Sequence and structural characteristics of the trypsin-resistant T6 surface protein of group A streptococci.

The gene for the trypsin-resistant surface T6 protein of Streptococcus pyogenes D471 (M type 6) was cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene (tee6) and its flanking regions was determined and found to include only one major open reading frame coding for a protein of 537 amino acids (Mr, 57,675). The N terminus of the deduced protein sequence exhibits features of a typical signal sequence, and the C-terminal segment was found to have a high degree of homology with the membrane anchor region of other gram-positive surface proteins, such as streptococcal M protein, wapA protein from Streptococcus mutans and staphylococcal protein A. A hexapeptide having the consensus sequence LPSTGE and located immediately upstream of the C-terminal hydrophobic segment showed the highest degree of conservation at both the protein and DNA levels, with nearly all reported surface proteins from gram-positive cocci. The amino acid composition of the T6 protein revealed 21% serine and threonine residues distributed nearly regularly throughout the molecule, and analysis of the secondary structure predicted a conformation composed of greater than 70% beta-sheet potential interrupted by beta-turns or random coils. Localization experiments in E. coli show very little T6 protein in the periplasmic space. When found here, however, this T6 protein had a molecular mass of 55 kilodaltons, similar to that extracted from the streptococci by nonionic detergent. Most of the T6 protein was found localized in the membrane fraction, where it was composed of a triple band of 60, 58, and 57 kilodaltons. The coexistence of streptococcal surface proteins which are either resistant (T protein) or sensitive (M protein) to proteolytic enzymes may offer a new dimension to the modulation of these antigens under specific biological conditions.     

Extensive sequence homology between IgA receptor and M proteins in Streptococcus pyogenes

Many strains of Streptococcus pyogenes are known to express a receptor for IgA. The complete nucleotide sequence of the gene for such a receptor, protein Arp4, has been determined. The deduced amino acid sequence of 386 residues includes a signal sequence of 41 amino acids and a putative membrane anchor region, both of which are homologous to similar regions in other streptococcal surface proteins. The processed form of the IgA receptor has a length of 345 amino acids and a calculated molecular weight of 39544. The N-terminal sequence of the processed form is different from that previously found for a similar IgA receptor isolated from a S. pyogenes strain of type M60. The sequence of protein Arp4 shows extensive homology to the C-terminal half of streptococcal M proteins, but not to the streptococcal IgG receptor protein G or staphlyococcal protein A. Apart from the membrane anchor, this homology includes a sequence of 119 amino acid residues containing three repeated units and a 54-residue sequence without repeats. The protein expressed in Escherichia coli is found in the periplasmic space, in which it constitutes the major protein. Protein Arp4 is the first example of a surface protein that has both immunoglobulin-binding capacity and structural features characteristic of M proteins.     

Sequence analysis of Enterococcus faecalis aggregation substance encoded by the sex pheromone plasmid pAD1

The location of the structural gene for aggregation substance on the sex pheromone plasmid pAD1 of Enterococcus faecalis was determined using an oligonucleotide deduced from the N-terminal amino acid sequence of the purified protein. The nucleotide sequence was determined for the corresponding region and two open reading frames (ORFs) could be identified. ORF1 codes for a small (Mr 13,160) acidic protein of unknown function. The gene for aggregation substance (named asa1) was found to code for a protein of 1296 amino acids (Mr 142,248). The protein has a signal peptide of 43 amino acids (the resulting Mr for mature aggregation substance is 137,429) and contains in its C-terminal region a proline-rich sequence, previously characterized as being involved in cell wall association, which is followed by a membrane anchor. The membrane anchor showed significant similarity to that of other Gram-positive organisms, but no other similarities to surface proteins from Gram-positive bacteria were found. In particular, no repeats on the DNA or protein level could be detected for pAD1-specific aggregation substance. The protein contains the amino acid motifs Arg-Gly-Asp-Ser and Arg-Gly-Asp-Val (once each), which, it is proposed, play a crucial role in adherence to eukaryotic cells.     

Isolation and characterization of the cell-associated region of group A streptococcal M6 protein.

DNA sequence analysis of the complete M6 protein gene revealed 19 hydrophobic amino acids at the C terminus which could act as a membrane anchor and an adjacent proline- and glycine-rich region likely to be located in the cell wall. To define this region within the cell wall and its role in attaching the molecule to the cell, we isolated the cell-associated fragment of the M protein. Assuming that the cell-associated region of the M protein would be embedded within the wall and thus protected from trypsin digestion, cells were digested with this enzyme, and the wall-associated M protein fragment was released by phage lysin digestion of the peptidoglycan. With antibody probes prepared to synthetic peptides of C-terminal sequences, a cell wall-associated M protein fragment (molecular weight, 16,000) was identified and purified. Amino acid sequence analysis placed the N terminus of the 16,000-molecular-weight fragment at residue 298 within the M sequence. Amino acid composition of this peptide was consistent with a C-terminal sequence lacking the membrane anchor. Antibody studies of nitrous acid-extracted whole bacteria suggested that, in addition to the peptidoglycan-associated region, a 65-residue helical segment of the C-terminal domain of the M protein is embedded within the carbohydrate moiety of the cell wall. Since no detectable amino sugars were associated with the wall-associated fragment, the C-terminal region of the M6 molecule is likely to be intercalated within the cross-linked peptidoglycan and not covalently linked to it. Because the C-terminal region of the M molecule is highly homologous to the C-terminal end of protein A from staphylococci and protein G from streptococci, it is likely that the mechanism of attachment of these proteins to the cell wall is conserved.     

The gene sequence and some properties of protein H. A novel IgG-binding protein

The gene for protein H, a novel bacterial cell wall protein with specific affinity for human IgG Fc, was cloned from a group A Streptococcus and expressed in Escherichia coli. Recombinant E. coli cells produced two forms of a human IgG Fc-binding protein, one with an apparent Mr of 42 kDa in a periplasmic fraction and the other with an apparent Mr of 45 kDa in a mixed fraction of cytoplasms and membranes. Both 42-kDa and 45-kDa protein preparations similarly bound to human IgG1 to IgG4, human IgG Fc, and rabbit IgG, but not to IgG of mouse, rat, bovine, sheep, goat, and human IgA, IgD, IgE, and IgM. The complete nucleotide sequence of the cloned 1.8-kb DNA fragment was determined. An open reading frame encoded a hypothetical protein of 376 amino acid residues (Mr = 42,498). The N-terminal amino acid sequence, consisting of 41 residues, which was removed post-translationally had typical characteristics of Gram-positive bacterial signal peptides. Thus, the mature form of protein H was suggested to consist of 335 residues (Mr = 38,162). There were 3 repeated sequences consisting of 42 residues that were highly homologous to those of protein Arp, an IgA-binding streptococcal cell wall protein, and streptococcal M6 and M24 proteins. The C-terminal amino acid sequence consisting of 93 residues, directly following the repeated sequences, was also highly homologous to that of M6 and M24 proteins. No sequence homology was found between protein H and protein A or protein G, two other IgG-binding bacterial cell wall proteins.     

Molecular evolution of streptococcal M protein: cloning and nucleotide sequence of the type 24 M protein gene and relation to other genes of Streptococcus pyogenes.

The structural gene for the type 24 M protein of group A streptococci has been cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene and the 3' and 5' flanking regions was determined. The sequence includes an open reading frame of 1,617 base pairs encoding a pre-M24 protein of 539 amino acids and a predicted Mr of 58,738. The structural gene contains two distinct tandemly reiterated elements. The first repeated element consists of 5.3 units, and the second contains 2.7 units. Each element shows little variation of the basic 35-amino-acid unit. Comparison of the sequence of the M24 protein with the sequence of the M6 protein (S. K. Hollingshead, V. A. Fischetti, and J. R. Scott, J. Biol. Chem. 261:1677-1686, 1986) indicates that these molecules have are conserved except in the regions coding for the antigenic (type specific) determinant and they have three regions of homology within the structural genes: 38 of 42 amino acids within the amino terminal signal sequence, the second repeated element of the M24 protein is found in the M6 molecule at the same position in the protein, and the carboxy terminal 164 amino acids, including a membrane anchor sequence, are conserved in both proteins. In addition, the sequences flanking the two genes are strongly conserved.     

The IgA-binding β antigen of the c protein complex of Group B streptococci: sequence determination of its gene and detection of two binding regions

The beta antigen of the lbc protein complex of Group B streptococci is a cell-surface receptor which binds the Fc region of human immunoglobulin A (IgA). Determination of the nucleotide sequence of the beta antigen gene shows that it encodes a preprotein having a molecular weight of 130,963 daltons and a polypeptide of 1164 amino acid residues that is typical of other Gram-positive cell-wall proteins. There is a long signal sequence of 37 amino acids at the N-terminus. Four of the five C-terminal amino acid residues are basic and are preceded by a hydrophobic stretch that appears to anchor the C-terminus in the cell membrane. To the N-terminal side of this hydrophobic stretch is a putative cell-wall-spanning region containing proline-rich repeated sequences. An unusual feature of these repeated sequences is a three-residue periodicity, whereby every first residue is a proline, the second residue is alternating positively or negatively charged, and the third residue is uncharged. The IgA-binding activity was approximately localized by expressing subfragments of the beta antigen as fusion proteins. Two distinct but adjacent DNA segments specified peptides that bound IgA, which indicates that the IgA-binding activity is located in two distinct regions of the protein.     

长角血蜱卵泡抑素相关蛋白的定性

参照GenBank中长角血蜱致病性Okayama株卵泡抑素基因的核苷酸序列(GenBank Accession No.DQ248886)设计合成一对引物,从本实验室保藏的单克隆洁净长角血蜱饥饿成蜱中快速提取总RNA,通过RT-PCR扩增出814bp的卵泡抑素基因,序列比对结果显示:与长角血蜱致病性Okayama株的核苷酸序列及氨基酸序列一致性分别为97.8%和99%,将其亚克隆到表达载体pGEX-4T-1中进行表达,GST融合重组蛋白预期分子量为57kD。表达重组蛋白经MagneGSTTM蛋白纯化系统纯化后作为抗原分别与抗不同发育阶段长角血蜱(卵、幼蜱、若蜱、成蜱)多克隆抗体作为一抗进行免疫印迹,结果表明:与长角血蜱卵制备的多克隆抗体有很强的免疫反应,而与其他发育阶段(幼蜱、若蜱、成蜱)饥饿长角血蜱制备的多克隆抗体反应性很弱。以上结果表明:长角血蜱卵泡抑素蛋白在长角血蜱产卵及卵成熟发育时期的表达水平较其他发育阶段(幼蜱、若蜱、成蜱)的蛋白表达水平高。     

Isolation of intact FNR protein (Mr 30 000) of Escherichia coli

FNR, the activator of anaerobic respiratory genes of Escherichia coli, has previously only been isolated as a protein of Mr 29,000, which lacks nine N-terminal amino acid residues. The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation. The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (Mr 30,000). Proteolysis only occurred during and shortly after disruption of the bacteria. The production of FNR (Mr 29,000) must therefore be regarded as an isolation artefact. The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane. Protease inhibitors directed against serine, cysteine or metalloproteases failed to prevent cleavage of FNR. In E. coli strain CAG627, proteolysis was greatly reduced making it possible to isolate FNR of Mr 30,000. The N-terminal sequence of FNR (Mr 30,000) was identical to that predicted from the fnr gene starting with the initiating methionine residue and including a four-cysteine cluster (16)Cys-X3-Cys-X2-Cys-X5-Cys(29).     

Cloning and sequence analysis of membrane-bound alkaline phosphatase cDNA of the silkworm, Bombyx mori.

The nucleotide sequence (1974 bp) of cDNA coding for membrane-bound alkaline phosphatases (m-ALP) of Bombyx mori was isolated. The cDNA clone contained an open reading frame encoding a polypeptide (547 amino acids), which contains a hydrophobic signal peptide of 36 amino acids and the mature protein of 511 amino acids (Mr = 56,163). We found a highly hydrophobic domain presumed to be a membrane anchoring region at the C-terminus. Comparing analysis between Bombyx m-ALP and mammalian and Escherichia coli ALPs suggested an evolutionary relationship of sharing a common ancestral gene.     

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs.     

Sequence analysis and characterization of a maize gene encoding a high-sulfur zein protein of Mr 15,000

We have isolated a gene coding for a sulfur-rich zein protein from a maize genomic library. The nucleotide sequence of this gene predicts a protein composed of 180 amino acids, including a 20-amino acid signal peptide. As is true of other zeins, there are no intervening sequences in the gene. Comparison of the nucleotide sequence of this gene with that of a homologous cDNA clone revealed only a single difference resulting in a valine/alanine substitution. The Mr 15,000 zein contains no repetitive nucleotide sequences and shows no homology with genes encoding the Mr 22,000 and 19,000 zeins. Circular dichroism analysis of the Mr 15,000 zein protein revealed that it is composed primarily of beta and turn structures. This gene has a short region of nucleotide sequence homology to the cysteine-rich domain of the Mr 27,000 zein, as well as the cysteine-containing barley B-hordein.     

cDNA clones completing the nucleotide and derived amino acid sequence of the alpha 1 chain of basement membrane (type IV) collagen from mouse

Six cDNA clones add 3549 nucleotides to the DNA sequence of the alpha 1 chain of basement membrane (type IV) collagen. Thus the complete nucleotide and derived amino acid sequence of the alpha 1 type IV collagen with 5007 nucleotides coding for 1669 amino acids with a calculated Mr of 160,827 is known. The six cDNA clones cover the putative N-terminal signal peptide, the 7 S region and two thirds of the helical region extending into the previously published murine nucleotide sequence [(1986) Gene 43, 301]. The protein sequence for 289 amino acids of the helical region adjacent to the 7 S region has not previously been published for any species.     

Sequence of glutamine synthetase from Salmonella typhimurium and implications for the protein structure

To aid in the interpretation of the 3.5 A resolution electron density map of glutamine synthetase (GS) from Salmonella typhimurium, the nucleotide sequence of the gene coding for this enzyme has been determined. The predicted sequence of 468 amino acids (Mr = 51,628) has been compared to the sequence and sequence fragments reported by others for GS of Anabaena and Escherichia coli. The homology between the pairs of sequences is sufficiently strong to suggest that the overall three-dimensional structures of the three GS are similar. The predicted positions of alpha helices are in moderately good agreement with the electron-density map.     

Tracking the evolution of the bacterial choline-binding domain: molecular characterization of the Clostridium acetobutylicum NCIB 8052 cspA gene.

The major secreted protein of Clostridium acetobutylicum NCIB 8052, a choline-containing strain, is CspA (clostridial secreted protein). It appears to be a 115,000-M(r) glycoprotein that specifically recognizes the choline residues of the cell wall. Polyclonal antibodies raised against CspA detected the presence of the protein in the cell envelope and in the culture medium. The soluble CspA protein has been purified, and an oligonucleotide probe, prepared from the determined N-terminal sequence, has been used to clone the cspA gene which encodes a protein with 590 amino acids and an M(r) of 63,740. According to the predicted amino acid sequence, CspA is synthesized with an N-terminal segment of 26 amino acids characteristic of prokaryotic signal peptides. Expression of the cspA gene in Escherichia coli led to the production of a major anti-CspA-labeled protein of 80,000 Da which was purified by affinity chromatography on DEAE-cellulose. A comparison of CspA with other proteins in the EMBL database revealed that the C-terminal half of CspA is homologous to the choline-binding domains of the major pneumococcal autolysin (LytA amidase), the pneumococcal antigen PspA, and other cell wall-lytic enzymes of pneumococcal phages. This region, which is constructed of four repeating motifs, also displays a high similarity with the glucan-binding domains of several streptococcal glycosyltransferases and the toxins of Clostridium difficile.     

Sequences of Escherichia coli uvrA gene and protein reveal two potential ATP binding sites

We have determined the nucleotide sequence of the uvrA gene of Escherichia coli. The coding region of the gene is 2820 base pairs which specifies a protein of 940 amino acids and Mr = 103,874. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the UvrA protein: the sequence of the first 7 NH2-terminal amino acids as well as the amino acid composition of the pure protein agreed with those predicted from the nucleotide sequence. By comparing the sequence of UvrA protein to the amino acid sequences of other ATPases, we found that two regions in the UvrA protein, separated from one another by about 600 amino acids, have the highly conserved G-X4-GKT(S)-X6-I(V) sequence found at the active sites of many, but not all, ATPases. Our findings suggest that UvrA protein may have two ATP binding sites.     

Differential immunogenicity of a DNA vaccine containing the Streptococcus mutans wall-associated protein A gene versus that containing a truncated derivative antigen A lacking in the hydrophobic carboxyterminal region

Two plasmid DNA constructs were obtained by cloning separately into the eukaryotic expression vector pcDNA3.1/V5-His-TOPO the wall-associated protein A (wapA) gene of Streptococcus mutans GS-5 or its truncated derivative antigen A (agA) gene encoding a known candidate antigen for dental caries vaccine. The immunogenicity of the two constructs, designated pcDNA-wapA and pcDNA-agA, was compared by intranasal immunization of two groups of mice using the cationic DMRIE-C (1,2-dimyristyloxypropyl-3-dimethylhydroxy ethyl ammonium bromide-cholesterol) as an adjuvant. Immunization with pcDNA-wapA or pcDNA- agA resulted in specific salivary IgA and systemic IgG antibodies to the target antigens after two doses given at 3-week intervals. Higher salivary IgA level was observed in the mice immunized with the pcDNA-wapA vaccine compared to those immunized with the pcDNA-agA vaccine. Furthermore, anti-WapA antibody inhibited S. mutans sucrose-dependent adherence suggesting a potential protection against S. mutans colonization of the tooth, while anti-AgA had no significant effect. Indeed, prediction and analysis of protein epitopes showed that WapA contains highly promiscuous MHC-II binding motifs in addition to those found in AgA. Immunodot assay confirmed that WapA bound biotin-labeled dextran, whereas AgA did not. These data indicated that full-length WapA is a better candidate vaccine antigen than the soluble AgA, which is truncated in the hydrophobic membrane and wall-spanning region.     

Conformational characteristics of the complete sequence of group A streptococcal M6 protein

M protein is considered a virulence determinant on the streptococcal cell wall by virtue of its ability to allow the organism to resist attack by human neutrophils. The complete DNA sequence of the M6 gene from streptococcal strain D471 has allowed, for the first time, the study of the structural characteristics of the amino acid sequence of an entire M protein molecule. Predictive secondary structural analysis revealed that the majority of this fibrillar molecule exhibits strong alpha-helical potential and that, except for the ends, nonpolar residues in the central region of the molecule exhibit the 7-residue periodicity typical for coiled-coil proteins. Differences in this heptad pattern of nonpolar residues allow this central rod region to be divided into three subdomains which correlate essentially with the repeat regions A, B, and C/D in the M6 protein sequence. Alignment of the N-terminal half of the M6 sequence with PepM5, the N-terminal half of the M5 protein, revealed that 42% of the amino acids were identical. The majority of the identities were "core" nonpolar residues of the heptad periodicity which are necessary for the maintenance of the coiled coil. Thus, conservation of structure in a sequence-variable region of these molecules may be biologically significant. Results suggest that serologically different M proteins may be built according to a basic scheme: an extended central coiled-coil rod domain (which may vary in size among strains) flanked by functional end domains.     

fhuC and fhuD genes for iron (III)-ferrichrome transport into Escherichia coli K-12.

The nucleotide sequence for a 1,900-base-pair region of the Escherichia coli chromosome that includes the genes fhuC and fhuD was determined. Within this sequence are two open reading frames: nucleotides 127 to 921 and nucleotides 924 to 1811. These coding regions specify a FhuC protein with an Mr of 28,423 and a mature FhuD protein with an Mr of 29,610. The deduced amino acid sequence of FhuC shows extensive homology with those of components of some bacterial transport systems which are peripheral proteins of the cytoplasmic membrane. Because the FhuD protein contains a typical signal sequence of 30 amino acids at the amino terminus and displays characteristics of a soluble protein, it may be exported into the periplasm.