isolation of cryptic plasmids from moderately halophilic eubacteria of the genus halomonas....

Mol Gen Genet (1995) 246:411-418 © Springer-Verlag 1995

Carmen Vargas • Rosario Fern/mdez-Castillo David C/movas • Antonio Ventosa • Joaquin J. Nieto

Isolation of cryptic plasmids from moderately halophUic eubacteria of the genus Halomonas. Characterization of a small plasmid from H. elongata and its use for shuttle vector construction

Received: 27 July 1994 / Accepted: 16 September 1994

Abstract Three cryptic plasmids have been isolated from moderately halophilic eubacteria belonging to three species of the genus Halomonas. These three plasmids were designated pilE1 (4.2 kb, isolated from H. elongata ATCC 33174), pHI1 (48 kb, isolated from "H. israelensis" ATCC 43985), and pHS1 (ca. 70 kb, isolated from H. subglaciescola U Q M 2927). Because of its small size, the plasmid pilE1 was selected for further characterization and construction of a shuttle vector for Halomonas strains, pilE1 was cloned into pBluescript KS and a detailed restriction map was constructed. Hy- bridization experiments excluded the existence of sequences homologous to pilE1 in total DNA from other strains of the genus Halomonas. Moreover, no DNA homology with pMH1, the only plasmid described so far from moderate halophiles, was found. Since pilE1 ap- peared to be unable to replicate in Escherichia coli cells, a number of mobilizable p i lE 1-derived hybrid plasmids were constructed that could be selected and maintained both in E. coli and in H. elongata. Finally, an improved shuttle vector, pHS15, was generated. The vector pHS15 contains an origin of replication from E. coli as well as one from H. elongata, a streptomycin resistance gene for positive selection in moderate halophiles, a number of unique restriction sites commonly used for cloning, and the mobilization functions of the broad host range IncP plasmid RK2. The vector pHS15 was readily mobilized by the RK2 derivative pRK2013 to all Halomonas strains tested so far. This is the first report on the development of a cloning vector useful for moderately halophilic eubacteria.

Key words Moderate halophiles • Halomonas elongata Plasmids • Cloning vectors

Communicated by W. Goebel

C. Vargas • R. Fernfindez-Castillo . D. C/movas • A. Ventosa J. J. Nieto ([~) Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain

Introduction

Moderately halophilic eubacteria are defined as those that grow optimally in media containing 3%-15% Na- C1 (Kushner 1978). They constitute a very heteroge- neous physiological group which includes a great variety of bacteria (Ventosa 1988). This microbial group, which plays an important role in the ecology of hypersa- line environments, represents an excellent example of adaptation to frequent changes in extracellular osmolality (Rodriguez-Valera 1986). In addition, they are also very interesting from a biotechnological point of view. Thus, many of them produce halophilic exoenzymes (amylases, nucleases, proteases, etc.) of commercial in- terest (Kamekura 1986) and some organic osmolytes named "compatible solutes", which could be used as stabilizers of enzymes and whole cells (Galinski 1989; Galinski and Tindall 1992). Nevertheless, although ex- tensive studies on their physiology and ecology have been reported (Kushner and Kamekura 1988; Ro- driguez-Valera 1986), their genetics remains a virtually unexplored field. No studies on DNA transfer mecha- nisms among these halophiles or between non- halophilic and moderately halophilic bacteria have been reported. Besides, studies on the isolation of mutants are extremely scarce (Kogut et al. 1992; Nieto et al. 1993), and suitable cloning vectors are not available so far. Since moderate halophiles exhibit the greatest salt tolerance found in prokaryotes, they might be consid- ered as very good candidates for the elucidation of the molecular biology of osmoregulation processes. In this respect, it is important to note that, to date, genetic analyses have only been performed in Enterobacteri- aceae and in salt-tolerant cyanobacteria (Csonka and Hanson 1991). Thus, it has been reported that changes in extracellular osmolality lead to changes in the expression of a number of genes, most of them encoding proteins involved in the synthesis or transport of compatible solutes (Csonka and Hanson 1991). A deeper knowledge of the genetics of moderate halophiles is therefore necessary to understand the molecular mecha-

412

nisms of osmoregulation. Furthermore, this knowledge will allow the genetic manipulation of these microorganisms for the overproduction of enzymes and solutes of potential industrial importance.

We are interested in the construction of cloning vectors based on endogenous plasmids from moderately halophilic eubacteria. Recently, we have shown that an autochthonous plasmid (pMH1) can be harboured by some moderate halophiles (Fern~indez-Castillo et al. 1992). We consider that members of the genus Halomonas display features very interesting for their genetic manipulation. Thus, they exhibit an extreme salt tolerance (0.3% to 32% NaC1) (Franzmann et al. 1988) and they can thrive in, and be isolated from virtually any saline environment. Moreover, they are very easy to grow in the laboratory, utilizing a wide variety of carbon compounds as sole carbon source, and possess wide-ranging biochemical versatility (Vreeland 1992). In this paper, we report the isolation of cryptic plasmids from three Halomonas species. A small plasmid isolated from H. elongata ATCC 33174, pilE1, has been selected for further molecular characterization and used for the development of a shuttle vector. This vector, the first reported for moderate halophiles, should facilitate the genetic analysis and expression of genes from Halomonas species.

Materials and methods

Bacterial strains and microbiological techniques

The eight culture collection strains belonging to the genus Halomonas used in this study are listed in Table 1. Strains 803 (Wood 1969), DH5cz (Hanahan 1983) and JM110 (Dam-, Yanisch- Perron et al. 1985) of Escherichia coli were used as hosts for transformation and conjugation procedures. All moderately halophilic strains were grown in saline medium (SWYE) containing 10% total salts to which 5 g/1 yeast extract (Difco) was added. The composition of the 10% total salt solution was as follows: 81 g/1 NaC1, 7 g/1 MgC12, 9.6 g/1 MgSO4, 0.36 g/1 CaCla, 2 g/1 KC1, 0.06 g/1 NaHCO3, 0.0026 g/1 NaBr (Nieto et al. 1989). The pH of the medium was adjusted to 7.2. The E. coli strains were grown in Luria medium (LB; Sambrook et al. 1989). Solid media were obtained by adding 20 g/1 Bacto-agar (Difco). Incubation was always at 37°C in an orbital shaker (New Brunswick Scientific Co., Edison, New Jersey, USA) at 200 rev/min. For E. coli, antibiotics were added when required as filter-sterilized concentrate in water or 70% ethanol at the standard appropriate concentrations (Sam- brook et al. 1989). The constructed plasmids were mobilized from E. coli to Halomonas and E. coli strains by triparental matings in

which the RK2 tra genes were provided in trans by the "helper" plasmid pRK2013 (Figurski and Helinski 1979). Aliquots (100 gl) of logarithmic cultures of each donor, helper, and recipient strain were mixed on a nitrocellulose filter on a plate of complex medium. For conjugations from E. coli to Halomonas, this complex medium was a modified SWYE medium in which the final per- centage of the total salt solution was decreased to 3% (to allow the growth of E. coli). LB medium was used for conjugations between E. coli strains. The filters were incubated overnight at 37 ° C. Halomonas transconjugants were selected on SWYE medium (on which E. coli does not grow) with the addition of 1 mg/ml streptomycin (to select for cells that had taken up the plasmid constructs). E. coli transconjugants were selected on LB medium with added ampicillin and streptomycin.

DNA isolation

Screening of the moderate halophiles for plasmids was performed using two complementary procedures, followed by agarose gel electrophoresis. Firstly, the presence of megaplasmids was tested by using a modification of the Eckhardt (1978) method described by Plazinski et al. (1985). Secondly, small- to medium-sized plasmids were screened for by using the alkaline lysis method (Morelle 1989). Prior to the lysis, cells were washed with 0.1% SDS in TE buffer (10 mM TRIS-HC1, 1 mM EDTA, pH 8.0). This washing step was found greatly to improve the isolation method, yielding cleaner plasmid DNA preparations, suitable for digestion with restriction enzymes. For large-scale preparation, alkaline lysis-isolated plasmid DNA was further purified through a Qiagen column (Qiagen, Dusseldorf, Germany). Plasmid DNA was isolated from E. coli using the "boiling" method described by Ausubel et al. (1989). Genomic DNA from E. coli and moderate halophiles was isolated as described elsewhere (Ausubel et al. 1989).

DNA manipulation

Plasmid and chromosomal DNAs were digested as recommended by the restriction enzyme manufacturer (Boehringer-Mannheim, Germany). Restriction fragments were separated on 0.8% agarose gels (Sigma) in TBE buffer (45 mM TRIS-borate, 1 mM EDTA, pH 8.0). Gels were stained with 0.5 gg/ml ethidium bromide for 10 min and photographed under 254 nm wavelength ultraviolet light. DNA ligations, preparation of competent E. coli cells and transformations were all performed according to Sambrook et al. (1989). For DNA hybridization, electrophoretically separated plasmid and genomic DNA fragments were transferred to nylon filters (Amersham) as described by Southern (1975). Colonies were lysed in situ and transferred to nylon filters as described by Sam- brook et al. (1989). The plasmid pilE1 was ct32p-radiolabelled using a Multiprime DNA labelling kit (Amersham). The solution used for the hybridizations contained 50% formamide, 3 x SSC (20 x SSC = 3 M NaC1, 0.3 M sodium citrate), 15 mM NaHaPO4, 15mM Na2HPO4, 10 x Denhardt solution (100 x Denhardt= 2% w/v BSA, 2% w/v Ficoll, 2% w/v polyvinylpyrolidone), 0.05% SDS, and 0.1 mg/ml salmon sperm DNA. Filters were pre-

Table 1 Strains of Halomonas used in this study. (ND not detected)

Microorganism Plasmid content Reference (kb)

H. elongata ATCC 33173 H. elongata ATCC 33174 H. halmophila ATCC 19717 "H. israelensis" ATCC 43985 H. subglaciescola UQM 2927 "'H. canadiana" ATCC 43984 H. halodurans ATCC 29629 H. meridiana DSM 5425

pMH1 (11.5) pilE1 (4.2) pMH1 (11.5) pHI1 (48) pHS1 (ca. 70) ND ND ND

Fern~mdez-Castillo et al. (1992) This study Fermindez-Castillo et al. (1992) This study This study This study This study This study

hybridized for 6 h at 42 ° C and hybridized with the ~z32p-labelled probe overnight at the same temperature. Washes were done at 42 ° C with 0.05% SDS, 2 x SSC,

Results

Isolation of cryptic plasmids from Halomonas strains

In a previous paper (Fernfindez-Castillo et al. 1992) we reported the isolation and partial characterization of the only plasmid described so far from moderate halophiles, pMH1. Plasmid pMH1, 11.5 kb in size, is present in two species of HaIomonas, H. elongata ATCC 33173 and H. halmophila ATCC 19717 (Table 1). In this study, other strains of the same genus were screened for the presence of plasmid DNA using two complementary procedures (see Materials and methods). When a modified Eckhardt method was utilized, no megaplasmids were detected in any of the strains tested (data not shown), including the two strains known to harbor pMH1. When the alkaline-lysis method was used, three strains were found to harbour one plasmid each. These plasmids were designed pilE1 (isolated from H. elongata ATCC 33174), pHI 1 (isolated from "H. israelensis" ATCC 43985), and pHS 1 (isolated from H. subglaciescola UQM 2927) (Table 1). The sizes of plasmids pilE1, pHI1 and pHS1 were estimated by restriction enzyme analysis and found to be 4.2, 48 and ca. 70 kb, respectively. Because of its small size, plasmid pilE1 was selected for further characterization and construction of a shuttle vector for Halomonas.

413

3.85 Sacl EcoRI o.oo - ~ ,Smal o.zo

3.80 BstXl ~ ~ - ~ ' ; H i n c l l oAs f ~

3.40 Eagl\ / X 3.3s Sac, _~ .... \

X / . . - ,.,,.3o z.zs Acc l " ~ ~ Hincl l 1.40

2.6o Xbal ~ " - Bal l l " ! - - \ Hincl l l .ss

2."4s Hincl l Xho 1.9o 2.35

Fig. 1 Restriction endonuclease cleavage map of the endogenous HaIomonas elongata ATCC 33174 plasmid pilE1

brook et al. 1989). Since overlapping dam methylation cannot occur in the pBluescript polylinker sequence, it was concluded that the XbaI site in pilE1 DNA must be followed by the sequence 5'-TC-3', which could com- plete the site recognized by Dam. This was confirmed when p H E l l was transformed into the D a m E.coli strain JM110, re-isolated from that strain, digested with XbaI and electrophoresed on an agarose gel. Two bands of the expected size were present, indicating that the XbaI site in pilE1 DNA had been modified by the E. coli Dam methylase. This result also suggests the absence of adenine methylation at the sequence 5'-GATC- 3' in H. elongata.

Physical map of pilE1

A preliminary restriction analysis of pilE1, prepared from H. elongata ATCC 33174, indicated that the plasmid possesses unique sites for BglII, EcoRI, PstI, XbaI, and XhoI. In order to construct a more detailed restriction map of p i l e 1, the EcoRI-digested plasmid was sub- cloned into pBluescript KS (Stratagene) to give plasmid pilE11. This derivative plasmid was purified and digested with a wide range of restriction enzymes, including those for which sites exist in the pBluescript polylinker. Additional unique restriction sites within pilE1 DNA were found for the enzymes BstXI, EagI, SacI, SaclI, and Sinai. The physical map of pi lE 1 is shown in Fig. 1.

Plasmid p H E l l has two XbaI sites, one in the pBlue- script polylinker and the second in the pilE1 DNA. However, XbaI-digested pilE 11 yielded only one band instead of the two bands expected. When digesting DNA prepared from E. coli, the restriction endonuclease XbaI can be inhibited by dam methylation if its recognition site in the target DNA (5'-TCTAGA-3') overlaps with the site recognized by the E. coli Dam methylase (5'-GATC-3'). This leads to methylation of the external adenine residue at the XbaI site, which cannot be cleaved by the restriction endonuclease (Sam-

Determination of DNA homology between pilE1 and DNA from Halomonas strains

To determine whether pilE1 shared homology with the DNA from other strains of the genus Halomonas, as well as with plasmid pMH1 (the only plasmid described so far from moderate halophiles), a Southern hybridization approach was adopted. For this purpose, 32p-labelled pilE1 was used as a probe against genomic DNAs prepared from eight Halomonas strains (including the parental strains of pilE1 and pMH1) as well as plasmid pMH1. pilE1 and total DNA from E. coli were included as positive and negative controls, respectively. Apart from the parental strain of pilE1, no hybridization signals were detected between pilE1 and DNA from any other Halomonas strains, including the two strains carrying pMH1 (H. elongata ATCC 33173 and H. halmophila ATCC 19717). Furthermore, no detectable homology between plasmids pilE1 and pMH1 was found, even when the hybridization experiment and washing were performed under the least stringent conditions (Fig. 2).

414

A 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5

B

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5

23.0 9.4 6.5 4.3

2.3 2.0

D

- •

Fig. 2 A Agarose gel electrophoresis of EcoRI + PstI-generated fragments of DNA from H. elongata ATCC 33174 (lane 2), H. elongata ATCC 33173 (lane 3), H. halmophila ATCC 19717 (lane 4), "H. canadiana" ATCC 43984 (lane 5), "H. israelensis" ATCC 43985 (lane 6), H. halodurans ATCC 29696 (lane 7), H. meridiana DSM 5425 (lane 8), H. subglaciescola UQM 2927 (lane 9), Es- cherichia coli 803 (lane 10), pilE1 (lane 12), and pMH1 (lane 14). Lane 15, pMH1 digested with EcoRI + HindIII; lane 1, HindIII- digested lambda DNA. B Hybridization of the above DNA samples with EcoRI-digested pilE1 DNA

Plasmid pilE1 is unable to replicate in E. coli cells

When cloned into pBluescript (as plasmid pHEll) , pilE1 could be stably maintained in E. coli. To check the possibility that pilE1 might carry antibiotic resistance genes which could be used as genetic markers, the E. coli strain DH5~ carrying the derivative plasmid pi lE 11 was screened on LB plates supplemented with a number of antibiotics. The following antibiotics were individually tested: cloramphenicol, erythromycin, gen- tamycin, kanamycin, nalidixic acid, neomycin, spectinomycin, streptomycin and tetracycline. Parallel controls were also carried out to exclude the generation of spon- taneous antibiotic-resistant mutants. No colonies grew on any of the selective media, suggesting that pilE1 does not carry any of the antibiotic resistance markers tested.

The ability of pilE1 to replicate in E. coli was as- sayed using a colony hybridization approach. The plasmid was introduced into two different E. coli strains, 803 and DH5~, by transformation (pBluescript was also used as a positive control for the transformation procedure itself). Transformants were plated onto non-selective media since no antibiotic resistance has so far been found associated with pilE1. Approximately 1000 colonies were selected and transferred to nylon filters. H. elongata ATCC 33174 was also included as a positive control. Filters were hybridized with pilE1 DNA. Ex- cept for the control colonies, no hybridization signals were detected, indicating that pilE1 was not present in the transformed E. coli cells and therefore that the plasmid is not able to replicate in this host (data not shown).

Construction of pHEl-derived hybrid plasmids

Four DNA fragments were used in the construction of pHEl-derived hybrid plasmids that could replicate in both E. coli and Halomonas strains: (i) the replicon pilE1, for the maintenance of the constructed plasmids in Halomonas; (ii) the E. coli high copy number plasmid pBluescript KS, which contains the functions necessary for replication in E. coli, a multiple cloning site and the ampicillin resistance marker. This marker is only useful when the plasmids are selected in E. coli since most moderate halophiles (including Halomonas strains) are resistant to this antibiotic (Nieto et al. 1993); (iii) a selectable marker for moderately halophilic eubacteria. The streptomycin resistance gene was chosen because I-Ialomonas and most moderately halophilic strains have been shown to be sensitive to this antibiotic (Nieto et al. 1993). The omega cassette, conferring both streptomycin and spectinomycin resistance, was isolated as a 1.95 kb HindIII fragment from plasmid pHP45-omega (Prentki and Krisch 1984); (iv) the mobilization region. A 3.1 kb ItindIII-SalI fragment from plasmid pNH- Kan/oriTwas the source of the functions necessary for mobilization. This fragment contains the origin of transfer region (oriT) of the broad-host-range plasmid RK2 (Hengen and Iyer 1992). Therefore, any plasmid carrying this oriT region can be transferred efficiently in a mating reaction where all other transfer functions are provided in trans by a separate helper plasmid (Ditta et al. 1980).

For the construction of pHEl-derived hybrid plasmids, these four DNA fragments were assembled as shown in Fig. 3. Firstly, the 1.95 kb HindIII fragment from plasmid pHP45-omega (containing the streptomycin/spectinomycin resistance gene) was cloned into pBluescript KS giving plasmid pKS-omega (Fig. 3A). Secondly, the 3.1 kb HindIII-SalI fragment from plasmid pNH-Kan/or iT (containing the oriT region) was cloned into HindIII + SalI-digested pKS-omega. The resulting plasmid, named pKS-omega-oriT (Fig. 3B), could be efficiently transferred from E. coli 803 to E. coli DH5~ in a triparental mating, demonstrating the effectiveness of the cloned oriTregion in mobilizing DNA.

415

A B

Bx Ea,N Spe Sm E Bx Ea,N Spe Sm E s~, Is¢,ll Xb I B I P I ~ S~, IS¢,, I Xb I B I P t ~V

,.,.. LLL' ' 1,1 L.LI ,Ill i 1 iI,lJ pBluescript ~ ~ p N H - K a n / o r i T

I Hindlu ~Sm/SpcR _L " + ,~m/Spc I Hindlll Sail Amp H R R

i pKS-om.9= ~ Hindll..Sall Vh, L ~ pKS'o°r?¢ga" '~L_ H o H . , - . in . . . " Co, , ,or ,

omega AmpR~ 4.gSkb ~ "- ~ BmSkb J

" ~ c pilE1 x - I / , , / ' ~ " ~ CotElori ~ S.He,Ae-V ':' or iT

Sm B ~ P SmE~ J Bg

K D,A X S,Hc,Ac

, pyE~ Sm p B.@E Bg e SmE , , ,, p H S 9 / p H S 1 4

(BglB) ESm p (Bg/,.,~pR~Sm k~ ,, J r" E pHSl 2

I kb / \ t I / \ \ /

s ~ \ / E . , . 3 \ " , i S v Sell J ~4 ~ " H R R %sm,s0o

C o I E , o r i t ' z ' z s k ' ~ : P h

D,A

S, Hc,

Fig. 3A-C Construction of mobilizable pHEl-derived plasmids that can replicate in both E. coli and H. elongata. Details of the construction are described in the text. Only EcoRI, PstI, Bg/II and Sinai sites are shown in pilE1 DNA. For the pairs pHS7/pHS8 and pHSg/pHS14, the orientations shown correspond to pHS7 and PHS9. Clones pHS8 and pHS14 have orientations opposite to PHS7 and pHS8, respectively. Sites in parentheses were lost in the construction of the plasmids. (SmR/Spc R streptomycin/spectinomycin resistance, Amp R ampicillin resistance)

When the plasmid was transferred in the same way to H. elongata, no streptomycin-resistant transconjugants were obtained, indicating that pKS-omega-oriTis not able to replicate in this host. Note that the orientation of the 1.95 kb HindIII omega cassette in plasmi d pKS- omega-oriTis the opposite to that of the same fragment in pKS-omega as mapped by SphI digestion (Fig. 3B). Finally, to provide the functions necessary for replica-

C

(Bg,B) I \

\

ESm p (Bg,B) pHSl 3 I I I )

Ikb I I / \ /

\ \ /KS)~ He'Ac

i l Z 2 S k b ~I, CoIEI ori

H~%,,. ~AC-Am~ Sph I | I i i i i I i i I i i i l l | l l ~ • ~ f f

S r n / S p c J, , , , , , HEv~b ~po~ BxS~l Sm Ea,N Scll

tion in Halomonas, plasmid pilE1 samples were individually digested with PstI, EcoRI and BglII, and sub- cloned into pKS-omega-oriT. The resulting plasmids were named pHS7/pHS8 (PstI cloning, both orientations), pHS9/pHS 14 (EcoRI cloning, both orientations), pHS12 (Bg/II-digested pilE1 cloned into the BamHI site of the pKS-omega-oriT polylinker), and pHS13 (Bg/II-digested pilE1 cloned into the BamHI site flanking the oriT region) (Fig. 3C). All six plasmids were transferred to the two H. elongata strains used in this study. In all cases, streptomycin-resistant transconjugants arose at an average frequency of 3x 10 3 transconjugants per donor cell, indicating that maintenance of the hybrid plasmids in these hosts was due to the presence of pilE1. Moreover, the EcoRI, PstI and Bg/II sites in pilE1 do not seem to interrupt any function essential for replication and maintenance of the plasmid in H. elongata.

416

1 2 3 4 5 6 7 8 9 10

23.0 9.4 6.5 4.3

2.3 20

Fig. 4 Plasmid DNA from transconjugants ofH. elongata ATCC 33174 carrying the plasmids pHS7 (lane 4, EcoRI digested), pHS8 (lane 5, EcoRI digested), pHS9 (lane 6, PstI digested), pHS14 (lane 7, PstI digested), pHS12 (lane 8, PstI digested), and pHS13 (lane 9, ItindIII+XbaI digested). Lane 3, EcoRI-digested pilE1 DNA. Lanes 1 and 10, ItindlII-digested lambda DNA. Lane 2, EcoRI + HindlII-digested lambda DNA

To determine whether the presence of pilE-derived plasmids in H. elongata ATCC 33174 caused the loss of the endogenous plasmid pilE1, plasmid DNA was prepared from transconjugants of this strain and digested with a restriction endonuclease that allowed differentia- tion of the restriction pattern of the derivative plasmids from that of pilE1 (Fig. 4). In all cases, these patterns were like those of the pHEl-derived plasmids (lanes 4- 9), indicating that, as expected, the derivative plasmids are incompatible with pilE1, which was lost when selection was carried out for transconjugants harbouring streptomycin-resistant plasmids. No rearrangements or modifications of any pHS plasmid were observed after transfer to Halomonas.

Construction of a pHEl-derived shuttle vector for Halomonas strains

Although any of the constructed pilE1 derivatives could be used as a vector, the deletion of some repeated restriction sites would greatly improve their suitability as a cloning vehicle. Plasmid pHS13 was selected since it possesses unique sites for BamHI, EcoRV, NotI, KpnI, SalI and SpeI. The region ofpHS13 located between the Sinai and EcoRV sites of the pBluescript polylinker was deleted (both flanking sites included) by Sinai + EcoRV digestion of pHS13, followed by DNA precipitation (to eliminate the Sinai to EcoRV sequence of the polylinker), blunt-end ligation, and transfer to competent E. coli DH5~ cells. Transformants were selected on LB supplemented with ampicillin and streptomycin, to ensure that the large 6.45 kb EcoRV-SmaI fragment of pHS 13 (containing the streptomycin/spectinomycin cassette, the or- i Tregion and part of p i lE 1) was present in the recombi- nant clones. Clones containing unique recognition sites for EcoRI and PstI were selected and checked for the

Xb Ac Ac Scll

oriT 17

H o H S 1 5 Hc

sPh--IUl "2.2 5 kb I~'Hc Sml /spR~' ~H

HBSpeXb Ea, N Scl lBx Scl

Fig. 5 Restriction map and diagram of the shuttle vector pHS15. It includes the entire 4.2 kb of pilE1 (empty area, all known restriction sites are shown), the 1.95 kb HindIII fragment containing the SmR/Spc a determinant (hatched area), the 2.95 kb E. coli plasmid pBluescript KS (including the ampicillin resistance determinant and the replication origin from E. coli), and the 3.1 kb HindI- II + SalI oriT region. This vector has unique BamHI, EcoRI, KpnI, Notl, PstI, SaII, Sinai, and SpeI sites. The restriction sites in the Bluescript polylinker are indicated as follows: A, ApaI; Ac AccI; B, BamHI; Bx, BstXI; C, ClaI; D, DraI; E, EcoRI; Ea, EagI; EV, EcoRV; H, HindII; Hc, HincII; K, KpnI; N, NotI; P, PstI; ScI, II, SacI, II; Sm, Sinai; Spe, SpeI

presence of the 6.45 kb EcoRV-SmaI fragment in the same orientation as pHS13. One of these resulting derivative plasmids was selected and named pHS15 (Fig. 5). This shuttle vector possesses unique recognition sites for BamHI, NotI, KpnI, SalI, and SpeI (in the two polylinkers derived from the pBluescript multiple cloning site), and EcoRI, PstI and SmaI (in pilE1 DNA). The PstI and EcoRI sites at least are suitable for cloning (as shown by the construction of the plasmids pHS7/pHS8 and pHS9/pHS14). Besides the two strains ofH. elongata tested, pHS15 was tested for mobilization and replication in three other species of Halomonas, H. subglaciescola, "H. israelensis", and H. halodurans. In all cases, streptomycin-resistant colonies arose at a frequency very similar to that obtained when plasmid pHS13 was transferred to H. eIongata. All these data support the use of pHS15 as a shuttle vector for Halomonas spp.

Discussion

The molecular genetics of moderately halophilic eubacteria is poorly understood. There are several reasons for this, including the lack of studies on genetic exchange systems such as conjugation, transformation or trans- duction, the paucity of reports on isolation of mutants (Kogut et al. 1992), and the absence of suitable plasmid

vectors. The isolation of autochthonous plasmids from these extremophiles, which could be used for the development of cloning vectors, should overcome some of these difficulties. Amongst the moderate halophiles, members of the genus Halomonas display several features that would be useful for genetic studies.

In this paper, we have demonstrated the presence of plasmid DNA in three strains of Halomonas using the alkaline lysis procedure. In a previous study (Fer- n/mdez-Castillo et al. 1992) we reported the isolation and partial characterization of the first plasmid described from moderate halophiles, pMH1. This plasmid was detected in four moderately halophilic strains, including two species of Halomonas, H. halmophila ATCC 19717 and H. elongata ATCC 33173. In contrast to the plasmids described in this work, pMH1 could not be detected by ethidium bromide staining of standard plasmid DNA preparations on agarose gels. However, pMH1 could be detected when these preparations were introduced into E. coli by transformation or in total DNA preparations by Southern blot analysis (Fer- nfindez-Castillo et al. 1992). The question remains why the yield of pMH1 was so low that it escaped detection by conventional procedures. One possibility is the presence in the strains that carry pMH1 of nucleases that rapidly degrade DNA after cellular lysis. A second ex- planation is close association of pMH1 with the cyto- plasmic membrane or with the chromosome in the parental strains but not in E. coli. Therefore, although pMH1 could have been used for the development of a cloning vector, our inability to visualize it following a conventional plasmid DNA preparation method led us to look for other plasmids that could be more easily manipulated. Among these plasmids, pilE1 (isolated from H. etongata ATCC 33174) was selected for further characterization and the construction of a shuttle vector for Halomonas strains. In fact, plasmids pMH1 (Fer- n/mdez-Castillo et al. 1992) and pilE1 (this study) seem to be very different from each other: pilE1 is unable to replicate in E. coli cells, while pMH1 could be maintained in this host; pilE1 could be isolated by a standard plasmid DNA preparation procedure whereas pMH1 could not. All these data are consistent with the fact that the two plasmids do not share DNA homology as judged by hybridization experiments. Attempts to as- sociate genetic determinants for antibiotic resistance with pilE1 were unsuccessful. It should be noted that ampicillin could not be tested since plasmid pilE11 car- ries the E. coli 13-1actamase gene (in pBluescript). More- over, it is conceivable that the cloning of pilE1 in pBluescript (to give plasmid pilE11) might have inser- tionally inactivated an antibiotic resistance gene. There- fore, pilE1 remains cryptic since no functions have so far been found to be encoded by this plasmid.

The small size of pilE1 made this plasmid particular- ly attractive for the construction of shuttle vectors that could be used in molecular studies on moderate halophiles. These chimeric pHS plasmids were generated in a number of steps. A resistance gene suitable for

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moderately halophilic bacteria was included by cloning the omega cassette carrying streptomycin/spectinomycin resistance into the conventional E. coIi vector pBluescript KS. Transformation systems have not been developed in moderate halophiles. Therefore, to enable these vectors to be mobilized from E. coli to Halomonas spp., the oriT of the broad-host-range IncP plasmid RK2 was incorporated. Finally, to ensure replication in Halomonas, the small plasmid pilE1 from H. elongata was included. Several pilE1 derivative plasmids were constructed to ensure that no vital functions in pilE1 had been disrupted by the cloning procedure. The constructed pHS plasmids could be mobilized from E. coli DH5~ to H. elongata by the RK2 derivative pRK2013 at frequencies comparable to those for normal RK2 transfer (Priefer et al. 1985), indicating the effectiveness of the system in mobilizing DNA. As shown in Fig. 4, when introduced into H. elongata ATCC 33174, pHE1- derived plasmids are incompatible with pilE1. Since selection was for transconjugants harbouring streptomycin-resistant plasmids, pilE1 was lost. However, further experiments will be necessary to compare the sta- bility of pilE1 and pHEl-derived plasmids in Halomonas under non-selective conditions. Since pHS plasmids were transferred to Halomonas from a Dam + E. coli strain (DH5~), they were presumably methylated at their XbaI site within pilE1 DNA. The existence of a restriction barrier between E. coli and Halomonas via methylation seems unlikely in view of the conjugation frequencies obtained. However, this possibility cannot yet be ruled out and should be explored in more detail. The existence of a restriction system similar to that of E. coli (Heitman and Model 1987), which recognizes and degrades DNA methylated by a methylase foreign to E. coli, has been also reported for the extreme halophile Haloferax volcanii (Holmes et al. 1991).

Finally, an improved cloning vector, pHS15, was constructed. This vector contains a number of unique restriction sites commonly used for cloning foreign DNA. Moreover, it could be mobilized and maintained in all the Halomonas species tested (H. elongata, H. halodurans, "H. israelensis", and H. subglaciescola). Therefore, pHS15 seems to be a good candidate for use as a cloning vector in the genetic analysis of Halomonas spp. Experiments are currently in progress to determine the host range of pHS15 with the aim of extending its use as a cloning vector to other Halomonas strains as well as to other moderately halophilic eubacteria.

A deeper knowledge of the genetics of moderate halophiles is necessary to understand the molecular ba- sis of salt tolerance and salt dependence in prokaryotes. In addition, the genetic manipulation of such extremophiles has great potential in biotechnology. In this paper, we demonstrate that genetic transfer between non-halophilic (E. coli) and moderately halophilic (Halomonas spp.) eubacteria is possible via conjugation. Moreover, we report the first cloning vector for moderate halophiles. This vector should constitute a useful tool for further studies on both fundamental and ap-

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plied aspects of the genetics of this group of extremophiles.

Acknowledgements We thank Dr. V. N. Iyer for providing the plasmid pNH-kan/oriT. Research in the authors' laboratory was supported by grants from the Commission of the European Com- munities (Generic Project "Biotechnology of Extremophiles", BIO-CT93-02734), Ministerio de Educacion y Ciencia, Spain (PB92-0670 and BIO94-0846-CE), and Junta de Andalucia.

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