The moderate thermophilic, green gliding bacterium Chloroflexus aurantiacus contains two alleles for chaperonins

Christin Vanberg, Alexandra Bjørk and Reidun Sirevåg.

Department of Biology, University of Oslo, Box 1031, Blindern, 0316 Oslo, Norway.

The chaperonins GroEL (Hsp60) and GroES (Hsp10), a subgroup of molecular chaperones, are found in all bacteria examined so far and are among the most conserved proteins known. Their function in the cell is to facilitate the folding of numerous polypeptides and to prevent protein aggregation under stress such as heat shock.

The phototrophic green bacterium Chloroflexus aurantiacus has an optimum temperature for growth of 55°C, but is capable of growth at temperatures up to 70°C. When cell free extracts of this organism grown at 45°C, 55°C, 60°C and 65°C were analyzed by SDS-PAGE, three distinct protein bands which increased in intensity with growth temperature were detected. These had molecular weights of 11.2 kD, 60 kD and 75 kD. After elution and purification of the 60kD band. The sequence of the N-terminal end of the purified 60 kD protein showed an identity of 55.6 % and a similarity of 76 % with GroEL from E.coli.

To further characterize this putative GroEL and its corresponding gene, a genomic library constructed in the lamda- ZAP Express vector was screened by a technique using PCR and a 600 bp fragment of the C. aurantiacus hsp60 gene kindly provided us by Gupta et al. (1). By this method a positive clone which contained an open reading frame corresponding to an operon with the two chaperonin genes groEL and groES was isolated. However, when the open reading frame was analyzed and compared to the previously determined amino acid sequence of the N-terminal end, 14 of the 45 amino acids were not identical. This prompted us to sequence the DNA fragment over again however with the same result.

In the prosess of the present work we became aware that a project which aim at sequencing the the whole genome of C. aurantiacus is in progress. This made it possible to perform a Blast search on the sequences available so far. In this way two open reading frames were found, one which had a 100% identity with that isolated by us from the genomic library, and a second which after translation to amino acids was identical to the N-terminal end of the putative GroEL islolated earlier.

The two operons will be described and the possible significance of the two alleles will be discussed.

1. Gupta RS, Mukhtar T, Singh B. (1999). Evolutionary relationships among photosynthetic prokaryotes (Heliobacterium chlorum, Chloroflexus aurantiacus, Cyanobacteria, Chlorobium tepidum and proteobacteria ) : implications regarding the origin of photosynthesis. Mol Microbiol Jun ; 32 (5) : 893-906.