Molecular cloning, expression, and characterization of the gene for the surface (HPI)-layer protein of Deinococcus radiodurans in Escherichia coli

Abstract

The HPI protein of Deinococcus radiodurans belongs to the ciass of surface layer proteins which form crystalline two-dimensional arrays on bacterial cell envelopes. We have cloned and expressed the gene for this protein of Mr about 100,000 by using plasmid pUC8 in Escherichia coli. As judged by immunoreaction with monospecific antibodies, apparent Mr, and limited proteolysis, a single clone contained the gene encoding the complete polypeptide on an 8.9-kilobase (kb) insert. The insert was reduced to a 5.7-kb HindIlI fragment, cloned in pUC18 in both orientations, and subjected to unilateral processive deletion with exonuclease III. The library of deletion derivatives was mapped and, in conjunction with protein immunoblotting of expressed polypeptides, was used to locate the positions of the structural gene and the Deinococcus promoter region that was responsible for expression of the HPI polypeptide. The HPI gene was confined to a stretch 2.95 kb in length.Regular surface layers (S-layers) of protein or glycoprotein are quite common among eubacteria as well as archaebacteria. A variety of functions, all related to interactions between the cell and its environment, has been ascribed to these layers (for recent reviews, see references 9 and 30).The structural and chemical characterization of the Slayer of Deinococcus radiodurans, the so-called HPI-layer (23), is particularly advanced. The HPI-layer has been investigated with a variety of electron microscopic techniques (6,7,14), and recently a determination of its three-dimensional structure to a resolution of 1.5 nm has been completed (4). Measurements of mass, performed with a scanning transmission electron microscope, in conjunction with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis have shown that the unit cell (or the morphological complex) contains six identical polypeptides, each with a molecular weight of about 100,000 (5).Our long-term objective is a high-resolution determination of the structure of the HPI-layer. Another line of investigation aims to elucidate the evolutionary relationships of eubacterial and archaebacterial S-layers. In any case, knowledge of the primary structure is indispensable. A molecular genetic approach to sequence analysis is particularly attractive, as it also creates a perspective for site-directed mutagenesis, which will be an important tool to address questions of function and assembly. Here we report the cloning of the gene for the HPI polypeptide and its characterization and expression in Escherichia coli. Using unilateral deletions generated with exonuclease III, we have mapped the position of the promoter region and the structural gene and constructed an ordered subset of deletion derivatives. MATERIALS AND METHODSMaterials and bacterial strains. Enzymes, nucleotides, M13 reverse sequencing primer, DNA-Mr markers, protein A-Sepharose, and Percoll were bought from Pharmacia P-L, * Corresponding author. D. radiodurans Sark (UWO 298) was kindly provided by R. G. E. Murray, University of Western Ontario, Lo...

Antigenic differences among Campylobacter fetus S-layer proteins

“…Indeed, by using the log-odds score of Dayhoff (11), the two different sequences displayed a mean homology score of 102. Virtually no identity is seen between either of the two C. fetus amino-terminal sequences and the sequences of other S-layer proteins that have been studied, however (16,25,35,36,50), and a search of the National Biomedical Research Foundation protein sequence library (27) failed to reveal any N-terminal sequences with significant homology to those of the C. fetus proteins, suggesting that they are proteins unique to this species.…”

“…Certainly the C. fetus S layer has an extremely fine structure, and a high degree of order can be seen only in patches. Also, oblique arrays have not commonly been reported, and detailed descriptions have been provided only for two Bacillus species (31,38) and Aquaspirillum (Spirillum) putridiconchylium (4,5 especially if C. fetus is comparable to other reported S-layerproducing organisms in which a single chromosomal gene codes for the S-layer protein (3,16,25,35,48,51). Protease digestion of the purified VC119 protein under nondenaturing conditions provided convincing evidence for the presence of two domains in the C. fetus S-layer protein, a large trypsin-, chymotrypsin-, and endoproteinase Glu-Cresistant core with an Mr of approximately 110,000 and a smaller enzyme-sensitive domain with an Mr of approximately 20,000.…”

Crystalline surface layers in procaryotes

“…Thus, sequence homology studies on S-layer proteins and characterization of the glycans of glycoproteins appear essential: these data will help to elucidate the taxonomical significance of S-layers as well as the structural, functional, and evolutionary relationships among S-layers and, possibly, between S-layer protomers and other pore-forming proteins (80; Sleytr and Messner, in press) (such as the regularly arrayed outer membrane proteins [porins] of gram-negative bacteria, which will aggregate only with a lipid bilayer [18]). Genetic approaches to sequence analysis have already been reported for S-layers of Bacillus brevis (89,90,100), Caulobacter crescentus (81) Deinococcus radiodurans (59,60), H. halobium (42), and Aeromonas salmonicida (6).…”

Conserved structures of cell wall protein genes among protein-producing Bacillus brevis strains

“…The morphological properties of S layers have been extensively characterized for a wide range of microorganisms (25)(26)(27), whereas the genes for S-layerforming proteins have been isolated from only a few microorganisms, such as Bacillus brevis 47 (35,37,39,41), Halobacterium halobium (11), Deinococcus radiodurans (18,19), Caulobactor crescentus (30), and Aeromonas salmonicida (4). Too little is known at present about the regulation of these genes to elucidate the mechanisms involved in the biosynthesis, transport, and assembly of Slayer proteins.…”