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1.
Effects of oxygen on pyruvate formate-lyase in situ and sugar metabolism of Streptococcus mutans and Streptococcus sanguis. 总被引:3,自引:5,他引:3
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The strictly anaerobic metabolism of sugar in strains of Streptococcus mutans and Streptococcus sanguis was studied because deep layers of dental plaque are strictly anaerobic. Galactose-grown cells of these streptococcal strains had higher pyruvate formate-lyase activity than did glucose-grown cells. Among these strains, two strains of S. mutans had a significantly higher pyruvate formate-lyase activity than did the others. This enzyme is extremely sensitive to oxygen, and even in situ the enzyme was inactivated by exposure of the cells to air. Lactate was less than 50% of the total end product of the strictly anaerobic incubation of the galactose-grown cells of S. mutans with excess glucose, and a significant amount of formate, acetate, and ethanol was produced through the catalysis of pyruvate formate-lyase. But the cells exclusively produced lactate when exposed to air for 2 min before the anaerobic incubation. The metabolism of sorbitol by S. mutans was seriously impaired by the exposure of the cells to oxygen, and the metabolic rate was reduced to less than 1/20 of that found under strictly anaerobic conditions because of the inactivation of pyruvate formate-lyase. S. sanguis produced a smaller amount of the volatile products from glucose than did S. mutans because of the low level of pyruvate formate-lyase. However, the pyruvate formate-lyase in situ in S. sanguis was less sensitive to oxygen than was that in S. mutans. Because of this low sensitivity, S. sanguis metabolized glucose more rapidly under aerobic conditions, whereas the rates of the aerobic and anaerobic metabolism of glucose by S. mutans were similar, which suggests that S. mutans rather than S. sanguis can sustain the rapid sugar metabolism in the deep layers of dental plaque. 相似文献
2.
Pyruvate formate-lyase (PFL) (formate acetyltransferase; EC 2.3.1.54) of oral streptococci is essential for metabolizing sugar into volatile compounds (formate, acetate, and ethanol). This enzyme is extremely sensitive to oxygen, and its activity is irreversibly inactivated by oxygen. When Streptococcus sanguis was anaerobically starved, a part of the active form of PFL was converted into a reversible inactive form that was tolerant of oxygen. This reversible inactive enzyme could be reactivated to the active enzyme by anaerobic sugar metabolism, with the recovery of volatile compound production. The PFL in Streptococcus mutans was not converted into an oxygen-tolerant inactive form by anaerobic starvation, and after exposure of the cells to oxygen the PFL could not be reactivated. These findings suggest that S. mutans can produce acids rapidly under anaerobic conditions because of its capacity to keep PFL active and that S. sanguis can protect its sugar metabolism from oxygen impairment because of its interconversion of PFL. 相似文献
3.
Characterization of the Streptococcus mutans pyruvate formate-lyase (PFL)-activating enzyme gene by complementary reconstitution of the In vitro PFL-reactivating system
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Yamamoto Y Sato Y Takahashi-Abbe S Takahashi N Kizaki H 《Infection and immunity》2000,68(8):4773-4777
The act gene was identified and an act mutant as well as the pfl mutant was constructed in Streptococcus mutans. Pyruvate formate-lyase (PFL) activity was regenerated with the mixture of the respective cell extracts from these mutants by complementary reconstitution of the in vitro reactivating system. The S. mutans act gene encoded the sole enzyme able to activate the PFL protein in this organism. 相似文献
4.
Cloning and expression of the gene encoding the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase of Streptococcus mutans. 总被引:4,自引:2,他引:2
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The fructose-1,6-diphosphate-dependent lactate dehydrogenase from Streptococcus mutans JH1000 was purified by a modification of published methods. The sequence of 27 amino-terminal amino acids was determined, which allowed us to construct a 17-base DNA probe that had 32-fold degeneracy. The probe was used to screen a genomic library in pBR322. Of 18 reactive clones, 1 was found that expressed lactate dehydrogenase (LDH) activity identical to that of S. mutans with regard to dependence on fructose-1,6-diphosphate, thermal inactivation profile, and inhibition by sodium oxamate. Extracts of this clone possessed a protein band that comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with purified LDH from JH1000. Compared with controls, the clone was shown to produce elevated amounts of L-(+)-lactic acid during growth in the presence of glucose, thereby indicating that the activity was expressed in vivo. This result was substantiated by demonstrating that the activity could complement a mutation in the fermentative D-(-)-LDH of Escherichia coli. Subcloning showed that the S. mutans LDH subunit is encoded by a 1.2-kilobase gene. Our ability to clone this gene is expected to have great practical significance in the construction of an effector strain for use in the replacement therapy of dental caries. 相似文献
5.
R. D. Klein R. A. Poorman M. A. Favreau M. H. Shea N. T. Hatzenbuhler S. C. Nulf 《Current genetics》1989,16(3):145-152
Summary The gene encoding invertase (INV) has been cloned from Schwanniomyces occidentalis. The enzyme consists of 533 amino acids, 8 potential glycosylation sites and has a 45 % identity with the invertase from Saccharomyces cerevisiae. The proenzyme has a 22 amino acid signal sequence that has a high -helical transmembrane potential which differs significantly from that predicted for the Saccharomyces cerevisiae enzyme. 相似文献
6.
7.
The complete nucleotide sequence of the Streptococcus mutans GS-5 scrB gene coding for sucrose-6-phosphate hydrolase activity was determined. A potential ribosome-binding site as well as promoter sequences were identified upstream from the gene. The deduced amino acid sequence of the enzyme suggested a molecular weight of 51,750, which is similar to that estimated for the enzyme isolated from strain GS-5. The enzyme is slightly acidic, with a pI of 5.9, and is a relatively hydrophilic protein. The nucleotide and amino acid sequences of the enzyme showed significant homology with those of the sacA protein from Bacillus subtilis. In addition, a region of amino acid homology with the S. mutans fructosyltransferase and B. subtilis levansucrase proteins was also detected. 相似文献
8.
Cloning and sequence analysis of a protective M-like protein gene from Streptococcus equi subsp. zooepidemicus. 总被引:4,自引:3,他引:4
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Streptococcus equi subsp. zooepidemicus, a Lancefield group C streptococcus, is a frequently isolated opportunist pathogen from a variety of animal hosts, including the horse. Previous studies have indicated that equine strains carry antigens with characteristics of the antiphagocytic M proteins on the Lancefield groups A and G streptococci. We have cloned a protective M-like protein gene (SzPW60) of an equine strain of S. equi subsp. zooepidemicus W60 and determined its sequence. This gene encodes a protein with a molecular weight of 40,123 which protects mice against subsp. zooepidemicus but not subsp. equi, stimulates antibodies which opsonize subsp. zooepidemicus but not equi, and reacts with antiserum to the protein of the parent strain. The predicted amino acid structure shows significant homology with the carboxy termini of groups A and G M proteins but no other homology. The M-like protein, although showing an extensive region of alpha helix, lacks the A, B, and C repeats found in group A M proteins and has a shorter signal sequence. A proline-rich region upstream from the LPSTGE motif contains 20 repeats of the tetrapeptide PEPK. The presence of this repeat region may account for the slow migration of the M-like protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 相似文献
9.
Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins. 总被引:6,自引:12,他引:6
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J S Sampson S P O'Connor A R Stinson J A Tharpe H Russell 《Infection and immunity》1994,62(1):319-324
Gene psaA, which encodes the Streptococcus pneumoniae 37-kDa protein, was cloned in Escherichia coli, and its complete nucleotide sequence was determined. Analysis of the sequence of the 2.4-kb cloned fragment revealed three open reading frames (ORFs). ORF2, which is 933 bp long, was identified as psaA. The two other ORFs identified flank psaA. ORF1, located upstream of psaA, is 836 nucleotides long and encodes a protein with a calculated molecular mass of 29,843 Da. The sequence for ORF3, located downstream of psaA, was only partially determined. Northern (RNA) blot analysis of pneumococcal RNA suggests that psaA is transcribed as part of a polycistronic message. Analysis of the primary structure of the protein encoded by this gene indicated significant similarity to two previously reported streptococcal proteins, SsaB (80% similarity) and FimA (92.3% similarity), from S. sanguis and S. parasanguis, respectively. These two homologous proteins have been shown to be associated with bacterial adhesion, and the possibility of a similar role for PsaA is hypothesized. 相似文献
10.
Cloning of the Streptococcus mutans gene encoding glucan binding protein B and analysis of genetic diversity and protein production in clinical isolates 总被引:6,自引:0,他引:6
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Mattos-Graner RO Jin S King WF Chen T Smith DJ Duncan MJ 《Infection and immunity》2001,69(11):6931-6941
Streptococcus mutans, the primary etiological agent of dental caries, produces several activities that promote its accumulation within the dental biofilm. These include glucosyltransferases, their glucan products, and proteins that bind glucan. At least three glucan binding proteins have been identified, and GbpB, the protein characterized in this study, appears to be novel. The gbpB gene was cloned and the predicted protein sequence contained several unusual features and shared extensive homology with a putative peptidoglycan hydrolase from group B streptococcus. Examination of gbpB genes from clinical isolates of S. mutans revealed that DNA polymorphisms, and hence amino acid changes, were limited to the central region of the gene, suggesting functional conservation within the amino and carboxy termini of the protein. The GbpB produced by clinical isolates and laboratory strains showed various distributions between cells and culture medium, and amounts of protein produced by individual strains correlated positively with their ability to grow as biofilms in an in vitro assay. 相似文献
11.
DNA sequence and in vitro mutagenesis of the gene encoding the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase of Streptococcus mutans. 总被引:3,自引:1,他引:2
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Previously, the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase gene of Streptococcus mutans JH1000 was cloned into Escherichia coli (J. D. Hillman, M. J. Duncan, and K. P. Stashenko, Infect. Immun. 58:1290-1295, 1990). In the present study, the nucleotide sequence of 1.29 kb of S. mutans DNA which contained the promoter and protein-coding region of the gene was determined. In vitro disruption of the gene was achieved by deletion of the promoter and a major portion of the protein-coding sequence. Subsequently, a tetracycline resistance gene from S. mutans was inserted at the deletion site as a marker for selection. In addition, evidence from Southern hybridization showed that S. mutans JH1000 contained a single copy of the lactate dehydrogenase gene. 相似文献
12.
目的克隆蓝氏贾第鞭毛虫Elp3基因,并应用生物信息学方法进行分析。方法根据蓝氏贾第鞭毛虫Elp3已知基因序列的特点设计引物,利用PCR技术扩增获得Elp3的核苷酸序列,连接到pET28a载体并测定序列。应用生物信息学方法分析Elp3基因的序列同源性与结构特征。结果扩增片段长度为1767bp,测序结果与蓝氏贾第鞭毛虫WB株(GL50830)同源性100%。可构建生物进化树,进行同源结构建模发现,71-362aa具有一个保守的S-腺甙基蛋氨酸区域,458-584aa具有组蛋白乙酰基转移酶区域。结论成功克隆了蓝氏贾第鞭毛虫Elp3基因,生物信息学分析发现,其在进化上与其它物种不属同一起源,具有SAM和HAT的结构和功能,这些发现为进一步研究其生物学功能提供了依据和线索。 相似文献
13.
14.
Sequence analysis of the glucosyltransferase A gene (gtfA) from Streptococcus mutans Ingbritt. 总被引:8,自引:7,他引:1
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The complete nucleotide sequence of a 2.4-kilobase fragment containing the glucosyltransferase A gene (gtfA) of Streptococcus mutans Ingbritt (serotype c) was determined. The gtfA gene contains 481 codons and specifies a protein of molecular weight 55,665. There is no evidence of a signal peptide in the protein or that the glucosyltransferase A enzyme is secreted. No sequence homologies were observed between the gtfA gene or protein and the gtfI or gtfB gene and its protein. 相似文献
15.
Nucleotide sequence analysis of the gtfT gene from Streptococcus sobrinus OMZ176. 总被引:3,自引:1,他引:2
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The gtfT gene and its upstream region isolated from the Streptococcus sobrinus OMZ176 chromosomal DNA were sequenced. The gtfT gene was preceded by a potential Shine-Dalgarno sequence. The gtfT gene product, glucosyltransferase (GTF), displays a typical gram-positive bacterial signal peptide sequence and both an active site peptide sequence and carboxy-terminal repeats typical of GTFs. The signal sequence is similar to those of other known GTF proteins. The putative active-site peptide sequence of this enzyme was DGIRVDAVD, which was different by one amino acid from the active-site peptide sequence derived from two different types of the S. sobrinus GTFs reported previously (G. Mooser, S. A. Hefta, R. J. Paxton, J. E. Shively, and T. D. Lee, J. Biol. Chem. 266:8916-8922, 1991). The gtfT gene product has three repeated sequences of 51 to 52 amino acids and a partial repeat of 18 amino acids. Another open reading frame (ORF) was detected in the region immediately upstream of the gtfT gene. The upstream ORF showed substantial DNA homology with the gtfS gene isolated from Streptococcus downei MFe28. The inferred amino acid sequence of the upstream ORF has four repeating units and has extensive homology with the repeated peptides coded by the S. downei gtfS gene. These results suggested that the gtfT gene was a typical gtf gene isolated from the mutans streptococci and that the two gtf genes were located in tandem on the chromosomal DNA of S. sobrinus OMZ176. 相似文献
16.
Cloning and nucleotide sequence of the gene encoding arginine deiminase of Mycoplasma arginini. 总被引:1,自引:1,他引:1
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T Ohno O Ando K Sugimura M Taniai M Suzuki S Fukuda Y Nagase K Yamamoto I Azuma 《Infection and immunity》1990,58(11):3788-3795
The existence of a mycoplasmal arginine deiminase which catalyzes the conversion of L-arginine to L-citrulline has been postulated. Here we show the partial amino acid sequence of arginine deiminase of Mycoplasma arginini and the complete nucleotide sequence of the arginine deiminase gene of M. arginini. The open reading frame deduced from this sequence consists of 1,230 bp encoding 410 amino acids. The mature form of this enzyme contains 409 amino acids after the deletion of the first methionine. In this open reading frame, TGA nonsense codons are used as tryptophan codons; this usage was verified by determination of the amino acid sequence. The molecular weight of the enzyme calculated from the deduced amino acid sequence is 46,372. Recently, the nucleotide sequence of the arginine deiminase gene of M. arginini was reported by Kondo et al. (K. Kondo, H. Sone, H. Yoshida, T. Toida, K. Kanatani, Y.-M. Hong N. Nishino, and J. Tanaka, Mol. Gen. Genet. 221:81-86, 1990). However, their sequence differed from ours in several places and especially at the C terminus. 相似文献
17.
Cloning and DNA sequence of the omc gene encoding the outer membrane protein-macromolecular complex from Neisseria gonorrhoeae. 总被引:8,自引:2,他引:6
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The omc gene, encoding the outer membrane protein-macromolecular complex (OMP-MC), was cloned in two pieces from Neisseria gonorrhoeae 2686. The 5' fragment of the omc gene included a promoter sequence, as indicated by its unregulated expression in Escherichia coli. Attempts to reconstruct an intact omc gene were unsuccessful, suggesting that expression of the complete OMP-MC protein was toxic to E. coli. Complete sequence determination revealed a coding sequence of 2,133 nucleotides; the deduced amino acid sequence indicated a mature protein of 687 amino acids with an NH2-terminal signal peptide of 24 amino acids. Analysis of the deduced amino acid sequence revealed that the NH2-terminal half of OMP-MC is generally hydrophilic, while the COOH-terminal portion contains alternating hydrophobic and hydrophilic regions. Serological analyses demonstrated that the NH2-terminal portion of OMP-MC is exposed on the gonococcal surface and the COOH-terminal portion is membrane associated. 相似文献
18.
Cloning and nucleotide sequence of the gene encoding the 136-kilodalton surface protein (muramidase-released protein) of Streptococcus suis type 2. 总被引:19,自引:0,他引:19
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We cloned and sequenced the gene encoding the muramidase-released protein (MRP) of a pathogenic Streptococcus suis type 2 strain to determine whether its amino acid sequence resembles that of proteins with known functions and to determine its function in virulence. The complete nucleotide sequence composing the gene and the regions flanking it was determined. The deduced amino acid sequence revealed the presence of a signal peptide at the N terminus and a cell envelope anchor at the C terminus, both of which resembled similar regions in several other surface proteins from gram-positive bacteria. The processed form of MRP has a length of 1,209 amino acids and a calculated molecular weight of 131,094. A highly repetitive region preceded the envelope anchor. The repeated units were preceded by a proline-rich stretch of amino acids (26 of 86). No overall homologies were observed between the amino acid sequence of MRP and protein sequences in the EMBL data bank. A particular region within the amino acid sequence, however, showed some similarity with the fibronectin-binding protein of Staphylococcus aureus. Binding of MRP to human fibronectin, however, could not be confirmed. 相似文献
19.
Sequence analysis of the gene for the glucan-binding protein of Streptococcus mutans Ingbritt. 总被引:26,自引:11,他引:15
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The nucleotide sequence of the gbp gene, which encodes the glucan-binding protein (GBP) of Streptococcus mutans, was determined. The reading frame for gbp was 1,689 bases. A ribosome-binding site and putative promoter preceded the start codon, and potential stem-loop structures were identified downstream from the termination codon. The deduced amino acid sequence of the GBP revealed the presence of a signal peptide of 35 amino acids. The molecular weight of the processed protein was calculated to be 59,039. Two series of repeats spanned three-quarters of the carboxy-terminal end of the protein. The repeats were 32 to 34 and 17 to 20 amino acids in length and shared partial identity within each series. The repeats were found to be homologous to sequences hypothesized to be involved in glucan binding in the GTF-I of S. downei and to sequences within the protein products encoded by gtfB and gtfC of S. mutans. The repeated sequences may represent peptide segments that are important to glucan binding and may be distributed among GBPs from other bacterial inhabitants of plaque or the oral cavity. 相似文献
20.
Cloning and sequence analysis of cytadhesin P1 gene from Mycoplasma pneumoniae. 总被引:43,自引:14,他引:29
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Mycoplasma pneumoniae cytadhesin P1 was purified by monoclonal antibody affinity chromatography followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal 18-amino-acid sequence of P1 was determined and used to design two synthetic oligonucleotides, a 14-mer corresponding to amino acids 1 to 5 and an 18-mer corresponding to amino acids 7 to 12. These oligonucleotides served as hybridization probes for the identification of the P1 gene by Southern blot analysis of M. pneumoniae DNA. The P1 gene was cloned into plasmid pUC19 and mapped by using appropriate restriction endonucleases. The DNA sequence of the entire P1 gene was determined by subcloning appropriate DNA fragments into bacteriophage M13 and sequencing the DNA by the dideoxy-chain-termination method. The P1 gene contains an open reading frame of 4,881 nucleotides coding for a protein of 1,627 amino acids with a calculated molecular weight of 176,288. Properties of the amino-terminal sequence suggest that protein P1 may be synthesized as a precursor with subsequent processing to a mature protein of a calculated molecular weight of 169,758. Potential antigenic sites were determined by hydrophilicity plots. A computer search revealed that part of the predicted P1 sequence is homologous to cytoskeletal keratin of mammalian species and human fibrinogen alpha chain precursor. These results demonstrate the uniqueness of P1 as a cytadhesin and virulence determinant. 相似文献