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.

Abstract

When the copper-resistant methanogen Methanobacterium bryantii BKYH was exposed to 1 mM Cu(II), it secreted approximately fourfold increased levels of three proteins, copper response extracellular (CRX) proteins. The members of the CRX protein trio had apparent molecular masses of 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-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of the three proteins were essentially identical, and antibodies raised against one of the trio reacted with all three proteins and with three other intracellular proteins with slightly higher molecular weights. The N-terminal amino acid sequence of one of these larger proteins was different from that of the secreted CRX proteins. The gene crx, which encodes the CRX proteins, was cloned and sequenced, and crx transcription was characterized. The crx sequence predicts that the encoded polypeptide is synthesized as a precursor with an N-terminal leader peptide, containing 28 amino acid residues, that is removed during the extracellular secretion of the CRX proteins. Transcription was initiated 274 bp upstream from the crx gene, producing an approximately 1.4-kb monocistronic transcript that was present in M. bryantii BKYH cells under all growth conditions but that increased approximately fourfold in vivo in response to Cu addition. The CRX proteins appear to be glycosylated, since they react with concanavalin A and neuraminidase, and to be the products of one gene that have different levels of posttranslational glycosylation. This is supported by very similar chromatographic and electrophoretic properties, identical N-terminal amino acid sequences, immunological cross-reactivities, and the detection of only one crx-related sequence by Southern blotting. Western blots (immunoblots) showed no evidence for CRX proteins in cell lysates of several other Methanobacterium strains.