Halotolerant or halophilic microorganisms, able to live in saline environments, offer a multitude of actual or potential applications in various fields of biotechnology (Wang et al., 2004; Margesin et al., 2001). These microorganisms are usually isolated from saline environments which are characterized by having high osmotic strength and low water activity. Most halophilic/halotolerant eubacteria cope with these conditions by accumulating small, highly water-soluble organic compounds, the so-called compatible solutes (Brandon, 2006; Dassarma and Arora 2001; Ciulla et al., 1997). Ectoines represent the predominant class of osmolytes in aerobic chemo-heterotrophic eubacteria of the family Halomonadaceae (Louis and Galinski, 1997; Wohlfarth et al., 1990). Members of the genus Halomonas tolerate high concentrations of sodium chloride (NaCl) as compared to other Gram-negative and rod-shaped bacteria (Brenner et al., 2005; Holt et al., 2005).
Ectoine is of vital importance to agriculture and the biotechnological and pharmaceutical industries. Infact ectoine and its derivatives are patented as moisturizers in cosmetics for the care of aged, dry, or irritated skin (Margesin and schinner, 2001; Wood et al., 2001). One of the most promising applications of ectoine is its use as a stabilizer in the polymerase chain reaction (PCR) and as an enzyme protectant against heat, freezing, and drying (Detkova and Boltyanskaya, 2006; Margesin and Schinner, 2001; Lippert and Galinski, 1992). Ectoine is biosynthesized from aspartic b-semialdehyde in three successive enzyme reactions. The genes responsible for these three enzymes are designated ectA, ectB, and ectC, which code for L-2,4-diaminobutyric acid acetyltransferase, L2-,4-diaminobutyric acid transaminase, and L-ectoine synthase respectively (Nakayama et al., 2000; Ono et al., 1999; Louis and Galinski, 1997; Peters et al., 1990).
The aim of this study was to isolate ectoine-producing Halomonas strains. Thus, a pair of common primers was designed to detect the ectC gene (encoding ectoine synthase) in bacterial species that belong to the genus Halomonas. For isolation of moderately halophilic bacteria, environmental samples were collected from various sites of a tannery factory in Isfahan, and the Persian Gulf, where the salt concentration is high. Samples included salted sheepskin and sheep intestine, wastewater and salt water. Samples from salted sheep skin and intestine were transferred to defined broth for enrichment and isolation. Wastewater and salt water were collected from a depth of 10 cm in sterile 500-ml bottles and transported on ice to the laboratory for isolation on the same day (Brent, 2002). The synthetic defined broth medium used was based on mineral salts medium containing (g/l): Nutrient broth, 8; MgSo4. 7H2O 9.6; MgCl2, 7; KCl, 2; CaCl2. 2H2O, 0.36; NaHCo3, 0.06; NaBr, 0.026 and NaCl, 50; pH 7.4 (Nieto et al., 1989). The synthetic defined agar medium was prepared as above but with the addition of 20 g/l of nutrient agar instead of nutrient broth. This medium was modified by addition of NaCl at a final concentration of 300 g/l. All chemicals were obtained from Merck, Germany. Media were sterilized at 121ºC for 15 min. Wastewater samples (5ml), saltwater samples (5ml), and swabs from salted sheep skin and salted sheep intestine were added to Erlenmeyer flasks containing 25 ml defined broth medium. Flasks were then incubated for 1 to 2 days at 28ºC on a rotary shaker (INFORS AG, Germany) operating at 150 rpm.
After enrichment, inoculum from the flasks was streaked out onto the defined solid medium and phenotypically varied colonies were purified on this medium. Phenotypically different colonies obtained from the plates were transferred to the defined solid medium containing various concentrations of NaCl (0-250 g/l). Most isolates were observed to grow in a wide range of NaCl concentrations. The optimum concentration of NaCl was determined by the microtitre plate method. Seed cultures were prepared by growing isolates in the defined broth medium for 24 h. A 50 ml sample of each seed culture was used to inoculate individual wells of a 96-well microtitre plate containing 200 ml of sterile synthetic broth medium with different concentrations of NaCl (0-250 g/l). The microtitre plates were then incubated at 28ºC for 24 h, followed by optical density measurements at 600 nm (OD600) using an ELISA reader (Stat fax 2100, Germany) (Emtiazi et al., 2005).
The78 bacterial isolates were identified by Gram staining and the presence of catalase, cytochrome oxidase, and their tolerance to high NaCl concentrations were also tested (Vreeland and Hochstein, 1993). The above tests were performed according to Bergey’s Manual of Determinative Bacteriology (Brenner et al., 2005; Holt et al., 1994). For the final identification of ectoine producing isolates, a 277 bp region located within the ectC gene was amplified using common primers (Cinagen, Iran).
The relevant ectC gene sequences available in the GenBank were aligned by using the MegAlign software package. These included sequences from 24 validly described Halomonas species. Based on this alignment, putative genus-specific primers were designed. Primers were designed in such a way to amplify a 277bp region of the ectC gene in the tentative Halmonas strains.
The primer were designed according to the methods available in http://www. Ncbi.NlmNih.Gov/entrez/ query and http://www.ebi. ac.uk/cgi-bin/ jobresults/clustalw/clustalw-20060710-515216.aln). The sequences for the forward and the reverse primers are as follows:
Y=C or T R=A or G B=C, G or T
H=A, C or T K=G or T D=A, G or T
DNA used for PCR was prepared as previously described (Sambrook and Russel, 2001). Multiple bands initially observed on the agarose gel were eliminated by changing the MgCl2 and primer concentrations and altering the annealing temperature (McPherson and Moller, 2000) (Fig. 1). The PCR reaction was finally optimized as follows: A 25 ml volume of reaction mixture contained 2 mM MgCl2, 10X PCR buffer (200 mM Tris, 500 mM KCl), 0.24 mM dNTPs, 60 pmol of each forward and reverse primer and one unit of Taq DNA polymerase and the final volume was adjusted to 25 ml by adding water. Amplification was carried out in a thermal cycler (Eppendorf AG 22331, Hamburg, Germany). After an initial denaturation at 94ºC for 2 min, 30 cycles of the three- step PCR amplification were completed, each consisting of denaturation at 98ºC for 20 sec, primer annealing at 57ºC for 1 min and primer extension at 72ºC for 1 min. The samples were incubated at 72ºC for 10 min at the end of amplification cycles to complete the extension reaction (Nakayama et al., 2000).
PCR products were separated by gel electrophoresis using a horizontal 1% (w/v) agarose gel (Sigma, St. Louis, MO, USA) in Tris-base-Ethylene diamine tetraacetic acid (TBE) buffer (80 volts for 1 h and 20 min). Gels were then stained in a solution of ethidium bromide and visualized with a UV transilluminator (UVP Inc., San Gabriel, CAS, USA).
The primary tests, i.e. Gram staining, oxidase and catalase tests and range of tolerance to NaCl (%), showed that out of the 78 isolates, 45 were Gram-negative rods, oxidase and catalase positive and all were able to tolerate a wide range of NaCl concentrations (Table 1). The microtiter plate method showed that from the 45 isolates, only those strains originating from the tannery factory showed the greatest growth (as OD600) at NaCl concentrations of 100-150 g/l (10-15%), where as the isolates from the Persian Gulf showed the greatest growth at NaCl concentrations of 50 g/l (5%). Such a difference is to be expected since the amount of NaCl in sea and lake water is approximatly 3-5% (Ventosa et al. 1998) but that in the tannery factory is much higher, being saturated in the salted intestine (Verma et al., 2001).
The results also showed that the growth of all isolates was at a minimum and almost equal, at a NaCl concentration of 250 g/l (25%). From the data obtained, it was deduced that these strains were closely related to the genus Halomonas.
Based on alignment of the ectC sequences belonging to the Halomonas species available in the GenBank, a primer pair (named ectione) was designed. The ectoine primer pair intended to amplify all Halomonas species capable of producing ectoine synthase. Out of the 78 isolates, 45 were identified by primary and biochemical tests as tentative Halomonas strains. The 45 isolates were then further investigated by applying the new PCR assay. PCR analysis showed that 34 of the 45 (75%) of the isolates belonged to the genus Halomonas, capable of producing ectoine synthase (Fig. 2). The specificity of the putative genus-specific PCR assays was determined by testing some of the bacteria previously identified as non-Halomonas strains and non-halophilic bacteria (Fig. 3). The novel genus-specific PCR assays indicated that several of the 45 isolates had been misidentified by the primary morphological and biochemical tests.
In this study, the Halomonas strains were easily and quickly differentiated from other genera by their ability to survive exposure to, and grow at very high (20%) NaCl concentration. All species tested were catalase positive, and most of them were also oxidase positive. Hence, we detected tentatively 45 isolates when belong to the Halomonas genus capable of producing ectoine synthase, but the specificity of the putative genus-specific anslysis showed that 34 of the 45 strains belonged to the genus Halomonas.
The types of osmotic solutes present in the cells depend greatly on the growth conditions such as, temperature, salinity, medium composition (Lamosa et al., 1998; Ventosa et al., 1998). Ectoine is found at high concentrations in different Halomonas species and is the dominant solute in cells grown in defined medium lacking glycine betaine, or its precursor choline, but in the presence of high NaCl concentrations (Ventosa et al., 1998; Wohlfarth et al., 1990). To attention that the synthetic defined solid medium is devoid of glycine betaine but whit high NaCl concentration, and results of PCR analysis, 34 isolates mentioned above tolerated high concentration of NaCl by producing ectoine.
Previous studies have used species specific primers, unable to detect and amplify the range of isolates demonstrated in this study. Nakayama et al. (2000) recognized ect genes in Halomonas elongata by special primers. They took a transgenic approach to investigate the function of ectoine as a compatible solute in plant cells. Louis et al. (1997) recognized ect genes in Marinococcus halophilus by special primers and cloned a 4.1-kb DNA fragment into Escherichia coli. The resulting clones exhibited accumulation of ectione and increased salt tolerance. The ectA, ectB, and ectC, were found to be located in the 4.1-kb DNA fragment. Jabber et al. (1992) and Min-Yu et al. (1993) while investigating on Halomonas strains, cloned the ect genes into E coli. The resulting clones exhibited tolerance and resistance to saline environments. The use of universal primers was not demonstrated in any of the above mentioned investigations. In this study, universal primers were designed for recognition of the ectC gene in the Halomonas species.
Ectoine is the dominant compatible solute in the Halomonadaceae, when grown at very high salt concnetrations. However, we found isolates with negative PCR results, proving that they do not belong to the Halomonas genus. Therefore, it is worthwhile mentioning that there also exists an alternative biosynthetic pathway for ectoine, isolates with glutamate (Ventosa et al., 1998; Louis and Galinski 1997). Some of the moderately halophilic bacteria also use different osmolytes to provide osmotic balance with the external medium (Ventosa et al., 1998).
We are grateful to Dr Amir Parviz Golbang for his assistance.