Stability of broad host range cloning vectors in the phototrophic bacterium Rhodospirillum rubrum

FEMS Microbiology Loners t~5 (ltm2) 7-12 It~92 Federation of European Microbiological Sociclic,, 1137S-IIIt~7/92/$1~5.1~ Published by Elsevier

FEMSI.E 1141t~6

Stability of broad host range cloning vectors in the phototrophic bacte riu m Rhodospirillum rubrum R u d o l f Saegcsscr, R o b i n G h o s h i and R c i n h a r d B a c h o f c n In~tittae tbr Phtnt !~iolok'y. Ziirwh, St~ttzerhznd

Recci'.ed 25 March Iqt~2 Rc',ision rccci','ed 4 Ma.', Iqt~2 Accepted 5 Ma~, Iqq2

Key words: Photosynthetic bacteria: Cloning vectors: Antibiotics: Rhodospirilhen ruhnon

i. S U M M A R Y The suitability of the broad-host range vectors pRK290, pVKI00 and p L A F R I respectively for use with the photosynthetic purple nun-sulphur bacterium, Rhodospirilhon rul>rum has been examined. All vectors are RK2 (RP4) derivatives (incompatibility group P-l) and contain the mob region of RK2. The three vectors could each bc transferred from Escherichia coil by triparcntal mating in the presence of a second E, ¢'o6 donor containing the helper plasmid pRK2013. The conjugation fregucncy was approx. 10 i in all cases, Selection of transconjugants was performed in the presence of tetracycline (pRK2t,~0, p L A F R I , pVKH)O) or kanamycin (pVKIO0). Southern-blotting experiments after many generations showed no rearrangements to have occurred, in the absence of antibiotic selection all three vectors were maintained tk~r at least 15 generations under acr-

I ('orrt~pondencc to: Present address: R. Ghosh. Dept. ,Jf

Microbiology. Biocenter. Klingelbergstr. 711.f.'|l 41156 Basel.

Switzerland.

obic chcmotrophic conditions but were rapid:y lost when the culture was grown phototrophieally (anaerobic).

2. I N T R O D U C T I O N The purple non-sulphur bacterium Rhodospiri//ton rttbrum has been the subject of intense activity lor the study of bacterial photosynthesis and microbial physiology for many years [t]. Although the biochemistry of R. n~hmm is as welldefined as for other species of phototrophic bacteria, e.g. Rho&~bacter sphaeroMes or R. capstdatus, the study of the genetic aspects has lagged behind the latter organisms. Recently a number of groups have begun to examine the genetics of R. rubmm and several genes (see refs. 2-4) have bcen cloned and sequenced. To date however, few data have been presented for the suitability of broad-host range vectors for the expression of genes from R. rttbrt~m within their normal host background. Whereas Hclinski and coworkcrs [5,6] showed that RK2 plasmids wcrc mobilizable to nine diffcrcnt

species of Gram-negative bacteria, and Olsen and Shipley [7] demonstrated that IncP plasmid transfer to R. rttbrum does occur, these studies involved naturally occurring plasmids and no description of copy number or plasmid stability for useful chining vectors was described. Scvcral cloning vectors have been described for Rb. cap.~'ulatus and Rb. ,whaeroides [8-10]. However since current taxonomic approaches place R. rubrum somewhat distant to Rb. capsulatus and Rb. sphaemides [11], we feel justified in examining rather elementary aspects of plasmid stability in the former organism explicitly. The three cloning vectors chosen, pRK290, pLAFRI and pVKI00 [5,12,13] (Fig. 1) are RK2 (incompatibility group P-la) derivatives and have been developed for a variety of Gram-negative bacteria. but of these only pLAFR1 and pRK290 have been widely used for photosynthetic bacteria so far[14-16]. These derivatives have several advantages: they have single well-defined cloning sites; they have suitable resistance markers for selection with slow-growing bacteria; and two of them, pLAFRI and pVKI00 (Fig. la) possess a cos site and are therefore well-suited for the construction of genomic banks [16,20]. A preliminary repolt of this work has been published [17].

3. MATERIALS AND METHODS 3.1. Growth of bacteria The wild-type strain R. rubrum SI was originally obtained from the German Collection of Microorganisms, Braunschweig, FRG. R. rubrum was grown at 3°C using Sistrom minimal medium A (without casamino acids) [18] either aerobically in the dark (chemotrophically) with vigorous shaking, or anaerobically in the light (phototrophically) in 100-ml flasks illuminated with light intensity of approx. 10 W / m 2. Growth on solid medium was performed using Sistrom minimal medium A supplemented with 1.5% agar (Difco). Cultures of R. mbrum containing the plasmid pRK290, or the cosmids pLAFRI or pVKI00, were selected using either 10/,tg/ml tetracycline or 50 g g / m l kanamycin, respectively. Cultures of E. colt containing pRK290 or pLAFR1 were

maintained on LB medium containing I[1/.tg/ml tetracycline and pRK2III3 or pVKl00 with 50 tzg/ml kanamycin. 3. 2. Mating procedure 5 ml of E. colt HBI01 contaio.ing either pRK290, oLAFRI, oVKII}0 or pRK2013 were grown to an OD~,~,~of approx. 11.8-1.5 at 37°C in LB medium. Cells (I ml) were washed twice by centrifugation with the same volume of I).9% (w/v) NaCI. Chemotrophically-grown cells of R. rubrum were harvested at an OD~,~,0 of approx. 1-1.5, pelleted and then resuspended in the same vollame of 0.9cA NaCI. For triparental mating, 2 × 10s cfu of chemotrophically-grown cells of R. nlbmm in 11.9% NaCI were mixed with E. colt HBI01 containing the plasmid (pRK290, pLAFRI or pVKi00) and a helper plasmid pRK2013 [6] at a ratio of IIII1:1: I. The mating mixture was pelleted and resuspended in 211 ptl 0.9% NaCI and plated immediately onto solid Sistrom medium supplemented with 3cA peptone and 1% yeast extract (conjugation medium). The plates were allowed to dry for 3[}-45 min at room temperaturc and then incubated for 6 h aerobically in the dark at 32°C. The selection of transconjugants was performed by streaking the cells from the conjugation plates onto Sistrom plates containing either 5 p.g/ml tetracycline for pRK290, pLAFRI and pVKI00 or 50 p.g/ml kanamycin for pVKl(111 alternatively. Counterselection was provided by the amino acid-auxotrophy of the E. colt HBI01 [19]. Incubation was performed either chemotrophically or phototrophically at 30°C for 5 days. After incubation the conjugation frequency was determined as the ratio of the number of transconjugants observed with antibiotic selection to the total number of viable R. rubrmn cells (without antibiotic selection). Reverse conjugation was performed by triparental mating using R. rubrmn as the donor and E. colt HBI01 as the recipient in the presence of the helper pRK2013. The mating procedure was as described above, and pRK290- or pVKl00-transconjugants of E. colt selected by growth on LB plates overnight at 37°C containing 10 p,g/ml tetracycline. Under

these conditions growth of R. rtthmm was not observed.

3.3. PlasmM mah~tenance The stabilities of the vectors pRK290, p L A F R I and pVKI00 were tested as follows: R, ruhrum cells, obtained from a preculture grown cithct ehemotrophieally or phototmphieally and containing the appropriate vector, were washed t~ice in 0.9% NaCI to eliminate tetracycline. The washed cells were used to inoculate Sistrom medium without tetracycline to give a final cell density of I - 2 × I1)" ml ) Samples from the growing culture were diluted in ().t)r:f NaCI and then spread onto solid Sistrom medium including 10 # g / m l tetracycline for thc plasmid pRK290 and the eosmids p L A F R I and pVKIII0 to select for vector-containing ccUs. Simultaneously, an equal volume was spread onto phttes without the antibiotic to determine the total number of viable cells. This procedure was followed up to 15 generations. The maintenance of the vectors in R. rubnmt was then determined as the ratio of the number of colonies observed in the presence of tetracycline compared with the number of cohmics observed in its absence. 3.4. PrtTmtxttion and analysi,~ ~t" I)NA Total DNA was isolated from R. r u h m m as follows: !111 ml of culture was pelleted by ccntrifugation and then resuspended in 1(1 ml 511 mM Tris" HCI pH 8.1), 20 mM EDTA Ilysis buffer). Lysozyme (I(1 mg in lysis buffer)was added to the resuspended cells lk~llowed by incubation at 37°C for ! h. Subsequently the lyscd cells were treated with DNAase-frcc RNAasc for 30-45 min, then with I(1 m g / m l pronase (preincubatcd in lysis buffer at 37°C for 1 h), Complete lysis was achieved by the addition of 1.5 ml 1(1c; ( w / v ) SDS within 5 min at 37°C. The total DNA was obtained by phenol extraction followed by ethanol precipitation according to Maniatis et al. [19], Plasmid DNA was isolated from E. coli by the above method followed by PEG precipitation and repeated CsCI eentrifugation according to Maniatis et al. [19]. Nick translation of the plasmid DNA was performed with [32P]-dCTP using the Boehringer

nick translation kit. Restriction digests, Southern blotting and hybridization were performed by standard methods [ 19]. Hybridization of restrictcd DNAs with [~-~P]-Iabellcd vector DNA was per-

,L ~

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pRK290 1 2 34 E,s NH E S

pVKlO0 56 E,S

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Kb

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17.3 t= - g g e 5.7

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pLAFR1 1 2 3 4 5 6 Kb E,S,~-t E S E,S

7

8

9 lO 11 12

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Fig. I. IA) Bread hint range cloning Vcclor~ used in this study,

Rc~,triction nile, arc abbreviated as h)llows: I-coRI, E; Ilmdlll. tl: Sail..%. (B) Southern blot analysisof the vectors pRK2q(), pVKI00 and pLAFRI in R. mhnmz SI. Lane I. i~,olatcd ',cctor rc~,tricted with EcoR| (E) and Sail (S) and hybridized aglthtM the same [32P]-Iabelled ~'¢ctor its probe. [.ant 2, h.~brh.]izittionof a A-tlind|ll (| l) restriction fragment to tht."indicated ,.ector. Lanes 3-5. genomic DNA containing the '.¢ctor indicated restricted ~,~,iththe restriction enzymes sho,a,n. Lane 6. hybridizationof restricted genomicDNA from vector-less R rubrum SI to the indicatedvector. For genomic DNA. 2.5-4 #g total DNA v,as digested for Southern blot imaly~,is. Lane 7~12. Autoradiographs of the hybridization with incrcasirtgconcentrationof the isolated vector (6.9. 13.7. 27.5.55. I Irk 2"1)ng). The autoradiographswere overexposed to detect minor species.

I0

Table I Frequencies of conjugation observed for wlrious plasmids from E. coli t o R. rtlbntm

Plasmid RP4 pLAFRI pRK290

Conjugation frequency ~' {I,1× |{I I 7,5x I0 2 6,2 X 10 - " 7,7x 10 z

pVKI0(I

" The conjugation frequency was calculated as the number of transconjugants/total number of viable R. n~hrum cells after conjugation. formed at 42°C in the presence of 50% formamide.

,L RESULTS A N D DISCUSSION In the present study we have established the basic methodology required for the transfer of genetic elements to the purple non-sulfur bacterium R. rubnml and their subsequent expression. Conjugation of R. rubrum with E. coil donors containing the plasmids pRK290, p L A F R I or

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GENERATION

NUMBER

15

Fig. 2. Plasmid maintenance in R. rubrum. Precultures were grown in selective liquid medium with 10 p.g/ml tetracycline either under aerobic, chemotrophic conditions (closed symbols) or anaerobic, phototrophic conditions (open symbols), and then used to inoculate I(1(Iml cultures grown similarlybut in the absence of antibiotic selection. At various times sanlpies of the cultures, growing without antibiotics, were plated plus or minus antibiotics as described in MATI~RIALS.V',:D METIIOL~S. The plasmids used were: RP4, D pRK2t~L ©: pLAFRI, o; and pVKI0iL A

pVKIIR) (Fig. l a ) w i t h subsequent selection with either 5 # g / m l tetracycline (pRK290, p L A F R I , pVKI(RI), or 5(I /.tg/ml kanamycin (pVKI00) at 30°C, yielded in all cases large numbers of transconjugants with a frequency of approx. Illin all cases (Table 1). This compares well with the frequency of transfer of RP4 into R. mbrum where both tra and mob regions are contained on a single plasmid. We have also observed reverse conjugation from R. rubmm to E. coli with a frequency sufficient to transfer the plasmid to the recipient directly using a single colony of R. rllbrlll?l. Southern-blotting analysis showed that the vectors pRK290, p L A F R I and p V K l 0 0 arc stable in R. rttbrum and do not undergo r e a r r a n g e m e n t s following conjugation (Fig. Ib). The two methods to detcrmine the copy number of the vectors p L A F R I, pVK Ill0 and pRK290, (a) by Cerenkov counting of the eluted [3,,p]. labelled hybridizing probe and (b) by densitometry were in good agreement giving 11-14 copies per R. ruhrum cell (results not shown). This calculation assumes the R. nlbrum genome to be similar in size to that of Rb. sphaeroides, which was determined recently to bc approx. 5000 kb [21]. For Rb. sF,haeroides the copy n u m b e r of a similar RK2-derivative, pRK404, is 6 - 8 u n d e r phototrophic conditions [9]. The higher value in R. rubrum may be due in part to an underestimation of the genome size. Finally the maintenance of the vectors pRK290, p L A F R I , and p V K l 0 0 in the absence of antibiotic selection under anaerobic, phototrophie and aerobic, chemotrophic conditions has been examined (Fig. 2). Interestingly, although all vectors are maintained well under aerobic conditions over at least 15 generations, a rapid loss of all vectors was observed in R. rubnm~ after l0 generations of anaerobic phototrophic growth in the absence of antibiotic selection.

ACKNOWLEDGEMENTS We thank Dr. Hans-Martin Fischer and Prof. H. Hennecke (Institute of Microbiology, ETH Ztirich) for the gifts of plasmids and strains and

for valuablc discussions. The generous financial support by the Swiss National Foundation (Grant 3.243.1)-85) is g r e a t l y a c k n o w l e d g e d .

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