APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1979, p. 956-964 Vol. 38, No. 50099-2240/79/11-0956/09$02.00/0

Plasmids in Escherichia coli Controlling Citrate-UtilizingAbility

NAOTAKA ISHIGURO,* CHIAKI OKA, YOSHIAKI HANZAWA, AND GIHEI SATO

Department of Veterinary Public Health, Obihiro University ofAgriculture and Veterinary Medicine,Obihiro 080, Japan

Received for publication 7 June 1979

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia colistrains isolated from pig sewage was transferred via conjugation to E. coli K-12.The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive andconcurrent with transfer of drug resistance. Weakly citrate-positive colonies werereadily obtained in conjugation experiments. Their Cit characters could betransmitted to the other E. coli strains at a similar frequency in the retransferexperiments, and the transconjugants obtained still showed same characteristicgrowth on Simmons citrate agar plates. The 19 thermosensitive plasmids confer-ring citrate utilization and drug resistance were Fi-, and 16 of these plasmidsbelonged to incompatibility group Hi. However, occasionally two conjugativeplasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization werealso obtained in the conjugation experiments, and they were Fi+ and compatiblewith 19 reference R plasmids. In the two citrate-positive E. coli strains, it wassuggested that the conjugative Cit plasmid showing Fi+ character and the morethermosensitive Hi plasmid conferring both the Cit character and drug resistancecoexisted in the strain. The characterization of citrate utilization plasmids derivedfrom pig farm sewage is discussed.

The existence of the biochemical charactersdetermined by transferable plasmids in the fam-ily Enterobacteriaceae has been recognized bymany workers (6, 9, 12). The IMViC tests areprobably the most important and useful tests fordifferentiation of coliforms into species and va-rieties. To test citrate utilization of the Entero-bacteriaceae, Simmons citrate agar is used rou-tinely. Ishiguro et al. (5) reported that 27 citrate-positive Escherichia coli variants were isolatedfrom domestic pigeons, pigs, cattle, and horses.Also, a preliminary study by Sato et al. (11)suggested that the citrate-utilizing ability in thevariants was controlled by transmissible plas-mids. Subsequently, we studied the isolation ofcitrate-positive E. coli strains from humans, var-ious domestic animals, feral birds, and environ-ments (N. Ishiguro and G. Sato, J. Hyg. Camb.,in press).

Recently, Smith et al. (14) reported that 15thermosensitive Hi plasmids derived from Sal-monella typhi strains and enterobacterial strainsfrom sewage and river water mediated citrateutilization in E. coli K-12. The citrate-utilizingability has always been found in association withthe thermosensitive H plasmid (11, 14). How-ever, few investigations have been carried out onthe relationship between citrate-utilizing abilityand R determinants in E. coli strains. We de-

tected 22 citrate-positive E. coli variants frompig farm sewage. Most of them carried conjuga-tive citrate-utilizing ability with drug resistance(Ishiguro et al., in press). The aim of this studywas to determine and to further characterizewhat relationship there is between the citrate-utilizing ability, its transfer, and R determinantsin the cells. This paper deals with the citrateutilization plasmids derived from 22 citrate-pos-itive E. coli strains isolated from pig sewagesamples in Japan.

MATERIALS AND METHODS

Bacterial strains. E. coli K-12 derivatives used inthis study are shown in Table 1. A list of the citrate-positive strains examined in this study, their drugresistance pattems, and their sources are given inTable 2. All 22 citrate-positive E. coli strains wereisolated from nine sewage samples obtained from fourpig farms during the survey. Eleven of 22 citrate-positive strains were obtained on selective media con-taining antibiotics. The details of the isolation proce-dure have been reported (Ishiguro et al., in press).These 22 strains were identified as typical E. coli by34 biochemical reactions, with the exception of citrateutilization.

Plasmids and phages. The reference R plasmidsused in this study are listed in Table 1. The RST10-1plasmid derived from E. coli KE10 was obtained afternitrosoguanidine treatment (16). The male specific

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TABLE 1. Bacteria andplasmids employed

Bacterial strain or plas- Relevant genetic erf-mid markers

ence

E. coli K-12ML1410ML1410-rifSG1

SG3

F- met nalrF- met nal' rifrRifampin-resistantmutant of HfrW1895

Rifampin-resistantmutant of E. coli921 met thr thileu lac hsdS

PlasmidRAl (A)' Tc, SmR40a (C) Su, Km, ApbR386 (FI) TcR100 (FII) Tc, Sm, Su, CmR124 (FIV) TcR144 (Ia) KmR391 (J) KmR387 (K) Sm, CmRN3 (N) Tc, Sm, CmRP4 (P) Tc, Km, ApRs-a (W) Sm, Su, Cm, KmR27 (Hi) TcRST10-1 (Hi) Sm, Su, Cm, Km 16R478 (H2) Tc, Cm, KmR446-b (M) Tc, SmR14 (0) Tc, Sm, Su, ApRtsl (T) KmR6k (X) ApR471a (L) Apa Parentheses indicate incompatibility group.

Ap, Ampicillin.

phages used were fl and f2, and phages A, T4, T7, andP1 were also used.

Media. Penassay broth (Difco) was used for con-jugation experiments. The selective media used forcitrate utilization were Simmons citrate agar (Eiken)plates (11) supplemented with methionine (50,g/ml)and either nalidixic acid (50,g/ml) or rifampin (50,g/ml) when necessary. BTB-lactose agar and deoxycho-late hydrogen sulfide-lactose agar (DHL, Eiken) were

used as the selective media for tetracycline (Tc, 25 jig/ml) and chloramphenicol (Cm, 25,g/ml), respectively(5). L broth (LB; 7, 8), LB agar, and soft agar were

used for growth and titration of phages. In this study,CaCl2 was added to LB or LB agar at a final concen-tration of 2.5 x 10' M. To examine drug resistance ofstrains or the resistance patterns of transconjugants,heart infusion agar (Eiken) was used as basal mediumfor susceptibility testing with Tc (25 Ag/ml), Cm (25,tg/ml), kanamycin (Km, 25,g/ml), and streptomycin(Sm, 12.5 ytg/ml), and Mueller-Hinton agar (Eiken)was used for sulfadimethoxine (Su, 800,ug/ml) (5).

Determination of drug resistance and conju-gation experiments. The methods used to determinedrug resistance or citrate-utilizing (Cit) ability of the22 strains were described previously (5). The methodsemployed for transfer experiments on citrate-utilizingability were those described by Sato et al. (11). Donorculture (0.1 ml) and recipient (1.0 ml) were added to4.5 ml of fresh Penassay broth. After 2 h of incubationat 37 or 25°C, 0.1-ml portions of mixture were inocu-lated on each selective medium. The transfer of drugresistance was examined similarly to transfer experi-ments of citrate-utilizing ability, using selective mediacontaining Tc and either nalidixic acid or rifampin.Simmons citrate agar plates used as selective mediawere incubated for 4 days at 37°C. Transfer frequen-cies were determined from 2-h matings at 37 or 25°C,measured as the number of transconjugants per donor.

TABLE 2. Twenty-two citrate-positive (Cit+) E. coli strains from pig sewage used in this studyTransfer Transfera

Pigfarm ~~~~~~~~~No.of of drugo irtPignar Unique strainfa Cit; E. resist- Transferred drug resist- of citrate

designa- U nno. Drug resistance pattern coti ance to ancea tiliza-tion no.is E.coition to E.stais 1.2ol coli K-12

A OH3117 Tc Su 1 - -OH3118 Tc Sm Su Cm 1 - -

B OH3119 Tc Km 1 + Tc Km +OH3120 Tc Sm Su Cm 1 + Tc Sm Su Cm +OH3121 Tc Sm Su 1 + Tc Sm Su +OH3122} Tc Km 11 + Tc Km +

OH3133 Tc Km 1OH3134 Tc Sm Su Cm 1 + Tc Sm Su Cm +

C OH3135 Tc Sm Su Cm Km 1 + Tc Sm Su Cm Km +

D OH3136 Tc Sm Su Cm Km 1 + Tc Sm Su Cm Km +OH3137 Tc Sm Su 1 + Tc +OH3138 Tc Sm Su 1 + Tc +

a Thermosensitive transfer.

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958 ISHIGURO ET AL.

The initial transfer of citrate-utilizing ability or drugresistance was performed between each citrate-posi-tive E. coli strain and the E. coli recipient strainML1410. Subsequent transfer between E. coli ML1410and ML1410-rif was made in the same way. To deter-mine transconjugant recipients and their characters,a total of 100 transconjugants from each selectionplate were patched on nonselective DHL agar platesand then replicated on antibiotic agar and Simmonscitrate agar containing methionine to determine un-selected markers.

Incompatibility tests. The compatibility propertyof the plasmids coding for the Cit ability or drugresistance was examined by the method of Datta (1).E. coli ML1410 was used as a donor strain, and arifampin-resistant mutant of this strain was used as arecipient. In each mating, colonies which appeared onthe selection plates were purified by successive single-colony isolations on the same selective medium, andthen the characters of purified colonies were tested forthe presence of incoming and resident plasmids.Transfer frequencies were determined from 2-h matingat 25°C, measured as the number of transconjugantsper donor. If both characters were present and stablein the transconjugants and were separately transfera-ble to another strain of E. coli K-12, both plasmidswere recorded as compatible.

Determination offertility inhibition and phageinhibition. The plasmid mediating drug resistance orthe Cit character under this study was introduced intoE. coli SG1, and the ability to repress production of Fpili was investigated by spot tests with phages fl andf2. If a lytic zone developed, the plasmid was regardedas Fi-, otherwise as the Fi+ type. Phage inhibitionexperiments were done as described by Taylor andGrant (15). The plasmid to be tested was transmittedto E. coli SG3 from ML1410 carrying the plasmids.The plasmid was tested for its ability to reduce boththe number of plaques and the plaque size of phagesA, T4, T7, and P1, using E. coli SG3 as an indicatorstrain.

RESULTSSources and properties of 22 citrate-pos-

itive E. coli strains. Twenty-two citrate-posi-tive E. coli strains were isolated from sewagesamples from four pig farms. Their growth onSimmons citrate agar at 370C was clearly visibleafter 2 days of incubation. As shown in Table 2,all 22 strains were resistant to antibiotics suchas Tc, Sm, Su, Cm, and Km. Except for three E.coli strains isolated from farms A and B, the Citcharacters were transmitted to E. coli ML1410.The Cit characters and drug resistance deter-minants were more efficiently transferred to E.coli ML1410 at 250C than at 370C, indicatingthat these Cit characters are as thermosensitiveas the R determinants (Ishiguro et al., in press).Therefore, subsequent mating experiments werecarried out at 250C. Transfer of neither the Citcharacter nor resistance markers of the threestrains (OH3117, OH3118, and OH3133) to E.

APPL. ENVIRON. MICROBIOL.

coli ML1410 was achieved under these condi-tions.Transfer experiments of the citrate-uti-

lizing character and drug resistance mark-ers. The transfer frequency of the Cit characteror drug resistance markers between Cit+ E. coliand E. coli ML1410 is shown in Table 3. Whenselection of drug resistance marker was madefor the Tc marker in each mating, the 19 donorstrains transferred the Cit character at frequen-cies ranging from >10-4 to <10-2 per donor cell.When selection was made for the ability to uti-lize citrate on Simmons citrate agar, the Citcharacter was transferred from each donor strainto E. coli ML1410 at equal frequencies. Thecharacters of transconjugants selected for resist-ance marker or for the Cit character are alsoshown in Table 3. The weak citrate-positivephenotype designated as Citw was obtained onreplicated Simmons citrate agar plates (Fig. 1)when selection of drug resistance marker wasmade for the Tc marker in each mating. TheseCitw colonies were picked, diluted in 0.05 mlsaline, and plated on nonselective medium. Thepurified colonies were examined for citrate uti-lization on Simmons citrate agar plates. Figure2 shows the growth features of the Citw colonieson Simmons citrate agar. No transconjugantsselected for the Cit character yielded the Citwphenotype. The significance of this phenomenonwill be discussed later in this report. In eachcase, the Cit transfer was accompanied by theacquisition of the drug resistance markers. Ofthe transconjugants selected for the Cit charac-ter, only one transconjugant that had receivedthe Cit character alone was obtained from onemating (Table 3).Table 4 shows the results of further transfer

of the Cit character and resistance markers froma Cit+ R+ transconjugant to E. coli ML1410-rif.When selection was made for the Tc marker, theCitw colonies appeared (see also Table 3). Itshould be noted that two colonies carrying theCit character alone were obtained in a furthermating (Table 4). The conjugative plasmids con-ferring the Cit character and resistance markerswere serially numbered as pOH. The transcon-jugants carrying the citrate utilization alone thatwere obtained in mating experiments (Tables 3and 4) possessed conjugative citrate-utilizingability showing thermosensitive transfer, andthese plasmids conferring the Cit character de-rived from OH3122 and OH3124 were designatedas pOH3122-1 and pOH3124-1, respectively.We were able to obtain strong citrate-positive

(Cit+) cells from weak citrate-positive pheno-types by passaging on Simmons citrate agarplates, and the resulting Cit+ clones were able to

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TABLE 3. Transfer of citrate-utilizing (Cit) characte,-and drug resistance markers to E. coli ML1410Characters of Characters of

Transferred re- Transfer Cit char Ratio of transconjugants transconjugantsfrequency transfer selected for drug selected for CitDonor m e .andCit o of drug re- quen fre- resistanceb utilization'

charkrandCiter ransistance quecit qenccharaer strs (R) (Ct+) R+/Cit+ Ci Cit Cit+ Cit+ Cit Cit

R+ R+ R+ i+ROH3119 Cit, TcKm 1 3 x 10-2 1 x 10-2 3.0 56 0 44 100 0 0OH3120 Cit, Tc Sm 1 5 x 10-2 3 x 10-3 16.7 7 0 93 100 0 0

Su CmOH3121 Cit, Tc Sm 1 2 x 10-2 1 x 10-2 2.0 100 0 0 100 0 0

SuOH3122 Cit, Tc Km 1 2 x 10-2 2 x 10-2 1.0 64 0 36 99 1 0OH3123 1 x 10-3 2 x 10-3 0.4 38 62

Cit, Tc Km 11 to to to to 0 to 100 0 0OH3132 2 x 10-2 1 x 10-2 10.0 72 28OH3134 Cit, Tc Sm 1 3 x 10-3 1 X 10-4 30.0 100 0 0 100 0 0

Su CmOH3135 Cit, Tc Sm 1 5 x 10-4 6 x 10-5 8.3 100 0 0 100 0 0

Su CmKm

OH3136 Cit, Tc Sm 1 8 x 10-3 2 x 10-3 4.0 72 0 28 100 0 0Su CmKm

OH3137 Cit, Tc 1 6 x 10-4 4 x 10-4 1.5 100 0 0 100 0 0OH3138 Cit, Tc 1 7 x 10-4 9 X 10-5 7.8 100 0 0 100 0 0a Transfer frequency was calculated as number of transconjugants per number of donor cells after a 2-h

mating at 25°C. Tc-selective media were used.b Number of colonies carrying drug resistance or the Cit character per 100 transconjugants tested. Cit+, The

strong citrate-positive phenotype colony; Citw, the weak citrate-positive phenotype colony.

*

FIG. 1. Growth of 100 transconjugant cells of E. FIG. 2. Growth of27 transconjugant cells ofE. colicoli ML1410 which were picked from Tc-selective ML1410 from spot inoculations on Simmons citrateplates onto a replicated Simmons citrate agar plate agar plate supplemented with methionine. The foursupplemented with methionine. The colonies observed cells (upper row) were picked from Cit+ phenotypeon theplate were designated as strong citrate-positive colonies onto a replicated Simmons citrate agarplate(Cit+) phenotype. The remainder showed growth on and have given good growth in 4 days at 37°C. TheSimmons citrate agar and were designated as weak remainder were obtained from Cit' phenotype colo-citrate-positive (Citw) phenotype. The plate was incu- nies; a small number of colonies is visible on eachbated at 37°C for 4 days. spot site.

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TABLE 4. Further transfer of the Cit character and drug resistance markers from E. coli ML1410 to E. coliML1410-rif at 25°C

No. of Ratio of Characters of transconju- Characters of transconju-Plasmid Ch f d°- o transfer gants selected for drug re- gants selected for the Citdesigna- aracter o do- fre- sistancea charactera

tion testedor quencytested R+/Cit+ Cit+ R+ Cit+ Citw R+ Cit+ R+ Cit+ Citw R+

pOH3119 Cit, Tc Km 1 28.6 76 0 24 100 0 0pOH3120 Cit, Tc Sm 1 0.2 100 0 0 100 0 0

Su CmpOH3121 Cit, Tc Sm 1 0.7 100 0 0 100 0 0

SupOH3122 Cit, Tc 1 150.0 38 0 62 100 0 0

KmpOH3123 Cit, Tc 1 9.0 74 0 26 100 0 0

KmpOH3124 Cit, Tc 1 26.7 46 0 54 98 2 0

KmpOH3125 0.1 76 24

Cit, TcKm 8 to to 0 to 100 0 0pOH3132 60.0 100 0pOH3134 Cit, Tc Sm 1 5.0 100 0 0 100 0 0

Su CmpOH3135 Cit, Tc Sm 1 2.9 100 0 0 100 0 0

Su CmKm

pOH3136 Cit, Tc Sm 1 0.3 100 0 0 100 0 0Su CmKm

pOH3137 Cit, Tc 1 1.1 100 0 0 100 0 0pOH3138 Cit, Tc 1 1.0 100 0 0 100 0 0

a Number of colonies carrying drug resistance or the Cit character per 100 transconjugants tested.

transfer the Cit character at a similar frequency(data not shown).Incompatibility tests. Conjugative plasmids

conferring the Cit character and drug resistancemarkers were tested for entry exclusion andincompatibility with the known Hi plasmidRST10-1 (Table 5). Since the incoming plasmidusually displaced the resident plasmid when se-lection was made for the incoming plasmid only,13 plasmids conferring citrate utilization andresistance markers were incompatible withRST10-1. Though single-colony purificationprocedures were carried out on selective platestwice in each mating, any transconjugant cloneobtained from four matings carried both theresistance markers of the incoming plasmid andthe Cit character. To examine the physical in-dependence of the Cit character or the recom-bination of the Cit character and the incomingR plasmid in the transconjugants, we studiedthe incompatibility between the reference Rplasmid (R27 or R478) and the plasmids in thetransconjugants obtained from the incompati-bility tests. The results of incompatibility testsare shown in Table 6. The phenotypes of strainscarrying both the resident plasmid and the Citcharacter were designated as RST10-1/Cit3122,RST10-1/Cit3124, RST10-1/Cit3125, or RST10-

1/Cit3132. The plasmids (pOH3122-1 andpOH3124-1) conferring the Cit character alone,which were obtained in the transfer experiments,were also tested for incompatibility with thereference R plasmids (Table 6). pOH3124-1 wascompletely compatible with R27 or R478. Theresults in Table 6 show that the resident RST10-1 plasmid in the case of RST10-1/Cit3122 andRST10-1/Cit3124 is always displaced by the in-coming R plasmids; however, all colonies ob-tained in these incompatibility tests had citrate-utilizing ability. These results suggest thatCit3122 or Cit3124, coding for citrate-utilizingability, independently maintain in the cells andwere compatible with Hi R plasmid. In spite ofthe introduction of R27 into a recipient carryingRST10-1/Cit3125 or RST10-1/Cit3132, any col-onies obtained in both cases carried all resist-ance markers of incoming and resident plasmidsand the Cit character (Table 6). The transcon-jugant clones carrying R27, RST10-1, and theCit character were designated as R27/RST10-1/Cit3125 or R27/RST10-1/Cit3132. To test forthe separate existence of the two or three plas-mids, the transconjugant clone was used as adonor, and separate selection plates were usedto test for transfer of each plasmid (Table 7).The pOH3124-1 was transferred separately to E.

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TABLE 5. Entry exclusion and incompatibility between RSTJO-I (Hi) andplasmids conferring drugresistance and the Cit character

Characters of purified trans-

Recipient of Phenotype No. of re- Marker Transfer No. of conjugantsbRST1O-1 of recipient cipiente selected frequency' tested RSTl0-1/ RST1O-1 Cit+

Cit+ only only

ML1410-rif 1 Cm 5 x 10-3 20 0 20 0ML1410-rif Cit, Tc Km 1 Cm 1 x 10-6 20 0 20 0(pOH3119)

ML1410-rif Cit, Tc Sm 1 Km 1xlO7 6 0 6 0(pOH3120) Su Cm

ML1410-rif Cit,TcKm 1 Cm 1 x 10-8 3 3 0 0(pOH3122)

ML1410-rif Cit,TcKm 1 Cm 9 x 10-8 5 0 5 0(pOH3123)

ML1410-rif Cit, Tc Km 1 Cm 5 x 10- 20 18 2 0(pOH3124)

ML1410-rif Cit, Tc Km 1 Cm 2 x i07 15 -6 9 0(pOH3125)

ML1410-rif(pOH3126)

Cit,TcKm 6ML1410-rif(pOH3131)

ML1410-rif Cit, Tc Km 1(pOH3132)

ML1410-rif Cit, Tc Sm 1

(pOH3134) Su CmML1410-rif Cit, Tc 1(pOH3137)

ML1410-rif Cit, Tc 1(pOH3138)

2 x 10-7Cm to 10

8 x 10-7

Cm 3 x 10-6 20

Km 1 x 10-' 6

Cm 2 x 10-8 8

Cm 3 x 10-8 3

0 10 0

2 18 0

0 6 0

0 8 0

0 3 0

a Transfer frequency was calculated as number of transconjugants per number of donor cells after a 2-hmating at 25°C.

b Number of strains carrying drug resistance or the Cit character among the transconjugants tested.

TABLE 6. Incompatibility tests for citrate-utilizing strain

Incom- Resistance phenotype and Cit character of trans-Ing pas- Resident Marker Transfer conjugantsdmida plasmidb selected frequency'

Tc' Sm' Su' Cm' Km' Cit+R27 pOH3124- Tc 3 x 10-3 20 0 0 0 0 20

1R478 pOH3124- Tc 6 x 10-3 20 0 0 20 20 20

1R27 RST10-1/ Tc 2 x 10-6 20 0 0 0 0 20

Cit3122R27 RST10-1/ Tc 2 x 10-6 20 0 0 0 0 20

Cit3124R27 RST10-1/ Tc 3 x 10-5 20 9 9 9 9 9

Cit3125R27 RST10-1/ Tc 4 x 10-7 20 8 8 8 8 8

Cit3132a E. coli ML1410 was used as the donor strain.bE. coli ML1410-rif was used as the recipient strain.'Transfer frequency was calculated as number of transconjugants per number of donor cells after a 2-h

mating period at 25°C.d Number of transconjugants carrying drug resistance or the Cit character per 20 tested.

coli W4573 at a similar frequency. The character and the other 17 standard R plasmids tested inbeing designated as Cit3122 or Cit3124 is re- this study showed that both the Cit character ofgarded as pOH3122-1 or pOH3124-1. Further- pOH3124-1 and the drug resistance markers ofmore, the incompatibility tests with pOH3124-1 R plasmids always coexisted stably (data not

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TABLE 7. Retransfer of resistance markers or the Cit character from transconjugants (ML1410-rif) carryingstandard R plasmids and the Cit character to E. coli W4573 at 25°C

Marker Transfer No. of trans- Character ofDonor selected frequency conjugants transconjugantsno. tested

ML1410-rif(R27) Tc 6 x 10-3 10/10 TcML1410-rif(R478) Tc 2 x 10-3 10/10 Tc Cm KmML1410- Cita 5 x 10-6 10/10 Cit+rif(pOH3124-1)

ML1410-rif(R27/ Tc 2 x 10-6 10/10 TcpOH3124-1) Cit 6 x 10-5 18/18 Cit+

ML1410-rif Tc 9 x 10-6 12/12 Tc Cm Km(R478/ Cit 3 x 10-5 19/19 Cit+pOH3124-1)

ML1410-rif(R27/ Tc 6 x 10-6 12/12 Tc Sm Su CmRST10-1/ Km Cit+pOH3125) Cm 5 x 10-6 14/14 Tc Sm Su Cm

Km Cit+Cit 2 x 10-6 20/20 Tc Sm Su Cm

Km Cit+a Cit, Simmons citrate agar was used as selective medium for citrate utilization in each experiment.

shown). In contrast, retransfer experiments us-

ing R27/RST10-1/Cit3125 showed that alltransconjugant clones contained drug resistancemarkers of R27 and RST10-1 and the Cit char-acter, whether the selection was for the Tc, Cm,or Cit marker, indicating that the markers oftwo R plasmids and the Cit character had be-come genetically linked (Table 7).

Fertility inhibition and phage inhibitiontests. The plasmids coding for both drug resist-ance markers and the Cit character or the Citcharacter alone were transmitted to the Hfrstrain SG1 from a Cit+ R+ or a Cit+ strain. Thetransconjugants obtained were purified by sin-gle-colony isolation on Simmons citrate agartwice. No lysis by phage fl or f2 on strain SG1(pOH3122-1 or pOH3124-1) having the Cit char-acter alone was found, indicating that the Ficharacter of pOH3122-1 or pOH3124-1 was Fi+(Table 8). However, the plasmids conferring theresistance marker and the Cit character derivedfrom 19 citrate-positive E. coli strains were Fi-.In the Cit+ E. coli strains of OH3122 andOH3124, these results indicate that the conju-gative citrate utilization plasmid showing Fi+character and the more thermosensitive H1 plas-mid conferring both the Cit character and drugresistance coexist in the strain.Table 8 shows the relative efficiency of plating

of bacteriophages tested on a Cit+ R+ or a Cit+strain of E. coli SG3. Two Cit+ R+ (pOH3120and pOH3121) strains gave a reduced titer com-pared with the Cit- R- strains when phage T4and P1 were plated. None of the other Cit+ R+plasmids, except two plasmids derived from twostrains isolated from farm B, inhibited develop-ment of some phages. Table 8 also shows the

incompatibility group of plasmids tested in thepresent study.

DISCUSSIONIn the present study, 19 conjugative citrate-

utilizing abilities out of the 22 citrate-positive E.coli strains were always transmitted to E. coliK-12 strains together with drug resistancemarkers at a lower temperature than 37°C. Incontrast, Citw R+ derivatives of Cit+ R+ strainscould be readily isolated when selection wasmade for resistance marker on transfer experi-ments. Since we have been able to obtain Cit+cells from Citw phenotype strains by passagingon Simmons citrate agar, and since the resultingCit+ strains carried conjugative citrate-utilizingability to other E. coli strains, we therefore feelthat the Citw phenotype is not due to the resultof the deletion of citrate-utilizing plasmid. Smithet al. (14) also reported that unstable mutants(Cit++ phenotype mutants) were found in allcultures harboring plasmids mediating citrateutilization. Probably this phenomenon origi-nates in either the function or expression ofcitrate-utilizing ability in E. coli strains.

It is known that Salmonella typhimuriumpossesses four inducible transport systems fortricarboxylic acids (citrate, isocitrate, cis-aconi-tate, and tricarballylate) (3, 4), but E. coli doesnot have any tricarboxylic acid transport system(4). We have observed that the E. coli ML1410carrying citrate-utilizing ability utilizes citrate,cis-aconitate, and tricarballylate as a source ofcarbon and energy on basal agar medium (un-published data). It is not yet clear whether theutilization of tricarboxylic acids by a citrate-uti-lizing plasmid is mediated by the inducible trans-

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TABLE 8. Bacteriophage inhibition byplasmids conferring drug resistance and the Cit character orconferring the Cit character alone

Plasmid Resistance pat- No. ofRi E

Cit+ RCit-)b Incdesignation acter mids group

tested A T4 T7 P1pOH3119 Cit, Tc Km 1 - 1 1 1 1 HipOH3120 Cit, Tc Sm Su 1 - 1 10-2_10-3 1 <10-8 Hi

CmpOH3121 Cit, Tc Sm Su 1 _ 1 10-2_10-3 1 <10-8 NTcpOH3122 Cit, Tc Km 1 - 1 1 1 1 HipOH3122-1 Cit 1 + 1 1 1 1 UTdpOH3123 Cit, Tc Km 1 - 1 1 1 1 HipOH3124 Cit, Tc Km 1 - 1 1 1 1 HipOH3124-1 Cit 1 + 1 1 1 1 UT

pOH31325 Cit, Tc Km 8 - 1 1 1 1 HipOH3134 Cit, Tc Sm Su 1 - 1 1 1 1 Hi

CmpOH3135 Cit,TcSmSu 2 _ 1 1 1 1 NTpOH3136 5 CmKm

p°H3137 Cit, Tc 2 - 1 1 1 1 HiaFi, Fertility inhibition.bE. coli SG3 was used in all experiments. EOP, Efficiency of plating, measured as described by Taylor and

Grant (15).c NT, Not tested.dUT, Untypable.

port system. If the plasmid-borne ability to uti-lize citrate were mediated by the inducible trans-port system, appearance of Citw phenotypestrains might be responsible for expression ofthe inducible transport system in E. coli strains.Our studies have confirmed the correlation

between the thermosensitive Hi plasmids andthe ability to utilize citrate and have furthersuggested that the Cit characters of most of theplasmids in this study lie on the Hi plasmid, asdescribed by Smith et al. (14). However, thoughwe have been unable to obtain Cit- R+ transcon-jugants, Cit+ R- transconjugants were obtainedfrom two mating experiments. These Cit+ R-strains possessed conjugative ability, and dataobtained showed that pOH3122-1 or pOH3124-1was Fi+ and compatible with 19 reference Rplasmids of a different incompatibility group, ascompared with pOH3122 or pOH3124, whichshowed Fi- character and belonged to group Hi.In some naturally occurring Cit+ E. coli strains,there is a possibility of the existence oftwo kindsof plasmids conferring citrate utilization in thestrain. The structural gene for citrate utilizationwas located on both conjugative and nonconju-gative plasmids. Since the plasmids conferringcitrate-utilizing ability were classified into twogroups, i.e., Fi+ class plasmids and Fi- classplasmids, the citrate-utilizing ability may be en-coded on a transposable genetic element. We arecurrently investigating this phenomenon in moredetail.

Our evidence also suggests that the recombi-nant plasmid of R27, RST10-1, and the Cit char-acter is formed in E. coli K-12 strains. It isconsidered that recombination may be impor-tant, because the thermosensitive R plasmidsare found most commonly in river water andsewage (2, 13; Hanzawa et al., unpublished data).There is the potential for spread of citrate-utiliz-ing ability associating with drug resistancemarkers in waters such as rivers, dams, andsewage. Utilization of citrate is an importantcharacteristic for the identification ofthe speciesbelonging to Enterobacteriaceae, and in partic-ular, confirmation of the presence of E. coli infoods and environmental samples by the IMViCtests (10). Since the R plasmid may play animportant role in the spread ofunusual biochem-ical characters mediated by plasmids, the pres-ent findings stress the importance of R plasmidto public health or food hygiene.

ACKNOWLEDGMENTSWe are grateful to K. Mise (Department of Microbiology,

Institute of Public Health, Tokyo), for bacteriophage P1 andbacterial strain. We also thank N. Terakado (National Insti-tute of Animal Health, Ibaragi) for reference R plasmids andmany helpful suggestions.

This work was supported in part by grants no. 348071 and311201 from the Scientific Research Fund of the Ministry ofEducation, Science and Culture of Japan.

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