JOURNAL OF BACTERIOLOGY, Oct. 1991, p. 6421-6427

Vol. 173, No. 20

0021-9193/91/206421-07$02.00/0 Copyright ©D 1991, American Society for Microbiology

Use of Cloned DNA Methylase Genes To Increase the Frequency of Transfer of Foreign Genes into Xanthomonas campestris pv. malvacearumt ROBERT DE FEYTERt AND DEAN W. GABRIEL* Plant Pathology Department, University of Florida, Gainesville, Florida 32611 Received 21 May 1991/Accepted 7 August 1991 In vitro-packaged cosmid libraries of DNA from the bacterium Xanthomonas campestnis pv. malvacearum were restricted 200- to 1,000-fold when introduced into Mcr' strains of Escherichia coli compared with restriction in the Mcr- strain HB101. Restriction was predominantly associated with the mcrBC' gene in E. coli. A plasmid (pUFRO52) encoding the XmaI and Xmall DNA methylases was isolated from an X. campestris pv. malvacearum library by a screening procedure utilizing Mcr+ and Mcr- E. coli strains. Transfer of plasmids from E. coli strains to X. campestris pv. malvacearum by conjugation was enhanced by up to five orders of magnitude when the donor cells contained pUFR052 as well as the plasmid to be transferred. Subcloning of pUFRO52 revealed that at least two regions of the plasmid were required for full modification activity. Use of such modifier plasmids is a simple, novel method that may allow the efficient introduction of genes into any organism in which restriction systems provide a potent barrier to such gene transfer.

Genetic analysis of many strains of bacteria is hindered by the presence of restriction-modification (RM) systems, which make it difficult to introduce or establish exogenous DNA in these strains. Many RM systems have been well characterized (4), and the genes encoding these have been cloned from numerous bacteria (16). The enzymes responsible for restriction and modification are usually site-specific endo-DNases (ENases) and DNA methyltransferases (MTases), respectively. RM systems have been classified according to gene and protein structure, cofactor dependence, and specificity of the binding and cleavage reactions (13). In the largest group, class II, the ENase and MTase act as separate proteins. Class II MTases transfer methyl groups from S-adenosylmethionine to either cytosine or adenine bases at specific sites on double-stranded DNA, while the corresponding ENases cleave unmodified DNA at sites of identical specificity. In contrast to most restriction ENases, some restriction systems recognize and cleave specifically modified DNA sequences. Three such restriction systems in Escherichia coli have been described. The McrA and McrBC systems restrict DNA modified at cytosines in the sequences CG and GC, respectively (22), while Mrr restricts DNA modified at adenine residues in the sequence GAC or CAG (12). The McrBC system, in particular, has been implicated in the restriction of methylated eucaryotic DNA in Mcr+ strains of E. coli. This can result in a significant reduction in the number of clones obtained when making genomic libraries of eucaryotic DNA and reduced representation of some DNA sequences in such libraries (21, 27). DNA isolated from bacteria generally does not suffer significant mcr-mediated restriction when introduced into E. coli, perhaps because most bacteria modify their DNA to a lesser extent than eucaryotes (20). However, DNA frag-

ments containing MTase genes from some bacteria are strongly selected against in Mcr+ or Mrr+ strains of E. coli (12, 22). This would be expected if the MTase methylates DNA sites which are subsequently recognized and cleaved by the Mcr or Mrr system. In this report, we show that cloned MTase genes, maintained initially in an Mcr- host, may be identified in a genomic library by their inability to be maintained in Mcr+ hosts. We have used this principle to isolate an MTase gene from the bacterium Xanthomonas campestris pv. malvacearum, the causal agent of bacterial blight of cotton. This organism is known to contain at least three restriction systems, which have been designated XmaI, XmaII, and XmaIII (9, 14). We have found that X. campestris pv. malvacearum strains are refractory to DNA introduction and have sought methods to improve the transfer frequency of new genes into these organisms. We have previously described small plasmids which function as stable shuttle vectors for Xanthomonas spp. (7). These can be introduced into several xanthomonads at frequencies of up to 10-3 per recipient by conjugation. However, in X. campestris pv. malvacearum the transfer frequency is 10-6 per recipient, and when inserts of 35 to 40 kb are cloned into these vectors, the transfer frequency is decreased to the order of 10-11 to 10-12. This makes it difficult to introduce cosmid-based libraries into strains of this pathovar. We report here the cloning of the XmaI and XmaIII DNA methylase genes and their use in an improved conjugation method to enhance the mating frequency by up to five orders of magnitude in X. campestris pv. malvacearum. MATERIALS AND METHODS

Bacterial strains, plasmids, and phage. The bacterial strains and plasmids used in this study are listed in Table 1. Strains ER1378, ER1381, ER1564, ER1565, and ER1648 were kindly provided by E. Raleigh. Bacteriophage XplaccI857 was obtained as an s+ revertant of Xgtll by plating on E. coli HB101 and was propagated on this strain. Media. E. coli strains were grown on LB medium (17) at

* Corresponding author. t Florida Agricultural Experiment Station Journal Series contri-

bution R-01593. t Present address: CSIRO-Division of Plant Industry, Canberra, ACT, Australia.

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DE FEYTER AND GABRIEL

TABLE 1. Bacterial strains and plasmids Relevant characteristics

Strain or plasmid

E. coli DH5at

F- endAl hsdR17 (rK- MK+) supE44 thi-1 recAl gyrA relAl

+80dlacZAM15 A(lacZYA-argF)U169

DH5aMCR

DH5a mcrA mcrBC mrr

ER1378 ER1381 ER1564 ER1565 ER1648 HB101

mcrA' mcrBCl mcrA' mcrBC', isogenic to ER1378 mcrAI272::TnIO mcrBC', isogenic to ER1381 mcrA]272::TnlO mcrBl, isogenic to ER1564 mcrAl272::TnlO A(mcrBC-hsd-mrr)2::zj-202::TnIO F- hsdS20 recAl3 ara-14 proA2 lacYl galK2 rpsL20(Smr) xyl-5 mtl-l supE44 X

X. campestris pv. malvacearum XcmH XcmN

XcmlO03 XcmlOO5 Plasmids pKT210 pLAFR3 pRK2073 pUC19 pUFA704 pUFRO34

pUFRO35 pUFRO39 pUFRO40 pUFRO41 pUFRO42 pUFRO43 pUFRO51 pUFRO52 pUFRO54 pUFRO55 pUFRO56 pUFRO59 pUFR060 pUFRO63 pUFRO70 pUFRO71 pUFRO72 pUFRO73

Source or reference

Bethesda Research Laboratories Bethesda Research Laboratories 22 22 22 22 22 3

10 10 6 This study

Spcr Rif' derivative of XcmN Spcr Rif' derivative of XcmH IncQ, Mob' Cmr Smr IncP, Mob' Tcr cosmid ColEl replicon, Tra+ Mob' spr, helper plasmid ColEl replicon, Apr lacZo+, MCS' Kmr cosmid clone containing 38-kb insert of XcmH DNA IncW, Kmr Mob' lacZa+ cosmid ColEl replicon, Mob- Cmr IncW, Gmr Kmr Mob' lacZa' IncW, Gmr Kmr Mob' lacZa+ Cosmid derivative of pUFRO40 IncW, Gmr Kmr Mob' lacZa+ Cosmid derivative of pUFRO42 IncP, Tcr lacZa' cosmid pUFRO34 with 42.4-kb insert of XcmH DNA, containing M XmaI and M XmaIII genes pUFRO51 containing 31.7-kb KpnI fragment from pUFRO52 Same as pUFRO54; KpnI fragment in reverse orientation pUFRO51 containing 26.6-kb XbaI fragment from pUFRO52 pUFRO40 containing 13.8-kb EcoRI fragment from pUFRO52 pUFRO59 deleted for 8.7-kb Hindill fragment pUFRO51 containing 8.9-kb BamHI fragment from pUFRO52 pUFRO40 containing 11.4-kb Hindlll fragment from pUFRO52 Derivative of pUFRO40 lack EcoRI site Cmr Smr pKT210 containing a 6-kb EcoRI fragment from pUFA704 pUFR063 deleted for 2.5-kb EcoRI fragment

1 23 25 28 10 7 7 6 6 This 6 This This This

This This This This This This This This This This

study

study study study study study study study study study study study study study

a MCS, multiple cloning site.

37°C, and X. campestris strains were grown at 30°C in PYGM medium (7). When appropriate, antibiotics were added at the following final concentrations (in micrograms per milliliter): chloramphenicol (Cm), 20; gentamicin (Gm), 1; kanamycin sulfate (Km), 20; rifampin (Rif), 75; spectinomycin (Sp), 35; streptomycin sulfate (Sm), 100; tetracycline (Tc), 15. For Lac' screening, 5-bromo-4-chloro-3indolyl-p-D-galactoside (X-Gal) was added at 40 pLg/ml and isopropyl-,-D-thiogalactopyranoside (IPTG) was added at 4 P,g/ml. Recombinant DNA methods. Plasmids were isolated by alkaline lysis methods from E. coli (2) and X. campestris (6) and purified by CsCl-ethidium bromide gradient fractionation when required (17). Xanthomonas total DNA was prepared essentially as described elsewhere (5). Restriction digests were carried out as recommended by the manufacturers except for XmaIII digests, which were incubated at

30°C for 16 h. All other recombinant DNA methods, including the construction of cosmid libraries, were those of Maniatis et al. (17). In vitro packaging of DNA was carried out with extracts from Stratagene (La Jolla, Calif.) and was performed as recommended by the manufacturer. E. coli strains were infected with phage particles after suspension of cells in sterile 10 mM MgSO4. Construction of plasmids. Cosmid pUFRO43 was constructed as follows. The smaller SaiI fragment containing the Gmr gene of pUFRO39 (7) was ligated to the larger SalI fragment of cosmid pUFRO34, forming pUFRO41. One SalI site was deleted by partial digestion of pUFRO41 with Sall, treatment with DNA polymerase I (Klenow fragment) plus deoxynucleotide triphosphates, and ligation with T4 DNA ligase, forming pUFRO43. This cosmid replicates stably at low copy number in both E. coli and Xanthomonas spp. and can accept up to 42 kb of DNA for in vitro

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parA

x

0.5

B FIG. 1. Schematic representation of cosmid pUFRO43, which was constructed as described in Materials and Methods. Arrows indicate direction of transcription when known. rep, replication origin from plasmid pSa; cos, phage packaging site; parA, partition locus; mob, conferring plasmid mobilization ability. Restriction sites: B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; K, KpnI; P, PstI; Sa, Sall; Sm, SmaI; Sp, SphI; S, SstI; Ss, SstII; X, XbaI; Xh, XhoI.

packaging. It has unique restriction sites for EcoRI, BamHI, KpnI, SstI, and Sall and is shown schematically in Fig. 1. Plasmid pUFRO51 was constructed by replacing the EcoRI-Hindlll polylinker of pLAFR3 with the polylinker from pUC19, introducing unique KpnI and XbaI sites within the IncP vector. Plasmid pUFRO71 was formed by digesting pUFRO40 with EcoRI, treating with DNA polymerase I (Klenow fragment) and deoxynucleotide triphosphates, and recircularization with T4 DNA ligase. Bacterial conjugation. Transfer of mobilizable plasmids from one E. coli strain to another was achieved by triparental conjugation (8) with pRK2073 as the helper plasmid (15) and from E. coli to rifampin-resistant Xanthomonas strains as follows. Mid-exponential-phase Xanthomonas cultures were harvested by centrifugation and resuspended in 0.05 volume of PYGM medium, and 10-,ll aliquots were spotted onto dry PYGM plates, up to 44 spots per plate. After excess liquid had soaked into the medium, 10 RI of a cell suspension of the E. coli donor and helper strains (each concentrated 10-fold from mid-exponential-phase cultures and mixed in a 1:1 ratio) was spotted onto the Xanthomonas spots. Plates were incubated at 30°C overnight to allow transfer. Cells from each zone were resuspended in 1.0 ml of sterile tap water, and suitable dilutions were plated onto PYGM agar supplemented with the appropriate antibiotics. Transconjugants appeared as yellow colonies after 3 days of growth. Frequencies of transfer were calculated as the number of transconjugants per recipient cell. As mentioned in the text, the helper plasmid in some conjugations was first introduced into the donor cells before conjugation with X. campestris pv. malvacearum.

X. campestris pv. malvacearum DNA is restricted in E. coli DH5a but not in HB101. In attempts to construct gene libraries of X. campestris pv. malvacearum strains, we noticed that some strains of E. coli were more efficient recipients of the X. campestris pv. malvacearum DNA than other E. coli strains. In one experiment, DNA from X. campestris pv. malvacearum XcmH was cut partially with Sau3A and size fractionated, and fragments of 35 to 40 kb were ligated to BamHI-cut, dephosphorylated DNA from cosmid pUFR034. The ligated DNA was packaged in vitro and used to transfect E. coli strains HB101 and DH5a. The number of kanamycin-resistant colonies obtained with HB101 was more than 1,000-fold greater than that with DH5a. When a AplaccI857 lysate was used to transfect the same cell samples, approximately equal numbers of plaques were obtained, showing that both strains were equally sensitive to X phage particles. To further investigate this phenomenon, Sau3A fragments of 15 to 20 kb from XcmH DNA were ligated to BamHI-cut, dephosphorylated pUFRO34 DNA, and the ligated DNA was introduced into DH5a by transformation. Approximately 104 kanamycin-resistant colonies were obtained per microgram of insert DNA; only about one-third of them were white on plates containing XGal, while the others were either pale blue or dark blue. When plasmids from 18 white (Lac-) colonies were examined, all had very small (