Characterization of Plasmids in Halobacteria

Transcription

1 JouRNAL OF BACTERIOLOGY, Jan. 1981, p /81/ $02.00/0 Vol. 145, No. 1 Characterization of Plasmids in Halobacteria F. PFEIFER,* G. WEIDINGER, AND W. GOEBEL Institut fur Genetik und Mikrobiologie, Universitat Wurzburg, D-8700 Wurzburg, West Gernany Extrachromosomal, covalently closed circular deoxyribonucleic acid has been isolated from different species of halobacteria. Three strains of Halobacterium halobium and one of Halobacterium cutirubrum, all of which synthesize purple membrane (Pum+) and bacterioruberin (Rub'), contain plasmids of different size which share extensive sequence homologies. One strain of Halobacterium salinarium, another one of Halobacterium capanicum, and two new Halobacterium isolates from Tunisia, which are also Pum+ Rub+, do not harbor covalently closed circular deoxyribonucleic acid but contain sequences, presumably integrated into the chromosome, which are similar if not identical to those of phh1, i.e., the plasmid originally isolated from H. halobium. Three other halophilic strains, Halobacterium trapanicum, Halobacterium volcanii, and a new isolate from Israel, do not carry phhl-like sequences. These strains are, by morphological and physiological criteria, different from the others examined and harbor plasmids unrelated to phhl. Halobacteria are obligate halophiles which are found in waters ofhigh salt concentrations under conditions of high light intensity and low oxygen tension. Three species of the genus Halobacterium have been commonly studied, namely, H. halobium, H. cutirubrum, and H. salinarium (for review see reference 1). Many of their morphological and physiological properties are similar, and of particular interest is their capability of synthesizing the purple membrane (Pum) which consists of bacteriorhodopsin, a chromoprotein composed of the protein opsin (Ops) and retinal (Ret) (5). By means of the purple membrane these three halophilic species are able to convert light energy into chemical energy. Other Halobacterium species are found which require lower salt concentrations for optimal growth, i.e., 2.0 to 2.5 M NaCl, in comparison to the 4.3 M NaCl needed by the other species. These halophilic isolates lack the purple membrane and gas vacuoles (Vac), but synthesize carotinoids, particularly bacterioruberin (Rub). We have previously shown that H. halobium carries a large plasmid, phhl, which seems to be involved in the genetic determination of gas vacuoles and possibly other characteristics of this bacterium (6, 9). To test whether the presence of such plasmid DNA is a general phenomenon among Halobacterium species, we have analyzed the DNA of several halobacteria strains. It was found that three strains of H. halobium and one of Halobacterium cutirubrum harbor covalently closed circular (CCC) DNA with extended sequence homologies. No 369 CCC DNA could be isolated from an H. salinarium strain, a Halobacterium capanicum strain, or the new isolates designated Halobacterium tunesiensis, all of which synthesize purple membrane and ruberin. Only one of these strains (H. tunesiensis A2) synthesizes gas vacuoles. However, these species carry sequences homologous to the plasmid phh1 either integrated into the chromosome or on extrachromosomal DNA which cannot be isolated in the CCC form. Strains of three other halobacteria species, Halobacterium trapanicum, Halobacterium volcanii, and a new isolate from Israel, which are morphologically and physiologically (Pum Vac) unrelated to the other strains examined, do not carry phh1 sequences, but harbor plasmids of different size and genetic information. The only exception is the halophilic isolate from IsraeL which does not seem to carry a plasmid. MATERIALS AND METIHODS Bacterial strains. All strains used are listed in Table 1. H. halobium NRC817 (Canadian Research Council, Ottawa), H. cutirubrum, H. capanicum, and H. salinarium were provided by D. Oesterhelt (Max- Planck-Institut fur Biochemie, Munich). H. halobium DSM670 and DSM671 were purified from strains which we obtained from the Deutsche Sammlung fur Mikroorganismen (Gottingen). H. volcanii and H. trapanicum were provided by W. Grant, University of Leicester (U.K.). The Israel isolate was provided by Aharon Oren, Jerusalem; the Tunisia isolates were isolated from salt crystals of the Chott-el-Djerid (southern Tunisia) in this laboratory. Source of reagents. All chemicals were obtained

2 370 PFEIFER, WEIDINGER, AND GOEBEL TABLE 1. Strains used Optimal Strain Genotypes salt concn (c%o) vac rub pum I. Rod-shaped halobacteria H. halobium NRC H. halobium DSM H. halobium DSM H. cutirubrum NRC34001 H. salinarium H. capanicum H. tunesiensis Al H. tunesiensis A II. Coccoid or coryneformic halobacteria H. trapanicum (coccoid) H. volcanii Israel isolate from Merck (Darmstadt) or Serva (Heidelberg). Nutrients and agar were obtained from Oxoid (Wesel). Radiochemicals used were from New England Nuclear Corp. (Boston, Mass.). The restriction enzymes EcoRI and PstI were kindly provided by H. Mayer, Braunschweig. HindIII was purchased from Biolabs, and nitrocellulose filters were obtained from Schleicher & Schiill (Dassel, W. Germany). Media and growth conditions. The strains were grown in salt medium, consisting of 4 M NaCl or 2 M NaCl, 0.12 M MgSO4, 0.03 M KCl, 0.01 M trisodium citrate, 0.5% Casamino Acids, and 1% peptone (Oxoid), ph 7.2. Cells were grown with shaking and illumination at 37 C for 7 days. Isolation of DNA. Cells were grown in 1-liter cultures at 37 C, harvested in the late logarithmic phase by centrifugation, suspended in basal salt (4 M NaCl, 0.12 M MgSO4, 0.03M KCl, 0.01 M trisodium citrate, ph 7.2), and lysed by the addition of sodium deoxycholate at a final concentration of M (30 mi, 0 C). After centrifugation of the lysed cells at 10,000 x g for 30 min, the cleared lysate was diluted with 2 volumes of water, and the DNA was concentrated by adding polyethylene glycol (PEG-6000) to a final concentration of 10% (4 C, 5 h). The precipitate was dissolved in TEN-buffer (0.04 M Tris-hydrochloride, M EDTA, and 0.05 M NaCl, ph 8.0), and plasmid DNA was separated by cesium chloride-ethidium bromide gradient centrifugation (TV850 rotor, rpm, 20 C, 20 h). After centrifugation, the CCC DNA was pooled, ethidium bromide was removed by isopropanol, and the DNA was dialyzed against 0.01 M Trishydrochloride (ph 7.5) M EDTA and concentrated by ethanol precipitation. Cleavage with restriction enzymes and agarose gel electrophoresis. Samples of 2 to 3 Ag of DNA were digested with PstI, HindIII, or EcoRI. Cleavage with PstI was carried out in 0.01 M Trishydrochloride M MgCl2 (ph 7.4); cleavage with EcoRI and HindIII was performed in 0.05 M NaCl- J. BACTERIOL M MgCl M Tris-hydrochloride (ph 7.5) for 1 h at 37 C in a total volume of 50 p1. Reactions were stopped by adding 5 pl of 0.07% bromophenol blue and 7% sodium dodecyl sulfate in 33% glycerol, and samples were layered on top of 1% agarose gels (Seakem). Electrophoresis was performed in Tris-phosphate buffer (0.02 M NaH2PO4, M EDTA, M NaCl, 0.04 M Tris, ph 8) and carried out at 3 V cm' at 6 C for 18 h. After electrophoresis, bands were visualized by staining with ethidium bromide and photographed under UV light. Transfer of DNA fragments to nitrocellulose filters and hybridization. Separated DNA fragments were transferred to nitrocellulose filters according to the Southern blotting technique (8) and hybridized with 3P-labeled complementary RNA (crna) as described (2). Isolation of purple membrane and ruberin. Cells were grown in 60-ml cultures, harvested in the stationary phase by centrifugation, and suspended in 0.5 ml of DNase solution (0.5 mg/ml). The lysate was dialyzed against 0.05 M Tris (ph 8.0) for 12 h and then layered on top of a 20 to 45% sucrose density gradient. After centrifugation (SW40 rotor, 39,000 rpm, 20 C, 6 h), the visible bands were pooled and measured in a spectrophotometer (350 to 700 nm). RESULTS Characterization of the halophilic strains. The halobacteria used in this study could be subdivided by their morphological appearance into two groups (Table 1). Group I included rod-shaped bacteria H. halobium strains NRC817, DSM670, and DSM671, H. cutirubrum NRC34001, H. salinarium, H. capanicum, and two new strains isolated from Tunisia. These two latter strains seem to differ from the known Halobacterium species by phenotypic and, as shown later, genotypic properties. We propose for them the designations Halobacterium tunesiensis Al and A2. All these strains require 4 to 4.5 M NaCl for optimal growth and synthesize bacterioruberin (Rub) and purple membrane (Pum). In addition, some of them are able to forn gas vacuoles (Vac). Group II contained the species H. trapanicum and H. volcanii and a newly isolated halophilic strain from Israel (A. Oren, Jerusalem). The bacteria are of coccoid or coryneformic shape, do not synthesize purple membrane or gas vacuoles, and are, with the exception of H. trapanicum, moderately halophilic (optimal growth at 2 M NaCi). However, they seem to be capable of producing bacterioruberin. Isolation and characterization of plasmids. The halobacteria were analyzed for CCC DNA as previously described. It was found that the three strains of H. halobium and the strains of H. cutirubrum, H. trapanicum, and H. volcanii contained CCC DNA of different molecular masses (Table 2). To compare the sequence

3 VOL. 145, 1981 TABLE 2. Plasmid-bearing strains Homol- Homol- ogy Strain Plasmid Mol wt ogy with (1O) with phv phh1 plasmids H. halobium phh NRC817 H. halobiun phh DSM670 H. halobiun phh DSM671 H. cutirubrum phc H. trapanicum pht pht H. vocanri phv phv homologies between these extrachromosomal DNAs, they were cleaved with the restriction endonucleases HindIu, PstI, and EcoRI (Fig. 1). The obtained fragments were then hybridized with 3P-labeled crna of phh1 (CCC DNA isolated from H. halobium NRC817) or nicktranslated 3P-labeled phh1 DNA according to the Southern procedure (8). The plasmids from H. halobium NRC817 (phh1) and DSM670 (phh2) exhibited very similar restriction patterns and extensive sequence homologies when hybridized with each other. The only identified difference between those two plasmids was insertions or rearrangements in the HindmI fragments Hi, H5, and H9, which led to an increase of 2.0, 6.8, and 0.8 megadaltons (Mdal), respectively. In addition, a small deletion in fragment H3 of plasmid phh2 was observed. H. halobium DSM671, which fails to produce gas vacuoles, contains a plasmid (phh3) considerably smaller (about 50 Mdal) than phh1 and phh2. The restriction patterns of phh3, obtained with HindIl, PstI, and EcoRI, indicated that this plasmid differs from phh1 and phh2 by one or more deletions (Fig. 1). The PstI fragments of phh3 also hybridized with fragments of phh1. A physical map of phh1 was constructed from the HindIHl and PstI fragments (G. Weidinger, F. Pfeifer, and W. Goebel, Methods Enzymol., in press). From this map it is obvious that most of the missing fragments of phh3 (compared to phh1) are on a continuous segment of phhl of about 50 Mdal, which is apparently deleted in phh3. In addition, phh3 suffered a smaller deletion of about 4 Mdal which is not continuous with the large deletion (Fig. 2). The extrachromosomal DNA (phcl) isolated from H. cutirubrum NRC34001 was similar in size to phh1 and phh2. The HindIII, PstI, and EcoRI restriction fragments of this PLASMIDS IN HALOBACTERIA 371 plasmid were in most cases indistinguishable in their electrophoretic mobility from those of phhl (Fig. 1). Even those fragments of phc1 which are different from phh1 fragments, however, hybridized with 32P-labeled crna from phh1. This indicates that some regions of phcl, despite homologous sequences, are differently arranged in this plasmid compared to phh1. When hybridization was performed between PstI fragments of phh1 and the 3P-labeled crna of phc1, the only fragment that was not visible on the autoradiogram was P8, indicating that the region of phh1 covered by P8 is missing in phcl. The two strains of H. trapanicum and H. volcanii carry plasmid DNA which can be separated by agarose gel electrophoresis into two species. In H. trapanicum one large plasmid, pht1 (80 Mdal), and a smaller one, pht2 (4 Mdal), were found. From H. vokanii, again, a large plasmid, phv1 (60 Mdal), and a small one, phv2 (4 Mdal), were isolated. No hybridization was observed with any of these plasmids when 3P-labeled crna of phh1 was used as hybridization probe. There also seemed to be no sequence homology between the two small plasmids, pht2 and phv2, despite their similar size. Furthermore, plamid pht2 carries two EcoRI sites but no PstI site, whereas phv2 carries a single PstI site but no EcoRI site (Fig. 1). A HlindMI Hind I Pst I FIG. 1. Restrictionpatterns ofthe isolatedplasmid DNA. Lanes I to 3: HindIII cleavage ofh. halobium plasmids phh3 (1), phh2 (2), phhi (3); Lanes 4 and 5: HindIII cleavage ofh. cutirubrum plasmidsphc (4) and phh1 (5); Lanes 6 to 8: PstI cleavage of H. volcanii and H. trapanicum plasmids phvi and phv2 (6), pht1 andpht2 (7), andphhi (8).

4 372 PFEIFER, WEIDINGER, AND GOEBEL J. BACTERIOL. H,n4l I PHH1 Pst, diiiiliyr 1 3 1i h1216 I IT ular-weight DNA. Taking advantage of these properties, we have recently isolated a large plasmid from a strain of H. halobium and demonstrated that it is identical with the AT-rich "satellite DNA" observed in this and other halobacteria strains by Moore and McCarthy (3, 4, 9) Ṗlasmid phh1 from H. halobium has clearly different molecular properties from p ids isolated from an H. salinarium strain by Simon (7). To test whether plasmids in different HalpHH 2 U detl jb8 phh 3 10(4 del d del et ion 120 phc 1 rear rancaeme n t s del. FIG. 2. Comparison of the maps of H. halobium plasmids phh1, phh2, and phh3 and H. cutirubrum plasmid phci. Solid lines represent regions of complete homology, yielding restriction fragments indistinguishable from phhi. Thin lines show homology with phh1, but the restriction fragments from this region are different from those ofphh1. The dashed lines represent fragments which are obviously deleted in the plasmids. summary of the molecular properties of the described plasmids is given in Table 2. Identification of phhl-like sequences in the total DNA of H. salinarium, H. capanicum and H. tunesiensis Al and A2. Whereas in four Halobacterium strains which are pum+, rub', and vac+ plasmids in CCC forn and with similar sequences were found, no such DNAs were isolated from H. salinarium, H. capanicum, or the two new H. tunesiensis strains. Nevertheless these strains are alsopum+, rub+, and vac+/vac (Table 1). Total DNA from these strains was therefore isolated and cleaved with HindIII. As shown in Fig. 3, defined bands were visible in the HindIII restriction patterns obtained with these DNAs which correspond in their electrophoretic mobility in part to DNA fragments obtained after cleavage of phhl with this restriction enzyme. Furthermore, hybridization of the HindIlI- or EcoRI-cleaved DNA of these four strains with 32P-labeled crna of phh1 DNA demonstrated extensive sequence homologies (Fig. 4). This indicates that these Halobacterium strains, from which no CCC DNA could be isolated, also carry sequences similar to those found on the plasmids in other Halobacterium strains. In contrast, halobacteria belonging to group II, i.e., H. trapanicum, H. vokcanui, and the halophilic isolate from Israel (Table 1), did not show such sequence homologies with phhl. DISCUSSION Halobacteria represent a unique class of microorganism which have adapted to extreme environmental conditions. Several lines of evidence suggest that these bacteria have several properties in common with other procaryotes now generally termed archaebacteria (10, 11). Their biochemical features differ from the other procaryotic organisms and resemble more those of eucaryotes. As almost nothing is known of the genetics of archaebacteria, the understanding of their evolution is difficult. We have chosen to study the genetics of halobacteria since pure Hi,o rs W.. 1 I..^.. i_...; s,.. 3* - l _ - _ - L L li _ E B 5_ B_ _ 5 _ W i _ E 8 1N E rir UI FIG. 3. HindIII digestion of total DNA isolated from halobacteria strains: H. halobium NRC817 (1), H. halobium DSM670 (2), H. halobium DSM671 (3), H. tunesiensis Al (4), H. tunesiensis A2 (5), H. salinarium (6), and H. volcanii (7). cultures of these microorganisms are already available or relatively easy to isolate from many sources, phenotypic mutants can be obtained at a high frequency (6, 9), growth occurs in simple, defined media, and the organims can be readily lysed, making possible isolation of high-molec- -d

5 VOL. 145, ] s.] s,!., FIG. 4. Hybridization patterns with labeled crna ofphhi and EcoRI-digested total DNA of halobacteria strains: H. halobium DSM670 (1), H. tunesiensis Al (2), H. tunesiensis A2 (3), H. salinarium (4), and H. capanicum (5). obacterium species are completely unrelated or whether they may carry common sequences, we carried out a more systematic analysis of plasmids in halobacteria. By morphological and biochemical criteria, our analyzed halobacteria strains seemed to fall into two groups. Those belonging to the first group are rod-shaped, are able to synthesize purple membrane and, in most cases, gas vacuoles, and are extreme halophiles. These bacteria originate from quite different geographical zones and are regarded as different species (H. halobium, H. cutirubrum, H. salinarium, and H. capanicum). Two new halophilic isolates from Tunisia also seem to belong to this group, based on these criteria. Half of the members of this group carry plasmids which can be isolated by standard techniques as CCC DNAs. These plasmids, albeit of different molecular weights, exhibit extensive sequence homologies. The data suggest that they may differ from each other only by insertions, deletions, or rearrangements in their sequences. The other halobacteria do not seem to carry CCC DNA. Sequences homologous to the plasmid sequences of the former halobacteria were, however, observed when total DNA of the plasmid-less strains was hybridized with plasmid DNA. As PLASMIDS IN HALOBACTERIA 373 shown by cleavage with HindIII of total DNA from plasmid-less strains, some of the fragments obtained were indistinguishable in size from those of phh1 of H. halobium NRC817. One of the HindIH fragments (H10) is of particular interest since it is conserved in all plasmids and in the total DNA of all plasmid-free strains of this group of halobacteria. However, most Hindm fragments obtained from total DNA of plasmid-less strains which hybridize to plasmid sequences are of a different size from those of phh1. This suggests that these DNA sequences are either differently arranged compared to phh1 or are integrated into different genetic environments. It cannot be decided from the present data whether the plasmid-like sequences of the plasmid-free halobacteria strains are integrated into the chromosome or are on extrachromosomal elements, not present in a stable CCC conformation. Interestingly, cleavage of total DNA with HindIII yields only a few distinct fragments, most of which hybridize with phh1, whereas the bulk of the DNA is not cleaved with the enzyme. Similar observations were also made with other restriction enzymes that recognize AT-rich sequences, whereas restriction enzymes specific for GC-rich DNA sequences cleave the total DNA of the halobacteria strains very efficiently (Pfeifer et al., manuscript in preparation). This is in agreement with the previous conclusion that the AT-rich satellite DNA represents plasmid sequences. No such plasmid sequences were found in extrachromosomal or chromosomal DNA from members of the second group of halobacteria. They differ in morphology (coccoid and coryneformic shape), by the absence of purple membrane and gas vacuoles, and in part by their more moderate halophilic behavior than the members of group I. Although two out of three tested halobacteria strains from this group carry CCC DNA, there is no indication that these plasmid DNAs exhibit any sequence imilarities to the plasmid sequences of group I halobacteria. It is not yet known whether the two large plasmids of H. trapanicum and H. volcanii share common sequences. The two small plasmids phtl and phv2, also isolated from these strains, did not show sequence homologies between each other. ACKNOWLEDGMENTS H. Schrempf is thanked for valuable discussions and C. Hughes for reading the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 105-All). LITERATURE CITED 1. Bayley, S. T., and R. A. Morton Recent developments in the molecular biology of extremely halophilic bacteria. Crit. Rev. Microbiol. 11:

6 374 PFEIFER, WEIDINGER, AND GOEBEL 2. de la Cruz, F., D. Muller, and W. Goebel A hemolysis determinant common to Escherichia coli Hly plasmids of different incompatibility groups. J. Bacteriol. 143: Moore, R. L, and B. J. McCarthy Characterization of the deoxyribonucleic acid of various strains of halophilic bacteria. J. Bacteriol. 99: Moore, R. L, and B. J. McCarthy Base sequence homology and renaturation studies of the deoxyribonucleic acid of extremely halophilic bacteria. J. Bacteriol. 99: Oesterhelt, D., and W. Stoeckenius Rhodopsinlike protein from the purple membrane of H. halobium. Nature (London) 233: Pfeifer, F., G. Weidinger, and W. Goebel Genetic variability in Halobacterium halobium. J. Bacteriol. J. BACTERIOL. 145: Simon, R. D Halobacterium strain 5 contains a plasmid which is correlated with the presence of gas vacuoles. Nature (London) 273: Southern, E. M Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: Weidinger, G., G. Klotz, and W. Goebel A large plasmid from Halobacterium halobium carrying genetic information for gas vacuole formation. Plasmid 2: Woese, C. R., and G. E. Fox Phylogenetic structure of the procaryotic domains: the primary kingdoms. Proc. Natl. Acad. Sci. U.S.A. 74: Woese, C. R., L J. Magrum, and G. E. Fox Archaebacteria. J. Mol. Evol. 11: