Effect of Porphyromonas gingivalis ATCC 33277 Vesicle on Adherence of Streptococcus mutans OMZ 70 to the Experimental Pellicle

The vesicles of Porphyromonas gingivalis ATCC 33277 strongly aggregated Streptococcus cricetus, S. rattus, and S. mutans, but poorly aggregated S. sobrinus. The adherence of S. mutans OMZ 70 to hydroxyapatite (HA) coated with whole saliva was increased in parallel with the quantity of the vesicles. The significant increase of adherence of S. mutans OMZ 70 by the vesicles was also observed on the HA coated with parotid saliva, submandibular saliva, serum, and type I collagen. These findings suggest that the vesicles may act as a bridge between mutans streptococcus and the tooth surface.     

Haemin inhibits the trypsin-like enzyme activity of Porphyromonas gingivalis W83

The effect of haemin and related porphyrins on the activity of the trypsin-like enzyme activity in cell sonicates of Porphyromonas gingivalis was examined using a spectrophotometric assay and by following the degradation of human IgG. Haemin was inhibitory in both assay systems and the effect was shown to be reversible. The high concentration of haemin accumulated by P. gingivalis may protect the organism against autodegradative effects of the trypsin-like protease.     

Interaction of a trypsin-like enzyme of Porphyromonas gingivalis W83 with antithrombin III

Abstract We have previously observed that trypsin-like activity in Porphyromonas gingivalis culture supernatants is inhibitable by the plasma arg-serpin antithrombin III (ATIII). This report demonstrates that a partially purified P. gingivalis trypsin-like enzyme ( M _r 47 000) is inhibited by ATIII with an association rate constant ( k _ass) of 5.65 × 10⁴ M⁻¹ s⁻¹ but does not form SDS-stable complexes. Heparin enhances the k _ass and stabilizes the complexes but in either case such inhibition is temporary and results in ATIII inactivation by reactive centre proteolysis between R³⁹³-S³⁹⁴. In the absence of heparin this is accompanied by N-terminal cleavage between K³⁹-I⁴⁰.     

Genome Sequence of the Welan Gum-Producing Strain Sphingomonas sp. ATCC 31555

Sphingomonas sp. strain ATCC 31555 can produce an anionic heteropolysaccharide, welan gum, which shows excellent stability and viscosity retention even at high temperatures. Here we present a 4.0-Mb assembly of its genome sequence. We have annotated 10 coding sequences (CDSs) responsible for the welan gum biosynthesis and 55 CDSs related to monosaccharide metabolism.     

Lysis of erythrocytes by the secreted cysteine proteinase of Porphyromonas gingivalis W83

The cysteine proteinase produced in the culture supernatant of Porphyromonas gingivalis was extensively purified. Haemagglutination type assays in which the enzyme was titrated against a fixed concentration of erythrocytes, showed that low levels of enzyme directly caused lysis of the red blood cells. However, using the same assay, the presence of stoichiometric amounts of the thiol blocking agent, 2,2'-dipyridyl disulphide (2-PDS) specifically inhibited the action of the enzyme or its haemagglutination with W83 cells or vesicles. In all cases, electron micrographs revealed that in the presence of 2-PDS the erythrocytes remained intact. Thiol activator free enzyme or aerated, inactivated enzyme had no effect on the red blood cells. These results show conclusively that the secreted cysteine proteinase of P. gingivalis causes lysis of erythrocytes and must now be regarded as a potent virulence determinant of P. gingivalis.     

Hemoglobinase Activity of the Lysine Gingipain Protease (Kgp) of Porphyromonas gingivalis W83

Porphyromonas gingivalis, an important periodontal disease pathogen, forms black-pigmented colonies on blood agar. Pigmentation is believed to result from accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocytic hemoglobin. The Lys-X (Lys-gingipain) and Arg-X (Arg-gingipain) cysteine proteases of P. gingivalis bind and degrade erythrocytes. We have observed that mutations abolishing activity of the Lys-X-specific cysteine protease, Kgp, resulted in loss of black pigmentation of P. gingivalis W83. Because the hemagglutinating and hemolytic potentials of mutant strains were reduced but not eliminated, we hypothesized that this protease played a role in acquisition of FePPIX from hemoglobin. In contrast to Arg-gingipain, Lys-gingipain was not inhibited by hemin, suggesting that this protease played a role near the cell surface where high concentrations of hemin confer the black pigmentation. Human hemoglobin contains 11 Lys residues in the alpha chain and 10 Lys residues in the beta chain. In contrast, there are only three Arg residues in each of the alpha and beta chains. These observations are consistent with human hemoglobin being a preferred substrate for Lys-gingipain but not Arg-gingipain. The ability of the Lys-gingipain to cleave human hemoglobin at Lys residues was confirmed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of hemoglobin fragments resulting from digestion with the purified protease. We were able to detect several of the predicted hemoglobin fragments rendered by digestion with purified Lys-gingipain. Thus, we postulate that the Lys-gingipain of P. gingivalis is a hemoglobinase which plays a role in heme and iron uptake by effecting the accumulation of FePPIX on the bacterial cell surface.     

Complete Genome Sequence of the Plant Pathogen Erwinia amylovora Strain ATCC 49946

Erwinia amylovora causes the economically important disease fire blight that affects rosaceous plants, especially pear and apple. Here we report the complete genome sequence and annotation of strain ATCC 49946. The analysis of the sequence and its comparison with sequenced genomes of closely related enterobacteria revealed signs of pathoadaptation to rosaceous hosts.Erwinia amylovora, a plant-associated member of the Enterobacteriaceae, causes fire blight, a devastating disease of rosaceous plants, especially pear and apple (6). The complete genome of Ea273 (ATCC 49946), a virulent strain isolated from an infected apple tree in New York State, was sequenced. Total DNA was extracted and prepared in pMAQ1 shotgun libraries. The complete shotgun sequence was obtained by using dye terminator chemistry in ABI 3730 automated sequencers and contains 88,457 reads (11.12-fold coverage), yielding a theoretical coverage of the genome of 99.99%. The sequence was assembled, finished, and annotated as described previously (1, 5), using Artemis (4) to collate data and facilitate annotation.The genome of E. amylovora consists of a circular chromosome of 3,805,874 bp and two plasmids, AMYP1 (28,243 bp) and AMYP2 (71,487 bp). Coding regions in the chromosome account for 85.1% of the total sequence, with 3,483 identified coding sequences (CDS). Two hundred fifty-four (7%) of the CDSs do not have any matches in current NCBI databases; 114 (3.3%) correspond to conserved hypothetical proteins. Forty-nine CDSs (1.4%) are similar to genes from mobile elements such as integrases, transposases, and bacteriophages, and 110 CDSs (3.2%) were classified as pseudogenes due to interruptions or truncations of the CDSs. The remaining 2,956 annotated CDSs include among other categories genes involved in biosynthesis of the cellular envelope and modifications of surface proteins (299 CDSs [11%]) and genes involved in signal transduction and regulation (228 CDSs [8%]). Seven rRNA operons and 78 tRNA sequences were identified in the chromosome; two new clusters were identified (AMY1550-1575 and AMY2648-2676) that resemble the T3SS-encoding SSR-1 island of Sodalis glossinidius (2), and four clusters that contain genes for biosynthesis of flagella, which based on their location might be regulated independently.The smaller plasmid, AMYP1, had been reported as pEA29 (3); its sequence is nearly identical to the one reported here. The larger plasmid, AMYP2, renamed pEA72 for consistency in nomenclature, contains 87 predicted CDSs, with two predicted mobile-element-related CDSs and one pseudogene. Among the CDSs with annotated functions are a cluster of genes (AMYP2_49 to AMYP2_62) that encode a putative type IV fimbrial system (pil genes).The genome of E. amylovora is only 3.8 Mb long, whereas most free-living enterobacteria, including plant pathogens, have genomes of 4.5 Mb to 5.5 Mb. Comparison of the genome of Ea273 with the sequenced genomes of 15 closely related enterobacteria identified 21 lineage-specific regions, which might be considered genomic islands. E. amylovora has many more predicted pseudogenes, relative to other enterobacteria with similar lifestyles. Given its size and the preponderance of pseudogenes, genome reduction may have occurred via mutational inactivation and subsequent deletion with the following consequences: E. amylovora has fewer genes involved in anaerobic respiration and fermentation than are found in typical related enterobacteria; this likely result in a reduced capacity to live in anaerobic environments.The genome sequence of E. amylovora has revealed clear signs of pathoadaptation to the rosaceous plant environment. For example, T3SS-related proteins are present that are more similar to proteins of other plant pathogens than to proteins of closely related enterobacteria. These include type III effectors, homologous to those of plant-pathogenic pseudomonads, which confer virulence to E. amylovora in plants, and a sorbitol-metabolizing cluster that may confer a competitive advantage for survival in rosaceous plants. The reduced genome size and erosion or loss of genes involved in anaerobic respiration and nitrate assimilation are remarkable, relative to other plant- and animal-pathogenic members of the Enterobacteriaceae.     

Draft Genome Sequence of the Quality Control Strain Enterococcus faecalis ATCC 29212

Enterococcus faecalis ATCC 29212, a vancomycin-sensitive strain, has been extensively used as a representative control strain for clinical and laboratory experiments. Here we report the draft genome and annotation of this strain, containing 3,027,060 bp, with a G+C content of 37.2% in 126 contigs (≥500 bp).     

Genome Sequence of the Human-Pathogenic Bacterium Vibrio vulnificus Type Strain ATCC 27562

Vibrio vulnificus, which is the causative agent of cholera, is a Gram-negative, curved, motile, and rod-shaped bacterium. Here, we present the draft genome sequence of the type strain, ATCC 27562, which was the first isolated Vibrio vulnificus strain.     

Complete Genome Sequence of the Copiotrophic Marine Bacterium Alteromonas macleodii Strain ATCC 27126T

The genome of Alteromonas macleodii strain ATCC 27126^T has been resequenced and closed into a single contig. We describe here the genome of this important and globally distributed marine bacterium.     

Protective Role of the PG1036-PG1037-PG1038 Operon in Oxidative Stress in Porphyromonas gingivalis W83

As an anaerobe, Porphyromonas gingivalis is significantly affected by the harsh inflammatory environment of the periodontal pocket during initial colonization and active periodontal disease. We reported previously that the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by an undescribed mechanism in P. gingivalis. DNA affinity fractionation identified PG1037, a conserved hypothetical protein, among other proteins, that were bound to the 8-oxoG lesion. PG1037 is part of the uvrA-PG1037-pcrA operon in P. gingivalis which is known to be upregulated under H₂O₂ induced stress. A PCR-based linear transformation method was used to inactivate the uvrA and pcrA genes by allelic exchange mutagenesis. Several attempts to inactivate PG1037 were unsuccessful. Similar to the wild-type when plated on Brucella blood agar, the uvrA and pcrA-defective mutants were black-pigmented and beta-hemolytic. These isogenic mutants also had reduced gingipain activities and were more sensitive to H₂O₂ and UV irradiation compared to the parent strain. Additionally, glycosylase assays revealed that 8-oxoG repair activities were similar in both wild-type and mutant P. gingivalis strains. Several proteins, some of which are known to have oxidoreducatse activity, were shown to interact with PG1037. The purified recombinant PG1037 protein could protect DNA from H₂O₂-induced damage. Collectively, these findings suggest that the uvrA-PG1037-pcrA operon may play an important role in hydrogen peroxide stress-induced resistance in P. gingivalis.     

Complete Genome Sequence of the Pyrene-Degrading Bacterium Cycloclasticus sp. Strain P1

Cycloclasticus sp. strain P1 was isolated from deep-sea sediments of the Pacific Ocean and characterized as a unique bacterium in the degradation of pyrene, a four-ring polycyclic aromatic hydrocarbon (PAH). Here we report the complete genome of P1 and genes associated with PAH degradation.     

Generation and characterization of a monoclonal antibody specific for the major thiol-activated cysteine proteinase of Porphyromonas gingivalis W83

An IgM monoclonal antibody specified for the thiol-activated proteinase of the oral pathogen Porphyromonas gingivalis W83 was generated. The antibody reacted with a single protein of approximate molecular mass 43 kDa in outer membrane preparations of P. gingivalis. Immuno-electron microscopy using the monoclonal antibody and immunogold labelling confirmed the cell surface location of the thiol-activated proteinase. The monoclonal antibody failed to detect any proteins in Western blot analysis of other closely related oral bacteria. The specificity of this monoclonal antibody to the thiol-activated proteinase of P. gingivalis should allow its use as a diagnostic tool for the rapid enumeration of P. gingivalis in clinical samples.     

Draft Genome Sequence of the Flocculating Zymomonas mobilis Strain ZM401 (ATCC 31822)

Zymomonas mobilis ZM401 is a flocculating strain which can be self-immobilized within fermentors for a high-cell-density culture to improve ethanol productivity, as well as high-gravity fermentation to increase ethanol titer, due to its improved ethanol tolerance associated with the morphological change. Here, we report its draft genome sequence.     

Genome Sequence of the Solvent-Producing Bacterium Clostridium carboxidivorans Strain P7T

Clostridium carboxidivorans strain P7^T is a strictly anaerobic acetogenic bacterium that produces acetate, ethanol, butanol, and butyrate. The C. carboxidivorans genome contains all the genes for the carbonyl branch of the Wood-Ljungdahl pathway for CO₂ fixation, and it encodes enzymes for conversion of acetyl coenzyme A into butanol and butyrate.Clostridium carboxidivorans strain P7^T (equivalent to ATCC BAA-624^T and DSM 15243^T) is an obligate anaerobe that can grow autotrophically with H₂ and CO₂ or CO (fixing carbon via the Wood-Ljungdahl pathway), or it can grow chemoorganotrophically with simple sugars (1). Acetate, ethanol, butanol, and butyrate are end products of metabolism.For slow-growing strict anaerobes such as Clostridium carboxidivorans, genome sequencing provides a rapid theoretical characterization of its metabolism compared to traditional methods. We isolated and amplified genomic C. carboxidivorans DNA using the Wizard genomic DNA purification kit (Promega, Madison, WI) and the REPLI-g kit (Qiagen). A single shotgun pyrosequencing run using a Genome Sequencer FLX system (454 Life Sciences, Branford, CT) resulted in 429,680 high-quality reads (mean read length, 231.6 bp) that were assembled using Newbler software (454 Life Sciences) into 225 contigs >500 bp long. Paired-end sequencing produced 111,154 reads (mean read length, 256.3 bp). Assembly of the paired-end and shotgun reads produced 73 scaffolds containing 216 large contigs with a mean sequence depth of 16.33 reads. PCR amplification and Sanger sequencing were conducted, followed by scaffold assembly using Sequencher (Gene Codes, Ann Arbor, MI). The 4.4-Mb final assembly has 33 scaffolds containing 69 contigs with a Phred-equivalent quality score of 40 or above (accuracy, >99.99%) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"ADEK00000000","term_id":"295883218","term_text":"ADEK00000000"}}ADEK00000000).The sequence was annotated using Annotation Engine (J. Craig Venter Institute) and manually curated using Manatee (http://manatee.sourceforge.net/). The genome has 29.7% G+C content and contains 4,174 protein-coding sequences, 3 rRNA operons, 1 tmRNA (dual tRNA-like and mRNA-like nature), 6 noncoding RNAs (ncRNAs), and 48 tRNA genes. (6). Comparison of 16S rRNA genes showed that C. carboxidivorans is closely related to Clostridium scatologenes ATCC 25775^T (97% sequence identity) and Clostridium drakei type strain SL1^T (99% sequence identity). C. carboxidivorans shares 94% 16S rRNA sequence identity with Clostridium ljungdahlii (4.6 Mb), another solventogenic species.Pathway analyses indicated that C. carboxidivorans is similar to other anaerobic acetogens, such as Moorella thermoacetica (8), in having an incomplete reductive tricarboxylic acid (TCA) cycle where fumarate reductase is absent. Like other acetogenic clostridia, C. carboxidivorans uses the Wood-Ljungdahl pathway for fixing carbon dioxide to organic carbon via acetyl coenzyme A (acetyl-CoA) (5). Two of these genes encode carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS), which form a complex to catalyze the carbonyl branch of the pathway for carbon fixation and acetyl-CoA production. C. carboxidivorans has genes that encode phosphotransacetylase and acetate kinase for converting acetyl-CoA into acetate, yielding ATP (2).C. carboxidivorans is unique among other known acetogenic clostridia because it can fix carbon via the Wood-Ljungdahl pathway and convert acetyl-CoA into butanol, which is more energy dense than ethanol. Both C. carboxidivorans and Clostridium acetobutylicum encode NADPH-dependent butanol dehydrogenase (74% identity) to convert acetyl-CoA into butanol (3, 4), but C. acetobutylicum cannot fix CO₂ or CO into acetyl-CoA. Conversely, C. ljungdahlii can fix CO and CO₂, but it lacks butanol dehydrogenase and cannot convert acetyl-CoA into butanol. Therefore, P7 includes beneficial properties of both these industrially important strains. The genome sequence of C. carboxidivorans P7 could potentially accelerate research allowing its industrial application for biofuel production or to enable some of its pathways to be used directly in synthetic biology for biofuel production.     

Structural analysis of the polysaccharide from the lipopolysaccharide of Porphyromonas gingivalis strain W50.

The lipopolysaccharide (LPS) of Porphyromonas gingivalis is an important pro-inflammatory molecule in periodontal disease and a significant target of the host's specific immune response. In addition, we recently demonstrated using monoclonal antibodies that the Arg-gingipains of P. gingivalis are post-translationally modified with glycan chains that are immunologically related to an LPS preparation from this organism. In the present investigation, we determined the structure of the O-polysaccharide of P. gingivalis W50 that was fully characterized on the basis of 1D and 2D NMR (DQF-COSY, TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-31P HXTOCSY) and GC-MS data. These data allowed us to conclude that the O-polysaccharide is built up of the tetrasaccharide repeating sequence: -->6)-alpha-D-Glcp-(1-->4)-alpha-L-Rhap-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Galp-(1--> and carries a monophosphoethanolamine residue at position C-2 of the alpha-rhamnose residue in a nonstoichiometric (approximately 60%) amount. These data indicate that the O-polysaccharide of P. gingivalis LPS is composed of an unusually modified tetrasaccharide repeating unit.