CLONING AND CHARACTERIZATION OF 2 GROESL OPERONS OF RHODOBACTER-SPHAEROIDES - TRANSCRIPTIONAL REGULATION OF THE HEAT-INDUCED GROESL OPERON

The nonsulfur purple bacterium Rhodobacter sphaeroides was found to co ntain two groESL operons. The groESL(1) heat shock operon was cloned f rom a genomic library, and a 2.8-kb DNA fragment was sequenced and fou nd to contain the groES and groEL genes. The deduced amino acid sequen ces of GroEL(1) (cpn60) and GroES(1) (cpn10) were in agreement with N- terminal sequences previously obtained for the isolated proteins (K. C . Terlesky and F. R. Tabita, Biochemistry 30:8181-8186, 1991). These s equences show a high degree of similarity to groESL genes isolated fro m other bacteria. Northern analysis indicated that the groESL(1) genes were expressed as part of a 2.2-kb polycistronic transcript that is i nduced 13-fold after heat shock. Transcript size was not affected by h eat shock; however, the amount of transcript was induced to its greate st extent 15 to 30 min after a 40 degrees C heat shock, from an initia l temperature of 28 degrees C, and remained elevated up to 120 min. Th e R. sphaeroides groESL(1) operon contains a putative hairpin loop at the start of the transcript that is present in other bacterial heat sh ock genes. Primer extension of the message showed that the transcripti on start site is at the start of this conserved hairpin loop. In this region were also found putative -35 and -10 sequences that are conserv ed upstream from other bacterial heat shock genes. Transcription of th e groESL(1) genes was unexpectedly low under photoautotrophic growth c onditions. Thus far, it has not been possible to construct a groESL(1) deletion strain, perhaps indicating that these genes are essential fo r growth. A second operon (groESL(2)) was also cloned from R. sphaeroi des, using a groEL(1) gene fragment as a probe; however, no transcript was observed for this operon under several different growth condition s. A groESL(2) deletion strain was constructed, but there was no detec table change in the phenotype of this strain compared to the parental strain.