Fast protein liquid chromatography of cell extract from methanol- or acetate-grown Methanosarcina thermophila resolved two peaks of CO dehydrogenase activity. The activity of one of the CO dehydrogenases was sixfold greater in acetate-grown compared with methanol-grown cells. This CO dehydrogenase was purified to apparent homogeneity (70 mumol of methyl viologen reduced per min per mg of protein) and made up greater than 10% of the cellular protein of acetate-grown cells. The native enzyme (Mr 250,000) formed aggregates with an Mr of approximately 1,000,000. The enzyme contained five subunits (Mrs 89,000, 71,000, 60,000, 58,000, and 19,000), suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The UV-visible spectrum suggested the presence of iron-sulfur centers. The electron paramagnetic resonance spectrum contained g values of 2.073, 2.049, and 2.028; these features were broadened in enzyme that was purified from cells grown in the presence of medium enriched with 61Ni, indicating the involvement of this metal in the spectrum. The pattern of potassium cyanide inhibition indicated that cyanide binds at or near the CO binding site. The properties of the enzyme imply an involvement in the dissimilation of acetate to methane, possibly by cleavage of acetate or activated acetate.

The nonsulfur purple bacterium Rhodobacter sphaeroides was found to contain two groESL operons. The groESL 1 heat shock operon was cloned from a genomic library, and a 2.8-kb DNA fragment was sequenced and found to contain the groES and groEL genes. The deduced amino acid sequences 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 sequences show a high degree of similarity to groESL genes isolated from other bacteria. Northern analysis indicated that the groESL 1 genes were expressed as part of a 2.2-kb polycistronic transcript that is induced 13-fold after heat shock. Transcript size was not affected by heat shock; however, the amount of transcript was induced to its greatest extent 15 to 30 min after a 40؇C heat shock, from an initial temperature of 28؇C, and remained elevated up to 120 min. The R. sphaeroides groESL 1 operon contains a putative hairpin loop at the start of the transcript that is present in other bacterial heat shock genes. Primer extension of the message showed that the transcription start site is at the start of this conserved hairpin loop. In this region were also found putative ؊35 and ؊10 sequences that are conserved upstream from other bacterial heat shock genes. Transcription of the groESL 1 genes was unexpectedly low under photoautotrophic growth conditions. Thus far, it has not been possible to construct a groESL 1 deletion strain, perhaps indicating that these genes are essential for growth. A second operon (groESL 2 ) was also cloned from R. sphaeroides, using a groEL 1 gene fragment as a probe; however, no transcript was observed for this operon under several different growth conditions. A groESL 2 deletion strain was constructed, but there was no detectable change in the phenotype of this strain compared to the parental strain.

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A.J. DUNCAN, C.B. BOTT, K.C. TERLESKY and N.G. LOVE.2000.GroEL is a ubiquitous constitutively synthesized protein that is also stress inducible. Activated sludge, which is a standard biological process used in wastewater treatment systems, is made up of a diverse microbial consortium. The synthesis of GroEL in activated sludge was significantly induced after heat (42 °C) shock. The increased level of GroEL expression was shown to be due to de novo protein synthesis. We have demonstrated a method which shows that stress proteins can be detected in activated sludge, and propose their use as specific indicators of system stress.