Molecular Microbiology (1992) 6(14), 1933-1941

Evasion of type I and type II DNA restriction systems by Inch plasmid Collb-P9 during transfer by bacterial conjugation Timothy D. Read, Angela T. Thomas and Brian M. Wiikins* Department of Genetics. University of Leicester, Leicester LEI 7RH, UK. Summary Transmission of unmodified plasmid Coi(b-P9 by bacterial conjugation is markedly resistant to restriction compared with transfer by transformation. One process atiowing evasion of type I and II restriction systems involves conjugative transfer of multiple copies of the plasmid. A more speciaiized evasion mechanism requires the Ard (alleviation of restriction of DNA) system encoded by Collb. The ard gene is transferred early in conjugation and specifically alleviates DNA restriction by all known families of type I enzyme, including EcoK. Collb has no effect on EcoK modification but this activity is impaired by multicopy recombinant plasmids supporting overexpression of ard. Genetic evidence shows that Ard protects Collb from EcoK restriction following conjugative transfer and that this protection requires expression of the gene on the immigrant plasmid. It is proposed that carriage of ard facilitates transfer of Collb between its natural enterobacterial hosts and that the route of DNA entry is important to the restriction-evasion mechanism.

Introduction Many bacterial plasmids specify conjugation systems that support transfer of DNA in single-stranded {ss) form from a donor to a recipient cell (Willetts and Wiikins. 1984). Conjugation systems have the capacity to transfer DNA between a wide range of bacterial genera, providing a route for the dissemination of bacterial genes in nature (Gormley and Davies. 1991; Mazodier and Davies. 1991; Trieu-Cuot et ai.. 1988). One potential barrier to promiscuous conjugation is imposed by the DNA restrictionmodification (R~M) systems commonly found in bacteria. The systems constitute a potential filter to heterospecific Received 25 February. 1992; revised and accepted 3 April, 1992. *For correspondence. Tel, (533) 523432: Fax (533) 523378.

gene flow, since a restriction endonuclease cleaves DNA when the nucleotide recognition sites lack modification conferred by the cognate DNA methyltransferase. R-M systems of enterobacteria may be distinguished as three types, according to subunit structure, cofactors, and distance between recognition and cieavage sites (Bickie, 1987; Wilson and Murray, 1991), Type I systems are the most complex and are currently divided into three families, exemplified by EcoK (la). EcoA (Ib), and EcoRi24 (Ic) {Bickie. 1987), Type la enzymes are the best characterized and are comprised of subunits encoded by the hsdR {restriction). hsdM {modification) and hsdS (specificity) genes. Activity of the complex as an endonuclease or methyltransferase depends on the methylation status of the specificity sites in duplex DNA: the enzyme will modify the second strand of hemimethylated DNA but acts as an endonuclease at a distant position when a recognition complex is formed at an unmethylated specificity site. Enzyme molecules do not recycle after the cleavage reaction. Type II R--M systems are exemplified by EcoRI, which is specified by some natural ColEI-related plasmids. and type III systems are represented by EcoPI. encoded by phage P I . Few generalizations can be made about the magnitude of restriction barriers to the conjugative transmission of DNA. Transfer of IncP plasmids and some shuttle vectors is acutely sensitive to restriction by some type II systems (Marra and Shuman, 1989; Trieu-Cuot et ai. 1991). In contrast, effective transmission of F-related plasmids is relatively resistant to type 1 restriction compared with infectious transfer of smaller double-stranded {ds) DNA of lambdoid phages (Boyer, 1971). One possible explanation of the differences is that specificity sites for some restriction enzymes have been eliminated from transmissible plasmids by mutation and selection (Guiney, 1984). Another explanation is that some conjugation systems cause breakdown of restriction mechanisms in the newly infected cell {Glover and Golson, 1965). Bacteriophages specify several different classes of proteins that protect infecting unmodified DNA from host restriction systems (Kruger and Bickie. 1983). The same may apply to conjugative plasmids. since IncN and Inch plasmids carry genes that alleviate restriction of DNA by some type I systems (Belogurov et al., 1985; Delver etal., 1991; Wiikins etai, 1991).

1934

T. D. Read, A. T. Thomas and B. M. Wilkins

In this paper we report the effects of EcoK and EcoRI on the transfer of the colicinogenic piasmid, Collb-P9, as part of a study of factors influencing the installation of conjugatively transferred Incil plasmids in the recipient cell. We show that the plasmid can evade restriction barriers to productive conjugation by two mechanisms. One is dependent on amplified DNA transfer from the donor cell, white the other involves an 'anti-restriction' gene carried by the plasmid and operative against type I restriction systems. Results Restriction of DNA transferred by conjugation and transformation The standard plasmid used was pLG221. This derivative of Collb-P9 (93 kb) carries a selectable kanamycin-resistance (Km") gene endowed by a Tn5 insertion, and a drd mutation that depresses the fertility inhibition system oontrolling expression of the conjugative transfer genes (Rees et al., 1987), Table 1 shows that pLG221 was restricted when introduced as dsDNA into rR,"" mR,' recipients by a chemical transformation procedure. Restriction in vivo is consistent with the presence on the piasmid of >20 sites that are cleaved in vitro by EcoRI endonuciease (Rees etai., 1987). Likewise, the transforming potential of unmodified pLG221 was substantially reduced when recipients specified EcoK, demonstrating that the plasmid contains nucieotide sequences recognized by this type I R-M system. In contrast, conjugative transmission of pLG221 was remarkably resistant to EcoK and EcoRI restriction; transconjugant yields in 60-min conjugations were reduced by less than fourfold relative to non-restrict* ing controls. It is noted that NTP14 specifies oolicin El and the donor strain was sensitive to this agent. Use of colicin El-resistant donors had no effect on the results shown. Interrupted matings showed a reduotion of transconjugant yield at early times in restricting conjugations (Fig. Table 1. Restriction of unmodified pLG221 transferred by transformation and conjugation. Yield Relative to Non-restricting Transfers Restriction system EcoRI EcoK

transformsnts

transconjugants 0,26 ± 0,04 0,65 ±0,15

EcoRI data were obtained using W31 iO{pLG22i) donors and C600N +/NTP14 as me recipient strains, EcoK data were obtained using RR1{pLG221) donors and C600N and 5KN recipients. Transconjuganl yields were determined after 60 min of mating; data show means and standard errors (n = 4}. Yields in non-restricting transfers were c, 5 x 10* Km" iransformants per ng DNA and c, 2 x 10^ Km" Nat" transconjugams per ml.

10 20 30 Interruption time (min)

40

Fig. 1. Kinetics of tfansmission of unmodified pLG221 {ard') to recipient cells specifying EcoK or EcoRI, Interrupted matings were performed usmg RR1 -donors. Recipient strains were 5KN (O. control), C600N (A) and 5KN(NTP14) (D), Al 12 min, a sample of Itie EcoRI-restricted mating was blended and incubated in the presence of SDS and nalidixic acid, prior to plating for transconjugants at the indicated limes (•).

1). Transconjugant production rapidly recovered after the initial delay, which amounted to 1-2 min and 6-8 min in EcoK and EcoRI-restricting conditions, respectively. Recovery from the delay required continued activity of donor cells, rather than an event in the recipients that rescues previously transferred DNA. This was established by separating conjugating cells in an EcoRI-resthcted mating by blending at 12 min and then incubating bacteria for up to 28 min prior to selection for transconjugants. SDS and nalidixic acid were added to the blended bacteria to prevent further conjugation: these agents block de novo formation of mating aggregates and inhibit the DNA transfer potential of drug-sensitive donors, respectively (Achtman et at., 1978; Witletts and Wilkins, 1984). The treatments arrested the increase of transconjugants in the restricted mating, indicating in turn that EcoRI-evasion is dependent on prolonged donor activity. Presumably continued activity of donor cells supports transfer of extra copies of the plasmid, leading to breakdown of the restriction system in the recipients. Transconjugant colonies formed in restricting matings generally carried normal plasmids. Plasmids were isolated from ten transconjugants generated early in rR,^ mRi"^ and in rK* mK* recipients and were found to be typical of pLG221 by their conjugative potential and the sizes of DNA fragments generated by in vitro digestion with endonucleases. fi/folecular analysts of the restriction alleviation gene on Collb Collbdrd carries a gene that alleviates FcoK restriction of phage X DNA (Table 2). This gene, originally called pra

Restriction evasion during conjugation Table 2. Alleviation of restriction of unmodified Xvirby Collbdnrfand an Afd" recombinani. Plasmid Present R-M system EcoK

Type

la la ECOfi. Ib EcoRI24 Ic EcoRI II EcoPI IN EccS

Strain C600 B251 WA2e99 5K(R124) C600(NTP14) RRI(PI)

None 1.4x10-* 1.5x10-^ 2.1x10-^ 8.9 X 10"^ 1.1x10-^ 2.8x10-=

pLG221

pLG290

2.3x10"^ NT NT NT 7.1x10"^ NT

0.95 0.8 0.92 1.0 NT 3.5x10-^

NT. not tested. Values show e.o.p, EcoRI data were obtained using k.K and are expressed relative to plaques formed on C600. All other tests involved X.O.

(plasmid-mediated restriction alleviation; Wilkins ef al., 1991). is now designated ard (alleviation of restriction of DNA) to be consistent with the nomenclature of Delver et ai. (1991). It is remarkable that Ard activity is enhanced by a drd mutation on the plasmid. since unmodified X {X.O) plated on C600 harbouring a Collb plasmid (pLG272) mXh an efficiency of plating (e.o.p.) of 3.7 x 10"^, in contrast to the value of 2.3 x 10'^ on C600 carrying pLG221. Enhancement of Ard function by inactivation of the fertility inhibition mechanism suggests that arc/contributes to the Collb conjugation system. The ard gene maps in the Collb leading region (Fig. 2), which is defined as the first segment of the plasmid to be transferred to the recipient cell following initiation of DNA transport at the adjacent origin of transfer {oriT) site. Recombinant plasmid pLG290, which carries ardon a 2.8 kb Sal\-Pst\ fragment of Collb, allowed >..O to form plaques on rK* mK* cells with high efficiency (Table 2). The recombinant also mitigated restriction of k by EcoB and representatives of the Ib and Ic families of type I restriction enzyme, namely EcoA and 5coR124. Ard conferred no protection on A. against restriction by the EcoRI and EcoPI systems. Analysis of the Ard system was facilitated by a set of Tn 1732 insertion mutants in the cloned 2.8 kb fragment. The transposon carries two useful EcoRI sites, each 15 bp in from a transposon terminus (Ubben and ^chmitt. 1986). The location of the Tn1732 insertions and of two Tn 5-insert ions in Co\\drd-1 indicate that ard is within a 1.4 kb region on the onT-proximal side of the Collb Sail site at co-ordinate 24.1 (Fig. 2). This region was dissected using pMS119 vectors, which contain an IPTG-inducibte tac promoter separated from transcriptional terminators by a multiple cloning site. Results summarized in Fig. 3 show that ard is contained in a 0.86 kb fragment, which is or/r-proximai to the Rsa\ site, and that the direction of transcription is from right to left towards oriT. The nucieotide sequence of this fragment was determined and found to contain a 498 bp open reading frame identical to that recently reported for ard by Delver efa/. (1991).

1935

Only part of ard is essential for alleviating restriction (Fig. 3). The 0.45 kb EcoRI-Smal fragment failed to specify Ard function when present in pMSi19 vectors (pTDR54 and pTDR55). However, the same fragment determined activity when inserted at EcoRI-Smal sites in pBluescript, giving pTDR35. One explanation is that the pBluescript recombinant specifies a functional fusion protein consisting of the W-terminal portion of (J-galactosidase encoded by the vector and the C-terminal region of Ard. This was verified by cloning the same 0.45 kb EcoRI-Smal fragment in pUCi9. giving pTDR36. Although in the same orientation as pTDR35. the insert in this recombinant is in a different reading frame relative to iacZ' in the vector and the plasmid is Ard". The fragment was also taken from pTDR35 by 6amHI-Sa/l cleavage and inserted into pUC18, producing pTDR40. In pTDR40 the fragment is maintained in the same reading frame relative to IacZ' as it is in pTDR35 and the recombinant is Ard"^. These results indicate that at least the 5' third of the gene is redundant for alleviating EcoK restriction of X, DNA.

Overproduction of Ard interferes with EcoK modification The Ard* recombinant ptasmid pLG290 impaired modification of DNA by EcoK. Single cycle growth experiments showed that progeny phages from X.K infection of C600(pLG290) were deficient in EcoK modification (Table

, 30

3

35

P P CP 1 i J I 1

P 1 *-

S

iii

oriT p

p p 1 1

1

'

esm 1 1

STS

'

1 13

S PE

1

1 1 1

ssb pLG290 s pLG291K

1

'•-.

1

1 15

A

1

i A l is

1.'

1

s

Sm

p

1

C ft P 1 1^

psiB

..

t-'"

1 30

E •-r

0 L_

1 1

Km' Fig. 2. Map of the Collb leading region showing insenions inactivating Ard function. Line i indicates Collb kb co-ordinales {Hees etal., 1967). Line 2 represents the largest EcoRI fragment of Collb and shows the origin of transfer {oriT) region, ttie single-stranded DNA-binding proiein [ssb) gene . and the plasmid SOS inhibition {psiB) gene. DNA transfer from onl is from left lo right, ssb and ps/S are Iranscribed in the leftward diredion. Two TnSinsertions in Collbd/'dthat have no effect on the Ard phenotype are shown (O). Line 3 indicates the derivation of Ihe 2.6 kb Collb fragment preseni in pLG290. The direction of Iranscnption from the /acZpromoter in the vector is indicated by the honzontal arrowhead. Tn 173.? insertions inpLG291 that inactivate (A) or have noeffecMA)on Ard are shown. The derivalion of pLG29i K is described in the text. The kilobase scale bar applies to pLG291 and pLG291 K. Cleavage sites are shown for Acd (A). C/al (C), £coRI (E), Psfl (P), Rsal (R), Salt (S), Smal (Sm).

1936

T. D. Read. A. T. Thomas and B. M. Wilkins

26.9 P

(E)

I

Sm

I

KCORF

Plasmid

E.o.p.

pTDR51

0.8

pTDRSO

0.6

pTDR52

1.0

pTOR53

0.02

pTDR54

0.0002

pTDR55

0.0001

pTDR35,40

t.O

pTOR36

0.0005

Fig. 3. Alleviation of EcoK restridion ot k.O by fragments of the Collb leading region. The rectangle at the top represents (he Collb segment from co-ordinaiGS 24.1 to 26.9 The leftward reading ORF corresponds lo ard. Values show e.o.p. ol ^.0 on IPTG-trealed C600 containing the indicated plasmid. pMSI 19EH was the vector for pTDR51, 52 and 54, and p M S n g HE for pTDRSO, 53 and 55, Arrowheads indicate the direction of transcription from the /acpromoter. Other vectors were pBluescript (pTDR35). pUCi9 (pTDR36) and pUClS (pTDR40), the arrowheads indicating transcription from the /ac promoter. Cleavage sites are shown for Pst\ (P), Rsa\ (R), San (S) and Smal (Sm). The EcoRI site (E) was introduced by TnT732insertion number 1 (Rg.2).

3), However, presence of the Collbdrdderivative pLG221 in C600 had no such effect. The implication that EcoK retains the capacity to modify DNA in the presence of pLG22i is further supported by data in Table 1. These show that pLG221 isolated from r^'m^"^ donor W3110 generated up to 10^ transformants per pg DNA in rK^nriK' recipient C600. If the ard gene on pLG221 were to cause breakdown of EcoK modification in the donor strain, the transforming DNA would require protection from EcoK restriction In the recipient, presumably through Ard activity determined by the immigrant plasmid. However, ard is unable to confer c/s-acting protection on transforming DNA, as shown by the restriction-sensitivity of pLG221 isolated from the r"m donor RR1. Table 3 shows a correlation between inhibition of EcoK modification activity, alleviation of EcoK restriction activity and the level of ard expression. The latter was determined by measuring |i-galactosidase specified by a IacZ promoter probe introduced at the Sma\ site in ard in the correct transcriptional orientation, fvlulticopy Ard"" plasmid pLG294 inhibited the two EcoK activities more effectively than Collbdrd plasmid pLG221. A derivative of pLG294 (named pLG295) carrying the arc/-/acZ fusion specified 30-fold more (i-galactosidase than an equivalent CoWhdrd plasmid (pLG296). No (i-galactosidase was determined by corresponding plasmids with the IacZ probe in the opposite transcriptional orientation to that of ard (data not shown). Some leading region genes are transiently induced in the recipient cell following conjugative transfer, as shown

using the /acZprobe (Jones etal.. 1992). Expression of arc/is regulated differently, because no zygotic induction of IacZ was detected in pLG296-mediated matings of MC4100 cells. Specific activity of (J-galactosidase was 1.1 immediately after mixing equal volumes of donor and recipient cells and rose to 2.0 after 60 min conjugation.

Conjugative properties of a Colltxird ard mutant Data already described indicate that Jn 1732 insertion number 3 (Fig. 2) lies in the 3' portion of ard. EcoRI sites near the ends of this insertion were used to construct a Collb mutant. Tn7732was first replaced with a non-transposable Km" cassette giving pLG291K (Fig. 2), The arc/::Km" mutation was then introduced into CoWbdrd-i using the gene replacement strategy. The resulting Collbdrd-t mutant, designated pLG292. was shown by EcoRI and Sail digestion to carry the insertion at the correct location in the leading region (Collb co-ordinate 25.2). The e.o.p. of X.0 on C600(pLG292) was 2.4 x 10"", confirming that the plasmid is Ard". Figure 4 shows the yield of pLG292 transconjugants in EcoK and EcoRl-restricting matings. Comparison with Fig. 1 indicates that pLG292 has the following properties. First, the plasmid shows no transmission defect under non-restricting conditions; hence, arc/is not essential for the conjugation process. However, transmission of pLG292 shows abnormal sensitivity to EcoK restriction and there was an extended delay before transconjugants were detected. This trend was observed in five different experiments. Recovery from the delay required prolonged conjugation, as shown by the abrupt arrest of transconjugant-formation when mating was interrupted and the separated cells were incubated in the presence of SDS and nalidixic acid prior to selection. In contrast, ard mutation had no effect on transconjugant production In EcoRIrestricting matings. This is consistent with tests showing

Table 3. Inhibition of EcoK restriction and modification at different expression leveSs of ard.

Plasmid

X..0 restriction

Expression kX modification (li-galactoslcJase)

pUC19-based pLG290 (aref) pLG294 (arcT) PLG295 (arc/: :/ac2)

0,95 0.23 5.4x10'^

0.03 0.7 1.0

NT 0 68

Collbtfrd-based pLG22l {anf) pLG296{a/-d::/acZ)

2.3x10"^ 2.4x10-''

1,0 1,0

0 22

Restriction values show e.o.p. of X.0 on C600N strains. Modification was measured using progeny phages from one cycle of growih of >..K in C600N harbouring the indicated plasmid; values show e.o.p. on C600N relative to 5KN. Expression shows [i-galactosidase (units per mg protein) determined in host strain MC4i00(Alac). NT, nol tested.

Restriction evasion during conjugation

1937

Ard function in conjugation is recA-independent

0

10 20 30 interruptron time (min)

Fig, 4. Kinetics of transmission of unmodified Collbdrd ard to recipients specifying EcoK or EcoRI. The donor was RRI {pLG292). Recipients were 5KN (O, coniroi), C600N (A) and 5KN(NTP14) (D). At 10 min. a sampie of the EcoK-restcicted mating was biended and incubated in the presence of SDS and nalidixic acid before piating (or transconjugants at the indicated times (A).

that ard alleviates restriction of X by type i but not type II systems (Table 2). The yield of transconjugants in a restricted mating never reached the value obtained in the controls, even after 60 mln of incubation (Table 1). The possibilities were eliminated that a substantial fraction of the recipient population was kilted or acquired some form of immunity to conjugation as a result of abortive rounds of plasmid transfer. The latter was established by mating RRI (pLG292) donors with C600N for 30 min. after which a twofold excess of C600(pLG273) donors were added. After a further 40 minutes of incubation naiidixic acidresistant (Nal"^} colonies were selected and scored for the presence of the Km" and tetracyciine-resistant (Tc*^) markers characteristic of pLG292 and pLG273. respectively. By these tests, 12% of the recipients at the 70 min sampling time carried pLG292 but 37% harboured pLG273 transferred in the second non-restricting challenge. These values imply that the deficiency cf transconjugants in restricting matings is due to lack of productive donor activity rather than of potential recipients.

Alleviation of EcoK restriction is known to be a cellular response that is inducible by DNA damage via a recAdependent process (Thoms and Wackernagel, 1982). Ard functions independently of the response, as shown by three lines of evidence. First, the plaque-forming ability of X.O on a recAl derivative of C600N was increased from 1.8x10"'' to 0.43 when the cells harboured pLG290. Second, presence of pLG290 in C600N recAl recipients increased the yield of pLG292 transconjugants following transfer of the ptasmid from RRI donors; after 10 min of conjugation, the transconjugant yield was 2000-fold higher when the recipients harboured the Ard* recombinant plasmid. Hence, Ard function in conjugation is recAindependent. Finally, the yield of C600N recAl transconjugants at 10 min was 200-foid higher when the plasmid transferred from RRI was pLG221 rather than pLG292. This demonstrates that the ard gene on the Collb plasmid is functional in the absence of RecA protein in the recipient cell.

Discussion At least two mechanisms contribute to the remarkable resistance of the Collb transfer system to representative type I and type II restriction enzymes. One involves a specialized gene, ard. operating to alleviate type I restriction systems. The second is a more general mechanism allowing Collb to evade both types of restriction system by a process requiring prolonged conjugation. Presumably, extended conjugation supports elevated levels of DNA transfer, since donor functions required fcr DNA

Requirement for Ard in the recipient cell Recombinant pLG290 determines a high level of Ard activity. When present in the recipient strain, pLG290 allowed the unmodified Collbdro'ard plasmid (pLG292) to evade completely the type I restriction barrier to productive transfer (Fig. 5). No protection was detected when pLG290 was harboured by the rj^mK donor strain cf pLG292. Hence. Ard product must be produced in the newly Infected recipient cell to allow conjugative evasion of EcoK restriction by Collb.

0

10 20 30 Interruptton time (min)

R Q . 5. Compiementation of Coilbdfrf ard by Ard' recombinant pLG290. Interrupted matings involved mixtures of RRI (pLG292} and C600N(pLG290) [V: R, recipienl], and of RR1(pLG292,pLG290) and C600N [ T ; D, donor]. Broken imes indicate data (rom Fig. 4: upper cun/e, RRI (pLG292) x 5KN as a non-restncting controi: lower curve, RRI (pLG292} v G600N as an EcoK-restricting mating.

1938

T. D. Read, A. T. Thomas and B. M. Wilkins

transport should be unaffected by the activity of a restriction enzyme in the recipient celi. it was shown previousiy that transfer of muitipie unit-length copies of Colib occurs when expression of genes on the immigrant piasmid is prevented, implying that the number of Coilb copies transferred is reguiated by some piasmid-encoded controi system expressed through the recipient ceii {Bouinois and Wiikins. 1978). We speculate thaf this controi signai is delayed in restricted matings. ailowing extra cycles of DNA transfer to occur. These may overwhelm the restriction barrier by substrate saturation. Transmission of large segments of the bacterial chromosome from Hfr donors may have the same effect (Boyer. 1971). Carriage of ard helps Coilb to evade EcoK restriction during conjugation. Presumably the gene acts iikewise against other fype I enzymes, since it alieviated restriction of phage X by representatives of ail three families of type I system. Although Ard activity is enhanced by derepression of the fertility inhibition system, the gene maps outside the defined transfer region on Collb in the leading region of the piasmid. We have determined by nucleofide sequencing an open reading frame (ORF) corresponding to ard, as defined by inactivating insertions and gene fusions. The sequence is the same as that recently reported by Delver et al. (1991). who predicted the product of ard to be a very acidic protein with a molecular mass of about 19 000. We have found no obvious simiiarity between ard and entries in the Ef^^BL Database or between predicted products examined by the TFASTA program. Our genetic data also indicate that the W-terminal third of Ard is inessential for aileviating restriction of phage A but this region of the protein may still have a physiologicai roie. The direction of transcription of arc/is towards oriT The same applies to two other leading region genes thought to play anciiiary roies in the instailation of the immigrant piasmid. These are ssb, encoding a ssDNA-binding protein, and psiB, specifying a product that inhibits induction of fhe baeteriai SOS response (Howland etai. 1989; Jones etal.. 1992), Expression of Coilb ssb and psiB is enhanced by derepression of the conjugation system, as is the case for ard. Compiementation of the Collbdrd ard mutant by a cloned ard* gene In the recipient, but not the donor cell. implies that the gene acts in conjugation following its expression in the newly infected cell. Presumably expression of the transferred arc/gene requires prior synthesis of the complementary DNA strand to provide the substrate for physioiogicaily correct transcription. This raises the timing problem of how Ard protein accumuiates in the infected cell before Ihe immigrant plasmid is irreversibly inactivated by the type I restriction enzyme. Any model must be compatible with the finding that carriage of ard in eis does not protect unmodified Coilb DNA from restriction when the plasmid is transferred by transformation.

This timing probiem is partly reconciled by the location of ard in the leading region, where it is one of the first genes to enter the recipient ceil. Transcription of the nearby ssb and psiB genes on CoWbdrd is strongly induced soon after conjugative transfer of the piasmid to the recipient celi. as shown by /acZtranscriptional fusions (Jones et ai, 1992). However, we have found no conjugative induction of the same /acZ promoter probe when carried in ard on pLG296. This plasmid determined a very low level of p-galactosidase. suggesting that ard is transcribed from a weak promoter. This is consistent with the features of the putative promoter identified in the nucieotide sequence by Deiver etai (1991). The implication is that relatively few moiecuies of Ard suffice to cause restriction evasion in conjugation but this needs to be confirmed by an immunologicai method when antibodies are avaiiabie. The mode of entry of DNA into the ceil may be important to restriction evasion. Bacteriophage T7 determines a restriction evasion mechanism specified by the 0,3 gene. This gene enters the cell early in infection and acts to inhibit the restriction and modification activities of EcoK, apparently by binding to the DNA-binding site of the enzyme (see Bandyopadhyay et ai. 1985), Moffatf and Studier (1988) have shown that T7 DNA does not become susceptible to degradation until several minutes after infection and yet gene 0.3 protein is made in this period. The preferred explanation is that the infecting phage DNA enters some compartment thaf is outside the inner cell membrane and accessible to RNA polymerases but not fo type I restriction enzymes. Possibly, conjugatively transferred Coilb DNA is targeted to a similar location that also supports complementary strand synthesis. tn this context it is noted that replication of infecting ssDNA of phage i^XMA appears to occur at a site that inciudes outer cell membrane markers, possibly providing direct access to the chromosomal replication apparatus (Kornberg and Baker. 1992). Entry of Collb via different routes in conjugation and transformation would explain the restriction-sensitivify of transforming DNA. How might Ard protein function to prevent restriction by type I enzymes? The system acts independently of RecA protein, showing thaf it Is unrelated to the host response of restriction alleviation induced by DNA damage (Day. 1977; Thoms and Wackernagel. 1982). Results showing that the protein alleviates DNA restriction by representatives of all known families of type I enzyme implies interference with a common reaction. Identification of the mode of action will require purified Ard protein. However, preferential inhibition of the cieavage response is suggested by the finding that Ard specified by Collb protects the transferred plasmid from restriction without preventing its eventual modification. Cleavage is thought to involve bidirectional ATP-stlmulated DNA translocation causing

Restriction evasion during conjugation neighbouring enzyme molecules to converge at the cleavage site (Studier and Bandyopadhyay, 1988). However, Ard specified by multicopy reccmbinant plasmids impairs modification of DNA by EcoK as well as restriction. Such an inhibitory eftect on DNA modification correlates with increased expression of ard. presumably owing to elevated gene copy number. At relatively high intracellular levels Ard might inhibit a feature of type I enzymes common to restriction and modification, such as binding of cofactors to the enzyme or binding of the enzyme to DNA. Ard may fail to impair modification during conjugation since there is no evidence that transcription of ard is increased when Colibdrdenters the recipient cell. Glover and Colson (1965) found that the restriction but not modification activity of a type I system transiently breaks down in recipient cells that have acquired unmodified F DNA by conjugation. This could reflect substrate saturation of the restriction enzyme or the presence of a restriction-alleviation gene on plasmid F. While the leading regions of Collb and F carry some highly conserved genes, including psiB and ssb (Jones et al., 1992), there is no obvious similarity between the nucieotide sequences of Collb ard and the F leading region from onTto ssb, given by Loh et ai (1989). No similarity has been detected between ard and other segments of the F plasmid by Southern hybridization at high stringency (P. Chilley, personal communication). We propose the following general model to describe evasion of type I restriction enzymes during Cotlb-mediated conjugation. The transferred DNA strand escapes recognition by these enzymes, which use dsDNA as substrate. Synthesis of the complementary DNA strand. thought to occur concurrently with DNA entry (Rees and Wiikins, 1990), potentiates expression of ard as a leading region gene. Competition then ensues between the nucleolytic activity of the type I enzyme and the alleviatory function of Ard. If the dsDNA is cleaved, the first round of DNA transfer aborts but it is followed by a second cycle of plasmid transfer to a cell that may already contain Ard protein. By promoting the effectiveness of early rounds of piasmid transfer, Ard increases the rate of productive transfer in a restricted mating. Inch ptasmids are maintained in enterobacterial genera closely related to Escherichia (N, Datta, personal communication) and strains of the same group of enterobacteria specify type I R-M systems (Daniel et ai., 1988). Thus, the carriage of ard by Inch plasmids may contribute a strategy that facilitates conjugative transmission of these elements between their different natural hosts.

1939

Table 4. Piasmids, Plasmid

Description

NTP14 CotEl repiicon, bla. cea, ecoRIR. ecoRIM. Ap" PLG221 IncM, Colibdrd-1, ard'. cib.JnS. Km" pLG272 IncM, Collb, arcr,cib:Jr\5. Km" pLG273 inch. CoWbdrd-1. ard-. cib::Jr\ 10. Tc"^ pLG290 pUCl9 ii(arer Sali-Pst\ 2.8 kb) A p " , [Fig. 2) pLG291 pBiuescript i.l{ard' Satt-Pst\ 2,8 kb), Ap'' PLG291K pLG29i.ard;:apM-M£coRI1,3kb)Ap^ Km" , (Fig. 2) pLG292 incM.Collbd/'d-f, a/-d.,-apM-;(£cc?RM.3kbat coord, 25,2), Km" pLG294 pUC19 U(Collbarcr, EcoRI (insert 1. coord. 25,5)-f?sal 0,86 Kb) Ap" pLG295 pLG2g4 ard::tacZ-Km" (4,7 kb at Smal site) Ap", Km" pLG296 Incli, Co\lbdrd-1 ard::tacZ•Km'^ (47 kb at coord. 25 0) Km*^ R124 IncFIV, rtsd^r. tra'. tet. Tc" Coord, indicates kiiobase coordinate on Collb (Fig, 2),

pLG221, pLG272 and pLG273 is given by Howland et al.. (1989). NTP14 (Smith et ai. 1976) and R124 (Firman et al., 1983) specify EcoRI and EcoR124. respectively. Vectors were pUCt8, pUC19 {Yanisch-Perron et ai. 1985) and pBluescript SK" (Stratagene Cloning Systems}. p M S n 9 E H and pMSI 19HE are derivatives of pJFi 19 expression vectors lacking the 1,33 kb Nru\~Nde\ fragment (Furste ef ai. 1986). pLG290 carries the 2,8 kb Collb fragment from pLG291, subcloned as a SamHI-Sa/l fragment, pLG291K carries the kanamycin-resistance cassette derived from pUC4K (Howland et al.. 1989), pLG294 is equivalent to pTDR53 (Fig, 3) except that the vector is pUCI 9; ard is in the opposite transcriptional orientation to the lac promoter in the vector, pLG295 carries a 4.7 kb lacZ-Km" cassette (Kokotek and Lotz, 1989) inserted by blunt-end ligation at Ihe Smal site at Collb co-ordinate 25.0; lacZ\s in the same transciptional orientation as ard.

Bacterial strains Donor host strains were RRI (re^me"; Bolivar et ai. 1977) and W3t10 (r^-rriK*; Arber and Dussoix, 1962), Recipient strains were Nal" mutants, designated by the suffix N, of C600 (rK*niK*; Arber and Dussoix, 1962) and its hsdfl derivative 5K (rK'^K"*; Hubacek and Glover, 1970). C600N recAl was constructed by PI contransduction of recAl with a selectable sf/::Tn JO mutation. Other strains were B251 (ra'niB'; Arber and Dussoix, 1962) and WA2899 (rA*mA*"; Arber and WautersWillems. 1970), RRI(PI) carried prophage P1c(Ts) TnS, MC4100 has a/ac deletion (Casadaban, 1976).

Insertionai mutagenesis and gene replacement Jn1732 mutants were obtained in RU4406 (Ubben and Schmitt, 1986), Tn5 mutagenesis and gene replacement using JC7623 (recBC sbc15 sbcC) were performed as described by Rees eM/. (1987).

Experimental procedures

Antibaoteriai agents

Ptasmids

The following were used at the indicated concentration:- ampicillin (Ap), 100 ng mi"''; kanamycin (Km), 10 fig mrV tetracycline (Tc). 7.5 ng ml"'; nalidixic acid (Nal), 25 ng m l " ' .

Plasmids are described in Table 4 and Fig, 3. The derivation of

1940

T. D. Read, A. T. Thomas and S, M. Wilkins

Transformation

Acknowledgements

A standard procedure was used, involving ice-cold competent cells in 100 mM CaCI^ and a 2-min heat shock at 42^0. Transformed cells were incubated at 37''C for 1 h in nutrient broth before plating on nutrient agar containing an appropriate selective antibiotic. DNA for transformation was isolated by the method of Birnboim and Doly (1979).

pLG294 and pLG295 were constructed by Viviana Corich. We thank Stuart Glover, Erich Lanka and Geraldine Willshaw for the gift of RI 24. pMS119EH and HE, and NTP14, respectively. We gratefully acknowledge helpful discussions with Bill Brammar, Louise Jones. Erich Lanka and Noreen Murray. T.D.R. was supported by a research studentship from the Medical Research Council.

Conjugation Bacteria from overnight cultures were grown in nutrient broth for three mass doublings toe, 2 x lO" organisms per ml. Mating mixtures contained equal volumes of donor and recipient cells and were incubated at 37"C. Mating was interrupted by vigorous blending and transconjuganfs were selected on nutrient agar containing Km and Nal. Nalidixic acid was also added to the phosphate butfer used for blending and dilution. Where indicated, interrupted bacteria were incubated in the presence of SDS (0-04%) and Nal prior to plating. Data shown in graphical form are mean values of at least two experiments. The lower limit of transconjugant detection was 10^ per ml of mating mixture.

Phage A assays All tests involved Xvir. X.0 was propagated on RR1 and k.K on C600. An e.o.p, of X.O on a strain determining a type I or III R-M system is expressed relative to plaque-forming ability on 5K. Plating cells were in mid-exponential phase of growth in nutrient broth, MgSO^ (10 mM) was added to plating cells and agar. When appropriate, IPTG was added at 1 mM to bacteria at least 1 h before plating. For single cycle growth tests, bacteria were grown in broth supplemented with MgSOj and maltose (0.2%), concentrated to c. 2 x 10^ per ml and mixed with phage at a multiplicity of infection of 0.05. After 15 min for adsorption, bacteria were washed to remove unadsorbed phage. diluted 100-fold in nutrient broth and incubated with aeration at 37'"'C. After 70 min the culture was treated with chloroform.

Assay of [i-galactosidase The method of Jones etal. (1992) was used.

DNA techniques Standard methods are described in Maniatis etal. (1982).

Nucieotide sequencing Sequencing was performed by the dideoxynucleotide chain termination method on denatured ds plasmid DNA using the T7 Sequencing Kit (Pharmacia-LKB Biotechnology). Primers were the M13 universal and reverse primers and an 18-mer complementary to a sequence within ard. Programs obtained from the Genetics Computer Group were used for data analysis (Devereux atal.. 1984).

References Achtman, M., Morelli, G., and Schwuchow, S. (1978) Cell-cell interactions in conjugating Escherichia coli: role of F piii and fate of mating aggregates. J Bacteriol i35\ 1053-1061. Arber, W.. and Dussoix, D. (1962) Host specificity of DNA produced by Escherichia coli. 1. Host controlled modification of bacteriophage X. JMolBlol5: 18-36. Arber, W., and Wauters-Willems, D. (1970) Host specificity of DNA produced by Escherichia coli. XII. The two restriction and modification systems of strain 15T~. Mot Gen Geriet 108:203-217. Bandyopadhyay. P.K., Studier, F.W.. Hamilton, D.L., and Yuan, R. (1985) Inhibition of the type I restriction-modification enzymes EooB and EcoK by the gene 0.5 protein ot bacteriophage T7. J Mo/S/o/182: 567-578, Belogurov, A.A,, Yussifov, T.N., Kotova. V.U., and Zavilgelsky, G.B. (1985) The novel gene(s) ARO of plasmid pKMIOI: alleviation of £coK restriction. Mol Gen Genet 198: 509-513. Bickle, T.A. (1987) DNA restriction and modification systems. In Escherichia coli and Salmonella typhimurium: Cellular and Moiecuiar Bioiogy. Neidhardt, F.C, Ingraham. J,L,, Low, K.B,, Magasanik, B., Schaechter, M., and Umbarger, H.E. (eds), Washington, DC: American Society for Microbiology, pp.692-696, Birnboim, H.C.. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant piasmid DNA. NucI Acids Res7: 1513-1523. Bolivar, F,, Rodriguez, R.L, Greene, P,J,, Betlach, M.C, Heynecker, H,L,, Boyer. H.W., Crosa, J,H,, and Falkow, S, (1977) Construction and characterisation of new cloning vehicles. II. A multipurpose cloning system. Gene 2: 95-113. Boulnois. G,J., and Wilkins. B.M. (1978) A Coil-specified product, synthesized in newly infected recipients, limits the amount of DNA transferred during conjugation of Escherichia coli K-12. J Bacteriol 133:1 - 9 . Boyer, H. (1971) DNA restriction and modification mechanisms in bacteria. Annu Rev Microbiol 25.153-176. Casadaban, M.J, (1976) Transposition and fusion of the lac genes to selected promoters in Escherichia co//using bacteriophage lambda and Mu. J Mol Biol WA: 541-555. Daniel, A.S.. Fuller-Pace; F.V., Legge, D.M., and Murray, N.E. (1988) Distribution and diversity of hsdgenes in Escherichia co//and other enteric bacteria. J Bacteriol M^: 1775-1782, Day, S.R, (1977) UV-induced alleviation of K-specific restriction of bacteriophage X. J Wra/21:1249-1251. Oelver. E.P,, Kotova, V.U.. Zavilgelsky, G.B., and Belogurov, A.A. (1991) Nucleotide sequence of the gene [ard) encoding the antirestriction protein of plasmid Collb-P9. J Bacteriol 173:5887-5892.

Restriction evasion during conjugation Devereux, J.. Haeberii. P., and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. NucI Acids Res 12: 387-395. Firman, K.. Creasey, W.A., Watson, G.. Price. C . and Glover, S.W. (1983) Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3. Moi Gen Gene/191:145-153. Furste, J.P.. Pansegrau, W., Frank. R,, Blbcker. H,, Scholz, P., Bagdasarian, M., and Lanka, E. (1986) Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene 48: 119-131. Glover, S.W.. and Colson, C. (1965) The breakdown ot the restriction mechanism in zygotes of Escherichia coii. Genet ResB: 153-155. Gormley. E.P., and Oavies. J. (1991) Transfer of plasmid RSF1010 by conjugation from Escherichia co//to Streptomyces iividans and Mycobacterium smegmatis. J Bacterioi 173:6705-6708, Guiney Jr, D.G. (1984) Promiscuous transfer of drug resistance in gram-negative bacteria. J Infect Dis 149: 320-329. Howland, C.J., Rees, CE.D., Barth, P.T.. and Wilkins, B.M. (1989) The ssb gene of plasmid Collb-P9. J. BacteriolMV. 2466-2473. Hubacek. J., and Glover. S.W. (1970) Complementation analysis of temperature-sensitive host specificity mutations in Escherichia coii. J Mol Biol 50:111 -127, Jones. A.L., Barth. P.T.. and Wilkins. B.M. (1992) Zygotic induction of plasmid ssb and psiB genes following conjugative transfer of Inch piasmid Collb-P9. Mol Microbioi 6: 605-613. Kokotek. W.. and Lotz, W. (1989) Construction of a lacZkanamycin-resistance cassette, useful for site-directed mutagenesis and as a promoter probe. Gene84:467-471. Kornberg, A., and Baker, T.A. (1992) DNA Replication, second edition. New York; W.H. Freeman and Co., p.577. Kruger, D.H., and Bickle, T.A. (1983) Bacteriophage survival: multiple mechanisms for avoiding the deoxyribonucteic acid restriction systems of their hosts. Microbioi Rev 47: 345-360. Loh. S., Cram, D., and. Skurray, R. (1989) Nucieotide sequence of the leading region adjacent to the origin of transfer on plasmid F and its conservation among conjugative plasmids. MoiGen Genef219:177-186. Maniatis, T., Fritsch, E.F., and Sambrook. J. (1982) Moiecuiar Cioning: A Laboratory Manuai. Cold Spring Harbor. New York: Cold Spring Harbor Laboratory. Marra, A., and Schuman, H.A, (1989) Isolation of a Legionella

1941

pneumophila restriction mutant with increased ability to act as a recipient in heterospecific matings. J Bacterioi 171: 2238-2240. Mazodier, P., and Davies. J. (1991) Gene transfer between distantly related bacteria. Annu Rev Genet 25:147-171. Moffatt, B.A., and Studier, F.W. (1988) Entry of bacteriophage T7 DNA into the cell and escape from host restriction. J Bac-

terioi ^70:2095-2^ 05. Rees. C.E.D., Bradley. D.E., and Wilkins, B.M. (1987) Organization and regulation of the conjugation genes of IncI, plasmid Collb-P9. Plasmid^8: 223-236. Rees, C.E.D., and Wilkins, B.M. (1990) Protein transfer into the recipient cell during bacteria! conjugation: studies with F and RP4, Mo/M/crofi/o/4:1199-1205. Smith, H.R,, Humphreys, G.O,, Willshaw, G.A., and Anderson. E.S, (1976) Characterisation of plasmids coding for restriction endonuclease EcoRi. /Wo/Gen GeneM43:319-325. Studier, F.W., and Bandyopadhyay, F.W. (1988) Model for how type I enzymes select cleavage sites in DNA. Proc NatiAcad

Sci USA 85: 4677^68-i. Thoms, B., and Wackernagel, W. (1982) UV-induced alleviation of X, restriction in Escherichia coii K-12: kinetics of induction and specificity of this SOS function. Moi Gen Gene/186: 111-117. Trieu-Cuot, P., Carlier, C. and Courvalin. P. (1988) Conjugative plasmid transfer from Enterococcus (aecalis to Escerichia coii. J Bacterion70\ 4388-4391. Trieu-Cuot, P., Carlier, C , Poyart-Salmeron, C . and Courvalin. P. (1991) Shuttle vectors containing a multiple cloning site and a lacZa gene for conjugal transfer of DNA from Escheriohia coii to Gram-positive bacteria. Gene 102: 99-104. Ubben, D.. and Sohmitt. R. (1986) Jn1721 derivatives for transposon mutagenesis, restriction mapping and nucieotide sequence analysis. Gene 41:145-152. Wilkins, B.M., Rees. C.E,, Thomas, AT., and Read, T.D. (1991) Conjugative events in the recipient cell affecting plasmid promiscuity. Piasmid25: 227. Wiiietts, N.. and Wilkins. B.M. (1984) Processing of plasmid DNA during bacterial conjugation. Microbiol RevAB: 24-41. Wilson, G.G.. and Murray, N.E. (1991) Restriction and modification systems. Annu Rev Genet 25: 585-627. Yanisch-Perron, C , Viera, T., and Messing, J. (1985) Improved Ml 3 phage cloning vectors and host strains: nucieotide sequences of the Mi3mp18 and pUCi9 vectors. Gene 33: 103-119.