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Identification and characterization of salt-inducible polypeptide in Paenibacillus sp., a moderately halophilic bacterium
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 100, No. 5, 573–575. 2005 DOI: 10.1263/jbb.100.573
© 2005, The Society for Biotechnology, Japan
Identification and Characterization of Salt-Inducible Polypeptide in Paenibacillus sp., a Moderately Halophilic Bacterium Ashrafaddin Sokhansanj,1 Ali Asghar Karkhane,2 and Ferdous Rastgar Jazii2* Department of Biology, Faculty of Sciences, University of Tehran, P.O. Box 14155-6455, Tehran, Iran1 and National Institute for Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14155-6343, Tehran, Iran2 Received 7 March 2005/Accepted 25 July 2005
In response to salt, Paenibacillus sp. strain XII expresses a 21.4 kDa polypeptide. N-terminal sequencing and sequence homology analysis indicate homology between the N-terminal sequence of the polypeptide and a segment of the N-terminus of the spore coat associated protein CotN of Oceanobacillus iheyensis, an extremely halotolerant bacteria of the deep-sea. [Key words: moderately halophilic bacteria, Paenibacillus, osmolyte, osmostress, salinity]
PAGE (11, 12). Following electrophoresis, the gels were stained with Coomassie Blue R250, destained and the target polypeptide band was cut out, put in a dialysis bag, eluted by electroelution in a submarine electrophoresis tank containing dialysis buffer composed of 192 mM glycine, 25 mM Tris–HCl, pH 8.3 at 5 mA constant current at 4°C overnight, and the protein was subsequently precipitated with 4 volumes of 100% cold acetone (13). The precipitate was rinsed with 100% cold methanol, air-dried and dissolved in phosphate buffered saline (PBS) for determination of molecular weight and estimation of isoelectric point (pI). Capillary isoelectric focusing was performed in 15-cm long glass tubes with a 1.5-mm internal diameter. Prefocusing was accomplished for 1 h at 200 V; subsequently, 5 µg of purified polypeptide was applied to each gel, covered with overlay buffer and the polypeptide was focused for 16 h at 500 V (14). At the end of focusing, gels were extruded, stained or used for pI determination. The pI was determined by cutting gels into 0.5-cm long pieces, each piece was placed in a separate tube containing 2 ml of distilled H2O, crushed, incubated at room temperature for 3 h and then the pH of each tube was measured (15, 16). For staining, gels were impregnated with distilled H2O until ampholine was completely diffused out and stained as above. Finally, N-terminal amino acid sequencing of the protein was performed using an Applied Biosystems 494 automated sequencer according to the method of Edman. To assess bacterial salt tolerance, the growth behavior of the bacterium was analyzed in response to increasing salt concentration in nutrient broth. As shown in Fig. 1, little or no bacterial growth could be observed in nutrient broth alone, which indicates the salt dependency of the bacterium. Growth started at a 1% NaCl concentration and increased up to a 5% NaCl concentration and the optimal growth occurred at a 2.5% NaCl concentration. This observation agrees well with what has been described for moderately halophilic bateria, which suggests the requirement of salt at a concentration ranging from 2.9% up to 14.6% of NaCl in culture
Moderately halophilic bacteria include a heterogeneous group of bacteria, which have adapted to survive in hypersaline environments. These organisms are capable of applying several molecular mechanisms to sense, respond, grow and reproduce in such a condition (1–3), among which are different transport systems for the cytoplasmic accumulation of osmolytes, such as the amino acids proline and glycinebetaine, from the surrounding environment and systems to synthesize osmolytes de novo and to export excess NaCl for the adjustment of cytoplasmic osmolarity (4, 5). Although many studies have so far been carried out on the genetics of moderate halophiles (6, 7), the cellular and molecular basis of osmoregulation in these group of bacteria is not yet well understood. Previous studies indicated that moderate halophiles are capable of maintaining their internal osmolarity and generate turgor in hypersaline environments by accumulating organic compatible solutes (8). This capability has enabled these bacteria to be distributed and occupy an important ecological niche in hypersaline environments (9), and they are excellent models for investigating the molecular basis of prokaryotic osmoadaptation, which has potential for application in biotechnology and applied microbiology (6–10). To study the molecular aspects of resistance to osmotic stress, in the present study, we focused on proteins that might be involved in osmoprotection in the moderate halophile Paenibacillus sp. strain XII, isolated from lake Oromieh, a high salt containing lake in the northwest of Iran. The bacterium was grown in nutrient broth (10 g/l meat extract, 10 g/l peptone, 5 g/l NaCl, pH 7.5–7.6), containing different amounts of NaCl (% w/v: 1%, 2.5%, 5%, 7.5%, 10%, 12.5% and 15%, final concentration) and the growth rate was monitored by examining the OD at 620 nm at different time intervals (0–45 h). Total bacterial proteins were extracted and applied to a 13% polyacrylamide gel and subjected to SDS– * Corresponding author. e-mail: [email protected] phone: +98-21-44580380 fax: +98-21-44580395 573
574
SOKHANSANJ ET AL.
J. BIOSCI. BIOENG.,
FIG. 1. Growth of Paenibacillus sp. strain XII. Cells were grown in the nutrient broth media containing the indicated concentration of NaCl. The growth was monitored by OD620. The obtimal growth was observed at 2.5% (0.43 M) NaCl. FIG. 3. Electrophoretic analysis of 21.4 kDa purified protein on 13% polyacrylamide gel for evaluating the purity of protein and efficiency of recovery. Lane 1, Molecular weight marker. The amount of protein applied was 20 µg in lane 2 and 10 µg in lane 3.
FIG. 4. Isoelectric focusing of the 21.4 kDa polypeptide shown in Fig. 2. Two parallel gels were run, one for staining and the other for determination of pI.
FIG. 2. Electrophoretic pattern of polypeptides of Paenibacillus sp. strain XII. Total protein were separated using a 13% SDS–polyacrylamide gel. Lanes 1 and 2, Two independent samples from cultures that were grown in the medium containing 7.5% NaCl (W/V); lanes 3 and 4, the corresponding cultures grown without NaCl.
medium (1, 2). Following the determination of the optimal salt requirement, the concentration of salt was progressively increased to identify the uppermost salt tolerance. Increasing the salt concentration up to 15% completely abolished bacterial growth, whereas for lower concentrations (10% and 12.5%), a minimal growth was observed after 28 h (Fig. 1), which might suggest adaptation to the condition. To study proteins that might possibly be involved in osmoresistance of Paenibacillus, the total proteins were extracted and analyzed by electrophoresis. By comparing the bacterial protein expression patterns after growth in different concentrations of salt, the expression of a 21.4 kDa polypeptide was observed to resume at a 7.5% NaCl concentration (Fig. 2). Being expressed in response to salt implies that this polypeptide is involved in osmoregulation or osmotolerance. Furthermore, it could also be suggested that at a 7.5% NaCl concentration, the organism senses and responds to a higher than optimal salt concentration. To assess whether the polypeptide is only salt inducible or might also be induced by other stresses, the bacterium was grown in medium containing the optimal salt concentration (2.5%) and a heat shock (42°C) was applied, and then protein extraction
and electrophoresis were carried out (data not shown). The induction of the polypeptide was only observed in response to salt and not to heat, which further indicates the salt inducibility of the polypeptide. The polypeptide was purified and further analyzed for determination of the molecular weight (Fig. 3), verifying the extent of the purity and the pI measured by applying isoelectric focusing. Subsequently, a pI of 4.8 was estimated (Fig. 4), which indicates the polypeptide is highly acidic. This feature of the polypeptide was used in combination with N-terminal sequencing in subsequent similarity searches (see below). Finally, N-terminal sequencing of the polypeptide was carried out and the sequence of 10 amino acids NH2-YFS DAETTNN-COOH was determined, which was used for deciphering the sequence similarity with that of other available protein sequences in data banks by applying BLAST programs. The ExPASy Proteomics tools (http://us.expasy.org/ tools/; accession nos. OB1302 or AP004597), and BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/; accession no. NP_692223) were used and the sequences with the highest similarity was determined. From the partial amino acid sequence, similarity searches, MW and pI, the salt-inducible polypeptide was found to be comparable with the spore coat associated protein (CotN) of Oceanobacillus iheyensis strain HTE831, an alkalophilic and extremely halotolerant bacterial strain of the deep-sea (5, 17). CotN has a molecular weight of 21.5 kDa and consists of 195 amino acids according to the UniProt database (http://www.ebi.uniprot.org/; accession no. Q8ERK0) and has an estimated pI of 3.81 based on ExPASy. The sequence homology was found with
VOL. 100, 2005
NOTES
a segment of amino acids from amino acid 28 to 37 from the N-terminus of CotN. Although some similarities are seen in some features between the two proteins, additional studies are required to determine the extent of similarity between the 21.4 kDa polypeptide and CotN. Searches for the biological function of CotN have so far not led us to an exclusive result; however, being a protein constituent of an extremely halotolerant and alkalophilic bacteria (17), it is possibly involved either structurally or functionally, in the salt tolerance of O. iheyensis. It is also possible that our identified polypeptide is also involved in osmoprotection using a similar mechanism. Further studies are required for validating the above propositions. It was previously shown that the deep-sea (5, 17) can be considered as a suitable source of halophilic bacteria; we have shown that such bacteria can be considered as a possible source of proteins that are expressed in response to salt. Such proteins might be involved in salt tolerance either structurally or functionally. Unraveling the true activity and application of the corresponding genes could have profound outcomes in biotechnology. REFERENCES 1. Ventosa, A., Nieto, J. J., and Oren, A.: Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev., 62, 504–544 (1998). 2. Ca’novas, D., Vargas, C., Csonka, L., Ventosa, A., and Nieto, J.: Osmoprotectants in Halomonas elongate: high-affinity betaine transport system and choline-betaine pathway. J. Bacteriol., 178, 7221–7226 (1996). 3. Eisenberg, H. and Wachtel, E. J.: Structural studies of halophilic proteins, ribosomes, and organelles of bacteria adapted to extreme salt concentrations. Annu. Rev. Biophys. Chem., 16, 69–92 (1987). 4. Galinski, E. A. and Trüper, H. G.: Microbial behaviour in salt-stressed ecosystems. FEMS Microbiol. Rev., 15, 95–108 (1994).
575
5. Takami, H., Takashi, Y., and Uchiyama, I.: Genome sequence of Oceanobacillus iheyensis isolated from the Iheya Ridge and its unexpected adaptive capabilities to extreme environment. Nucleic Acid Res., 30, 3927–3935 (2002). 6. Fernández-Castillo, R., Vargas, C., Nieto, J. J., Ventosa, A., and Ruiz Berraquero, F.: Characterization of a plasmid from moderately halophilic eubacteria. J. Gen. Microbiol., 138, 1133–1137 (1992). 7. Kunte, H. J. and Galinski, E. A.: Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata. FEMS Microbiol. Lett., 128, 293–299 (1995). 8. Kushner, D. J. and Kamekura, M.: Physiology of halophilic eubacteria, p. 109–138. In Rodriguez-Valera, F. (ed.), Halophilic bacteria, vol. I. CRC Press, Boca Raton, FL (1988). 9. Rodriguez-Valera, F.: The ecology and taxonomy of aerobic chemoorganotrophic halophilic eubacteria. FEMS Microbiol. Rev., 39, 17–22 (1986). 10. Ventosa, A. and Nieto, J. J.: Biotechnological applications and potentialities of halophilic microorganisms. World J. Microbiol. Biotechnol., 11, 85–94 (1995). 11. D’Souza, S., Atlekar, W., and D’Souza, S. F.: Adaptive response of Haloferax mediterranei to low concentrations of NaCl (<20%) in the growth medium. Arch. Microbiol., 168, 68–71 (1997). 12. Laemmli, U. K.: Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227, 680– 685 (1970). 13. Johnstone, A. and Thorpe, R.: Immunochemistry in practice, 3rd ed., p. 230–235. Blackwell Science, Oxford (1996). 14. Coligan, J. E., Kruisbeek, A., Margulies, D., Shevach, E. M., and Strober, W. (ed.): Current protocols in immunology, p. 8.5.1–8.5.5. John Wiley and Sons, NY (1994). 15. Bollag, D. and Edelstein, S.: Protein methods, p. 169. WileyLiss Publication, New York (1991). 16. Andrews, A. T.: Electrophoresis, theory, techniques and biochemical and clinical applications, p. 267. Clarendon Press, Oxford (1990). 17. Lu, J., Nogi, Y., and Takami, H.: Oceanobacillus iheyensis gen. no., sp. nov., a deep-sea extremely halotolerant and alkalophilic species isolated from a depth of 1050 m on the Iheya Ridge. FEMS Microbiol. Lett., 205, 291–297 (2001).
© 2005, The Society for Biotechnology, Japan
Identification and Characterization of Salt-Inducible Polypeptide in Paenibacillus sp., a Moderately Halophilic Bacterium Ashrafaddin Sokhansanj,1 Ali Asghar Karkhane,2 and Ferdous Rastgar Jazii2* Department of Biology, Faculty of Sciences, University of Tehran, P.O. Box 14155-6455, Tehran, Iran1 and National Institute for Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14155-6343, Tehran, Iran2 Received 7 March 2005/Accepted 25 July 2005
In response to salt, Paenibacillus sp. strain XII expresses a 21.4 kDa polypeptide. N-terminal sequencing and sequence homology analysis indicate homology between the N-terminal sequence of the polypeptide and a segment of the N-terminus of the spore coat associated protein CotN of Oceanobacillus iheyensis, an extremely halotolerant bacteria of the deep-sea. [Key words: moderately halophilic bacteria, Paenibacillus, osmolyte, osmostress, salinity]
PAGE (11, 12). Following electrophoresis, the gels were stained with Coomassie Blue R250, destained and the target polypeptide band was cut out, put in a dialysis bag, eluted by electroelution in a submarine electrophoresis tank containing dialysis buffer composed of 192 mM glycine, 25 mM Tris–HCl, pH 8.3 at 5 mA constant current at 4°C overnight, and the protein was subsequently precipitated with 4 volumes of 100% cold acetone (13). The precipitate was rinsed with 100% cold methanol, air-dried and dissolved in phosphate buffered saline (PBS) for determination of molecular weight and estimation of isoelectric point (pI). Capillary isoelectric focusing was performed in 15-cm long glass tubes with a 1.5-mm internal diameter. Prefocusing was accomplished for 1 h at 200 V; subsequently, 5 µg of purified polypeptide was applied to each gel, covered with overlay buffer and the polypeptide was focused for 16 h at 500 V (14). At the end of focusing, gels were extruded, stained or used for pI determination. The pI was determined by cutting gels into 0.5-cm long pieces, each piece was placed in a separate tube containing 2 ml of distilled H2O, crushed, incubated at room temperature for 3 h and then the pH of each tube was measured (15, 16). For staining, gels were impregnated with distilled H2O until ampholine was completely diffused out and stained as above. Finally, N-terminal amino acid sequencing of the protein was performed using an Applied Biosystems 494 automated sequencer according to the method of Edman. To assess bacterial salt tolerance, the growth behavior of the bacterium was analyzed in response to increasing salt concentration in nutrient broth. As shown in Fig. 1, little or no bacterial growth could be observed in nutrient broth alone, which indicates the salt dependency of the bacterium. Growth started at a 1% NaCl concentration and increased up to a 5% NaCl concentration and the optimal growth occurred at a 2.5% NaCl concentration. This observation agrees well with what has been described for moderately halophilic bateria, which suggests the requirement of salt at a concentration ranging from 2.9% up to 14.6% of NaCl in culture
Moderately halophilic bacteria include a heterogeneous group of bacteria, which have adapted to survive in hypersaline environments. These organisms are capable of applying several molecular mechanisms to sense, respond, grow and reproduce in such a condition (1–3), among which are different transport systems for the cytoplasmic accumulation of osmolytes, such as the amino acids proline and glycinebetaine, from the surrounding environment and systems to synthesize osmolytes de novo and to export excess NaCl for the adjustment of cytoplasmic osmolarity (4, 5). Although many studies have so far been carried out on the genetics of moderate halophiles (6, 7), the cellular and molecular basis of osmoregulation in these group of bacteria is not yet well understood. Previous studies indicated that moderate halophiles are capable of maintaining their internal osmolarity and generate turgor in hypersaline environments by accumulating organic compatible solutes (8). This capability has enabled these bacteria to be distributed and occupy an important ecological niche in hypersaline environments (9), and they are excellent models for investigating the molecular basis of prokaryotic osmoadaptation, which has potential for application in biotechnology and applied microbiology (6–10). To study the molecular aspects of resistance to osmotic stress, in the present study, we focused on proteins that might be involved in osmoprotection in the moderate halophile Paenibacillus sp. strain XII, isolated from lake Oromieh, a high salt containing lake in the northwest of Iran. The bacterium was grown in nutrient broth (10 g/l meat extract, 10 g/l peptone, 5 g/l NaCl, pH 7.5–7.6), containing different amounts of NaCl (% w/v: 1%, 2.5%, 5%, 7.5%, 10%, 12.5% and 15%, final concentration) and the growth rate was monitored by examining the OD at 620 nm at different time intervals (0–45 h). Total bacterial proteins were extracted and applied to a 13% polyacrylamide gel and subjected to SDS– * Corresponding author. e-mail: [email protected] phone: +98-21-44580380 fax: +98-21-44580395 573
574
SOKHANSANJ ET AL.
J. BIOSCI. BIOENG.,
FIG. 1. Growth of Paenibacillus sp. strain XII. Cells were grown in the nutrient broth media containing the indicated concentration of NaCl. The growth was monitored by OD620. The obtimal growth was observed at 2.5% (0.43 M) NaCl. FIG. 3. Electrophoretic analysis of 21.4 kDa purified protein on 13% polyacrylamide gel for evaluating the purity of protein and efficiency of recovery. Lane 1, Molecular weight marker. The amount of protein applied was 20 µg in lane 2 and 10 µg in lane 3.
FIG. 4. Isoelectric focusing of the 21.4 kDa polypeptide shown in Fig. 2. Two parallel gels were run, one for staining and the other for determination of pI.
FIG. 2. Electrophoretic pattern of polypeptides of Paenibacillus sp. strain XII. Total protein were separated using a 13% SDS–polyacrylamide gel. Lanes 1 and 2, Two independent samples from cultures that were grown in the medium containing 7.5% NaCl (W/V); lanes 3 and 4, the corresponding cultures grown without NaCl.
medium (1, 2). Following the determination of the optimal salt requirement, the concentration of salt was progressively increased to identify the uppermost salt tolerance. Increasing the salt concentration up to 15% completely abolished bacterial growth, whereas for lower concentrations (10% and 12.5%), a minimal growth was observed after 28 h (Fig. 1), which might suggest adaptation to the condition. To study proteins that might possibly be involved in osmoresistance of Paenibacillus, the total proteins were extracted and analyzed by electrophoresis. By comparing the bacterial protein expression patterns after growth in different concentrations of salt, the expression of a 21.4 kDa polypeptide was observed to resume at a 7.5% NaCl concentration (Fig. 2). Being expressed in response to salt implies that this polypeptide is involved in osmoregulation or osmotolerance. Furthermore, it could also be suggested that at a 7.5% NaCl concentration, the organism senses and responds to a higher than optimal salt concentration. To assess whether the polypeptide is only salt inducible or might also be induced by other stresses, the bacterium was grown in medium containing the optimal salt concentration (2.5%) and a heat shock (42°C) was applied, and then protein extraction
and electrophoresis were carried out (data not shown). The induction of the polypeptide was only observed in response to salt and not to heat, which further indicates the salt inducibility of the polypeptide. The polypeptide was purified and further analyzed for determination of the molecular weight (Fig. 3), verifying the extent of the purity and the pI measured by applying isoelectric focusing. Subsequently, a pI of 4.8 was estimated (Fig. 4), which indicates the polypeptide is highly acidic. This feature of the polypeptide was used in combination with N-terminal sequencing in subsequent similarity searches (see below). Finally, N-terminal sequencing of the polypeptide was carried out and the sequence of 10 amino acids NH2-YFS DAETTNN-COOH was determined, which was used for deciphering the sequence similarity with that of other available protein sequences in data banks by applying BLAST programs. The ExPASy Proteomics tools (http://us.expasy.org/ tools/; accession nos. OB1302 or AP004597), and BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/; accession no. NP_692223) were used and the sequences with the highest similarity was determined. From the partial amino acid sequence, similarity searches, MW and pI, the salt-inducible polypeptide was found to be comparable with the spore coat associated protein (CotN) of Oceanobacillus iheyensis strain HTE831, an alkalophilic and extremely halotolerant bacterial strain of the deep-sea (5, 17). CotN has a molecular weight of 21.5 kDa and consists of 195 amino acids according to the UniProt database (http://www.ebi.uniprot.org/; accession no. Q8ERK0) and has an estimated pI of 3.81 based on ExPASy. The sequence homology was found with
VOL. 100, 2005
NOTES
a segment of amino acids from amino acid 28 to 37 from the N-terminus of CotN. Although some similarities are seen in some features between the two proteins, additional studies are required to determine the extent of similarity between the 21.4 kDa polypeptide and CotN. Searches for the biological function of CotN have so far not led us to an exclusive result; however, being a protein constituent of an extremely halotolerant and alkalophilic bacteria (17), it is possibly involved either structurally or functionally, in the salt tolerance of O. iheyensis. It is also possible that our identified polypeptide is also involved in osmoprotection using a similar mechanism. Further studies are required for validating the above propositions. It was previously shown that the deep-sea (5, 17) can be considered as a suitable source of halophilic bacteria; we have shown that such bacteria can be considered as a possible source of proteins that are expressed in response to salt. Such proteins might be involved in salt tolerance either structurally or functionally. Unraveling the true activity and application of the corresponding genes could have profound outcomes in biotechnology. REFERENCES 1. Ventosa, A., Nieto, J. J., and Oren, A.: Biology of moderately halophilic aerobic bacteria. Microbiol. Mol. Biol. Rev., 62, 504–544 (1998). 2. Ca’novas, D., Vargas, C., Csonka, L., Ventosa, A., and Nieto, J.: Osmoprotectants in Halomonas elongate: high-affinity betaine transport system and choline-betaine pathway. J. Bacteriol., 178, 7221–7226 (1996). 3. Eisenberg, H. and Wachtel, E. J.: Structural studies of halophilic proteins, ribosomes, and organelles of bacteria adapted to extreme salt concentrations. Annu. Rev. Biophys. Chem., 16, 69–92 (1987). 4. Galinski, E. A. and Trüper, H. G.: Microbial behaviour in salt-stressed ecosystems. FEMS Microbiol. Rev., 15, 95–108 (1994).
575
5. Takami, H., Takashi, Y., and Uchiyama, I.: Genome sequence of Oceanobacillus iheyensis isolated from the Iheya Ridge and its unexpected adaptive capabilities to extreme environment. Nucleic Acid Res., 30, 3927–3935 (2002). 6. Fernández-Castillo, R., Vargas, C., Nieto, J. J., Ventosa, A., and Ruiz Berraquero, F.: Characterization of a plasmid from moderately halophilic eubacteria. J. Gen. Microbiol., 138, 1133–1137 (1992). 7. Kunte, H. J. and Galinski, E. A.: Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata. FEMS Microbiol. Lett., 128, 293–299 (1995). 8. Kushner, D. J. and Kamekura, M.: Physiology of halophilic eubacteria, p. 109–138. In Rodriguez-Valera, F. (ed.), Halophilic bacteria, vol. I. CRC Press, Boca Raton, FL (1988). 9. Rodriguez-Valera, F.: The ecology and taxonomy of aerobic chemoorganotrophic halophilic eubacteria. FEMS Microbiol. Rev., 39, 17–22 (1986). 10. Ventosa, A. and Nieto, J. J.: Biotechnological applications and potentialities of halophilic microorganisms. World J. Microbiol. Biotechnol., 11, 85–94 (1995). 11. D’Souza, S., Atlekar, W., and D’Souza, S. F.: Adaptive response of Haloferax mediterranei to low concentrations of NaCl (<20%) in the growth medium. Arch. Microbiol., 168, 68–71 (1997). 12. Laemmli, U. K.: Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227, 680– 685 (1970). 13. Johnstone, A. and Thorpe, R.: Immunochemistry in practice, 3rd ed., p. 230–235. Blackwell Science, Oxford (1996). 14. Coligan, J. E., Kruisbeek, A., Margulies, D., Shevach, E. M., and Strober, W. (ed.): Current protocols in immunology, p. 8.5.1–8.5.5. John Wiley and Sons, NY (1994). 15. Bollag, D. and Edelstein, S.: Protein methods, p. 169. WileyLiss Publication, New York (1991). 16. Andrews, A. T.: Electrophoresis, theory, techniques and biochemical and clinical applications, p. 267. Clarendon Press, Oxford (1990). 17. Lu, J., Nogi, Y., and Takami, H.: Oceanobacillus iheyensis gen. no., sp. nov., a deep-sea extremely halotolerant and alkalophilic species isolated from a depth of 1050 m on the Iheya Ridge. FEMS Microbiol. Lett., 205, 291–297 (2001).