Novel Plasmids from Alkaliphilic Halomonads

Plasmid 41, 268 –273 (1999) Article ID plas.1999.1400, available online at http://www.idealibrary.com on

SHORT COMMUNICATION Novel Plasmids from Alkaliphilic Halomonads Steven A. Fish, 1 Andrew W. Duckworth, and William D. Grant Department of Microbiology and Immunology, University of Leicester, Maurice Shock Building, P.O. Box 138, University Road, Leicester LE1 9HN, United Kingdom Received August 17, 1998; revised February 9, 1999 Seventeen alkaliphilic halomonads were examined for the presence of plasmids. Of these, eight strains harbored one or more from 5.3 to 33 kb in size, the first plasmids to be identified from an alkaliphilic halomonad source. Restriction and hybridization analysis revealed three strains that maintained an identical 5.9-kb plasmid which we named pAH1, two that had an identical 33-kb plasmid, and three others, of which one carried two plasmids of 5.3 and 15 kb, the former being designated pAH2. The two final strains maintained plasmids of 15 and 20.5 kb. Restriction mapping of both pAH1 and pAH2 indicated that they have a number of unique restriction sites and are of a small enough size to make them suitable for vector construction. © 1999 Academic Press

Alkaliphilic halomonads are a group of organisms which form a significant part of the microbial population in the soda lakes of the Kenyan Tanzanian Rift Valley, particularly the northern Lakes Bogoria, Elmenteita, Nakuru, and Sonachi. They have also been isolated from the hypersaline southern Lake Magadi (Duckworth et al., 1996). A key factor responsible for the alkalinity in these locations is the dearth of magnesium and calcium cations due to their insolubility as carbonate minerals in the alkaline environment (Grant et al., 1990). This generates conditions rich in carbonate and chloride anions, resulting in salinity with an elevated pH in the range 8 –12 (Jones et al., 1998). Although other alkaliphiles have been extensively investigated, particularly the alkaliphilic bacilli (Horikoshi, 1998), the alkaliphilic halomonads are an uninvestigated biotechnological resource. Growth characteristics which make these organisms particularly attractive are their rapid growth rates, which would be an advantage in the production of desirable compounds, and their ability to grow at high pH, which could decrease production costs in an industrial process by reducing contamination. In common with halomonads from neutral 1

To whom correspondence should be addressed.

0147-619X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

268

environments (Ca´novas et al., 1996, 1997), this group of organisms produces compatible solutes (E. Galinski and K. Karla, personal communication), organic compounds which maintain the osmotic balance within the cell. These compounds can protect against heating, freezing, and high salt concentrations and it has been suggested that they have applications in the biotechnology industry as protecting agents for enzymatic processes (Galinski, 1993). These strains must also harbor genes responsible for the production of alkaline stable enzymes and polymers that might be of equal interest for the biotechnology industry. Alkaliphilic microorganisms have made a large impact on a number of industrial processes. Detergents may contain proteinases, lipases (Grant et al., 1990), or cellulases (Fukumori et al., 1985) from an alkaliphilic source. Detergents are alkaline and contain sequestering compounds for calcium ions. As the soda lakes have low calcium ion concentrations, this may make enzymes from the alkaliphilic halomonads particularly suitable for use in these products combined with their intrinsic stability at high pH. Alkaliphilic enzymes also have applications in leather tanning and in the cosmetic, pharmaceutical, and food industries

SHORT COMMUNICATION

(Grant et al., 1990). Although no alkaliphilic halomonad bacteria, compounds, or enzymes are currently used in any industrial processes, the value in further investigation is in their novelty, which could lead to future applications. Before any of the alkaliphilic halomonads can be investigated and utilized in biotechnological terms, molecular biology tools need to be developed. Currently there are a number of plasmids isolated from the neutrophilic halomonads which have subsequently been developed into vectors (Vargas et al., 1995; Mellado et al., 1995). However, there is no previously published documentation identifying alkaliphilic halomonad plasmids or genetic exchange in these organisms. To aid their future manipulation, 17 strains were screened for the presence of endogenous plasmids which could be used as a basis for vector construction. The alkaliphilic halomonads were routinely cultured in Horikoshi I medium (Horikoshi, 1971) supplemented with 4% sodium chloride. Plasmid DNA was extracted using the alkaline lysis method (Sambrook et al., 1989) and further purified with phenol– chloroform treatment. In some cases a cleared lysate was difficult to achieve; this was rectified by a longer spin at

TABLE 1 Size Determination of Plasmids Isolated from the Alkaliphilic Halomonads Using Restriction Endonucleases Strain

Plasmid size (kb)

Designation

19N1 21M1 24B1 29C1 29C1 35E2 75C4 25B1 27M1

5.9 5.9 5.9 5.3 15.0 33.0 33.0 20.5 15.0

pAH1 pAH1 pAH1 pAH2 pAH3 pAH4 pAH4 pAH5 pAH6

Note. pAH1 was digested with BamHI, SstI, HindIII, SalI, XhoI, and EcoRV. pAH2 was digested with EcoRV, PstI, XbaI, ClaI, SalI, and HincII. pAH3 was digested with BamHI, EcoRV, and HindIII. pAH4 was digested with BamHI, EcoRV, HindIII, and SalI. pAH5 was digested with BamHI, ClaI, EcoRV, and PstI. pAH6 was digested with ClaI, PstI, and SalI.

269

FIG. 1. Restriction maps. (Top) Plasmid pAH1 isolated from strains 19N1, 21M1, 24B1. (Bottom) pAH2 isolated from strain 29C1.

higher rpm after the phenol extraction step or the use of Qiagen plasmid purification columns. Of the 17 alkaliphilic halomonads screened for the presence of plasmids, 8 contained one or more types (Table 1). Of these, the smallest (5.9 and 5.3 kb) were selected for restriction mapping, their small size aiding in any future vector construction (Fig. 1). The larger of the two was designated pAH1 (alkaliphilic halomonad 1) and the smaller, pAH2. Phylogenetic analysis of 16S rRNA genes had previously revealed a relatively close relationship between soda lake isolates and members of the genera Halomonas and Chromohalobacter (Duckworth et al., 1996). Tree construction (Fig. 2) confirmed the soda lake isolates, which together with known Halomonas and related Chromohalobacter spp. formed three distinct clusters (Duckworth et al., in preparation).

270

SHORT COMMUNICATION

FIG. 2. Unrooted phylogenetic tree of 16S rRNA genes of halophilic and alkaliphilic Halomonas isolates. The least-squares algorithm of Fitch and Margoliash (1967) was used to construct the tree using an evolutionary distance matrix (Jukes and Cantor, 1969). Values at modes indicate the percentage of occurrence in 100 boot-strapped trees. Accession numbers of 16S rRNA genes are listed in Duckworth et al. (1996), with the exception of Halomonas pantelleriense X93493 and Halomonas desiderata X92417.

Plasmid restriction mapping of DNA from strains 19N1, 21M1, and 24B1 using identical enzymes revealed they all maintained the same type of plasmid (Fig. 1, top). These strains are closely related, as determined by phylogenetic analysis (Fig. 2), although each strain was isolated from a different site (Table 2). The mo-

lecular biological data are thus complementary to the phylogenetic tree. Strains 35E2 and 75C4 also maintain a 33-kb plasmid of the same type, as determined by restriction analysis with a variety of the same enzymes (data not shown). Again when the phylogenetic analysis is compared to the plasmid data there is agreement

SHORT COMMUNICATION TABLE 2 Origins of the Alkaliphilic Halomonads Isolate

Location

Sample type

8B1 24B1 25B1 65B4 WB2 WB4 WB5 35E2 44E3 WE5 21M1 27M1 19N1 28N1 12C1 29C1 75C4

Bogoria Bogoria Bogoria Bogoria Bogoria Bogoria Bogoria Elmenteita Elmenteita Elmenteita Magadi Magadi Nakuru Nakuru Sonachi Sonachi Sonachi

Littoral mud/water Littoral mud/water Littoral mud/water Mud on shore line Littoral mud/water Littoral mud/water Littoral mud/water Dried lake mud Littoral mud/water Littoral mud/water Littoral mud/water Littoral mud/water Littoral mud/water Littoral mud/water Littoral mud/water Littoral mud/water Littoral mud/water

(Fig. 2). Strains 25B1 and 27M1 were found to contain plasmids of 20.5 and 15 kb, respectively, while 29C1 had two plasmids of 5.3 and 15 kb, determined using a modified method from Hintermann et al. (1981). Both 15-kb plasmids had different banding patterns when digested with the same enzymes (data not shown), illustrating their difference. The plasmid DNA maintained in these microorganisms might indicate that important or essential functions are encoded on them, which could relate to the environmental conditions these strains tolerate. In any case, the 5.3-kb plasmid isolated from strain 29C1 and the 5.9-kb plasmid from strains 19N1, 21M1, and 24B1 have a number of unique restriction sites and this makes them both suitable for vector construction. The DIG high prime labeling and detection kit (Boehringer Mannheim) was used to label probes and detect hybridization. A DNA probe of plasmid from strain 19N1 hybridized as strongly with plasmid DNA from 21M1 and 24B1 as it did to itself (Fig. 3A). Identical results were obtained with probes derived from plasmid DNA isolated from 21M1 and 24B1 (data not shown). This, combined with the restriction data, strongly suggests that all three

271

organisms maintain an identical 5.9-kb plasmid. The 19N1 plasmid probe (pAH1) has low or no homology to plasmids from strains 35E2 or 75C4 nor to the plasmid pBR328 (Fig. 3A). When plasmid DNA from the other strains was probed with pAH1, low level hybridization was observed with 25B1 and 27M1 and the 5.3-kb plasmid from 29C1; however, strong hybridization was seen with the 15-kb plasmid from 29C1, suggesting a much higher degree of homology (Fig. 3B). This also illustrates the difference between both 15-kb plasmids, as one hybridized much more strongly to the probe than the other. The same probe did not hybridize to pHE1, a plasmid isolated from Halomonas elongata (Vargas et al., 1995), a neutrophilic halomonad; this indicates that pAH1 is in a new class of halomonad plasmids. A probe of plasmid DNA derived from 35E2 hybridized only to itself and plasmid DNA from 75C4 (Fig. 3C), which together with the restriction data indicates that these two strains maintain an identical plasmid. Therefore, the plasmids isolated so far from the alkaliphilic halomonads fall into two distinct and separate groups, which are in turn different from pHE1 from H. elongata. The fact that so many strains harbor plasmids in this group might suggest a degree of gene transfer through the bacterial population in response to the stringency of the alkaline or salt conditions. Indeed the hybridization results go some way to confirming this. An expansion on the current study may involve hybridization experiments between genomic and plasmid DNA from the 17 strains. Curing experiments might also help us understand the phenotypic impact of these plasmids on the alkaliphilic halomonads. An important future goal is to initially sequence the two small plasmids and perhaps later attempt the sequencing of the larger plasmids. As the first alkaliphilic halomonad plasmids isolated and characterized, these may carry novel genes which could have industrial applications, or they may harbor genes which when disrupted allow the elucidation of survival mechanisms at high pH or high salt concentrations. As such, the ability

272

SHORT COMMUNICATION

FIG. 3. Slot blot hybridization analysis. (A) Plasmids isolated from alkaliphilic halomonad strains 19N1, 21M1, 24B1, 35E2, and 75C4 and the plasmid pBR328 were probed with pAH1 from strain 19N1. (B) Plasmids isolated from alkaliphilic halomonad strains 25B1, 27M1, 29C1a (5.3 kb), 29C1b (15 kb), 35E2, 75C4, and 19N1 and the neutrophilic halomonad plasmid pHE1 from Halomonas elongata were probed with pAH1 from strain 19N1. (C) Same as (B) except probed with pAH4 from strain 35E2.

of the alkaliphilic halomonads to grow in alkaline conditions and the availability of a number of plasmids make them an interesting target for further investigation. ACKNOWLEDGMENTS We thank Ms. N. Denning for the initial work on strain 19N1. This study was supported by a grant from the European Commission (Generic Project “Biotechnology of Extremophiles” BIO-4-CT96-0488).

REFERENCES Ca´novas, D., Vargas, C., Csonka, L. N., Ventosa, A., and Nieto, J. J. (1996). Osmoprotectants in Halomonas elongata: High-affinity betaine transport system and cholinebetaine pathway. J. Bacteriol. 178, 7221–7226.

Ca´novas, D., Vargas, C., Iglesias-Guerra, F., Csonka, L. N., Rhodes, D., Ventosa, A., and Nieto, J. J. (1997). Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes. J. Biol. Chem. 272, 25794 – 25801. Duckworth, A. W., Grant, W. D., Jones, B. E., and van Steenberger, R. (1996). Phylogenetic diversity of soda lake alkaliphiles. FEMS Microbiol. Ecol. 19, 181– 191. Fitch, W. M., and Margoliash, E. (1967). Construction of phylogenetic trees: A method based on mutational distances as estimated from cytochrome C sequences of general applicability. Science 155, 279 –285. Fukumori, F., Kudo, T., and Horikoshi, K. (1985). Purification and properties of a cellulase from alkaliphilic Bacillus spp. No. 1139. J. Gen. Microbiol. 131, 3339 – 3345.

SHORT COMMUNICATION Galinski, E. A. (1993). Compatible solutes of halophilic eubacteria: molecular principles, water-solute interaction, stress protection. Experientia 49, 487– 496. Grant, W. D., Mwatha, W. E., and Jones, B. E. (1990). Alkaliphiles: ecology, diversity and application. FEMS Microbiol. Rev. 75, 255–270. Hintermann, G., Fischer, H. -M., Crameri, R., and Hu¨tter, R. (1981). Simple procedure for distinguishing CCC, OC and L forms of plasmid DNA by agarose gel electrophoresis. Plasmid 5, 371–373. Horikoshi, K. (1971). Production of alkaliphilic enzymes by alkaliphilic microorganisms. Part I. Alkaline proteases produced by Bacillus No. 221. Agric. Biol. Chem. 36, 1407–1414. Horikoshi, K. (1998). Alkaliphiles. In “Extremophiles: Microbial Life in Extreme Environments” (K. Horikoshi and W. D. Grant, Eds.), pp. 155–179. Wiley–Liss, New York. Jones, B. E., Grant, W. D., Duckworth, A. W., and Owenson, G. G. (1998). Microbial diversity of soda lakes. Extremophiles 2, 191–200.

273

Jukes, T. H., and Cantor, C. R. (1969). Evolution of protein molecules. In “Mammalian Protein Metabolism” (H. N. Munro, Ed.), Vol. 3, pp. 21–132. Academic Press, New York. Mellado, E., Nieto, J. J., and Ventosa, A. (1995). Construction of novel shuttle vectors for use between moderately halophilic bacteria and Escherichia coli. Plasmid 34, 157–164. Sambrook, K. J., Fritsch, E. F., and Maniatis, T. (1989). “Molecular Cloning: A Laboratory Manual,” 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Vargas, C., Ferna´ndez-Castillo, R., Ca´novas, D., Ventosa, A., and Nieto, J. J. (1995). Isolation of cryptic plasmids from moderately halophilic eubacteria of the genus Halomonas. Characterization of a small plasmid from H. Elongata and its use for shuttle vector construction. Mol. Gen. Genet. 246, 411– 418. Communicated by D. R. Helinski