Detection of GroEL in activated sludge: a model for detection of system stress.

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 degrees 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.

Cloning and characterization of two groESL operons of Rhodobacter sphaeroides: transcriptional regulation of the heat-induced groESL operon.

The nonsulfur purple bacterium Rhodobacter sphaeroides was found to contain two groESL operons. The groESL1 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 GroEL1 (cpn60) and GroES1 (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 groESL1 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 degrees C heat shock, from an initial temperature of 28 degrees C, and remained elevated up to 120 min. The R. sphaeroides groESL1 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 groESL1 genes was unexpectedly low under photoautotrophic growth conditions. Thus far, it has not been possible to construct a groESL1 deletion strain, perhaps indicating that these genes are essential for growth. A second operon (groESL2) was also cloned from R. sphaeroides, using a groEL1 gene fragment as a probe; however, no transcript was observed for this operon under several different growth conditions. A groESL2 deletion strain was constructed, but there was no detectable change in the phenotype of this strain compared to the parental strain.

Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides.

Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography. In the presence of 1 mM ATP, the chaperonin 10 and chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation. The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for chaperonin 60 and 60,000 for chaperonin 10. Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment. When cells were grown photoheterotrophically or chemoheterotrophically, chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein. The N-terminal amino acid sequence of each protein was determined; chaperonin 60 of R. sphaeroides showed 72% identity to E. coli chaperonin 60 protein, and R. sphaeroides chaperonin 10 showed 45% identity with E. coli chaperonin 10. R. sphaeroides chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R. sphaeroides chaperonin 10, but not E. coli chaperonin 10. The E. coli chaperonin 60 ATPase activity was inhibited by chaperonin 10 from both R. sphaeroides and E. coli.

Ferredoxin requirement for electron transport from the carbon monoxide dehydrogenase complex to a membrane-bound hydrogenase in acetate-grown Methanosarcina thermophila.

Cell extracts from acetate-grown Methanosarcina thermophila contained CO-oxidizing:H2-evolving activity 16-fold greater than extracts from methanol-grown cells. Following fractionation of cell extracts into soluble and membrane components, CO-dependent H2 evolution and CO-dependent methyl-coenzyme M methylreductase activities were only present in the soluble fraction, but addition of the membrane fraction enhanced both activities. A b-type cytochrome(s), present in the membrane fraction, was linked to a membrane-bound hydrogenase. CO-oxidizing:H2-evolving activity was reconstituted with: (i) CO dehydrogenase complex, (ii) a ferredoxin, and (iii) purified membranes with associated hydrogenase. The ferredoxin was a direct electron acceptor for the CO dehydrogenase complex. The ferredoxin also coupled CO oxidation by CO dehydrogenase complex to metronidazole reduction.

Purification and characterization of a ferredoxin from acetate-grown Methanosarcina thermophila.

A ferredoxin, which functions as an electron acceptor for the CO dehydrogenase complex from Methanosarcina thermophila, was purified from acetate-grown cells. It was isolated as a trimer having a native molecular weight of approximately 16,400 and monomer molecular weight of 4,888 calculated from the amino acid composition. The ferredoxin contained 2.80 +/- 0.56 Fe atoms and 1.98 +/- 0.12 acid-labile sulfide. UV-visible absorption maxima were 395 and 295 nm with monomeric extinction coefficients of epsilon 395 = 12,800 M-1 cm-1 and epsilon 295 = 14,460 M-1 cm-1. The A395/A295 ratio ranged from 0.80 to 0.88. There were 5 cysteines per monomer but no methionine, histidine, arginine, or aromatic amino acids. The N-terminal amino acid sequence showed a 4-cysteine cluster with potential to coordinate a Fe:S center. The protein was stable for 30 min at 70 degrees C, but denatured during incubation at 85 degrees C.

EPR properties of the Ni-Fe-C center in an enzyme complex with carbon monoxide dehydrogenase activity from acetate-grown Methanosarcina thermophila. Evidence that acetyl-CoA is a physiological substrate.

The carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila was further studied by EPR spectroscopy. The as purified enzyme exhibited no paramagnetic species at 113 K; however, enzyme reduced with CO exhibited a complex EPR spectrum comprised of two paramagnetic species with g values of g1 = 2.089, g2 = 2.078, and g3 = 2.030 (signal I) and g1 = 2.057, g2 = 2.049, and g3 = 2.027 (signal II). Isotopic substitution with 61Ni, 57Fe, or 13CO resulted in broadening of the EPR spectra indicating a Ni-Fe-C spin-coupled complex. Pure signal II was obtained following treatment of the CO-reduced enzyme with acetyl-CoA but not by addition of acetyl phosphate or CoASH. Acetate-grown cells were highly enriched in acetate kinase (EC 2.7.2.1) and CoASH-dependent phosphotransacetylase (EC 2.3.1.8) activities. These results suggest acetyl-CoA is a physiological substrate for the carbon monoxide dehydrogenase complex synthesized in acetate-grown cells of M. thermophila.

Isolation of an enzyme complex with carbon monoxide dehydrogenase activity containing corrinoid and nickel from acetate-grown Methanosarcina thermophila.

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.