Cloning, characterization and functional analysis of groESL operon from thermophilic cyanobacterium Synechococcus vulcanus

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Abstract

Genes encoding 10 914 Da and 58 267 Da polypeptides homologous to groES and groEL of Escherichia coli were cloned and sequenced from a thermophilic cyanobacterium, Synechococcus vulcanus. The deduced amino acid sequence of the GroEL protein was much more homologous to GroELs of other cyanobacteria which accompany GroES than another GroEL homolog of S. vulcanus (GroEL2) reported previously (M. Furuki, N. Tanaka, T. Hiyama, and H. Nakamoto, Biochim. Biophys. Acta 1294 (1996) 106–110). We designate the gene as groEL1 to distinguish it from the non-operon forming groEL2 gene. A 9-base pair inverted repeat sequence (TTAGCACTC-N9-GAGTGCTAA) was located upstream of the promoter region of groEL1, which was absent in groEL2. Southern blot analysis indicated that only one groESL1 operon was present in the genomic DNA of S. vulcanus. The amount of the bicistronic, 2.3 kb transcript of groESL1 operon increased 30-fold within 30 min upon heat shock. The increase was completely inhibited by chloramphenicol, suggesting the involvement of heat-induced production of a polypeptide. Introduction of the cloned groEL1 gene into a groEL defective mutant of E. coli resulted in the complementation of heat sensitivity, which contrasted with the previous result with groEL2.

Introduction

Heat shock proteins (HSPs) are present ubiquitously and are some of the most highly conserved protein families known [1]. HSP families often include constitutive as well as heat-inducible members [1]. This is because they represent a class of proteins that have general and essential cellular functions, e.g., promotion of assembly, disassembly and translocation of proteins in addition to providing tolerance against heat stress [2]. HSP60s, also referred to as chaperonin 60 (abbreviated as cpn60) or GroEL, comprise one group of HSP families, and are found in the cytosol of bacteria. Homologs are also found in eukaryotic cell organelles such as mitochondria and chloroplasts. Unlike E. coli and most bacteria, several bacterial species have been shown to contain more than one groEL gene [3]. They include Gram-positive bacteria, Streptomyces albus, Mycobacterium leprae and M. tuberculosis, and Gram-negative bacteria of the Rhizobiaceae family, Rhizobium meliloti, R. leguminosarum and Bradyrhizobium japonicum 4, 5, 6, 7, 8. Chloroplasts contain two distinctly different types called α and β [9]. Two types of groEL-like genes were also found in cyanobacteria, Synechocystis sp. PCC 6803 10, 11, 12. This is of particular interest in terms of the endosymbiont hypothesis for the origin of chloroplasts. The hypothesis postulates that chloroplasts originated from a cyanobacterium that became associated with an originally non-photosynthetic host cell.

Recently, we showed the presence of two kinds of GroEL-like polypeptides in a thermophilic cyanobacterium, S. vulcanus, and cloned a gene which encoded one of the GroELs [13]. The gene, groEL2, did not accompany a groES gene. Expression of the gene was observed upon heat shock treatment both in S. vulcanus and in an E. coli mutant to which the groEL2 gene had been introduced. But, over-expression of GroEL2 in this groEL defective mutant did not result in the complementation of heat sensitivity. In this paper, we show the complete nucleotide sequence of the groESL1 operon, whose groEL1 gene was expressed to a much higher extent than groEL2 upon heat shock [13]. We further show that the introduction of this gene for a thermophilic cyanobacterial homologue (GroEL1) can restore heat tolerance in the E. coli mutant. Characteristics of the gene structure and possible heat-induced regulation mechanisms will also be discussed.

Section snippets

Organisms and culture conditions

Cells of S. vulcanus (kindly provided by Dr. Yorinao Inoue of RIKEN) were cultured photoautotrophically either at 50 or 55°C with a light intensity of 30 μE/m2/s in the medium used originally for another thermophilic cyanobacterium Synechococcus elongatus [14]. The culture was aerated with air supplemented with 5% CO2. For heat shock studies, cultures were incubated at 63°C under the same aeration and light conditions. The E. coli K-12 strains, JM109, W3110 and NRK117 (groEL44, zje::Tn10, kindly

Amino acid sequences of 63 kDa and 14 kDa polypeptides

The amino-terminal amino acid sequence of GroEL1 was previously determined as AKRIIYNENARRALEKGMDILAESVAV [13]. In the present study, we further determined the internal sequence of GroEL1 to design oligonucleotide primers for PCR. One of the lysylendopeptidase fragments of the 63 kDa polypeptide gave an internal sequence (SMTTELEVT) which happened to be identical with an internal sequence of the GroEL from Synechococcus sp. PCC 7942. The amino-terminal sequence of the 14 kDa polypeptide

Discussion

In the present study, we confirmed that there are at least two kinds of groEL genes in a thermophilic cyanobacterium, S. vulcanus. In a mesophilic cyanobacterium, Synechocystis sp. PCC 6803, two groEL-like genes referred to as groEL and cpn60, have been isolated 10, 12. As in the case of cpn60, groEL2 of S. vulcanus has no groES-like gene in its immediate upstream and is expressed as a monocistronic mRNA [13]. The present gene (groEL1) is organized in an operon with groES as in the case of groEL

Acknowledgements

The authors are grateful to Professor Yorinao Inoue of RIKEN for kindly providing S. vulcanus and its genomic library, and to Professor Koreaki Ito of Kyoto University for generously providing NRK117. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (No. 06259202) from the Ministry of Education, Science and Culture, Japan.

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