PURIFICATION OF THE COPPER RESPONSE EXTRACELLULAR PROTEINS SECRETED BY THE COPPER-RESISTANT METHANOGEN METHANOBACTERIUM-BRYANTII BKYH AND CLONING, SEQUENCING, AND TRANSCRIPTION OF THE GENE ENCODING THESE PROTEINS

When the copper-resistant methanogen Methanobacterium bryantii BKYH wa s exposed to 1 mM Cu(II), it secreted approximately fourfold increased levels of three proteins, copper response extracellular (CRX) protein s, The members of the CRX protein trio had apparent molecular masses o f 40.8, 42.3, and 42.9 kDa and were purified together from the culture supernatant and separated from each other by sodium dodecyl sulfate-p olyacrylamide gel electrophoresis, The N-terminal amino acid sequences of the three proteins were essentially identical, and antibodies rais ed against one of the trio reacted with all three proteins and with th ree other intracellular proteins with slightly higher molecular weight s, The N-terminal amino acid sequence of one of these larger proteins was different from that of the secreted CRX proteins, The gene err, wh ich encodes the CRX proteins, was cloned and sequenced, and err transc ription was characterized, The err sequence predicts that the encoded polypeptide is synthesized as a precursor with an N-terminal leader pe ptide, containing 28 amino acid residues, that is removed during the e xtracellular secretion of the CRX proteins, Transcription was initiate d 274 bp upstream from the err gene, producing an similar to 1.4-kb mo nocistronic transcript that was present in M. bryantii BKYH cells unde r all growth conditions but that increased approximately fourfold in v ivo in response to Cu addition, The CRX proteins appear to be glycosyl ated, since they react with concanavalin A and neuraminidase, and to b e the products of one gene that have different levels of posttranslati onal glycosylation. This is supported by very similar chromatographic and electrophoretic properties, identical N-terminal amino acid sequen ces, immunological cross-reactivities, and the detection of only one e rr-related sequence by Southern blotting, Western blots (immunoblots) showed no evidence for CRX proteins in cell lysates of several other M ethanobacterium strains.