Compatibility and molecular properties of plasmid Rms 149 in Pseudomonas aeruginosa and Escherichia coli

PLASMID

3, l-6 (1980)

Compatibility and Molecular Properties of Plasmid Pseudomonas aeruginosa and Escherichia R. W. HEDGES*

AND

Rms 149 in co/i

G. A. JAcoavt

*DepurtmentofBacteriology, RoyalPostgraduate MedicalSchool, Du Curie Road, London WI2 OHS, England, and tMassachu.setts General Hospitnl, Boston. Massachusetts 02114 Received July 5, 1979 Rms149, the unique plasmid of incompatibility group P-6 in the Pseudomonas ueru,qinosa classification, can be conjugally transferred into Escherichin co/i by mobilization with a broad host range IncP-1 plasmid without change in its molecular properties. In the new host species, it was stably inherited and proved to be compatible with members of all incompatibility groups so far defined. We propose the definition of a new incompatibility group, IncG, for this plasmid in E. coli.

Rms149 is a plasmid conferring resistance to carbenicillin, gentamicin, spectinomycin, streptomycin, and sulfonamides (Sagai et al., 1975). It is the prototype, and only known member, of group P-6 in the Pseudomonas plasmid incompatibility classification (Sagai et al., 1976; Jacoby, 1977; Jacoby and Shapiro, 1977). Originally, Rms149 was considered to be conjugally self-transmissible between strains of Pseudomonas aeruginosa (though not to Escherichia coli), but subsequent studies have shown that the plasmid is nonconjugative, the intraspecific transfer being mediated by a generalized transducing phage (G. A. Jacoby, T. R. Korfhagen, and L. Sutton, Abstr. 18th Intersci. Conf. Antimicrob. Ag. Chemother. 1978, No. 93). Since this phage is inactive on E. coli K-12, the lack of Rms149 transfer to E. co/i does not imply an inability to replicate in this species. Recently, it has been shown that Rms149 can be mobilized for transfer to E. coli and that in the new host it can replicate so as to ensure its efficient inheritance (Hedges and Matthew, 1979). In this paper we show that Rms149 is compatible with plasmids of all the E. coli incompatibility groups, and thus is the prototype of a new group that we designate IncG. R811 was proposed as the prototype of a

group with this letter assignment (Hedges, 1974) but that nomination was subsequently withdrawn (Matthew and Hedges, 1976) so there is no conflict of designation.’ MATERIALS

AND METHODS

Bacteria. Pseudomonas aeruginosa: PU21, i/v, ieu, str, rf, R plasmid free (Jacoby, 1974). Escherichia cofi K12: 553 met, pro; C6001ac, leu, thi, thr; C600-1 NalR mutant of C600; ES-2 trpE5, rif; all plasmid free. Plasmids. For plasmids used, see Table 1. Plasmid transfer, stability, and incompatibifity . Techniques for determinations of plasmid transfer, stability, and incompatibility have been described (Datta et al., 1971; Dennison, 1972; Coetzee et al., 1972 Jacoby , 1974). Agarose gel electrophnresis. The method used for agarose gel electrophoresis was described by Korfhagen et al. (1978). ’ RX11 is not a single plasmid but a complex consisting of a small nontransmissible plasmid, determining TEM-1 &lactamase, apparently similar to those described by numerous authors (e.g., Milliken and Clowes, 1973) and a transmissible plasmid without resistance markers which has not been studied further. Since there is thus no evidence that R8ll has unique compatibility properties, the designated of this plasmid as prototype for IncG must lapse. 1

0147-619X/80/010001-06$02.00/0 Copyright 62 1980 by Academic Press, IX. All rights of reproduction in any form rc~crvcd.

HEDGES AND JACOBY TABLE I PLASMIDSSHOWN

Plasmid

Incompatibility group

RP4 pUZ8 R751 pMG5 CAM RIP64 R1162 Rms163 Rms148 FP2 SAL pSR1 RPI-1 R716

P-l P-l P-l P-2 P-2 P-3 P-4 P-5 P-7 P-8 P-9 P-10 P-11 Unclassified

RAl R57b- 1 R386 R136 ColB R124 F&c R27 R478 R144 R621a R721 TPl14 R391 R387 R446b N3T R724 R75 1 pUZ8 R3OOB Rts 1 R769 R388 R711b R48Se TP228

A-C A-C FI FII FIII FIV FV Hl H2 Iff IY 16 12 J K M N 0 P P

PlCm R946b

Y Unclassified’

TO BE COMPATIBLEWITH

Relevant phenotype”

Rms149

Reference

(A) Plasmids tested in P. aeruginosu

Q T V W Xd Xd Xd

Cb, Km, Tc Km, Tc, Hg TP Ak, Km, Su, Tm, Hg, Pma, Ter Cam, Uv, Ter Cb, Cm, Gm, Su, Tm, Hg Sm, Sir” Cm, Su, Tc Sm’ Hg, Pma Sal Gm, Km, Su, Tm, Hg, Pma Cb’ Sm, Hg (B) Plasmids tested in E. coli Su, Tc Cm, Su Tc Tc Cba Tc Lac Tc Cm, Km, Tc, Asr, Hg, Ter Cib, Km, Tc Tc Sm, TP Km Km, Hg Cm, Sm Sm, Tc Tc Cm, Sm, Su, Tc TP Km, Tc, Hg Sm, Su Km Cm, Km, Sm, Su SK TP Km su Km, Sm, Su, Tc Cm Km

Jacoby and Shapiro (1977) Hedges and Matthew (1979) Jacoby and Shapiro (1977) Jacoby and Shapiro (1977) Jacoby and Shapiro (1977) Jacoby and Shapiro (1977) Jacoby and Shapiro (1977) Jacoby and Shapiro (1977) Jacoby and Shapiro (1977) Jacoby and Shapiro (1977) Jacoby and Shapiro (1977) Jacoby et al. (1978) Jacoby and Shapiro (1977) Jacoby and Shapiro (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob ef al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Taylor and Grant (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et nl. (1977) Jacob et al. (1977) Hedges and Matthew (1979) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) Jacob et al. (1977) J. Frost and H. Smith (personal communication) Jacob et al. (1977) S. Levy (personal communication)

(2Phenotypic designations follow Novick et al. (1976) with the addition of resistance to arsenate or arsenite (Asr), to phenylmercuric acetate (Pma), to tellurate or tellurite (Ter), and ability to degrade camphor (Cam).

PROPERTIES OF Rms149

Plasmid isolation and restriction enzyme digestion. Plasmid DNA was isolated by

3

MOLECULAR WT OF XEcoRI FRAGMENTS IO6

ethidium bromide-cesium chloride equilibrium density centrifugation, dialyzed, and digested with EcoRI (New England Biolabs) as described by Jacoby et al. (1978). An ecoR1 digest of phage A DNA served as a standard (Thomas and Davis, 1975). RESULTS Compatibility

Properties

in P. aeruginosa

As previously reported (Sagai et al., 1976; Jacoby, 1977), Rms149 proved compatible with representative plasmids of Pseudomonas Inc groups P-l, P-2, P-3, P-4, P-5, P-7, and P-8, justifying its assignment to a distinct Inc group, P-6. Rms149 was also compatible with plasmids belonging to more recently defined Inc groups P-9, P-10, and P11 and with unclassified plasmid R716. The FIG. 1. Agarose gel electrophoresis of EcoRI digests plasmids with which Rms149 has been of DNA purified from: A, P. aeruginosa PU21 shown to be compatible are listed in Table 1. (Rmsl49); B, E. co[i C600-1 (Rmsl49); C, bacteriophage A.

Compatibility

properties

in E. coli K12

Rms149 was transferred into E. coli K12 strain E5-2 using pUZ8 as the mobilizing plasmid (Hedges and Matthew, 1979). In all cases tested (over 50) the E. coli transconjugants contained both Rms149 and pUZ8 as separate plasmids. Transfer from these strains to C600- 1, selecting with ampicillin, produced a majority of clones carrying both plasmids but about 5% of transconjugants carried only Rms149. One of these strains was used in the studies described below. No transfer of resistance markers to E. coli or P. aeruginosa recipients was observed from C600-1 (Rms149) (transfer fre-

quency less than 10Wgper donor per hour). The plasmids listed in Table 1 were transferred into C600-1 (Rms149). In all cases the efficiency of transfer was approximately what would have been expected for transfer into a plasmid-free recipient, so that there was no indication that Rms149 determined entry exclusion toward these plasmids. Transfer of the various plasmids into C600-1 (Rms149) showed no tendency to eliminate the resident plasmid, and in all cases both plasmids were stably maintained during growth of transconjugant clones.

* Since the markers of Rms149 and Rl162 overlap, compatibility was tested by demonstrating by agarose gel electrophoresis the retention of Rl162 (molecular weight 5.5 x 10’) in transconjugants receiving Rms149. c Retention of Rmsl48 or RPl-1 in transconjugants receiving Rms149 was tested by the resistance these plasmids display toward phage B39 (Jacoby, 1977). d The status of group X is being reexamined. Present evidence suggests that it may be heterogenous, containing perhaps three truly incompatible groups (Bradley, 1977, 1978; N. Datta, unpublished; J. Frost and H. Smith, unpublished; M. Nugent, unpublished). We have, therefore, tested Rms149 against representatives of each of the groups previously confounded as group X. e A derivative of R485 carrying transposon Tn7 (Barth et al., 1976) was used in compatibility testing. ’ R946b has not been assigned to any group. It may be the prototype of a new group.

4

HEDGES TABLE

AND JACOBY

2

P. aeruginosa COORDINATION OF E. co/i AND PLASMID CLASSIFICATION: INCOMPATIBILITY GROUP DESIGNATION E. coli

P. aeruginosa

A-C F complex G H complex I complex J K M N 0

P-3 -(I

i T V W X Y -( -d

P-6

-b P-l P-4 -h P-2 P-5 P-7 P-8 P-9 P-10 P-11

” (-) indicates that transfer of plasmids between E. coli and P. aeruginosa by conjugation has not been observed (Dattaand Hedges, 1972; Jacoby, 1975,1977). However, Dunican and Tiemey (1973) reported transmission of an FII R plasmid to P. aeruginosa and Mergeay and Gerits (1978) showed that F’ plasmids (IncFI) can be transferred to a strain of P. jhorescens. * At least one N group plasmid and three W group plasmids are transmissible to P. aeruginosa (Jacoby, 1975) but are unstable. So far as they have been tested plasmids found in Pseudomonas are compatible with these plasmids (Jacoby, 1977). Since IncN or IncW plasmids have not yet been found to occur naturally in Pseudomonas, no corresponding IncP number has been assigned. c IncP-9 R plasmids have not been transmitted to E. coli by conjugation but the TOL plasmid which belongs to IncP-9 (White and Dunn, 1979) has been transferred to E. coli where it reportedly is incompatible with plasmids belonging to groups FI, P, and Q (Benson and Shapiro, 1978). d The Cb determinant of RPl-1 (Tnl) can be transferred to E. coli but exists there not on a plasmid but integrated into the chromosome (Richmond and Sykes, 1972).

Thus, Rms149 was compatible with all the plasmids tested. Molecular

Properties of Rmsl49

By agarose gel electrophoresis the estimated molecular weight of Rms149 in P. aeruginosa or in E. coli was 36 x 10fi. Digestion with EcoRI nuclease followed by electrophoresis produced four bands of molecular weights 12,8.4,4.0, and 2.0 x lo6 (Fig. 1; the 2.0 x lo6 band of track A was evident in the original photograph). The EcoRI fragmentation patterns of Rms149 from P. aeruginosa or E. coli were identical. It appears, therefore, that Rms149 exists in similar, or identical, configurations in the two hosts. DISCUSSION

Independent systems have been devised for the classification of plasmids of the Enterobacteriaceae in E. coli and for those of Pseudomonadaceae in P. ueruginosa (Bryan et al., 1973; Shahrabadi et al., 197.5; Datta, 1975; Sagai et al., 1976; Jacoby, 1977). Since many types of plasmid are incapable of transfer between bacteria of the two families (Datta and Hedges, 1972; Jacoby, 1975, 1977), it is not possible to coordinate the two classifications completely by direct tests although indirect tests, such as measurement of DNA homology, are possible (Shahrabadi et al., 1975). The results presented in this paper demonstrate that Rms149, the prototype of group P-6 in the Pseudomonas classification, is able to replicate efficiently and express its resistance determinants in E. coli and that it is compatible with all the plasmids we have tested. We have, therefore, nominated it as the prototype of incompatibility group G. Table 2 summarizes the present coordination of incompatibility groups in E. coli and P. ueruginoscr . ACKNOWLEDGMENTS We are grateful to Drs. Jennifer Frost and Henry Smith, Public Health Laboratory Service, Colindale,

5

PROPERTIES OF Rms149 London, for plasmids and unpublished information. We also thank Lorraine Sutton for expert assistance. This work was supported, in part, by Grant PCM75-03932 from the National Science Foundation. REFERENCES

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