JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 377-384Copyright © 1977 American Society for Microbiology

Vol. 132, No. 2Printed in U.S.A.

Pseudomonas aeruginosa Mutants Resistant to UreaInhibition of Growth on AcetanilideMARY GREGORIOU,* PAUL R. BROWN, AND RENEE TATA

Department of Biochemistry, King's College London, Strand, London WC2R 2LS, England

Received for publication 12 May 1977

Pseudomonas aeruginosa Al 3 was able to grow in medium containingacetanilide (N-phenylacetamide) as a carbon source when NH4+ was thenitrogen source but not when urea was the nitrogen source. AIU mutantsisolated from strain Al 3 grew on either medium. Urease levels in bacteriagrown in the presence of urea were 10-fold lower when NH4, or acetanilide wasalso in the medium, but there were no apparent differences in urease or itssynthesis between strain Al 3 and mutant AIU 1N. The first metabolic step inthe acetanilide utilization is catalyzed by an amidase. Amidases in several AIUstrains showed altered physicochemical properties. Urea inhibited amidase in atime-dependent reaction, but the rates of the inhibitory reaction with amidasesfrom the AIU mutants were slower than with Al 3 amidase. The purifiedamidase from AIU 1N showed a marked difference in its pH/activity profilefrom that obtained with purified Al 3 amidase. These observations indicatethat the ability of strain AIU 1N and the other mutants to grow on acetanilide/urea medium is associated with a mutation in the amidase structural gene;this was confirmed for strain AIU IN by transduction.

The amidase (acylamide amidohydrolase, EC3.5.1.4) from Pseudomonas aeruginosa cata-lyzes the hydrolysis of aliphatic amides to givethe corresponding acid and ammonia. Sub-strates for this enzyme are the short-chainaliphatic amides formamide, acetamide, andpropionamide, higher-molecular-weight amidesbeing poor substrates (9). Strain Al 3 is amutant constitutive for amidase synthesis andis able to utilize acetanilide as the sole carbonsource for growth. Strain Al 3 produces analtered amidase with increased activity to-wards acetanilide compared with that of thewild-type enzyme (4). Thus, growth of thisstrain on acetanilide occurs because the alteredamidase it produces catalyzes the hydrolysis ofacetanilide to acetate and aniline:('}I3\ t 6II,"CIL () OI t 11,N

Acetate serves as a growth substrate, but nei-ther the nitrogen nor the carbon of the anilinemolecule is available to the organism. In theoriginal medium used to select strain Al 3,nitrogen was supplied as NH4+. We have foundthat, when urea is substituted for NH4+ as thenitrogen source, with acetanilide as the solecarbon source, strain Al 3 is unable to grow. Inthis paper we describe the isolation of mutants(AIU strains) from strain Al 3 that are able to

utilize urea as the sole nitrogen source whenacetanilide provides carbon. To explain thegrowth properties of strain Al 3 and the AIUmutants under these conditions, we performedexperiments to test the following possibilities.(i) Strain Al 3 does not produce a urease eitherthrough absence of the gene coding for such anenzyme or through a defect in the regulation ofits synthesis. Growth of the AIU mutantsmight then arise by generation of a ureaseactivity through changes in a preexisting en-zyme. Alternatively, a defect in regulation ofurease synthesis in strain Al 3 could have beenrepaired in the AIU mutants. (ii) Urea inhibitsutilization of the acetanilide by strain Al 3,and this inhibition is relieved in the AIU mu-tants.

Previous reports on whether urease is pro-duced by P. aeruginosa are conflicting, butStewart (13) found that urease activity wasdetectable in 50 isolates of the genus Pseudom-onas and was repressed by free NH4+ in grow-ing cultures. A mechanism along the lines of iiwas suggested by the work of Kelly and Korn-berg (10), who found that urea was a noncom-petitive inhibitor of wild-type amidase (K,, 1.1mM) with propionamide as a substrate.

MATERIALS AND METHODS

Organisms. The parent P. aeruginosa strain usedin our studies was Al 3. Strain Al 3 is constitutive

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378 GREGORIOU, BROWN, AND TATA

for the production of an altered amidase with in-creased activity towards acetanilide; it was selectedfrom strain L 10, which is magno-constitutive forthe synthesis of wild-type amidase (4). The amidase-negative strain RT lAm 21 was selected from strainRT 1 by the fluoroacetamide method of Clarke andTata (5); it gives partial cross-reaction with antise-rum to wild-type amidase and has a low rate (ca. 1in 101) of spontaneous reversion to amidase positive.RT 1 is an acetanilide-utilizing mutant isolatedfrom L 100 (a strain magno-constitutive for wild-type amidase) after ICR 191E mutagenesis (P. R.Brown, A. Heaton, and R. Tata, unpublished data).

Bacteriophage and transduction. Lysates of bac-teriophage F 116 (8) were prepared and transductionwas performed as described by Brammar et al. (2).

Media. All media were prepared from minimalsalts medium (1) with or without 0.1% (NH4)2SO4(NH4+) as indicated in the text. Solid media con-tained 1% Difco Noble Agar. Potassium lactate(0.3%) and sodium succinate (0.3%) were autoclavedwith the medium. Other carbon and nitrogensources were added aseptically to the autoclavedmedium at the concentrations shown: acetanilideAl, 0.1%; formamide, 0.1%; acetamide, 0.1%; urea(U), normally 0.05%, except in AI/5U medium whereit was supplied at 0.25%.

Isolation of AIU mutants. Strain Al 3 was grownovernight in 5 ml of nutrient broth and suspendedin 5 ml of dilution buffer (2), and 0.1-ml sampleswere plated on Al/U medium. Mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine was effected byplacing a few crystals of nitrosoguanidine in thecenter of the plate. Mutant colonies were picked offafter 6 days of incubation at 37°C, grown overnightin nutrient broth, and reisolated as single coloniesby diluting and plating on Al/U medium. MutantAI5U 1N was obtained after nitrosoguanidine mu-tagenesis of strain Al 3 on AI/5U medium.

Bacterial extracts. Extracts were prepared in0.05 M tris(hydroxymethyl)aminomethane (Tris)buffer (pH 7.2) as described previously (3). Proteinwas estimated by the method of Lowry et al. (11),with bovine serum albumin as a standard.Enzyme assays. Amidase activity was assayed

by adding enzyme to 1.0 ml of 0.5 mM 4-NO2-acet-anilide in 0.05 M Tris buffer (pH 7.2) at 37°C andfollowing the formation of 4-NO2-aniline at 400 nmin an SP 800 recording spectrophotometer (UnicamInstruments Ltd.). Urease activity was assayed byadding enzyme to 1 ml of 20 mM urea in 0.05 M Trisbuffer (pH 7.2) at 37TC; 0.1-ml portions were re-moved at timed intervals and assayed for ammoniaby the ninhydrin method described previously (3).Amidase purification. Amidases from strains Al 3

and AIU IN were purified from cells grown in 20liters of lactate/NH4, medium, using the methoddescribed previously (3) for purifying wild-type ami-dase but omitting the heat treatment step. Centerfractions from the amidase peak eluted from thediethylaminoethyl-Sephadex column were bulked,concentrated by ultrafiltration, and further purifiedby fractionating 2-ml portions (10 to 15 mg of pro-tein) on a G-200 Sephadex column (75 by 1.5 cm)eluted with 0.05 M Tris (pH 7.2) containing 1 mM

ethylenediaminetetraacetate and 1 mM mercapto-ethanol. Fractions shown by polyacrylamide discon-tinuous electrophoresis (6) at loadings of 50 ,ug ofprotein to be homogeneous for amidase were usedfor experimental purposes.

Immunological methods. Rabbit antiserum topurified Al 3 amidase was obtained by giving two0.5-ml injections (intramuscular) of amidase (4 and9 mg), emulsified with 0.5 ml of complete Freundadjuvant, in 0.05 M Tris buffer (pH 7.2). Injectionswere spaced by 2 weeks, and the animal was bled 2weeks after the second injection. Immunodiffusionexperiments were done in agar gels on microscopeslides as described previously (3). Quantitative es-timation of amidase cross-reacting material wasdone by radial immunodiffusion (12) on slides (76by 51 mm) coated with agar (5 ml) containingantiserum (6%, vol/vol). Samples (10 gl) were placedin 3-mm-diameter wells, and the diameters of theprecipitin rings obtained after 48 h at 4°C weremeasured with a traveling microscope. Calibrationcurves were obtained for each gel by using purifiedAl 3 amidase over the range 1 to 10 /ig.

Electrophoresis. Extracts (10 ,pl) were subjectedto electrophoresis in duplicate on a sheet (15 by 9cm) of Cellogel (Oxoid Ltd.) for 1 h in an electropho-resis tank (Colab Labs Ltd.). The gel was cut intwo, so that each half contained one of the samplesof each extract run. Acetamidase and urease activi-ties were detected by overlaying the two halveswith filter papers soaked in acetamide solution (100mM) and urea solution (20 mM), respectively, incu-bating at 37°C for 1 h, removing the paper, andreplacing it with paper soaked in Nessler reagent.Yellow bands indicated the position of the enzymes.Electrophoresis buffers used were 0.05 M potassiumphosphate (pH 6.0) and 0.05 M sodium barbitone(pH 8.5).

Chemicals. Urea was Aristar grade from BDH.4-NO2-acetanilide (BDH) was recrystallized twicefrom ethanol. Other amides were recrystallized asdescribed previously (3).

RESULTS

AIU mutants. Twenty-three mutants grow-ing on Al/U medium were selected for furtherstudy. The spontaneous mutants were labeledAIU 1 through 6; those obtained after nitroso-guanidine mutagenesis were labeled AIU INthrough AIU 17N. Of these strains, the one westudied most extensively was AIU IN. StrainAI5U IN is a nitrosoguanidine-induced mu-tant selected on AI/5U medium. All of thesestrains produced clearly visible colonies on Al/U medium after 48 h of incubation at 37°C.Only strain A15U 1N grew on AI/5U medium.Strain Al 3 produced no visible growth on Al/U or Al/5U medium. Strain Al 3 produced novisible growth on Al/U or Al/5U medium evenafter prolonged incubation.Growth properties of strain Al 3. The

growth properties of strain Al 3 on a variety of

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UREA-RESISTANT MUTANTS OF P. AERUGINOSA 379

solid media were examined to investigatewhether the failure of strain Al 3 to grow on

Al/U medium was due primarily to an inabilityto obtain nitrogen from urea or an inability toobtain carbon from acetanilide. The media usedand the growth properties of strains Al 3 andAIU 1N are shown in Table 1. The same growthproperties of strain Al 3 were observed in thecorresponding liquid media: with a 1% inocu-lum strain Al 3 was fully grown in lactate/U,lactate/U/NH4+, lactate/NH4+, lactate/Al/U,and lactate/AI/NH4+ liquid media after 15 h ofshaking at 37°C; growth on AI/NH4+ was slower,reaching maximum bacterial density after 24 hof incubation. No growth in Al/U or AI/U/NH4+ medium was observed even after 96 h ofincubation. Growth of strain Al 3 on lactate/Umedium indicated that this strain was able toutilize urea as a nitrogen source, presumablythrough the activity of a urease. The ability ofstrain Al 3 to grow on lactate/Al/U mediumindicated that utilization of urea was not in-hibited by acetanilide. Since strain AIU 1N,selected on Al/U medium, also grew on AI/U/NH4+ medium, this implied that the inabilityof strain Al 3 to grow on either medium was

attributable to a single cause. Comparing thefailure of strain Al 3 to grow on either Al/U orAI/U/NH4+ medium with its ability to grow on

AI/NH4+ medium suggested that urea was in

some way blocking utilization of acetanilide.Urease activities. To confirm that these

strains did synthesize urease and to see

whether urease synthesis in strain Al 3 differedfrom that in the mutants, extracts preparedfrom strain Al 3 and a selection of mutantstrains grown in lactate/NH4+ medium were

assayed for urease activity. Urease activitywas detected in all of the extracts, and the spe-cific activities for strain Al 3 and mutants were

all about the same (Table 2). Similar values

TABLE 1. Growth properties of strains AI 3 andAIU IN on solid media containing various

combinations of lactate, acetanilide, urea, and NH4+

GrowthaCarbon source Nitrogen source

AI 3 AIU 1N

Lactate NH4+ + +Lactate Urea + +Lactate Urea + NH4+ + +Acetanilide NH4+ + +Acetanilide Urea - +Acetanilide Urea + NH4+ - +Acetanilide + Urea + +

lactate

a +, Clearly visible colonies after 48 h of incuba-tion at 37°C; -, no visible growth after 96 h ofincubation at 37°C.

TABLE 2. Amidase and urease activities of extractsofAI 3 and various AIU mutants, prepared from

cells grown in lactateINH4+ medium

Urease activity Amidase activityaStrain nmol of.NH41acii (nmol of 4-NO,-ani-Str(mo of pro4tin) line/min per mg ofper mg Of protein) protein)

AI 3 23.8 596AIU iON 29.3 866AIU 11N 21.4 128AIU 12N 23.8 123AIU 13N 28.8 134AIU 14N 11.9 173AIU 15N 15.3 151AIU 16N 32.6 146AIU 17N 36.5 766

a Amidase activities were calculated by using E40)= 11,500 for 4-NO2-aniline.

(not shown) were obtained with strain L 10, theparent of strain Al 3 (producing wild-type ami-dase), and GN 8, an amidase-negative strain.Urease activity was insensitive to avidin (100gg/ml) in the assay mixture, indicating thatthe enzyme was not a biotin-dependent ureaamidohydrolase (7). The effect of growth condi-tions on the level of urease activity was inves-tigated by measuring urease activities in ex-tracts of strains Al 3 and AIU 1N grown over-night in lactate media containing various com-binations of acetanilide, urea, and NH4+ (Table3). With urea alone as the nitrogen source, thespecific urease activities were 10-fold higherthan those obtained when either NH4' or acet-anilide was also present in the medium. Sinceremoval of low-molecular-weight material fromthe extracts by passage through a column of G-25 Sephadex left the relative urease activitiesunaffected, it seemed unlikely that the de-creased urease activities were due to inhibitionof enzyme activity by contaminating NH4+, oracetanilide. The observations indicated thaturease synthesis was repressed by NH4+ andacetanilide or its metabolic products. Undernone of these growth conditions were signifi-cant differences in urease activities betweenstrains AIU 1N and Al 3 observed, so it seemedunlikely, though not impossible, that thegrowth of strain AIU 1N and the other mutantscould be explained in terms of a change in theregulation of urease synthesis.Amidase activities. The growth properties of

strain Al 3 suggested that urea might preventgrowth on Al/U medium by blocking utilizationof the acetanilide. The amidase of strain Al 3,which catalyzes the first step in acetanilidemetabolism, seemed a likely target for ureainhibition (urea had been reported as a non-competitive inhibitor of the wild-type enzyme

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380 GREGORIOU, BROWN, AND TATA

TABLE 3. Amidase and urease activities of extracts of strains AI 3 and AIU 1N prepared from cells grown inlactate medium containing various combinations of acetanilide, urea, and NH,"

activity Amidase activityCarbon source Nitrogen source Strain (nmol of NH,/min (nmol of 4 NOe anio

permgof prti line/min per mg ofper mg of Protein ) protein)

Lactate NH+ Al 3 23 596AIU 1N 18 604

Lactate Urea Al 3 199 637AIU 1N 251 709

Lactate Urea + NH;t Al 3 19 49AIU 1N 21 210

Lactate + acetanilide Urea Al 3 36 21AIU 1N 21 81

Doubling times for both strains were 102 min in lactate/urea/acetanilide medium and 93 min in allother media.

[10]). To obtain an indication whether such aninhibition occurred in strain Al 3 and was re-

lieved in the mutants through a change affect-ing amidase, the regulation of amidase synthe-sis and the physicochemical properties of ami-

dases in some of the mutants were comparedwith those of strain Al 3. Amidase activity wasmeasured by an assay developed by M. J.Smyth and P. R. Brown (unpublished), employ-ing 4-NO2-acetanilide as a substrate. Understandard conditions, the activity of the wild-type amidase was 4% that of the Al 3 amidase.Specific amidase activities of strain Al 3 and a

selection of mutants were measured in extractsprepared from cells grown in lactate/NH4- me-

dium (Table 2). For some of the mutants ami-

dase activity was three- to fourfold less than in

strain Al 3, but in others, including AIU IN,the activity was the same or slightly greater.The effects on the specific amidase activity

of the presence of different combinations ofNHR, urea, and acetanilide in the lactategrowth medium were compared for strains Al3 and AIU 1N (Table 3). A striking feature ofthe results was the low amidase activity ob-served in cells grown in the presence of acetan-ilide plus urea or of NH4- plus urea comparedwith the levels observed when urea or NH4+was present separately. Thus, for cells grown

in the presence of trea, there appeared to be a

correlation between amidase and urease activi-ties in that, under conditions in which urease

activity was repressed, amidase activity was

also low. Since neither NH,' nor urea aloneappeared to affect amidase synthesis in strainAl 3, these observations could not all be ex-

plained easily in terms of repression of enzymesynthesis. Furthermore, estimations of ami-

dase cross-reacting material in the extracts of

strain Al 3 gave values of 78 and 76 ,ug/mg ofprotein for the lactate/U/NH4+ and lactate/Ucultures, respectively, and a lower value of 40,ug/mg of protein for the lactate/U/acetanilideculture. These results indicated that the ob-served reductions in amidase levels were attrib-utable to inhibition of enzyme activity coupled,in the case of the culture grown in the presenceof acetanilide, with some repression of' enzymesynthesis. A consistent difference observed be-tween strain Al 3 and AIU 1N was that ami-dase activities of the latter strain grown in thepresence of urea but under conditions of ureaserepression were fourfold greater than thoseobtained for strain Al 3. The significance ofthese results became apparent with subsequentexperiments but pointed to amidase as theorigin of the differences between parent andmutants.Growth on amide media. For strain Al 3,

which synthesizes amidase constitutively, therange of amides that can serve as sole carbonand/or nitrogen sources for growth reflects thesubstrate specificity of the amidase. To exam-ine amidases in the mutants for changes insubstrate specificity, growth of mutants wasexamined and compared with that of strain Al3 on solid media containing different amides assole carbon and/or nitrogen sources. Growthon acetamide and Al/NH4+ media showed nodifferences, but all AIU mutants gave littleobservable growth on succinate/formamide me-dium compared with strain Al 3, which grewwell after 48 h of' incubation at 370C. Thisobservation that formamide could not serve asa nitrogen source for the mutants implied thatcatalytic activity of their amidases towardsformamide was reduced compared with that ofAl 3 amidase.

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UREA-RESISTANT MUTANTS OF P. AERUGINOSA 381

Electrophoretic mobility of AIU IN and Al3 amidases and ureases. The mobilities ofamidases from strains Al 3 and AIU 1N werecompared by the electrophoresis of extracts onCellogel at pH 6.0 and 8.5. Amidase was de-tected by staining the gel for acetamidase activ-ity. No differences in electrophoretic mobilitybetween amidases from the two strains wereobserved. On the same gel, the mobilities forureases produced by the two strains were com-pared. Urease moved more slowly than ami-dase at both pH values; there was no differencein urease mobilities between the two strains.Immunological reactions of amidases. Ex-

tracts from mutant strains were tested by dou-ble diffusion in agar gels for reaction withantiserum to the purified Al 3 amidase. Eachmutant gave a precipitin line that fused com-pletely with the line given by an extract ofstrain Al 3; no spurring was observed, so nostructural differences between amidases weredetectable by this method.Heat inactivation of amidases. Wild-type

amidase is stable at 60CC for 10 min, but Al 3amidase loses 40% of its activity under theseconditions (4). The heat stabilities of amidasesfrom several mutants were compared with thatof Al 3 amidase at 57 and 62°C by using extractsas a source of enzyme.Heat inactivation wasan apparent first-order process (Fig. 1 shows atypical semilog plot), and results comparingthe rates of inactivation are given as first-orderrate constants in Table 4. These data indicatedthat in some strains amidase was more heatlabile than in strain Al 3; however, in somemutants, including AIU IN, no difference wasdetectable. Where differences were observed,they indicated structural changes in the ami-dases; however, since these experiments were

"I4Al 3

i_\0

0 2 4 6 aMINUTES at 62 C

FIG. 1. Heat inactivation of AI 3 and AIU 14Namidases at 62°C.

TABLE 4. Rate constants for heat inactiuation ofamidases in cell extracts at 57 and 62°C

Strain

Al 3...............................AIU IN ...........................AIU 3N ...........................AIU 4N ...........................Al 3...............................AIU 11N ..........................AIU 12N ..........................AIU 14N ..........................

k (min-')

0.03 at 57C0.03 at 57C0.20 at 57C0.03 at 57TC0.04 at 62TC0.18 at 62TC0.13 at 62TC0.18 at 62°C

performed with cell extracts, the possibility ofa change in other factors in the cell affectingheat stability of the enzyme could not be ruledout.

Inhibition of amidase by urea. When thesusceptibility of the Al 3 amidase to urea inhi-bition was investigated, urea was found toinhibit activity, as Kelly and Kornberg (10)had found for the wild-type enzyme. We, how-ever, discovered that the inhibition was timedependent. Typical time courses for inhibitionof amidase activity in extracts of Al 3 and twomutants, AIU 1N and AI5U 1N, by 1 mM ureaare shown in Fig. 2. The inhibition rates weredependent on urea concentration, but it wasstriking that for a given concentration of ureaall of the mutants tested gave slower rates ofinactivation than did strain Al 3. Plots of ln(u,1uo) against time were essentially linear over10 min; so for convenience in comparing resultsfor strain Al 3 and the mutants, the inactiva-tion rates have been expressed, on the basis ofthe slope of the semilog plot, as T1,2 values,where T1,2 is the time taken to inhibit enzymeactivity by 50%. T112 values obtained for Al 3extract and a selection of mutants all at thesame protein concentrations are given in Table5. In mutant AI5U 1N, which had been selectedon AI/5U medium containing five times theurea concentration used for isolating the othermutants, amidase showed notably greater re-sistance to urea inhibition than in all the othermutants tested. In contrast the wild-type ami-dase from strain L 10 showed greater sensitiv-ity to urea inhibition than did strain Al 3amidase.Although the extracts were prepared from

cells grown in lactate/NH4+ medium in whichurease activity was repressed, it was difficultto ascertain the possible influence of the pres-ence of a low level of urease on the time courseof amidase inhibition by urea. To eliminatethis and to establish that the observed effectswere directly related to the properties of theamidases, the effect of urea on purified ami-dases from strain AI 3 and one of the mutants,

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382 GREGORIOU, BROWN, AND TATA

LAIU IN

MINUTES

FIG. 2. Progressive inhibition of AI 3, AIU IN,and AI5U 1N amidases bv 1 mM urea. A 20-,uamount of 50 mM urea was added to 1 ml of extract(0.6 mg of protein) in 0.05 M Tris (pH 7.2) equili-brated at 37°C. Portions (100 ud) were assaVed foramidase actiuity (see text). Activity is expressed aspercent activity of untreated extract assayed in thepresence of 0.09 mM u1rea.

TABLE 5. T, 2 values for urea inhibition of amidaseactiuity in seueral strains and purified AI 3 and

AIU IN amidasesa

Ti 2 (min)Extract

0.8 mM" 1.0 mM 10 mMAl 3 16.7 3.6 0.7L10 _ b 1.7 -AIU 1N - - 19.2AIU 4N 1,020 - -AI5U 1N - - 60.3

Purified Al 3 - 3.5 -

amidasePurified AIU 1N - 150amidase

aUrea concentration.b Not determined.

strain AIU IN, was investigated. The purifiedenzyme preparations that had no detectableurease activity gave the same results as theextracts had done, showing a time-dependentreaction with urea that was much slower forthe AIU IN amidase than for the Al 3 amidase.Semilog plots for urea inactivation of the puri-fied enzymes at 1 and 50 mM urea are shown

in Fig. 3, and T, 2 values are included in Table5.The observation that inhibited amidase was

present in extracts prepared from cells grownin lactate/U/NH4+ and lactate/U/acetanilidemedia suggested that inhibition of amidaseactivity by urea was not readily reversible.This was tested by measuring the rate of regen-eration of activity from urea-inactivated en-zyme. Al 3 amidase was inactivated by incubat-ing at 37°C with 2 mM [14C]urea for 1 h. The14C-labeled inactive enzyme was separated fromfree urea on a column of G-25 Sephadex equili-brated with 0.05 M Tris buffer (pH 7.2) andrun at 4°C. At this temperature, the inactiveenzyme was stable, regaining no activity after48 h; at 37°C, activity was slowly regained,reaching 100% of the control value after 30 h.A detailed account of urea inhibition will bepublished elsewhere (M. Gregoriou and P. R.Brown, manuscript in preparation).

Effect of pH on Al 3 and AIU amidaseactivities. Except for exhibiting a reduced rateof reaction with urea, few of the other proper-ties of the AIU IN amidase examined in ex-tracts showed significant differences from Al 3amidase; however, comparison of the pH/activ-ity profiles of the two purified enzymes pro-vided additional evidence for a structuralchange in the AIU 1N enzyme. AIU 1N ami-dase showed a steep decline in activity as thepH was reduced from 8 to 6, whereas over thesame range Al 3 amidase showed a slightincrease in activity (Fig. 4). A pH/activityprofile for the amidase of strain AI5U 1N was

L AIU 1N

SOmM UREA

FIG. 3. Semilog plot of inhibition ofpurified AI 3and AIU 1N amidases bv 1 and 50 mM urea.

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UREA-RESISTANT MUTANTS OF P. AERUGINOSA 383

0.6

0.5

'j0.4-,

-C

0.3

0.2

6.0 7.0 pH o.u 9.0

FIG. 4. pH/actiuity profile for purified AI 3 andAIU IN amidases. Assays were performed in 0.5mM 4-NO,-acetanilide buffered with 0.05 M Soren-sen phosphate (pH 6.45 to 8.20) and 0.05 M sodiumbarbitone buffer (pH 8.20 to 9.40) containing 1 mMethylenediaminetetraacetate and 1 mM mercaptoeth-anol, at 37°C.

obtained with an extract as a source of enzyme.This closely resembled the profile given by an

extract of Al 3 (which gave a result indistin-guishable from that obtained with the purifiedAl 3 amidase). This observation indicated thatan alteration in pH sensitivity of amidase was

not a necessary consequence of an alterationresulting in a reduced rate of reaction withurea.

Transduction of the amidase structuralgene of strain AIU 1N. A phage F 116-mediatedtransduction was performed to investigatewhether repair of a defective amidase struc-tural gene in a recipient by recombination withthe amidase gene of AIU 1N bestowed upon

the recombinant the ability to grow on Al/Umedium. The recipient used was the amidase-negative strain, RT lAm 21, chosen because itcarried the negative mutation in its amidasestructural gene as evidenced by its productionof material giving partial cross-reaction withantiserum to amidase; furthermore, it ex-

hibited a very low reversion rate to amidasepositive. Two transduction experiments were

done: one with AIU 1N as the donor and theother a control, with strain Al 3 as the donor.Amidase-positive transductants were selectedby plating on AI/NH4+ medium. Fifty transduc-tants from a transduction with strain Al 3 as

the donor and 25 with strain AIU 1N as thedonor were tested for their ability to grow on

Al/U medium. All of the AIU iN-derived trans-ductants grew; none of those derived fromstrain Al 3 did. To check that the properties ofthe transductants were attributable to inherit-ance of the parental amidase gene, tests were

made on the amidases produced by two Al 3-derived (Tr A120, Tr A124) and two AIU 1N-

derived transductants (Tr UlS, Tr U1T); thesetransductants were selected randomly. Ami-dase activity in extracts prepared from lactate/NH4+-grown transductants was tested for sen-sitivity to pH and to urea inhibition (Table 6).The AIU 1N amidase gave a ratio of 0.3:1 whenamidase activities at pH 6.0 and 8.0 were com-pared, differentiating it from Al 3 amidase,which gave a ratio 1.2:1. Clearly, the amidasesin the transductants have the pH sensitivitycharacteristic of the donor strain. Likewise,sensitivity to inhibition by 1 mM urea wassimilar for the donor and corresponding pair oftransductants. Thus, the sole feature distin-guishing strain AIU 1N from strain Al 3 intheir different growth properties on Al/U me-dium appeared to be located in the amidasegene, although the possibility of the involve-ment of another factor specified by a gene veryclosely linked to the amidase gene could not beexcluded by this experiment.

DISCUSSIONA plausible explanation for the inability of

strain Al 3 to grow on Al/U medium emergesfrom the results. In medium containing urea,the presence of acetanilide or NH,+ repressedurease synthesis, and under these conditions a15-fold reduction in amidase activity was ob-served (Table 3). The reduction in amidaseactivity presumably occurs through the reac-tion of the enzyme with urea. Inhibition of Al3 amidase activity in this way would, thus,probably prevent the hydrolysis of acetanilidein Al/U medium. In medium containing ureabut without acetanilide or NH4,, amidase ac-tivities of Al 3 cultures were normal, probablybecause the high level of urease activity leadsto rapid removal of urea from the medium,forestalling its inhibitory reaction with ami-dase.The only apparent difference between strains

Al 3 and AIU 1N is a structural alteration in

TABLE 6. Properties of amidases from amidase-positive transductants obtained with AI 3 and AIU

1N as donors

Amsat Inactivation by 1 mMStan Amidase activity ueStrain pH 6/pH 8 urea

Al 3 1.2 4AIU 1N 0.3 160Tr AI20a 1.3 6.9Tr A124a 1.3 19.8Tr U1Sb 0.4 195Tr U1Tb 0.4 260

a Al 3 donor.b AIU 1N donor.

VOL. 132? 1977

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384 GREGORIOU, BROWN, AND TATA

the AIU 1N amidase, which significantly re-

duces the rate of its interaction with urea. Theamidase activity of strain AIU IN in mediumcontaining urea but under conditions in whichurease activity was repressed was fourfoldhigher than the amidase activity of strain Al 3under similar conditions. This must reflect thereduced rate of interaction between the AIU1N amidase and urea, so growth of strain AIU1N on Al/U medium occurs because the strainretains sufficient active amidase on this me-

dium to catalyze acetanilide hydrolysis at an

adequate rate to support growth.That a similar explanation accounts for the

growth of all the AIU mutants on Al/U mediumhas not been formally proven; however, since

all show some evidence for the production ofaltered amidases, it does seem likely that in

every case a structural change in the amidasereducing the rate of reaction with urea hasoccurred. An alternative mechanism for sur-

vival on this medium might be through theproduction of high urease levels, and we are

screening new mutants for this property. Sincemany of the amidases of the mutant strainsexamined displayed different sets of properties,it would seem that any one of several amino

acid substitutions can fulfil the twin require-

ments of reducing affinity for urea while con-

serving catalytic activity towards acetanilide.A feature that all mutants have in common is

a reduced ability to grow on succinate/formam-ide medium, which is indirect evidence that allof them produce amidases with reduced activitytowards formamide. This was established withthe purified AIU 1N amidase for which no

formamidase activity was detectable. The asso-

ciation between reduced activity towards form-amide and reduced affinity for urea and theclose structural similarities between these twocompounds suggest that urea and formamideinteract with amidase at the same site. De-tailed studies of the reactions of the Al 3 andAIU IN amidases with urea and related com-

pounds will be published elsewhere (Gregoriouand Brown, in preparation).

ACKNOWLEDGMENTS

We are grateful to the Science Research Council for theaward of a research studentship to M.G.

LITERATURE CITED

1. Brammar, W. J., and P. H. Clarke. 1964. Induction andrepression of Pseudomonas aeruginosa amidase. J.Gen. Microbiol. 37:307.

2. Brammar, W. J., P. H. Clarke, and A. J. Skinner. 1967.Biochemical and genetic studies with regulator mu-tants of the Pseudomonas aeruginosa 8602 amidasesystem. J. Gen. Microbiol. 47:87-102.

3. Brown, J. E., P. R. Brown, and P. H. Clarke. 1969.Butyramide-utilizing mutants of Pseudomonasaeruginosa 8602 which produce an amidase with al-tered substrate specificity. J. Gen. Microbiol. 57:273-285.

4. Brown, P. R., and P. H. Clarke. 1972. Amino acidsubstitution in an amidase produced by an acetani-lide-utilizing mutant of Pseudomonas aeruginosa. J.Gen. Microbiol. 70:287-298.

5. Clarke, P. H., and R. Tata. 1973. Isolation of amidase-negative mutants of Pseudomonas aeruginosa by apositive selection method using an acetamide ana-logue. J. Gen. Microbiol. 75:231-234.

6. Davis, B. J. 1964. Disc electrophoresis. II. Method andapplication to human serum proteins. Ann. N.Y.Acad. Sci. 121:404.

7. Hodson, R. C., S. K. Williams ll, and W. R. Davidson,Jr. 1975. Metabolic control of urea catabolism inChlamydomonas reinhardi and Chlorella pyrenol-dosa. J. Bacteriol. 121:1022-1035.

8. Holloway, B. W., J. B. Egan, and M. Monk. 1960.Lysogeny in Pseudomonas aeruginosa. Aust. J. Exp.Biol. Med. Sci. 38:321-330.

9. Kelly, M., and P. H. Clarke. 1962. An inducible ami-dase produced by a strain of Pseudomonas aerugi-nosa. J. Gen. Microbiol. 27:305-316.

10. Kelly, M., and H. L. Kornberg. 1964. Purification andproperties of acyltransferases from Pseudomonasaeruginosa. Biochem. J. 93:557-566.

11. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

12. Mancini, G., A. 0. Carbonara, and J. F. Heremans.1965. Immunochemical quantitation of antigens bysingle radial immunodiffusion. Immunochemistry2:235-254.

13. Stewart, D. J. 1965. The urease activity of fluorescentpseudomonads. J. Gen. Microbiol. 41:169-174.

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