MATERIALS AND METHODS

Sampling and isolation of low temperature-adapted bacteria: Seawaters were collected by water samplers attached to CTD from Makasar Strait, Indonesia (Fig. 1) during INSTANT Cruise on R/V Baruna Jaya I.

Fig. 1:	Sampling sites of deep-sea waters at makasar strait, Indonesia

Isolation of bacterial isolates: Preparation of water samples and isolation of bacterial isolates were carried out according to a method previously described (Radjasa et al., 2001). Isolation was performed onboard on R/V Baruna Jaya.

Psychrophily test: Psychrophily test was conducted at the laboratory of Marine Microbiology, Department of Marine Science, Diponegoro University. Test was conducted to classify the low temperature-adapted bacteria based on the ability to grow at two different temperatures, 4 and 20°C (Radjasa et al., 2001). Those grow only at 4°C are regarded as psychrophilic bacteria while those grow both at 4 and 20°C are regarded as psychrotrophic bacteria, respectively (Morita, 1975).

Repetitive-PCR: Molecular based-works including DNA extractions, rep-PCR, and PCR were carried out at the laboratory of Marine Biotechnology, Department of Marine Science, Diponegoro University. Whereas, the DNA sequencings were performed at the Molecular Biology Laboratory, Agency for the Assessment and Application Technology (BPPT), in Jakarta, Indonesia.

For Rep-PCR, BOX A1R (5'-CTACggCAAggCg AcgC Tg ACg-3') (Versalovic et al., 1994) were used. The REP 1R-I and REP 2-I primers contain the nucleotide inosine (I) at ambiguous positions in the REP consensus (34). PCR reaction contained of 1 μL DNA template (diluted 100x), 1 μL primer, 7, 5 μL Megamix Royal dan sterile water up to total volume of 15 μL. Amplifications were performed with a thermal cycler model Gene Amp PCR System 9700 with the following temperature profiles: initial denaturation at 95°C for 5 min; 30 cycles of denaturation (92°C for 1 min), annealing (50°C for 1.5 min), extension (68°C for 8 min); and final extension at 68°C for 10 min. Five microliter aliquot PCR products were run using elektrophores on 6% acrilamide gel by using 1xTBE buffer.

Grouping of isolates: Grouping was carried out by making matrixes from the positions of bands on the gel which were then analyzed by using Free Tree program by using UPGMA method for constructing the tree. Resampling was performed by bootstrapping with 1000 replications.

DNA extraction, PCR amplification and sequencing of 16S rRNA gene fragments: DNA extractions, PCR amplification of partial 16S rRNA gene of bacterial strains, purification of PCR products and subsequent sequencing analysis were performed according to the method of Radjasa et al. (2007). The determined DNA sequences of strains were then compared for homology to the BLAST database.

RESULTS

Temperature profiles: As seen in the Fig. 2, there were temperature drops in the depth of 400 m and below that indicated the presence of psychrosphere having water temperatures lower than 10°C.

Fig. 2:	The temperature profile of waters from different depths and location

Table 1:	Psychrotrophic isolates collected from different depths and stations
Sign means gram negative bacteria

Psychrophilic isolates: Psychrophily test indicated that all isolates were able to grow both at 4 and 20°C, therefore they were regarded as psychrotrophic bacteria (Table 1).

Rapid grouping of psychrotrophic isolates: Based on the repetitive-PCR results and constructed dendrogram of the isolates, 6 groups were created and 6 different isolates representing different groups (Fig. 3) were further selected for DNA sequencings.

Sequencing of representative psychrotrophic isolates: The results of DNA sequencing of the representative isolates based on PCR repetitive approach are presented in the following Table 2.

Fig. 3:	Dendrogram of psychrotrophic isolates (PSM 1.5, PSM 2.2, PSM 2.4, PSM 2.7, PSM 2.8, PSM 3.4 were further selected for DNA sequencing)

Table 2:	Characterization of representative psychrotrophic isolates

Table 3:	Grouping of Psychrotrophi isolates

Generic composition of psychrotrophic isolates: As indicated in the Table 3, the members of genus Psychrobacter are the most dominant group followed by Pseudomonas and Vibrio groups.

DISCUSSION

In a study aimed at determining the diversity of low temperature-adapted bacteria, we investigated marine bacteria isolated from deep-sea waters of Makasar strait, Indonesia. Our attention was focused on the occurrence of low temperature-adapted bacteria followed by PCR-based approach for estimating the richness of psychrotrophic bacteria.

Understanding the indigenous low temperature-adapted bacteria has important implications for analyses of microbial function and biogeochemical processes in the deep-sea environments as well as their biotechnological potentials.

It is interesting to note that 4 out of 6 representative strains chosen based on rep-PCR, belonged to the genus Psychrobacter. Members of the genus Psychrobacter are known to be obligate or facultative psychrophiles and have been isolated from a wide range of habitats (Shivaji et al., 2005), including food, clinical samples, skin, gills and intestines of fish, sea water, penguin colonies in Antarctica, Antarctic sea ice and Japan Trench sediments (Bozal et al., 2003; Bowman et al., 1997; Maruyama et al., 1997; Yumoto et al., 2003).

Romanenko et al. (2004) isolated two species of Pyschrobacter from coastal sea ice and sediments of the Sea of Japan and proposed new species Psychrobacter maritimus and Psychrobacter arenosus. Furthermore, Shivaji et al. (2005) isolated the member of genus Psychrobacter from cyanobacterial mat samples collected from various water bodies in the McMurdo Dry Valley region of Antarctica and proposed new species, namely Psychrobacter vallis and Psychrobacter aquaticus, respectively.

One representative isolate obtained showed closest similarity to Pseudomonas sp. The genus of Pseudomonas is widely distributed in the marine environment and considered one of the most abundant group of marine γ-proteobacteria. Furthermore, Zeng et al. (2004) reported that 10 psychrotrophic bacteria isolated from the west Pacific deep-sea sediments were affiliated to the Psychrobacter, Pseudoalteromonas and Pseudomonas genera in the γ-Proteobacteria group. Radjasa et al. (2001) also reported that Pseudomonas and Halomonas, specifically occurred in surface water of the North-western Pacific ocean.

The member of genus Vibrio was also found in the present study. Pseudoalteromonas, Photobacterium and Vibrio, were common to surface and deep-sea waters of the North-western Pacific. Overall, the members of Vibrionaceae appear to be dominant in both habitats (Radjasa et al., 2001). Urakawa et al. (1999a), used Restriction fragment length polymorphism (RFLP) for grouping 136 natural isolates collected from inshore areas of Japan, mainly in winter, belonging to the family Vibrionaceae. Five RFLP groups (groups I to V) were obtained. Group I was found to be corresponded to Vibrio splendidus-like strains. It was confirmed that this group was not only found in Otsuchi Bay, but also in broad coastal areas of Japan.

Furthermore, Urakawa et al. (1999b) performed typing and identification of 60 marine psychrophilic and psychrotrophic vibrios isolated from the north-western Pacific Ocean and coastal environment of Japan performed by restriction fragment length polymorphism analysis. Fifteen operational taxonomic units (OTUs) by digestion with four restriction endonucleases (HhaI, DdeI, RsaI and Sau3AI) were obtained. Significant differences were observed in OTU composition between isolates from the deep sea and coastal areas. Vibrio marinus and Photobacterium species were the dominant culturable vibrios in the deep sea areas, while Vibrio splendidus like species were the dominant culturable vibrios in a coastal area of Japan.

In conclusion, the rep-PCR method is useful for rapid grouping of marine pyshcrotrophic bacteria from seawater of Makasar strait, Indonesia and offers an alternative technique for grouping of big numbers of marine bacterial isolates.

ACKNOWLEDGMENTS

This work was partly supported by the grants from INSTANT project and Ministry of Research and Technology, Indonesia. Special thanks to the Captain and crews of R/V Baruna Jaya I, for their skillful helps during the sampling.