. In the GasPak anaerobic jar (Becton Dickinson and Co.), the wild-type strain PAOI grew on arginine-yeast extract medium in 3 to 5 days; mutants could be isolated that were unable to grow under these conditions. All mutants (except one) were defective in at least one of the three enzymes of the arginine deiminase pathway (arcA, arc), and arcC mutants) or in a novel function that might be involved in anaerobic arginine uptake (arcD mutants). The mutations arcA (arginine deiminase), arcB (catabolic ornithine carbamoyltransferase), arcC (carbantate kinase), and arcD were highly cotransducible and mapped in the 17-min chromosome region. Some mutations in the arc cluster led to low, noninducible levels of all three arginine deiminase pathway enzymes and thus may affect control elements required for induction of the postulated arc operon. Two fluorescent pseudomonads (P. putida and P. fluorescens) and P. mendocina, as well as one PAO mtitant, possessed an inducible arginine deiminase pathway and yet were unable to grow fermentatively on arginine. The ability to use arginine-derived ATP for growth may provide P. aeruginosa with a selective advantage when oxygen and nitrate are scarce.
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...
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