Host Range, Stability and Compatibility of Broad Host ... - Science Direct

System. App!. Microbio!. 20, 173-181 (1997) © Gustav Fischer Verlag

Host Range, Stability and Compatibility of Broad Host-RangePlasmids and a Shuttle Vector in Moderately Halophilic Bacteria. Evidence of Intrageneric and Intergeneric Conjugation in Moderate Halophiles CARMEN VARGAS, MARIA J. CORONADO, ANTONIO VENTO SA and JOAQUIN J. NIETO Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain

Received September 23,1996

Summary Broad-host-range plasmids, belonging to the incompatibility groups IncP (pVK102), IncQ (pKT230), IncW (pGV1l24), and IncN (pCU1), as well as pHS15 (a shuttle vector based on an endogenous plasmid of the moderate halophile Halomonas elongata) were transferred by conjugation from E. coli to moderately halophilic strains of the genera Chromohalobacter, Deleya, Halomonas, Vibrio and Volcaniella, to test their utility as cloning vectors for these extremophiles. Several factors affecting the efficiency of conjugation (cell growth phase, mating time, ratio donor:recipient, and composition and salinity of the mating medium) were evaluated. The highest frequencies (from 10-1 to 10-4 transconjugants/recipient cell) were achieved when exponentially grown donor and recipient cells were mixed and incubated for 16 h at 30°C on SW-2 saline medium at a ratio 1:1 (for Deleya, Halomonas and Volcaniella strains) or SW-5 saline medium at a ratio 2:1 (for Chromohalobacter and Vibrio strains). Whereas IncP, IncQ, and pHS15 plasmids were able to replicate in all strains tested, the IncW plasmid pGVl124 was only maintained in H. elongata, and pCUl could not be established in any of the moderate halophiles tested. The IncQ plasmid pKT230 was the most stably maintained in the majority of the moderate halophiles. The shuttle vector pHS15 revealed to be compatible with each of the broad-host-range plasmids assayed. Finally, both intra generic and intergeneric conjugation between moderate halophiles have been demonstrated by using the self-transmissible IncP plasmid RK2. Transfer of RK2 between Halomonas spp. was observed at 2-7% total salts, yielding the highest transfer frequencies (from 1.2x10-J to 2.8x10-4 ) on SW-2 medium. Intergeneric conjugation on SW-2 between Halomonas and other moderate halophiles such as Volcaniella (transfer frequency of 2x01 0-5 ) and Deleya (5xlO-5 ) were also found. This is the first report on the existence of conjugation between moderately halophilic bacteria. Key words: Moderate halophiles - Conjugation - Plasmid transfer - Cloning vectors

Introduction Halophilic microorganisms are defined as those which require high NaCl concentrations to grow and survive (KUSHNER, 1978). Besides the extremely halophilic aerobic archaea (the so-called halobacteria), the moderately halophilic bacteria are the most important group of microorganisms adapted to thrive in hypersaline environments. These extremophiles are defined as those prokaryotes which grow optimally in media containing 3 to 15% NaCI (KUSHNER and KAMEKURA, 1988), and constitute a very heterogeneous physiological group, including a great variety of bacteria (VENTOSA, 1994). Due to their abundance in hypersaline habitats, this microbial

group plays an important role in the ecology of such extreme environments, representing an excellent example of adaptation to frequent changes in extracellular osmolality (SODE and TATARA, 1993). Besides, these extremophiles have gained considerable importance in recent years due to their interesting biotechnological applications and potentialities. Thus, many of them produce halophilic exoenzymes such as amylases, nucleases, and proteases of potential commercial interest (KAMEKURA, 1986), and the majority accumulate a variety of organic osmolites, named "compatible solutes", which could be utilized as stabilizers and protecting agents for industrial

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enzymes and whole cells (GALINSKI, 1993). In addition, their use in enhanced oil recovery processes or in the degradation of industrial residues and toxic chemicals that can pollute hypersaline habitats have been claimed (VENTOSA and NIETO, 1995). Furthermore, since moderate halophiles exhibit a wide salt tolerance among prokaryotes, they may be considered as excellent biological models for the elucidation of the molecular biology of bacterial osmoregulation processes. Despite extensive studies concerning their physiology (KUSHNER and KAMEKURA, 1988) and ecology (RoDRIGUEZ-VALERA, 1986), little information is still available on their genetics, hampering the genetic manipulation of these extremophiles for biotechnological purposes and the understanding of the molecular basis of their salt tolerance and salt dependence. In fact, reports on the isolation of stable mutants (KOGUT et al., 1992; NIETO et al., 1993; KUNTE and GALINSKI, 1995), or the presence of autochthonous plasmids (FERNANDEZ-CASTILLO et al., 1992; VARGAS et al., 1995; MELLADO et al., 1995) are very scarce. Moreover, genetic transfer procedures such as transformation or transduction have not been so far developed for these halophiles. Very recently, we have located and characterized the minimal replication origin of the cryptic plasmid pCM1 from Chromohalobacter marismortui (MELLADO et al., 1995) , and developed the first shuttle vector (pSH15) for moderate halophiles from a small (4.2-kb) cryptic plasmid from Halomonas elongata ATCC 33174 (VARGAS et al., 1995). This vector (which contains a streptomycin-resistance gene, the E. coli plasmid pBluescript, and the RK2 oriT region) could be mobilized by the RK2 derivative pRK2013 to Halomonas spp., demonstrating that genetic transfer between non-halophilic (Escherichia coli) and moderately halophilic (Halomonas spp.) bacteria is possible via conjugation (VARGAS et al., 1995). By using this vector, we have very recently shown that the ice nucleation gene inaZ of Pseudomonas syringae is efficiently expressed in several moderately halophilic species, demonstrating its utility as a reporter gene for promoter activity and gene expression studies in these bacteria (ARVANITIS et al., 1995). So far, pHS15 has been shown to replicate in both E. coli and species of the genera Halomonas (H. elongata, H. halodurans, H. meridiana, and H. subglaciescola) (VARGAS et al., 1995), Deleya (D. halophila) and Volcaniella (V: eurihalina) (ARVANITIS et al., 1995). However, quantitative studies about transfer frequencies and stability of maintenance in these hosts were not approached. Otherwise, Vibrio costicola has been traditionally considered as the most representative organism within the group of moderately halophilic bacteria and in fact, the majority of physiological studies on this group of extremophiles have been carried out by using this species (KUSHNER, 1978; KUSHNER and KAMEKURA, 1988). We have reported that this moderate halophile predominates in ponds and salterns with 10% to 25% (w/v) salts (RoDRIGUEZ-VALERA et al., 1985; GARCIA et al., 1987). However, no cloning vectors useful for genetic studies in this interesting moderate halophile have been thus developed. Since V. costicola shows a very eurihyaline response and

many studies concerning its physiological adaptation to saline media have been carried out (KUSHNER and KAMEKURA, 1988), the availability of cloning vectors that would permit the development of genetic studies in this halophile focused on osmoregulatory mechanisms is clearly needed. Besides shuttle vectors, which are generally high copynumber plasm ids and are restricted in host range, broadhost-range vectors (usually low copy number, apart from IncQ plasmids) also represent an important alternative in genetic studies (SCHMIDHAUSER et al., 1988). In this work, a comparative study on plasmid host-range, stability, and compatibility in moderate halophiles, including V. costicola, have been done by using the shuttle vector pHS15 and broad-host-range plasmids of Gram-negative bacteria belonging to the incompatibility groups IncP, IncQ, IncW, and IncN. Besides, conjugation between representative species of moderate halophiles has been tested. The results presented herein demonstrate, for the first time, the existence of both intrageneric and intergeneric conjugation between moderately halophilic bacteria.

Materials and Methods Bacterial strains, plamids, media, and growth conditions: The moderately halophilic strains used as recipients in this study were spontaneous rifampicin-resistant mutants isolated from the corresponding culture collection strains. These were Chromohalobacter marismortui ATCC 17056, Deleya halophila CCM 3662, "Halomonas canadiana" ATCC 43984, H. elongata ATCC 33173, H. elongata ATCC 33174, H. halodurans ATCC 29629, "H. israelensis" ATCC 19717, H. meridiana DSM 5425, H. subglaciescola UQM 2927, Vibrio costicola NCIMB 701, and Volcaniella eurihalina ATCC 49336. The E. coli donor strains and plasmids transferred to the moderate halophiles are listed in Table 1. All moderate halophiles were grown at 30°C in a saline medium (SW-10) containing 10% (w/v) total salts and 0.5% (w/v) yeast extract (Difco) (NIETO et al., 1989). E. coli strains were grown at 37°C in Luria medium (LB) (SAMBROOK et al., 1989), which contains 1 % NaC!. The pH of the media was adjusted to 7.2. Liquid cultures were shaken at 200 rpm in an orbital shaker. Solid media were obtained by adding 2% (w/v) Bacto Agar (Difco). For matings, other final concentrations of total salts (7, 5, 3, or 2 %) in the SW medium (SW-7, SW-5, SW-3, and SW-2, respectively) and of

Table 1. E. coli strains and plasmids used in this study. Strain

Plasmid

Relevant plasmid features'

HB101 S17-1 S17-1 HBlOl HB101 S17-1

RK2 pVKl02 pKT230 pGVl124 pCUl pHS15

Ap', Tc', Km', Tra+, Mob+, IncP Tc', Km', Tra-, Mob+, IncP Km', Sm', Tra-, Mob+, IncQ Sm', Cm', Tra-, Mob+, IncW Cm', Tra+, Mob+, IncN Sm', Spc', Ap', Tra-, Mob+8

Abbreviations: Ap - ampicillin; Cm - chloramphenicol; Km kanamycin; Spc - spectinomycin; Sm - streptomycin; T c - tetracycline; Tra+ - conjugal transfer functions; Mob+ - mobilization functions; r - resistant. a

Genetic transfer in moderate halophiles NaCl (2%) in the LB medium (LB-2) were also used. When required, media were supplemented with ampicillin (Ap), chloramphenicol (Cm), kanamycin (Km), rifampicin (Rp), spectinomycin (Spc), streptomycin (Sm), or tetracycline (Tc) at a determined concentration. Conjugal transfer of plasmids: This was done by filter matings on solid media. Biparental matings were performed to transfer all plasmids except pGV1124 (IncW), which was assisted by the tra genes of the IncN plasmid pCU1 in a triparental mating. Plasmids RK2 (IncP) and pCUl (IncN) are self-transmissible. All other plasm ids were mobilized in trans by the RK2 functions integrated in the genome of E. coli S17-1 (SI\101\ et aI., 1983). Respective donor and recipient cell cultures were incubated with the corresponding antibiotics, harvested, mixed at a determined ratio, washed twice with liquid mating medium, and resuspended in 100 pi of the same medium. The mating mixture was then placed on the surface of a sterile 0.45 pm pore-membrane filter onto a plate of the corresponding solid mating medium and incubated at 30°C (different conjugation times were used). The cells were then resuspended in 20 % (w/v) sterile glycerol and, after appropriate dilutions, plated onto SW-2 (for Halomonas, Deleya, or Volcaniella strains) or SW-5 (for Chromohalobacter or Vibrio strains) media, containing rifampicin (to counterselect the donor strain) and a suitable antibiotic to select for transconjugants carrying the plasmid. We have previously reported that the susceptibility of moderate halophiles to many antimicrobials is greatly affected by the salinity of the medium (CORONADO et aI., 1995). Thus, selective media were individually designed according to the strain and the minimal inhibitory concentration (MIC) of the antibiotic in the corresponding medium. As a control for spontaneous mutation, both parental strain were plated on the same selective media. To estimate the total number of recipient cells, appropriate dilutions of the mating mixture were also plated onto SW-2 or SW-5 media with rifampicin. The efficiency of conjugation (expressed as transfer frequency) was given as the number of transconjugants per final number of surviving recipient cells. Plasmid stability and compatibility analysis: The sta bility of plasmids was assessed by subculturing exponentially growing cultures of each transconjugant in non-selective SW-1 0 medium for a period equivalent to 80 generations. Subcultures were made at approximately 8-h intervals. For each subculture, the number of generations occurred was estimated (DURLAND and HELINSKI, 1987) and samples were diluted and plated for single colonies on antibiotic-free SW-10 plates. To determine the percentage of plasmid retention, 100 randomly selected colonies were replicated onto SW-2 (for Haiomonas, Deleya, or Voicaniella strains) or SW-5 (for Chromohalobacter of Vibrio

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strains) media containing the appropriate concentration of antibiotics. The existence of incompatibility between the plasmid pHS15 and each of the broad-host-range plasmids used in this study was investigated. Plasmids pVK 1 02 (IncP), pKT230 (IncQ) and pGV1124 (IncW) were independently transferred from E. coli to H. elongata ATCC 33174 cells carrying pHS15. Mixture matings were plated onto SW-2 medium containing Sm + Tc, Sm + Km, or Sm + Cm (to select for transconjugants carrying pHS15 + pVK102, pHS15 + pKT230 or pHS15 + pGVl124, respectively). In all cases, the presence of plasmids in the recipient strain was confirmed by alkaline-lysis isolation, enzyme digestion, and agarose gel electrophoresis (SAMBROOK et aI., 1989).

Results Determination of optimal conditions for conjugation between E. coli and moderate halophiles To establish the optimal conditions for the matings, several factors affecting the conjugation transfer were examined. These included the cell growth phase (mid-exponential or early-stationary), the mating time (4, 8, or 16 h), the ratio donor:recipient (1 :1, 1:2, 1:4, 1:8,2:1,4:1, and 8:1) and the composition and salinity of the medium used for the mating (SW-2, SW-3, SW-5, LB, or LB-2). E. coli S17-1 carrying the plasmid pKT230 and H. elongata ATCC 33173 were selected for this study as donor and recipient strains, respectively. By using preliminary test conditions (ratio donor:recipient 1:1, SW-3 as the mating medium, and incubation at 30°C for 16 h) the transfer frequency estimated when exponentially grown cells (OD 600 = 0.4,10 8 cells/ml) were used as donor and recipient (3x103) was drastically reduced (5xl0 a'2 ) when the same cells were grown up to early-stationary phase (overnight cultures). Moreover, transfer frequencies increased with increasing mating times, yielding better transfer frequencies (2x10- 1) with 16 h of mating than with 8 h (2xlO-4 ), or 4 h (8.5xl0-5 ). Therefore, to optimize the medium and the ratio donor:recipient, exponentially grown cultures and 16 h of incubation time were always employed. Table 2 shows the effect of the composition and salinity of the mating medium on the conjugation efficiency

Table 2. Effect of the composition and salinity of the mating medium on the conjugation efficiency between E. coli S17 -1 (pKT230) and H. elongata ATCC 33173 at different ratios donor:recipient." Media

LB LB-2 SW-2 SW-3 SW-5

Ratio donor:recipient 1:1

1:2

1:4

1:8

2:1

4:1

8:1

6.3x10-5b 5.6xlO-5 2.1x10-1 3.1x10-1 5.4xl0-5

8.9x10-7 1.3xlO--4 2.6xlO-2 2.5x10-4 2.5xl0- 5

5.6x10-7 7.6x10--4 7.9xl0-1 5.6x10-5 1.3x10-5

2.0xl0- 7 3.9xl0-4 2.4x10-3 2. 7x1 0-5 7.8x10-6

1.7xl0-7 2.6x10-2 3.0xlO-3 8.4x10-4 4.4x10-4

2.8x10-7 2.3xl0- 2 3.0xl0-3 l.lx10-3 4.0x10-4

1.0x10-7 2.0x10-2 5.6xlO-3 2.9x10-1 3.8x10-4

" Exponentially grown donor and recipient cells were mixed and incubated at 30°C for 16 h. b Transfer frequencies are expressed as number of transconjugants per recipient cell. LB = 1 % NaCl, LB-2 NaCl, SW-3 = 3% NaCI, and SW-5 = 5% NaCI.

=2 % NaCl; SW-2 = 2 %

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between E. coli and H. elongata when different ratios donor:recipient were assayed. It is noteworthy that, regardless of the medium used for the mating, transfer frequencies were not significantly improved when a 4-fold or a 8-fold excess of E. coli donor cells was used instead of a 2-fold excess. Likewise, similar transfer frequencies were calculated when either a 4-fold excess or a 8-fold excess of H. elongata cells were used as recipient. With regard to the culture medium used for the conjugation, the lowest transfer frequencies were found when matings were made on LB medium, varying from 6.3xlO-s to lxlO-7. As the salinity of the LB medium was raised up to 2% NaCl (LB-2), improved conjugation frequencies were detected, especially when a 2-fold excess of E. coli donor cells were used (2.6xlO-Z). SW-2 medium was found to be the optimal medium for conjugation between E. coli and H. elongata, resulting in the highest transfer frequency (2xl 0- 1) when the donor and recipient culture cells were mixed at a ratio 1:1. Instead, when the ratio donor:recipient was 1:2, the transfer frequency in the same medium was about lO-fold lower (2.6xlO-Z). This can be also considered a very good transfer efficiency, comparable to that obtained when LB-2 was used as a mating medium, and donor and recipient cells were mixed at a ratio 2:1 : A remarkable influence of salinity on the conjugation efficiency was observed when transfer frequencies found

with SW-2, SW-3, and SW-S media were compared. Thus, for the same ratio donor:recipient (1:1), if compared with the frequency obtained in SW-2 (2xlO- I ), the frequency of transfer decreased 100-fold (3xlO- 3 ) when using SW-3 and about lO.OOO-fold (S.3xlO-S) when using SW-S. Improved conjugation efficiencies in SW-3 and SW-S media (varying from S.6xlO- 3 to 3.8xl0-4) were found when an excess of E. coli donor cells was added to the mating mixture.

Host-range of pHS15 and broad-host range plasmids in moderate halophiles By using optimal conditions for conjugation, several plasmids based on IncP, IncQ, and IncN replicons, as well as the shuttle vector pHSlS, were transferred from E. coli to several moderately halophilic bacteria to test their ability to replicate in these extremophiles. As shown in Table 3, plasmids pVKl02 (IncP), pKT230 (IncQ) and pHSlS were able to replicate in all strains tested. In contrast, the IncW plasmid pGVl124 could only be maintained in the two H. elongata strains, and the IncN plasmid pCUl could not be established in any of the moderate halophiles tested. In all cases when transfer occurred, the moderately halophilic strains expressed all antibiotic resistance genes carried by the respective plasmids. When

Table 3. Host-range, among moderate halophiles, of plasmids belonging to the incompatibility groups IncP, IncQ, IncW, and IncN, as well as of pHS15, a shuttle vector based on an endogenous cryptic plasmid of H. elongata ATCC 33174'. Recipient strain

Conjugation frequencyb pVK102 (IncP)

pKT230 (IncQ)

pGV1124 (IncW)

C. marismortui ATCC 17056 D. halophila CCM 3662

3.3x10-4

7.2x10-3

3.2x10-3

8.3x10-2

2.5x10-1

6.3x10-4

pCUl (lncN)

pHS15

"H. canadiana"

2.0x10-3

2.4x10-z

2.9xl0-3

ATCC 43984 H. halodurans ATCC29629

6.2x10-z

2.1x10-1

2.5x10-3

"H. israelensis"

5.0x10-1

3.3x10-z

8.3x10-2

7. 6x10-z

9.5x10-z

4.2xl0-6

1.6x10-1

3.2x10-3

5.0x10-z

3.9x10-6

4.0x10-2

6.8x10-3

7.4x10-2

1.4xlO-l

5.0x10-z

3.9xlO-3

1.1x10-2

7.0x10-4

1.3x10-3

1. 7x10-4

6.0x10-3

5.0x10-2

4.1x10-2

ATCC 19717 H. elongata ATCC 33173 H. elongata ATCC 33174 H. meridiana DSM 5425 H. subglaciescola UQM2927 V. costicola

NCIMB 701 V. eurihalina

ATCC 49336 Exponentially grown donor and recipient cells were mixed and incubated at 30°C for 16 h. Conjugations involving Deleya, Halomonas , and Volcaniella strains were made on saline SW-2 medium by using a ratio donor:recipient of 1:1. Conjugations involving Chromohalobacter and Vibrio strains were made on saline SW-5 medium by using a ratio donor:recipient of 2:1.

a

b Transfer frequencies are expressed as number of transconjugants per recipient cell.

Genetic transfer in moderate halophiles

177

pHS15

120

a

10 c .2 80

--

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....

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Fig. 1. Analysis of the stability of plasmids pHS15, pKT230, and pVK102 in several moderately halophilic strains. The assay is described in Materials and Methods section. Percent plasmid retention means the percentage of the cells population which still harbours the plasmid.

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90

C. marismortui ATCC17056 -+ D. halophila CCM3662

..,... H. elongata ATCC33173 meridiana DSM5425

H. elongata ATCC33174

V.eurihalina ATCC49336

Number of generations pKT230

120

pV K102

120

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120.--------------------------,

using IncP, IncQ plasmids, or pHS15, transfer frequencies varied slightly depending on the individual strain and the plasmid used, but they ranged from 10-1 to 10-4 transconjugants/recipient cell. In marked contrast, very low conjugation frequencies were found in those experiments involving transfer of pGVl124 to H. elongata strains.

Plasmid stability analysis The stability of pHS15 and the IncP, IncQ, and IncW cloning vectors was assayed and compared up to approximately 80 generations. For this purpose, seven representative strains of the genera Chromohalobacter, Deleya, Halomonas, Vibrio, and Volcaniella, containing the above cited plasmids, were selected (Figure 1). According to our results, the hybrid plasmid pHS15 cannot be stably maintained, in the absence of selective pressure, for long time in any of the moderate halophiles tested, including H. elongata ATCC 33174 (the parental strain of pHEl, the endogenous plasmid used for pHS15 construction). In any case, the highest stability of pHS15 occurred in the parental strain (5 % loss after 50 generations, 60% loss after 80 generations) and the lowest in V. costicola (90% loss after 15 generations, and the plasmid was completely lost after 40 generations) and in D. halophila (80% loss after 20 generations, and the plasmid was completely lost after 60 generations). In the other moderate halophiles tested, the stability of maintenance of pHS15 varied depending on the host, showing after 40 generations a 50% loss in H. elongata ATCC 33173 and C. marismortui, and a 20% loss in H. meridiana and V. eurihalina. In all four cases, pHS15 was lost after 80 generations. The IncQ plasmid vector pKT230 was found to be stably inherited for more than 80 generations in all strains tested, except in V. costicola, where it was completely lost after 20 generations (Figure IB). On the contrary, the IncP plasmid pVKI02 was highly unstable, being completely lost after about 25-50 generations in most moderately halophilic hosts, with the exception of C. marismortui (less than 5% loss after 80 generations) (Figure lC) . Plasmid pVKI02 is a mini-RK2 derivative, in which some of the functions of the parental plasmid have been deleted. Therefore, to test the possibility that those functions could be necessary for the stable maintenance of IncP plasmids in moderate halophiles, we transferred the self-transmissible parental plasmid RK2 to H. elongata ATCC 33173 and tested its stability in this host. As well as pVKI02, plasmid RK2 was readily lost after about 18 generations (Figure 2). Maintenance of plasmid pGVl124 in H. elongata was observed to be very unstable (it was lost after 48 generations under nonselective conditions) (Figure 2). Plasmid compatibility determination

The existence of incompatibility between the shuttle vector pHS15 and each of the broad-host-range plasmids tested was determined by individual transfer of pVKI02,

1-

RK2 -+- pGV1124 I

c

o

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Q)

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5

10 15 20 25 30 35 40 45

Number of generations Fig. 2. Stability of the broad-host range plasmids RK2 and pGV1124 in H. elongata ATCC 33174. The assay is described in Materials and Methods section. Percent plasmid retention means the percentage of the cell population which still harbours the plasmid.

pKT230, and pGVl124 from E. coli to H. elongata ATCC 33174 carrying pHS15. This strain is free of the native plasmid pHEl, since pHS15 contains the entire pHEl sequence and, therefore, both replicons are incompatible (VARGAS et aI., 1995). Comaintenance of pHS15 and each of the broad-host-range plasmids was determined by selection fo r the antibiotic resistance markers linked to both plasmids. Restriction enzyme analysis of plasmid DNA extracted from the transconjugants confirmed the presence of both plasmids in H. elongata ATCC 33174 (data not shown). The shuttle vector pHS15 showed to be compatible with each of the broadhost-range vectors assayed.

Conjugation between moderate halophiles To test the existence of conjugation between moderately halophilic bacteria, the strain H. elongata ATCC 33173 carrying the self-transmissihle IneP plasmid RK2,

and a spontaneous rifampicin-resistant mutant of the same culture collection strain, were used as donor and recipient, respectively. Exponentially grown donor and recipient cells were grown at the same salinity as the mat-

Genetic transfer in moderate halophiles

ing medium. To ensure the maintenance of the plasmid RK2 in the donor cells, kanamycin was added to the growth media (50 fIg/ml to SW-2, 100 fIg/ml to SW-S, 200 fIg/ml to SW-7, and 300 fIg/ml to SW-10). Donor and recipient cultures were mixed at a ratio of 1:1 and then incubated at 30°C for 16 h on the surface of solid SW media. The highest conjugation frequencies were achieved when the mating mixture was incubated on SW2 medium (conjugation frequencies from 1.2xl0-3 to 2.8x10-4). When the salinity of the mating medium was raised up to 5% or 7%, conjugation was also observed although the transfer frequencies were lower, ranging from 1.4x10-S (on SW-S) to 2.Sxl0-7 (on SW- 7). No genetic transfer by conjugation was observed on SW-10 medium, although the assay was repeated five times. Finally, to check the occurrence of intergeneric conjugation between moderate halophiles, the self-transmissible plasmid RK2 was transferred from H. elongata to spontaneous rifampicin-resistant mutants of Volcaniella eurihalina and Deleya halophila. The same range of salinity as above was used. Transfer of RK2 was also found, although less efficiently than in intra generic matings. Thus, transfer frequencies on SW-2 were 2.0x10-S, for V. eurihalina, and S.Ox10-S for D. halophila. At salinities higher than SW-2, transconjugants were not detected in five different mating experiments.

Discussion Moderately halophilic bacteria are excellent models to study the molecular basis of prokaryotic osmoregulation, and they have new promising potentialities for biotechnological and bioremediation processes. Although some recent advances on the genetics of moderate halophiles have been achieved (VARGAS et aI., 1995; MELLA DO et aI., 1995; ARVANITIS et aI., 1995), the knowledge of how these extremophiles, which usually inhabit hypersaline environments, exchange genetic information in their natural habitat is still very limited. In fact, genetic transfer mechanisms such as transformation or phage transduction have not been reported in these bacteria, and conjugation has only been demonstrated for a few of them, when E. coli cells were used as donors of the plasmid transferred to a recipient moderately halophilic strain. Thus, shuttle vectors based on endogenous plasmids isolated from moderate halophiles (VARGAS et aI., 1995; MELLADO et aI., 1995), or the broad-host-range plasmid RK2 (KUNTE and GALINSKI, 1995) have been transferred from E. coli to some moderately halophilic bacteria, but not to V. costicola, considered as the most representative species of this physiological group. Besides, the existence of conjugation between these extremophilcs had not been so far shown. In this work, conjugational genetic transfer in moderately halophilic bacteria has been investigated by using two complementary approaches . Firstly, conjugation between E. coli and a wide range of moderate halophiles has been standarized and used to test the host range, stability, and compatibility of the shuttle vector pHS15 and

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of a number of broad-host-range plasmids of Gram-negative bacteria. Secondly, the existence of conjugation between moderately halophilic bacteria themselves has been demonstrated, for the first time. Amongst all conjugation assay parameters examined to optimize conjugation between E. coli and H. elongata, the age of donor and recipient cultures appeared to be the most critical factor, yielding much better transfer frequencies when exponentially grown cells were used. LB did not appear to be suitable for genetic transfer between E. coli and moderate halophiles, regardless of the ratio donor:recipient used. This is probably due to the fact that, although LB is a complex medium, it has a lower salinity than that in which moderate halophiles usually grow (2-20% total salts). The best transfer frequencies were found when media containing 2% NaCI (SW-2 or LB-2) were used for matings, indicating that they are suitable for conjugation between E. coli and moderately halophilic species with low requirements for salts (e.g. Halomonas, Volcaniella, or Deleya). As the salinity of the mating medium increased, the frequency of conjugation between E. coli and H. elongata decreased. This agrees with previous reports on the conjugation efficiency of E. coli, showing that an increased salt concentration correlated with a decreased conjugation efficiency of this bacterium (SODE and TATARA, 1993). This can be explained by the fact that, although E. coli can tolerate moderate salinities, the conditions of osmotic stress, to which moderate halophiles are well adapted, partially impair the viability of the non-halophilic E. coli cells. In summary, for genetic transfer between E. coli and those moderate halophiles which grow well in SW-2 or LB-2, such as Halomonas, Deleya, or Volcaniella, we propose the following mating conditions: OD 6oo = 0.4, 16 h of mating at 30°C on SW-2 (ratio donor:recipient 1:1) or LB-2 (ratio donor:recipient 2:1) media. For moderate halophiles with more stringent requirements for salts as Vibrio or Chromohalobacter (VENTOSA, 1994), conjugations may be made on SW-5 media but using a ratio donor-recipient 2: 1 to compensate the reduced viability of E. coli cells in this saline medium. Plasmids from the IncP, IncQ, and IncW groups have been shown to have a particulary broad range among Gram-negative bacteria (SCHMIDHAUSER et aI., 1988). The data presented herein on the transfer of the plasmids pVKI02 (IncP) and pKT230 (IncQ) to moderately halophilic strains support this general statement. However, except for H. e!ongata, no evidence of conjugal transfer of the IncW vector pGV1124 to moderate halophiles was detected. Even for H. e!ongata, transfer frequencies were not much above background levels due to spontaneous antibiotic-resistant mutants. It is worth mentioning that pGVl124 was mobilized from E. coli to moderate halophiles by using the IncN plasmid peUl as a "helper". It is known that the IncN "helpers" are not as efficient as the IncP ones to promote transfer of DNA (SCHMIDHAUSER et aI., 1988). Therefore, we can not rule out the possibility that transfer of the IncW plasmid pGV1124 to other moderate halophiles apart from H. elongata occurred, although below the level of detection.

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The shuttle vector pHS15 was relatively unstable in all moderately halophilic strains tested. The lowest stability of maintenance was found for the IncP plasmids RK2 and its derivative pVKI02 (except for C. marismortui), and the highest for the IncQ plasmid pKT230 (except for V. costicola). Since plasmids pHE1 and pHS15 utilize the same replication system, they are incompatible and thereby the transconjugant of H. elongata ATCC 33174 carrying pHS15 has lost the endogenous plasmid pHE1 (VARGAS et ai., 1995) which, therefore, does not influence pHS15 stability. It has been reported that joint replicons may, in some cases, reduce the stability of the resulting vector. Therefore, the relative unstability of the hybrid plasmid pHS15 in moderately halophilic strains might be a consequence of the incorporation of the E. coli plasmid pBluescript. However, it is also possible that pHE1 itself does not have mechanisms to ensure its correct segregation to daughter H. elongata cells. D. halophila CCM 3662, H. elongata ATCC 33173 and V. costicola NCIMB 701 contain the same 11.5-kb-sized plasmid, pMH1 (FERNANDEZ-CASTILLO et ai., 1992) and C. marismortui ATCC 17056 harbours a 17.5-kb narrow-host-range plasmid, pCM2 (MELLADO et ai., 1995). These plasmids could have a negative effect on pHS15 stability. The reason why the IncQ plasmid pKT230 was rapidly lost in V. costicola under nonselection conditions is unclear. However, since the endogenous plasmid pMH1 is also harboured by other moderately halophilic strains (FERNANDEZ-CASTILLO et ai., 1992) in which pKT230 is stably maintained, pKT230 instability in V. costicola must be due, not to the presence of pMH1 but to the absence of important regions in the chromosome of this moderate halophile. Conversely, our results indicate the lack, in most moderately halophilic hosts but not in Chromohalobacter, of essential functions for the stably maintenance of IncP plasmids. This result was unexpected, since the RK2 group of plasmids have been reported to be stably maintained in most Gram-negative hosts (SCHMIDHAUSER et ai., 1988). Recent studies have reported the instability of plasmid RK2 in other Gram-negative bacteria such as Chromatium vinosum (PATTARAGULWANI and DAHL, 1995). Plasmid pGV1124 is a derivative of the IncW plasmid pSa. It has been reported that mini-pSa plasmids lack the stable maintenance properties of the parental plasmid (SCHMIDHAUSER et ai., 1988). This fact, or the absence of regions necessary for plasmid stability in H. elongata, may account for the instability found in this host. Vectors with a broad-host-range have made a significant contribution to our knowledge about the genetics of many processes in Gram-negative bacteria. In parallel to the use of broad-host-range vectors, shuttle vectors have become very important tools in molecular genetics. Our results indicate that both IncQ and IncP plasmids can be particulary useful as cloning vectors for moderate halophiles. Moreover, the utility of pHS15 has been extended to other species of Halomonas as well as to other moderate halophiles, such as C. marismortui and V. costicola. Up to now, no cloning vectors for V. costicola

have been developed. The use of IncP, IncQ, or pHS15 may facilitate the study of the genetics of this representative moderate halophile. As expected, pHS15 was compatible with all the broad-host-range vectors assayed. This compatibility will allow genetic studies (for example, complementation or strain improvement) that use DNA fragments cloned into pHS15 and a broad-hostrange plasmid that are maintained together in moderately halophilic hosts. A limitation of the use of IncP plasmids, or pHS15, in these extremophiles involves their apparent instability in most strains tested. However, since even extremely unstable replicons can be maintained in a growing bacterial population by antibiotic selection, the importance of the stability of maintenance to the utility of IncP or pHS15 as cloning vectors seems to be irrelevant. On the other hand, in this study both intrageneric and intergeneric conjugation between moderately halophilic bacteria have been demonstrated, for the first time, by using the self-transmissible plasmid RK2. However, our results indicate that as the salinity of the mating medium increases, the frequency of RK2 transfer decreases. In fact, no transfer of this plasmid could be detected on saline SW-10 medium. The reason for this is unknown. It does not seem probable that moderately halophilic bacteria are uncapable of exchanging genetic information at 10% total salts, since these are actually the conditions that predominate in their natural habitats. Rather than the cells, it seems that the failure to conjugate at high salinity comes from the plasmid used. At this respect, it would be very interesting to test in future if any of the high molecular weight plasmids isolated in our laboratory from moderate halophiles (VARGAS et ai., 1995) are conjugative and, if so, whether or not they are able to mediate genetic transfer in the hypersaline conditions of the natural environments where moderately halophilic bacteria optimally grow and thrive.

Acknowledgements M. J. CORONADO is supported by a fellowship from the Spanish Ministerio de Educaci6n y Ciencia. This work was supported financially by the EC Generic Projects "Biotechnology of Extremophiles" (contract BI02-CT93-0274), and "Extremophiles as Cell Factories" (contract BI04-CT96-0488) the Spanish Ministerio of Educaci6n y Ciencia (PB 92-0670, BI0940846-CE, and PB93-0920), and the Junta de Andalucia.

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