Relationships between autonomous and integrated forms of a tetracycline resistance plasmid in Staphylococcus aureus

PLASMID2, 216224(1979)

Relationships Tetracycline

between Autonomous and Integrated Resistance Plasmid in Staphy/ococcus

Forms of a aureus

SERBAN IORD~NESCUANDMARIETTASURDEANU Institute

Cantacuzino,

Bucharest

35, Rumania

Accepted September 10, 1978 Recombinant plasmids carrying apparently the complete genome of a small staphylococcal plasmid, pT181, or of its temperature-sensitive replication mutant, pSA0301, were isolated and characterized; in these recombinants, pTl81 or pSA0301 were considered as “integrated” into the other plasmid, inasmuch as they seem to have a subsidiary role in the replication of the respective recombinant plasmids. Using these recombinants, the incompatibility relationships between integrated and autonomous forms of the same plasmid were studied. The results obtained showed that, although integrated plasmids express their incompatibility toward autonomous ones, they are not susceptible to the incompatibility manifested by an autonomous or another integrated plasmid. No differences were observed between pT181 and pSA0301 in their response to the incompatibility manifested by recombinant plasmids. The expression of the incompatibility of an integrated plasmid did not require the function of the repC gene, involved in plasmid autonomous replication. Moreover, the pT181 repC+ gene seems not to be expressed when pT181 is integrated into another plasmid in that the integrated form does not complement autonomous pSA0301 for replication at nonpermissive temperature.

“Plasmid incompatibility is the inability of two different plasmids to coexist stably in the same host cell in the absence of continued selection pressure” (Novick et al., 1976). Incompatibility is manifested not only between autonomously replicating plasmids but also between integrated and autonomous plasmids, belonging to the same incompatibility group (DeVries et al., 1975; Cabello et al., 1976;Novick and Schwesinger, 1976).Though there seemsto be no difference in the specificity of plasmid interactions in the two situations, there is, however, no definite proof that the mechanisms involved are the same (Novick and Schwesinger, 1976; Ptister et al., 1976). Plasmids carry at least a rep gene encoding a product required for their autonomous replication (Jacob et al., 1963; Wyman and Novick, 1974) as well as a determinant(s), inc, responsible for their incompatibility specificity (Novick, 1967; DeVries et al., 1975; Willetts, 1974). The two determinants 216 0147-619X/79/020216-09$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

have been shown to be in close linkage in many different plasmids (Novick, 1967; Timmis et al., 1975). However, there are still few studies on their possible functional interactions (Novick and Schwesinger, 1976). The present work was undertaken in an attempt to determine the relationships between integrated and autonomous forms of a small staphylococcal plasmid, as well as to initiate the study of possible functional interactions between the determinants responsible for autonomous replication and incompatibility properties of this plasmid. MATERIALS

AND METHODS

Organisms. The phenotypes and derivations of the plasmids used in this study are presented in Table 1 and their pedigrees in Fig. 1. Staphylococcus aureus strain NCTC 8325 and its recombination-deficient deriva-

AUTONOMOUS

AND INTEGRATED

217

PLASMIDS

TABLE 1 PLASMIDS Plasmid number

Phenotype0

pT181 pSA0301 pE194 pSA2800 pSA2400 pSA2402 pSA2403 pSA2500 pSA2502 pSA2504 pSA4500 pSA4501 pSA4502 pSA3202 pSA4802

Tc Inc3 Tc Inc3 Tsr Em Incll Tc Inc4 Cm Tc Inc3 Inc5 Incl Cm” Tc” Cm Tc Inc3 Inc5 Inc8 Tc” CmS Tc Em Inc3 Inc 11 Tc” Ems Cm’Tc” Inc3- Inc4+ IncS+ Inc8+ CmrTcs Inc3+ Inc4-IncS+ Inc8+

Derivation Naturally occurring (Iordanescu, 1976b) Tsr mutant of pT181 (Iordanescu, 1976b) Naturally occurring (Iordanescu, 1976a) Recombinant, pSA0302xpC221 (Iordanescu ef al., 1978) Recombinant, pSA2100~pT181~ Cm” mutant of pSA2400b TcS mutant of pSA2400* Recombinant, pSA2100xpSA0301* Tc” mutant of pSA25006 Cm” mutant of pSA25OOb Recombinant, pSA0301~pE194~ Tc” mutant of pSA45OW Em” mutant of pSA45006 TcS mutant of pSA3200 (Iordanescu, 1979) Inc3+Inc4- derivative of pSA3202*

a The phenotype notation is in accord with the recommendations of Novick et al. (1976). This paper.

b

tive RN981 (8325-4 his-7 recAl) (Wyman 1974), kindly supplied by Dr. R. Novick, were used as hosts for plasmids. Phage 80 ICY(Iordanescu, 1975) was used in all transduction experiments.

et al.,

Transduction

and incompatibility

tests.

The methods employed have been described elsewhere (Iordanescu, 1975, 1977). A diagram of compatibility-incompatibility relationships between the plasmids studied in this work is presented in Fig. 2. Kinetics of loss of tetracycline resistance (Tc’) plasmids from heteroplasmid strains. The method is presented elsewhere

(Iordanescu, 1979). To ensure the independent nature of the plasmids carried by such strains, a recA- host was always used. Besides, the possibility to transduce separately each plasmid from these strains to a plasmid-negative recipient was tested. This test does not provide, however, an absolutely certain proof for the physical independence of the plasmids carried by heteroplasmid strains, since the transducing phages are known to carry recombination functions that are active in plasmid recombination (Wyman et al., 1974).

Ultraviolet

irradiation

of phage lysates.

Phage lysates diluted to about lo9 plaque forming units (PFU)/ml in phage buffer (Novick, 1963) were gently shaken during irradiation with a 15 W germicidal lamp. The phage survival was estimated by the ratio of PFU after and before irradiation. Yellow, dim light was always used after mixing irradiated phage with cells. RESULTS Isolation and Properties Plasmids

of Recombinant

Plasmid pT181, encoding Tc resistance, has been shown previously to be cotransducible with pSA2100, a cointegrate plasmid-conferring streptomycin and chloramphenicol resistance (Iordanescu, 1975) and some of the cotransductants were proved to carry T&m’ recombinant plasmids; the Sm’ marker of pSA2100 was always lost in this process (Iordanescu, 1977). Such a Tc’Cm’ recombinant plasmid pSA2400, was selected for further studies. Similarly, another recombinant plasmid, pSA2500, was isolated after cotransduction of pSA2 100

218

IORDANESCU

AND SURDEANU

A.

pSA0501 ,c1;4

(SmrInc5) (CmrI”c8)

cotr. I pSA2100

pSA3202 Inc4

(CmrSm”Inc5

Inc8)

(TcsCmrSms 111~5 11~8)

plasmid plasmid

cotr. pTl81

pSA4802

with (TcrInc3)

(TcsCmrSms Inc3 Inc5

pSA2407 In;3

(TcsCmrSms Inc5 IncS)

111~8)

B pT181

(TcrInc3) NTG 1

pSA0301

Tcr111c3 plasmid

repC-

(TcrInc3 cotr. pE194

uv tr. ApSA4500

repC-1 with (EmrIncll)

I (TcrEmrInc3

Incll)

NTG

pSA4501 (TCSEflF 11x3 1~11)

d/h

pSA4502 (TcrEmS Inc3 111~11)

FIG. 1. Pedigree of plasmids. (A) Recombinants obtained between different Tc’ plasmids and pSA2100 and their derivatives. (B) pSA4500 and its derivatives. Cotr., obtained by cotransduction; UV tr., obtained by irradiation of transducing phage; NTG, obtained by mutagenesis with N-methyl-N’-nitro-N-nitrosoguanidine.

with pSA0301, a temperature-sensitive (Tsr) mutant of pT181 (IordBnescu, 1976b). No loss of pSA2400 or pSA2500 from the host cells was observed after about 12 cell generations either at 32 or 43°C. The recombinant plasmids could be transduced with phage 80pa with a frequency of about 5 x lo-VPFU, the linkage between the two resistance markers being 100%. Cm” and TcS mutants were isolated from

both recombinants (Table 1) to allow the study of their relationships with other plasmids. pSA2400 and pSA2500 were found to have the same incompatibility behavior toward pSAO501 and pC194 (Table 2), as has been reported for the parental plasmid, pSA2100 (IordSinescu, 1975). In addition, both recombinants presented also a unilateral incompatibility toward pT181 and pSA0301, being able to eliminate these plas-

AUTONOMOUS

\

AND INTEGRATED

I

-\I

pSA3200

Id

219

PLASMIDS

&iA03'11-

-I

~SA4802

I

2. Compatibility-incompatibility relationships. ( t, ) Compatible; (-++ ) unilaterally ) bilaterally incompatible; (& self-incompatible (absence of arrows, absence of data).

mids efficiently from recipients, but not to be displaced by them in crosses in the opposite direction (Table 2). In order to determine whether the entire genome of pT181 or pSA0301 was present in the recombinant plasmids, pSA2400 and pSA2500, the possibility of recovering these plasmids from the recombinant ones was TABLE 2 INCOMPATIBILITY

TESTS WITH PLASMIDS pSA2500

pSA2400 AND

Donor plasmid

Resident plasmid

pSA2400 pSA2500 pSAO501

pSAO501 pSAO501 pSA2400 pSA2500 pT181 pSA2402 pT181 pSA2504 pSA2403 pSA2502 PC194 pSA2403 PC194 pSA2502 pSA2402 pSA2504

pSA2403 pSA2502 pT181 pSA2402 pSA2504 PC194

Transductants with both markers/tested transductants O/8 O/8 518 l/7 0116 O/8 O/8 O/8 718 8/8 O/8 l/8 O/8 O/8 218 O/8

tested. For this purpose, phage preparations transducing pSA2400 or pSA2500 were ultraviolet irradiated at rather high doses (phage survival 10P5)and used in transduction to a plasmid-negative recipient, with selection for Tc resistance at 32°C. About 0.2% of the transductants obtained in these experiments were found to be Cm sensitive. All the Tc’ plasmids isolated in this way from pSA2400 were stably maintained both at 32 and 43°C and were compatible with pC194. However, most of them were still incompatible with pSAO501; only one out of eight tested Tc’ plasmids was identical with pT181 in incompatibility tests, i.e., compatible with pSA0501 but still displaced by pSA2403. On the other hand, six Tc’ plasmids isolated from pSA2500 were Tsr and compatible with pC194, and one of them was also compatible with pSAO501. On the basis of the data presented above it can be concluded that all of the three known genetic determinants tet, inc3, and rep2 of pT 181 and pSA0301 were integrated in the recombinant plasmids pSA2400 and pSA2500, respectively, and this integration seems to be very stable. Another recombinant plasmid, pSA4500, was isolated following the cotransduction of pSA0301 and pE194, a plasmid-conferring

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IORDANESCU

AND SURDEANU

inducible erythromycin (Em) resistance (Iordanescu, 1976a), to a plasmid-negative recipient. pSA4500, which seems to be a cointegrate plasmid, carried both Tc and Em resistance markers, and its maintenance in the host followed a pattern similar to pE 194: no loss at 32°C (less than 5 x 10m4per cell generation) and rate of loss around 3.5 x 1O-2 per cell generation at 43°C (Iordanescu, 1976a).Therefore, at least at the nonpermissive temperature for pSA0301 (43”C), the cointegrate replication had to be controlled by the pE 194 element. pSA4500 could be transduced by phage 8Opa with a frequency of about 6 x 10-Y PFU, with a 100%linkage of the two markers. Using Tc” and Em” mutants isolated from pSA4500 (Table l), its incompatibility with both parental plasmids was ascertained and demonstrated in each case to have a unilateral character: neither pT181 (pSA0301) nor pE194 could displace pSA4500 when it was carried by the recipient strain (Table 3). Besides, it should be mentioned that genetically marked derivatives of pSA4500 showed an apparently less stringent incompatibility toward one another than that usually seen between small S. aUreUSplasmids (see Table 3). This finding was not studied further. A Tc’Em” derivative was obtained from pSA4500 by ultraviolet irradiation of the transducing phage. The respective Tc’ plasmid proved to be Tsr and compatible with pE194. Therefore, it seems that pSA4500 TABLE

3

INCOMPATIBILITYRELATIONSHIPSBETWEEN AND ITSPARENTAL PLASMIDS

Donor plasmid

Resident plasmid

pSA450 1

pT181 pSA4502 pEl94 pSA4501 pSA4501 pSA4502

pSA4502 pT181 pE194

pSA4500

Transductants with both markers/tested transductants O/8 618 O/8 418 717 818

TABLE INCOMPATIBLITY

4

TESTS WITH

PLASMIDS

pSA4802 AND pSA3202

Donor plasmid

Resident plasmid

pSA4802

pT181 pSA2800 pT181 pSA2800 pSA4802 pSA3202 pSA4802 pSA3202

pSA3202 pT181 pSA2800

Transductants with both markers/tested transductants O/8 8/8 16116 018 818 8/8 8/8 818

also carries all the three determinants ret, inc3, and repC of pSA0301. Another Inc3+ recombinant plasmid, pSA4802, was obtained in the following manner: cotransduction of pSA2100 (Sm’Cm’ Inc5 In&) with pC221 (Cmr Inc4) and pSA0302 (Tc’ defective) (Iordanescu, 1979) gave rise to a new complex recombinant, pSA3200 (T&m’ Inc4 Inc5 Inc8 Inc3RepC+). This plasmid was shown to carry the tet and repC determinants of pT181 but not to express Inc3 incompatibility specificity (Iordanescu 1979). A Tc” mutant of pSA3200 was obtained, pSA3202$and this was in turn cotransduced with pT181 with selection for the Tc’ and Cm’ markers. Several of the Tc’ and Cm’ cotransductants were then subjected to serial subculture on nonselective media, with replica plating to check for the presence of the two markers. Ultimately, Tc”Cm’ derivatives were isolated in this manner and testing of several of these showed that all were carrying Cm’Inc3+ plasmids; pSA4802 is one of these. Further study showed that pSA4802 differed from pSA3202 not only in that it had acquired incompatibility toward pT 181 but also in that it no longer expressed Inc4 specificity, as tested with pSA2800 (Tc’ Inc4) (see Table 4). The loss of Inc4+ accompanied the acquisition of Inc3+ phenotype in two other independently isolated derivatives of pSA3202. Due to the present lack of information on the exact

AUTONOMOUS

AND INTEGRATED

221

PLASMIDS

from RecA- strains harboring also either pSA2403 (repC+) or pSA2502 (rep(F). The results of these studies (Fig. 3) showed that the rate of loss of pT181 in the presence of either recombinant plasmid was not affected by the incubation temperature. Moreover, there was no significant differences between pSA2403 and pSA2502 .concerning the rate of elimination of a coexistent pT181 plasmid. No loss of the Cm’ recombinant I I plasmids pSA2403 and pSA2502 was ob2 3 4 5 6 7 8 9 10 11 12 1 Cellgenerot1ons served in these experiments. A second set of experiments was perFIG. 3. Kinetics of loss of pT181 from strains formed to evaluate the role of repC in the SA6OO-RN981(pT181,pSA25-02),SA683-RN981(pT181, response to the Inc3 incompatibility expSA2403) at permissive (32°C) and restrictive (42°C) temperatures. Symbols: (0) SA683 at 32 and (0) pressed by the recombinant plasmids 42°C; (A) SA600 at 32 and (A) 42°C pSA2403 and pSA2502. Strains harboring each of the four possible plasmid pairs were nature of the Inc3- phenotype of pSA3202 tested for loss of Tc resistance at 32°C. It (Iordanescu, 1979) it is not possible yet to was observed (Fig. 4) that the rate of loss ascertain the process involved in pSA4802 of pSA0301, in the presence of pSA2403 or isolation. pSA4802 might have originated pSA2502, was similar to or even lower than either by deletion of the pC221 material that of pT181. Therefore, it appears that the present on pSA3202, assuming that this repC locus has little, if any effect on response material had been inserted in the region to Inc3 incompatibility, at least in this specific responsible for the Inc3 character, so in- situation. Since the rate of loss of pSA0301 in these activating it, or the Inc3+ phenotype could have been acquired by recombination be- experiments did not exceed a value of 3.5 tween pT181 and pSA3202, leading also to x 10-l per cell generation, a third aspect could be studied, namely the expression of elimination of the pC221 segment. the integrated pT181 repC gene on the reIncompatibility Relationships between Integrated and Autonomous pT181 and pSAO301 Plasmids In order to study in more detail the incompatibility behavior of integrated toward autonomous plasmids, derivatives of the RecA- strain RN981, carrying either pSA2403 or pSA2502, together with pT181 or pSA0301 were constructed. The study of the kinetics of loss, in different conditions, of the plasmids carried by these strains might provide answers to several questions. Initially, a test was performed to ascertain the role of the pT181 repC gene in the expression of Inc3 incompatibility by recombinant plasmids. Therefore, the kinetics of loss of pT181 was followed at permissive (32°C) and restrictive (42°C) temperatures

IO1

2

3

4

5

6

7

8

9

loll

12

Cell genemtlons FIG. 4. Kinetics of loss of TcR plasmids at 32°C from strains SAWRN981(pT181, pSA2502), SA683RN981(pT181, p&42403), SA685-RN981(pSA0301, pSA2403), and SA686-RN981(pSAO301), p&42502). Symbols: (0) SA683, (A) SA600; (X) SA686; (m) SA685.

222

IORDANESCU

AND SURDEANU

TABLE 5 combinant plasmid, pSA2403. The rationale of this test was as follows. The complemenRELATIONSHIPS BETWEEN pSA4501 AND pSA2403 tation of repC- (Tsr) mutants for mainteOR pSA2502 nance in the host cell at restrictive temperaTransductants with ture has been demonstrated using an Inc3Donor Resident both markers/tested repC+ derivative plasmid, pSA3202 (Iorplasmid plasmid transductants danescu , 1979). Therefore, a complementa16116 tion effect would be expected to occur also pSA4501 pSA2403 15115 pSA2502 if the repC+ gene of the integrated pT181 818 pSA4501 is fully expressed by pSA2403 in this sit- pSA2403 pSA4501 818 pSA2502 uation; if so, the loss of pSA0301 from strains carrying also pSA2403 should be not much higher at 42°C than at 32°C. When this was in the kinetics of loss of pSA0301 at restricstudied, however, the effect of a coexisting tive temperature when the coexisting pSA2403 plasmid on the rate of loss of plasmid was either pSA2403 or pSA4802 pSA0301 at restrictive temperature was (Fig. 5). These results suggest that the presfound to be quite limited, if any (Fig. 5). As ence on the integrated plasmid of Inc3 deexpected, pSA2502 did not improve the terminant is associated with lack of repC stability of pSA0301 at restrictive temper- complementation; in absence of Inc3, comature . plementation can be observed. Since, at least The apparent absence of RepC product in this plasmid system, complementation of synthesis by pSA2403 might be a conse- replication defects was observed also bequence of the integration process affecting tween autonomous, incompatible plasmids this gene or could be due to a specific con- (unpublished results), it seemslikely that the trol mechanism. In an attempt to differentiate lack of complementation observed with these alternatives, the Inc3+ plasmid repC+ Inc3+ integrated plasmids was due to pSA4802 was used. Since both pSA3202 and lack of repC+ allele expression in this sitpT181 carry functional repC+ genes, what- uation. ever the process leading to pSA4802 was, it seems rather unlikely to affect the repC Relationships between Replicons Carrying the Same Integrated gene. There was no significant differences

Plasmid

123456789 Cell gensmtions

FIG. 5. Kinetics of loss of pSA0301 at 42°C from strains SA318-RN98l(pSA0301), SA685-RN981(pSA0301, pSA2403), SA686-RN981(pSA0301, pSA2502) and SA769-RN981(pSA0301, pSA4802). Symbols: (0) SA318; (0) SA685; (X) SA686; (A) SA769.

Since pSA2400 and pSA2500 share with pSA4500 only the integrated pTl81 (pSAO301) plasmid, these recombinants were used to study the relationships between distinct replicons carrying the same integrated plasmid. The results of incompatibility tests proved the compatibility of pSA4501 with either pSA2403 or pSA2502 (Table 5). As a further test of this conclusion, the stability of plasmids in clones carrying both pSA4501 and pSA2403 or pSA2502 was studied. No significant loss of any of these plasmids was observed. DISCUSSION

The incompatibility manifested by plasmids integrated in the host genome (Dubnau

AUTONOMOUS

AND INTEGRATED

and Maas, 1%8; Novick and Schwesinger, 1976) is by necessity only unilateral, while for plasmids integrated in other extrachromosomal elements the incompatibility might be expressed by the elimination of either the integrated or autonomous plasmid. However, at least for the Tc’ plasmid used in this study, the integrated plasmids expressed their incompatibility toward the same autonomous plasmids but were not susceptible to the incompatibility of either autonomous plasmids or another integrated plasmid. The ability of integrated pSA0301 plasmid, carrying a repC- (Tsr) mutation, to manifest a quantitatively similar incompatibility toward an autonomous plasmid at permissive and restrictive temperature, suggests that the RepC function is not required for incompatibility. In fact, not even the presence of a repC gene seems to be necessary for the expression of incompatibility, as pSA2200, which was proved to lack repC, still could manifest incompatibility toward pT181 (Iordanescu, 1979). These data, correlated with the repC expression in an Inc3derivative (Iordanescu, 1979) show that either the rep or the inc determinant of pT181 may fulfill its function in the absence of the other. The independence of rep and inc determinants, both genetically and functionally, has been reported also for a staphylococcal penicillinase plasmid (Novick and Schwesinger, 1976). A difference in copy numbers, even at permissive temperature, between wild-type penicillinase plasmids and their Tsr mutants has been reported (Rush et al., 1975).Though such data are not available for pT181 and pSA0301, it seems interesting to note that these two plasmids were eliminated with a similar rate at permissive temperature by Inc3+ recombinant plasmids. There seems to be no data on the mechanisms which might be involved in the control of the expression of rep or inc plasmid determinants. The complementation of pSA0301 for maintenance in the host cell at restrictive temperature was taken as an in-

PLASMIDS

223

dicator for the expression of the repC gene of a recombinant plasmid. The difference observed in this regard between the Inc3plasmid pSA3202 and the Inc3+ plasmids, pSA2403 and pSA4802, all of which carry the repC+ gene, suggests that the Inc3+ character of the integrated plasmid prevents in some way the occurrence of the complementation effect. This could be achieved either by preventing the function of the RepC product or by repression of RepC product synthesis. The first possibility seems rather unlikely, since it would imply an ability to discriminate the RepC product of the autonomous and integrated plasmids. Moreover, since an integrated plasmid is supposed not to use its own replicative system, the repression of its rep gene might be expected. Anyway, the effect of the inc+ determinant or a nearby sequence of the integrated plasmid would be exerted only in cis, since a tram effect, affecting to the same extent the function of the repC gene of a coexisting autonomous plasmid would be expected to lead to a much higher rate of loss of this plasmid, than that observed experimentally. Therefore, the putative mechanism controlling rep function suggested by the present data, might be active only in special conditions (e.g., integrated plasmids) and would not necessarily play a role in incompatibility interactions; besides, there is yet no proof that the same determinant is involved in both situations. It should be recalled, that a complementation of plasmid replication defects by an Inc+ integrated plasmid has been previously reported for the F plasmid (Cuzin and Jacob, 1965),but a posteriori, these data are difficult to interpret (Wyman and Novick, 1974). For a full understanding of the present results, it would be important to have a direct evaluation of the replication rate of autonomous Tc’ plasmids when coexisting with Inc3+ recombinants, especially as for an integrated IncC penicillinase plasmid it was suggested that its incompatibility toward an autonomous plasmid would be rather due to

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IORDANESCU AND SURDEANU

an impairment of the segregation process, with only a slight reduction in the replication rate (Novick and Schwesinger, 1976). ACKNOWLEDGMENT The skillful technical assistance of Mary Kovary is gratefully acknowledged.

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In “Microbiology1974” (D. Schlessinger, ed.), pp, 166-170. Amer. Sot. for Microbial., Washington, D. C. DUBNAU, E., AND MAAS, W. K. (1968). Inhibition of replication of an F’lac episome in Hfr cells of Escherichia co/i. J. Bacterial. 95, 531-539. IORDXNESCU, S. (1975). Recombinant plasmid obtained from two different, compatible staphylococcal plasmids. J. Bacterial. 124, 597-601. IORDANESCU, S. (1976a). Three distinct plasmids originating in the same Staphylococcus aureus strain. Arch. Roum. Pathol. Exp. Microbial. 35, 111-118. IORDANESCU, S. (1976b). Temperature-sensitive mutant of a tetracycline resistance staphylococcal plasmid. Arch. Roum. Pathol. Exp. Microbial. 35, 257-264. IORDXNESCU,S. (1977). Relationships between cotransducible plasmids in Staphylococcus aureus. J. Bacterial. 129, 71-75. IORDANESCU,S., SURDEANU,M., DELLA LATTA, P., AND NOVICK, R. (1978). Incompatibility and molecular relationships between small staphylococcal

plasmids carrying the same resistance marker. P/asmid 1, 468-479.

IORDANESCU,S. (1979). Incompatibility-deficient derivatives of a small staphylococcal plasmid. Plusmid 2, 207-215. JACOB, F., BRENNER, S., AND CUZIN, F. (1963). On the replication of DNA in bacteria. Cold Spring Harbor

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NOVICK, R. P. (1%3). Analysis by transduction of mutations affecting penicillinase formation in Staphylococcus aureus. J. Gen. Microbial. 33, 121-136. NOVICK, R. P. (1%7). Penicillinase plasmids of Staphylococcus

aureus. Fed. Proc. 26, 29-38.

NOVICK, R. P., CLOWES,R. C., COHEN, S. N., CURTISS, R., III, DATTA, N., AND FALKOW, S. (1976). Uniform nomenclature for bacterial plasmids: A proposal. Bacterial. Rev. 40, 168- 189. NOVICK, R. P., AND SCHWESINGER,M. (1976). Independence of plasmid incompatibility and replication control functions in Staphylococcus aureus. Nature (London)

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PFISTER,A., DEVRIES,J. K., AND MAAS, W. K. (1976). Expression of a mutation affecting F incompatibility in the integrated, but not the autonomous state of F. J. Bacterial. 127, 348-353. RUSH, M., NOVICK, R., AND DELAP, R. (1975). Detection and quantitation of Staphylococcus aureus penicillinase plasmid DNA by reassociation kinetics. J. Bacterial. 124, 1417-1423. TIMMIS, K., CABELLO, F., AND COHEN, S. N. (1975). Cloning, isolation and characterization of replication regions of complex plasmid genomes. Proc. Nat. Acad. Sci. USA 72, 2242-2246.

WILLETTS, N. (1974). Mapping loci for surface exclusion and incompatibility on the F factor of Escherichia coli K12. J. Bacterial 118, 778-782. WYMAN, L., GOERING, R. V., AND NOVICK, R. P. (1974). Genetic control of chromosomal and plasmid recombination in Staphylococcus aureus. Genetics 76, 681-702. WYMAN, L., AND NOVICK, R. P. (1974). Studies on plasmid replication: IV, Complementation of replication-defective mutants by an incompatibility-deficient plasmid. Mol. Gen. Genet. 135, 149-161.