OF BACTERIOLOGY, Feb. 1992, p. 1197-1204 0021-9193/92/041197-08$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 174, No. 4

JOURNAL

Restriction Endonuclease Mapping of the H12 Incompatibility Group Plasmid R478 KENNETH F. WHELAN AND EMER COLLERAN* Department of Microbiology, University College Galway, Galway, Ireland Received 28 October 1991/Accepted 16 December 1991

A restriction map of the 272-kb IncHI2 plasmid R478 was constructed by using the enzymes ApaI, XbaI, Sail, and XhoI. The map was derived from cloned restriction fragments from R478 inserted into cosmid and plasmid vectors as well as from double-digestion analysis of R478 and R478 miniplasmids. All previously known resistance determinants were cloned from R478, and their positions were located on the restriction map. A region involved in incompatibility was cloned and mapped. The location of a previously unreported arsenite resistance gene was also determined. The genes encoding tellurite resistance, colicin B resistance, and phage inhibition were found to be associated with a 6.7-kb Sail fragment of R478.

The first reported isolation of a plasmid of the H incompatibility group was from the chloramphenicol-resistant Salmonella typhi strain responsible for the 1972 Mexican typhoid epidemic (2). IncH plasmids have since been shown to encode for multiple drug and metal resistances in bacteria of the family Enterobacteriaceae (1, 31, 36). Plasmids from this group are large, with molecular masses of up to 200 MDa (36, 37). Two major IncH plasmid groups, designated IncHI and IncHII, are now recognized (6). IncHI plasmids are temperature sensitive for conjugative transfer, having an optimum rate of transfer at 26 to 30°C (25, 36). IncHII plasmids differ from IncHI plasmids in that they are nonthermosensitive for transfer, are not repressed for pilus synthesis, and transfer at high frequencies (6). On the basis of DNA-DNA hybridization studies, the IncHI group of plasmids is further divided into three subgroups, IncHIl, IncHI2, and IncHI3 (42). Subgroups IncHIl and IncHI2 both have many plasmids, but there is only one plasmid, MIP233, in the IncHI3 subgroup (27). MIP233 has been reported to encode for pili dissimilar to those encoded for by all other IncH plasmids (5), and it has been suggested that this plasmid may need reclassification (32). (For reviews on IncH plasmids, see references 18 and 32.) Plasmids belonging to the IncHII and IncHI2 groups encode for a larger spectrum of characteristics than IncHIl plasmids. IncHI2 and IncHII plasmids inhibit the development of some DNA phages, such as lambda, T5, and T7, but not T4 or P1 (37). This phenomenon is known as bacteriophage inhibition (Phi). Another characteristic mediated by IncHI2, but not IncHIl, plasmids is resistance to colicin B (PacB). Carriage of IncHI2 plasmids by Escherichia coli strains has been shown to confer protection from the normally lethal action of colicin B (24). Within the IncH group of plasmids, this trait was originally thought to be exclusive to IncHI2 plasmids, but more recent studies have indicated that IncHII plasmids also express the PacB determinant (19). Resistance to tellurite (Ter) is commonly mediated by IncHI2 and IncHII plasmids but has not yet been shown to be encoded by plasmids of the IncHIl subgroup. The Ter determinants from the IncHI2 plasmid pMER610 (16) and the IncHII plasmid pHH1508a (39, 40) have been cloned and analyzed. *

Mercury resistance (Hgr) in IncH plasmids has also been found, but it is not confined to any particular subgroup (6, 18). The spectrum of mercury resistance varies within the IncHI2 group of plasmids. The plasmids R826 and R828 have been reported to carry broad-spectrum Hg resistance (28), while the plasmids R478 (31) and pMER610 (15) mediate narrow-spectrum Hg resistance. Restriction endonuclease maps have been generated for the 208-kb IncHII plasmid pHH1508a (44) and the 182-kb IncHIl plasmid R27 (21, 33, 34). To date, a restriction map has not been reported for a representative member of the IncHI2 subgroup. R478 is a well-documented plasmid of the IncHI2 subgroup. It was first isolated in the United States in 1969 from a clinical isolate of Serratia marcescens (13). It is temperature sensitive for transfer and encodes a variety of antibiotic and metal resistance determinants (Table 1). In this paper, we describe the construction of the first restriction map of R478 and indicate locations for several of the genes on this plasmid. MATERIALS AND METHODS

Bacterial strains, plasmids, cosmids, and media. All strains, plasmids, and cosmids used in this study are listed in Table 1. E. coli JM109 was used for plasmid cloning by using the vector pUC13. Transformants were isolated on Luria-Bertani (LB) medium containing 20 ,ug of 5-bromo-4-chloro-3indolyl-o-D-galactopyranoside per ml, 20 jig of isopropyl-PD-thiogalactopyranoside (Sigma Chemical Co. Ltd.) per ml, and 100 jig of ampicillin per ml (38). E. coli DH1 was used for all other experiments, unless otherwise stated, and all plasmid clones and deletion plasmids were ultimately transformed into this strain. The plasmid R478 was maintained in E. coli J53-2. Antibacterial agents were used at the following concentrations: ampicillin, 100 jig/ml; kanamycin, 100 ,ug! ml; chloramphenicol, 30 ,ug/ml; nalidixic acid, 30 ,ug/ml; tetracycline, 20 ,ug/ml; mercuric chloride, 80 jig/ml; potassium tellurite, 30 ,ug/ml; and rifampin, 50 ,ugIml. All antibacterial plates were made from tryptone soya agar or brain heart infusion agar (Oxoid Ltd.), unless otherwise indicated. Resistance to arsenate was detected by using 3.5% (wt/ vol) sodium arsenate in LB agar plates. Resistance to arsenite was examined by using 0.5% sodium arsenite (12) in brain heart infusion agar plates. Resistance to both arsenic compounds was tested by examining colony formation on

Corresponding author. 1197

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TABLE 1. Bacterial strains, plasmids, and cosmids used in this study Relevant characteristics"

Designation

E. coli strains DH1

JM109 J53-2

Plasmids pUC13 R478

Cosmids pSUP205 pHC79 pUCKW pHCKW1 pHCKW2

F- recAl endAl gyrA96 thi-J hsd17 supE44 relAl AendAl recAl gyrA96 thi hsdRJ7 relAl supE44 A(lac-proAB) (F' traD36 proAB lacIq ZAM15) proA metF Rif

A

Reference

,Psti

11 45

3

Ap Asa Asi Cm Km Hg PacB Phi Tc Te IncHI2

38

Cm Tc Ap Tc Ap Ap Ap

30 14 This study This study

B

13, 36

This study

a Ap, ampicillin resistance; Asa, resistance to sodium arsenate; Asi, resistance to sodium arsenite; Cm, chloramphenicol resistance; Km, kanamycin resistance; Hg, resistance to mercuric chloride; PacB, resistance to colicin B-producing E. coli strains; Phi, phage inhibition; Tc, tetracycline resistance; Te, resistance to potassium tellurite; IncHI2, incompatibility with H12 subgroup plasmids. Resistances were determined as indicated in Materials and Methods.

the agar plates, using the strain J53-2 containing plasmid and cosmid clones. DNA isolation, manipulation, and analyses. DNA manipulations were performed according to standard published protocols (20, 22). Plasmid DNA was isolated by the Birnboim and Doly method (4). R478 and miniplasmid DNA and all vector DNA were further purified by CsCl-ethidium bromide density gradients. DNA fragments of >30 kb were analyzed on 0.3 to 0.4% agarose gels in Tris-acetate buffer. The gels were subjected to electrophoresis for up to 40 h with periodic changes of the running buffer (33, 44). Sizes of restriction fragments were determined by using undigested phage lambda DNA and lambda DNA digested with the enzymes SmaI, HindIII, XhoI, and ApaI. Sizes of fragments greater than 48 kb were calculated from double digests of selected cosmid clones and miniderivatives of R478 (see Fig. 3). For cosmid cloning, E. coli DH1 was transfected by using packaging kits (Amersham) as recommended by the manufacturer. Restriction enzymes (Sall, BamHI, EcoRI, KpnI, HindIll, XhoI, Sacl, AvaI, PstI, XbaI, ApaI, and SmaI) were obtained from Boehringer Mannheim UK or Amersham International, plc. T4 DNA ligase was from Amersham. Construction of cosmid vectors. Three cosmid vectors, pUCKW, pHCKW1, and pHCKW2, were constructed and used for cloning XbaI, ApaI, and Sall fragments from plasmid R478. Approximately 70% of the restriction fragments generated from R478 by these three enzymes were 20 kb or larger (Table 2). The vectors were specially constructed for this work, since most available cosmid vectors do not have XbaI or Apal sites for insertion of fragments generated by using these enzymes. The vector pUCKW was prepared by the insertion of a 0.4-kb PstI fragment containing the cos site from the cosmid vector pSUP205 into the plasmid vector pUC13 (Fig. 1A). The vector allowed cloning of XbaI or Sall fragments of

ba

C

FIG.

1.

X

l

Cosmid vectors constructed for this study. (A) pUCKW

constructed from pUC13 (light area) and the cos site from (dark area); (B)

pHCKW1

pSUP205

constructed from a 3.35-kb BamHI-SalI

fragment of R478 (dark area) inserted into the cosmid pHC79 (light area); (C) pHCKW2 constructed by insertion of a 6.8-kb BamHI fragment

from

R478

(dark

area)

into

pHC79

(light

area).

The

insertion sites into the original vectors as well as sites for enzymes

ApaI, Sail, and XbaI

approximately prepared

by the

are shown.

kb or less. The vector pHCKW1 was insertion of a 3.35-kb BamHI-SalI fragment

48

from R478 (coordinates 68.4 to 71.8 in Fig. 2) into the BamHI and Sail sites of the cosmid pHC79. This 3.35-kb fragment has an internal ApaI site (Fig. iB), thus allowing insertion of ApaI fragments of approximately 41 kb or less. The third cosmid vector constructed was pHCKW2. This vector was

made by the insertion of a BamHI site of pHC79 (Fig.

R478, but its location

on

6.8-kb BamHI fragment into the iC). This 6.8-kb fragment is from the map was not determined. The

RESTRICTION MAP OF R478

VOL. 174, 1992

TABLE 2. Sizes of XbaI, Apal, and Sall restriction fragments of R478 Size (kb)b of:

Fragmenta

A B C D E F G H I J K L M N 0 P

Q R Total

Sall

ApaI

XbaI

60.0 51.0 30.0 28.0 24.0 14.8 13.6 10.4 9.0 6.7 6.7 4.5 4.3 3.05 2.05 1.6 1.6 0.5

65.5 40.5 36.5 29.6 20.5 15.9 13.0 11.3 8.4 6.7 6.5 5.3 4.9 3.2 2.4 1.85 0.7

63.5 57.0 44.5 34.6 21.5 19.0 13.8 11.0 5.4 1.2 0.6

271.8

272.7

272.1

a Fragments are designated alphabetically in decreasing order of size, with A being the largest fragment. b Sizes of fragments were determined with reference to lambda phage DNA uncut and cut with various enzymes. Sizes of fragments >48 kb were determined as described in Results.

vector pHCKW2 allowed the insertion of XbaI fragments of up to 38 kb. Construction of miniplasmids of R478. R478 plasmid DNA was digested with the enzymes Sall and ApaI separately overnight. The DNA samples were then ligated as described previously (33). The ligated DNA preparations were electroporated into E. coli DH1 with a Bio-Rad gene pulser by using the high-efficiency protocol described by Dower et al. (9). Colicin B resistance and bacteriophage inhibition detection. The presence of the colicin B resistance determinant (PacB) was assayed, as described previously, by using E. coli W3110 carrying the plasmid pColB-K260 (24). This strain mediates colicin B production, and E. coli strains which are sensitive to colicin B fail to grow around chloroform-lysed colonies of the strain (24). Bacteriophage inhibition (Phi) was determined by comparing the efficiencies of plating of the phages T5 and T4 in E. coli DH1 containing various plasmid and cosmid clones and deletions thereof (37). RESULTS Restriction enzyme digests of R478. Of the 11 enzymes used for digestion of R478 plasmid DNA, only 3 were found to be suitable for the construction of a restriction map. These three enzymes, Sall, ApaI, and XbaI, produced 18, 17, and 11 fragments, respectively, from R478. The sizes of these fragments are shown in Table 2. A fourth enzyme, XhoI, was used in double digests with each of the above three enzymes, and the restriction fragment patterns from these digests indicated that there were three sites for XhoI in R478. These sites were later confirmed in the R478 derivatives. From the sizes of the fragments produced by Sall, ApaI, and XbaI, it was determined that the size of R478 was approximately 272 kb, or 177 MDa. Restriction enzyme map of R478. Isolation of pure plasmid

1199

DNA from the plasmid R478 was difficult and never produced the amounts of DNA required for partial digestion and size fractionation which would have facilitated easier mapping of this plasmid. Hence, plasmid DNA from R478 was directly cloned into the constructed cosmid vectors with the aim of isolating particular large fragments. The details of all the relevant cosmid and plasmid clones as well as miniplasmids used in the generation of the map of of R478 are shown in Table 3. Several of the cosmid clones listed in Table 3 contain more than one fragment. Because of the method of cloning used, contiguity was not assumed for cosmid clones containing multiple inserted fragments. Two particular clones, pKFW95 and pKFW96, contain several fragments, but the fragments of interest in each clone, SalI-D (28.0 kb) and Sall-C (30.0 kb), respectively, could not have inserted into the cosmid vector pUCKW (3.0 kb) without the insertion of other smaller fragments. The restriction map of R478 was deduced by single- and double-digestion analysis of cosmid and plasmid clones and by similar analysis of R478 and the miniderivatives of R478. Additional map data were obtained by deleting selected fragments from cosmid and plasmid clones. All plasmid clones were mapped in finer detail with at least three other enzymes (not shown). Figure 2 shows the relative positions of Sall, ApaI, XbaI, and XhoI sites in R478. XhoI sites were detected in the clones pKFW80, pKFW81, and pKFW91. It was not possible to work out the orientation of the XbaI-J and -K fragments within the map of R478. Fragments of less than 0.5 kb were not considered in the restriction map. Miniderivatives of R478. As the restriction map of R478 was developed, it became clear that part of the plasmid was not inserted into any of the cosmid vectors, and it was therefore assumed that several of the largest fragments overlapped in this region. This region thus required the generation of miniderivatives of the plasmid R478. No miniplasmids were obtained from the Apal digests of R478. Three types of miniplasmids were obtained from the Sall digests. One type was resistant to chloramphenicol (Cmr), a second type mediated tetracycline resistance (Tcr), and a third type expressed tellurite resistance (Te). Only two of these smaller derivatives of R478 were analyzed in detail in order to complete the restriction map. These were a Cmr miniplasmid, designated pKFW72, which consisted of the 4.5-kb SalI-L fragment and the 51.0-kb SalI-B fragment from R478, and a Tcr miniplasmid, designated pKFW74, which consisted of the 60.0-kb Sail-A and 51.0-kb Sail-B fragments from R478. The orientations of the Sall fragments in pKFW74 and pKFW72 were noncontiguous in relation to the orientations shown for these fragments in Fig. 2. Restriction analysis of both these derivatives enabled accurate sizing of five of the largest (>48-kb) fragments indicated in Table 2. The size of the sixth fragment, XbaI-A, was estimated from overlapping clones (Table 3). Figure 3 shows fragments, in the size range of 9 to 48 kb, for restriction digests of pKFW72 and pKFW74. Incompatibility and replication loci. DNA colony blotting using the IncHI2 incompatibility probe pULB2433 showed homology with a 1.9-kb EcoRI fragment of R478 inserted into the plasmid vector pUC13. This plasmid clone, designated pKFW10, also exhibited strong incompatibility with the plasmid R478. The probe also hybridized to the miniplasmid pKFW72 but not to the plasmid clone pKFW65. This result indicated that the incompatibility determinant (inc) was located between coordinates 0 and 32.5 (Fig. 2). Since there were no Sal, XbaI, ApaI, or XhoI enzyme sites in this

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WHELAN AND COLLERAN

TABLE 3. R478 plasmid clones, cosmid clones, and deletion plasmids Designation

Resistance profile"

Plasmid clones pKFW1 pKFW3 pKFW4 pKFW5 pKFW25 pKFW37 pKFW44 pKFW64 pKFW65

Ap Tc Ap Hg Ap Te Ap Cm Ap Ap Ap Ap Ap

Cosmid clones pKFW80 pKFW81 pKFW82 pKFW90 pKFW91 pKFW9Se pKWF96e

Ap Ap Ap Ap Ap Ap Ap

Miniplasmids pKFW72 pKFW74

Cm Tc

Cm PacB Cm Te Phi Asa Asi Tc

Hg Phi Te

Vector

pUC13

Fragment(s) insertedb

Coordinates (kb)'

pUC13 pUC13

4.5-kb HindIll 15.7-kb PstI 12.5-kb BamHl 4.6-kb PstI XbaI-G XbaI-E Sall-E Sall-I 20.9-kb BamHI

pHCKW1 pHCKW1 pHCKW1 pHCKW2 pUCKW pUCKW pUCKW

ApaI-C, ApaI-K ApaI-B ApaI-D XbaI-D XbaI-C, XbaI-J, Xbal-K SalI-D, Sail-F, Sail-N Sail-C, Sail-K, SalI-J

95.5-132.0, 89.0-95.5

Sail-B, SalI-L Sail-B, Sail-A

0-51.0, 122.7-127.2 0-51.0, 212.0-272.0

pUC13 pUC13 pUC13 pUC13 pUC13 pUC13

NAf' NA

231.3-235.8 132.1-147.8 102.7-115.2

NDd 232.7-246.5 192.1-213.6 187.8-211.0 110.5-119.5 32.6-53.5

165.2-205.7 205.7-235.3 43.0-77.6

149.9-190.4, 190.-192.2 131.5-159.5, 173.0-187.8, 159.5-162.6 71.8-101.8, 51.0-58.7, 101.8-108.5

a As listed in footnote a in Table 1. b For the enzymes XbaI, Sall, and ApaI, the fragments are designated as shown in Fig. 2. For all other enzymes, the size of the inserted fragment is indicated. I R478 map coordinates shown in Fig. 2. dND, not determined. I Fragments in these clones are noncontiguous with the restriction map of R478 (Fig. 2) but are indicated in the order of insertion into cosmid pUCKW. f NA, not applicable.

region, another enzyme had to be used to further refine the location of the inc region. The enzyme chosen for this purpose was Sacl. A plasmid DNA bank of pKFW72 was generated by using the vector pUC13 and various restriction enzymes, including Sac. The restriction map of pKFW72 and the relevant clones used in the generation of this map are shown in Fig. 4. The inc determinant was found to span the coordinates 10.8 to 12.7 in Fig. 2 and 4. The plasmid derivatives pKFW72 and pKFW74 from R478 are self-replicating, and they have the Sall-B fragment in common. Therefore, it was concluded that a region involved in replication on R478 is within this fragment. By using the enzyme Sacl, plasmid DNA from pKFW72 was subjected to the same procedure as was used for the generation of miniderivatives of R478. Only one miniplasmid from pKFW72 was obtained, and this was designated pKFW109 (Fig. 4). This plasmid was found to contain a deletion of 22.6 kb (coordinates 20.6 to 43.2 in Fig. 4). It was concluded that an origin of replication in pKFW109 must be located in one of the two remaining areas, i.e., those spanning the coordinates 0 to 20.6 and 43.2 to 51.0 (Fig. 2 and 4). Localization of tellurite resistance, colicin B resistance, and bacteriophage inhibition determinants. Several of the cosmid and plasmid clones from R478 mediated resistance to potassium tellurite (Ter) and colicin B (PacB) and also expressed bacteriophage inhibition (Phi). By deleting various fragments from some of these clones, it was possible to locate all three determinants on the map of R478. Table 4 shows the details of the clones and the genes they expressed. Tellurite resistance was detected in all clones containing the SalI-J fragment or part thereof, and it was therefore concluded that the Ter determinant must be totally within this fragment. Colicin B resistance was expressed by cells containing the cosmid clone pKFW80. The XhoI-C fragment from pKFW80

was deleted to generate pKFW80b. This deletion resulted in loss of the SalI-L fragment as well as parts of the SalI-M and -P fragments. Unlike the parent pKFW80, the pKFW80b derivative was chloramphenicol and kanamycin sensitive, although it still retained the Ter, Phi, and PacB determinants (Table 4). By deleting Sall fragments from pKFW80, it was found that the SalI-J fragment and part of the Sall-C fragment (coordinates 89.0 to 101.8 in Fig. 2) were required for the expression of the PacB trait. Both fragments were also present in the cosmid clone pKFW96, but this clone was sensitive to colicin B. Further analysis of pKFW96 revealed that the orientation of the SalI-J and Sall-C fragments was different from the orientation shown in Fig. 2, and hence part of the PacB determinant must span the SalI site at coordinate 101.8 (Fig. 3). By using the bacteriophage T5, phage inhibition was exhibited by several clones (Table 4). All of these clones contained part or all of the SaiI-J fragment. Deletion of Sall fragments from pKFW80 also generated pKFW80c, which had lost all Sall fragments except the SaiI-J and part of the Sall-C fragment. The level of phage inhibition expressed by pKFW80c was higher, by a factor of 107, than the level of phage inhibition exhibited by all other clones which expressed the Phi trait. No decrease in efficiency of plating was detected for the control phage T4 on any of the clones listed in Table 4. It was deduced from these results that the Phi determinant is linked to the SalI-J fragment of R478. Localization of the tetracycline, chloramphenicol, kanamycin, and mercury resistance genes. Tetracycline resistance (Tcr) was mediated by the cosmid pKFW82, the plasmid pKFW1, and the miniplasmid pKFW74 (Table 3). It was concluded that the Tcr determinant was located between coordinates 231.3 and 235.8 (Fig. 2), which is the region encompassed by the smallest Tcr clone, pKFW1 (Table 3).

VOL. 174, 1992

RESTRICTION MAP OF R478

IncHI2

A B C D E F G H I J

1201

K L M N

48.533.3-

23.1-

15.2-

9.4Hg CmKm FIG. 2. Restriction and genetic map of R478. Restriction enzyme sites and fragments are indicated in the outer circles. These sites are positioned with respect to the Sall site at coordinate 0, and the map is numbered by distance (in kilobases) from that site. The restriction fragments are labelled in decreasing order of size, with A being the largest fragment. Locations of the mercury (Hg), tellurite (Te), arsenite (Asi), arsenate (Asa), tetracycline (Tc), chloramphenicol (Cm), kanamycin (Km), and colicin B (PacB) resistance genes and phage inhibition (Phi) and IncHI2 plasmid incompatibility (IncHI2) determinants were identified as described in the text.

Chloramphenicol resistance (Cmr) was found in all clones containing the 4.5-kb SalI-L fragment, while kanamycin resistance was found in all clones containing the 1.6-kb Sall-P fragment. There is an XhoI site within the 1.6-kb SalI-Q fragment which distinguishes it from the Sall-P fragment. Both the SalI-L and -P fragments are located side by side in the map of R478 (Fig. 2). Mercury resistance (Hgr) was mediated by the clones pKFW3 and pKFW95. Expression of this resistance was not easily obtainable without prior exposure of clones to a subinhibitory level of mercuric chloride (