Penicillinase plasmids of Staphylococcus aureus: Restriction-deletion maps

PLASMID

2, 109- 129 (1979)

Penicillinase

Plasmids of Staphy/ococcus Deletion Maps

aureus: Restriction-

R.P. NOVICK, E. MURPHY, T. J.GRYCZAN, E. BARON,AND I. EDELMAN Department

of Plasmid Biology,

The Public Health Research Institute New York, New York 10016

of The City of New York, Inc.,

Accepted September 11, 1978 The derivation of physical-genetic maps of two Staphylococcus aureus penicillinase plasmids-pI258 [28.2 kilobases (kb)] and pII147 (32.6 kb)-is described. The maps are based on data obtained by recombination, deletion, restriction endonuclease digestion, molecular cloning, and insertional inactivation. Evidence is presented for the existence of at least three separate operons transcribed in different directions. Data are presented to show that these plasmids are closely related physically as well as genetically to several others with which they can recombine. Physical mapping studies of one recombinant have helped to pinpoint structural differences between the two parental plasmids.

This laboratory has isolated and extensively characterized a series of R plasmids from Staphylococcus aureus encoding /3lactamase production and resistance to a number of inorganic ions and erythromycin (Novick and Richmond, 1965; Novick and Roth, 1968; Peyru et al., 1969). Three of these, which may be regarded as prototypes of a very large class, have been studied in most detail. These three, ~1258, ~1524, and ~11147, have molecular weights ranging from 18to 21 x 106,share many of the same genetic markers, and are closely related by the criterion of genetic recombination (Novick, 1967a; Novick and Richmond, 1965). In this report we present comparative physical-genetic maps obtained by a combination of deletion analysis, recombination mapping, and restriction endonuclease fragmentation and insertional inactivation for two of these three plasmids and a preliminary EcoRI fragmentation pattern, for the purpose of comparison, for the third. The information obtained is then used to analyze the structure of a heterogenic plasmid recombinant and so pinpoint the sites of the recombination event by which it was formed.

MATERIALS

AND METHODS

Organisms and culture conditions. The bacterial strains (all S. aureus) and plasmids used in this study are listed in Table 1. CY broth and GL agar were formulated as in Novick and Brodsky (1972). Bacterial strains were stored in CY broth at -75°C. Cultures were grown at 37°C (or at 32°C for repA- mutants) either on GL agar or in CY broth with shaking. Concentrations of inorganic salts and antibiotics in agar medium used for scoring recombinants were as listed in Novick and Roth (1968). New plasmid deletions were generated by selection against the mad locus (Smith and Novick, 1972). Genetic tests. Transduction was with phage $11 and was carried out as described previously (Novick, 1967b). Plasmid metal ion and antibiotic resistance loci were scored as previously described (Novick and Roth, 1968). p-Lactamase inducibility was scored by staining with PNCB (Novick and Richmond, 1965) replicas on nonselective medium and on the same medium supplemented with the /3-lactamase inducer, 2(2’ - carboxyphenyl)benzoyl - 6 - amino -

109

0147-619X/79/010109-21$02.00/0 Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

110

NOVICK

ET AL.

TABLE BACTERIAL

STRAINS

1 AND PLASMIDS MOkCUh3*

weight ( x 103

Strain

Plasmid

RN1464 RN1834 RN492

pRN3037 pRN3091 pRN1033

~I258 bla-401 cadA mer-I4 osi-13 pI258 blaI443 ma-33 ermB20 repAl ~I524 b/al/

21.2”

RN1465 RN1599 RN1468 RN1472 RN1501 RN453

pRN2003 pRN2048 pRN2049 pRN2050 pRN2057 pRN3038

pII 147 blaNO0 pII14789Pfblal pII147AlCO[codA+oso] pII147AlOl[mer-+b/o] pII147AlOZ[mer-wsa] ~1258 bhI443

21.8” 21.8 Il.50 9.80 6.3” 1W

p1258A88[bla] p1258A90(mer-ranr] pI258A93[mer-rbla] pI258A94[mer+anr] pI258A97[mer+bla] pI258698[mer+blo] pI258A116[ermB] pIZSSAlZO[asa asi am] pII147[mcrI~-tblal:pI258[asa-termB], pRN4011A441bla ermB]’ p1524Ix08[EcoA::TN551]’

18’ 9.3c 1W 7.4c 11.5’ l(F 15.26 15d 24.Td 16.4d

RN2460 RN2462 RN2465 RN2466 RN2469 RN2470 RN2491 RN1853 RN25P RN741 RN47 RN53 RN647

;RN3169 pRN3172 pRN3173 pRN3176 pRN3 177 pRN3186 pRN4011 pRN4074 pRN4007 pRN4008

Plasmid genotype

pII147Il109(EcoA::TRf NaturaUy occurring

]

pRN258J

Source or derivation Novick (1974); Novick and Roth (1968) Novick (1974); Novick and Rofh (1%8) Novick (1%3); Smith and Novick (unpublished data); D. Zouzias (personal communication) (formerly PCI: rri-1) Smith and Novick (1972) (formerly pi-300) Smith and Novick (1972) (formerly pi-300) Smith and Novick (1972) (formerly pi-300) Smith and Novick (1972) (formerly pi-300) Smith and Novick (1972) (formerly @3OU) Novick and Richmond (1%5) (formerly $443); Rosh er 01. (1%9) From pRN3038, this paper From pRN3038, this paper From pRN3038, this paper From pRN3038, this paper From pRN3038, this paper From pRN3038, this paper From pRN3038, this paper Lindberg and Novick (1973) Recombinant pII147 x ~1258 (Novick, 1%7a) Ultraviolet-induced deletion Translocation of ermB from chromosome to plasmid (R. Novick ef al., 1978) Peyru e, al., 1%9

n From contoor length in the electron microscope. ’ The values origidy published for these plasmids were based on a $X174 standard molecular weight that has since been shown to be a slight underestimate. The values listed here have been corrected according to the now accepted +X174 molecular weight (Sanger er al., 1977). c From sedimentation rate in neutral sucrose. * From sum of molecular weights of endonuclease fragments as determined from relative electrophoretic mobilities in agarose. c All strains are derivatives of NTCC8325 (which has no known plasmid in its natural state) except for RN2591, which is a derivative of MS256 and RN647, which is a derivative of strain 147. This is the original host strain for plasmid ~1258 and, in addition, for a hitherto unknown tetracycline resistance plasmid, pRN258. ‘Note that square brackets are used lo denote parts of plasmids that are deleted. present in recombinants, or inserted by translocation.

Restriction endonuclease fragments are penicillanic acid (CBAP) (Leggate and Holmes, 1967). The repA allele (Wyman and designated basically as suggested by Smith Novick, 1974) was scored by staining and Nathans (1973). The full designation of any fragment includes the plasmid from CBAP replicas at 32 and 43°C. RepAcolonies were p-lactamase positive at 32”C, which it came, the enzyme(s) that produced negative at 43°C; repA+ were positive at it, and a letter. The designations of fragments involving deletions include the fragboth temperatures. Plasmid incompatibility was scored as ment(s) involved followed by the deletion described by Peyru ef al. (1969) and stability number. Cofragments are lettered according to the primary fragments from which as described by Richmond (1966). Nomenclature. Plasmid nomenclature is they were derived. Thus, pI258EcoA-XbaB as described in Novick et al. (1976). Dele- is the cofragment derived from EcoA and tions are indicated by a A. Each deletion XbaB. DNA preparations. Plasmid DNA was has a unique number, which follows the A, and the extent of the deletion is indicated prepared by a modification of existing in square brackets. Generally, the first and methods (Clewell and Helinski, 1969; last markers deleted are listed in clockwise Novick and Bouanchaud, 1971) as follows: Organisms (grown on an agar surface or in order (Fig. 6) separated by an arrow.

MAPPING

OF STAPHYLOCOCCAL

CY broth) were concentrated by centrifugation to -5 x 101O/mlin 10 mM EDTA, pH 7.0. Two volumes of acetone and 2 vol of 95% ethanol were then added and the suspension was held at 0°C for at least 10 min. Ten volumes of 0.1 M NaCl + 10 mM EDTA, pH 7.0, were then added, the suspension was held an additional lo-20 min at 0°C with occasional mixing, pelleted by centrifugation, and resuspended in 0.1 M NaCl + 50 mM EDTA, pH 7, at a cell density of -2.5 x lOlo organisms/ml. Lysostaphin (Schwarz/Mann) was then added (15 pg/ml) and the suspension was incubated for 15 min at 37°C. This enzymatic removal of the cell wall at this stage does not result in lysis. Instead, a structure remains that is microscopically recognizable as a coccus. This structure, which we refer to as an “acetone spheroplast,” is sensitive to lysis by proteolytic enzymes and detergents such as Sarkosyl or sodium dodecyl sulfate but not by osmotic shock. It is most probably a matrix of partially denatured membrane proteins, although we have no direct proof of this. Lysis at this stage is accomplished by the rapid addition of an equal volume of 2% Sarkosyl in water. The resulting viscous lysate is then “cleared” by centrifugation (49,000g for 40 min), treated with RNase Tl (1 unit/ml) + pancreatic RNase (50 p.g/ ml) for 1 h at 45°C followed by Pronase (selfdigested for 30 min at 37”C), 500 pg/ml, for 30 min at 45°C. The Pronase treatment is repeated, sodium acetate is added to a final concentration of 0.3 M, and the DNA is precipitated with 2 vol ethanol. After at least 2 h at -2o”C, the precipitate is pelleted by centrifugation, redissolved in 0.1 M NaCl + 50 mM EDTA, and subjected to one or two cycles of dye-buoyant density centrifugation in CsCl + ethidium bromide (Radloff et al., 1967). After isopropanol extraction of the ethidium bromide, the DNA is dialyzed against 10 mM NaCl + 1 mM EDTA, pH 7, and then stored at 4°C. Approximate molecular weights of the newly isolated plasmids with deletions were

PLASMIDS

111

determined by band sedimentation of radioactively labeled plasmid DNA in neutral sucrose gradients (see Novick and Bouanchaud, 1971, for labeling conditions, etc.). In a few cases, molecular weights were estimated from the sum of the molecular weights of restriction endonuclease fragments. Molecular weights of some of the plasmids were determined previously by contour length measurements in the electron microscope (Rush et al., 1969; Smith and Novick, 1972). Coliphage A DNA, obtained from Miles Laboratories, was used to generate restriction endonuclease fragments of known molecular weights. Restriction endonucleases. EcoRI, BglII, XbaI, HpaI, HindIII, XmaI, and BumHI were used in these studies. They were for the most part obtained commercially and were used with buffer systems as specified by the manufacturer. We would like to acknowledge gifts of BumHI from F. Young, XmaI from B. Weisblum, and Hind111 from D. Nathans. Electrophoretic separations were done in agarose slabs (BioRad or Seakem), usually with 0.7 or 0.8% agarose in Tris-borate buffer (Greene et al., 1974). Gels were stained with ethidium bromide (1 pg/ml in water), destained in water, and photographed either with 35mm plus-X (Kodak) or with type 665 (Polaroid), with uv transillumination and red and yellow filters (Kodak gel filters 23A and 8). Molecular weights of endonuclease fragments were estimated from their electrophoretic mobilities in comparison with published values for the molecular weights of EcoRI and Hind111 coliphage A fragments (Helling et al., 1974; Thomas and Davis, 1975; and Murray and Murray, 1975) and of the three smallest S. aureus plasmid ~1258 EcoRI fragments (Chang and Cohen, 1974). As will be seen in the various gel patterns, fragments smaller than about 1.0 kilobase (kb) are usually not visualized. In general, these were visualized on separate gels with larger amounts of DNA and higher agarose

112

NOVICK ET AL.

FIG. 1. Genetic maps of ~I258 and pII147 based on previous deletion analysis (Novick, 1967a; Smith and Novick, 1972). Groups of markers in parentheses were known to be located in the indicated regions but had not been ordered.

content (1.4%). Their molecular weights were determined by comparison with mobilities of EcoRi-Hind111 codigest fragments of coliphage A. RESULTS Genetic Maps

Figure 1 shows the genetic maps of two prototype plasmids, ~1258 and ~11147. The map of ~1258 is based on a combination of deletion and recombination mapping data some of which have previously been published (Novick, 1967a; Smith and Novick,

1972). We give here a brief summary of some of the recombinational data that have not been published and are necessary to establish the sequence of certain genes. The recombinational mapping was performed by transductional crosses between ~1258 derivatives multipy marked by ethylmethane sulfonate-induced mutations (Novick and Roth, 1968). A typical cross is diagrammed in Fig. 2. In this cross, the donor plasmid was pRN3037, the recipient, pRN3091. Transductants were doubly selected for cad+ and ermB+. As doubly resistant colonies were mixtures of recom-

repA-

FIG. 2. Mapping by recombination gradients. Heavy circles represent parental plasmid genomes. Thin solid

lines represent regions selected for and therefore obligatory. Dotted lines represent unselected regions, and possible crossovers are indicated by arrows. Data are given in the upper part of Table 2. In this figure, distances between markers are arbitrary.

MAPPING OF STAPHYLOCOCCAL

binants and incompatible heteroplasmid cells, they were purified by streaking on nonselective medium with incubation at 32°C the permissive temperature for the repA- allele. Three single colonies from each streak were picked and scored for all markers by replica plating with a 36-100~ replicator. When all three did not match, the majority class was taken as representative of the clone. In the rare cases where all three were different, the colony was discarded. Since an odd number of crossovers between a pair of circular genomes produces a heterozygous dimeric circle whereas an even number produces a homozygous monomer, it was necessary to establish which of these was the predominant outcome of interplasmid recombination. This distinction was worked out on the basis of the p-lactamase stain, since this stain per-

113

PLASMIDS

mits a visual evaluation of the presence of persistent heteroplasmid cells (i.e., products of odd number of reciprocal crossovers). As shown in Table 2, the vast majority of recombinants had one or the other of the parental p-lactamase genotypes. These are clearly homozygous since blaI+ and blaZ+ are both dominant. The few blaZ+ blaZ+ recombinants could either have been heterozygotes or the result of recombination between the blaZ and blaZ loci. These were all entirely stable for the p-lactamase phenotype-one would have expected a considerable frequency of segregation if they had been heterozygotes. Moreover, most could be shown to be homozygous for at least one other plasmid marker. For this reason, we regard the occurrence of two (or a higher even number of) crossovers as inevitable. Previous mapping studies (Novick,

TABLE 2 RECOMBINATIONAL

Selection

MAPPING

Recombinants”

Cd-Em

Cd+

Cd+Pc+

Cd+Pc+I-

Cd+Pc+IIAsa-

Cd+Pc+I+Asa-Asi+

Ab B A+B

119 104 223

53 98 151

11 98 109

0 88 88

0 44 44

Cd-Em

Em+

Em+Rep+

A B A+B

119 104 223

94 33 127

Cd-Asa

Asa+

Asa+Asi-

Em+Rep+Hg60 9 69 Asa+Asi-Em+

A B A+B

86 39 125

82 28 110

78 28 106

Cd-Asa

Cd+

Cd+Pc+

Cd+Pc+I-

A B A+B

86 39 125

39 20 59

Asa+Asi-Em+Rep+ 71 27 98

Asa+Asi-Em+Rep+Hg53 21 74

9 15 24

a Phenotypes are as in Novick et al. (1976). I, Inducibility for penicillinase. Numbers are actual numbers of recombinants scored. b A and B are results for reciprocal crosses.

114

NOVICKET AL.

1967a; Smith and Novick, 1972) had established the (circular) order, cad-bfa-asaIn this particular cross, it ermB-inc-mer. was possible to map the blal and Z loci with respect to cad and asa, the asa and asi loci with respect to bla and ermB, and the repA locus with respect to mer and ermB. The data on the frequencies of various recombinant classes are listed in Table 2. Because of the requirement for paired crossovers (circular interference), it was simplest to score these crosses on the basis of a gradient of recombination, as illustrated in Fig. 2. The selected markers are cad+ and ermB+, or cad+ and asa+. For the former, to restore monomeric plasmid circles there has to be one crossover between cad and ermB and the second between ermB and cad. Considering first the cadA-ermB crossover, which can take place in location a, b, c, d, or e, one can see that the total of Cd’ Em’ recombinants (a + b + c + d + e) is greater than the total of Cd’, PLY, Em’ (b + c + d + e) which in turn is greater than the total of Cd’, PC’, II, Em’ (c + d + e). This result establishes the order cadA-blaZ-bfal (otherwise, the Cd’I-Em’ class would have been greater than the Cd’I-Pc’Em’ class). By a similar argument, the orderasa-asi-ermB was established, and by considering the second crossover, from ermB to cad, the order ermB-repA-mer was established. We have not succeeded in obtaining mutants affecting antimony resistance, encoded by a gene that is closely linked to arsenate and arsenite on the basis of deletions (see Fig. 6). Arsenate resistance is inducible (Novick and Roth, 1%8) and for reasons discussed below we consider it highly probable that the arsenate-arsenite-antimony region is organized as an operon, one that is transcribed in the clockwise direction as the map (Fig. 1) is written. The organization and mapping of the mercury resistance locus is discussed below. The genetic map of pII147 has been determined previously by deletion and recombination mapping (Novick, 1967a; Smith

and Novick, 1972). However, for both maps, there are still a number of loci for which only approximate positions are known. These include bismuth and lead resistances (linked to cad on ~1258 by deletion mapping) and cadB of ~11147, closely linked to repA (Smith and Novick, 1972). On the pII147 map, lea and bis are closely linked to cadB but could also be duplicated, with a second set linked to cadA (Smith and Novick, 1972). The replication region, previously referred to as mc or mcr (Novick, 1967a), includes the determinant of incompatibility specificity (Novick, 1967a), the repA locus (Wyman and Novick, 1974), and, by implication, the origin of replication (ori). Restriction

Maps

Prototype plasmids. In Fig. 3a is shown the agarose gel electrophoresis patterns for EcoRI digests of the three prototype plasmids, ~1524, ~11147, and ~1258, in lanes 2, 4, and 6, respectively. In addition, in lanes 3 and 5 are shown the EcoRI patterns of derivatives of ~1524 and pII147 with insertions of Tn.551 (Em) (Pattee et al., 1977). The three prototype plasmids have two fragments in common, according to mobilities, with lengths of 6.3 and 2.1 kb, respectively. These are indicated as C and D in lane 6 and are, in fact, very probably identical for all three plasmids; each plasmid has a single BamHI site and EcoRI-BamHI codigests have revealed that this site, in each case, is very close to the center of the 2.1-kb common fragment; each plasmid has two XmaI sites and EcoRI-XmaI codigests have revealed that, in each case, both are in the common 6.3-kb fragment and at corresponding positions, about 4.8 kb apart. The results of XmaI and BamHI digestion and codigestion for pII147 are shown in Fig. 3b, lanes 7-10. The identity of this region has been confirmed by electron microscopic examination of heteroduplex molecules (E. Murphy, unpublished). The single Barn HI site, present on all

b

7 8

9 IO

II 12 13

FIG. 3(a). EcoRI patterns of prototype plasmids. Density gradient-purified supercoiled plasmid DNA was digested with EcoRI and subjected to horizontal slab gel electmphoresis in Tris-borate buffer on 0.7% agarose. Gels were run at 35 V for 16 h, stained with ethidium bromide, then destained and photographed. Lane (1) coliphage hDNA; (2) ~I524 plasmid DNA; (3) pRN4007-p1524fll08[EcoA::Tn551] (5.2-kb insertion in fragment A); (4) pII147 DNA; (5) pRN4008-PII147fl108[EcoA::Tn55Z ] (5.2-kb insertion in fragment A); (6).~1258 DNA. (b) Restriction endonuclease patterns of pII147 and derivatives. Plasmid DNA, purified by two cycles of dye-buoyant density centrifugation, was incubated with the restriction endonuclease under standard conditions, then subjected to agarose gel (0.8%) electrophoresis in ‘Iris-borate buffer. The gels were stained with ethidium bromide then destained and photographed. Lane (1) coliphage A DNA digested w;ith EcolU; (2) pII147 DNA digested with EcoRI; (3) pRN2048 (A99[bla]) with EcoRI; (4) pRN2049 (AlOO[cadA+asa]) with EcoRI; (5) pRN2050 (AlOI[mer~~lu]) with EcoRI; (6) pRN2051 (A102[mer+asa]) with EcoRI; (7) pII147 DNA digested with EcoRI; (8) pII147 DNA digested with XmaI; (9) pII147 DNA codigested with XmaI and EcoRl (incomplete digest; the two smallXmaI-EcoRI cofragments, both ~1.0 kb, are not visible on this gel); (10) pII147 DNA codigested with XmaJ and BarnHI; (11) pRN4011 DNA digested with EcoRI [arrows indicate open circular and closed circular DNA of a previously unknown tetracycline resistance plasmid (that lacks any EcoRI sites) that was present in this strain (a derivative of MS258-see Table l)]; (12) pII147 digested with EcoRI; (13) pRN4087 (A44[6la-+ermB]) digested with EcoRI.

a

123456

I23456

116

NOVICK

ET AL.

TABLE EcoRI FRAGMENTS

OF

3

~11147, ~1524, AND DERIVATIVE?

~11147 Fragments

pII147 pRN2048 pRN2049 pRN2050 pRN2051 pRN4011 pRN4074 pRN4008

pII147A99[b/a J pIIl47AlOO[cadA-wsa] pIIl47AlOl[mer~bla] pI1147AlM[mer+asa] pII147 x ~I258 recombinant pRN401 lA44[bla+ermB] pII147IMI9[E~oA::Tn55l]~

G (2.1)

B (6.3)

D (4.4)

A (7.7)

C (6.1)

F (2.5)

x

x

x

x

x

x

x

x x x x x x

x x x

x x

x

x

x x x

x x x

x x x x x x x

x

A (10.3) x pI524Il108[EcoA::Tn551]

B (9.7) x x

x*

x x x

~I524 Fragments

~1524 pRN4007

in kb)

(3.4)

E Genotype

(lengths

x (lengths

x

New fragment (length)

AA99 (7.4 kb) D/CA100 (1.8) B/AA101 (1.2) G/CA102 (3.3) p1258A-pI1147C AICA44 (6. l)c A::Tn55l(CIIO9)

(12.9)

3Al4.3Bi 2,7,12 3Bi3 3814 3B/J 3Bi6 3Bill 3Bil3 3Al5

A::TnSSl(fL108)

(15.5)

3Ai2 3Al3

(10.6)*

in kb)

C (6.3)

D (2.4)

E (2.1)

x x

x 7.

x x

F (1.0) x x

’ Summary of results of gel electrophoresis as shown in Figs. 2 and 3. “x” signifies the presence of a fragment. 0 Recombinant fragment. ’ Could have a short segment of ~1258 EcoA at clockwise end (see Fig. 7). d Insertions are signified by a Greek omega followed by a serial insertion number and by an identification of the insertion nature, in square brackets.

three plasmids at a homologous location, has been chosen as the origin for kilobase coordinates of these plasmids as shown in Fig 6. pII147 and its derivatives. In Fig. 3b (lanes 2,7, and 12) is the agarose gel electrophoresis pattern for EcoRi digests of pRN2033 (a mutant of pII147), showing the seven fragments of 7.7, 6.3, 6.1, 4.4, 3.5, 2.5, and 2.1 kb, respectively. In lanes 3-6 are shown the EcoRI patterns of four pII147 derivatives with deletions of varying extents. The results of this experiment are tabulated in Table 3. From these results one can obtain an approximate idea of the overall orientation of the seven EcoRI fragments. A glance at the deletion data (Table 3) will show that fragments A and D are adjacent since they are codeleted in two cases where four of the remaining five are retained (B, E, F, and G in pRN2049, and C, E, F, and G in pRN2050). E and F are also adjacent since they are the only two intact fragments remaining in pRN205 1. G must be adjacent to E-F and C and B must be on opposite sides of the E-F-G group since B

Refer to gel pattern, Fig/lane

as to location

and

and C are deleted independently and each can be deleted without affecting G. The location of G with respect to B, C, E, and F was determined by codigestion of the wild-type plasmid with BamHI and XmaI. This treatment gave one very large fragment that did not separate from undigested DNA and two smaller ones of 4.8 and 2.0 kb, respectively (see Fig. 3b, lane 10). Since the 4%kb fragment corresponds to the distance between the two XmaI sites on the EcoB fragment, the 2.0-kb fragment must correspond to a BarnHI-XmaI cofragment . Since G has the only BumHI site, this places G unequivocally adjacent to B. Since B but not G is deleted along with A and D in pRN2050, G must be between B and E-F rather than between B and A-D. Since, as mentioned, C can be deleted without B or G and B without C or G, C must be between A-D and E-F. The G-E-F region of the plasmid could have two possible orders: G(2.1 kb) , E(3.5), F(2.5) I I

MAPPING OF STAPHYLOCOCCAL

or G(2.1) , F(2.5) , E(3.5) . 8 I This region was analyzed by partial EcoRI digestion (results not shown). Among the partial digestion products of pRN2003 one of 5.6 kb was found. No partial product of 4.6 or of 6.0 kb was detected, and so it appears more likely that the order is G-E-F than G-F-E. We regard this conclusion as somewhat uncertain, however, due to the relatively small differences in the molecular lengths of the partial digestion products. From the previous data, EcoRI fragments A and D are between B and C in either of two orders: B-A-D-C, or B-D-AC. In order to orient these two fragments, we made use of pRN4074, a deletion derived from the recombinant plasmid, pRN4011. The derivation of these two plasmids is described in detail below and is shown in Fig. 7. Note that the A44 deletion [induced by uv irradiation (Novick, 1967a)] eliminated all of the counterclockwise end of the ~1258 contribution, terminating in a region that is unequivocally derived from pII147 (see Fig. 7). Consequently, it is valid for restriction analysis of this region. As shown in Fig. 3b (lane 13) and in Table 3, it has EcoRI fragments B, D, E, F, and G plus a junction fragment of 6.1 kb (this cannot be EcoC since EcoC was replaced by ~1258 DNA as a consequence of the recombination event and, moreover, would have been deleted). Since at least part of EcoA is deleted and EcoD is intact, the order BD-A-C is unequivocally established and therefore, the overall fragmentation map is B-D-A-C-F-E-G (see Fig. 6a). Given the EcoRI fragment map of ~11147, the composition of the EcoRI junction fragments generated in the deletion mutants can now be determined. pRN2049 is deleted for fragments EcoD, A, and C. Since fragments EcoB and F are intact, the new 1.8-kb fragment must be composed of portions of fragments EcoD and C, and is therefore listed as EcoDICAlOO in Table 3. Similarly, plasmid pRN2050 is deleted for fragments

PLASMIDS

117

EcoB, D, and A. Since fragments EcoG and C are intact, the junction fragment must be composed of portions of EcoB and A and therefore is listed as EcoB/AAlOl. Plasmid pRN2051 is deleted for fragments EcoG, B, D, A, and C. Since fragments E and F are intact, the 3.3-kb junction fragment must be composed of portions of fragments EcoG and C and is thus listed as EcoGICA102. Since the BamHI site on EcoG is deleted in this plasmid, this new fragment must contain 5 1.0 kb of the EcoG fragment and ~2.3 kb of the EcoC fragment. ~1258 and its derivatives. As first demonstrated by Chang and Cohen (1974) and confirmed here, ~1258 has four EcoRI fragments, 12.8, 7.0, 6.3 and 2.1 kb, respectively (see Fig. 3). A series of deletions of this plasmid were generated by selection against the mud locus (Smith and Novick, 1972). The fragmentation patterns of several of these plus the parental plasmid after digestion with EcoRI and electrophoresis on 0.8% agarose gel are shown in Fig. 4. These results are summarized in Table 4. Several of these deletions have only the EcoA fragment plus a junction fragment (Fig. 4, lane 11). Some of these latter were found to be sensitive to BarnHI, thus placing fragments A and D in contiguity. Since fragment C has the only two XmaI sites, XmaI-BamHI codigestion was performed to localize fragment C with respect to D. The result here was similar to that seen with pII147-one very large fragment and two smaller ones of 4.8 and 2.0, respectively-thus establishing that C and D are adjacent (data not shown). This result gives the order A-B-C-D. Several of the deletions (as exemplified by A94, Fig. 4, lane 14) have retained only a single EcoRI site. These have molecular lengths of about 10 kb. As they have retained erythromycin resistance, which is on the A fragment (see below), they must be A-B or A-D composites. This was resolved by EcoRI-XbaI codigestion. XbaI digestion of ~I258 also gives rise to four fragments (Fig. 4, lane 3 and Table 4).

118

ET AL.

NOVICK

TABLE

4

RESTRICTIONENDONUCLEASEFRAGMENTSOF ~1258 AND DERIVATIVES Plasmid Digestion fragments

Length in kW

PI258

pRN3 169 (not shown)

6.3 7.0 12.8 2.1

(4A/1,5/ 7,lO)C x x x x

EcoRI C B A D New

Xbal A B C D New (kb) EcoRI-Xbol EcoC EcoB-XbaA EcoB-XbaB EcoA-XbaB XbaC XbaD EcoA-XbnA EcoD New (kb)

0 0 x 0 DlBA9Q (2.O)d

12.4 11.2 3.9 0.7

6.3 2.4 4.6 6.6 3.9 0.7 1.6 2.1

(4N3) x x x x

(4N2) x x x x x x x x

pRN3173

pRN3176

pRN3177

pRN3172

(4Al14)

(not shown)

(4A/8)

(4A/ll)

0 0 0 0 D/AA94 (10.1)

0 0 x 0 D/B897

(4A/lO) 0 0 x x AIBA98 (8.2)

AlBA.91 (9.5)

(5.8) (4Ao.5) 0 0 0 0 x x x

(4A/9) 0 0 0 x x x x

(4Ai12) 0 0 0 x x x x

EcoDIEcoBA98

(1.2)

AA116 (7.6)

(4A/13) 0 0 x

EcoBIEcoDA93 (1.9)

(not shown) x 0 0 x B/CA1 16 (10.1) (not shown)

EcoA-XboBl XboCAll6 (5.1)

(513) 0 0 0 x x 0 C/AA94 (7.0)

(515) x x 0 0 0

EcoRI-&/II EC&-BglB Bg/F EC&-BglA EcoB EcoA-&/A &ID Bg/E EcoA-BglC EcoD-BglC EcoD-BglB New (kb)

4.9 0.6 0.8 7.0 4.8 2.0 1.4 4.6 0.8 1.3

W8) x x x x x x x x x x

Hpd B D A C New (kb)

7.3 4.2 10.5 6.2

(T/12,15) x x x x

(not shown) 0 0 0 x B/AA94 (3.9)

(not shown) 0 0 0 x BIAA97 (10.9)

(501) x x x x x

(not shown) 0 0 0 0 0

(not shown) 0 0 0 0 x

1.2 4.2 0.9 7.0 2.6

(4A/4)

x x 0 x

(4.4)

6.2 0.6 12.6 2.0 1.4 5.4

EcoRW~ol EC&-HpnB HpoD EC&-HpaA ECOB EcoA-HpoA

(1.2)

(4AJ16) 0 0 x x AiBA94

&/II B F A D E C New (kb)

x x x x x x

DIBA98

0 0 x 0 D/BA93 (1.9)

(not shown)

pRN3201

0”

0 B/AA,,“,

(14.6)

(4.5)

EcoDIEcoAXboBA94

P&9)

0 0 x 0

pRN3 186

(514) 0 0 0 x x 0 C/AA98 (10.0)

A/&l6 (10.8)

(not shown) x x 0 0 AlCAll6 (11.3)

(4A/6) x 0 x BAl2:, (4.w x x 0 0 x x x x XbaBA120

(6.4)

(6.3)

MAPPING OF STAPHYLOCOCCAL

119

PLASMIDS

TABLE 4-Continued Plasmid Digestion tkagments HpoC Eco A-Hpa

EcoD

B

Length in kbb 6.2 4.0 2.1

PI258

pRN3 169

pRN3173 x x 0 EcoDIEcoAHpa AA94

x x x

New

pRN3176

pRN3177

pRN3 172

pRN3 186

pRN3201

x 7. 0 EcoBIEcoD

A97 (4.5)

(0.4) (not seen) a Presence of a particular fragment is indicated by a “x”; absence by a zero. Blank spaces indicate that no data are available. b Lengths are generally uncertain to the extent of about +O. 1 kb. c Notation in parentheses refers to tigureilane in which the corresponding gel pattern is shown. d Length in kilobases.

EcoRI-XbaI codigestion of ~1258 and these nearest to C and D produce Eco-Xba codeleted derivatives (Fig. 4) revealed that fragments whose sum cannot be less than three of the fourXba1 sites are in the EcoA 3.6 kb (see Table 4), the 12.4-kb XbaA fragment, the other in EcoB (Table 4). Since fragment must be the one that encompasses the EcoC and D fragments lack XbuI sites EcoC and D. The 11.2-kb XbuB fragment and since the XbuI sites in B and A that are must be adjacent to XbuA, encompassing

123456

A

pI 258 Eco RI

7 8910

II 1213 I4 IS I6

+

3 CB

D -w

FIG. 4. EcoRI and XbuI patterns. (A) Agarose gel electrophoresis patterns of ~1258wild type and several deletions after single and double digestion with EcoRI and XbuI. Lane (1) ~1258DNA digested with EcoRI; (2) ~1258 DNA codigested with EcoRI and XbaI; (3) ~1258 DNA digested with XbaI; (4) pRN3201 (Al2O[asu-asi-ant]) digested with EcoRI; (5) pRN3201 (A120[asa-usi-ant]) codigested with EcoRI and XbuI; (6) pRN3201 (A120[ma-d-ant]) digested with XbaI; (7) coliphage A DNA digested with EcoRI; (8) pRN3177 (A98[mer+blu]) digested with EcoRI; (9) pRN3177 (A98[mer+blu]) codigested with EcoRI and XbuI; (10) pRN3177 (A98[mer+blu]) digested with XbaI; (11) pRN3172 (A93[mer+bla]) digested with EcoRI; (12) pRN3172 (A93[mer+blu]) codigested with EcoRI and XbaI; (13) pRN3 172 (A93[mer+blu]) digested with XbuI; (14) pRN3 173 (A94[mer+usu]) digested with EcoRI; (15) pRN3173 (A94[mer-+usu]) codigested with EcoRI and XbaI; (16) pRN3173 (A94[mer-+usa]) digested withXba1. The second largest band in lanes 9, 10, 12, 13, 15, and 16 is the XbuC-D partial digestion product. This site seems to be somewhat less sensitive to the enzyme than the other XbuI sites (see Fig. 4B).

120

NOVICKET AL.

the 4.6-kb EcoB-Xba cofragment. This assignment was confirmed by an examination of pRN3201, a plasmid, containing a 4.9-kb deletion (8120) affecting the EcoAEcoB junction. This deletion was confined to XbaB (see Fig. 4 and Table 4). The two remaining XbaI fragments were oriented by means of a deletion affecting ermB only (pRN3181-see Table 4 and Fig. 6). This deletion eliminated XbaI fragments B and C so that these are therefore adjacent. The XbaI fragmentation map is thus A-BC-D (in clockwise order with respect to the EcoRI map; see Fig. 6b). This order was confirmed by examination of the four XbaI subfragments of EcoA, as generated by an EcoRI-XbaI codigest of pRN3177, a ~1258 deletion that has only EcoA plus a small junction fragment. These subfragments have molecular lengths of 6.6, 3.9, 0.7, and 1.6 kb, respectively. The first and last of these are not present in XbaI digests and so must be the two EcoRI-XbaI cofragments. An Xba-BamHI codigest of ~1258 revealed that the BamHI site (in EcoD) in XbaI fragment A, giving subfragments of 2.7 and 9.7 kb. This result could have been obtained only if the 1.6-kb Eco-Xba cofragment of EcoA was adjacent to EcoD. Deletion No. 94 when codigested with EcoRI and XbaI was found to retain the 1.6-kb cofragment but not the 6.6-kb cofragment. Therefore, A94 is an EcoAEcoD composite, retaining the EcoRI site at 27.1 kb (Fig. 6) but not that at 14.3 kb. In Fig. 6b are also shown the locations of the cleavage sites for the restriction endonuclease BgfII. Digestion with this enzyme gives rise to six fragments with molecular lengths of 12.6,6.2,5.4,2.0, 1.4, and 0.6 kb, respectively (Table 4 and Fig. 5, lanes 2, 10, and 18). Two of these are close to the ermB locus in EcoA since they are absent in deletion No. 116. This 5.2-kb deletion eliminates BglII fragments A, D, and E (Fig. 5, lane 5). Since BglD and E do not contain EcoRI sites, (Fig. 5, lane 10) they must be contained within EcoA. Since BglA is larger than 5.2 kb, D and E must be adjacent and

flanked by A. The contiguity of D and E was confirmed by an examination of several deletions (e.g. A94 and A98), which contain only BglD and E plus a junction fragment (see Table 4 and Fig. 5, lanes 3 and 4). Since A is the only BgfII fragment large enough to encompass the entire EcoB fragment (which lacks a BglII site), A must lie on the counterclockwise side of D and E, as shown in Fig. 6B. Another BglII site is in EcoD, very close to the single BamHI site, as BgfII-BamHI codigestion reduces the molecular length of BglB by 0.3 kb. These gel patterns are shown in Fig. 5 and summarized in Table 4 and Fig. 6b. The precise orientation of the BglII map with respect to the EcoRI sites was determined by an EcoRI-BglII codigest (Table 4 and Fig. 5, lane 9). The largest fragment in the codigest is EcoB, followed by three fragments with lengths ranging from 4.6 to 4.9 kb. These are all EcoRI-BglII cofragments, two of which must come from EcoA on the basis of their size. This places two of the BglII sites in EcoA at about 19.1 and 21.1 kb, respectively, as shown in Fig. 6b. BglB, as noted above, has the unique BamHI site about 0.3 kb from one of its ends (Fig. 5, lane 1). Analysis of ~1524 (E. Murphy, unpublished) has revealed two BgfII fragments that correspond precisely with ~1258 BglB and F. These two are located entirely within the ~1524 region corresponding to ~1258 EcoC and D (27.1 to 7.3 kb on the map, Fig. 6b; it will be recalled that this region is identical, by available criteria, for all three prototype plasmids). The orientation of ~1258BglB and F is therefore taken to be the same as that of the corresponding ~1524 fragments and is as shown in Fig. 6b. This is consistent with the location of the unique BamHI site in fragment B. This places BgfC between D-E and B. The orientation of BglD and E was established by an examination of plasmid pRN4007, which carries an insertion of the translocon Tn.551 derived from ~1258. This in-

MAPPING OF STAPHYLOCOCCAL

I 2 3 4 5

6 7 8 9

PLASMIDS

121

IO il 12 13 14 15 16 17 18

FIG. 5. BglII and HpaI patterns for ~1258and derivatives. Endonuclease digestion and agarose gel electrophoresis were performed as described above. For interpretation, see text and Table 4. Lane (1) ~1258codigested with BglII and BumHI; (2) ~1258digested with &HI; (3) pRN3173 (A94[rner+asa]) digested withBglI1; (4) pRN3177 (A98[mer+bla]) digested with BglII; (5) pRN3186 (A116[ermB]) digested with BglII; (6) coliphage A DNA digested with f&RI; (7) coliphage A DNA digested with EcoRI; (8) ~1258 digested with EcoRI; (9) ~1258 codigested with BglII and EcoRI; (10) ~1258digested with BglII; (11) ~1258DNA digested with EcoRI; (12) ~1258 DNA codigested with EcoRI and HpaI; (13) ~1258 DNA digested with HpaI; (14) coliphage A digested with EcoRI; (15) coliphage A digested with HpaI; (16) ~1258 DNA digested with HpoI; (17) ~1258 DNA codigested with HpaI and BglII; (18) ~1258DNA digested with BglII; (19) ~1258DNA codigested with HpaI and BamHI. Note: Light bands in lanes 3,4, and 5 are partial digestion products. BglII fragment F of PI258 does not appear in any of these patterns. TheBglII site at 27.9 kb is somewhat insensitive: ABglB-C partial digestion product is seen in lane 17 (just under BglA) and HpuB is incompletely digested (lane 16).

sertion, which seems to correspond precisely with the deletion in pRN3186 (A116) (Novick et al., 1978), carries with it the HpaI site at kb 16.9 (see below), the XbaI site at kb 20.9, and the two BglII sites at 19.1 and at 20.5 or 21.1, respectively. Digestion of pRN4007 with BglII yields a fragment that comigrates with BglD; therefore, this fragment must be the one that is carried by the translocon. This places the uncertain BglII site at 21.1 kb and so establishes the order as A-D-E-C-B-F, so completing the fragmentation map. Restriction endonuclease HpaI also recognizes four sites on pI258, giving rise to

four fragments as listed in Table 4. The locations of these sites were established by codigests as shown in Fig. 5 and by examination of two deletions (not shown). In Fig. 5, lane 17, a HpaI-BglII codigest of ~12% is shown. It can be seen (by comparison with lane 18, BglII alone) that BglD and E do not contain &a1 sites and are contained within HpaC. In Fig. 5, lane 19, is shown the BamHI-&a1 codigest of ~I258 from which it can be seen (by comparison with lane 16, HpaI alone) that the BamHI site is in HpaB giving rise to subfragments of 2.2 and 5.1 kb, respectively. Since EcoB lacks an &a1 site, HpaA must be placed

122

NOVICK ET AL,

FIG. 6. Physical-genetic maps. (a) ~11147.Reading from the outside: kilobase coordinates reading clockwise from theBamH1 site at 8 o’clock and referring to restriction sites as indicated: EcoRI, 0; BarnHI, 0; XmaI, V. Heavy outer pair of circles represents genetic map with marker symbols as indicated; EcoRI fragmentation map is next, followed by four deletions as listed in Table 3. Extent of deletions is indicated by dashed lines: terminuses of deletions with respect to the physical map are approximate. (b) ~1258.Reading from the outside: Numbers are kilobase coordinates reading clockwise from theBamH1 site at 8 o’clock. Heavy arrows represent probable direc-

MAPPING OF STAPHYLOCOCCAL

PLASMIDS

123

so as to encompass EcoB since it is the only types in having two cadmium resistance HpaI fragment large enough. Deletion No. loci, cudA and cudB (Smith and Novick, 116 eliminates the HpaA-HpaC junction at 1972). Deletions 100 (pRN2049) and 101 16.9 kb, and so these fragments must be ad- (pRN2050) establish that all or part of cudA jacent. HpaD could be between B and A or is on EcoD and that arsenate is on EcoA or between B and C. The former location was C (see Table 3, and Fig. 3b, lanes 4 and 5). established by that A94, which has only It is clear, from the results of A102, that HpuB plus a junction fragment (Table 4), the mcr (maintenance, compatibility, and and lacks HpuD (not shown). Further, the replication) region is in the G-E-F secresults with this deletion show that HpuC is tion of the plasmid since this plasmid is encompassed by EcoA which therefore has stable and replicates autonomously. The two of the four HpuI sites. Since EcoB and only identifiable genetic marker carried by EcoD lack HpuI sites, the other two must the A102 plasmid (Fig. 3b, lane 6) is the region, and so this region is be in EcoC. Of HpuA, B and D, only D is cudB-b&lea small enough to fit within EcoC; moreover, also located in the G-E-F section. InasHpuA and B both have EcoRI sites. Since much as cudA (at or near the B-D junction) HpuB is placed by itsBumH1 site, the over- is in the region that appears to be identical for all three plasmids, it is considered likely all order is A-C-B-D. that lea and bis are duplicated along with cud in pII147 since they are closely linked Orientation of Restriction and Genetic to the single cud locus in the other two plasMaps mids. The physical-genetic map as curpIZl47. pII147 plasmid pRN2048, which rently envisioned is shown in Fig. 6a. has a deletion of about 0.3 kb affecting p~1258. In their cloning experiment, Chang lactamase activity (Smith and Novick, and Cohen (1974) found that both fragments 1972), has an EcoRI pattern similar to that EcoA and EcoB of ~I258 expressed pof pRN2003 (Fig. 3, lane 3) except that the lactamase activity in Escherichiu coli. In A fragment migrates slightly faster. This dif- our hands, none of the deletions that reference in mobility corresponds to the 0.3- tained an intact EcoA fragment but lost kb deletion. Since no other fragment is af- most of B (A90,93,94, 97, or 98) expressed fected, we conclude that an essential part of any /3-lactamase activity in S. uureus. the p-lactamase locus is on fragment A. It Therefore, we conclude that the only pis also probable that an essential part of the lactamase locus on ~1258 that is active in mercury resistance determinant is on frag- S. uureus is on theEcoB fragment. Whether ment B since the two deletions that do not there is a silent p-lactamase locus on the affect fragment B do not affect mercury ~1258 EcoA fragment corresponding to the resistance (pRN2048, and pRN2049), one attributed by Chang and Cohen (1974) whereas the two that do affect mercury re- to the ~I258 EcoA fragment in the pI258sistance, A101 and 8102, both eliminate part pSClO1 chimera, pSCll2, seems at present of this fragment. No other fragment shows doubtful; this locus is not only not expressed this correlation. in S. uureus but also cannot mutate to expII147 is unique among the three proto- pression either spontaneously or following tion of transcription for the mer and asa operons. Symbols represent restriction endonuclease recognition sites: EcoRI, l ;Xma, V;HpaI, O;BglII, O;XbaI, l ;BamHI, 0. Heavyouterpairofcircles representsgenetic map with marker symbols as indicated; cud, asn, and met23 are evidently divided by EcoRI digestion as indicated. Similar information for other genes and restriction enzyme sites is not available, and so their location should be regarded as approximate. Fragmentation maps for EcoRI, Xba, BglII, and HpaII are indicated next, followed by a series of seven deletions, as listed in Table 4. In each case, the retained portion ofthe plasmid is indicated by a heavy line. The presence or absence of the BarnHI site at O/28.2 kb is indicated for the five outer deletions (see text).

124

NOVICK ETAL

nitrosoguanidine mutagenesis (Edelman and Novick, unpublished data). Unfortunately, pSCl12 is unstable in E. cofi; it has undergone one or more structural rearrangements since its isolation (S. Cohen, personal communication), and so experiments such as transforming it back into S. aureus or checking the identity of the S. aureus DNA it contains are at present impractical. All three of the prototype S. aureus penicillinase plasmids encode resistance to organic and inorganic mercury compounds (Richmond and John, 1964). Mercury resistance is inducible (Zouzias et al., 1973), and Weiss and co-workers (1977) have recently shown that at least two separate enzymes are involved (mercuric reductase and organomercurial hydrolase), that these seem to be coordinately induced, and that singlepoint mutations commonly abolish both enzyme activities. Thus, mercury resistance probably involves an operon of at least three cistrons merA (reductase), merB (hydrolase), and merR (control). We have found recently that TN551(Em) (Pattee et al., 1977) can inactivate mercury resistance by insertion (Novick ef al., 1978). These studies made use of ~16187, a naturally occurring plasmid that is apparently identical with ~1258 except that it lacks the erythromycin resistance determinant, ermB, of the latter; it has EcoRI, HpaI, and BglII fragmentation patterns, including codigests, that are indistinguishable from those of ~1258 All6 (see below and Table 4) and it was used as a target for Tn551(Em) translocation (Novick et al., 1978). Three of these insertions, all in ~16187 EcoD, inactivate resistance to organic but not inorganic mercury compounds. Therefore, the hydrolase gene (med) must be at least partly in EcoD. Since these insertions are likely to be polar, it is probable that the hydrolase gene is distal to the reductase. Two other insertions, both in ~16187EcoC, inactivate both resistances, placing the reductase gene in fragment C. This conclusion is consistent with the recent finding of Lofdahl et al. (1978) who cloned the ~1258

fragments in S. aureus and found that fragment EcoC carried inorganic mercury resistance (resistance to organomercurials was not tested). These findings in conjunction with the results of Weiss ef al. (1977) suggest that the mercury operon is transcribed in the counterclockwise direction (see Fig. 6b). Plasmid ~1258, but not the other two, encodes constitutive erythromycin resistance. Deletion No. 116 confined to the ~1258 EcoA fragment, abolishes erythromycin resistance; deletion No. 94, with part of the A fragment and not more than 1.0 kb of D, retains erythromycin resistance. Seven other deletions affecting only erythromycin resistance have been examined and all lack a 5.2-kb segment of the EcoA fragment (data not shown). In view of the recent finding that the ~1258 ermB locus is translocatable in S. aureus (Pattee et al., 1977) and that translocations of ermB (Tn551) also involve a 5.2-kb length of DNA (Novick et al., 1978), it seems likely that these deletions all involve a discrete segment of DNA, namely the translocatable element itself. On the basis of the following argument, we suggest that the arsenate-arseniteantimony resistance region, of which arsenate resistance is demonstrably inducible (Novick and Roth, 1968) is an operon transcribed in the clockwise direction (Fig. 6) and spans the EcoRI site at 14.3 kb. Deletion No. 120 is sensitive to arsenate, arsenite, and antimony and lacks a 4.9-kb segment spanning this EcoRI site (see Fig. 4). Deletion No. 90, which has lost all three resistances, Asa, Asi, and Ant, retains the EcoA-EcoB junction; therefore, at least part of the Asa-Asi-Ant region is on EcoB. Deletion No. 93, however, which has retained all three of these markers, cannot have more than 0.9 kb derived from EcoB (it has a 1.9-kb junction fragment and has retained the BamHI site). As it seems unlikely that the entire group of three genes can be located in a region that is probably shorter than 0.9 kb, we consider it likely that A90 has eliminated a promoter or other

MAPPING

OF STAPHYLOCOCCAL

positive control element rather than the entire group of three structural genes. On the basis of this consideration, we regard it as probable that the three genes are under common control and are transcribed in clockwise direction. The cloning experiments of Lofdahl et al. (1978) support this conclusion and suggest that the arsenate locus itself spans the AB junction: Clones carrying fragment A but not B expressed arsenite but not arsenate resistance; clones carrying fragment B alone expressed neither (antimony resistance was not tested). The mcr region (including ori, repA, and incf) is evidently on the A fragment beyond kb 21.1 (A116[ermB] eliminates the BglII site at bk 21.1 but does not affect the mcr function). One of the Tn55Z (Em) insertions (Novick et al., 1978) has resulted in a deletion of the entire D fragment-in particular, it has lost the D-A junction. Again, mcr functions do not seem to be affected. Finally, in view of the location of at least part of the cadA locus on pII147 EcoD, it is extremely likely that at least part of cad is on ~1258EcoB, since the two are almost certainly identical in this region. Here again, the cloning results with ~1258 fragments (Liifdahl et al., 1978)are instructive: Neither the clones carrying EcoC alone or those carrying EcoB alone express cadmium resistance, thus it appears that the cadmium resistance locus spans the ~1258 B-C junction (equivalent to pII147 EcoB-D). Two other genes in this region, his and lea, were not scored by Lofdahl et al. (1978) and so their location remains uncertain. pI258-pIIl47 recombinant. Although they are in different incompatibility groups, ~1258 and pII147 recombine readily, giving rise to what appear to be homozygous recombinants (Novick and Richmond, 1965; Novick, 1967a). However, inasmuch as there are four known genetic differences between them (see Fig. l), it seemed likely that these heterogenic recombinants, unlike homogenic ones, would be structurally different from either parent and that their study

PLASMIDS

125

might be enlightening with respect to plasmid structure and the recombination mechanism. Accordingly, we determined the EcoRI endonuclease fragmentation patterns of a number of PI258 x pII147 recombinants. A thorough analysis of these is in progress and will be reported elsewhere. For present purposes, we shall describe the EcoRI pattern for one of these, pRN4011, and for a derivative with a large deletion. The former illustrates some unusual features of the structures of the two plasmids and the latter was used to determine the orientation of pII147 fragments EcoA and D. From the genetic maps of pII147 and ~1258 and the genetic differences between them it was clear that pRN4011 could have been formed by two crossovers (see Fig. 7); since it has the bla locus of the pII147 parent and the asa-asi-ant region of the ~1258 parent, one of these must have been between bla and asa [for example, at location (b) in Fig. 71. Since it has the ermB locus of ~1258 and the inc2 and cadB loci of ~11147, the second, in this case, must have been between ermB and the cadB-inc region [for example, at location (d) in Fig. 71. Thus, we were not surprised to find that pRN4011 has six of the seven pII147 EcoRI fragments, namely, A, B, D, E, F, and G, but no recognizable pI258-specific EcoRI fragment, as shown in Fig. 3, lane 7. As noted above, the region of pII147 containing EcoB and G is probably identical to the region of ~1258 containing ~1258 EcoC and D, so that strictly speaking, the origin of this region, which was unmarked in the cross, must be regarded as unproved. However, we choose to make the simplifying assumption that only two crossovers occurred and so we list this region as being derived from ~11147. The largest fragment in pRN4011 is new and is therefore recombinant. Since all of the seven EcoRI sites in pRN4011 are derived from ~11147, this new fragment must replace pII147 fragment C. As can be seen from Fig. 7, it is possible to line up the ap-

126

NOVICK ET

AL

FIG. 7. Formation of heterogenic recombinants, pRN4011 and pRN4087. The ~1258(inner) and pII147 (outer) maps have been aligned so as to maximize known and suspected regions of homology. Regions of known or suspected nonhomology are represented by substitution or insertion loops. Symbolic notations are as in Fig. 6. In addition, crossovers are represented by arrows: (b) crossover between bla and asa involved in formation of pRN4011; (c) second crossover giving rise to an em B- recombinant; (d) second crossover involved in formation of pRN4011, an ermB+ inc2 recombinant; (f) second crossover giving rise to an ermB+ incl recombinant. Approximate terminuses of the deletion A44, giving rise to pRN4074 are at (a) and (e).

parently homologous regions by assuming the existence of substitution and insertion loops approximately as indicated. One of these loops, the ermB loop, corresponds to the translocatable element Tn551. Because of this, we have indicated separate insertion loops for ermB and for asi-ant. This configuration is supported by the isolation of other recombinants (not shown) that have the asi-ant region of ~12% but not the ermB locus. The second crossover in these could have occurred at location (c) in Fig. 7.

DISCUSSION

In this report, several types of mapping data have been combined to produce composite physical-genetic maps of two prototype staphylococcal penicillinase plasmids that have been extensively studied in this and other laboratories for many years. The map of a third member of this group, ~1524, has certain rather special features and will be presented elsewhere. Recombination mapping has been, in

MAPPING

OF STAPHYLOCOCCAL

general, rather unrewarding in plasmid genetics, and the detailed analysis of plasmid genomes has waited until the newer physical methods became available. There are two main reasons for this: circular interference and input disparities. The recombination gradient analysis described here circumvents these and makes possible precise long-distance mapping as well as the determination of order for closely spaced markers. Although our data suggest that all recombinants are the result of an even number of crossovers, our statistics do not permit an evaluation of the circular interference models of Steinberg and Stahl (1967) in which recombination products with an odd number of crossovers are (a) chewed up by a “hungry tiger,” (b) raised to the next higher even number by a “benevolent father,” or (c) simply forbidden. The deletion-restriction approach leaves certain ambiguities in the precise location of genetic markers. This is because the available deletions do not happen to have cut at all of the critical sites. A large number of additional deletions could be obtained; possibly this might result in greater detail. Probably a better approach would be that of using the fragments separately in transformation, either with or without molecular cloning. As noted, this approach has been initiated by Lofdahl et al. (1978) and is being continued in our laboratory with particular reference to the region of the plasmids containing the essential genes (the mcr region). Precision of the mapping assignments has been greatly improved by insertional inactivation; thus far, only a few insertion mutants have been isolated and the insertions have not yet been precisely localized. Nevertheless, they have permitted a preliminary analysis of the organization of the mercury resistance operon and promise to provide a good deal of additional insight into the detailed organization of the plasmid genome. Preliminary restriction analysis of recombinants formed between heterogenic plasmids by conventional recombination has

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provided a good deal of insight into the structural differences that correspond to genetic differences between these plasmids. In particular, it is clear, as shown in Fig. 7, that an extensive region, from the EcoRI site at 27.1 kb (~1258) to beyond the EcoRI site at 7.3 kb has been conserved. Since the p-lactamase regions and the arsenate locus are presumably homologous then there must be an insertion/deletion of some 5 kb between the EcoRI site at 7.3 kb and that at 14.3 kb (~1258). We have assumed that this insertion/deletion involves an en bloc segment and have placed it as shown in Fig. 7 since this location allows it to include the EcoRI site at 11.7 kb (pII147), for which there is no corresponding site on ~1258. As pII147 lacks both arsenate and antimony resistance, we considered it likely that there would be a second insertion/deletion corresponding to these two loci as indicated in Fig. 7. The location and size of the ermB loop have been determined with some precision as this region has been found to be transposable (Novick et al., 1978). Perhaps the most interesting region is that from about 24-30 kb (~11147).This part of the plasmid contains the mcr region and we have good reason to believe that the two plasmids are nonhomologous in this region: They are compatible (i.e., they have different inc determinants (Novick, 1967a) and they do not cross-complement for the repA function (Wyman and Novick, 1975)). Moreover, there are two nonhomologus EcoRI sites (as 25.7 and 28.2 (~11147))in this region as well as the cadB-&s-lea segment, present only on ~11147. We have arbitrarily shown this as an unequal substitution because that is the simplest configuration. A preliminary heteroduplex analysis (M. Schwesinger, personal communication) is roughly consistent with this diagrammatic representation. There appears to be an unequal substitution rather than an insertion/deletion at 11.7 kb (~11147); there is, however, no visible nonhomology corresponding to the arsenate-antimony region; there are two closely spaced substitution loops corre-

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sponding to the mcr-cadB region and the ermB transposon loop is as expected. These results will be reported separately. The fact that these three plasmids, one from Japan (~1258) and two from Great Britain share an identical region of over 8.4 kb, suggests a strong evolutionary relationship among them. We have recently extended these observations to an additional series of plasmids isolated in Great Britain in the 1960s (Dyke and Richmond, 1967) and have found several groups with strikingly similar EcaRI patterns. These studies will be extended to strains from other parts of the world and will be the subject of a separate report. It seems worth emphasizing, in conclusion, that these maps have been derived by a combination of no less than five different mapping strategies: recombination, deletion, restriction, insertion, and molecular cloning. These strategies complement one another nicely, any one providing information that would be much more difficult to obtain by the others. ACKNOWLEDGMENTS Expert technical assistance by Roberta Brodsky and skilled secretarial assistance of Annabel Howard and Kay Kelly are gratefully acknowledged. This work was supported in part by National Institutes of Health Grant GM 14372 and by American Cancer Society Grant VC 229 (both to R. P. N.).

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