Analysis and nucleotide sequence of the genes encoding the surface‐layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis

Abstract

The genes (slgA) encoding the surface-layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis were cloned and sequenced. The nucleotide sequences of these genes differ at only nine positions, resulting in three amino acid differences.In both organisms, the transcription start site was localized by primer extension analyses. The DNA sequence at this site conforms to the promotor box B motif for promotors of archaea. 24 nucleotides upstream of the transcription start is an A + T-rich region, which closely resembles the consensus box A motif of promoters of methanogens. Ribosome binding sites are exactly complementary to the 3' end of the 16s rRNA of these met hanogens.Both slgA genes encode for a precursor of the mature surface-layer protein containing 593 amino acid residues with a putative N-terminal signal sequence of 22 amino acid residues. The deduced protein sequences contain 20 sequon structures representing possible carbohydrate-binding sites.In comparison with other surface-layer proteins, these obtained from the two hyperthermophilic methanogens contain unusually high amounts of isoleucine, asparagine and cysteine residues. Predicted secondary structures have a high content of fl-sheet structure (44%) and only 7% a-helix structures.Glycosylated proteins fulfil a variety of important functions in eukaryotes [l, 21, whereas in prokaryotes glycoproteins have been found almost exclusively in the cytoplasmic membrane or in the surface layers [3 -61.The surface layers consist of protein or glycoprotein subunits, which assemble into mono-layered crystalline arrays. Surface layers are widespread among bacteria and archaea and they can be part of complex cell walls or they can form the only cell wall layer. Surface layers are involved in maintaining shape, cell protection and cell adhesion and they function as molecular barriers and sieves.In extremely thermophilic bacteria, surface layers are directly exposed to the extreme environments and cannot be stabilized by chemical components as is the case with cytoplasmatic proteins [7]. The molecular basis for stability, heat resistance and evolution of the extremely thermophilic surface layer (glyco-) The genes encoding surface-layer proteins have been sequenced from four prokaryotes [8 -1 ll. Recently, we reported on the biosynthesis of the glycan strands of the surface-layer glycoprotein of the methanogen Methanothermus fervidus [ 12, 131. In this paper we describe the cloning and sequencing of the encoding genes of the surface layer glycoproteins of the hyperthermophilic methanogens M t . fervidus and Methanothermus sociabilis in order to obtain more information on the primary structure of extremely thermophilic surface-layer glycopro teins. [19]. Restriction enzymes were purchased from Boehringer Mannheim o r from BRL. The M-MuLV reverse transcriptase, DNase I and the universal and reverse primer for sequencing were from Boehringer Mannheim; T4 DNA ligase and exonuclease BAL31 from BRL; and exonuclease 111 a...

Posttranslational Protein Modification inArchaea

“…While the glycosylated nature of S-layer proteins has been proposed in many archaeal species, experimental proof for this posttranslational modification is limited to the S-layer glycoproteins of Halobacterium salinarum (246), Haloferax volcanii (421), Haloarcula japonica (299), Methanothermus fervidus (204), Methanothermus sociablis (41), Sulfolobus spp. (146), and components of the S-layer of Staphylothermus marinus (345).…”

S-Layer Proteins

“…According to sequence analyses, the isoelectric points (pIs) of most S-layer proteins are in the weakly acidic pH range. An exception was found for the S-layer proteins of Methanothermus fervidus (17) and for different lactobacilli (11), which possess pIs of 8.4 and 9 to 11, respectively. According to circular-dichroism measurements, approximately 20% of the amino acids are organized as ␣ helices and about 40% occur as ␤ sheets.…”

Posttranslational Processing ofMethanococcus voltaePreflagellin by Preflagellin Peptidases ofM. voltaeand Other Methanogens

“…In addition, the leader peptide is not followed by a stretch of hydrophobic amino acids as always found in archaeal flagellins (8) but instead has acidic or basic amino acids in 8 of the first 21 positions of the mature protein. The S-layer gene and protein of Methanothermus fervidus, another flagellated methanogen, have also been studied (5). In this case, the leader peptide has a typical bacterium like leader peptide of 22 amino acids with an Ala-Gly-Ala sequence preceding the cleavage site.…”