Synthesis and function of Actinomyces naeslundii T14V type 1 fimbriae require the expression of additional fimbria-associated genes

The nucleotide sequence of the chromosomal DNA flanking the Actinomyces naeslundii (formerly A. viscosus) T14V type 1 fimbrial structural subunit gene (fimP) was determined. Six open reading frames (ORFs), in the order 5' ORF3, ORF2, ORF1,fimP, ORF4, ORF5, ORF6 3', were identified. ORF1 encoded a protein of 408 amino acid residues (Mr = 39,270) and had significant sequence homology with the A. naeslundii T14V type 1 and A. naeslundii WVU45 type 2 fimbrial structural subunits. An in-frame fusion of ORF1 to the malE gene of the expression vector, pMAL-c2, yielded a protein that was immunostained with antibodies raised against the maltose binding protein and A. naeslundii T14V whole bacteria. Digestion of the fusion protein with factor Xa released a protein (apparent molecular mass of 34 kDa) that was immunostained only with the antibody directed against A. naeslundii T14V whole bacterial cells. Integration plasmids carrying a kanamycin resistance gene (kan) that was used to substitute for ORF1 or for DNA fragments internal to the coding region of the other five ORFs were used to transform A. naeslundii T14V. Neither type 1 fimbriae nor the 65-kDa fimbrial structural subunit was detected in mutants obtained by allelic replacement of ORF1 or ORF2. Mutants obtained by allelic replacement of ORF3 or ORF4 expressed only the 65-kDa fimbrial structural subunit. These mutants did not bind, in vitro, to proline-rich proteins that serve as the receptors for Actinomyces type 1 fimbriae. In contrast, a mutant in which the integration plasmid DNA had been inserted at a site close to the carboxyl terminus of ORF6 expressed type 1 fimbriae and had adherence properties similar to those observed in the wild-type strain. These results demonstrate the existence of additional genes near fimP that are likely to be involved in the synthesis and function of cell surface fimbriae of A. naeslundii T14V.     

Antigenic relationships among oral Actinomyces isolates, Actinomyces naeslundii genospecies 1 and 2, Actinomyces howellii, Actinomyces denticolens, and Actinomyces slackii

Antigenic relatedness among human strains of oral Actinomyces and similar isolates from cattle has been analyzed by agglutination and immunoblotting. Whole cell agglutination placed A. viscosus serotype II, A. naeslundii serotypes II and III, Actinomyces NV, and strains from numerical taxomonic clusters C1, C2, C3, C4, and C6 into a single group. A. viscosus serotype I cross-reacted weakly with this group. A naeslundii serotype I strains and the cattle isolates Actinomyces denticolens and Actinomyces howellii were distinct. The agglutination results for A. slackii were equivocal. Immunoblots of cell wall extracts developed with non-absorbed sera showed cross-reactivity (23% to 90% antigenic similarity) among all of the strains tested, including A. israelii. The range of antigenic similarities among the group which included strains of A. viscosus serotype II, the A. naeslundii serotypes, and clusters C1, C2, C3, C4, and C6 was from 39% to 89%. Immunoblotting showed that A. howellii and A. denticolens were between 39% and 72% similar to A. naeslundii and A. viscosus. Absorption of antisera with A. israelii cell walls removed antibodies recognizing antigens common to Actinomyces and made the sera more specific. Immunoblotting with absorbed sera supported the grouping and separation of strains shown by agglutination. In some cases, serotypes could be included into a specific taxonomic cluster. A. naeslundii serotype II and Actinomyces NV most closely resembled cluster C1 strains, and A. naeslundii serotype III resembled cluster C1 strains, and A. naeslundii serotype I and A. viscosus serotype I were included into clusters C5 and C7, respectively. The results support a recent proposal that strains of A. viscosus serotype II, A. naeslundii serotypes II and III, and Actinomyces NV be included into A. naeslundii genospecies 2, that A. naeslundii serotype I should be designated A. naeslundii genospecies 1, and that A. viscosus serotype I should be retained distinct from A. naeslundii, as A. viscosus.     

Establishment and distribution of Actinomyces viscosus and Actinomyces naeslundii in the human oral cavity

The intraoral establishment and proportional distribution of suspected periodontal pathogens Actinomyces viscosus and Actinomyces naeslundii were studied using a recently developed differential plating medium, CNAC-20. Saliva and dental plaque samples were collected from 108 subjects ranging in age from infants to young adults; tongue and buccal mucosa samples were collected from only the adult subjects. Catalase-negative A. naeslundii was isolated from 40% of the predentate infants' and almost all other subjects' saliva samples. It predominated among CNAC-20 isolates in the saliva of subjects of all age groups, in the plaques of young children, and in the adult tongue samples. In contrast, catalase-positive A. viscosus was not isolated from predentate infant samples, and its frequency of isolation increased slowly with age (greater than 50% detection by age 7). A. viscosus was isolated in highest relative proportions from dental plaque and buccal mucosa samples. The two closely related species A. viscosus and A. naeslundii apparently differ in respect to factors determining the host age at which they colonize and their relative intraoral distribution in humans.     

Isolation of Actinomyces viscosus from two patients with clinical infections

The isolation of Actinomyces viscosus from two patients is described. One was a case of multiple myeloma, the organism being found on blood culture; the other was a patient with a submandibular abscess. These are believed to be the first such isolations of A. viscosus in this country.     

Cloning and expression of sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from the leech, Macrobdella decora

Sialidase L is a NeuAcalpha2-->3Gal linkage-specific sialidase that releases 2,7-anhydro-NeuAc instead of NeuAc from sialoglycoconjugates (Chou, M.-Y., Li, S.-C., Kiso, M., Hasegawa, A., and Li, Y.-T.(1994) J. Biol. Chem. 269, 18821-18826). A 2. 5-kilobase cDNA of sialidase L was cloned by a combination of methods based on polymerase chain reactions. The composite cDNA sequence reveals an open reading frame coding for 762 amino acids, including a putative 28-residue signal peptide at the N terminus that is similar to the signal sequence of the Clostridium septicum sialidase. The result suggests that sialidase L is a secretory enzyme. The coding sequence excluding the putative signal peptide of sialidase L was overexpressed in Escherichia coli. The purified recombinant enzyme was characterized to be as active as the enzyme isolated from the leech. It also possessed the strict NeuAcalpha2-->3Gal linkage specificity and released the unique cleavage product, 2,7-anhydro-NeuAc from sialoglycoconjugates. The deduced amino acid sequence of sialidase L exhibits little similarity with other reported sialidases. However, sialidase L contains a conserved "FRIP region" and four repeating "Asp box" motifs that align well with the corresponding positions of bacterial sialidases. The predicted beta-strand structures near the conserved motifs of sialidase L are similar to those of Salmonella typhimurium sialidase. Several conserved single amino acid residues of bacterial sialidases, including those known to be involved in the active site of Salmonella enzyme, are conserved in the deduced amino acid sequence of sialidase L. This observation suggests that part of the catalytic mechanism of sialidase L may be similar to the ordinary sialidase.     

Inhibition of a Porphyromonas gingivalis colonizing factor between Actinomyces viscosus ATCC 19246 by monoclonal antibodies against recombinant 40-kDa outer-membrane protein

1. Porphyromonas gingivalis, an important pathogen in human periodontal disease, aggregates with Actinomyces viscosus ATCC 19246. 2. Monoclonal antibodies (mAbs) against purified recombinant 40-kDa outer-membrane protein (r40-kDa, OMP) of P. gingivalis 381 inhibited its coaggregation with A. viscosus ATCC 19246 in a dose-dependent manner. 3. Five mAb clones against r40-kDa OMP were selected. The isotype of the five was IgG1. 4. Pg-ompA2 inhibited the coaggregation of several strains of P. gingivalis with A. viscosus ATCC 19246 cells.     

Cytogenetic analysis of thymocytes during early stages after irradiation in mice with different susceptibilities to radiation-induced lymphomagenesis

The small nanH gene encoding the neuraminidase from Clostridium perfringens ATCC 10543 was cloned in JM109 using pUC19 as a vector. Sequence analysis revealed an ORF encoding 382 amino acids without a signal peptide sequence. Four regions of amino-acid sequence, 71-82, 140-151, 208-219, and 255-266 constituted four repeated and conserved sequence motifs-Ser-X-Asp-X-Gly-X-Thr-Trp-, the "Asp boxes." When compared, the nanH polypeptides of C. perfringens ATCC 10543 and Salmonella typhimurium LT12 shared 33% sequence identity and 60% similarity if conservative replacements were included. The homology-modeled structure of C. perfringens NanH showed the same folding topology as the x-ray three-dimensional structure of NanH in S. typhimurium LT12. Amino acid residues Arg37, Arg56, Asp62, His63, Asp100, Glu230, Asp247, Tyr347, and Glu362 located around the pocket of modeled C. perfringens small nanH were superimposed with the active-site pocket of S. typhimurium LT12, nanH. The catalytic amino-acid residues as well as the role of the "Asp boxes" have not been characterized for C. perfringens and S. typhimurium. In this study, Asp100, Glu230, and Asp62 were found to be involved in the catalytic activity of C. perfringens small nanH with immunoreactive properties and site-directed mutagenesis analysis. Four "Asp-box" motifs were found remote from the active-site pocket. Mutational and immunoreactive analysis of the highly conserved amino acids located in the "Asp boxes" suggest that these highly conserved residues are important in maintaining the tertiary structure of NanH. The results of this study provide some knowledge for the design of new inhibitors of small neuraminidase.     

Rapid sequence evolution of the mammalian sex-determining gene SRY

In mammals, induction of male sex determination requires the Y-chromosome gene SRY. SRY encodes a protein with a central 'high mobility group' domain (HMG box) of about 78 amino acids. HMG boxes are found in a wide variety of proteins that bind to DNA with high affinity but differing degrees of sequence specificity. The human SRY protein binds to linear DNA with sequence specificity and to cruciform DNA structures without sequence specificity. The DNA-binding activity of the SRY protein resides in the HMG box and mutations in this region are associated with sex reversal in XY females. No function has been ascribed to the portions of the SRY protein outside the HMG box. SRY belongs to a family of genes that are related by sequence homology within the DNA-binding domain: the genes most similar to SRY (> 60%) have been named SOX genes (SRY box genes). None of the known SOX genes is homologous to SRY outside the HMG-box region. Although SRY is an important developmental regulator, its sequence is poorly conserved between species apart from the HMG-box domain. Here we investigate the coding sequence of SRY in primates and find that evolution has been rapid in the regions flanking the conserved domain. The high degree of sequence divergence and the frequency of non-synonymous mutations suggest either that the majority of the coding sequence has no functional significance or that directional selection has occurred.     

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a noncoding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3'-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the 'large' sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species.     

Comparison of circumsporozoite proteins from avian and mammalian malarias: biological and phylogenetic implications

The circumsporozoite (CS) protein of malaria parasites (Plasmodium) covers the surface of sporozoites that invade hepatocytes in mammalian hosts and macrophages in avian hosts. CS genes have been characterized from many Plasmodium that infect mammals; two domains of the corresponding proteins, identified initially by their conservation (region I and region II), have been implicated in binding to hepatocytes. The CS gene from the avian parasite Plasmodium gallinaceum was characterized to compare these functional domains to those of mammalian Plasmodium and for the study of Plasmodium evolution. The P. gallinaceum protein has the characteristics of CS proteins, including a secretory signal sequence, central repeat region, regions of charged amino acids, and an anchor sequence. Comparison with CS signal sequences reveals four distinct groupings, with P. gallinaceum most closely related to the human malaria Plasmodium falciparum. The 5-amino acid sequence designated region I, which is identical in all mammalian CS and implicated in hepatocyte invasion, is different in the avian protein. The P. gallinaceum repeat region consists of 9-amino acid repeats with the consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region II-plus, which is associated with targeting the invasion of liver cells, is also conserved in the avian protein. Phylogenetic analysis of the aligned Plasmodium CS sequences yields a tree with a topology similar to the one obtained using sequence data from the small subunit rRNA gene. The phylogeny using the CS gene supports the proposal that the human malaria P. falciparum is significantly more related to avian parasites than to other parasites infecting mammals, although the biology of sporozoite invasion is different between the avian and mammalian species.     

The plastid genome of the cryptophyte alga, Guillardia theta: complete sequence and conserved synteny groups confirm its common ancestry with red algae

Previously, we have purified and characterized DNA helicase III from the yeast Saccharomyces cerevisiae [Shimizu, K. and Sugino, A. (1993) J. Biol. Chem. 268, 9578-9584]. Here, we have further characterized DNA helicase III activity. It was found that the combined action of the helicase III, yeast DNA topoisomerase I (yTop I), and yeast RPA protein on a covalently closed, circular DNA generates a highly underwound DNA species that has been called form I* or form U. Furthermore, these underwound structures can be accessed by yeast DNA polymerase I (alpha)-primase to initiate DNA synthesis. These reactions mimic in vivo initiation of chromosomal DNA replication. In order to clone the gene encoding DNA helicase III, a partial amino acid sequence of the purified DNA helicase III polypeptide was determined. Using a mix oligonucleotides synthesized based on the amino acid sequence of the helicase, we cloned the gene encoding the helicase III and found it to be identical to YER176W (HEL1) on chromosome V. The amino acid sequence predicted from the nucleotide sequence of the gene has conserved DNA helicase domains that are highly homologous to those of DNA helicases required for DNA replication. However, complete deletion of the gene from the chromosome did not result in any growth defect, suggesting that the gene product is not required for DNA synthesis or that it is functionally substituted by other helicase(s). Furthermore, the deletion strain does not exhibit sensitivity to any DNA-damaging reagents, although it is hypersensitive to calcofluor white, hygromycin, and papulacandin.     

Isolation and characterization of human fast skeletal beta troponin T cDNA: comparative sequence analysis of isoforms and insight into the evolution of members of a multigene family

A cDNA encoding human fast skeletal beta troponin T (beta TnTf) has been isolated and characterized from a fetal skeletal muscle library. The cDNA insert is 1,000 bp in length and contains the entire coding region of 777 bp and 5' and 3' untranslated (UT) segments of 12 and 211 bp, respectively. The 3' UT segment shows the predicted stem-loop structure typical of eukaryotic mRNAs. The cDNA-derived amino acid sequence is the first available sequence for human beta TnTf protein. It is encoded by a single-copy gene that is expressed in a tissue-specific manner in fetal and adult fast skeletal muscles. Although the human beta TnTf represents the major fetal isoform, the sequence information indicates that this cDNA and the coded protein are quite distinct from the fetal and neonatal TnTf isoforms reported in other mammalian fetal muscles. The hydropathy plot indicates that human beta TnTf is highly hydrophilic along its entire length. The protein has an extremely high degree of predicted alpha-helical content involving the entire molecule except the carboxy-terminal 30 residues. Comparative sequence analysis reveals that the human beta TnTf shares a high level of sequence similarity in the coding region with other vertebrate TnTf and considerably reduced similarity with slow skeletal and cardiac TnT cDNAs. The TnT isoforms have a large central region consisting of amino acid residues 46-204 which shows a high sequence conservation both at the nucleotide and amino acid levels. This conserved region is flanked by the variable carboxy-terminal and an extremely variable amino-terminal segment. The tropomyosin-binding peptide of TnT, which is represented by amino acid residues 47-151 and also includes a part of troponin I binding region, is an important domain of this central segment. It is suggested that this conserved segment is encoded by an ancestral gene. The variable regions of vertebrate striated TnT isoforms reflect the subsequent addition and modification of genomic sequences to give rise to members of the TnT multigene family.     

Cloning and characterization of a putative human RNA helicase gene of the DEAH-box protein family

A human RNA helicase gene, DBP1, was cloned by PCR methodsusing degenerate oligonucleotide primers corresponding to highly conserved motifs among known members of the DEAH-box protein family. The full-length DBP1 contains 3028 nucleotides and codes for a protein of 813 amino acids with a calculated mol. wt. of 92723 daltons. The predicted amino acid sequence shares extensive homology with Prp2, Prp16, and Prp22 proteins, which are required to splice mRNA precursors in budding yeast. The protein encoded by DBP1 has RGD, RD, and HS(A/T) repeat motifs close to the N-terminus. Southern blot analysis suggested the presence of a homologue of the DBP1 genes in other species, and Northern blot analysis showed that DBP1 is expressed ubiquitously in various human organs investigated. The DBP1 gene was found to be on chromosome 4p15.3 and encodes a putative nuclear ATP-dependent RNA helicase.     

Molecular cloning and characterization of a plasma membrane-associated sialidase specific for gangliosides

Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation, and transmembrane signaling. A mammalian sialidase located in plasma membranes is unique in specifically hydrolyzing gangliosides, suggesting crucial roles in regulation of cell surface functions. Here we describe the cloning and expression of a cDNA for the ganglioside sialidase, isolated from a bovine brain cDNA library based on the amino acid sequence of the purified enzyme from bovine brain. This cDNA encodes a 428-amino acid protein containing a putative transmembrane domain and the three Asp boxes characteristic of sialidases and sharing 19-38% sequence identity with other sialidases. Northern blot and polymerase chain reaction analyses revealed a general distribution of the gene in mammalian species, including man, and the mouse. In COS-7 cells transiently expressing the sialidase, the activity was found to be 40-fold that of the control level with ganglioside substrates in the presence of Triton X-100, and the hydrolysis was almost specific to gangliosides other than GM1 and GM2, both alpha2-->3 and alpha2-->8 sialyl linkages being susceptible. The major subcellular localization of the expressed sialidase was assessed to be plasma membrane by Percoll density gradient centrifugation of cell homogenates and by immunofluorescence staining of the transfected COS-7 cells. Analysis of the membrane topology by protease protection assay suggested that this sialidase has a type I membrane orientation with its amino terminus facing to the extracytoplasmic side and lacking a signal sequence.     

Cell-mediated immune response to products of Actinomyces viscosus cultures

Hartley strain guinea pigs were sensitized with 0.5 ml of concentrated cell-free Actinomyces viscosus culture supernatant fluids mixed with Freund complete adjuvant. Fourteen to 16 days later the animals were challenged by intradermal injection with 0.1 ml of the culture supernatant, and the reactions were observed at 4, 8, 16, 24, and 48 h. Peritoneal exudate cells from sensitized animals were used for determination of migration inhibition factor, and guinea pig peripheral blood served as a source of cells for determining the induction of mitogenesis by antigenic material. Skin responses were consistently positive to challenge with the test material, whereas reactions to noninoculated culture medium were negative. Sensitized cells, challenged with antigen, resulted in 60% or greater inhibition of migration of indicator cells in migration inhibition factor experiments. Tests for mitogenesis showed a greater than fourfold increase in isotope uptake when sensitized cells were challenged with test material. The data are consistent with the suggestion that A. viscosus culture supernatants contain substances that induce cell-mediated immune responses in guinea pigs.     

Nucleotide sequence of the gene for ribosomal protein S17 from Dictyostelium discoideum

The nucleotide sequence of the gene for the Dictyostelium homologue of eukaryotic ribosomal protein S17 has been assembled from cDNA and genomic DNA clones. The predicted primary structure of the S17 protein displays a similar level of sequence identity with its counterparts from higher eukaryotes (53%) as other Dictyostelium ribosomal proteins. Although Dictyostelium genes usually are organized in a rather simple manner, the rps17 gene harbors two introns. One of them, located immediately 3' from the ATG initiator codon, appears to be ubiquitously conserved in eukaryotic rps17 genes.