the genetic organization of arginine biosynthesis in pseudomonas aeruginosa

Molec. gen. Genet. 154, 7 - 2 2 (1977)

© by Springer-Verlag 1977

The Genetic Organization of Arginine Biosynthesis in Pseudomonas aeruginosa

Dieter Haas* and Bruce W. Holloway

Department of Genetics, Monash University, Clayton, Victoria 3168, Australia

Anton Schamb6ck** and Thomas Leisinger Mikrobiologisches Institut, Eidgen6ssische Technische Hochschule, ETH-Zentrum, CH-8092 Ziirich, Switzerland

Summary. Six loci coding for arginine biosynthetic enzymes in Pseudomonas aeruginosa strain PAO were identified by enzyme assay: argA (N-acetylglutamate synthase), argB (N-acetylglutamate 5-phosphotransferase), argC (N-acetylglutamate 5-semialdehyde dehydrogenase), argF (anabolic ornithine carbamoyltransferase), argG (argininosuccinate synthetase), and argH (argininosuccinase). One-step mutants which had a requirement for arginine and uracil were defective in carbamoylphosphate synthase, specified by a locus designated car. To map these mutations we used the sex factor FP2 in an improved interrupted mating technique as well as the generalized transducing phages F l l6L and G101. We confirmed earlier studies, and found no clustering of arg and car loci. However, argA, argH, and argB were mapped on a short chromosome segment (approx. 3 rain long), and argF and argG were cotransducible, but not contiguous.

N-Acetylglutamate synthase, the enzyme which re- plenishes the cycle of acetylated intermediates in ornithine synthesis of Pseudomonas, appears to be essential for arginine synthesis since argA mutants showed no growth on unsupplemented minimal medium.

Introduction

A considerable number of genes has been mapped on the chromosome of Pseudornonas aerugbzosa but only in a few cases are the corresponding gene prod-

* Present address: Institut Pasteur, 25, rue du Docteur Roux, F-75224 Paris, France ** Present address: Institut ffir Molekularbiologie I, Universit~it Zfirich, H6nggerberg, CH-8049 Z/irich, Switzerland

Please send correspondence to: Professor T. Leisinger, Mikrobio- logisches Institut der ETH, ETH-Zentrum, CH-8092 Zfirich, Switzerland

ucts known (Holloway, 1975; Loutit, 1969; Matsu- moto and Tazaki, 1975 ; Day, Potts and Clarke, 1975). Pseudomonads have long attracted the interest of biochemists, yet the genetic basis of the biochemical versatility of Pseudomonas has been explored in a very limited number of cases (Clarke and Ornston, 1975).

In this study we have investigated the gene-enzyme relationships in the arginine pathway of P. aeruginosa. Figure 1 summarizes the enzymatic steps involved in the pathway and the genetic symbols used. In earlier work, several arginine loci were located on the P. aeruginosa chromosome (Pelnberton and Holloway, 1972a) and arginine auxotrophs were classified into transduction groups (Fargie and Hol- loway, 1965; Feary, Williams, Calhoun and Walker, 1969; Isaac and Holloway, 1972). Although the chromosomal locations of these transduction groups were not determined, it was apparent that the distribution of arginine loci was quite different from that found in Escherichia coli, Salmonella typhimurium or Bacillus subtilis.

Using conjugation and transduction techniques we now have mapped in P. aeruginosa six different genes coding for arginine biosynthetic enzymes and a locus specifying carbamoylphosphate synthase. In each case, the genetic mutations were identified by enzyme assay and, where possible, correlated with arg loci described previously. We have thus established, for the first time, the genetic organization of a biosynthetic pathway in P. aeruginosa. Our primary aim is to provide a basis for further genetic and biochemical investigations of anabolic and catabolic pathways in P. aeruginosa.

Materials and Methods

Bacterial Strains. All mutants used in this study were derived from P. aeruginosa strain PAO1 (ATCC15692) and are listed in Table 1.

8 D. Haas et al. : Arginine Genes in Pseudomonas aeruginosa

argJ

~-Arginine -- -- h

l argH I I

~-Argininosuccinate J I

l i

I L-Citrull ine-~----J

~ (2 L-Ornithine

N 2 - A c e t y l - ~ - o r n ± ± h ± n e

a.rgD l

~-Acetyl-L-glutamate 5-semiaTdehyde

angC l

N-Acetyl-~-glutomyl- 5-phosphate

argB I

~-Acetyl-~-glutamate

Pyrimidines

(1) l

Carbamoylphosphate

t+ car (3)

Glutamine + HCO~ + ATP (NH~)

~ ~ -~ ~ C o A

org__AA

L-Glutamate Acetyl-CoA =

Fig. 1. Arginine metabolism in P. aeruginosa. The following gene designations for the biosynthetic pathway are used: argA, N-acetylglutamate synthase (acetyl-CoA: L-glutamate N-acetyltransferase EC 2.3.1.1); argB, N-acetylglutamate 5-phosphotransferase (ATP: N-acetyl-L- glutamate 5-phosphotransferase EC 2.7.2.8); argC, N-acetylglutamate 5-semialdehyde dehydrogenase (N-acetyl-L-glutamate 5-semialdehyde: NADP + oxidoreductase (phosphorylating) EC 1.2.1.38); argD, N2-acetylornithine 5-aminotransferase (NZ-acetyl-b-ornithine: 2-oxogluta- rate aminotransferase EC 2.6.1.11); argE, acetylornithinase (N2-acetyl-L-ornithine amidohydrolase EC 3.5.1.16); argF, anabolic ornithine carbamoyltransferase (carbamoylphosphate : L-ornithine carbamoyltransferase EC 2.1.3.3); argG, argininosuccinate synthetase (L-citrulline: L-aspartate ligase (AMP-forming) EC 6.3.4.5); argH, argininosuccinase (L-argininosuccinate arginine-lyase EC 4.3.2.1); argJ, ornithine acetyltransferase (N2-acetyl-b-ornithine : b-glutamate N-acetyltransferase EC 2.3.1.35) ; car, carbamoylphosphate synthase (glutamine) (ATP: carbamate phosphotransferase (dephosphorylating, amldotransferring) EC 2.7.2.9). We use the symbol car for carbamoylphosphate synthase as proposed by Mergeay et aI. (1974) and adopted by Bachmann, Low and Taylor (1976) for the Escherichia coli linkage map. Note that car has also been used for carotenoid pigmentation m Rhizobium lupini (Heumann et al., 1971). A summary of feedback inhibition mechanisms operating in the arginine biosynthetic pathway is given by Haas and Leisinger (I974). Arginme catabolism via the "dihydrolase" pathway is indicated by dotted lines. The enzymes involved are: (1) arginine deiminase (L-arginine iminohydrolase EC 3.5.3.6); (2) catabolic ornithine carbamoyltransferase (carbamoylphosphate L-ornithine carbamoyltransferase EC 2.1.3.3); and (3) carbamate kinase (ATP : carbamate phosphotransferase EC 2.7.2.2)

Media and Growth Conditions. Nutrient yeast broth (NYB), nutrient agar (NA) (Stanisich and Holloway, 1972), minimal medium (MM) (Vogel and Bonner, 1956) and medium P (P) (Leisinger, Haas and Hegarty, 1972) have been described. The concentration of amino acids was 1 mM when used as supplements and 20 mM when used as the sole carbon and nitrogen source. P. aeruginosa

was grown at 37 ° C. Solid MM contained 1.5% Difco agar. Toler- ance to aeruginocin AR41 was scored as described by Mills and Holloway (1976). Cells used for enzyme assays were grown in Fernbach flasks on a rotary shaker and harvested in the late exponential phase at a cell density of approx. 109 cells/ml. Wet cells were stored at - 2 0 ° C.

D. Haas et al. : Arginine Genes in Pseudomonas aeruginosa 9

Table 1. Strains of P. aeruginosa

Strain Genotype Sex Origin, method of construction, or reference

PAO1 prototroph, chl-2 FP Holloway (1969) PAO12 pur-136, Ieu-8, chl-2 F P - Pemberton and Holloway (1972a) PAO18 pro-64, put-66 F P - Krishnapillai (1971) PAO21 trpA1, argF3 FP argF derivative of strain trpEl (Calhoun et al., 1973) PAO25 argFlO, leu-lO F P - Haas and Holloway (1976); derived from PAO317 PAOI77 met-28, ilv-202, argBl, str-1 FP- Haas and Holloway (1976) PAO229 met-28, ttp-6, lys-12, his-4, pro-82, ilv-226, str-43 FP Streptomycin-resistant derivative of PAO222

(Watson and Holloway, 1976) PAO287 met-28, ilv-202, argB1, str-1, his-12, ese-4 FP Stanisich and Holloway (1969); derived from PAO177 PAO296 argB13 FP Isaac and Holloway (1972) PAO297 argHl4 F P - Isaac and Holloway (1972) PAO298 argB15 F P - Isaac and Holloway (1972) PAO303 argB18 FP Isaac and Holloway (1972) PAO304 argF19 FP This study PAO305 argG27 FP Isaac and Holloway (1972) PAO307 argC54 FP Isaac and Holloway (1972) PAO309 argG30 FP Isaac and Holloway (1972) PAO311 argC55 F P - Isaac and Holloway (1972) PAO315 argG9 FP Isaac and Holloway (1972) PAO317 argFlO FP Issac and Holloway (1972) PAO332 argB18, lys-60 FP lys derivative of PAO303 PAO362 argH32 FP Isaac and Holloway (1972) PAO364 argB34 FP Isaac and Holloway (1972) PAO366 argA36 FP Isaac and Holloway (1972) PAO369 argF39 F P - This study PAO372 argH32, lys-58 FP lys derivative of PAO362 PAO378 argC76 FP This study PAO381 leu-38, str-7 FP2 + Stanisich and Holloway (1969) PAO417 trpB4, car-9 F P - car derivative of strain trpF4 (Calhoun et al., 1973) PAO477 met-28, ilv-202, argB1, str-1, hal-12 F P - NAL-res is tant derivative of PAO177 PAO512 argH32, lys-58, hal-7 F P - NAL-resis tant derivative of PAO372 PAO513 argB18, lys-60, hal-8 FP NAL-resis tant derivative of PAO332 PAO516 car-161, thi-1, pur-66 FP , Pro + C a r - recombinant of PAO834 (R68.45) x PAO18

R PAO517 car-161, thi-1 F P - Pur * t ransductant of PAO516 x Fl16C4 (Watson and

Holloway, 1976) propagated on PAO1 PAO522 pur-136, argF2 FP , Leu + Arg - recombinant of M78(R68.45) x PAOI2;

R - Fl16 ~, G10P PAO540 his-5075, cys-5605, argA171 F P - J. Govan; arg derivative of GMA253 (Mee and Lee, 1969) PAO639 his-5039, argC38 F P - arg derivative of GMA039 (Mee and Lee, 1967) PAO642 trpB4, car-9, ilv-219, nal-9 FP ilv NAL-resis tant derivative of PAO417 PAO824 pur-66, his-151, argA163, ese-14 F P - Pemberton and Holloway (1972a) PAO834 pur-66, his-151, pyrB21, thi-1, car-161, ese-I4 FP Pemberton and Holloway (1972a); arg-161 renamed car-I61;

Fl16 r, G101 r PAO850 pur-66, his-151, pyrB21, thi-1, car-160, ese-I4 FP Pemberton and Holloway (1972a); arg-160 renamed car-160 PAO952 argA127 F P - This study PAO953 argA147 FP This study PAO954 met-9011, amiE200, argD17 FP Voellmy and Leisinger (1976) PAO955 argA127, lvs-61 FP lvs derivative of PAO952 PAO956 argA, lys-61, hal-13 F P - NAL-resis tant derivative of PAO955 PAO1715 pur-136, leu-8, r(f-1, chl-3, tolA12 F P - Mills and Holloway (1976) PTOI3 trp-6 FP2 + Stanisich and Holloway (1969) M78 trp-1, argF2, aerR2, str FP Kageyama (1970); Fl16 r, G101 ~ GMA153 pur-66, his-35 FP2 + GMA153 (Mee and Lee, 1967); made male by conjugational

transfer of FP2 from PTO13

Genotype symbols are the same as those used in E. coli (Bachmann, Low and Taylor, 1976) except that str signifies streptomycin resistance, chl chloramphenicol resistance, ese resistance to phage E79, ami inability to utilize acetamide as a carbon and nitrogen source. Note that trp alleles are designated according to the nomenclature used by Crawford (1975); trpB (formerly trpF, Calhoun et al.,. 1973) and trpA (formerly tJT~E, Calhoun et al., 1973) mutants are defective in t ryptophan synthase (13- and c~-subunit, respectively)


Mutant Isolation. Most arg mutants used in this study were isolated by Isaac and Holloway (1972) and Pemberton and Holloway (1972a). These mutants were induced with N-methyl-N'-nitro-N'- nitrosoguanidine (NG). More recently, we have preferred to use ethyl methane sulfonate (EMS) as the mutagen and mutants were isolated after contraselection with 2 mg carbenicillin/ml (Watson and Holloway, 1976) in liquid MM containing the supplements required for the parental strain. The spontaneous argA mutants PAO952 and PAO953 were found following the enrichment technique described by Ornston, Ornston and Chou (1969). Mutants resistant to high levels of nalidixic acid (NAL) were obtained by plating approx. 5 x l0 s cells on NA containing 2 mg NAL/ml.

Construction of Donor Strains. Strains carrying the sex factor R68.45 were isolated as described previously (Haas and Holloway, 1976). Transfer of the sex factor FP2, which confers resistance to mercuric ions (Loutit, I970), was accomplished as follows: Over- night cultures of an FP2 + donor (2 ml) and an FP recipient (0.1 ml) were incubated in 6 ml NYB with gentle aeration at 37 ° C for 6 h. Plasmid transconjugants were isolated and purified on selective MM containing 4 pg HgC12/ml.

Matings oll the Plate were performed as described by Stanisich and Holloway (1972).

Interrupted Matings. This procedure is based on that of Carey and Krishnapillai (1974) and similar techniques are being used in E. eoli genetics (Miller, 1972; Zipkas and Riley, 1976). The NAL-resistant recipient is grown to early stationary phase in NYB, centrifuged and resuspended in MM (adjusted to pH 7.4 with NaOH and containing 0.5% w/v glucose). The cell density is approx. 2 x 109 cells/ml. The FP2 + donor cells (sensitive to NAL) are grown to exponential phase (about 5x 108cells/ml) with good aeration, centrifuged and resuspended gently in 1/4 volume of NYB. To initiate the mating, 1.5 ml of donor cells are mixed with 6.0 ml of recipient cells in a 250 ml Erlenmeyer flask. At intervals, 0.6 ml samples are removed, pipetted into 0.3 ml MM (pH 7.4) containing 1500 pg NAL/ml and shaken vigorously with a Vortex mixer for 10 s. Aliquots of 0.2 ml are plated in duplicate on selective MM (pH 7.4) containing 1200 gg NAL/ml (for analysis of early markers) or 500 gg NAL/ml (for markers entering later than 15 min). The first sample is taken immediately after initiation of the mating (the zero min control). Sampling intervals of 2 3 min are usually satisfactory. All operations are carried out at 37 ° C with prewarmed materials. The donor: recipient ratio is approx. 1:4. If the volume of the mating mixture is increased, a larger flask is used to keep the cells in a shallow layer which prevents pellicle formation. The MM is brought to pH 7.4 in order to avoid precipitation of NAL.

Transduction. Fresh overnight NYB cultures of PAO cells (viable count approx. 2 x 109 cells/ml) were centrifuged, resuspended in TNM buffer (Mee and Lee, 1967) and mixed with an equal volume of bacteriophage F116L (Krishnapillai, 1971) in TNM buffer (titre approx. 5 x 109 pfu/ml). After adsorption for 15 rain at 37 ° C without aeration, the transducing mixture was centrifuged to remove unadsorbed phage and resuspended in TNM buffer. Samples of 0.2 ml were plated on selective media. An analogous procedure was used with bacteriophage G101 (Holloway and van de Putte, 1968), except that the time for adsorption was 30 rain. Transduc- tants were scored after 2 4 days of incubation at 37 ° C. Prototroph reduction transduction was used essentially as described by Fargie and Holloway (1965) to establish whether phenotypically similar mutations are closely situated on the chromosome. Prototroph reduction was standardized for the transducing ability of phage preparations (Mee and Lee, 1967) as follows: phototroph reduc- t ion- a/b x c/d x 100 (%).

a, number of Arg ÷ transductants obtained with phage propagated on arg mutant, b, number of Arg ÷ transductants obtained with phage propagated on the wild type strain PAO1. c, number of Aux + transductants obtained with phage propagated on PAO1 (the auxotrophlc (aux) mutation is unlinked to the arg mutation studied; wherever possible, arg aux doubly marked recipients were used), d, number of Aux + transductants obtained with phage propagated on arg Aux + mutant.

The generalized transducing phages G101 and F l l 6 L were also used to measure cotransduction frequencies of closely linked, phenotypically different markers. Individual stocks of Fl16 and its variant F116L appear to give frequencies of cotransfer of met-28 and ilv-202 ranging from 10% to 37% (Krishnapillai, 1971 ; Stani- sich and Richmond, 1975; Day et al., 1975; Dunn and Holloway, 1971) for reasons which are not clear. The F l l 6 L stock used in this study gave a cotransduction value of 14%-17% for met and ilv (average of reciprocal crosses).

Preparation of Cell Extracts. All operations were carried out at 0-4 ° C. Wet cells were suspended in buffer (composition as specified below) at 20% w/v and disrupted by sonication. Follow- ing removal of cell debris by centrifugation at 30,000 x g for 20 min, the crude extracts were either dialyzed overnight against the extrac- tion buffer or passed through a Sephadex G-25 column. Extracts were prepared in the following buffers: 0.1 M Tris-HC1 pH 7.8, 5 mM 2-mercaptoethanol for the assay of N-acetylglutamate synthase and ornithine acetyltransferase; 0.1 M Tris-HC1 pH 7.5 for argininosuccinate synthetase; 10 mM imidazole-HC1 pH 7.2, 15% v/v glycerol, 1 mM EDTA, 1 mM dithiothreitol, 2 m M phenylmethylsulfonyl fluoride for carbamoylphosphate synthase; 0.1 M potassium phosphate pH 7.0, 2 mM 2-mercaptoethanol for all other enzymes.

The Protein Concentration of extracts was estimated by the method of Lowry et al. (1951).

Enzyme Assays. N-Acetylglutamate synthase (argA enzyme) was assayed by the method of Haas, Kurer and Leisinger (1972).

N-Acetylglutamate 5-phosphotransferase (argB enzyme) was measured by the ferric chloride method as described by Haas and Leisinger (1975a).

N-Acetylglutamate 5-semialdehyde dehydrogenase (argC enzyme) was determined by the method of Vogel and McLellan (1970a). Extensive dialysis of cell extracts was essential for the specificity of the assay. The protein concentration in the incubation mixture was ca. 0.3 mg/ml.

N2-Acetylornithine 5-aminotransferase (argD enzyme) was assayed by the procedure of Voellmy and Leisinger (1975).

Acetylornithinase (argE enzyme) was measured by the method of Vogel and McEellan (1970b). The protein concentration was about 0.3 mg/ml in the assay and the incubation time 15-30 rain.

Ornithine acetyltransferase (argJ enzyme) was assayed as described by Haas, Kurer and Leisinger (1972).

Anabolic ornithine carbamoyltransferase (argF enzyme) was determined as described by Theil, Forsyth and Jones (1969) except that 0.1 M Tris-HC1 pH 8.5 was used as a buffer. At pH 8.5 the activity of the catabolic ornithine carbamoyltransferase was negligi- ble (Stalon, Ramos, Pi6rard and Wiame, 1967). The protein concentration in the assay was about 0.1 mg/ml when cells had been grown in NYB. The incubation time was 15 min.

Argininosuccinate synthetase (argG enzyme) was assayed by a radioassay using L-[14C]citrulline as a substrate. L-Argininosuccin- ate was converted to an anhydride at acidic pH (probably predom- inantly to the anhydride I described by Ratner and Kunkemueller, 1966) and isolated by high voltage paper electrophoresis. The reaction mixture contained, in a final volume of 50 pl : 60 mM Tris-HC1 pH 8.7, 10 mM L-aspartate-Na2, 10 mM ATP-Na2, 10 mM MgC12,

D. Haas et al. : Arginine Genes in Pseudomonas aeruginosa i1

2 mM L-[ureido-t¢C]citrulline (3 pCi/~tmol), extract (0.1-0.2 mg protein) and 200 mM fumarate-Na2 to inhibit hydrolysis of L-argininosuccinate by argininosuccinase present in crude extracts. Controls without ATP were used as blank values. The incubation at 35 ° C was started by the addition of ATP and, after 20 min, terminated by the addition of 20 pl 0.75 M formic acid. The samples were boiled for 15 min and precipitated protein was removed by centrifugation. Samples of 5 gl were spotted on to Whatman No. 3MM paper and subjected to high-voltage electrophoresis in 0.75 M formate buffer pH 2.2 at 110 V/cm for 15 min. In each run the following unlabelled markers were included: h-citrulline, L-argininosuccinate, L-argininosuccinate anhydride (obtained by boiling argininosuccinate in 0.75 M formate pH 2.2) and L-arginine. After electrophoresis, the marker substances were localized by spraying with 0.2% (w/v) ninhydrin in acetone. The paper was dried at 100 ° C for 2 min. The sample area corresponding to the anhydride was cut out and the radioactivity measured by liquid scintillation count- ing in 4 g PPO+I00 mg POPOP/1 toluene with an efficiency of 90%. Under the experimental conditions, the anhydride moved towards the cathode. Citrulline moved less rapidly and was well resolved from the anhydride. Formation of arginine was not observed. Formation of argininosuccinate was a linear function of time up to 30 min of incubation.

Argininosuccinase (argH enzyme) was assayed by the procedure of TheiI, Forsyth and Jones (1969), at a protein concentration of about 1 mg/ml.

Carbamoylphosphate synthase (car enzyme) was measured by a modification of the method of Pi6rard, Glansdorff and Yashphe (1972). The reaction mixture contained: 60 mM Tris-HCl pH 8.7, 10 mM ATP-Na2, 10 mM MgC12, 5 mM L-ornithine, 10 mM KCI, 10 mM [14C]NaHCO3 (0.05 pCi/pmol), 200 units of ornithine carbamoyltransferase (partially purified from E. coli by the method of Legrain, Halleux, Stalon and Glansdorff, 1972), crude extract (0.5-1.5 mg protein) and either 5 mM L-glutamine or 12 mM NH4CI• The total volume was 1.0 ml. Blanks contained neither glutamine nor NH4CI. The reaction was started by the addition of extract. After incubation for 20 min at 30 ° C the reaction was stopped by the addition of 1 ml 0.5 M trichloroacetic acid in 2 M HC1. Excess [14C]NaHCO3 was removed and [14CJcitrulline determined as described by Pihrard et al. (1972). Enzymatic activity was a linear function of incubation time up to 20 min and of protein concentration below 0.6 mg/ml. Carbamoylphosphate synthase was very labile in 0.2 M imidazole buffer pH 7.2, but sufficiently stable in the buffer described above in "preparation of cell extracts ".

Chemicals. The sources of most chemicals have been given in previous communications (Haas etal., 1972; Haas and Leisinger, 1975a; Voellmy and Leisinger, 1975; Haas and Holloway, 1976). In addition, the following chemicals were obtained commercially: [14C]NaHCO3 and L-[ureido-14C]citrulline from New England Nu- clear, carbamoylphosphate.Li z from Fluka (Buchs, Switzerland), NZ-acetyl-L-ornithine and L-argininosuccinate-Ba from Sigma.

Results

In terrupted M a t i n g s

A range o f m a r k e r s was l o c a t e d on the P. aeruginosa c h r o m o s o m e by i n t e r r u p t e d ma t ings . E x i s t i n g in te r -

r u p t e d m a t i n g t e c h n i q u e s us ing the sex f a c t o r F P 2

( L o u t i t a n d M a r i n u s , 1968 ; P e m b e r t o n a n d H o l l o w a y ,

1972 b; C a r e y a n d K r i s h n a p i l l a i , 1974) were m o d i f i e d

to inc rease the i r r e so lu t ion . Since the r e c o m b i n a t i o n

4000 f

g 500

\ &O0

c

.~ 300 E O 200 -

r ~

100

,,k

0 0 15.0

i •

t • /

/ /

i i

/ i

/ / t

. . ~ 1 ' . ~. 1 '. I I

75 10.0 12,5 Time (minutes)

Fig. 2. Interruption of mating by nalidixic acid. PAO642 (trpB4, car-9, ilv-219, hal-9) and PAO381 (leu-38, str-7, FP2 +) were mated as described for interrupted matings in "Materials and Methods ". At different times, NAL was added to samples of the mating mixture to give a final concentration of 500 gg/ml. • = NAL added at t =0 rain; • =NAL added at t = 10 min (arrow); ~, NAL added at t - 1 4 rain; 0.2 ml samples were plated on medium selective for ilv + recombinants and containing 1200 ~tg NAL/ml

f r e q u e n c y in F P 2 + x F P 2 - m a t i n g s is less t h a n 10 . 4

pe r d o n o r cell f o r m a r k e r s m a p p i n g la te r t h a n a b o u t

10 m i n f r o m the o r ig in o f FP2 , e f fo r t s h a v e been

m a d e to m a x i m i z e the n u m b e r o f cells p l a t e d at e a c h

s a m p l i n g t ime ( D a y et al., 1975) a n d to m i n i m i z e the

b a c k g r o u n d n u m b e r o f co lon ie s due to r e m a t i n g on

the p la te a f t e r i n t e r r u p t i o n . In the p r o c e d u r e de-

s c r ibed in " M a t e r i a l s a n d M e t h o d s " we use N A L

to s top c o n j u g a l c h r o m o s o m e t ransfe r . D o n o r cells

a re sens i t ive to N A L . R e c i p i e n t cells a re res i s tan t

to h igh levels o f N A L ( > 2 0 0 0 g g / m l ) a n d ca r ry

m a r k e r s w i t h l ow s p o n t a n e o u s r e v e r s i o n f requenc ies .

The e x p e r i m e n t i l l u s t r a t ed by F i g u r e 2 shows tha t

a f te r i n t e r r u p t i o n N A L ef fec t ive ly p r e v e n t s f u r t h e r

f o r m a t i o n o f r e c o m b i n a n t s . S a m p l e s were t a k e n f r o m

a m a t i n g m i x t u r e cons i s t i ng o f e x p o n e n t i a l l y g r o w i n g

F P 2 + d o n o r cells ( P A O 3 8 1 ) a n d r ec ip i en t cells in

t h e s t a t i o n a r y phase (PAO642) . I n t e r r u p t i o n o f m a t -

ing was a c h i e v e d by s h a k i n g the s amples v i g o r o u s l y fo r 10 s in the p re sence o f N A L (500 pg /ml ) . S a m p l e s

were p l a t e d on m e d i u m select ive fo r ilv ÷ r e c o m b i -

nan t s a n d c o n t a i n i n g 1200 lag N A L / m l . V e r y few ilv + r e c o m b i n a n t s were f o r m e d w h e n N A L was a d d e d im-

m e d i a t e l y a f te r i n i t i a t i on o f p a i r i n g (zero m i n sam-

ple) : the b a c k g r o u n d n u m b e r o f co lon i e s was a b o u t 5 pe r plate . Th is b a c k g r o u n d was n o t e x c e e d e d in

o t h e r e x p e r i m e n t s a n d was o f t en zero . W h e n the con-

j u g a l m i x t u r e was i n t e r r u p t e d at 10 ra in , a b o u t

350 ilv + r e c o m b i n a n t s pe r 10 8 d o n o r cells were re-

c o v e r e d ; this n u m b e r r e m a i n e d c o n s t a n t fo r at leas t


Table 2. Enzymological analysis of arginine auxotrophs of P. aeruginosa PAO

Enzyme Strata Allele Mutagen a Growth Specific enzyme activity Comments medium u (~tmol x h i x mg-1)

Carbamoylphosphate PAO1 cal .+ - NYB 0.30/0.030 synthase PAO417 car-9 c EMS NYB 0.007/0.006

PAO834 car-16] NG NYB 0.005/0.006 PAO850 car-160 N G NYB 0.023/0.014

N-Acetylglutamate PAO1 argA ÷ -- MM 0.27 synthase P 0.28

PAO366 argA36 NG MM 0 ( < 0.003) PAO540 argA171 EMS P 0 ( < 0.003) PAO824 argA163 NG P 0 ( < 0.003) PAO952 argA127 c Spont. P 0 ( < 0.003) PAO953 argA147 Spont. P 0 ( < 0.003)

N-Acetylglutamate PAO1 argB + - MM 1.50 5-phosphotransferase P 1.86

PAO 177 argB1 NG P 0.06 PAO296 argB13 NG MM 0 ( < 0.02) PAO298 a r g B l 5 NG MM 0 ( < 0.02) PAO303 argB18 c NG MM 0.03 PAO364 argB34 NG MM 0.05

N-Acetylglutamate PAO1 argC + - P 1.01 5-semialdehyde PAO307 argC54 c NG P 0.02 dehydrogenase PAO311 argC55 NG P 0.02

PAO378 argC76 NG n.d. n.d. PAO639 argC38 EMS n.d. n.d.

Anabolic ornithine PAO1 argF + - NYB 2.36 carbamoyltransferase PAO2I argF3 EMS NYB 0 (< 0.08)

PAO25 argFlO ~ NG NYB 0 (< 0.08) PAO304 argF19 NG NYB 0 (< 0.08) PAO369 argF39 NG NYB 0.08 PAO522 argF2 NG NYB 0 (< 0.08)

Argininosuccinate PAO1 argG + - NYB 0.165 synthetase PAO305 argG27 NG NYB 0 ( < 0.010)

PAO309 argG30 NG NYB 0 (< 0.010) PAO315 argG9 ~ NG NYB 0 ( < 0.010)

Argininosuccinase PAO1 a r g H + - P 0.I4 PAO297 a r g H i 4 NG P 0.01 PAO362 argH32 ~ NG P 0.01

First figure: glutamine-dependent activity; second figure: NH4 ÷ -dependent activity

Enzyme defect in PAO378 and PAO639 inferred from genetic data

n.d. = Not determined Mutagen used to induce the arg or car mutation

b Growth medium in which ceils were grown for enzyme assay. MM and medium P were supplemented with appropriate amino acids at 1 mM. The carbon and nitrogen source in medium P was 20 mM L-arginine c We recommend these alleles as reference alleles

4 m i n a f t e r a d d i t i o n o f N A L . By con t r a s t , the n u m b e r

o f i l v + r e c o m b i n a n t s rose sha rp ly to app rox . 4000

w h e n i n t e r r u p t i o n was p e r f o r m e d at 14 min . In a sub-

s e q u e n t e x p e r i m e n t (Fig. 3) t i m e o f e n t r y o f i l v was

f o u n d to be 7 8 min .

T h e cell c o n c e n t r a t i o n in o u r i n t e r r u p t e d m a t i n g s was a p p r o x . 2 x 10 9 ce l l s /ml , thus h i g h e r t h a n tha t

u sed by L o u t i t a n d M a r i n u s (1968) a n d P e m b e r t o n

a n d H o l l o w a y (1972b) . As n o t e d by M a r i n u s a n d L o u t i t (1969), t i m e o f en t ry cu rves are p a r a b o l i c r a t h e r t h a n s t ra igh t l ines a t h igh cell densi t ies . F i g u r e s

3 a n d 4 s h o w c lea r ly t h a t f o r m a t i o n o f r e c o m b i n a n t s

was n o t a l inea r f u n c t i o n o f t ime, b u t r e c o m b i n a n t

n u m b e r s i n c r e a s e d g r a d u a l l y a n d p rog re s s ive ly for sev-

eral m i n u t e s . T i m e o f en t ry is de f i ned by the ear l ies t t ime a t wh ich r e c o m b i n a n t s a p p e a r a b o v e back-

g r o u n d (Low, 1973), S a m p l e s o f m a t i n g m i x t u r e s were

i n t e r r u p t e d at i n t e rva l s o f 2 3 m i n ; this t i m e i n t e rva l

was suf f ic ien t to a l l o w a s ign i f i can t inc rease in c o l o n y n u m b e r s in the ear l ies t p o r t i o n o f en t ry t i m e curves

fo r m a r k e r s m a p p i n g n o t l a te r t h a n a b o u t 30 m i n f r o m the F P 2 or ig in . S h o r t e r s a m p l i n g in t e rva l s d id n o t i m p r o v e the p rec i s ion o f en t ry t i m e d e t e r m i n a -

t i ons excep t fo r ve ry ear ly marke r s .


In earlier experiments (Stanisich and Holloway, 1969 ; Pemberton and Holloway, 1972 b) time of entry data were usually obtained by extrapolating a straight port ion of interrupted mating curves to the abscissa. Sampling intervals were 5-10 min. As will be discussed later, this procedure has led to somewhat inaccurate assignments of entry times for a number of markers.

We present the data on car and arg loci in the order in which they map on the PAO linkage group.

Carbamoylphosphate Synthase

Loutit (1952) observed that mutants of P. aeruginosa which required both arginine and uracil were frequent among survivors in cultures irradiated with x-rays. Since one period of irradiation was used these mutants probably arose f rom one mutational event. In E. coli and other bacteria the simultaneous requirement for arginine and pyrimidines is due to a defect in carbamoylphosphate synthase (Pi6rard et al., 1976). This enzyme catalyzes the formation of carbamoylphosphate f rom bicarbonate, ATP and glutamine (Fig. 1). Ammonia can replace glutamine as an amino group donor in vitro (Mergeay et al., 1974). The specific activity of the P. aeruginosa carbamoylphosphate synthase was ten times higher with glutamine than with ammonia as a substrate under our assay conditions (Table 2), and ammonia probably has little if any function in carbamoylphosphate synthesis in vivo.

Strain PAO417 is an EMS-induced mutant which requires arginine + uracil or citrulline + uracil for growth. Arginine alone or uracil alone do not allow growth. As expected, a defect in carbamoylphosphate synthase was found in this biauxotrophic mutant. The mutat ion was designated car-9. Pemberton and Hol- loway (1972a) described two arginine auxotrophic mutations, arg-161 (in PAO834) and arg-160 (in PAO850), which mapped in the early region of the chromosome. Both mutants were derived f rom a strain with a pyrimidine requirement (pyrB21) and responded to either citrulline or arginine in the presence of uracil. As shown in Table 2, the two mutants had a greatly reduced carbamoylphosphate synthase activity and, therefore, were renamed car-161 and car-

160, respectively. The three car mutants described here were affected

in the glutamine-dependent as well as in the ammonia- dependent carbamoylphosphate synthase activity. On the basis of the enzyme assay data (Table 2) we cannot distinguish between two possible types of mutation, carA (coding for a glutamine-binding subunit) and carB (coding for an ammonia-dependent carbamoylphosphate synthase subunit) as found in E. coli (Mer- geay et al., 1974).

Table 3. Transduction analysis of car strains

Recip- Marker lent selected strain

F116L donor strain

PAO417 PAO517 PAO850 PAO1

PAO417 car-9 0 (<l) 5 (2) 2 (2) 124 trpB4 0 226 61 98

PAO516 car-161 0(<2) 0(<1) 0(<3) 59 pur-66 229 1300 a 0 294

PAO850 car-160 0(<3) 0(<1) 0(<4) 46 pur-66 155 760" 0 214

Each figure represents the number of transductants per plate (averages of duplicates). Prototroph reduction in car + , indicated in parentheses, was calculated as detailed in "Materials and Methods"

Approximately

Transductional analysis showed that car-9, car-

160 and car-161 are closely linked mutations since car + transductants were not recovered or were very rare in any car× car cross (prototroph reduction < 4 % , see Table 3). Mills and Holloway (1976) de- monstrated that a locus conferring tolerance to aeruginocin AR41, tolA12, is highly cotransducible with car-160. Using the generalized transducing phage F l l 6 L we found 86%, 89%,and 84% cotransduction between tolA12 and car-9, car-160, and car-161, respectively, confirming that the car loci are closely linked. In each cross, selection was made for car +

and 400 transductants were scored. A chromosomal location for car was obtained by

interrupted matings. The entry time of car-9 was 9 _+ 1 min and that of ilv-219 was 8_+1 min (mean of 3 experiments), PAO642 being the recipient and PAO381 the donor. Figure 3 shows a typical experiment. The entry time of ilv-219 is in good agreement with the value obtained by Marinus and Loutit (1969) for ilvB, to which ilv-219 appears closely linked by proto t roph reduction (J. Carrigan, personal commu- nication). No cotransduction was detected between ilv-219 and car-9.

Arginine induces the enzymes of t h e " dihydrolase" pathway (indicated by dotted lines in Fig. 1), arginine deiminase, the catabolic ornithine carbamoyltransferase and carbamate kinase (Ramos, Stalon, Pi6rard and Wiame, 1967). The first two enzymes degrade arginine to ornithine, carbamoylphosphate and ammonia. Carbamate kinase further catabolizes carbamoylphosphate in the presence of ADP to give ammonia, bicarbonate and ATP. Carbamate kinase activity could be due to carbamoylphosphate synthase (Raij- man and Jones, 1973). Induction kinetics of carbamate kinase activity in P.fluorescens, however, suggested that carbamate kinase is distinct f rom carbamoylphosphate synthase (Ramos et al., 1967). The


300 .m o

o

~ 200

i 100 n , -

o o lO 20 30 40

Time (minutes)

Fig. 3. Time of entry kinetics of ilv-219, car-9 and trpB4. An interrupted mating was performed as described in "Materials and Methods ". PAO642 (ilv-219, car-9, trpB4, nal-9) was the recipient, PAO381 (leu-38, str-7, FP2 +) the donor, o =ilv + ; • =car + ; • = trpB +

car mutants of P. a e r u g m o s a allow a more direct experimental approach to the problem. If carbamoylphosphate synthase has carbamate kinase activity in vivo, then we would expect carbamoylphosphate to accumulate in car mutants grown on arginine as the sole carbon and nitrogen source (conditions of induction, Voellmy and Leisinger, 1975) and hence the pyrimidine requirement should disappear. However, strain PAO517 ( car -161 , thi-1) was unable to grow on medium P containing 20 mM L-arginine (and traces

of thiamine), whereas the wild-type PAO1 grew well on this medium. Addition of uracil was necessary to allow growth of PAO517. Similarly, car -9 and car-

160 mutants needed uracil to grow on arginine. Thus, we conclude that in our car mutants carbamoylphosphate was not produced from arginine in quantities sufficient to satisfy the requirement in pyrimidine synthesis. At present we cannot rule out the possibility that in our car mutants only the biosynthetic function of a hypothetical single "amphibol ic" carbamoylphosphate synthase was defective and carbamate kinase activity was left intact, but the current evidence suggests that two enzymes are present in P s e u d o -

m o n a s , one catalyzing the biosynthesis of carbamoylphosphate and the other responsible for the utilization of carbamoylphosphate. An analogous specialization has been established for P s e u d o m o n a s ornithine carbamoyltransferases (Stalon et al., 1967).

In conclusion, we have identified three closely linked mutations affecting carbamoylphosphate synthase. We do not know, however, whether these car

mutations occur in the same or in different functional units and whether further loci unlinked to car -9 are involved in the synthesis of carbamoylphosphate.

N - A c e t y l g l u t a m a t e S y n t h a s e

The first enzyme of the arginine pathway, N-acetylglutamate synthase, catalyzes the acetylation of L-glutamate with acetyl-CoA (Maas, Novelli and Lipmann, 1953). In P. a e r u g i n o s a this reaction serves to replen- ish the pool of N-acetyl-L-glutamate and other N-

Table 4. Transduction analysis of argA strains

Recipient Marker F116L donor strain strain selected

PAO366 PAO540 PAO824 PAO952 PAO953 PAO1

PAO366 argA36 0 (< 1) 4 (12) 2 (15) 2 (5) 2 (5) 36

PAO540 argA171 2 (< 1) 0 (< 1) 4 (8) 4 (2) 4 (2) 198 his-5075 820 a 1 35 139 161 142

PAO824 argA163 0 (< 1) 0 (<2) 0 (<6) 1 (2) 0 (<2) 46 his-151 98 28 0 29 41 31

PAO952 argA127 3 (1) 0 (< 1) 1 (2) 0 (< 1) 1 (1) 143

PAO953 argA147 1 (< 1) 1 (1) 0 (<2) I (1) 1 (1) 124

PAO303 argB18 250 63 26 81 83 69

Numbers of transductants per plate (averages of duplicates) are given. Transductants of PAO303, PAO540, PAO952, and PAO953 were scored after 2 days, those of PAO366 after 3 days and those of PAO824 after 4 days. In all transductions with PAO824 the plates selective for Arg + showed about 20 additional very small colonies, perhaps indicative of spontaneous suppression ofargA163. Prototroph reduction in argA + (values in parentheses) is calculated by the formula given in "Materials and Methods" using argBi8 as a standard when no second auxotrophic marker was available in an argA stram a Approxlmately

600

500

c 400 0 -0

00 0

\ 300

t--

- 200 ,,13 E O u

rr 100

113 1

0 10 40

b 0 "~

0 10

I

20 30 Time (minutes)

600

500 if)

c "

~0o

0

\ 300 u l

200 ..13 E 0 u

rr 100

20 30

Time (minutes)

D. H a a s et al. : Arg in ine Genes in Pseudomonas aeruginosa 15

40

600

500

t - O &00

\ 300 L~

200

100

c 0

/f 10 20 30 40

Time (minutes)

Fig. 4a -e . Time of ent ry kinet ics of argA, argH, argB and lys. In t e r rup t ed ma t ings us ing PAO381 as a d o n o r were car r ied out as descr ibed in " M a t e r i a l s and M e t h o d s " . The recipients were: a PAO956 (argA127, lys-61, nal-13), b PAO512 (argH32, lys-58, nal-7), e PAO513 (argB18, lys-60, nal-8), o =argA + ; zx = a r g H + ; [] =argB + ; • = l y s +

acetylated compounds which participate in the reaction cycle producing L-ornithine (Udaka, 1966; Haas et al., 1972). The regulation of N-acetylglutamate synthase activity appears to be complex: inhibitors include the endproducts 5-arginine, putrescine and spermidine and the reaction product N-acetyl-L-glutamate (Haas and Leisinger, 1974).

Mutants deficient in N-acetylglutamate synthase were found among spontaneous and mutagen-induced ornithine auxotrophs. Five argA mutants were examined in some detail. None had any detectable N-acetylglutamate synthase activity (Table 2) and none was able to grow on minimal media without ornithine or arginine. We conclude that the enzyme is essential for arginine synthesis and that during growth on minimal media there is no alternate source of N-acetyl-L- glutamate.

Strain PAO1 can utilize N-acetyl-L-glutamate as

the sole carbon and nitrogen source; argA mutants, paradoxically, cannot. Possibly, N-acetylglutamate has to be deacetylated outside the cytoplasm to be taken up. This model is supported by the finding that argA mutants did not grow with N-acetylglutamate as a supplement in minimal media, but spontaneous derivatives could be selected which did respond to this compound and therefore may have gained an N-acetylglutamate permease activity.

The five argA mutations appeared closely linked but distinct on the basis of prototroph reduction transduction (Table 4).

In the FP2-mediated interrupted mating PAO956 (argA, lys, na/)xPAO381 (leu, str, FP2 +) argA entered at 18 19 min, slightly earlier than the reference marker lys, whose entry time was 20 _+ 1 min (mean of 6 experiments). The kinetics of this experiment are shown in Figure 4a. Confirmation that in


P A O 9 5 6 a r g A is the p r o x i m a l m a r k e r w i th respec t

to the F P 2 o r ig in was o b t a i n e d by ana lys i s o f ear ly

a r g A + a n d l y s + r e c o m b i n a n t s : the a r g A + r e c o m b i -

n a n t s a p p e a r i n g b e t w e e n 21 a n d 25 m i n ra re ly co in-

Table 5. AnaIysis of argA + , argH + , argB + and lys + recombinants for coinheritance of the unselected marker. Recombinants appearing early in the interrupted matings listed below were picked, spotted on to the selective medium and analyzed for coinheritance of the second, unselected markers by replica plating on to MM

Mating Selected Time of Unse- Coinher- Number marker inter- lected itance of

ruption marker of unse- colonies (min) lected tested

marker (%)

(a) argA127 21 lys-61 14 35 PAO956 23 28 100 x PAO381 lys-61 21 argA127 88 16 (Fig. 4a) 23 69 32

25 78 100

(b) argH32 21 lys-58 80 10 PAO512 24 51 41 x PAO381 27 63 49

(Fig. 4b) lys-58 21 argH32 86 14 24 85 52 27 92 50

(c) lys-60 21 argB18 5 20 PAO513 24 19 80 x PAO381

argB18 21 lys-60 50 6 (Fig. 4c) 24 75 20

27 73 58

Table 6. Gene order in the 17 20 rain regmn of the chromosome PAO955 (argA127, lys-61) and GMA153 (pur-66, his-35, FP2 +) were mated on the plate. Selection was made for argA + and lys+; hzs-35 was used as an unselected marker. The genotypes of 200 argA + and lys + recombinants were determined and the gene order (FP2- origin)-his-35 (=hisII) argA127-lys-61 was deduced

FP2 his-35 - a Donor ~ r r igA +

lys-61 + F

Recipient I I I his-35 + argA lys-61

Marker Recombinant Frequency Minimal seiected genotype (%) number of

crossovers required

argA127

lys-61

his + arg + lys + 36 2 his- arg + lys + 28 2 his + arg + lys 21 2 his- arg + lys 15 2

his arg + lys + 48.5 2 his + arg + lys + 25 2 his + arg lys + 24.5 2 his- arg- lys + 2 4

arg A

I 0 (< 0./.)

(a)

o (< o.3) (a)

a r g H lys

[ I 45 2.5

(a) (b) 42 1.2

4 [a) (b)

o (<o.2) 4

(c)

CTg B

i

ID

Cross Donor Re- cipient

Selected Un- Number marker selected of trans-

markers ductants scored

(a) F116L.PAO952 PAO372

(b) Fll6L.PAO1 PAO513

(c) G10I.PAO303 PAO362

argH32 argA127. 261 lys-58

lys-58 argA127, 338 argH32

argB18 lys-60 160 lys-60 argB18 250

argH32 argB18 600

Fig. 5. Transductional linkage between markers in the 20min region. Cotransduction values are given in per cent. Arrowheads point to the selected markers. Letters in parentheses refer to the cross as detailed below

he r i t ed l y s + , b u t the m a j o r i t y o f the ear ly l y s + r e c o m -

b i n a n t s h a d r ece ived the a r g A + al lele (Tab le 5 a).

A t h r e e - f a c t o r c ross (Tab le 6) e s t ab l i shed tha t

a r g A is l o c a t e d b e t w e e n h i s - 3 5 ( = h i s l I , M e e a n d Lee,

1967) a n d lys . T h e en t ry t ime o f h i s H was 17_+2

ra in ( m e a n o f 3 expe r imen t s , d a t a n o t shown) .

A r g i n i n o s u c c i n a s e

I saac a n d H o l l o w a y (1972) f o u n d tha t m u t a n t s

b l o c k e d in a r g i n i n o s u c c i n a s e ( a r g H ) b e l o n g e d to one

t r a n s d u c t i o n g roup . W e h a v e c o n f i r m e d the n a t u r e

o f the e n z y m e defec t in two m u t a n t s , P A O 2 9 7 a n d

P A O 3 6 2 ( T a b l e 2). P A O 5 1 2 ( a r g H , lys , ha l ) was used to o b t a i n a m a p l o c a t i o n o f a r g H . In an i n t e r r u p t e d

m a t i n g wi th an F P 2 - d o n o r a r g H a n d l y s b o t h e n t e r e d

a t a b o u t 20 ra in (Fig. 4b) . The f r equenc i e s o f c o i n h e r -

i t ance o f the unse l ec t ed m a r k e r in ea r ly r e c o m b i n a n t s

sugges t t h a t l y s is the dis ta l m a r k e r r e l a t ive to the

F P 2 o r ig in ( T a b l e 5 b). In F 1 1 6 L - t r a n s d u c t i o n , a r g H a n d l y s were a b o u t

4 0 % l inked. By con t r a s t , a r g H a n d a r g A were n o t co t r ansduc ib l e . T r a n s d u c t i o n a l l i nkage d a t a are sum-

m a r i z e d in F i g u r e 5.


N-acetylglutamate 5-phosphotransferase

Four ornithine-requiring mutants, PAO296, PAO298, PAO303 and PAO364, were placed into one transduction group by holloway and Isaac (1972), but no enzyme defect was found. We have now re-examined these mutants by a specific assay for N-acetylglutamate 5-phosphotransferase (Haas and Leisinger, 1975 a) : they lack or have greatly reduced N-acetylglutamate 5-phosphotransferase activity (Table 2). An additional mutation, argB1 (in PAO177) was classified into the same group on the basis of enzyme assay and prototroph reduction transduction with argB18 (in PAO303).

The time of entry of argB was established in a strain with a lys mutation as a reference marker, like in previous experiments. Fig. 4c demonstrates that argB18 in PAO513 entered at about 21 min, approximately 1 min later than lys. This marker order was confirmed by analysis of early recombinants (Ta- ble 5c). Stanisich and Holloway (1969) reported that time of entry of arg-i in PAO287 (now argB1) was 1 min. In a control experiment (data not shown) we found that argB1 (in PAO177) entered at 21 min as did argB18. Possible reasons for this discrepancy will be discussed later. The present genetic data are consis- tent with there being one argB locus. This is in agreement with the finding that N-acetylglutamate 5-phosphotransferase is an oligomeric enzyme composed of similar, possibly identical subunits (Haas and Leis- inger, 1975a).

argB and lys are sufficiently close to be cotransducible by F116L; a cotransduction frequency of approx. 2% was found (Fig. 5).

trp- 6

I

q

62 (a)

36

(b)

argC

I

11

(c)

5

(b)

(c) 29

(a}

str -/.3

I

Cross Donor Re- Selected Un- Number cipient marker selected of trans-

markers ductants scored

(a) F116L.PAO229 PAO307 argC54 t~T-6, 400 str-43

(b) Fl l6L.PAO307 PAO229 trp-6 argC54, 400 str-4 3

(c) FI16L.PAO229 PAO307 str-43 argC54, 400 trp-6

Fig. 6. Transductional linkage between markers in the 35 min region. Linkage is expressed in per cent cotransduction. Arrow- heads indicate the selected markers. Letters in parentheses refer to the crosses hsted below. When selection for str was made, the transducing mixture was poured in 2.5 ml soft agar on NA plates; after 3 h of incubation at 37 ° C the plates were overlaid with 2.5 ml soft agar containing 10rag streptomycin/m1 (delayed selection). The agar contained 0.2% KNO3 to enhance growth of colonies in the layers

N-Acetylglutamate 5-semialdehyde Dehydrogenase

Strains PAO307 and PAO311, two ornithine auxotrophs in the same transduction group (Isaac and Holloway, 1972), were deficient in the third enzyme of the pathway, N-acetylglutamate 5-semialdehyde dehydrogenase (Table 2). A locus coding for this enzyme, argC54, was found to be cotransducible with str-2 and trp-6 by Pemberton and Holloway (1972a). The position of argC relative to trp-6 and str was established by F116L transduction. Each marker was selected in turn and the inheritance of the two unselected markers scored. Cotransduction values sum- marized in Figure 6 indicate a gene order trp-6- argC-str. The orientation of the three genes with respect to the FP2 origin is not known.

The arg loci in PAO378 and PAO639 were cotransducible with trp-6 (40% and 46%, respectively) and therefore were classified as argC (Table 2). The

arg-6 mutation described by Loutit (1969) is likely to be an argC mutation as well because of its high linkage to str.

Pemberton and Holloway (1972a) and Carey and Krishnapillai (1975) have estimated that the entry time of trp-6 is 33-35 min. Based on these data we infer that argC is located in the 35 min region of the chromosome.

Anabolic Ornithine Carbamoyltransferase

P. aeruginosa has two distinct ornithine carbamoyltransferases : an anabolic enzyme which converts ornithine to citrulline and is repressible by arginine and a catabolic enzyme which degrades citrulline to ornithine and carbamoylphosphate and is inducible by arginine (Stalon etal., 1967; Isaac and Holloway, 1972). As shown by Stalon, Ramos, Pi~rard and Wiame (1972) for P.fluorescens, the two enzymes

18

Table 7. Transduction analysis of argF strains

D. Haas et al. : Arginine Genes in Pseudomonas aeruginosa

Recipient Marker F 116L donor strain strain selected

PAO21 PAO25 PAO304 PAO369 PAO522 PAO1

PAO21 argF3 0 (< I) 2 (1) 1 (1) 1 (< 1) 13 (2) 240 trpA1 0 107 111 298 461 170

PAO25 argFlO 1 (1) 0 (<2) 3 (1) 1 (1) 0 (< 1) 58 leu-lO 136 0 332 146 670 75

PAO304 argF19 1 (1) 0 ( < 1) 0 ( < 1) 0 ( < 1) 9 (6) 73

PAO369 argF39 1 (1) 3 (2) 0 (< 1) 0 (< 1) 3 (1) 103

PAO522 argF2 2 (3) 1 (1) 4 (4) 3 (4) 0 (< 1) 76 pur-136 108 144 171 138 0 124

PAO417 car-9 49 60 96 64 94 47

The figures represent numbers of transductants per plate (average of duplicates). The reduction in argF + formation (figures in brackets) was calculated as described in "Materials and Methods'. The car-9 marker of PAO417 was used for standardization wherever the argF strain did not carry an additional auxotrophic marker

funct ion unidirectionally owing to their regulatory properties.

Five P A O mutants which responded to either citrulline or arginine, but not to ornithine as a supplement, lacked anabolic ornithine carbamoyl t ransferase activity (Table 2). Characteristically, the growth re- sponse o f these mutants on M M plates was stronger to arginine than to citrulline, which might be taken up less efficiently than arginine. Two argF mutants , PAO25 and PAO522, were examined in more detail for their growth behaviour. They showed no growth on M M plates after 2 days in the absence o f arginine or citrulline; after p ro longed incubat ion some back- g round growth was noticed. This behaviour indicates that the catabolic ornithine carbamoyltransferase , present in PAO25 and PAO522, has little if any biosynthetic function.

The five argF mutat ions tested were closely linked but distinct on the basis o f p ro to t roph reduct ion t ransduct ion (Table 7). Thus, it appears that in P. aeruginosa there is one locus coding for a biosynthetic ornithine carbamoyl t ransferase as, for example, in E. coli B, but unlike E. coli K-12 where the enzyme is coded for by two genes, argF and argI (Legrain et al., 1972).

Carey and Krishnapillai (1975) found an entry time o f 55 + 7 rain for argF2. The gene order leu-38- pur-66--argF-leu-lO was established by M a t s u m o t o and coworkers (Mat sumoto and Tazaki, 1975; H. Matsumoto , personal communicat ion) .

Argininosuccinate Synthetase

Argininosuccinate synthetase activity was missing in strains PAO305 , PAO309 and PAO315 (Table2) ;

these argG mutants belong to one t ransduct ion group (Isaac and Hol loway, 1972). Wat son (J. Watson, personal communica t ion) found that in P. aeruginosa strain P A T argG and argF were cotransducible. The same linkage occurs in strain P A O : when F116L was grown on PAO25 (argI O and argG + t ransductants o f PAO315 were selected on M M plus citrulline (do- nor phenotype selection), 45% of the t ransductants formed large colonies and grew on unsupplemented MM. Thus their genotype was argF+argG +. 55% of the t ransductants appeared as small colonies on M M plus citrulline and did not grow on MM. Their genotype was argF argG + and their slow growth on M M plus citrulline was expected since argF mutants do no t utilize citrulline efficiently as a supplement. The 55% cot ransduct ion between argF and argG indicates that these two genes are close, but not contiguous. The orientat ion o f argF-argG relative to other markers is no t known.

N 2 -Acetylornithine 5-aminotransferase

NZ-Acetylornithine 5-aminotransferase is induced by arginine and in addi t ion to its biosynthetic funct ion this enzyme has ornithine aminotransferase activity used to catabolize ornithine (Voellmy and Leisinger, 1975). A mutan t defective in N2-acetylornithine 5- aminotransferase, PAO954 (argD17), can grow on M M in the absence o f arginine since at least one other enzyme, 4-aminobutyra te aminotransferase, can catalyze the t ransaminat ion o f N-acetylglutamate 5- semialdehyde and is present at levels sufficient to per- mit normal growth in M M (Voellmy and Leisinger, 1976). As yet, we have not been able to detect linkage


of argD to any marker in the 0 60 min region of the PAO chromosome.

Ornithine Acetyltransferase and Acetylornithinase

N2-Acetyl-L-ornithine is converted to L-ornithine by two Pseudomonas enzymes in vitro: ornithine acetyltransferase and acetylornithinase. Their relative contribution to ornithine synthesis in vivo is not known as no mutants lacking these enzymes have been described to date. Among 26 ornithine-requiring auxotrophs all had normal ornithine acetyltransferase and acetylornithinase activity and all could be placed in one of three classes, argA, argB or argC. In yeast, a defect in ornithine acetyltransferase can result in ornithine auxotrophy (Wiame and Dubois, 1976) and a carboxypeptidase, presumably with little biosynthetic function, seems responsible for the acetylornithinase activity measured in vitro (Degryse, 1974). Feary et al. (1969) reported that ornithine auxotrophs of P. aeruginosa strain PAO could be classified into four transduction groups and hence one group could conceivably be defective in ornithine acetyltrausferase (argJO. No enzyme assays, however, have been carried out with the mutants described by Feary et al.

Are the activities of N-acetylglutamate synthase and ornithine acetyltransferase the property of one enzyme ? This was suggested to be the case in Chlorella by Morris and Thompson (1975), who found that both activities were not separated during partial purification. In P. aeruginosa, by contrast, the two enzymes are separable by gel-filtration (T. Leisinger, unpublished observations) and the five argA mutants examined in some detail have normal ornithine acetyltransferase activity (Table 2). However, until argJ mutants become available, we cannot rule out the possibility that in Pseudomonas the two enzymes share a common acetyltransferase subunit.

Discussion

Six loci coding for arginine biosynthetic enzymes and one locus controlling carbamoylphosphate synthase have been identified and mapped on the chromosome of P. aeruginosa strain PAO, as illustrated by Fig. 7. We knew from earlier work (Fargie and Holloway, 1965; Feary et al., 1969; Isaac and Holloway, 1972) that arg mutations are not clustered in P. aeruginosa and we have now corroborated this finding by estab- lishing the map position of most genes coding for arginine biosynthesis. Mutations in argD and argJ remain to be mapped and the role of argE in arginine biosynthesis is unclear at present.

( met- 9011 leu-10

hex-9001 { n a l - 7

argG ( argF

(pur-66 leu-38

pro-64 FP2 ]

0 5

15

- 6 0 20

- 5 5 25

40 35 I i

30 -5O

4 5 i

pro-82 t rp-6 argC ) s t r

- - i l v - 2 1 9 (thi-1)

- - cor-9 tolA )

- - hisI cys-5605

- - hisIl

- - argA orgH ) [ys -12 orgB )

- - pur-136

trpA - - trpB )

ilv-202 met-28 ) pyr8

Fig. 7. Chromosome map of P. aeruginosa PAO. This map summarizes results presented in this paper and includes some unpublished results. It is based on earlier versions (Pemberton and Holloway, 1972a; Holloway, 1975). Markers whose position is indicated by a bar were mapped by interrupted matings using the sex factor FP2. Brackets indicate cotransduction with F116L or G101. Proto- troph reduction transductions (data not given) have established that the following groups of markers are closely linked: his-4, his-151 and his-35 (=hisII, Mee and Lee, 1967); lys-12, lys-58, lys-60, lys-61 as well as lys-53 (Pemberton and Holloway, 1972a); ilv-2]9 and ilv-226 (=ilvB/C, Marinus and Loutit, 1969); leu-8 and leu-38, his-5075 and his-5039 are hisI mutations (Mee and Lee, 1967, i969), hal-7, nal-8, hal-9, hal-12 and hal-13 are mutations conferring high level resistance to NAL; they were 22-32% linked with hex-9001 in F116L-transduction. The chromosomal location of hex-9001 was determined by Matsumoto (personaI communica- tion). The thi-1 mutation was mapped near car (Pemberton and Holloway, 1972a; Carey and Krishnapillai, 1974), but its precise position was not investigated in this study

All arg loci described in this study are linked to other markers, either in transduction or in R68.45- mediated conjugation (data obtained with R68.45 will be presented elsewhere). The sex factor R68.45 transfers short pieces of chromosome from a wide range of origin s i tes-usual ly the length of the fragment inherited is less than 10 min of the genetic m a p - a n d hence this plasmid can be used like a large generalized transducing phage (Haas and Holloway, 1976). Gen- etic manipulation of the arg genes by transduction and mobilization with R68.45 has thus become possible and should facilitate further studies on arginine metabolism in Pseudomonas.


Some arg loci might serve as useful reference markers as the localization of genes on the PAO chromosome continues. By means of a refined interrupted mating technique we have mapped a number of genes in the 0-30 rain region of the chromosome and as a consequence the position of some markers given in an earlier map (Pemberton and Holloway, 1972a) had to be revised. Markers which have been assigned a new map location include argB (formerly arg-1, arg-18), car (arg-160, arg-161), argA (arg-163), hisH (his-151), argH (arg-32), ih,B/C (ilv-226, ilv-261, iiv- 219) and pur-136 (mapped at about 25 rain by interrupted mating, data not shown). All lys mutations in strain PAO examined to date appear to be located at 20 rain; lys-53, previously reported to map at 8 min (Pemberton and Holloway, 1972a), was found to be very closely linked to lys-12 by prototroph reduction transduction (data not given). --

Most, but not all, markers whose map position has been revised displayed an earlier time of entry in previous experiments (Stanisich and Holloway, 1969; Pemberton and Holloway, 1972a). These au- thors used the virulent phage E79 to interrupt conjugation. It is possible that lysis by phage E79 is not as effective as NAL in preventing a second round of mating after interruption and, therefore, a rela- tively high background number of recombinants may have given the false impression of an early entry time in some cases. An additional difficulty in interpreting interrupted matings using phage E79 is that the loci conferring resistance to phage E79 (ese) in recipients were not mapped (Stanisich and Holloway, 1969; Pemberton and Holloway, 1972a; Day et al. 1975). If a donor transfers an allele specifying sensitivity to phage E79 before a marker under investigation, then the number of recombinants for that marker is expected to be reduced. Any factors which lower the recovery of recombinants will tend to delay the entry time. The loci specifying resistance to NAL are located in the 60 min region of the chromosome (Fig. 7) and are therefore unlikely to interfere with the recovery of markers mapped in this study.

In the interrupted matings described here, time of entry curves were nonlinear with respect to time. As in E. coli matings (Low, 1973), this nonlinearity was more pronounced for markers entering after about 20 min than for early markers. This is one reason why it is difficult to determine accurately the entry time of late markers. Another reason is that the recovery of recombinants in FP2 matings de- creases markedly with increased distance of a marker from the FP2 origin and the detection of the earliest recombinants formed becomes a problem. To over- come these difficulties, we need sex factors differing from FP2 in their origin of chromosome transfer or

giving higher recoveries of recombinants than FP2. A search for such conjugative plasmids is under way.

From a taxonomic point of view it is interesting to compare the arrangement of arg genes in different bacterial species. There is a considerable degree of homology between E. coli and Salmonella typhimurium ." both have an argECBH cluster and the remaining arg genes are scattered (Vogel et al., 1971 ; Sanderson, 1972). There is evidence that the arrangement of arg loci in Klebsiella pneumoniae is quite similar to that in E. coli (Matsumoto and Tazaki, 1970). In other Enterobacteriaceae such as Proteus species and Serra- tia marcescens an argECBGH cluster is found (Pro- zesky, Grabow, van der Merwe and Coetzee, 1973; Matsumoto, Hosogaya, Suzuki and Tazaki, 1975). In Bacillus subtilis there are probably gene clusters coding for the first five and last two enzymes (Baum- berg, 1976). As we have shown in this study, the arg genes of P. aeruginosa are widely scattered and thus their arrangement is different from that found in Enterobacteriaceae and Bacillus. Major differences are also observed in the regulation of arginine biosynthesis: All arginine biosynthetic enzymes are repressed by arginine in E. coli K-12 (albeit not in E. coli B; Vogel et al., 1971), while in P. aeruginosa and P. putida only one enzyme, the anabolic ornithine carbamoyltransferase, is strongly repressed by arginine (Isaac and Holloway, 1972; Voellmy and Leis- inger, 1972; Condon, Collins and O'Donovan, 1976). In P. aeruginosa the main control exerted by end prod- ucts of the arginine pathway appears to act the level of enzyme activity in that both the first and the second enzyme of the pathway are subject to feedback inhibition (Haas and Leisinger, 1974; 1975 b).

Enterobacteriaceae and Bacillus do not have an ornithine acetyltransferase; by contrast, Pseudomonas belongs to a group of microorganisms which possess this enzyme and hence recycle the acetyl group in the pathway (Udaka, 1966). Since the presence of an ornithine acetyltransferase (argJ; see Fig. 1) conserves en- ergy required for arginine synthesis and since euka- ryotic organisms such as yeast, Neurospora and green algae have this enzyme, Pseudomonas has been consid- ered a more highly evolved form of bacterium than enteric bacteria and Bacillus (Udaka, 1966). It may be, however, that during evolution, clustering of arginine biosynthetic genes occurred in some organisms (like enteric bacteria) and that this process provided a basis for efficient control of enzyme levels by repression; as a consequence, ornithine acetyltransferase and feedback inhibition of the second enzyme of the pathway, N-acetylglutamate 5-phosphotransferase, may have become redundant and were lost.

We do not know the selective forces involved in the evolution of the arginine pathway in various mi-


croorganisms. We do know that such selection has resulted in widely varying gene arrangements and control mechanisms. These evolutionary processes can hardly be studied experimentally. However, the recent discovery of plasmids having a broad host range (IncP-1) opens up a new experimental approach to comparative studies on gene expression in microorganisms. Fragments of chromosomal D N A can be in- corporated into such plasmids and in this way struc- tural and regulatory genes can be transferred from one bacterial genus to another. It is now experimentally possible for Pseudomonas arg genes to be in- troduced into E. coli and vice versa and their expression and regulation may be studied in the new host.

Acknowledgements. We wish to thank John Watson, Chris Scott, Joy Brake and U. Sidler for prowdiug unpublished data and some of the bacterial strains used in this investigation and Linda Bell for her enthusiastic technical assistance. One of us (D.H.) was supported by a fellowship of the Swiss National Science Founda- tion. The project was supported in part by the Australian Research Grants committee (at Monash University) and by grant no. 3.5090.75 from the Swiss National Science Foundation (at the ETH).

References

Bachmann, B.J., Low, K.B., Taylor, A.L.: Recalibrated linkage map of Escherichia coli K~12. Bact. Rev. 40, 116 167 (1976)

Baumberg, S.: Genetic control of arginine metabolism in proka- ryotes. In: Second International Symposium on the Genetics of Industrial Microorganisms (K.D. Macdonald, ed.), pp. 36%389. London: Academic Press 1976.

Calhoun, D.H., Pierson, D.L., Jensen, R.A.: The regulation of tryptophan biosynthesis in Pseudomonas aerugh2osa. Molec. gen. Genet. 121, 117--132 (1973)

Carey, K.E., Krishnapillai, V. : Location of prophage H90 on the chromosome of Pseudomonas aerugmosa strain PAO. Genet. Res. 23, 155-164 (1974)

Carey, K.E., Krishnapillai, V. : Chromosomal location of prophage J51 in Pseudomonas aeruginosa strain PAO. Genet. Res. 25, 179-187 (1975)

Clarke, P.H., Ornston, L.N. : Metabolic pathways and regulation. In: Genetics and biochemistry of Pseudomonas (P.H, Clarke and M.H. Richmond, eds.), pp. 191 340. London: J. Wiley & Son 1975

Condon, S., Collins, J.K., O'Donovan, G.A.: Regulation of arginine and pyrimidine biosynthesis in Pseudomonas putida. J. gen. Microbiol. 92, 375-383 (1976)

Crawford, I.P. : Gene rearrangements in the evolution of the tryptophan synthetic pathway. Bact. Rev. 39, 87-120 (1975)

Day, M., Ports, J.R., Clarke, P.H.: Location of genes for the utilization of acetamide, histidine and proline on the chromosome of Pseudomonas aerughmsa. Genet. Res. 25, 71-78 (1975)

Degryse, E. : Evidence that yeast acetylornithinase is a carboxypeptidase. FEBS Lett. 43, 285-288 (1974)

Dunn, N.W., Holloway, B.W.: Transduction and host controlled modification. The role of the phage. In: Informative molecules in biological systems (L.G.H. Ledoux, ed.), pp. 223-233. Am- sterdam : North-Holland Pubhshing Company 1971

Fargie, B., Holloway, B.W. : Absence of clustering of functionally related genes in Pseudomonas aeruginosa. Genet. Res. 6, 284-299 (1965)

Feary, T.W., Williams, B., Calhoun, D.H., Walker, T.A. : An anal-

ysis of arginine requiring mutants in Pseudomonas aeruginosa. Genetics 62, 673-686 (1969)

Haas, D., Holloway, B.W.: R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa. Molec. gen. Genet. 144, 243-251 (1976)

Haas, D., Kurer, V., Leisinger, T.: N-Acety]glutamate synthetase of Pseudomonas aeruginosa. An assay in vitro and feedback inhibition by arginine. Europ. J. Biochem. 31, 290-295 (1972)

Haas, D., Leisinger, T.: Multiple control of N-acetylglutamate synthetase from Pseudomonas aeruginosa: synergistic inhibition by N-acetylglutamate and polyamines. Biochem. biophys. Res, Commun. 60, 42-47 (1974)

Haas, D., Leisinger, T.: N-Acetylglutamate 5-phosphotransferase of Pseudomonas aeruginosa. Purification and ligand-directed as- sociation-dissociation. Europ. J. Biochem. 52, 365-375 (1975a)

Haas, D., Lelsinger, T : N-Acetylglutamate 5-phosphotransferase of Pseudomonas aerug#~osa. Catalytic and regulatory properties~ Europ. J. Biochem. 52, 377-383 (1975b)

Heumann, W., Piihler, A., Wagner, E.: The two transfer regions of the Rhizobium lupini conjugation. I. Fertility factor elimina- tion and one way transfer. Molec. gen. Genet. 113, 308-315 (1971)

Holloway, B.W. : Genetics of Pseudomonas. Bact. Rev. 33, 419-433 (1969)

Holloway, B.W.: Genetic organization of Pseudomonas. In: Gen- etics and biochemistry of Pseudomonas (P.H. Clarke and M.H. Richmond, eds.), pp. 133 161. London: J.Wiley & Son 1975

Holloway, B.W., van de Putte, P. : Lysogeny and bacterial recombination. In: Replication and recombination of genetic material (W.J. Peacock and R.D. Brock, eds.), pp. 175 183. Australian Academy of Science 1968

Isaac, J.I-I., Holloway, B.W.: Control of arginine biosynthesis in Pseudomonas aeruginosa. J. gen. Microbiol. 73, 427438 (1972)

Kageyama, M.: Genetic mapping of a bacteriocinogenic factor in Pseudomonas aeruginosa. I. Mapping of pyocin R2 factor by conjugation. J. gen. appl. Microbiol. 16, 523-530 (1970)

Krishnapillai, V. : A novel transducing phage. Its role in recognition of a possible new host-controlled modification system in Pseu- domonas aerug#7osa. Molec, gen. Genet. 114, 134 143 (1971)

Legrain, C., Halleux, P., Stalon, V., Glansdorff, N.: The dual genetic control of ornithine carbamoyltransferase in Escherichia coli. A case of bacterial hybrid enzymes. Europ. J. Biochem. 27, 93 102 (1972)

Leisinger, T., Haas, D., Hegarty, M.P. : Indospicine as an arginine antagonist in Eseherich~a coli and Pseudomonas aeruginosa. Bio- chim. biophys. Acta (Amst.) 262, 214-219 (1972)

Loutit, J.S.: Studies on nutritionally deficient strains of Pseudo- monas aerugmosa. I. The production by x-rays and the isolation of nutritionally deficient strains. Aust. J. Exp. Bml. reed. Sci. 30, 287-294 (1952)

Loutit, J.S.: Investigation of the mating system of Pseudomonas aerug#2osa strain 1. IV. Mapping of distal markers, Genet. Res. 13, 91 98 (1969)

Loutit, J.S.: Investigation of the mating system of Pseudomonas aeruginosa strain 1. VI. Mercury resistance associated with the sex factor (FP). Genet. Res. 16, 179-184 (1970)

Loutit, J.S., Marinus, M.G.: Investigation of the mating system of Pseudomonas aerug#7osa strain 1. IL Mapping of a number of early markers. Genet. Res. 12, 37-44 (1968)

Low, B. : Rapid mapping of conditional and auxotrophic mutations in Escherichta coli K-12. J. Baet. 113, 798-812 (1973)

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275 (1951)

Maas, W.K., Novelli, G.D., Lipmann, F. : Acetylation of glutamic acid by extracts ofEseherichia coli. Proc. nat. Acad. Sci. (Wash.) 39, 1004 1007 (1953)

22 D. Haas et al. : Arginme Genes in Pseudomonas aeruginosa

Marinus, M.G., Loutit, J.S. : Regulation of isoleucine-valine biosynthesis in Pseudomonas aeruginosa. I. Characterisation and mapping of mutants. Genetics 63, 547-556 (1969)

Matsumoto, H., Hosogaya, S., Suzuki, K., Tazaki, T.: Arginlne gene cluster of Serratia marceseens. Jap. J. Microbiol. 19, 35-44 (1975)

Matsumoto, H., Tazaki, T. : Genetic recombination in Klebsiella pneumoniae. An approach to genetic linkage mapping. Jap. J. Microbiol. 14, 129-141 (1970)

Matsumoto, H., Tazaki, T.: Serotypic recombination in Pseudo- monas aeruginosa. In : Microbial drug resistance (S. Mitsuhashi and H. Hashimoto, eds.), pp. 281-290. University of Tokyo Press 1975

Mee, B.J., Lee, B.T.O. : An analysis of histidine requiring mutants in Pseudomonas aeruginosa. Genetics 55, 709-722 (1967)

Mee, B.J., Lee, B.T.O.: A map order for hisI, one of the genetic regions controlling histidine biosynthesis in Pseudomonas aeruginosa, using the transducing phage F116. Genetics 62, 687~696 (1969)

Mergeay, M., Gigot, D., Beckmann, J., Glansdorff, N., Pi6rard, A.: Physiology and genetics of carbamoylphosphate synthesis in Escherichia coli K12. Molee. gen. Genet. 133, 299 316 (1974)

Miller, J.H.: Experiments in molecular genetics, pp. 86-90. New York: Cold Spring Harbor Laboratory 1972

Mills, B.J., Holloway, B.W.: Mutants of Pseudomonas aeruginosa that show specific hypersensitivity to ammoglycosides. Antimi- crob. Agents Chemother. 10, 411-416 (1976)

Morris, C.J., Thompson, J.F, : Acetyl coenzyme A-glutamate acetyltransferase and N2-acetylornithine-glutamate acetyltransferase of Chlorella. Plant. Physiol. 55, 960-967 (1975)

Ornston, L.N., Ornston, M.K., Chou, G. : Isolation of spontaneous mutant strains of Pseudomonas putida. Biochem. biophys. Res. Commun. 36, 179 184 (1969)

Pemberton, J.M., Holloway, B.W. : Chromosome mapping in Pseu- domonas aeruginosa. Genet. Res. 19, 251-260 (1972a)

Pemberton, J.M., Holloway, B.W. : A mutant of Pseudomonas aeruginosa with increased conjugational ability. Aust. J. exp. Biol. meal. Sci. 50, 577 588 (1972b)

Pi6rard, A., Glansdorff, N., Gigot, D., Crabeel, M., Halleux, P., Thiry, L. : Repression of Eseherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways. J. Bact. 127, 291-301 (1976)

Pi6rard, A., Glansdorff, N., Yashphe, J. : Mutations affecting uridine monophosphate pyrophosphorylase or the argR gene in Escher# chia coli. Molec. gen. Genet. 118, 235-245 (1972)

Prozesky, O.W., Grabow, W.O.K., van der Merwe, S., Coetzee, J.N.: Arginine gene clusters in the Proteus-Providence group. J. gen. Microbiol. 77, 237-240 (1973)

Raijman, L., Jones, M.E.: Carbamate kinase. In: The Enzymes (P.D. Boyer, ed.), Vol. IXB, pp. 97-119. New York: Academic Press 1973

Ramos, F., Stalon, V., Pi6rard, A., Wiame, J.M.: The specialization of the two ornithine carbamoyltransferases of Pseudo- monas. Biochim. biophys. Acta (Amst.) 139, 98 106 (1967)

Ratner, S., Kunkemueller, M. : Separation and properties of argininosuccinate and its two anhydrides and their detection in biological materials_ Biochemistry 5, 1821-1832 (1966)

Sanderson, K.E. : Linkage map of Salmonella typhimurium, edition IV. Bact. Rev. 36, 558 586 (1972)

Stalon, V., Ramos, F., Pi6rard, A., Wiame, J.M. : The occurrence of a catabolic and an anabolic ornithine carbamoyltransferase in Pseudomonas. Biochim. biophys. Acta (Amst.) 139, 91-97 (1967)

Stalon, V., Ramos, F., Pi6rard, A., Wiame, J.M.: Regulation of the catabolic ornithine carbamoyltransferase of Pseudomonas

fluorescens. A comparison with the anabolic transferase and with a mutationally modified catabolic transferase. Europ. J. Biochem. 29, 25-35 (1972)

Stanisich, V., Holloway, B.W. : Conjugation in Pseudomonas aeruginosa. Genetics 61, 327-339 (1969)

Stanisich, V.A., Holloway, B.W. : A mutant sex factor of Pseudo- monas aeruginosa. Genet. Res. 19, 91-108 (1972)

Stanisich, V.A., Richmond, M.H. : Gene transfer in the genus Pseu- domonas. In: Genetics and biochemistry of Pseudomonas (P.H. Clarke and M.H. Richmond, eds.), pp. 163-190. London: J. Wiley & Son 1975

Theil, E.C., Forsyth, G.W., Jones, E.E. : Expression of the arginine regulon of Escherichia coli W: evidence for a second regulatory gene. J. Bact. 99, 269-273 (1969)

Udaka, S. : Pathway-specific pattern of control of arginine biosynthesis in bacteria. J. Bact. 91, 617M21 (1966)

Voellmy, R., Leisinger, T.: Regulation of enzyme synthesis in the arginine system of Pseudomonas aeruginosa. J. gen. Microbiol. 73, xiii 0972)

Voellmy, R., Leisinger, T. : Dual role for N2-acetylornithine 5-aminotransferase from Pseudomonas aeruginosa in arginine biosynthesis and arginine catabolism. J. Bact. 122, 799-809 (1975)

Voellmy, R., Leisinger, T. : Role of 4-aminobutyrate aminotransferase in the arginine metabolism of Pseudomonas aeruginosa. J. Bact. 128, 722-729 (1976)

Vogel, H.J., Bonner, D.M.: Acetylornithinase of Escherichia coli: partial purification and some properties. J. biol. Chem. 218, 97-106 (1956)

Vogel, H.J., McLellan, W.L.: N-Acetylglutamic y-semialdehyde dehydrogenase (Escherichia col O. Methods Enzymol. 17A, 255-260 (1970a)

Vogel, H.J., McLellan, W.L. : Acetylornithinase (Escherichia colO. Methods Enzymol. 17A, 265-269 (1970b)

Vogel, R.H., McLellan, W.L., Hirvonen, A.P., Vogel H.J.: The arginine biosynthetic system and its regulation. In: Metabolic regulation (H,J. Vogel, ed.), Vol. 5, pp. 463488. New York: Academic Press 1971

Watson, J.M., Holloway, B.W. : Suppressor mutations in Pseudo- monas aeruginosa. J. Bact. 125, 780-786 (1976)

Wiame, J.M., Dubois, E.L.: The regulation of enzyme synthesis in arginine metabolism of Saccharomyces cerevlsiae. In: Second International Symposium on the Genetics of Industrial Mi- croorganisms (K.D. Macdonald, ed.), pp. 391406. London: Academic Press 1976

Zipkas, D., Riley, M. : Simplified method for interruption of conjugation in Eseheriehia coll. J. Bact. 126, 559-560 (1976)

Comnmnicated by G.A. O'Donovan

Received February 18, 1977

the genetic organization of arginine biosynthesis in pseudomonas aeruginosa

Documents