Isolation and characterization of a novel strain of Natrinema containing a bop gene

Isolation and characterization of a novel strain of Natrinema containing a bop gene 1 first-author 1 †E-mail:wumin@cls.zju.edu.cn †E-mail:wumin@cls.zju.edu.cn wumin@cls.zju.edu.cn 2 School of Life Sciences, Zhejiang University, Hangzhou 310027, China School of Life Sciences, Zhejiang University, Hangzhou 310027, China School of Life Sciences, Fudan University, Shanghai 200433, China School of Life Sciences, Fudan University, Shanghai 200433, China A novel member of extremely halophilic archaea, strain AJ2, was isolated from Ayakekum Lake located in Altun Mountain National Nature Reserve of Xinjiang Uygur Autonomous Region in China. The strain AJ2 requires at least 10% (w/v) NaCl and grows 10% to 30% (optimum at 20%). Phylogenetic analysis based on 16S rDNA sequence comparison revealed that strain AJ2 clustered to three Natrinema species with less than 97.7% sequence similarities, suggesting AJ2 is a novel member of Natrinema . A bacteriorhodopsin-encoding ( bop ) gene was subsequently detected in the AJ2 genome using the polymerase chain reaction technique. The cloning and sequencing of a 401 base pairs fragment indicated the deduced amino acid sequence of bop from AJ2 is different from that reported for bacteriorhodopsins. This is the first reported detection of a bop gene in Natrinema . Halophilic archaea Phylogeny Bacteriorhodopsin INTRODUCTION 1 Extremely halophilic archaea belonging to the order Halobacteriales had been isolated from various hypersaline environments such as the Dead Sea, the Great Salt Lake, sabkhas, and natural or artificial salterns (Radax et al., 2001 2001 ). Western China has many natural salt lakes, providing extremely abundant halophilic archaea resources. A novel haloarchaeal strain AJ2 containing a bop gene was documented in reports of molecular investigation on microbiological resources regarding their adverse effect on the environment. Bacteriorhodopsin (BR), found in some extremely halophilic archaea, is a retinal-binding membrane protein with seven membrane-spanning segments and functions as a light-driven proton pump (Oesterhelt and Stoeckenius, 1971 1971 ; Luecke et al., 1999 1999 ; Mukohata et al., 1999 1999 ). Their especial photoelectric properties and high thermal stability of BR proteins are useful in such as optical memory (Wise et al., 2002 2002 ), artificial retina (Frydrych et al., 2000 2000 ), photon switch, and so on (Lanyi and Pohorille, 2001 2001 ; Margesin and Schinner, 2001 2001 ). Because of the limitation of site-directed method, looking for native BR proteins, which could offer the best properties for materials improvement, become the available method today (Wise et al., 2002 2002 ). In this study, we isolated and characterized a novel strain of extremely halophilic Altun Mountain archaeon containing a bacteriorhodopsin-encoding ( bop ) gene. This is the first reported detection of a bop gene in Natrinema . MATERIALS AND METHODS 1 Archaeal strains and growth media 2 Strain AJ2 was isolated from water samples collected from Ayakekum Lake (37° N, 89° E, at altitude of 3880 m) located in Altun Mountain National Nature Reserve in Western China. Reference strain Halobacterium salinarium ( halobium ) R1M1 was obtained as a gift from Li Qing-guo (Shanghai, China). The strains were routinely cultured aerobically at 37 °C in rich medium (Oesterhelt and Stoeckenius, 1974) containing (L −1 ) 250 g NaCl, 20 g MgSO 4 ·7H 2 O, 3 g trisodium citrate dihydrate, 2 g KCl, 0.26 g CaCl 2 , 10 g peptone (Oxiod L37). The pH was adjusted to 7.0 with NaOH. And slants and plates were prepared by adding 20 g aga/L. Growth characteristics 2 Optimal salt concentration conditions of strain AJ2 were determined by growing in DSMZ Meduim 97 regulated with 0, 5, 10, 12.5, 15, 17.5, 20, 22.5, 25 and 30% (w/v) NaCl. The pH tolerance was also tested in rich medium with pH values 3, 4, 5, 6, 7, 8, 9, 10, 11. All of the optimal conditions tests groups were incubated at 37 °C for every three days until day 14, and cell growth was monitored by OD 600 . Cell shape was examined by optical microscopy (Olympus model BX40 microscope). DNA extraction 2 Genomic DNAs from archaeal strains were extracted using the classic genomic DNA isolation kit (Sangon) according to the instructions of the manufacturer. 16S rDNA sequence PCR ampification 2 The 16S rDNA sequence primers, designed according to Gupta et al.( 1983 1983 ), were rDNA22F (5′-ATTCCGGTTGATCCTGC-3′, positions 6–22) and rDNA1521R (5′-AGGAGGTGATCCAGCCGCAG-3′, positions 1540–1521). The E. coil 16S rRNA sequence was used as the position reference. The PCR thermal cycling conditions were as follows: 25 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min. Cloning and sequencing of PCR products 2 The desired PCR products were purified using the UNIQ-10 column DNA gel extraction kit (Sangon) according to the instructions of the manufacturer. The purified fragments were treated with T4 DNA ligase (MBI), then ligated to pUCm-T (Sangon), and transformed into Escherichia coli DH5α. Ampicillin (100 μg/ml) and a blue-white selection were used to identify the positive transformants. The plasmids were extracted using the alkaline lysis method. Subsequently, plasmids were digested by PstI and electrophoresed in agarose gel (1.5%) to select recombinants harboring plasmids with the correct insert of the 16S rDNA sequence. PCR amplifications, as done under above conditions, were performed using approximately 50 ng plasmid DNA as the template. Sequences were determined by automated dideoxynucleotide methods with the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer) on an ABI Prism 377XL DNA sequencer. The M13 universal sequencing primers (M13F, 5′-GTAAAACGACGGCCAGT-3′, and M13R 5′-GGAAACAGCTATGACCATG-3′) were used in the sequencing reaction. Analysis on sequence data, and phylogenetic tree construction 2 The 16S rDNA sequence of strain AJ2 was fitted in the halophilic archaeal alignment with Clustal X 1.8, and a few minor corrections were made manually. Analyses were carried out with PHYLIP (version 3.6a3) (Felsenstein, 2003 2003 ). The DNADIST programme was used to construct a Jukes and Cantor evolutionary distance matrix, then phylogentic tree was constructed using the neighbor-joining method with randomized input order. Bootstrap analysis (1000 replications) was performed using the additional programs SEQBOOT and CONSENSE. Identification of bop gene 2 The bop PCR primers, designed on the basis of the conserved protein sequences PLLLLDL and KVGFGFI (Otomo et al., 1992 1992 ; Wang et al., 2000 2000 ), were bop331F (5′-CCGCTG(CT)TG(CT)TG(CT)T(AC)GACCTCG-3′, positions 310–331) and bop686R (5′-AGGATGA(GA)(CG)CCGAA(CG)CCGACCTT-3′, positions 707–686). The Hbt. salinarium bop gene sequence was used as the position reference. The PCR amplification was performed in a 100 μl reaction mixture, for 25 cycles with denaturation at 94 °C for 1 min, annealing at 60 °C for 1 min and extension at 72 °C for 1 min. PCR products were examined by gel electrophoresis in 4% agarose gel using the generulerTM 100 bp DNA ladder plus markers (Sangon) for size comparisons of PCR products. Cloning and sequencing of PCR products protocols, as described above, were used. RESULTS 1 Growth characteristics and other features 2 At 37 °C, strain AJ2 grows at pH 6.0 to pH 8.0, and in media containing 10%–30% NaCl (pH 7.4) with an optimum at 20% NaCl. No growth was detected at NaCl concentrations below 10%. Microscopy indicated that cells lysed in less than 6% NaCl. In actively growing liquid cultures, the cells of strain AJ2 were rod shaped but were pleomorphic under unfavorable conditions. Colonies of stain AJ2 were red, circular and convex. Phylogenetic analysis of 16S rDNA sequence 2 The 16S rDNA sequence of strain AJ2, 1474 bp was determined. The GenBank accession number was AY208972. Phylogenetic tree based on 16S rDNA sequence was constructed using the neighbor-joining method with bootstrap values calculated from 1000 trees subsequently (Fig. 1 1 ). On the phylogenetic tree strain AJ2 clustered to the three described Natrinema species. Similarity percentages between the AJ2 sequence and the type strains of Nnm. pallidum (NCIMB 777 and NCIMB 784), Nnm. pellirubrum and Nnm. versiforme were 97.1%, 97.1%, 96.6% and 97.7%, respectively. Detection and analysis of bop gene by PCR 2 The nucleotide sequence of the partial bop gene from strain AJ2 (GenBank accession No. AY279548), 401 bp in total, was sequenced and the deduced amino acid sequence was compared to the previously determined sequence of Hbt. salinarium , which was the first discovered as BR protein, and strain arg-4, which processes the highest similarity sequence with AJ2 among all extremely halobacterial archaea (Fig. 2 2 ). The result of sequence alignment confirmed that the nucleotide fragment from PCR reaction is just the part of the bop gene that encodes the retinal protein from helix C to helix G. The hydropathic indices from helix C to G region of BR proteins (Fig. 3 3 ) were calculated according to the method of Kyte and Doolittle ( 1982 1982 ). The similar hydropathic profiles between Hbt. salinarium BR and retinal protein of strain AJ2, as illustrated in Fig. 3 3 , could mean that both proteins fold their polypeptide chains in a similar way. DISCUSSION 1 The results showing that cells of strain AJ2 lyse in low NaCl concentration ( < 6%) medium, morphology and growth characteristics agreed with descriptions of Natrinma (McGenity et al., 1998 1998 ; Xin et al., 2000 2000 ), AJ2 clustered to the three described Natrinema species in phylogenetic tree based on 16S rDNA sequence, and the cluster was supported by a high bootstrap value (100%), indicating strain AJ2 is a member of Natrinma belonging to extremely halophilic archaea. Moreover, compared to 16S rDNA sequences among Natrinma strains, AJ2 had more than 95% and less than 97.7% sequence similarity with others. According to the current classification suggestion, strains whose 16S rDNA sequence homology values is below about 97.5% are related at the species level (Stackebrandt and Goebel, 1994 1994 ). Therefore, AJ2 is a novel member of Natrinma . The fragment of bop gene encoding BR protein from helix C to helix G contains the main gene primary structure, and this part of protein domain include the main part of amino acid residues which line the retinal binding pocket or proton channel. Accordingly, the partial protein sequence from helix C to helix G region is more conservative than the whole protein sequence. However, compared to BR protein sequences from different strains, about 94.7% of amino acid residues are identical between strain AJ2 and arg-4, but less than 60% homology exists between AJ2 and other reported strains (Ihara et al., 1999 1999 ; Mukohata et al., 1999 1999 ). The results indicated strain AJ2 should be closer to the strain arg-4, which belong to Haloterrigena , than to other strains, such as Hbt. salinarium . The phylogenetic tree (Fig. 1 1 ) also makes clear this viewpoint. Among different genera of Halobacteriaceae , five have been found to possess bop gene: Halobacterium , Haloarcula , Halomicrobium , Halorubrum and Haloterrigena (Ihara et al., 1999 1999 ; Mukohata et al., 1999 1999 ). This is the first reported detection of a bop gene in Natrinema . The study extends the range of strains in other genera that harbor bop gene, and provides the base for using of species and BR proteins resources. References R Dunn , J McCoy , M Simsek , A Majumdar , SH Chang , UL RajBhandary , HG Khorana . The bacteriorhodopsin gene . Proc Natl Acad Sci USA , 1981 . 78 6744 - 6748 . . The bacteriorhodopsin gene J Felsenstein . PHYLIP . 2003 . 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Natrinema versiforme sp. nov., an extremely halophilic archaeon from Aibi salt lake, Xinjiang, China 10.1631/jzus.2005.B0142 Porject (No. 30370029) supported by the National Natural Science Foundation of China 2004-05-13 2004-10-28 2005-01-20 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 6B