Isolation and expression in Escherichia coli of a Xanthomonas oryzae recA-like gene

Gene, 132 (1993) 113-l 18 0 1993 Elsevier Science Publishers

GENE

B.V. All rights

reserved.

113

0378-l 119/93/$06.00

07264

Isolation and expression in Escherichia coli of a Xanthomonas oryzae red-like gene (Recombinant DNA; RecA; DNA repair; mutagen resistance; rice pathogen; RecA antigenically related protein)

Siritida Rabibhadana”, Sangpen Chamnongpol”, and Skorn Mongkolsuka*b

Janine E. Trempy”, Nicholas P. Ambulos Jr.d

aDepartment of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand; bLaboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, Thailand: ‘Department of Microbiology, Oregon State University, Cowallis, OR 97331, USA; and dUniuersity of Maryland Baltimore County, Catonsville, MD 21228, USA Received by A.M. Chakrabarty:

23 December

1992; Revised/Accepted:

1 March/IS

March

1993; Received at publishers:

13 May 1993

SUMMARY

The recA gene from the bacterium Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen, was cloned based on its ability to complement DNA repair defects of Escherichia coli recA- mutants. The Xoo recA was localized to a 1.3-kb Sau3AI-XhoI fragment and, when cloned into pBR322, specifies increased methylmethanesulfonate and mitomycin C resistance to E. coli recA mutants and allows h red- gum- to plaque on an E. coli recA- host. An E. coli recA- strain harboring a plasmid containing the Xoo recA-like gene was shown to produce a 40-kDa protein which cross-reacted with an anti-E. coli RecA antibody. A similar molecular mass protein to RecA has been detected in several Xanthomonas pathovars using an anti-E. coli RecA antibody. Furthermore, the cloned Xoo recA was shown to hybridize to genomic DNA from various Xanthomonas pathovars, but not to genomic DNA from other bacteria species under high-stringency hybridization conditions. These results indicate the isolation of the Xoo recA gene.

INTRODUCTION

Xanthomonas spp. are a group of bacteria which are pathogenic to many economically important plant species. Xanthomonas campestris is also economically important; it produces byproducts such as xanthan gum, Correspondence to: Dr. S. Mongkolsuk, Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Rd., Bangkok 10400, Thailand. Tel. (66-2) 245-5650; Fax (66-2) 246-3026. Abbreviations: A, absorbance (I cm); Ab, antibody(ies); Ap, ampicillin; bp. base pair(s); cfu, colony-forming unit(s); Cm, chloramphenicol; kb, kilobase or 1000 bp; LB, Luria-Bertani (medium); MC, mitomycin C; MMS, methylmethanesulfonate; nt, nucleotide(s); p, plasmid; PAGE, polyacrylamide-gel electrophoresis; pfu, plaque-forming unit(s); pv, pathovar; RecA, recombination protein; recA, gene encoding RecA, a, resistance/resistant; SDS, sodium dodecyl sulfate; Tc, tetracycline; UV, ultraviolet: X., Xanthomonas; X.C., X. campestris; X.0, X. oryzae: Xoo, X. o. pv. oryzae; [I, denotes plasmidcarrier state.

a food thickening agent. Xanthomonas oryzae pv. oryzae (Xoo) is the causative agent of leaf blight, a devastating bacterial disease of rice. A difficulty in controlling infection is that Xoo, like other bacterial plant pathogens, has a high frequency of phenotypic variation. The presence of highly repetitive DNA sequences (Leach et al., 1990) and repeated structure of pathogenicity (pth) and avirulence (aur) genes (Swrup et al., 1992; Bonas et al., 1989), coupled with an active general recombination system, may play a direct role in phenotypic variation in Xanthomonas and hence in its pathogenicity. A general recombination pathway mediated by a RecA-like protein presents a mechanism for this variation. The phenotypic variation could generate new Xanthomonas variants which may overcome plant resistance genes and lead to disease development. Furthermore, the lack of a recA_ mutant in this important family of plant pathogens has hampered many types of genetic manipulations.

114 Escherichia

The

characterized properties ATPase

coli

protein of this

activity,

RecA

(Walker, enzyme

protein

include

and an activity in cellular

that facilitates

functions

gous recombination,

induction

induction,

to DNA-damaging

ation

responses

of stable

DNA

is highly

conserved

species (Miller acterized

among

and Kokjohn,

and Roberts, 1987). The gene

divergent

bacterial

1990). In support

from more than 16 bacterial

Kokjohn,

1990; Martinez-Salazar

of this, and char-

species (Miller and

et al., 1991). In most

recA genes have been identified by virtue to complement functional deficiencies in

cases, bacterial of their ability an E. coli recA-

recA genes have been cloned

ilar strategy genomic

mutant.

was used to isolate

library.

A small aliquot

ing the Xoo genomic colonies

by complementa-

defects of E. coli recA mutants.

tion of pleiotropic

containing

agents, and initi-

of the E. coli recA have been isolated

analogues

1984).

such as homolo-

(Phizicky

(b) Isolation of Xoo recA Many

an

proteo-

(Walker,

1982; Weinstock, even

well-

of DNA repair, prophage

replication

1980; Little and Mount,

a

a recombinase,

lytic cleavage of LexA and h CI repressors RecA is involved

is

1984). The multifunctional

library

100 ug Ap/ml

A sim-

the Xoo ret from the of DHSclMCR

harbor-

was plated on LB agar media and 5 ug MC/ml.

arose after overnight

incubation.

Eight MCR

Upon restreak-

ing on media containing

100 ug Ap/ml and 5 ug MC/ml,

four of the eight colonies

maintained

plasmids

were isolated

ping (Birnboim

and analyzed

and Doly,

clones possessed

the MCR phenotype; by restriction

1979). Plasmids

map-

from all four

the same 2.2-kb Sau3AI restriction

frag-

ment, and thus only one clone was used for further

char-

acterization. was

The plasmid

named

pSM-Al.

observed when media containing

isolated No

from this transformant

MCR

transformants

were

DHSclMCR[pBR322] was plated 100 ug Ap/ml and 5 ug MC/ml.

on

The aim of present study was to clone Xoo recA and detect it by complementation of E. coli recA_ mutants, express this gene in E. coli, immunologically it, and assess the RecA protein

characterize

and gene homologies

in

several Xanthomonas pathovars.

(c) Characterization of XOOrecA The putative Xoo recA plasmid Al,

was purified

and

used

clone, designated

pSM-

E. coli recA

to transform

mutant strains DHSa, HBlOl, and BW368, selecting for ApR. Twenty transformants of each strain were restreaked to media

containing

restreaked EXPERIMENTAL

AND DISCUSSION

en-resistance

(a) Construction of a genomic library from Xoo Xoo was grown in modified SB media at 28°C with aeration; chromosomal the method of Pitcher library

was constructed

DNA was isolated according to et al. (1989). The Xoo genomic according

to the procedures

Silhavy et al. (1984). Xoo isolate 8707 chromosomal 50 ug was partially

digested

with Sau3AI

under

of

system

phosphatase. The Xoo genome conwhich can be cleaved by the E. (Ehrlich

was plasmid

or MC. All

that the mutag-

encoded.

In a paral-

lel experiment, pBR322 was transformed into the same E. coli strains: all control transformants were sensitive to both MMS and MC. Similar results were observed in experiments in which MC was used (data not shown). These data indicate that the 2.2-kb Sau3AI pSM-Al encodes Xoo recA.

fragment

in

condi-

9 kb were electroeluted. The electroeluted DNA was ligated into pBR322 which had been BamHI digested and

coli MCR

phenotype

MMS

grew, indicating

DNA

tions which gave a majority of DNA fragments in the 1.5-9 kb range. The digested DNA was separated on a 0.7% agarose gel, and DNA fragments between 1.5 and

treated with alkaline tains 5methylcytosine

Ap and either

transformants

et al., 1987). To overcome

potential problems with transformation efficiencies, the Xoo genomic library in plasmid pBR322 was propagated

(d) Mutagen dose-response survival curve To demonstrate that the Xoo recA-like

gene encoded

on pSM-Al was capable of complementing a recA- E. coli strain, we compared the survival efficiencies of E. coli BW368 harboring pBR322 with the same host harboring pSM-Al. To do this, plasmid-containing cells were grown in LB-Ap to AeOOnm = 0.7 and serially diluted. Then 0.1 ml of each dilution was spread on plates containing Ap and various concentrations of either MMS or MC. After overnight growth

at 37°C cell survival

was calculated

as the

in E. coli DHSctMCR. It has been observed that the allows mcrA_, mcrBC_, mrr- phenotype of DHSaMCR

percentage of cfu on media containing mutagen plus Ap versus cfu on media containing Ap alone. As a positive

higher

control, included.

a wild-type E. coli LE392[pBR322] was Plasmid pSM-Al was able to restore a level of

mutagen

resistance

transformation

unpublished

observation;

efficiencies de Feyter

of Xoo

DNA

and Gabriel,

(SM., 1991).

Transformants were selected on LB agar plates containing 50 ug Ap/ml; 7 x lo3 ApR transformants were pooled.

recA-

to a recA_

and recA+ host containing

host intermediate pBR322 (Fig. 1).

to a

115

.OOl .OC!Ol,

.

r

0.00

.

0.01

,

.

0.02

,

.

,

0.03 MMS

.

0.04

,

.

0.05

7 0.06

(%)

B

100 10 1 .L 2 ZJ e

1 .l .Ol DO1 0001

.OOOOl, 0.0

.

, 0.2

_

,

BW368[pBR322] or BW368[pSM-Al] and E. coli LE392[pBR322] were grown in TBMM (Gottesman et al., 1981). Each culture (100 ~1) was mixed with an aliquot of h red-gum- phage and incubated at 37°C for 15 min. Each h-infected culture was serially diluted, and each dilution was plated on LB with 3 ml of LB semisoft agar and incubated overnight at 37°C. We observed that h red-gam- did not plaque on BW368[pBR322] but did plaque on BW368[pSM-Al] with a plaquing efficiency nearly equal to plaquing on a rec+(LE392) host (Table I). Plasmid pSM-Al restores repair functions to a recA_ host and also complements recombination functions needed to allow h replication.

.

0.4

,

.

, 0.8

. 1 .o

MC (pg,::) Fig. 1. Mutagen dose-response survival curves of + E. coli recA_ BW368[pBR322] +; BW368[pSM-Al] (squares); and * wildtype E. co/i LE392[pBR322] (crosses) on plates containing various concentrations of MMS (A) and MC(B) Both bacterial cultures were grown in 1 ml of LB broth supplemented with 50 ug Ap/ml. Then 0.1 ml of serial dilutions of both cultures was plated on media containing 100 pg Ap/ml and specified concentrations of either MMS or MC. After incubation overnight, survival efficiencies are expressed as the cfu/ml ratio on plates containing mutagen or no mutagen,

(e) Plaquing h red-gam- on BW36?3[pSM-Al] Phage h replication in E. coli proceeds through either of two independent pathways. One pathway involved the formation of dimeric phage h DNA with concomitant inhibition of host exonucleases by h-encoded proteins Red and Gam. The second pathway requires a functional host recA product to produce packageable DNA from concatenates (Weinstock, 1987). Thus, h phage will not plaque on E. coli if both replication pathways are blocked by using a recA- host and a Red- Gam- h phage. This observation has been exploited for the purpose of cloning ret-like genes in a h red-gum- vector and selecting for recombinant phage which will plaque on a ret- host (Barcak et al., 1989; Martinez-Salazar et al., 1991). E. coli

(f) Immunological characterization of the XOOrecA product Polyclonal Ab generated against the E. coli RecA will cross-react with Ret-like proteins from organisms as evolutionarily disparate as Bacillus subtilis (Marrero and Yasbin, 1988). Ab to E. coli RecA protein was used to screen Western blots of total cellular protein extracts prepared from Xoo cells. A protein of approximately 40 kDa was detected using this Ab, which is similar in size to E. coli RecA (Fig. 2). To determine if pSM-Al encoded the RecA-like protein, total cellular extracts were prepared from Xoo, recA-deleted strain BW368 harboring pBR322 or pSM-Al, and LE392[pBR322] and analyzed by Western blot. The Ab reacted with a 40-kDa protein from LE392[pBR322], Xoo, and BW368[pSM-Al] but not BW368[pBR322] (Fig. 2). Plasmid pSM-Al encodes a protein which is antigenically related to E. coli RecA. (g) Localization of XOOrecA within the 2.2-kh insert in pSM-Al Restriction enzyme deletion analysis was performed to localize the complementing activity of pSM-A 1. HindIIIand EcoRV-mediated deletions (pSM-H and pSM-E, respectively) in the plasmid resulted in the loss of complementation of ret function (Fig. 3). Insertion of the CmR gene (cat) within the XhoI site to generate pSM-C did TABLE

I

Plaque formation of a h red- gam- phage on E. coli recA+ and recAhost harboring Xoo ret gene Host strain[plasmid]”

RecA”

pfu/ml’

LE392[pBR322]

+ + +

6.70 x lo6

BW368[pSM-Al] BW368[pBR322]

1.14 x lo6
“For plasmid pSM-Al see Fig. 3. bE. co/i LE392 as a recA+ host, BW 368 as a recA_ host, and pSMAl carry ret gene of Xoo on a plasmid. ‘Performed as described in section e.

116 not affect the ability of the plasmid to complement recA functions. Thus, Xoo recA was localized to a 1.3-kb Sau3AI-XhoI fragment. (h) Detection of RecA-like

proteins in various

Xanthomonas spp. The section f results indicate that immunological techniques are useful tools for studying the expression and for the isolation of recA in Xanthomonas. Total cellular proteins prepared from various Xanthomonas spp. were used in Western blot analysis, probing with anti-E. coli RecA antibody. The results (Fig. 4) showed a 40-kDa protein from all Xanthomonas spp. tested cross-reacted with the Ab. This confirms not only the presence of RecA homologues but also implicates the use of immunological techniques in the isolation and characterization of RecA from other Xanthomonas spp. (i) Detection of analogous Xoo vecA sequences in various Xanthomonas spp. Gene recA has been shown to be highly conserved Fig. 2. Western blot analysis of E. coli recA- strains expressing the Xoo reed-like gene. O.l%SDS-12%PAGE was performed according to the methods of Laemmli (1970), and the proteins were subsequently electroblotted onto nitrocellulose membrane (Towbin et al., 1979). The membrane was blocked by incubation for 2 h in TBS-Blotto (Johnson et al., 1984). After rinsing with TBS-Blotto, the membrane was incubated for 60 min with TBS-Blotto-diluted (l/1000) anti-E. coli RecA Ab (Marrero and Yasbin, 1988). The filter was then washed three times for 10 min in TBS-Blotto followed by incubation with TBS-Blottodiluted (l/7500) goat anti-rabbit IgG conjugated to alkaline phosphatase. The filter was washed 3 times for 10 min with TBS-Blotto and 10 min in TBS. Alkaline phosphatase detection was done according to the supplier’s recommendations (Promega, Madison, WI, USA). Lanes: 1, BW368[pBR322]; 2, BW368[pSM-Al]; 3, Xoo isolate 8707; 4, LE392[pBR322]. TBS is 10 mM TrisHCl/lS mM NaCl pH 7.4, and TBS-Blotto is TBS with 5% nonfat dry milk.

B/S

RV

ia

ih

S/B

pSM-AI

MMS ’

pSM-RV

MMS ’

pSM-H

MMS ’

pSM-C

MMS R

Fig. 3. Restriction

map and localization

among evolutionary distant microbes (Miller and Kokjohn, 1990). To test the relatedness between Xoo recA and other bacteria recA, chromosomal DNA prepared from several Xanthomonas, E. coli, Pseudomonas, Salmonella, and Klebsiella were analyzed by Southern blot hybridization using the 0.74-kb SalI-XhoI fragment containing a portion of Xoo recA from pSM-AI as a probe. Under high stringency conditions which permitted detection of 80% or greater homologous sequences, the probe only hybridized to DNA from Xanthomonas spp. and not to DNA from other bacteria (data not shown). The results confirmed that the probe was of Xoo origin and that the recA sequence is highly conserved among different Xanthomonas spp. but less so compared with other bacteria. Furthermore, the hybridization patterns

of the gene encoding

the Xoo

RecA-like protein. Deletions extending within the Xoo Sau3AI fragment were constructed by deleting DNA between the Hind111 or EcoRV sites from pSM-Al to generate pSM-H and pSM-RV, respectively. Plasmid pSM-C was constructed by insertion of a Sal1 cassette containing a Cm’ gene (cat; Close and Rodriguez, 1982) into a XhoI restriction site within the cloned insert. B/S and S/B, BamHI/Sau3AI cloning junction; H, HindIII; RV, EcoRV, Sa, SalI; Xh, XhoI.

Fig. 4. Immunological detection of RecA analogues in various Xanthomonas spp. O.l%SDS-12%PAGE, Western blot, and Ab reactions were carried out essentially as described in Fig. 2. Total protein (30 ug) from eight different Xanthomonas pathovars were loaded into each well. Lanes: 1, X.C. pv. campestris; 2, Xoo isolate PX086; 3, X.0. pv. oryzicolar; 4, X.C. pv. phaseoli; 5, Xx. pv. translucens; 6, X.C. pv. citri; 7, X.C. pv. vesicatoria; 8, X. malthophilia; 9, E. coli[LE392]. The positions of protein molecular weight markers in kDa are shown. The IO-kDa protein bands which cross-reacted with the anti-E. coli RecA Ab is indicated by an arrowhead.

117 revealed strong hybridization signals to a 1%kb Hind111 fragment among Xoo and X.0. pv. oryzicolar strains which were absent in the Xanthomonas spp. tested (Fig. 5). This hybridization pattern may be useful in differentiating X.0. from other Xanthomonas spp. (j) Conclusions

We have cloned and characterized recA from a genomic library of Xoo DNA. The cloned ret has the following properties: (I) Complementation of DNA repair defects of E. coli recA mutants by virtue of the ability of the cloned ret to enhance resistance to MMS and MC. (2) Complementation of recombination functions of E. coli ret mutants by virtue of the ability of the cloned ret to allow h red-gam- to plaque on a ret- host. (3) Antigenically related to E. coli RecA as demonstrated by Western blot analysis using an antibody generated against E. coli RecA. (4) The 0.74-kb SalI-XhoI DNA fragment encoding ret

hybridizes only to DNA from Xanthomonas spp. and not from other bacteria tested. Further characterization of Xoo recA may lead to a better understanding and facilitate genetic analysis of the pathogenic mechanisms of Xoo. An understanding of the general recombination system of Xanthomonas might also allow further exploitation of the economic potential of Xanthomonas byproducts.

ACKNOWLEDGEMENTS

Some of the initial work was carried out in Professor P.S. Lovett’s laboratory. We thank Jim Marks for critical reading of the manuscript and R. Yasbin for the gift of anti-RecA Ab. The research work was partially supported by a UNDP grant to Chulabhorn Research Institute, a NIH grant GM42925 to P.S.L., and an OSU center for Gene Research and Biotechnology grant to J.E.T.

REFERENCES

123

4

5

6

kb

9.46.6--

4A-

23-

blot analysis for the relatedness of recA among DNA (5 ug) was digested with HindHI, electrophoresed on 1% agarose gel, and subsequently blotted onto a nylon membrane (Maniatis et al., 1982). The blot was probed with 0.74-kb SalI-XhoI C3’P]fragments containing the coding region of Xoo recA from pSM-Al. The hybridization was performed at 65°C overnight according to Maniatis et al. (1982). The blot was washed twice in 0.5 x SSC at 65°C for 15 min each before the autoradiogram was performed overnight. Lanes: 1, Xoo isolate 8707; 2, Xoo isolate PXO86; 3, X.0. pv. oryzicola; 4, X.C. pv. campestris; 5, X.C. pv. phaseoli; 6, X.C. pv. oesicatoria. Positions of M, markers are given on the left margin, as in the conserved 1.8-kb Hind111 fragments (see arrow) which hybridized to the Xoo recA probe. SSC is 0.15 M NaCl/0.015 M Na,citrate pH 7.6. Fig. 5. Southern

Xanthomonas spp. Bacterial chromosomal

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