| 1. |
Asanuma N,
Hino T,
( 2003 ) Molecular characterization of HPr and related enzymes, and regulation of HPr phosphorylation in the ruminal bacterium Streptococcus bovis. PMID : 12610726 : DOI : 10.1007/s00203-003-0516-9 Abstract >>
Molecular properties of HPr, enzyme I, and HPr kinase in Streptococcus bovis, and the regulation of HPr phosphorylation were examined. The genes encoding HPr (ptsH) and enzyme I (ptsI) were found to be cotranscribed. Two transcriptional start sites were detected in a region upstream of the HPr kinase gene (hprK). HPr kinase had both HPr-phosphorylating and HPr-dephosphorylating activities. The importance of phosphorylation of Ser-46 in HPr was shown by using a mutant HPr in which Ser-46 was replaced by Ala. When S. bovis was grown in glucose-limited medium, the amount of seryl-phosphorylated HPr (HPr-[Ser-P]) decreased drastically as the growth rate decreased. In contrast, the amount of histidyl-phosphorylated HPr (HPr-[His-P]) increased gradually as the growth rate decreased. The amount of HPr kinase did not greatly change with the growth phase, whereas the intracellular P(i) concentration increased as the growth rate decreased. HPr-[Ser-P] decreased as the intracellular P(i) increased as a consequence of inhibition of HPr kinase activity by P(i) and simultaneous enhancement of HPr-[Ser-P] phosphatase activity by P(i). Thus, it is conceivable that the ratio of HPr-[Ser-P] to HPr-[His-P] is regulated by the bifunctional activity of HPr kinase in response to intracellular P(i) concentration.
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2. |
Teng LJ,
Hsueh PR,
Tsai JC,
Chen PW,
Hsu JC,
Lai HC,
Lee CN,
Ho SW,
( 2002 ) groESL sequence determination, phylogenetic analysis, and species differentiation for viridans group streptococci. PMID : 12202549 : DOI : 10.1128/jcm.40.9.3172-3178.2002 PMC : PMC130726 Abstract >>
The full-length sequences of the groESL genes (also known as cpn10/60) of Streptococcus anginosus, Streptococcus constellatus, Streptococcus gordonii, and Streptococcus sanguis and the near full-length sequence of the groESL genes of Streptococcus intermedius, Streptococcus bovis, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, and Streptococcus salivarius were determined. The lengths of the groES genes from the 10 species listed above ranged from 282 to 288 bp, and the full-length sequences of groEL determined for 4 species (S. anginosus, S. constellatus, S. gordonii, and S. sanguis) revealed that each was 1,623 bp. The intergenic region (spacer) between the groES and groEL genes varies in size (15 to 111 bp) and sequence between species. The variation of the groES sequences among the species tested was greater (62.1 to 95.1% nucleotide sequence identities) than that of the groEL sequences (77.2 to 95.2% nucleotide sequence identities). Phylogenetic analysis of the groES and groEL genes yielded evolutionary trees similar to the tree constructed by use of the 16S rRNA gene. The intraspecies variation of the spacer was minimal for clinical isolates of some species. The groESL sequence data provide an additional parameter for identification of viridans group streptococcal species.
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3. |
Poyart C,
Quesne G,
Trieu-Cuot P,
( 2002 ) Taxonomic dissection of the Streptococcus bovis group by analysis of manganese-dependent superoxide dismutase gene (sodA) sequences: reclassification of 'Streptococcus infantarius subsp. coli' as Streptococcus lutetiensis sp. nov. and of Streptococcus bovis biotype 11.2 as Streptococcus pasteurianus sp. nov. PMID : 12148636 : DOI : 10.1099/00207713-52-4-1247 DOI : 10.1099/00207713-52-4-1247 Abstract >>
The taxonomic dissection of the Streptococcus bovis-Streptococcus equinus group was carried out upon obtaining sequences for the manganese-dependent superoxide dismutase gene (sodA) of the type strains of S. bovis, Streptococcus caprinus, S. equinus, Streptococcus gallolyticus, Streptococcus infantarius, Streptococcus macedonicus and Streptococcus waius. The sodA sequences of 29 streptococcal strains of animal and human origin that were related to S. bovis were also sequenced. A phylogenetic analysis of the sodA sequences revealed that the S. bovis-S. equinus group comprises five different clusters that correspond to five distinct species. The type strains of S. bovis and S. equinus were associated in the same cluster, corresponding to the species S. equinus. The type strains of S. caprinus, S. gallolyticus, S. macedonicus and S. waius were associated in the same cluster, which defined a single species containing S. gallolyticus and its junior synonym S. caprinus, and S. macedonicus and its junior synonym S. waius. The two subspecies thought to constitute the species S. infantarius, namely S. infantarius subsp. infantarius and 'S. infantarius subsp. coli', were located in two distinct clusters. One of these clusters defined the species S. infantarius and included the type strain of S. infantarius subsp. infantarius. The other cluster defined 'S. infantarius subsp. coli', leading to the proposal of its reclassification as the novel species Streptococcus lutetiensis (NEM 782T = CIP 106849T). The remaining cluster comprised all of the strains previously identified as belonging to S. bovis biotype 11.2, leading to the proposal to reassign these strains to the novel species Streptococcus pasteurianus (NEM 1202T = CIP 107122T). The results of the phylogenetic analysis were confirmed by DNA-DNA hybridization experiments, thus demonstrating that sequence databases of defined DNA targets, such as sodA, may constitute a valuable alternative approach for modern bacterial systematics.
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4. |
Asanuma N,
Hino T,
( 2002 ) Molecular characterization and expression of pyruvate formate-lyase-activating enzyme in a ruminal bacterium, Streptococcus bovis. PMID : 12089014 : DOI : 10.1128/aem.68.7.3352-3357.2002 PMC : PMC126763 Abstract >>
To clarify the significance of the activation of pyruvate formate-lyase (PFL) by PFL-activating enzyme (PFL-AE) in Streptococcus bovis, the molecular properties and gene expression of PFL-AE were investigated. S. bovis PFL-AE was deduced to consist of 261 amino acids with a molecular mass of 29.9 kDa and appeared to be a monomer protein. Similar to Escherichia coli PFL-AE, S. bovis PFL-AE required Fe(2+) for activity. The gene encoding PFL-AE (act) was found to be polycistronic, and the PFL gene (pfl) was not included. However, the act mRNA level changed in parallel with the pfl mRNA level, responding to growth conditions, and the change was contrary to the change in the lactate dehydrogenase (LDH) mRNA level. PFL-AE synthesis appeared to change in parallel with PFL synthesis. Introduction of a recombinant plasmid containing S. bovis pfl and the pfl promoter into S. bovis did not affect formate and lactate production, which suggests that the activity of the pfl promoter is low. When the pfl promoter was replaced by the S. bovis ldh promoter, PFL was overexpressed, which caused an increase in the formate-to-lactate ratio. However, when PFL-AE was overexpressed, the formate-to-lactate ratio did not change, suggesting that PFL-AE was present at a level that was high enough to activate PFL. When both PFL-AE and PFL were overexpressed, the formate-to-lactate ratio further increased. It is conceivable that LDH activity is much higher than PFL activity, which may explain why the formate-to-lactate ratio is usually low.
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5. |
Nakamura M,
Ogata K,
Nagamine T,
Tajima K,
Matsui H,
Benno Y,
( 2001 ) The replicon of the cryptic Plasmid pSBO1 isolated from Streptococcus bovis JB1. PMID : 11375657 : DOI : 10.1007/s002840010252 Abstract >>
The cryptic plasmid pSBO1 (3904 bp) was isolated from Streptococcus bovis JB1. pSBO1 contained an open reading frame (ORF) that is homologous to sequences encoding the replication protein (Rep) in pEFC1 (isolated from Enterococcus faecalis), pSK639 (Staphylococcus epidermidis), pLA103 (Lactobacillus acidophilus), and pUCL287 (Tetragenococcus halophila). In addition, four 22-bp direct repeats (DRs) were located upstream of the putative replication gene (rep) of pSBO1. Recombinant plasmids (pSBE10 and pSBE11) containing the DRs and putative rep of pSBO1 replicated in S. bovis 12-U-1 and no8 strains. This result indicates that the putative rep encoded Rep and that the replicon of pSBO1 contained the DRs and the rep. Gel shift assays showed that the Rep of pSBO1 bound the 22-bp DRs.
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6. |
Iwamoto M,
Hino T,
( 1999 ) Structure and transcriptional regulation of the gene encoding pyruvate formate-lyase of a ruminal bacterium, Streptococcus bovis. PMID : 10206694 : DOI : 10.1099/13500872-145-1-151 Abstract >>
The gene (pfl) encoding pyruvate formate-lyase (Pfl) from Streptococcus bovis was sequenced. The deduced amino acid sequence of Pfl was similar to Streptococcus mutans Pfl, and included the conserved regions necessary for free-radical formation and a catalytic site. The Pfl of S. bovis appeared to be a free-radical-containing enzyme because of its dioxygen sensitivity and its amino acid sequence similarity with the Escherichia coli enzyme. The pfl mRNA of S. bovis was approximately 2.3 kb and was transcribed in a monocistronic fashion. When cells were grown in batch culture at pH 6.9, the level of pfl transcript increased as the growth phase changed from exponential growth to stationary phase. This result was in constrast to the previous observation that the level of lactate dehydrogenase (Ldh) mRNA decreased during the later stages of growth. Continuous culture experiments conducted at pH 6.9 under glucose-limited and ammonia-limited conditions revealed that pfl mRNA was decreased by an excess supply of glucose, as well as by a high growth rate. On the contrary, ldh mRNA increased when excess glucose was supplied and the growth rate was high. The amount of pfl mRNA in cells was lower at pH 4.5 than pH 6.9, whereas the level of ldh mRNA was higher at pH 4.5. This result was consistent with the amounts of Pfl and Ldh in cells and the proportion of formate and lactate produced. These results support the hypothesis that S. bovis regulates Pfl and Ldh synthesis at the transcriptional level in response to growth conditions.
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7. |
Nigutova K,
Morovsky M,
Pristas P,
Teather RM,
Holo H,
Javorsky P,
( 2007 ) Production of enterolysin A by rumen Enterococcus faecalis strain and occurrence of enlA homologues among ruminal Gram-positive cocci. PMID : 17241363 : DOI : 10.1111/j.1365-2672.2006.03068.x Abstract >>
Purification and partial characterization of an extracellular bacteriocin produced by the ruminal isolate Enterococcus faecalis II/1 and determine the frequency of occurrence of enterolysin A structural gene within the ruminal cocci. Bacteriocin produced by E. faecalis II/1 was purified to homogeneity. Purified bacteriocin exhibited a single band on sodium dodecylsulphate polyacrylamide gel electrophoresis with an apparent molecular weight of about 35 kDa. The amino acid sequence of the first 30 amino acids of purified bacteriocin was identical with the enterolysin A sequence. The DNA sequence of the nearly complete E. faecalis II/1 bacteriocin structural gene was identical to the enterolysin A gene sequence, confirming that this bacteriocin is identical to enterolysin A, a cell wall-degrading bacteriocin from E. faecalis LMG 2333. Enterolysin A structural genes were detected in approximately one-sixth of the Gram-positive ruminal cocci examined by PCR using primers targeting the enterolysin A structural gene. Bacteriocin produced by E. faecalis II/1 is identical to enterolysin A. Enterolysin A structural gene homologues are frequently encountered in rumen enterococcal and streptococcal bacterial strains. This is the first evidence of a large heat-labile bacteriocin produced by rumen E. faecalis strain, enlarging the number and types of known anti-bacterial proteins produced by rumen bacteria.
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8. |
Asanuma N,
Hino T,
( 2006 ) Presence of NAD+-specific glyceraldehyde-3-phosphate dehydrogenase and CcpA-dependent transcription of its gene in the ruminal bacterium Streptococcus bovis. PMID : 16553827 : DOI : 10.1111/j.1574-6968.2006.00111.x Abstract >>
To know the role of NADP+-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN) in Streptococcus bovis, the molecular properties and transcriptional control of the gene encoding GAPN (gapN) were examined. The GAPN in S. bovis was deduced to consist of 476 amino acids with a molecular mass of 51.1 kDa. The gapN gene was transcribed in a monocistronic fashion. GAPN synthesis appeared to be regulated at the transcriptional level in response to changes in growth conditions. In a mutant that lacks the ccpA gene encoding catabolite control protein A (CcpA), the gapN-mRNA level was lower than in the parent strain. A binding site of CcpA was found in the upper region of gapN. These results suggest that transcription of gapN is regulated through CcpA. Overexpression of GAPN in S. bovis did not affect the growth rate or formate-to-lactate ratio, suggesting that the flux in the glycolytic pathway is unlikely to be altered by GAPN activity. Streptococcus bovis GAPN was NADP+ dependent, but not phosphate dependent. In addition, S. bovis did not have other NADPH-producing systems such as the hexose monophosphate pathway and NADPH:NAD+ oxidoreductase. Therefore, GAPN may play an important role in NADPH production in S. bovis.
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9. |
Itoh Y,
Kawamura Y,
Kasai H,
Shah MM,
Nhung PH,
Yamada M,
Sun X,
Koyana T,
Hayashi M,
Ohkusu K,
Ezaki T,
( 2006 ) dnaJ and gyrB gene sequence relationship among species and strains of genus Streptococcus. PMID : 16487673 : DOI : 10.1016/j.syapm.2005.12.003 Abstract >>
The dnaJ and gyrB nucleotide sequences were determined for members of the genus Streptococcus. The average similarity between the species tested was 76.4% (69.7-100%) for dnaJ and 75.9 (70.1-98.7%) for gyrB. These data indicated that the dnaJ and gyrB genes are more divergent and more discriminatory than the 16S rDNA gene. Furthermore, the variation in the dnaJ nucleotide sequences among the mitis group was greater than that of the gyrB nucleotide sequences, especially between Streptococcus pneumoniae and Streptococcus mitis. Subsequently, the high discrimination power of dnaJ within the mitis group was confirmed. Thus, we conclude that the dnaJ and gyrB genes are efficient alternative targets for the classification of the genus Streptococcus, and that dnaJ is suitable for phylogenetic analysis of closely related Streptococcus strains.
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10. |
Asanuma N,
Yoshii T,
Hino T,
( 2004 ) Molecular characterization of CcpA and involvement of this protein in transcriptional regulation of lactate dehydrogenase and pyruvate formate-lyase in the ruminal bacterium Streptococcus bovis. PMID : 15345406 : DOI : 10.1128/AEM.70.9.5244-5251.2004 PMC : PMC520867 Abstract >>
A ccpA gene that encodes global catabolite control protein A (CcpA) in Streptococcus bovis was identified and characterized, and the involvement of CcpA in transcriptional control of a gene (ldh) encoding lactate dehydrogenase (LDH) and a gene (pfl) encoding pyruvate formate-lyase (PFL) was examined. The ccpA gene was shown to be transcribed as a monocistronic operon. A catabolite-responsive element (cre) was found in the promoter region of ccpA, suggesting that ccpA transcription in S. bovis is autogenously regulated. CcpA required HPr that was phosphorylated at the serine residue at position 46 (HPr-[Ser-P]) for binding to the cre site, but glucose 6-phosphate, fructose 1,6-bisphosphate, and NADP had no effect on binding. Diauxic growth was observed when S. bovis was grown in a medium containing glucose and lactose, but it disappeared when ccpA was disrupted, which indicates that CcpA is involved in catabolite repression in S. bovis. The level of ccpA mRNA was higher when cells were grown on glucose than when they were grown on lactose, which was in line with the level of ldh mRNA. When cells were grown on glucose, the ldh mRNA level was lower but the pfl mRNA level was higher in a ccpA-disrupted mutant than in the parent strain, which suggests that ldh transcription is enhanced and pfl transcription is suppressed by CcpA. The ccpA-disrupted mutant produced less lactate and more formate than the parent, probably because the mutant had reduced LDH activity and elevated PFL activity. In the upper region of both ldh and pfl, a cre-like sequence was found, suggesting that the complex consisting of CcpA and HPr-[Ser-P] binds to the possible cre sites. Thus, CcpA appears to be involved in the global regulation of sugar utilization in S. bovis.
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11. |
Asanuma N,
Yoshii T,
Hino T,
( 2004 ) Molecular characteristics of phosphoenolpyruvate: mannose phosphotransferase system in Streptococcus bovis. PMID : 15297922 : DOI : 10.1007/s00284-003-4232-0 Abstract >>
To elucidate the regulatory mechanism of catabolite control in Streptococcus bovis, we investigated the molecular properties and gene expression of the mannose-specific phosphoenolpyruvate (PEP)-dependent sugar: phosphotransferase system (PTS). The mannose PTS gene cluster (man) was found to comprise a gene encoding enzyme (E) II AB (manL) and genes encoding EIIC (manM), EIID (manN), and a putative regulator (manO). The gene cluster (man operon) was transcribed from one transcriptional start site, which was located 40 bp upstream of the manL start codon. However, two transcriptional start sites were found between manN and manO in primer extension analysis, and the manO may be transcribed independently from the man operon. The man operon and manO were constitutively transcribed without being affected by culture conditions, such as the sugar supplied (glucose, galactose, fructose, maltose, lactose, sucrose, or mannose), growth rate, or pH.
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12. |
Hill JE,
Penny SL,
Crowell KG,
Goh SH,
Hemmingsen SM,
( 2004 ) cpnDB: a chaperonin sequence database. PMID : 15289485 : DOI : 10.1101/gr.2649204 PMC : PMC509277 Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
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13. |
Drancourt M,
Roux V,
Fournier PE,
Raoult D,
( 2004 ) rpoB gene sequence-based identification of aerobic Gram-positive cocci of the genera Streptococcus, Enterococcus, Gemella, Abiotrophia, and Granulicatella. PMID : 14766807 : DOI : 10.1128/jcm.42.2.497-504.2004 PMC : PMC344509 Abstract >>
We developed a new molecular tool based on rpoB gene (encoding the beta subunit of RNA polymerase) sequencing to identify streptococci. We first sequenced the complete rpoB gene for Streptococcus anginosus, S. equinus, and Abiotrophia defectiva. Sequences were aligned with these of S. pyogenes, S. agalactiae, and S. pneumoniae available in GenBank. Using an in-house analysis program (SVARAP), we identified a 740-bp variable region surrounded by conserved, 20-bp zones and, by using these conserved zones as PCR primer targets, we amplified and sequenced this variable region in an additional 30 Streptococcus, Enterococcus, Gemella, Granulicatella, and Abiotrophia species. This region exhibited 71.2 to 99.3% interspecies homology. We therefore applied our identification system by PCR amplification and sequencing to a collection of 102 streptococci and 60 bacterial isolates belonging to other genera. Amplicons were obtained in streptococci and Bacillus cereus, and sequencing allowed us to make a correct identification of streptococci. Molecular signatures were determined for the discrimination of closely related species within the S. pneumoniae-S. oralis-S. mitis group and the S. agalactiae-S. difficile group. These signatures allowed us to design a S. pneumoniae-specific PCR and sequencing primer pair.
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14. |
Asanuma N,
Yoshii T,
Hino T,
( 2004 ) Molecular characteristics and transcription of the gene encoding a multifunctional alcohol dehydrogenase in relation to the deactivation of pyruvate formate-lyase in the ruminal bacterium Streptococcus bovis. PMID : 14676990 : DOI : 10.1007/s00203-003-0638-0 Abstract >>
To clarify the deactivation mechanism of pyruvate formate-lyase (PFL) and its role in the regulation of fermentation in Streptococcus bovis, the molecular properties and genetic expression of multifunctional alcohol dehydrogenase (ADHE) were investigated. S. bovis was found to have ADHE, which was deduced to consist of 872 amino acids with a molecular mass of 97.4 kDa. The ADHE was shown to harbor three enzyme activities: (1) alcohol dehydrogenase, (2) coenzyme-A-linked acetaldehyde dehydrogenase that catalyzes the conversion of acetyl-CoA to ethanol, and (3) PFL deactivase. Similar to Escherichia coli ADHE, S. bovis ADHE required Fe2+ for its activity. The gene encoding ADHE (adhE) was shown to be monocistronic. The level of adhE mRNA changed in parallel with the mRNA levels of the genes encoding PFL (pfl) and PFL-activating enzyme (act) as the growth conditions changed, although these genes are independently transcribed. Synthesis of ADHE, PFL-activating enzyme, and PFL appears to be regulated concomitantly. Overexpression of ADHE did not cause a change in the formate-to-lactate ratio. It is conceivable that ADHE is not significantly involved in the reversible inactivation of active PFL under anoxic conditions. Partition of the flow from pyruvate appears to be mainly regulated by the activities of lactate dehydrogenase and PFL.
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15. |
Xiao H,
Chen X,
Chen M,
Tang S,
Zhao X,
Huan L,
( 2004 ) Bovicin HJ50, a novel lantibiotic produced by Streptococcus bovis HJ50. PMID : 14702402 : DOI : 10.1099/mic.0.26437-0 Abstract >>
A bacteriocin-producing strain was isolated from raw milk and named Streptococcus bovis HJ50. Like most bacteriocins produced by lactic acid bacteria, bovicin HJ50 showed a narrow range of inhibiting activity. It was sensitive to trypsin, subtilisin and proteinase K. Bovicin HJ50 was extracted by n-propanol and purified by SP Sepharose Fast Flow, followed by Phenyl Superose and Sephadex G-50. Treatment of Micrococcus flavus NCIB8166 with bovicin HJ50 revealed potassium efflux from inside the cell in a concentration-dependent manner. The molecular mass of bovicin HJ50 was determined to be 3428.3 Da. MS analysis of DTT-treated bovicin HJ50 suggested that bovicin HJ50 contains a disulfide bridge. The structural gene of bovicin HJ50 was cloned by nested PCR based on its N-terminal amino acid sequence. Sequence analysis showed that it encodes a 58 aa prepeptide consisting of an N-terminal leader sequence of 25 aa and a C-terminal propeptide domain of 33 aa. Bovicin HJ50 shows similarity to type AII lantibiotics. Chemical modification using an ethanethiol-containing reaction mixture showed that two Thr residues are modified.
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16. |
Hinse D,
Vollmer T,
Erhard M,
Welker M,
Moore ER,
Kleesiek K,
Dreier J,
( 2011 ) Differentiation of species of the Streptococcus bovis/equinus-complex by MALDI-TOF Mass Spectrometry in comparison to sodA sequence analyses. PMID : 21247715 : DOI : 10.1016/j.syapm.2010.11.010 Abstract >>
The Streptococcus bovis/equinus complex is a heterogeneous group within the group D streptococci with important clinical relevance regarding infective endocarditis, sepsis and colon carcinoma. The taxonomic identification of species and sub-species of this complex, by the standard methods remains difficult. In the present study, we compared the cluster analysis of 88 strains of species of the S. bovis/equinus complex by sequence analysis of the manganese-dependent superoxide dismutase gene (sodA) and by Matrix Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). We observed a high congruence of strain grouping by MALDI-TOF MS in comparison with sodA sequence analyses, demonstrating the accuracy and reliability of MALDI-TOF MS in comparison to DNA sequence-based method. By generating mass spectra for each species and sub-species, we were able to discriminate all members of the S. bovis/equinus complex. Furthermore, we demonstrated reliable identifications to the species level by MALDI-TOF MS, independently of cultivation conditions.
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17. |
Asanuma N,
Kanada K,
Arai Y,
Yoshizawa K,
Ichikawa T,
Hino T,
( 2010 ) Molecular characterization and significance of phosphoenolpyruvate carboxykinase in a ruminal bacterium, Streptococcus bovis. PMID : 20513959 : Abstract >>
To gain knowledge about the significance of phosphoenolpyruvate (PEP) carboxykinase (PCK) in Streptococcus bovis, the sequence of the gene encoding PCK (pck) was determined. Transcriptional analysis indicated that the pck is transcribed in a monocistronic fashion. The level of pck-mRNA was higher when cells were grown on lactose than on glucose, suggesting that PCK synthesis increases when the growth rate is low. The pck-mRNA level was higher in a mutant lacking ccpA, which encodes the catabolite control protein A (CcpA), than in the parent strain, suggesting that pck transcription is suppressed by CcpA. S. bovis PCK showed oxaloacetate (OAA)-decarboxylating activity, but no PEP-carboxylating activity (reverse reaction). In S. bovis, OAA was speculated to be produced from PEP via pyruvate. Disruption of pck in S. bovis resulted in decreased growth rate and cell yield. When a pck-disrupted mutant was grown in a medium lacking amino acids, the lag phase was longer and the cell yield was lower than the case of the parent strain. These results suggest that pck is involved in the initiation of growth, including the induction of amino acid synthesis and energy metabolism.
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18. |
Glazunova OO,
Raoult D,
Roux V,
( 2010 ) Partial recN gene sequencing: a new tool for identification and phylogeny within the genus Streptococcus. PMID : 19880633 : DOI : 10.1099/ijs.0.018176-0 Abstract >>
Partial sequences of the recN gene (1249 bp), which encodes a recombination and repair protein, were analysed to determine the phylogenetic relationship and identification of streptococci. The partial sequences presented interspecies nucleotide similarity of 56.4-98.2 % and intersubspecies similarity of 89.8-98 %. The mean DNA sequence similarity of recN gene sequences (66.6 %) was found to be lower than those of the 16S rRNA gene (94.1 %), rpoB (84.6 %), sodA (74.8 %), groEL (78.1 %) and gyrB (73.2 %). Phylogenetically derived trees revealed six statistically supported groups: Streptococcus salivarius, S. equinus, S. hyovaginalis/S. pluranimalium/S. thoraltensis, S. pyogenes, S. mutans and S. suis. The 'mitis' group was not supported by a significant bootstrap value, but three statistically supported subgroups were noted: Streptococcus sanguinis/S. cristatus/S. sinensis, S. anginosus/S. intermedius/S. constellatus (the 'anginosus' subgroup) and S. mitis/S. infantis/S. peroris/S. oralis/S. oligofermentans/S. pneumoniae/S. pseudopneumoniae. The partial recN gene sequence comparison highlighted a high percentage of divergence between Streptococcus dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis. This observation is confirmed by other gene sequence comparisons (groEL, gyrB, rpoB and sodA). A high percentage of similarity was found between S. intermedius and S. constellatus after sequence comparison of the recN gene. To study the genetic diversity among the 'anginosus' subgroup, recN, groEL, sodA, gyrB and rpoB sequences were determined for 36 clinical isolates. The results that were obtained confirmed the high genetic diversity within this group of streptococci.
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19. |
Glazunova OO,
Raoult D,
Roux V,
( 2009 ) Partial sequence comparison of the rpoB, sodA, groEL and gyrB genes within the genus Streptococcus. PMID : 19620365 : DOI : 10.1099/ijs.0.005488-0 Abstract >>
Phylogenetic analysis and species identification of members of the genus Streptococcus were carried out using partial sequence comparison of the 16S rRNA gene (1468-1478 bp), rpoB, encoding the beta subunit of RNA polymerase (659-680 bp), sodA, encoding the manganese-dependent superoxide dismutase (435-462 bp), groEL, encoding the 60 kDa heat-shock protein (757 bp), and gyrB, encoding the Beta subunit of DNA gyrase (458-461 bp). For the first time, most species within the genus Streptococcus were represented in the study (65 strains, representing 58 species and nine subspecies). Phylogenies inferred from rpoB, sodA, gyrB and groEL sequence comparisons were more discriminative than those inferred from 16S rRNA gene sequence comparison, and showed common clusters. The minimal interspecies divergence was 0.3, 2.7, 0, 2.5 and 3.4 % for the 16S rRNA gene, rpoB, sodA, gyrB and groEL, respectively. In general, groEL partial gene sequence comparison represented the best tool for identifying species and subspecies and for phylogenetic analysis.
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20. |
Liu G,
Zhong J,
Ni J,
Chen M,
Xiao H,
Huan L,
( 2009 ) Characteristics of the bovicin HJ50 gene cluster in Streptococcus bovis HJ50. PMID : 19202107 : DOI : 10.1099/mic.0.022707-0 Abstract >>
Bovicin HJ50 is a new lantibiotic containing a disulfide bridge produced by Streptococcus bovis HJ50; its encoding gene bovA was reported in our previous publication. To identify other genes involved in bovicin HJ50 production, DNA fragments flanking bovA were cloned and sequenced. The bovicin HJ50 biosynthesis gene locus was encoded by a 9.9 kb region of chromosomal DNA and consisted of at least nine genes in the following order: bovA, -M, -T, -E, -F, ORF1, ORF2, bovK and bovR. A thiol-disulfide oxidoreductase gene named sdb1 was located downstream of bovR. A knockout mutant of this gene retained antimicrobial activity and the molecular mass of bovicin HJ50 in the mutant was the same as that of bovicin HJ50 in S. bovis HJ50, implying that sdb1 is not involved in bovicin HJ50 production. Transcriptional analyses showed that bovA, bovM and bovT constituted an operon, and the transcription start site of the bovA promoter was located at a G residue 45 bp upstream of the translation start codon for bovA, while bovE through bovR were transcribed together and the transcription start site of the bovE promoter was located at a C residue 35 bp upstream of bovE. We also demonstrated successful heterologous expression of bovicin HJ50 in Lactococcus lactis MG1363, which lacks thiol-disulfide oxidoreductase genes; this showed that thiol-disulfide oxidoreductase genes other than sdb1 are not essential for bovicin HJ50 biosynthesis.
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21. |
Asanuma N,
Yoshizawa K,
Hino T,
( 2009 ) Properties and role of glyceraldehyde-3-phosphate dehydrogenase in the control of fermentation pattern and growth in a ruminal bacterium, Streptococcus bovis. PMID : 19034572 : DOI : 10.1007/s00284-008-9326-2 Abstract >>
To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD(+)-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP(+)-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD(+) ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.
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22. |
Asanuma N,
Kanada K,
Hino T,
( 2008 ) Molecular properties and transcriptional control of the phosphofructokinase and pyruvate kinase genes in a ruminal bacterium, Streptococcus bovis. PMID : 18565772 : DOI : 10.1016/j.anaerobe.2008.05.004 Abstract >>
Molecular properties of pyruvate kinase (PYK) and phosphofructokinase (PFK) in Streptococcus bovis and transcriptional control of the two enzymes were examined. Sequence analysis indicated that the PYK gene (pyk) clusters with the PFK gene (pfk) and several other genes. It was demonstrated that the pyk and pfk are cotranscribed and their transcription appeared to be regulated at the transcriptional level in response to the sugars supplied. The intracellular pyk-mRNA level was lower in a catabolite control protein A (CcpA)-disrupted mutant than in its parent strain, and a binding site of CcpA was found in the upstream region of pfk. These results suggest that pfk-pyk transcription is enhanced by CcpA. A recombinant pyk-overexpressing strain showed approximately five-fold higher PYK activity, but it did not affect the growth rate or formate-to-lactate ratio significantly, suggesting that the flux in the glycolytic pathway is not altered by an increase in PYK activity.
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23. |
Tao J,
Diaz RK,
Teixeira CR,
Hackmann TJ,
( 2016 ) Transport of a Fluorescent Analogue of Glucose (2-NBDG) versus Radiolabeled Sugars by Rumen Bacteria and Escherichia coli. PMID : 27096355 : DOI : 10.1021/acs.biochem.5b01286 Abstract >>
Fluorescent tracers have been used to measure solute transport, but transport kinetics have not been evaluated by comparison of radiolabeled tracers. Using Streptococcus equinus JB1 and other bacteria, the objective of this study was to determine if a fluorescent analogue of glucose (2-NBDG) would be transported with the same kinetics and transporters as [(14)C]glucose. We uniquely modified a technique for measuring transport of radiolabeled tracers so that transport of a fluorescent tracer (2-NBDG) could also be measured. Deploying this technique for S. equinus JB1, we could detect 2-NDBG transport quantitatively and within 2 s. We found the Vmax of 2-NBDG transport was 2.9-fold lower than that for [(14)C]glucose, and the Km was 9.9-fold lower. Experiments with transport mutants suggested a mannose phosphotransferase system (PTS) was responsible for 2-NBDG transport in S. equinus JB1 as well as Escherichia coli. Upon examination of strains from 12 species of rumen bacteria, only the five that possessed a mannose PTS were shown to transport 2-NBDG. Those five uniformly transported [(14)C]mannose and [(14)C]deoxyglucose (other glucose analogues at the C-2 position) at high velocities. Species that did not transport 2-NBDG at detectable velocities did not possess a mannose PTS, though they collectively possessed several other glucose transporters. These results, along with retrospective genomic analyses of previous 2-NBDG studies, suggest that only a few bacterial transporters may display high activity toward 2-NBDG. Fluorescent tracers have the potential to measure solute transport qualitatively, but their bulky fluorescent groups may restrict (i) activity of many transporters and (ii) use for quantitative measurement.
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24. |
Jans C,
de Wouters T,
Bonfoh B,
Lacroix C,
Kaindi DW,
Anderegg J,
Böck D,
Vitali S,
Schmid T,
Isenring J,
Kurt F,
Kogi-Makau W,
Meile L,
( 2016 ) Phylogenetic, epidemiological and functional analyses of the Streptococcus bovis/Streptococcus equinus complex through an overarching MLST scheme. PMID : 27329036 : DOI : 10.1186/s12866-016-0735-2 PMC : PMC4915170 Abstract >>
The Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises seven (sub)species classified as human and animal commensals, emerging opportunistic pathogens and food fermentative organisms. Changing taxonomy, shared habitats, natural competence and evidence for horizontal gene transfer pose difficulties for determining their phylogeny, epidemiology and virulence mechanisms. Thus, novel phylogenetic and functional classifications are required. An SBSEC overarching multi locus sequence type (MLST) scheme targeting 10 housekeeping genes was developed, validated and combined with host-related properties of adhesion to extracellular matrix proteins (ECM), activation of the immune responses via NF-KB and survival in simulated gastric juice (SGJ). Commensal and pathogenic SBSEC strains (n = 74) of human, animal and food origin from Europe, Asia, America and Africa were used in the MLST scheme yielding 66 sequence types and 10 clonal complexes differentiated into distinct habitat-associated and mixed lineages. Adhesion to ECMs collagen I and mucin type II was a common characteristic (23 % of strains) followed by adhesion to fibronectin and fibrinogen (19.7 %). High adhesion abilities were found for East African dairy and human blood isolate branches whereas commensal fecal SBSEC displayed low adhesion. NF-KB activation was observed for a limited number of dairy and blood isolates suggesting the potential of some pathogenic strains for reduced immune activation. Strains from dairy MLST clades displayed the highest relative survival to SGJ independently of dairy adaptation markers lacS/lacZ. Combining phylogenetic and functional analyses via SBSEC MLST enabled the clear delineation of strain clades to unravel the complexity of this bacterial group. High adhesion values shared between certain dairy and blood strains as well as the behavior of NF-KB activation are concerning for specific lineages. They highlighted the health risk among shared lineages and establish the basis to elucidate (zoonotic-) transmission, host specificity, virulence mechanisms and enhanced risk assessment as pathobionts in an overarching One Health approach.
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25. |
Zhang J,
Feng Y,
Teng K,
Lin Y,
Gao Y,
Wang J,
Zhong J,
( 2014 ) Type AII lantibiotic bovicin HJ50 with a rare disulfide bond: structure, structure-activity relationships and mode of action. PMID : 24814218 : DOI : 10.1042/BJ20131524 Abstract >>
Lantibiotics are ribosomally synthesized antimicrobial peptides containing unusual amino acids. As promising alternatives to conventional antibiotics, they have a high potential for alleviating the problem of emergent antibiotic resistance, with possible applications in many industries that have antibacterial demand. Bovicin HJ50 is a type AII lantibiotic, the largest group of lantibiotics, comprising a linear N-terminal region and a globular C-terminal region. Interestingly, bovicin H50 has a disulfide bond that is rare in this group. Owing to limited information about the spatial structures of type AII lantibiotics, the functional regions of this type and the role of the disulfide bond are still unknown. In the present study, we resolved the solution structure of bovicin HJ50 using NMR spectroscopy. This is the first spatial structure of a type AII lantibiotic. Bovicin HJ50 exhibited high flexibility in aqueous solution, whereas varied rigidities were observed in the different rings with the conserved ring A being the most rigid. The charged residues Lys??, Asp?? and Lys??, as well as the essential disulfide bond were critical for antimicrobial activity. Importantly, bovicin HJ50 showed not only peptidoglycan precursor lipid II-binding ability, but also pore-forming activity, which is significantly different from other bacteriostatic type AII lantibiotics, suggesting a novel antimicrobial mechanism.
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26. |
( 1997 ) Cloning, sequence, and expression of the L-(+) lactate dehydrogenase of Streptococcus bovis. PMID : 9142744 : Abstract >>
The ldh gene encoding the fructose-1,6-diphosphate-dependent L-(+) lactate dehydrogenase from the ruminal bacterium Streptococcus bovis was cloned and sequenced. A genomic library of S. bovis JB1 DNA was constructed in lambda ZAP II and screened by use of a heterologous probe derived from the cloned Streptococcus mutans ldh gene. Several clones were isolated that contained a common 2.9-kb fragment as determined by restriction analysis. Nucleotide sequence analysis revealed a 987-bp open reading frame with extensive homology to Streptococcus thermophilus and S. mutans ldh nucleic acid and amino acid sequences. Expression of the cloned S. bovis ldh gene in Escherichia coli was confirmed by the ability to complement the ldh mutation of E. coli FMJ39, by using an in-gel activity screen and by enzymatic assay. Increased LDH activity was observed in S. bovis JB1 containing the cloned ldh genes on a multicopy plasmid.
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27. |
( 1996 ) Purification and characterization of a malic enzyme from the ruminal bacterium Streptococcus bovis ATCC 15352 and cloning and sequencing of its gene. PMID : 8702261 : PMC : PMC168054 Abstract >>
Malic enzyme (EC 1.1.1.39), which catalyzes L-malate oxidative decarboxylation and pyruvate reductive carboxylation, was purified to homogeneity from Streptococcus bovis ATCC 15352, and properties of this enzyme were determined. The 2.9-kb fragment containing the malic enzyme gene was cloned, and the sequence was determined and analyzed. The enzymatic properties of the S. bovis malic enzyme were almost identical to those of other malic enzymes previously reported. However, we found that the S. bovis malic enzyme catalyzed unknown enzymatic reactions, including reduction of 2-oxoisovalerate, reduction of 2-oxoisocaproate, oxidation of D-2-hydroxyisovalerate, and oxidation of D-2-hydroxyisocaproate. The requirement for cations and the optimum pH of these unique activities were different from the requirement for cations and the optimum pH of the L-malate oxidative decarboxylating activity. A sequence analysis of the cloned fragment revealed the presence of two open reading frames that were 1,299 and 1,170 nucleotides long. The 389-amino-acid polypeptide deduced from the 1,170-nucleotide open reading frame was identified as the malic enzyme; this enzyme exhibited high levels of similarity to malic enzymes of Bacillus stearothermophilus and Haemophilus influenzae and was also similar to other malic enzymes and the malolactic enzyme of Lactococcus lactis.
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28. |
Cotta MA,
Whitehead TR,
( 1993 ) Regulation and cloning of the gene encoding amylase activity of the ruminal bacterium Streptococcus bovis. PMID : 7679887 : DOI : 10113/24580 PMC : PMC202076 Abstract >>
Streptococcus bovis is an important starch-degrading ruminal bacterium that has been implicated as being important in the etiology of a number of ruminal pathologies associated with diets high in grains. Previous studies with S. bovis have shown that amylase production was influenced by the growth substrate, but the nature of this regulation was not determined. The current study was conducted to better describe the regulatory phenomena and gain a better understanding of the molecular characteristics of this activity. Nutritional experiments demonstrated that the presence of starch or the starch-derived disaccharide maltose was required for maximum amylase production. Subsequent time-course experiments showed that amylase synthesis was induced by maltose and repressed by glucose, cellobiose, and fructose, while inulin and lactose had little effect on enzyme accumulation. The effects of the added antibiotics rifampin and tetracycline were consistent with transcriptional control of amylase synthesis. Analysis of S. bovis cells grown on glucose or maltose showed that they contained similar low levels of cyclic AMP, indicating that it was unlikely that regulation of amylase synthesis was mediated through a mechanism involving this nucleotide. The amylase gene from S. bovis JB1 was cloned and expressed in Escherichia coli. The amylase produced in E. coli was of lower molecular weight than that synthesized by S. bovis and had catalytic characteristics different from those of S. bovis amylase. When the gene was introduced back into S. bovis JB1, only one form of amylase activity was detected, indicating that the entire gene was present on this insert. The use of the amylase gene as a genetic probe for identification of S. bovis strains is discussed.
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29. |
Whitehead TR,
Cotta MA,
( 1995 ) Identification of intracellular amylase activity in Streptococcus bovis and Streptococcus salivarius. PMID : 7532504 : Abstract >>
The ruminal bacterium Streptococcus bovis has been demonstrated to produce an extracellular amylase activity. We previously reported on the cloning of a gene from S. bovis encoding for what was initially believed to be the extracellular amylase. DNA sequence analyses indicated that the amylase produced by the cloned gene did not match the N-terminus amino acid sequence of the purified extracellular amylase and contained no apparent leader sequence for secretion. Analyses of crude extracts demonstrated the presence of an intracellular amylase in S. bovis JB1 that differed in molecular weight (56,000) from that of the extracellular amylase (70,000). The 56,000 molecular weight amylase was identical to the amylase produced by Escherichia coli containing the cloned amylase gene. Low levels of intracellular amylase activity were also detected in other strains of S. bovis and also Streptococcus salivarius. Introduction of the plasmid pVA838 containing the cloned amylase gene into S. bovis and S. sanguis resulted in enhanced intracellular amylase production by both organisms. The amylase gene has been sequenced, and analysis of the deduced amino acid sequence for the amylase indicates a high degree of similarity with secreted amylases from Bacillus species.
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30. |
Whitehead TR,
Cotta MA,
( 1993 ) Development of a DNA probe for Streptococcus bovis by using a cloned amylase gene. PMID : 7691873 : DOI : 10113/24632 PMC : PMC265766 Abstract >>
Streptococcus bovis is a normal inhabitant of the rumen but has been implicated as a causative agent for ruminal lactic acidosis and related problems. While rarely isolated from humans, S. bovis has been identified as a causative agent for endocarditis, meningitis, and septicemia. Recent reports have also suggested a correlation between human colonic carcinoma and increased levels of S. bovis. Identification of S. bovis strains of human origin has been problematic because of variations in results of biochemical tests compared with results for ruminal strains. We have tested a cloned amylase gene from the ruminal strain S. bovis JB1 as a potential DNA probe for rapid and accurate identification of S. bovis strains from all sources. DNAs from strains identified as S. bovis, of both human and ruminal origin, were found to hybridize with the probe under stringent conditions. The probe also hybridized with variants of S. bovis that did not grow on starch. The probe did not hybridize with DNA isolated from other bacteria of human colonic and ruminal origin, including Bacteroides thetaiotaomicron, Bacteroides ruminicola, Butyrivibrio fibrisolvens, and Enterococcus faecalis but did demonstrate hybridization with Streptococcus salivarius.
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31. |
( 2012 ) Use of tuf as a target for sequence-based identification of Gram-positive cocci of the genus Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus. PMID : 23181410 : DOI : 10.1186/1476-0711-11-31 PMC : PMC3533577 Abstract >>
Accurate identification of isolates belonging to genus Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus at the species level is necessary to provide a better understanding of their pathogenic potential, to aid in making clinical decisions, and to conduct epidemiologic investigations,especially when large blind samples must be analyzed. It is useful to simultaneously identify species in different genera using a single primer pair. We developed a primer pair based on the tuf gene (encoding elongation factor) sequence to identify 56 Gram-positive cocci isolates. The target sequences were amplified from all 56 samples. The sequencing results and the phylogenetic tree derived from the partial tuf gene sequences identified the isolates as three enterococcal species, two lactococcal species, two staphylococcal species, and six streptococcal species, as well as eight isolates that were novel species of the genus Streptococcus. Partial gene sequence analysis of the sodA, dnaK, and 16S RNA genes confirmed the results obtained by tuf gene sequencing. Based on the uniform amplification of the tuf gene from all samples and the ability to identify all isolates at both the genus and species levels, we conclude that the primer pair developed in this research provides a powerful tool for identifying these organisms in clinical laboratories where large blind samples are used.
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32. |
( 1998 ) Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase. PMID : 9431917 : PMC : PMC124804 Abstract >>
We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodA(int)) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus, S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus, S. canis, S. cricetus, S. downei, S. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. equinus, S. gordonii, S. iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis, S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodA(int) fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of their sodA(int) fragments on the phylogenetic tree of the sodA(int) fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.
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33. |
( 1997 ) Purification, characterization, and nucleotide sequence of an intracellular maltotriose-producing alpha-amylase from Streptococcus bovis 148. PMID : 9406414 : PMC : PMC168820 Abstract >>
An intracellular alpha-amylase from Streptococcus bovis 148 was purified and characterized. The enzyme was induced by maltose and soluble starch and produced about 80% maltotriose from soluble starch. Maltopentaose was hydrolyzed to maltotriose and maltose and maltohexaose was hydrolyzed mainly to maltotriose by the enzyme. Maltotetraose, maltotriose, and maltose were not hydrolyzed. This intracellular enzyme was considered to be a maltotriose-producing enzyme. The enzymatic characteristics and hydrolysis product from soluble starch were different from those of the extracellular raw-starch-hydrolyzing alpha-amylase of strain 148. The deduced amino acid sequence of the intracellular alpha-amylase was similar to the sequences of the mature forms of extracellular liquefying alpha-amylases from Bacillus strains, although the intracellular alpha-amylase did not contain a signal peptide. No homology between the intracellular and extracellular alpha-amylases of S. bovis 148 was observed.
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