FEMS Microbiology Letters 59 (1989) 269-274 269 Published by Elsevier

FEM 03576

Transfer of broad host-range plasmids to sulphate-reducing bacteria

Bridget Powell 1,2, M a x Merg eay 1 and Nicholas Chr is tof i 2

l Department of Biology, SCK/CEN, Center of Studies for Nuclear Energy, Mol, Belgium, and 2 Department of Biological Sciences, Napier College, Edinburgh, Scotland, U.K.

Received 24 November 1988 Revision received 18 January 1989

Accepted 31 January 1989

Key words: Desulfovibrio; Transfer of IncP1 plasmids; Mobilisation of IncQ plasmids; IncP1 mediated retrotransfer (hermaphroditism)

1. SUMMARY 2. INTRODUCTION

The broad-host-range, IncQ, plasmid R300B (Sm, Su) has been stably transferred to two strains of sulphate-reducing bacteria (Desulfovibrio sp. 8301 and Desulfovibrio desulfuricans 8312), using the IncP1 transfer system of the helper plasmid pRK2013 and cocultivation of sulphate-reducing bacteria with facultative anaerobes in media pro- vided with sulphate and nitrate ions as electron acceptors. R300B was transferred at a frequency of 10 -2 to 1 per acceptor cell. The Smg marker was expressed in both sulphate-reducing bacteria strains while the Su R was expressed only in strain 8301. R300B can also be transferred back to E. coli strains provided with IncP1 plasmids taking advantage of the retrotransfer ability of these plasmids. This occurs at a frequency up to 10 - 4

by recipient E. coli cell.

Correspondence to: M. Mergeay, Laboratoire de G6nrtique & Biotechnologie, Departement voor Leefmilieu & Energie, S.C.K.--C.E.N., B-2400 Mol, Belgium.

Sulphate-reducing bacteria (SRB) are a major group of anaerobic bacteria including both gram- negative and gram-positive genera. Due to their almost universal presence in soils, lake sediments and resting waters, they play an important role in geochemical cycles as in corrosion processes [1]. These rather exacting anaerobes are still difficult bacteria to work with from the genetic point of view [1]. DNA from different species of SRB has been cloned into Escherichia coli [2-4]; Voordouw et al. [5] used DNA from Desulfovibrio vulgaris (Hildenborough), cloned into E. coli to isolate the D. vulgaris hydrogenase gene; the gene was iso- lated but the gene product was found to be inac- tive. Li et al. [3] and Fons et al. [2] used the strategy of trying to complement pyrF and pro mutations in E. eoli strains using DNA from D. uulgaris (Hildenborough) and D. desulfuricans (Norway), respectively.

Other work on the genetics of the SRB involved a study of the homology of the structural nif DNA from Klebsiella pneumoniae with DNA from

0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies

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various SRB species; homology was shown to occur with 13 diazotrophic strains of Desulfo- vibrio, and in 3 strains of D. vulgar&, the nif genes were shown to be plasmid-borne [6].

The aim of this study was to determine if broad host range, antibiotic resistance plasmids such a R300B could be transferred into SRB strains from E. coli [7-10]. A medium suitable for co-culture of an obligate anaerobic bacterium (SRB) and aerobic facultative anaerobes (E. coli and Alealigenes eutrophus) was developed.

3. MATERIAL A N D METHODS

3.1. Strains The bacterial strains used are listed in Table 1.

Sulphate-reducing bacteria were cultured in an anaerobic glove-box at 30 ° C.

The donor strains of E. coli and Alcaligenes eutrophus containing broad host-range plasmids were constructed by conjugation [11]. A. eutrophus strains are counterselected in crosses with SRB, by virtue of their sensitivity to trimethoprim. The counter-selection of E. coli donors was achieved via auxotrophy.

3.2. Media Media used for the culture of SRB were pre-

pared freshly for each experiment. Medium E [1] is a rich, solid medium for the growth of the SRB. On this medium the colonies appear black due to FeS. The addition of nitrate to this medium, in the form of KNO3, makes it suitable as a medium for co-culture of SRB and facultative anaerobic bacteria such as E. coil and A. eutrophus, the nitrate providing an alternative electron acceptor for these species.

Medium C [1] is a clear liquid medium used for the mass culture of SRB. Medium 9 is a defined minimal medium for SRB which grow as grayish- yellowish colonies (FeS is not formed as the Fe is chelated by EDTA). This medium is used as a basic selection medium in crosses and for making viable counts of SRB. The formula for this medium was supplied by Dr. T.A. Hansen.

Medium 9: Na2SO4, 2.84 g; KH2PO 4, 0.2 g; MgC12 . 6H20, 0.4 g; NaCI, 1.16 g; NH4C1, 0.27 g; CaC12-2H20, 0.15 g; KCI, 0.37 g; trace ele- ments 1 ml; water 940 ml; agar 15 g.

Trace elements." EDTA, 5.0 g; FeSO 4. 7H20, 2.0 g; ZnSO4- 7H20, 0.1 g; MnC1 z - 4H20, 0.03 g; H3BO3, 0.3 g; CoC12 • 6H20, 0.2 g; CuC12, 0.01 g;

Table 1

Bacterial strains used in this work

Bacterial species Strain Chromosomal markers Plasmid and Origin or designation relevant markers a source

Escherichia coil CM578 leu, purE, trp, his, argG, ilv, pRK2013 ( K m / N m , Tra + ) [15] This paper (CSH57 Rif g ) metA or B, thL ara, lacY, gal,

malA, xyl, rntl, rpSL (Sm a) R300B (IncQ, 8.7 kb; Sm, Su, Mob + ) rpo (Rif g ) [161

CM404 leu, pro, thL lacY, hsr, hsm pRK2013 ( K m / N m ; Tra + ) [15] (HB101 (pRK2013)) recA, rpsL (Sin R) HB101 leu, pro, thi lacY, hsr, hsm

recA rpsL (Sm R) Alcaligenes AEI61 prototrophic pMOL30 (238kb, Cad, Cob, Zin, [17,18] eutrophus Cup, Hg(Tn4380 )

pMOL4 (RP4: :Tn4380) ( lncPl , 69 kb; Kin, Tc, Ap, Hg (Tn4380)

Desulfovibrio sp. NCMIB 8301 195 kb cryptic plasmid N C I M B b D. desulfuricans NCMIB 8312 none NCIMB

Tn4380 is a 9 kb transposon containing mercury resistance genes [17]. b NCIMB: National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

NiC12 • 6H20, 0.02 g; N a 2 M o O 4 • 2H20, 0.03 g; water to 1 litre.

Other solutions: (add per litre) Sodium lactate 1 M (heat sterilized) 20 ml; N a H C O 3 1M (heat sterilized), 30 ml; NazS 0.5 M (heat sterilized in O z free water), 4 ml; HCI 1 M, approx. 8 ml. Vitamins (filter sterilized) 0.5 ml. (vitamins: Gibco MEM + Vitamin solution (100 × ) Cat. Nr. 043- 1120).

3.3. Filter mating A membrane filter mating technique was used

in all the crosses. 1.8 ml of a suspension of recipi- ent Desulfovibrio cells (cultures of 5 and 18 days old were used) was mixed with 0.2 ml of an overnight culture of donor cells. This mixture was immediately filtered onto a 0.45 /~m pore-size, Millipore filter. The filter was incubated overnight on Medium E supplemented with 1.5 g/1 K N O 3, under anaerobic conditions at 30 o C.

After incubation the cells were removed from the filters by vortexing in 1 ml of 10 -2 M MgSO 4 and plated after appropriate dilutions.

3.4. Plasmid extraction and DNA hybridization Plasmid extraction from the SRB transcon-

jugants, grown on medium C, was carried out using a rapid screening technique which is a com- bination of a lysozyme disruption method :[12] followed by the extraction according to Kado and Liu [13]. The main point is that all the incubations (lysozyme, N a O H / S D S ) occurred on ice.

Using this technique a previously unreported plasmid was found in the strain NCIMB 8301 which corresponds in size to the plasmid found in the strain D. vulgaris NCIMB 8303 [14].

Plasmid extraction of E. colt strains was done following the procedure of Kado and Liu [13]. D N A - D N A hybridization was carried out using 32p labelled nicktranslated D N A of R300B as probe and GeneScreen Plus Membranes (NEN Research).

4. RESULTS

4.1. Matings between Desulfovibrio and aerobic donors

The matings were carried out following the filter mating procedure described. Viable counts,

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after matings, indicate that the concentration of nitrate in the medium is not inhibitory to the growth of the SRB [1]. The level of H2S produced by the SRB does not seem to be inhibitory to the aerobic donor strains.

In the mating Desulfovibrio 8301 × A. eutrophus AE161, IncP1 mediated transfer of resistance to mercury compounds was found to occur at maxi- mal frequency up to 1 transconjugant per recipi- ent or by donor. The Met a transconjugants were purified and checked to be free of A. eutrophus, but they turned out to be unstable as they had lost the resistance to merbromine after the 3rd purifi- cation without having expressed any other marker of pMOL4. In the same mating, no expression of zinc resistance (a marker carried by pMOL30, a plasmid of AE161) [18] was found.

In the crosses between SRB and E. coli strain CM578 (carrying R300B and pRK2013) stable Sm r transconjugants were found at very high fre- quencies up to 5.10 -2 per recipient 8301 and up to 1 per recipient 8312. Sm R transconjugant col- onies were purified and shown to be free of E. coli; they were also tested for the unselected marker of R300B, resistance to sulfamides. They were streaked onto Medium 9 plates containing 100 /ag/ml of sulfadiazine, to which original recipient strains are sensitive. The transconjugants of 8301 (R300B) were found to be resistant while the transconjugants of 8312 (R300B) were still sensi- tive to sulfadiazine. This observation might indi- cate a taxonomic divergence between strains 8301 and 8312.

The transconjugants were checked for the sta- bility of the acquired phenotypes: it was done by replica plating of transconjugants grown on non- selective Medium 9 onto selective plates contain- ing streptomycin or sulfadiazine. In both 8301 (R300B) and 8312 (R300B), 100% of colonies grown on Medium 9 grew on selective plates after replica plating.

The transconjugants were grown in Medium C and Medium C + S m 50 # g / m l and a plasmid extraction was carried out on the cultures. R300B was found to be present in all the tested transcon- jugants but was seen as faint bands which were better revealed by further hybridization with a labeled R300B probe.

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4.2. Retromobilization experiments Having successfully introduced R300B into the

SRB, it was decided to try to recover the plasmid in E. coil by doing a further mating and relying on the phenomenon of retrotransfer or plasmid capture [19]. The plasmid pRK2013 shares with IncP1 plasmids the hermaphroditic properties lin- ked to their transfer mechanism and was therefore used here. For this experiment, the strain CM404 bearing pRK2013 was used as a mating partner for 8301 (R300B) and 8312 (R300B). From these matings E. coli transconjugants becoming re- sistant to sulfadiazine were selected in aerobic conditions (in order to counterselect SRB). In both cases transconjugants were found: at a frequency of 2 -10 - 4 with 8301 (R300B) as a 'donor ' and at a frequency of 4 . 1 0 -6 with 8312 (R300B) as a 'donor': the E. coil retrotranscon- jugants were purified and grown in nutrient broth for extraction of plasmids by the method of Kado and Liu. In all the tested transconjugants, R300B was detected in agarose gel electrophoresis and remained unchanged in size. Crosses were also carried out between 8301 (R300B) and a plasmid free strain of E. coli (HB101) to see if the large native plasmid of 8301 was capable of mobilizing R300B. No Su R colonies were found after this cross. Thus, pRK2013 is solely responsible for the retrotransfer of R300B in E. coli. The system also paves the way for a two ways gene exchange between SRB and facultative aerobic strains.

and IncQ plasmids into the 8 subgroup of the purple bacteria which include Desulfovibrio de- sulfuricans, Desulfotobacter postgatei and also Myxococcus xanthus, Bdellovibrio stolpii [21]. Pur- ple bacteria (so classified according to Woese [21]) constitute the bulk of gram-negative bacteria as well as of recipients of broad host range plasmids [22]. The 8 subgroup is the less explored subgroup of the purple bacteria and also the less known from the genetic point of view. Moreover, the unability of IncP1 plasmids (represented in this work by pMOL4) to self replicate in the tested strains of Desulfovibrio goes along with a similar observation made ifi Myxococcus xanthus [22].

The reported experiments also demonstrate that, using this system, the barrier between facultative anaerobes and strict anaerobes can be crossed. This barrier has previously been crossed between Bacteroides and E. coli but using mobilisation of an ' in vitro' constructed shuttle vector [23-25]. The transfer of R300B in SRB seems thus to be the very first report of a natural plasmid trans- ferred in soil anaerobes and opens the way for the genetic analysis of SRB. It emphasizes again the remarkable potential of broad host range plasmids (as IncQ and IncP1 plasmids) regarding gene dis- semination [22,19]: it is especially relevant as far as environmental release of recombinant DNA is concerned.

A C K N O W L E D G E M E N T S

5. DISCUSSION

A mating technique has been developed for the SRB whereby plasmids may be screened for their ability to transfer, replicate and express resistance markers, using aerobic facultative bacteria as donors. Using this technique successful matings between two Desulfovibrio strains and as donors, E. coli strain CM578 and A. eutrophus AE161, were carried out.

The results of the crosses with E. coli CM578 confirm the ability of IncP1 transfer systems to work with Desulfovibrio strains and they show that the IncQ replication system also works. They give an interesting extension of range for IncP1

This work was partially supported by the E.E.C. Biotechnology Action Program. We are grateful to Drs. T.A. Hansen (Rijksuniversiteit Groningen) and L. Diels for providing precious information, A. Toussaint and D. Springael for criticisms and suggestions. Expert technical assistance of A. Adriaensen-Ryngaert, J. Cools and J. Gerits is gratefully acknowledged.

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