A novel beta-N-acetylglucosaminidase from Streptomyces thermoviolaceus OPC-520: gene cloning, expression, and assignment to family 3 of the glycosyl hydrolases

A beta-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an Mr of 66, 329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned beta-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical beta-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable beta-glucosidase activity, revealed homology with microbial beta-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of beta-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60 degreesC and pH 5.0, and the apparent Km and Vmax values for p-nitrophenyl-beta-N-acetylglucosamine were 425.7 microM and 24.8 micromol min-1 mg of protein-1, respectively.     

Follicular vascularity--the predictive value of transvaginal power Doppler ultrasonography in an in-vitro fertilization programme: a preliminary study

The glnA gene from the human pathogen Streptococcus agalactiae was cloned from a genomic library prepared with the lambda phage vector lambdaDASHII. A 4.6-kb DNA fragment of one of the recombinant phages was subcloned in pUC18. This Escherichia coli clone expressed a 52-kDa protein encoded by a 1,341-bp open reading frame. The nucleotide sequence of the open reading frame and the deduced amino acid sequence shared a significant degree of homology with the sequences of other glutamine synthetases (GS). The highest homology was between our deduced protein and GS of gram-positive bacteria such as Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus. Plasmids with the cloned streptococcal glnA were able to complement E. coli glnA mutants grown on minimal media. Rabbit antisera to streptococcal GS recombinant protein recognized not only the recombinant protein but also a similar-sized band in mutanolysin extracts of all group B streptococcal strains tested, regardless of polysaccharide type or surface protein profile. The amino acid sequence of the deduced protein had similarities to other streptococcal cell-surface-bound proteins. The possible functional role of the immunological features of streptococcal GS is discussed.     

Structure of the beta-galactosidase gene from Thermus sp. strain T2: expression in Escherichia coli and purification in a single step of an active fusion protein

The nucleotide sequence of both the bgaA gene, coding for a thermostable beta-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (Mr, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the beta-galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric beta-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70 degrees C and a Km value of 1.6 mM with o-nitrophenyl-beta-D-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70 degrees C.     

Isolation and characterization of a satellite DNA family in the Saccharum complex

EaCIR1, a 371-bp Erianthus-specific satellite DNA sequence, was cloned from TaqI restricted genomic DNA after agarose-gel electrophoresis. This sequence has 77% homology with a 365-bp satellite of Helictotrichon convolutum and 72% homology with a 353-bp tandem repeat sequence from Oryza sativa. PCR primers defined in the conserved regions of these repetitive sequences were used to isolate other satellite DNAs in different representatives of the Saccharum complex: SoCIR1 in Saccharum officinarum, SrCIR1 in Saccharum robustum, SsCIR1 and SsCIR2 in Saccharum spontaneum, and MsCIR1 in Miscanthus sinensis. EaCIR1 and SoCIR1 were localized to subtelomeric regions of the chromosomes by fluorescence in situ hybridization. Southern hybridization experiments, using two representatives of this repeat sequence family as probes, illustrated contrasting species-specificity and demonstrated the existence of similar repetitive elements in sorghum and maize.     

Staphylococci express a receptor for human transferrin: identification of a 42-kilodalton cell wall transferrin-binding protein

Staphylococcus aureus and the coagulase-negative staphylococci are commonly responsible for peritonitis in renal patients undergoing continuous ambulatory peritoneal dialysis. To simulate growth conditions in vivo, staphylococci isolated from peritoneal infections were cultured in used human peritoneal dialysate (HPD). Immunoblotting experiments using cell wall preparations from these staphylococci revealed the presence of the host iron-binding glycoprotein transferrin bound to S. aureus, S. epidermidis, S. capitis, S. haemolyticus, and S. hominis but not to S. warneri or S. saprophyticus. Similar results were obtained by incubating broth-grown staphylococci with human transferrin, although, in contrast to S. aureus, the coagulase-negative staphylococci bound more transferrin after growth in iron-restricted broth. To determine whether the staphylococci express a saturable specific receptor for human transferrin, the interaction of human 125I-transferrin with the staphylococci was examined. Both S. aureus and S. epidermidis bound the radiolabelled iron-saturated ligand in a time- and concentration-dependent manner. From competition binding assays, the affinity (Kd) and number of receptors were estimated for S. epidermidis (Kd, 0.27 microM; 4,200 receptors per cell) and S. aureus (Kd, 0.28 microM; 4,200 receptors per cell). S. epidermidis but not S. aureus receptor activity was partially iron regulated. Human apotransferrin and iron-saturated transferrin and rabbit and rat transferrins competed equally well for the staphylococcal receptor. Bovine and porcine transferrins and ovotransferrin as well as human and bovine lactoferrins were much less effective at competing with human transferrin. Treatment of whole staphylococci with protease abolished transferrin binding, indicating the involvement of cell surface protein. Western blots (immunoblots) of cell wall preparations probed with human transferrin revealed the presence of a 42-kDa transferrin-binding protein common to both S. aureus and S. epidermidis. On Western strip blots, the binding of human transferrin to this protein was blocked by labelled human transferrin but not by albumin, immunoglobulin G, or bovine transferrin or ovotransferrin. To assess the conservation of the 42-kDa transferrin-binding protein, cell wall proteins of S. epidermidis, S. haemolyticus, S. capitis, S. hominis, S. warneri, and S. saprophyticus were Western blotted and probed with human transferrin. Only S. warneri and S. saprophyticus lacked the 42-kDa wall protein, consistent with their inability to bind transferrin. These data show that the staphylococci express a specific receptor for human transferrin based at least in part on a common 42-kDa cell wall protein.     

N-Acetyltransferase 2 (NAT2) and Glutathione S-Transferase μ (GSTM1) in Bladder-cancer Patients in a Highly Industrialized Area

The gene encoding the 33 kDa piroplasm surface protein of Theileria sergenti isolated in Korea was cloned and the nucleotide sequence was determined by dideoxy chain termination method. The cloned gene corresponds to 869 bp encoding an open reading frame 283 amino acids. Comparison of the sequence between Korean and Japanese isolates showed 99.4% homology rate in the nucleotide sequence and 98.9% homology rate in the amino acid sequence.     

Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii

A Coxiella burnetii Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by [35S]methionine labeling, autoradiography, and immunoblotting. The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies. The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed. A single open reading frame (ORF) with a size of 1,968 bp was identified. The ORF encodes a protein containing 656 residues and having a molecular weight of 70, 800. The -10 promoter sequence was shown to be identical with the consensus heat shock sigma32 promoter sequence. The base composition at the presumed -35 region revealed an EcoRI site in the expected region, which is assumed to be located at the border of the cloned fragment. The gene was expressed in Escherichia coli as an intact protein. The C. burnetii 71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family. A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g., Legionella pneumophila and Francisella tularensis, as well as E. coli DnaK, is more than 80%. The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins. The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy. The dual localization was verified by Western blot analysis of proteins in C. burnetii cell fractions, using purified antibodies directed to the 71-kDa protein.     

Characterization and biological properties of a new staphylococcal exotoxin

Staphylococcus aureus strain D4508 is a toxic shock syndrome toxin 1-negative clinical isolate from a nonmenstrual case of toxic shock syndrome (TSS). In the present study, we have purified and characterized a new exotoxin from the extracellular products of this strain. This toxin was found to have a molecular mass of 25.14 kD by mass spectrometry and an isoelectric point of 5.65 by isoelectric focusing. We have also cloned and sequenced its corresponding genomic determinant. The DNA sequence encoding the mature protein was found to be 654 base pairs and is predicted to encode a polypeptide of 218 amino acids. The deduced protein contains an NH2-terminal sequence identical to that of the native protein. The calculated molecular weight (25.21 kD) of the recombinant mature protein is also consistent with that of the native molecules. When injected intravenously into rabbits, both the native and recombinant toxins induce an acute TSS-like illness characterized by high fever, hypotension, diarrhea, shock, and in some cases death, with classical histological findings of TSS. Furthermore, the activity of the toxin is specifically enhanced by low quantities of endotoxins. The toxicity can be blocked by rabbit immunoglobulin G antibody specific for the toxin. Western blotting and DNA sequencing data confirm that the protein is a unique staphylococcal exotoxin, yet shares significant sequence homology with known staphylococcal enterotoxins, especially the SEA, SED, and SEE toxins. We conclude therefore that this 25-kD protein belongs to the staphylococcal enterotoxin gene family that is capable of inducing a TSS-like illness in rabbits.     

Sequence analysis of a cryptic plasmid from Flavobacterium sp. KP1, a psychrophilic bacterium

A cryptic plasmid found at high copy number was isolated from Flavobacterium sp. KP1, a psychrophilic Gram-negative bacterium, cloned, and sequenced. The sequence will appear in the DDBJ/EMBL/GenBank databases under the accession number AB007196. The pFL1 plasmid is 2311 nucleotides in length with 32.7% GC content, and shows a distinctive nucleotide sequence without homology to other plasmids of similar length. The plasmid contains two open reading frames of significant length, ORFI and ORFII. ORFI encodes a protein similar to the replication proteins found in Gram-negative bacterial plasmids, Bacteroides fragilis plasmid pBI143 and Zymomonas mobilis plasmid pZM2. The putative translation product of ORFII shows homologies with plasmid recombination proteins found mainly in Gram-positive bacterial plasmids such as Staphylococcus aureus plasmid pT181.     

cDNA cloning and characterization of a rat spermatogenesis-associated protein RSP29

RSP29, a protein secreted by rat round spermatids, stimulates the secretory function of Sertoli cells in the testis. By making use of the N-terminal sequence homology of RSP29 and a human protein hDP1 that we had previously isolated, we cloned the full length cDNA sequence that encodes RSP29. The entire amino acid sequence of RSP29 showed significant homology with that of hDP1, which was later identified as glyoxalase II. Southern analysis showed that the RSP29 protein sequence is highly conserved in eukaryotes and possibly in prokaryotes. The RSP29 mRNA is expressed in many tissues but has an extremely high abundance in testis. These data suggest that RSP29 may have an important function in most tissues of enkaryotic organisms. The high expression of RSP29 in testis and its stimulatory effects on Sertoli cells suggest that RSP29 could be especially important in the regulation of spermatogenesis.     

Cloning and sequence analysis of a protective M-like protein gene from Streptococcus equi subsp. zooepidemicus

Streptococcus equi subsp. zooepidemicus, a Lancefield group C streptococcus, is a frequently isolated opportunist pathogen from a variety of animal hosts, including the horse. Previous studies have indicated that equine strains carry antigens with characteristics of the antiphagocytic M proteins on the Lancefield groups A and G streptococci. We have cloned a protective M-like protein gene (SzPW60) of an equine strain of S. equi subsp. zooepidemicus W60 and determined its sequence. This gene encodes a protein with a molecular weight of 40,123 which protects mice against subsp. zooepidemicus but not subsp. equi, stimulates antibodies which opsonize subsp. zooepidemicus but not equi, and reacts with antiserum to the protein of the parent strain. The predicted amino acid structure shows significant homology with the carboxy termini of groups A and G M proteins but no other homology. The M-like protein, although showing an extensive region of alpha helix, lacks the A, B, and C repeats found in group A M proteins and has a shorter signal sequence. A proline-rich region upstream from the LPSTGE motif contains 20 repeats of the tetrapeptide PEPK. The presence of this repeat region may account for the slow migration of the M-like protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity

Small granules of horseshoe crab hemocytes contain two known major antimicrobial substances, tachyplesin and big defensin (S5), and at least five protein components (S1 to S6), with unknown functions. In the present study, we examined the biological properties and primary structure of a small granular component S2, named tachycitin. This component was purified from the acid extract of hemocyte debris by two steps of chromatography. The purified tachycitin was a single chain protein with an apparent M(r) = 8,500 on Tricine-SDS-polyacrylamide gel electrophoresis. Ultracentrifugation analysis revealed tachycitin to be present in monomer form in solution. Tachycitin inhibited the growth of both Gram-negative and -positive bacteria, and fungi, with a bacterial agglutinating property. Moreover, tachycitin and big defensin acted synergistically in antimicrobial activities. The amino acid sequence and intrachain disulfide bonds of tachycitin were determined by amino acid and sequence analyses of peptides produced by enzymatic cleavages. The mature tachycitin consisted of 73 amino acid residues containing five disulfide bonds with no N-linked sugar. A cDNA coding for tachycitin was isolated from a hemocyte cDNA library. The open reading frame coded for an NH2-terminal signal sequence followed by the mature peptide and an extension sequence of -Gly-Arg-Lys at the COOH-terminus, which is a putative amidating signal. The COOH-terminal threonine amide released after digestion of tachycitin with lysylendopeptidase was identified. The NH2-terminal 28 residues of tachycitin shows sequence homology to a part of chitin-binding regions found in antifungal chitin-binding peptides, chitin-binding lectins, and chitinases, all of which have been isolated from plants. Tachycitin showed a specific binding to chitin but did not bind with the polysaccharides cellulose, mannan, xylan, and laminarin. Tachycitin may represent a new class of chitin-binding protein family in animals.     

Enhancing regional perinatal care: a clinical traineeship for perinatal nurses in a predominantly rural area

The gene nprM encoding the calcium-dependent extracellular proteinase from Bacillus megaterium ATCC 14581 was cloned in the vector pBR322 and expressed in Escherichia coli HB101. The DNA sequence of the cloned 3.7 kb fragment revealed only one open reading frame consisting of 1686 bp with a coding capacity of 562 amino acid residues. A predicted Shine-Dalgarno (SD) sequence was observed 9 bp upstream from the presumptive translation start site (ATG). A possible promoter sequence (TAGACG for the -35 region and TATAAT for the -10 region) was found about 69 bp upstream of the ATG start site. The deduced amino acid sequence exhibited a 24 amino acid residue signal peptide and an additional polypeptide 'pro' sequence of 221 amino acids preceding the putative mature protein of 317 amino acid residues. Amino acid sequence comparison revealed 84.5% homology between the mature protein and that of a thermolabile neutral protease from B. cereus. It also shares 73% homology with the thermostable neutral proteases of B. thermoproteolyticus and B. stearothermophilus. The zinc-binding sites and the catalytic residues are completely conserved in all four proteases. NprM has a temperature optimum of 58 degrees C, a pH optimum of between 6.4 and 7.2, and is stimulated by calcium ions and inhibited by EDTA. These results indicate that the enzyme is a neutral (metallo-) protease.     

A high molecular weight protein from Staphylococcus intermedius cross-reacts with Staphylococcus aureus enterotoxin antibodies

Enterotoxin production by Staphylococcus species other than Staphylococcus aureus has been reported. Staphylococcus strains (104 in toto) representing twelve species and subspecies were examined for enterotoxins using a commercial staphylococcal enterotoxin ELISA immunoassay (TECRA, International Bioproducts). Staphylococcus intermedius (24 strains) and S. aureus (7 strains) were positive with this test. Western blots of S. aureus exoproteins demonstrated proteins of approximately 30 kD, consistent with known staphylococcal enterotoxins. The major antigen in all S. intermedius strains, a 75 kD protein, was not analogous to previously described staphylococcal enterotoxins. This protein was unique to S. intermedius. Gel filtration data indicate that the protein is a subunit of a larger protein in vivo. The 75 kD protein cross-reacts with several enterotoxin antibodies. It is unclear whether the protein is a toxin, but its homology with S. aureus enterotoxins may indicate a shared toxic region, or this protein may create false positive results in screening for enterotoxin.     

Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli

Phosphoglycerate mutase is an essential glycolytic enzyme for Zymomonas mobilis, catalyzing the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate. The pgm gene encoding this enzyme was cloned on a 5.2-kbp DNA fragment and expressed in Escherichia coli. Recombinants were identified by using antibodies directed against purified Z. mobilis phosphoglycerate mutase. The pgm gene contains a canonical ribosome-binding site, a biased pattern of codon usage, a long upstream untranslated region, and four promoters which share sequence homology. Interestingly, adhA and a D-specific 2-hydroxyacid dehydrogenase were found on the same DNA fragment and appear to form a cluster of genes which function in central metabolism. The translated sequence for Z. mobilis pgm was in full agreement with the 40 N-terminal amino acid residues determined by protein sequencing. The primary structure of the translated sequence is highly conserved (52 to 60% identity with other phosphoglycerate mutases) and also shares extensive homology with bisphosphoglycerate mutases (51 to 59% identity). Since Southern blots indicated the presence of only a single copy of pgm in the Z. mobilis chromosome, it is likely that the cloned pgm gene functions to provide both activities. Z. mobilis phosphoglycerate mutase is unusual in that it lacks the flexible tail and lysines at the carboxy terminus which are present in the enzyme isolated from all other organisms examined.     

The mutK gene of Vibrio cholerae: a new gene involved in DNA mismatch repair

A new gene, mutK, of Vibrio cholerae, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified. The product of the mutK gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of Escherichia coli mutS, mutL, mutU, and dam mutants. The spontaneous mutation frequency of a chromosomal mutK knockout mutant was almost identical to that of wild-type V. cholerae cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent. The complete nucleotide sequence of the mutK gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the mutK gene product. The mutK gene product has no significant homology with any of the proteins deposited in the EMBL data bank. ORF2, located upstream of mutK, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the E. coli vsr gene. ORF1, located farther upstream of mutK, has more than 80% homology with a major cold shock protein found in several bacteria. Downstream of mutK, a partial ORF having 60% homology with an RNA methyltransferase has been identified. The mutK gene has recently been positioned in the ordered cloned DNA map of the genome of the V. cholerae strain from which the gene was isolated (10).     

Cloning and characterization of a major allergen of the house dust mite, Dermatophagoides pteronyssinus, homologous with glutathione S-transferase

A major allergen of the house dust mite, Dermatophagoides pteronyssinus, has been identified and characterized from a lambda gt11 cDNA library of the mite. IgE antibodies from the sera of allergic patients that recognise the cloned polypeptide bind to an approximately 26 kDa polypeptide on a Western blot of reduced mite polypeptides. Nucleotides sequencing of the clone revealed a 219 amino acid open reading frame encoding a protein with a derived molecular mass of 25,589 Da and a pI of 6.3. Comparison of the deduced amino acid sequence with amino acid sequence databanks revealed a strong homology with glutathione S-transferases. The nucleotide sequence of the clone displayed a strong homology with the active glutathione binding site of glutathione transferases and contained all but one of the 19 positionally conserved amino acid residues found in glutathione transferases. The cloned polypeptide was expressed in Escherichia coli and affinity-purified on glutathione agarose.     

Ultrastructural organization and regulation of a biomaterial adhesin of Staphylococcus epidermidis

Coagulase-negative staphylococci have emerged as important pathogens in infections associated with intravascular devices. Microbial adherence to biomaterial surfaces is a crucial step in the pathogenesis of these infections. Staphylococcal surface proteins (herein referred to as SSP-1 and SSP-2) are involved in the attachment of Staphylococcus epidermidis 354 to polystyrene. In the present study we show that the adhesin protrudes from the cell surface as a fimbria-like polymer. Furthermore, in vitro proteolytic cleavage of SSP-1 produces an SSP-2-like protein which coincides with a loss of adhesive function. SSP-1 expression is down-regulated in a phenotypical variant of S. epidermidis 354 whereas SSP-2 expression is not. These results could suggest that proteolytic cleavage is a key to the regulation of the adhesive state of S. epidermidis in vivo.