Abstract
The gene (icl) encoding cold-adapted isocitrate lyase (ICL) of a psychrophilic bacterium, Colwellia psychrerythraea, was cloned and sequenced. Open reading frame of the gene was 1,587 bp in length and corresponded to a polypeptide composed of 528 amino acids. The deduced amino acid sequence showed high homology with that of cold-adapted ICL from other psychrophilic bacterium, C. maris (88% identity), but the sequential homology with that of the Escherichia coli ICL was low (28% identity). Primer extension analysis revealed that transcriptional start site for the C. psychrerythraea icl gene was guanine, located at 87 bases upstream of translational initiation codon. The expression of this gene in the cells of an E. coli mutant defective in ICL was induced by not only low temperature but also acetate. However, cis-acting elements for cold-inducible expression known in the several other bacterial genes were absent in the promoter region of the C. psychrerythraea icl gene. The substitution of Ala214 for Ser in the C. psychrerythraea ICL introduced by point mutation resulted in the increased thermostability and lowering of the specific activity at low temperature, indicating that Ala214 is important for psychrophilic properties of this enzyme.
Similar content being viewed by others
References
Britton KL, Abeysinghe IS, Baker PJ, Barynin V, Diehl P, Langridge SJ, McFadden BA, Sedelnikova SE, Stillman TJ, Weeradechapon K, Rice DW (2001) The structure and domain organization of Escherichia coli isocitrate lyase. Acta Cryst D57:1209–1218
Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR (2002) Low-temperature extremophiles and their applications. Curr Opin Biotechnol 13:253–261
Cozzone AJ (1998) Regulation of acetate metabolism by protein phosphorylation in Escherichia coli. Annu Rev Microbiol 52:127–164
D’Aoust JY, Kushner DJ (1972) Vibrio psychroerythrus sp. n.: classification of the psychrophilic marine bacterium, NRC 1004. J Bacteriol 111:340–342
Deming JW, Somers LK, Straube WL, Swartz DG, Macdonell MT (1988) Isolation of an obligately barophilic bacterium and description of a new genus, Colwellia gen. nov. Syst Appl Microbiol 10:152–160
Duilio A, Madonna S, Tutino ML, Pirozzi M, Sannia G, Marino G (2004) Promoters from a cold-adapted bacterium: definition of a consensus motif and molecular characterization of UP regulative elements. Extremophiles 8:125–132
Fields PA, Somero GN (1998) Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes. Proc Natl Acad Sci USA 95:11476–11481
Geolette D, Damien B, Blaise V, Depiereux E, Uversky VN, Gerday C, Feller G (2003) Structural and functional adaptation to extreme temperatures in psychrophilic, mesophilic, and thermophilic DNA ligase. J Biol Chem 278:37015–37023
Gerday C, Aittaleb M, Arpigny JL, Baise E, Chessa JP, Garsoux G, Petrescu I, Feller G (1997) Psychrophilic enzymes: a thermodynamic challenge. Biochim Biophys Acta 1342:119–131
Ishii A, Suzuki M, Sahara T, Takada Y, Sasaki S, Fukunaga N (1993) Gene encoding two isocitrate dehydrogenase isozymes of a psychrophilic bacterium, Vibrio sp. strain ABE-1. J Bacteriol 175:6873–6890
Kandror O, Deleon A, Goldberg AL (2002) Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for vaiability at low temperatures. Proc Natl Acad Sci USA 99:9727–9732
Kenan C (1998) Use of bacteriophage lambda recombination function to promote gene replacement in Escherichia coli. J Bacteriol 180:2063–2071
Kornberg HL (1966) The role and control of the glyoxylate cycle in Escherichia coli. Biochem J 99:1–11
Kornberg HL, Krebs HA (1957) Synthesis of cell constitutents from C2-units by a modified tricarboxilic acid cycle. Nature 179:988–991
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Qoronfleh MW, Debouck C, Keller J (1992) Identification and characterization of novel low-temperature-inducible promoters of Escherichia coli. J Bacteriol 174:7902–7909
Sælensminde G, Halskau Ø Jr, Helland R, Willassen NP, Jonassen I (2007) Structure-dependent relationships between growth temperature of prokaryotes and the amino acid frequency in their proteins. Extremophiles 11:585–596
Sahara T, Suzuki S, Tsuruha J, Takada Y, Fukunaga N (1999) cis-Acting elements responsible for low-temperature-inducible expression of the gene coding the thermolabile isocitrate dehydrogenase isozyme of a psychrophilic bacterium, Vibrio sp. strain ABE-1. J Bacteriol 181:2602–2611
Sambrook J, Russell D (2001) Molecular Cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor
Shine J, Dalgano L (1974) The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346
Siddiqui KS, Cavicchioli R (2006) Cold-adapted enzymes. Annu Rev Biochem 75:403–433
Takada Y, Ochiai T, Okuyama H, Nishi K, Sasaki S (1979) An obligatory psychrophilic bacterium isolated on the Hokkaido coast. J Gen Appl Microbiol 25:11–19
Tanabe H, Goldstein J, Yang M, Inouye M (1992) Identification of the promoter region of the Escherichia coli major cold shock gene, cspA. J Bacteriol 174:3867–3873
Vanni P, Giachetti E, Pinzauti G, McFadden BA (1990) Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comp Biochem Physiol 95B:431–458
Watanabe S, Takada Y (2004) Amino acid residues involved in cold adaptation of isocitrate lyase from a psychrophilic bacterium, Colwellia maris. Microbiology 150:3393–3403
Watanabe S, Takada Y, Fukunaga N (2001) Purification and characterization of a cold-adapted isocitrate lyase and a malate synthase from Colwellia maris, a psychrophilic bacterium. Biosci Biotech Biochem 65:1095–1103
Watanabe S, Takada Y, Fukunaga N (2002a) The cold-inducible icl gene encoding thermolabile isocitrate lyase of a psychrophilic bacterium, Colwellia maris. Microbiology 148:2579–2589
Watanabe S, Yamaoka N, Fukunaga N, Takada Y, (2002b) Purification and characterization of a cold-adapted isocitrate lyase and expression analysis of the cold-inducible isocitrate lyase gene from the psychrophilic bacterium Colwellia psychrerythraea. Extremophiles 6:397–405
Yumoto I, Kawasaki K, Iwata H, Matsuyama H, Okuyama H (1998) Assignment of Vibrio sp. strain ABE-1 to Colwellia maris sp. nov., a new psychrophilic bacterium. Int J Syst Bacteriol 48:1357–1362
Acknowledgments
We sincerely thank Dr. Isao Yumoto of the National Institute of Advanced Industrial Science and Technology for the donation of C. psychrerythraea.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by K. Horikoshi.
Rights and permissions
About this article
Cite this article
Sato, Y., Watanabe, S., Yamaoka, N. et al. Gene cloning of cold-adapted isocitrate lyase from a psychrophilic bacterium, Colwellia psychrerythraea, and analysis of amino acid residues involved in cold adaptation of this enzyme. Extremophiles 12, 107–117 (2008). https://doi.org/10.1007/s00792-007-0115-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00792-007-0115-9