A mutant of Pseudomonas aeruginosa PAO lacking arginine deiminase activity (arcA) was isolated by screening for a derivative of an arcB mutant (deficient in catabolic ornithine carbamoyltransferase) that did not excrete citrulline under conditions of limited aeration. The arcA mutation was highly cotransducible with arcB. In Pseudomonas aeruginosa, the arginine deiminase pathway converts arginine to ornithine and carbamoylphosphate, which serves to form ATP from ADP (8). The three enzymes of

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... s pathway, arginine deiminase (EC 3.5.3.6), catabolic ornithine carbamoyltransferase (EC 2.1.3.3), and carbamate kinase (EC 2.7.2.2), are coordinately induced by the limitation of oxygen tension or the depletion of the carbon and energy source (6). Mutants of strain PAO that fail to use arginine as the only carbon source aerobically appear to be unaffected in the arginine deiminase pathway enzymes (7; A. Mercenier, Ph.D. thesis, Universite Libre de Bruxelles, 1980); the precise defects in these mutants are unknown. A mutant blocked in the catabolic ornithine carbamoyltransferase (arcB) grows normally on arginine under aerobic conditions (1) and, similarly to the wild-type strain, converts arginine to putrescine in vitro via the arginine decarboxylase pathway (5). After growth under oxygen limitation in the presence of arginine, the arcB mutant PA0630 excretes large amounts of citrulline in the stationary phase (1). We have now used this property to isolate a mutant lacking arginine deiminase activity (arcA). A growing culture of strain PA0630 (arcB9) was subjected to mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (to 1% survival), diluted into fresh nutrient yeast broth (Difco nutrient broth, 8 g/liter; Difco yeast extract, 5 g/ liter; and NaCl, 8 g/liter), cultivated at 37°C with shaking for 6 h, and plated on minimal medium P containing 20 mM glutamate and 20 mM arginine (3). Single colonies were picked off to inoculate master plates and microtiter plate wells containing 100 ILI of nutrient yeast broth + 5 mM arginine. The microtiter plates were sealed with adhesive foil to ensure conditions of limited aeration and incubated at 37°C for 48 h. Citrulline excretion was detected by the Archibald color reaction (as described below). A total of 3,300 clones from five mutagenized cultures was examined. One clone that did not excrete detectable amounts of citrulline was found to have no arginine deiminase activity in toluenized cells and crude extracts and was named PA0990. Several citrulline nonexcreters had gained catabolic ornithine carbamoyltransferase activity, probably by reversion of the arcB mutation, but had normal arginine deiminase activity and were not kept. The arcA mutation was mapped by FP2-mediated conjugation close to arcB, between hisIf and argA (Fig. 1A) . In these experiments arcA and arcB were unselected markers and detected in recombinant clones by enzyme assay. To do this, we purified the recombinants to single colonies and grew them, in triplicate, to the stationary phase by using sealed microtiter plates. Cells were permeabilized with 2 ,ul of toluene (exposure 30 min at 37°C). Arginine deiminase was assayed in the first well by the addition of 25 ,ul of 50 mM arginine and 1 M citrate buffer (pH 5.5) and by incubation at 37°C for 2 h. Catabolic ornithine carbamoyltransferase was tested in the second well by the addition of 25 p.1 of 50 mM omithine, 50 mM carbamoylphosphate, 50 mM potassium phosphate, and 0.75 M imidazole buffer (pH 7.5) and by incubation at 37°C for 1 h. Controls for citrulline excretion in the third well received 25 p.l of water. Citrulline production was visualized with 100 p.l of color reagent (0.3% [wt/vol] diacetylmonoxime in ethanol-H3PO4-H2SO4 [2:9:3, by volume]) and incubation at 85°C for 90 min in the dark. A bright orange color indicated citrulline production. The arcBl(Su) allele, which is tightly linked to arcB9, specifies a modified catabolic ornithine carbamoyltransferase capable of citrulline synthesis in vivo and suppresses specifically muta-787 on May 9, 2020 by guest