Modification of EcoRI restriction sites by Caulobacter vibrioides

Gene, 17 (1982) 163-166 Elsevier Biomedical Press

Modification

163

of EcoRI restriction sites by Caulobacter vibrioides

(Plasmid RPl; endonuclease susceptibility; stalked bacteria)

David R. Scholl *, R. Bruce Patterson Jr. and Joseph D. Jo&k Department of Zoology and Microbiology and The College of OsteopathicMedicine, Ohio lJniversity,Athens, OH45701 (U.S.A.) (Received October 14th, 1981) (Accepted November 9th, 1981)

SUMMARY

of EcoRI digestion profiles of plasmid RPl isolated from Caulobacter vibrioides WS48 and Escherichia coli CSH29 demonstrated that EcoRI sites were modified by WS48. A comparison

INTRODUCTION

Two types of DNA modification in bacteriophagehost systems are recognized: hypermodiflcation of DNA, as seen with E. coli phage T4, resulting from the phage-specified methylation of host-derived cytosines (Warren, 1980); and modification of specific nucleotide sequences, affecting the susceptibility of DNA to site-specific restriction endonucleases (Nathans and Smith, 1975). The possibility that Caulobacter vibrioides WS48 might modify EcoRI sites was suggested during characterization of its phage $6. We found that the double-stranded DNA of this phage was EcoRI insen* Present address: Roche Institute of Molecular Biology, Nutley, NJ 07110 (U.S.A.). Address reprint requests to D.R.S. Abbreviations: cfu, colony-forming units; EtBr, ethidium bromide; SDS, sodium dodecyl sulfate.

0378-1119/82/0000-0000/$02.75

0 Elsevier BiomedicalPress

sitive and also that WS48 chromosomal DNA was resistant to this enzyme. Gross chemical modification of phage $6 DNA was not detected by enzymatic digestion and subsequent paper chromatopaphy. However, the possibility of specific minimal modification of the DNA at EcoRI sites could not be excluded. Evidence for methylation of Caulobacter DNA has been reported, but the functional relationship between the methylated nucleotides and restriction endonuclease activity was not addressed (Degnen and Morris, 1973). It was reasonable to assume that if phage $6 DNA was being modified by WS48 then other DNA introduced into this host should undergo similar modification. We selected plasmid RPl, since Alexander and Jollick (1977) previously reported the transfer of this broad-host-range plasmid from Pseudomonas aenrginosa and E. coli to Caulobacter, and since Grinsted et al. (1977) reported the presence of only a single EcoRI site in RPl .

164

MATERIALS

AND METHODS

(d) Isolation

(a) Organisms

of plasmid RPl

Plasmid RPl DNA was extracted from CSH29 and WS48 by a modification of the method of Birnboim

Bacterial strains used in this study, i.e. Caulobacter vibrioides WS48*, Escherichia coli CSH29 and Pseuplasmid RPl ,

domonas aeruginosa PA067, containing as well as bacterial growth conditions detail by Hua et al. (1981).

are described in

Bacteriophage

propaga-

tion was by the agar layer method (Adams, 1959).

and Doly (1979).

Cells were collected

from 1 liter

broth cultures (approx. lo9 cfulml) by centrifugation and the cell pellets from each culture were resuspended

in 36 ml of 50 mM glucose,

10 mM EDTA

and 25 mM Tris, pH 8.0. After adding 80 ml of 0.2 M NaOH and 1% SDS, the solutions were thoroughly mixed, cooled on ice for 10 min and 40 ml of 3 M

(b) Mating procedure

potassium

acetate (pH 4.8) was added. The bacterial

lated on L-agar (per liter: 10 g tryptone, 5 g yeast extract, 0.5 g NaCl, 2 ml 1 N NaOH, 15 g agar and 10

chromosomal DNA precipitate was removed by centrifugation and the supernatant was filtered through sterile Whatman 3 MM filter paper. Plasmid DNA and contaminating RNA in the supernatant were precipitated with 100 ml of isopropanol at -20°C. The plasmid DNA precipitate was rinsed in cold 70% ethanol and air-dried. The dried precipitate was dissolved in 3.8 ml of 10 mM Tris, pH 7.5, 10 mM EDTA to which was added 0.4 ml of a stock EtBr solution (5 mg/ml). This solution was brought to a density of 1.72 g/cm3 by addition of CsCI. The CsClplasmid mixture was centrifuged for 40 h at 17”C, 40 000 rev./min in a Beckman SW 50.1 rotor. Plasmid bands were collected and diluted with 4 ~01s. of distilled water followed by precipitation with 2

ml of a sterile 20% glucose solution)

~01s. of cold 95% ethanol.

Plasmid RPl transfer was by conjugation. Donor and recipient cell titers were adjusted to approximately 3 X lOa cfu/ml, mixed at a donor/recipient ratio of 1 : 10 and then incubated for 3 h, at the optimum growth temperature of the recipient. Mating mixtures were then diluted and plated on appropriate selective media. In the case of transfer of RPl from PA067 to CSH29, transcipients were isolated on minimal thiamine

medium containing tryptophan (40 pg/ml), (5 pg/ml) and kanamycin (2O/.&ml). Plas-

mid RPl was also transferred from C. vibrioides WS48 to CSH29. Transcipients in this cross were iso-

benicillin

(25 /&ml).

containing

Counter-selection

car-

of the CauZo-

batter donor was effected by its inability

to grow on

L-agar. In all cases, newly isolated transcipients

were

tested for kanamycin, tetracycline and carbenicillin resistance as well as susceptibility to the RPl-specific phages PRRl

and PRDl .

(c) Preparation Bacteriophage

of phage $6 DNA $6 suspensions

containing

1O’* pfu/

ml were treated with five cycles of Tris-EDTA-saturated phenol extractions. Phenol was removed from the aqueous DNA fraction by exhaustive dialysis.

The plasmid DNA pellet

was rinsed in cold 70% ethanol, air-dried and dissolved in 200 1.11TNE buffer (50 mM Tris, pH 7.5, 100 mM NaCl and 5 mM EDTA). RNase was added to give 100 E.cg/mland the solution incubated

at 37°C

for 1 h. The solution was brought to 0.5% SDS and protease was added to give 100 pg/ml. Incubation at 37°C was continued for 1 h. The RNased, proteased plasmid preparation was extracted with 1 vol. of phenol and then reprecipitated in cold 95% ethanol. The plasmid precipitate was rinsed with 70% ethanol, air-dried and finally dissolved in 1 mM EDTA, 10 mM Tris, pH 7.5, prior to agarose gel electrophoresis or restriction endonuclease digestion. (e) Agarose gel electrophoresis

* C. vibrioides WS48 is the new designation of C. vibrioides CV6 described in previous publications. The change is made to conform to a uniform Caulobacter strain designation scheme proposed by Dr. B. Ely.

Horizontal gels containing 0.8% agarose were made up in a low salt buffer containing 40 mM Tris-acetate and 2 mM EDTA adjusted to pH 7.9. Gel runs were usually 200 volt-hours constant voltage. Gels were stained with EtBr and photographed using a 300 nm UV light source and Polaroid type 667 film.

165

(f) Restriction

endonuclease

All restriction

endonucleases

Research Laboratories conditions

were

E. coli. The same three bands representing

digestion were from Bethesda

(BRL). Digestion

those

described

buffers and

in the

enzyme

product profile supplied by BRL.

the three

plasmid forms are seen in lane c, whereas only a single band representing linear RPl is seen in lane f. To demonstrate

that our EcoRI digestion protocols were

correct and that the Caulobacfer plasmid RF’1 preparation did not affect restriction we included

an internal

endonuclease

X DNA control

activity with RPl

from Caulobacter in an EcoRI digest. This control is in lane a and shows the three forms of RPI as seen in RESULTS AND DISCUSSION

lanes b and c, as well as the expected

EcoRI frag

ments generated from the h DNA. RPl plasmid DNA isolated from Pseudomonas or Escherichia contains

Lane d contains

the same Caulobacter RPl prepa-

a single EcoRI site and a single

ration as found in the first three lanes; however, it has

Hind111 site (Grinsted et al., 1977), the cleavages of which may be detected by a change in electrophoretic

been treated with HindIII. Hind111 digestion was included because although the site base sequence dif-

mobility as a result of the change from forms I and/or II to linear form III. Fig. 1 shows an agarose gel elec-

fers from that of EcoRI, the base compositions of the sites are identical. The bands representing forms I

trophoresis of EcoR.I and Hind111 digests of plasmid Rpl isolated from both Caulobacter and Escherichia, as well as of phage @6 and h DNA, and appropriate controls. The results indicate that plasmid RPl isolated from Caulobacter is not cleaved by EcoRI,

and II are no longer present and a heavy form III band is seen, as would be expected of a plasmid with

whereas that isolated from Escherichia is linearized. Both plasmid preparations are cleaved by HindIII. Lanes b and e, containing untreated RPl from Caulobacfer and Escherichia, respectively, show the same three bands representing the three plasmid forms. Lane c showsEcoRI-treated plasmid RPl from Caulobatter, and lane f shows EcoRI-treated plasmid from

a single Hind111 site. As expected, in lane g, Hind111 digestion of the E. coli plasmid also results in the change of forms I and II to linear form III. Lanes h, i, and k contain phage $6 DNA. Lane h, the untreated control, lane i the EcoRI digest and lane k the Hind111 digest, are identical in electrophore tic mobility. To assure that the EcoRI resistance of plasmid RI’1 derived from C. vibrioides WS48 was due to a host-specified modification and not the result of selection for a mutant plasmid, we also established the EcoRI restriction pattern of RPl that had been introduced

into E. coli CSH29 from WS48. Although

the EcoRI

did not cleave the plasmid isolated from

WS48, the EcoRI susceptibility

was reestablished

by

transfer of RI’1 from WS48. Fig. 2 shows an agarose gel electrophoresis of EcoRI and Hind111 digests of plasmid RPl isolated from E. coli that had received the plasrnid from WS48 (lanes d-f) and from Pseudomonas (lanes g-i), as well as RPl from WS48 (lanes a-c). Lanes a, d, and g represent the untreated controls. Lanes b, e, and h are the EcoRI-treated samples and lanes c, f, and i are the HindIII-treated Fig. 1. Agarose-gel electrophoresis of restrictionendonuclease digests of plasmid RPl from C. vibrioidesWS48 and E. coli CSH29 and of 06 DNA. a, RPl from WS48 + h DNA + EcoRI; b, RPl from WS48 untreated control; c, RPl from WS48 + EcoRI; d, RPl from WS48 +HindIII; e, RPl from CSH29 untreated control; f, RPl from CSH29 + EcoRI; g, RPl from CSH29 +HindIII; h, ~6 DNA untreated control; i, 06 DNA + EcoRI; k, @6 DNA + HindIII.

samples. The results described in this communication indicate that C. vibrioides WS48 has the capacity to modify the EcoRI site of plasmid RPl. The resistance of phage $6 DNA could be due to the modification of EcoRI sites or the absence of this specific sequence in the DNA. Further study will be required to deter-

date whether

a functional

correlation

exists between

these two observations.

ACKNOWLEDGEMENTS

We thank Dr. Christine discussions

K. Shewmaker

for helpful

and Ms. Mary C. Walker for excellent

technical assistance.

Fig. 2. Agarose-gel electrophoresis of restrictionendonuclease digests of plasmid RPl from C. vibrioides WS48 (a-c) and E. coli CSH29 transcipients derived from matings with WS48-containing RPl (d-t) and Paeruginosa PA067containing RPl (g-i), a, RPl from WS48 untreated control; b, RPl from WS48+EcoRI; c, RPl from WS48+ifindIII; d, RPl from CSH29 untreated control; e, RPl from CSH29+ EcoRI; f, RPl from CSH29+HindIII; g, RPl from CSH29 untreated control; h, RPl from CSH29 + EcoRI; i, RPl from CSH29 +HindIII.

mine which case exists. Additionally, this study does not identify the nature of the modification. The capacity of WS48 to modify EcoRI restriction endonuclease sites is, however, to the best of our knowledge, the first report of a sequence-specific modification of DNA in Caulobacter. This finding is of additional interest since the report by Hua et al. (198 l), which showed functional modification of the plasmid RPl-specified pilus by C. vibrioides WS48. Further study is required to eluci-

REFERENCES Adams, M.H.: Bacteriophages, pp. 134-162. Interscience, New York, 1959. Alexander, J.L. and Jollick, J.D.: Transfer and expression of Pseudomonas plasmid RPl in Caulobacter. J. Gen. Microbiol. 99 (1977) 325-331. Birnboim, H.D. and Doly, J.: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7 (1979) 1513-1523. Degnen, S.T. and Morris, N.R.: Deoxyribonucleic acid methylation and development in Caulobacter bacteroides. J. Bacterial. 116 (1973) 48-53. Grinsted, J., Bennett, P.M. and Richmond, M.H.: A restriction enzyme map of the R plasmid RPl. Plasmid 1 (1977) 34-37. Hua, T.-C., Scholl, D.R. and Jollick, J.D.: Functional modification of the plasmid RPl-specified pilus by Caulobatter vibrioides. J. Gen. Microbial. 124 (1981) i19128. Nathans, D. and Smith, H.P.: Restriction endonucleases in the analysis and restructuring of DNA molecules. Annu. Rev. Biochem. 44 (1975) 273-293. Warren, R.A.J.: Modified bases in bacteriophage DNAs. Annu. Rev. Microbial. 34 (1980) 137-158. Communicated

by A.M. Skalka.