变形链球菌GGDEF结构域基因的克隆表达及功能初步研究
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摘要
龋病是人类最常见的细菌感染性疾病之一，其发病率高，发病涉及范围广。变形链球菌(Streptococcus mutants)是人类主要的致龋菌，研究表明，变形链球菌拥有大量的毒力因子参与了其在牙面的黏附、聚集、菌斑生物膜的形成、产酸耐酸以及与宿主之间的交互作用。同时，口腔环境中的外界条件如pH、温度、营养以及机体的免疫因子等会随着人体的各种生理病理活动而发生变化，菌斑生物膜中各种微生物之间也随时处在生存竞争之中。因此，如何感应外界环境信号的变化来调节这些毒力因子的转录、翻译、表达，对变形链球菌的生存及致龋都是至关重要的。随着变形链球菌基因组测序的完成，越来越多的毒力因子的功能已逐渐明确，但对一种或几种毒力因子的抑制并不能在体内有效地阻止龋病的进程。因此，对调节这些蛋白的表达及作用的信号网络的研究不但有助于对龋病的发生发展的深入了解，而且针对这些调节因子的干预作用也有望成为龋病预防的新途径。
近年来，大量细菌的全基因组测序完成，比较基因组学研究发现一类含有GGDEF结构域的蛋白在各类细菌中几乎普遍存在，进一步研究发现其功能主要是参与c-di-GMP(bis-(3’-5’)-cyclic dimericguanosine monophosphate，单磷酸鸟苷环二聚体)的合成。c-di-GMP近来已被证实是原核细胞信号转导中一类新的第二信使，已有的研究表明它的作用涉及细菌细胞分化、生物膜形成、毒力因子的表达、细胞间通讯以及与宿主细胞间相互作用等多个方面，是细菌生存和代谢中的关键性调节因子之一，但其在变形链球菌中的研究尚属空白。在变形链球菌的生命活动中是否存在c-di-GMP的作用途径?变形链球菌基因组中存在着编码类似GGDEF结构域蛋白的基因，其功能如何?对该蛋白的干预是否会影响表型的改变和毒力因子的表达?对这些问题的回答将有助于加深对细菌信号转导途径的理解，也为探索新的防龋方法提供了依据。
本研究共分为三个部分：
第一部分：变形链球GGDEF结构域基因的克隆、原核表达、纯化及活性分析
常规厌氧培养变形链球菌UA159，提取基因组DNA作为模板，根据GenBank序列设计引物，PCR法扩增gcp基因除信号肽序列外的全长编码区基因片段，连接T载体后转化E coli感受态细胞，挑取阳性克隆，鉴定后测序，结果与GenBank序列完全一致。
将测序正确的gcp基因片段定向插入原核表达载体pP_(RO)EX HTb，构建原核表达质粒pP_(RO)EX HTb-gcp，转化E.coli DH5α感受态细胞，IPTG诱导表达融合蛋白GGDEF，即Gcp蛋白。SDS-PAGE检测表达产物，结果在约77KDa处出现新生蛋白条带，超声裂菌的上清中可见新生蛋白带，表明成功诱导出Gcp蛋白的可溶性表达。
使用发酵罐进行表达菌的大体积培养和诱导，超声裂菌，通过金属螯合亲和层析(Ni~(2+)-NTA介质)纯化获得重组表达的Gcp蛋白；western-blot实验证实纯化蛋白可以特异性地与抗His抗体反应。通过HPLC实验结果表明成功获得了有活性的Gcp融合蛋白，该蛋白具有明显促进c-di-GMP合成的能力。
第二部分：变形链球菌GGDEF结构域基因失活菌株的构建、鉴定及生物学特性的检测。
以变形链球菌UA159基因组DNA为模板，PCR扩增873bp的gcp基因内部序列，连接T载体后将该内部片段定向插入自杀载体pVA8912，酶切鉴定，发现PCR产物及插入片段大小与预期值相符，表明成功构建了打靶载体pVA8912/gcp；将鉴定正确的质粒转化变形链球菌UA159株，挑取阳性克隆，提取基因组DNA，用PCR结合酶切鉴定发现gcp基因失活株基因组中gcp基因内部成功插入了打靶载体片段。结果表明成功构建了变形链球菌gcp基因失活株，为该基因功能的研究奠定了基础。
厌氧培养变形链球菌gcp基因失活株，在不同时间点测定OD_(600)值，绘制生长曲线，发现gcp基因失活株和野生菌的生长曲线无显著性差异，表明gcp的基因失活对变形链球菌的生长无显著影响。
利用pH5．0～7．0的系列BHI液体培养基厌氧培养gcp基因失活株和野生菌48 h，测量培养前后的pH，观察其产酸性；测量OD_(600)值，观察其耐酸性。结果发现其产酸及耐酸特性有明显差异，说明gcp的基因失活对变形链球菌的产酸耐酸具有显著影响。
将gcp失活菌和野生菌悬液转入无菌的96孔酶标板中厌氧培养48 h，黏附菌使用结晶紫溶液染色，乙醇／丙酮混合液显色，测量OD_(575)值，以定量反映生物膜形成量。结果发现gcp失活菌生物膜形成量显著低于野生菌，表明gcp可能与变形链球菌生物膜形成能力有关。
荧光标记gcp失活菌和野生菌，与唾液包被的羟基磷灰石粉末共同孵育后，测定羟基磷灰石沉淀的荧光值，比较粘附率的差异，发现gcp失活菌的黏附率(19．7％±0．91)低于野生菌(29．5％±2．9)，差异有显著性(P<0．05)；说明gcp失活菌的黏附特性明显的下降。
总之，本研究发现gcp的功能主要是与变形链球菌细菌的毒力密切相关，Gcp蛋白通过c-di-GMP信号通路影响变形链球菌的致龋能力。这一研究成果，为揭示变形链球菌致龋的分子机理以及探索新的防龋方法提供了实验依据。
Dental caries is one of the most common bacterial infections in human beings. Streptococcus mutans has shown to be the causative agent of this disease. Research into the etiology of dental caries and caries prevention established the key role of S. mutans cariogenic factors in caries occurrence and progression. The main virulence factors associated with cariogenicity include adhesion, acidogenicity, and acid tolerance. Streptococcus mutants (S. mutans) is thought to be one of the primary causative agents of dental caries depends on a biofilm lifestyle. Even though dental plaque consists of numerous bacterial species, but the presence of S. mutans has been consistently linked with the production of dental caries.
Recently, as the completion of the majority bacterials genomic sequencing, according to the research of comparative genomics, GGDEF is widespread in bacteria but is not found the outside bacterial kingdom, The observation that GGDEF domains are often associate with domains involved in signal perception or signal transduction, argued for the existence of a dedicated regulatory network that converts a variety of different signals into the production of the second messenger c-di-GMP. Recent studies showed that it has effects on bacterial cell differentiation、biofilm formation、virulence factor expression、cell-cell communication and host cell interaction, etc.. It is one of key regulatory factors in bacterium exist and metabolism , however, the research in its streptococcus mutans is still a blank. Is there any way of c-di-GMP acting existing in streptococcus mutans vital movement? What are functions of the genes similar to GGDEF in streptococcus mutans gene group? Does the intervention to this protein affect the changes of phenotypic and expression of virulence factor? The answers of these questions will help to strengthened the comprehension of bacterium signal transduction pathway, and provide the evidence for exploring new anticarious methods。This study will give evidence on the effect of c-di-GMP on s.mutans metabolism and cariogenic activity, which might theoreticallyindicate a new way in caries prevention.
This research consists of three parts as follows.
PartⅠCloning, prokaryotic expression of S. mutans gcp gene andpurification of the recombinant Gcp protein
S. mutans UA159 was routinely cultured at an anaerobic atmosphere and its genomic DNA was isolated. The encoding region of gcp, excluding the signal sequence, was amplified by polymerase chain reaction (PCR). The relevant fragment was ligated with a pMD-19T simple vector before transformed into E. coli competent cells. Positive clones were selected and sequenced. The sequencing result was found completely consistent with that in GenBank.
The fragment was then inserted into a prokaryotic expression vector pP_(RO)EX HTb such that the coding sequence of the gcp fragment was in-frame with an N-terminal His_6 tag, to construct the plasmid pP_(RO)EX HTb-gcp. The plasmid was then used to transform E. coli DH5αcompetent cells. Positive clones were selected and identified by enzyme restriction analysis, and then induced by IPTG to express the His-tagged GGDEF fusion protein, named Gcp protein. The supernatant of cell lysate was analyzed by SDS-PAGE. A new band with 77KDa appeared in the SDS-PAGE gel and was approximately the same as expected, which indicated that we successfully obtained the soluble expression of Gcp protein in E. coli.
By fermenter, We get large amount of recombinant Gcp fusion protein. The harvested cells were sonificated and the supernatant of cell lysate were purified by Ni~(2+)-NTA affinity chromatography. The purified rGcp protein was utilized to test interaction with c-di-GMP by HPLC. Its reactivity to anti-His antibody was tested by western-blot. The results indicated that we successfully obtained the purified rGcp protein possessing activity.
PartⅡPartⅡConstruction and identification of an insertion mutant of gcp and examination of its cariogenic properties
A 873bp internal fragment of the gcp gene was amplified from S. mutans UA159 chromosomal DNA by PCR and ligated with the pMD-19T vector. After enzyme restriction analysis, the fragment was subcloned into the suicide vector pVA8912 to give plasmid pVA8912/gcp. S. mutans UA159 cells were transformed with plasmid pVA8912/gcp using natural transformation method. The positive clones were selected and their genomic DNA was isolated. The correct location of the insertion was confirmed by PCR and enzyme restriction analysis, as well as western-blot analysis. The results showed that plasmid integrated into the gcp gene region on the chromosome. These results indicated that the gcp mutant was successfully constructed.
In order to examine the cariogenic properties of gcp mutant, several tests were performed.
The growth, acidogenicity and cariogenicity of the gcp mutant and wildtype cultures were evaluated during the 48 h inoculation. The results showed that the gcp mutant was indistinguishable from the wildtype with respect to growth rate,but distinguishable with acidogenicity and acidodurance.
The abilities of gcp mutant and wildtype cells to form stable biofilms were assessed by growing the cells in 96-well, flat-bottom microtiter plates in an aerobic atmosphere for 48 h. The adherent bacteria were stained with crystal violet and the bound dyes were extracted by using ethanol/acetone (8:2, v/v) mixture. Biofilm formation was then quantified by measuring the optical density of the solution at 575 nm. The results showed that the biofilms formed by the mutant had reduced numbers of cells compared to the wild type, which indicated that Gcp protein might contribute to the S. mutans biofilm formation in some degree.
Biofilm structures of the gcp mutant and wildtype strains on enamel chips were observed by scanning electron microscopy (SEM). Upon inspection, gcp mutant has a noticeable difference in biofilm structure from that of the wild type. The wild type biofilms have a thick and confluent appearance, with cells regularly arranged. However, the gcp mutant biofilms are very thin and seem much more heterogeneous, with large gaps and more long-chains in the biofilm. This phenomenon suggested the important role of Gcp protein in S. mutans biofilm structure.
To investigate the adhesion properties of gcp mutant and wildtype strains, the bacteria cells were labeled with a fluorescent indicator (BCECF/AM) and incubated with saliva-coated hydroxyapatite (SHA). Bacteria adhered to the SHA were measured using a spectrofluorometer. The result showed a significantly lower adherence percentage in the gcp mutant group (19.7%±0.91) as compared with the wild type strains (29.5%±2.9). After the labeled wild type bacteria were co-incubated with anti-Gcp antibody and SHA, the adherence percentage was decreased compared to the non-antibody controls (P <0.05). The test indicated that GGDEF domain played a role in the S. mutans adhesion to SHA .
近年来，大量细菌的全基因组测序完成，比较基因组学研究发现一类含有GGDEF结构域的蛋白在各类细菌中几乎普遍存在，进一步研究发现其功能主要是参与c-di-GMP(bis-(3’-5’)-cyclic dimericguanosine monophosphate，单磷酸鸟苷环二聚体)的合成。c-di-GMP近来已被证实是原核细胞信号转导中一类新的第二信使，已有的研究表明它的作用涉及细菌细胞分化、生物膜形成、毒力因子的表达、细胞间通讯以及与宿主细胞间相互作用等多个方面，是细菌生存和代谢中的关键性调节因子之一，但其在变形链球菌中的研究尚属空白。在变形链球菌的生命活动中是否存在c-di-GMP的作用途径?变形链球菌基因组中存在着编码类似GGDEF结构域蛋白的基因，其功能如何?对该蛋白的干预是否会影响表型的改变和毒力因子的表达?对这些问题的回答将有助于加深对细菌信号转导途径的理解，也为探索新的防龋方法提供了依据。
本研究共分为三个部分：
第一部分：变形链球GGDEF结构域基因的克隆、原核表达、纯化及活性分析
常规厌氧培养变形链球菌UA159，提取基因组DNA作为模板，根据GenBank序列设计引物，PCR法扩增gcp基因除信号肽序列外的全长编码区基因片段，连接T载体后转化E coli感受态细胞，挑取阳性克隆，鉴定后测序，结果与GenBank序列完全一致。
将测序正确的gcp基因片段定向插入原核表达载体pP_(RO)EX HTb，构建原核表达质粒pP_(RO)EX HTb-gcp，转化E.coli DH5α感受态细胞，IPTG诱导表达融合蛋白GGDEF，即Gcp蛋白。SDS-PAGE检测表达产物，结果在约77KDa处出现新生蛋白条带，超声裂菌的上清中可见新生蛋白带，表明成功诱导出Gcp蛋白的可溶性表达。
使用发酵罐进行表达菌的大体积培养和诱导，超声裂菌，通过金属螯合亲和层析(Ni~(2+)-NTA介质)纯化获得重组表达的Gcp蛋白；western-blot实验证实纯化蛋白可以特异性地与抗His抗体反应。通过HPLC实验结果表明成功获得了有活性的Gcp融合蛋白，该蛋白具有明显促进c-di-GMP合成的能力。
第二部分：变形链球菌GGDEF结构域基因失活菌株的构建、鉴定及生物学特性的检测。
以变形链球菌UA159基因组DNA为模板，PCR扩增873bp的gcp基因内部序列，连接T载体后将该内部片段定向插入自杀载体pVA8912，酶切鉴定，发现PCR产物及插入片段大小与预期值相符，表明成功构建了打靶载体pVA8912/gcp；将鉴定正确的质粒转化变形链球菌UA159株，挑取阳性克隆，提取基因组DNA，用PCR结合酶切鉴定发现gcp基因失活株基因组中gcp基因内部成功插入了打靶载体片段。结果表明成功构建了变形链球菌gcp基因失活株，为该基因功能的研究奠定了基础。
厌氧培养变形链球菌gcp基因失活株，在不同时间点测定OD_(600)值，绘制生长曲线，发现gcp基因失活株和野生菌的生长曲线无显著性差异，表明gcp的基因失活对变形链球菌的生长无显著影响。
利用pH5．0～7．0的系列BHI液体培养基厌氧培养gcp基因失活株和野生菌48 h，测量培养前后的pH，观察其产酸性；测量OD_(600)值，观察其耐酸性。结果发现其产酸及耐酸特性有明显差异，说明gcp的基因失活对变形链球菌的产酸耐酸具有显著影响。
将gcp失活菌和野生菌悬液转入无菌的96孔酶标板中厌氧培养48 h，黏附菌使用结晶紫溶液染色，乙醇／丙酮混合液显色，测量OD_(575)值，以定量反映生物膜形成量。结果发现gcp失活菌生物膜形成量显著低于野生菌，表明gcp可能与变形链球菌生物膜形成能力有关。
荧光标记gcp失活菌和野生菌，与唾液包被的羟基磷灰石粉末共同孵育后，测定羟基磷灰石沉淀的荧光值，比较粘附率的差异，发现gcp失活菌的黏附率(19．7％±0．91)低于野生菌(29．5％±2．9)，差异有显著性(P<0．05)；说明gcp失活菌的黏附特性明显的下降。
总之，本研究发现gcp的功能主要是与变形链球菌细菌的毒力密切相关，Gcp蛋白通过c-di-GMP信号通路影响变形链球菌的致龋能力。这一研究成果，为揭示变形链球菌致龋的分子机理以及探索新的防龋方法提供了实验依据。
Dental caries is one of the most common bacterial infections in human beings. Streptococcus mutans has shown to be the causative agent of this disease. Research into the etiology of dental caries and caries prevention established the key role of S. mutans cariogenic factors in caries occurrence and progression. The main virulence factors associated with cariogenicity include adhesion, acidogenicity, and acid tolerance. Streptococcus mutants (S. mutans) is thought to be one of the primary causative agents of dental caries depends on a biofilm lifestyle. Even though dental plaque consists of numerous bacterial species, but the presence of S. mutans has been consistently linked with the production of dental caries.
Recently, as the completion of the majority bacterials genomic sequencing, according to the research of comparative genomics, GGDEF is widespread in bacteria but is not found the outside bacterial kingdom, The observation that GGDEF domains are often associate with domains involved in signal perception or signal transduction, argued for the existence of a dedicated regulatory network that converts a variety of different signals into the production of the second messenger c-di-GMP. Recent studies showed that it has effects on bacterial cell differentiation、biofilm formation、virulence factor expression、cell-cell communication and host cell interaction, etc.. It is one of key regulatory factors in bacterium exist and metabolism , however, the research in its streptococcus mutans is still a blank. Is there any way of c-di-GMP acting existing in streptococcus mutans vital movement? What are functions of the genes similar to GGDEF in streptococcus mutans gene group? Does the intervention to this protein affect the changes of phenotypic and expression of virulence factor? The answers of these questions will help to strengthened the comprehension of bacterium signal transduction pathway, and provide the evidence for exploring new anticarious methods。This study will give evidence on the effect of c-di-GMP on s.mutans metabolism and cariogenic activity, which might theoreticallyindicate a new way in caries prevention.
This research consists of three parts as follows.
PartⅠCloning, prokaryotic expression of S. mutans gcp gene andpurification of the recombinant Gcp protein
S. mutans UA159 was routinely cultured at an anaerobic atmosphere and its genomic DNA was isolated. The encoding region of gcp, excluding the signal sequence, was amplified by polymerase chain reaction (PCR). The relevant fragment was ligated with a pMD-19T simple vector before transformed into E. coli competent cells. Positive clones were selected and sequenced. The sequencing result was found completely consistent with that in GenBank.
The fragment was then inserted into a prokaryotic expression vector pP_(RO)EX HTb such that the coding sequence of the gcp fragment was in-frame with an N-terminal His_6 tag, to construct the plasmid pP_(RO)EX HTb-gcp. The plasmid was then used to transform E. coli DH5αcompetent cells. Positive clones were selected and identified by enzyme restriction analysis, and then induced by IPTG to express the His-tagged GGDEF fusion protein, named Gcp protein. The supernatant of cell lysate was analyzed by SDS-PAGE. A new band with 77KDa appeared in the SDS-PAGE gel and was approximately the same as expected, which indicated that we successfully obtained the soluble expression of Gcp protein in E. coli.
By fermenter, We get large amount of recombinant Gcp fusion protein. The harvested cells were sonificated and the supernatant of cell lysate were purified by Ni~(2+)-NTA affinity chromatography. The purified rGcp protein was utilized to test interaction with c-di-GMP by HPLC. Its reactivity to anti-His antibody was tested by western-blot. The results indicated that we successfully obtained the purified rGcp protein possessing activity.
PartⅡPartⅡConstruction and identification of an insertion mutant of gcp and examination of its cariogenic properties
A 873bp internal fragment of the gcp gene was amplified from S. mutans UA159 chromosomal DNA by PCR and ligated with the pMD-19T vector. After enzyme restriction analysis, the fragment was subcloned into the suicide vector pVA8912 to give plasmid pVA8912/gcp. S. mutans UA159 cells were transformed with plasmid pVA8912/gcp using natural transformation method. The positive clones were selected and their genomic DNA was isolated. The correct location of the insertion was confirmed by PCR and enzyme restriction analysis, as well as western-blot analysis. The results showed that plasmid integrated into the gcp gene region on the chromosome. These results indicated that the gcp mutant was successfully constructed.
In order to examine the cariogenic properties of gcp mutant, several tests were performed.
The growth, acidogenicity and cariogenicity of the gcp mutant and wildtype cultures were evaluated during the 48 h inoculation. The results showed that the gcp mutant was indistinguishable from the wildtype with respect to growth rate,but distinguishable with acidogenicity and acidodurance.
The abilities of gcp mutant and wildtype cells to form stable biofilms were assessed by growing the cells in 96-well, flat-bottom microtiter plates in an aerobic atmosphere for 48 h. The adherent bacteria were stained with crystal violet and the bound dyes were extracted by using ethanol/acetone (8:2, v/v) mixture. Biofilm formation was then quantified by measuring the optical density of the solution at 575 nm. The results showed that the biofilms formed by the mutant had reduced numbers of cells compared to the wild type, which indicated that Gcp protein might contribute to the S. mutans biofilm formation in some degree.
Biofilm structures of the gcp mutant and wildtype strains on enamel chips were observed by scanning electron microscopy (SEM). Upon inspection, gcp mutant has a noticeable difference in biofilm structure from that of the wild type. The wild type biofilms have a thick and confluent appearance, with cells regularly arranged. However, the gcp mutant biofilms are very thin and seem much more heterogeneous, with large gaps and more long-chains in the biofilm. This phenomenon suggested the important role of Gcp protein in S. mutans biofilm structure.
To investigate the adhesion properties of gcp mutant and wildtype strains, the bacteria cells were labeled with a fluorescent indicator (BCECF/AM) and incubated with saliva-coated hydroxyapatite (SHA). Bacteria adhered to the SHA were measured using a spectrofluorometer. The result showed a significantly lower adherence percentage in the gcp mutant group (19.7%±0.91) as compared with the wild type strains (29.5%±2.9). After the labeled wild type bacteria were co-incubated with anti-Gcp antibody and SHA, the adherence percentage was decreased compared to the non-antibody controls (P <0.05). The test indicated that GGDEF domain played a role in the S. mutans adhesion to SHA .
引文
[1] Hugoson A, Koch Q Gothberg C, Helkimo AN, Lundin SA, Norderyd O, SjodinB, Sondell K.Oral health of individuals aged 3-80 years in Jonkoping, Swedenduring 30 years (1973-2003). n. Review of clinical and radiographic findings. SwedDent J. 2005,29(4): 139-155.
[2] World Health Organization (2003): The World Oral Health Report 2003. Geneva:WHO.
[3] WHO releases new report on global problem of oral diseases:http://www.who.int /mediacentre/news /releases/2004/pr15/en/print.html
[4]全国牙病防治指导组．第二次全国口腔健康流行病学抽样调查．第1版，北京：人民卫生出版社．1999．
[5]赵连香．变形链球菌毒力因子的研究职业与健康，2003,19(11)：45-47．
[6]Loesche WI. Role of Streptococcus mutants in human dental decay.Microbiol Rev.1986; 50:53-380
[7] Howard K. Kuramitsu. Virulence properties of oral bacteria: impact of molecular biology. Curr Issues Mol Biol. 2001,3: 35-36.
[8]刘筱娣．变形链球菌生物膜形成相关基因的研究进展 牙体牙髓牙周病杂志2006；16(9)：532-535
[9]Fuqua WC et al. Annu Rev Genet,2001,35: 439-468
[10]Martin CA.Therapeutic frontiers: preventing and treating infectious diseases byinhibiting bacterial quorum sensing. Eur J Clin Microbiol Infect Dis. 2008 Mar 6
[11]KleerebezemM et al. Mol Microbiol, 1997, 24(5): 895-904
[12] Deziel E etal. Proc Nail Acad Sci USA, 2004, 101(5): 1339-1344
[13]MashburnLM et al. Nature, 2005, 437: 422-425
[14]Xavier KB et al. Nature, 2005, 437(7059): 750-753
[15]宋水山等．生物技术，2005，15(1)：7-10
[16]Manefield M et al. Microbiology, 2002, 148(Pt4): 1119-1127
[17]Chun CK et al. Proc Natl Acad Sci USA , 2004, 101(10): 3587-3590
[18]宋水山等．细菌小分子的信号转导．生命的化学，2007，27(5)：396-398
[19]Moore. W. E. C et al. The bacteria of periodontal diseases.Periodontol.20005:66-67
[20]Socransky, S.S et al. Evidence of bacterial etiology:a historicalperspective.Periodontol.2000 5:7-25
[21]Facklam R et al. What happened to the streptococci: overview of taxonomic andNomenclature changes. Clin. Microbiol. Rev. 2002,15: 613-630
[22]Kolenbrander. P. E. Oral microbial communities:biofilms,interactions,andgenetic systems. Annu. Rev. Microbiol. 2000,54: 413-437
[23]Marsh, P. D. Dental plaque as a microbial biofilm. Caries Res. 2004,38:204-211
[24]Whittaker, C.J. et al. Mechanisms of adhesion by oral bacteria. Annu. Rev.Microbiol. 1996,50: 513-552
[25]Weerkamp, A et al. Antagonistic substances produced by streptococci fromhuman dental plaque and their significance in plaque ecikogy. Caries Res. 1977b, 11:245-56
[26]Weerkamp, A. et al. Bacteriocins as factors in the in vitro interaction betweenoral streptococci in plaque. Infect.Immun. 1977a, 16: 773-780
[27]Bassler, B.L Cell-to-cell communication in bacteria. Cell. 1999,109:421-424
[28]Shapiro, J. A.Thinking about bacterial populations as multicellular organisms.Annu. Rev.Microbiol. 1998,52: 81-104
[29]Nealson, K.H. et al. Cellular control of the synthesis and activity of the bacterial luninescent system.J. Bacteriol. 1970, 104:313-322
[30]Davies, D.G. et al. The involvemont of cell-to cell signals in the development of a bacterial biofilm. Science. 1998, 280:295-298
[31]Givskov, M. et al. Eukaryotic interference with homoserine lactone-mediaterd prokaryotic signaling. J.Bacteriol. 1996, 178:6618-6622
[32]Lee, M.S. and D.A. Morrison. Identification of a new regulator in Streptococcus pneumoniae linking quorum sensing to competence for genetic transformation. J.Baceriol. 1999, 181:5004-5016
[33]Li,Y-H. et a.l Natural genetic transformation of Streptococcus mutans growing in biofilms. J.Bacteriol. 2001b, 183:879-908
[34]Ji, G. et al. Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. Proc. Natl. Acad. Sci. USA. 1995, 92:12055-12059
[35]Bassler, B. L. Small talk: Cell-to-cell communication in bacteria. Cell. 2002, 109:421-424
[36]Podbielski, A and B. Kreikemeyer. Cell density-dependent regulation:basic principles and effects on the virulence of Gram-positive cocci. Int. J. Infect. Dis.2004, 8: 81-95
[37]Sturme, M. et al. Cell to cell communication by autoinducing peptides in grampositive bacteria. Antonie van Leeuwenhoek. 2002, 81: 233-243
[38]Bassler, B. L. et al. Multiple signaling systems controlling expression of luminescence in Vibro harveyi: sequence and function of genes encoding a second sensory pathway. Mol. Microbiol. 1994,13: 273-286
[39]Merritt, J. et al. Mutation of luxS affects biofilm formation in Streptococcrs mutans. Infec. Immun. 2003, 71: 1972-1979
[40]Yoshida, A. et al. LuxS-based signaling affects Streptococcus mutans biofilmformation. Appli. Environ. Microbiol. 2005,71: 2372-2380
[41]Wen, Z. T. and R. A. Burne. LuxS- mediated signaling in Streptococcus mutansis involved in regulation of acid and oxidative stress tolerance and biofilm formation.J. Bacteriol. 2004,186: 2862-2691
[42]Jayaraman, G C. et al. Transcriptional analysis of the Streptococcus mutanshrcA, grpE and dnakK genes and regulation of expression in response to heat shockand environmental acidification. Mol. Microbiol. 1997,25: 329-341
[43]Li, Y. et al. Cell density modulates acid adaptation in Streptococcus mutans:implications for survival in biofilms. J. Bacteriol. 2001a, 183: 6875-6884
[44]Li, Y. et al. A quorum-sensing signaling system essential for genetic competencein Streptococcus mutans is involved in biofilm formation. J. Bacteriol. 2002b, 184:2699-2708
[45]Wang, B. Y. and H. K. Kuramitsu. Interactions between oral bacteria: inhibitionof Streptococcus mutans bacteriocin production by Streptococcus gordonii. Appl.Environ. Microbiol. 2005,71: 345-362
[46]Li, Y. et al. Novel two-component regulatory system involved in biofilmformation and acid resistance in Streptococcus mutans. J. Bacteriol. 2002a, 184:6333-6342
[47]Luo, P. et al. CotnX is a unique link between multiple quorum sensing outputsand competence in Streptococcus pneumoniae. Mol. Microbiol. 2003,50: 623-633
[48] Luo, P.and D, A, Morrison. Transient association of an alternative sigma factor,comX, with RNA polymerase during the period of competence for genetictransformation in Streptococcus pneumoniae. J. Bacteriol. 2003,185: 349-358
[49]Ajdic, D. et al. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. USA. 2002, 22: 14434-14439
[50]Senadheera, M. D. et al. A VicR/K signal transduction system in Streptococcus mutans affects gtfB, gtfC and ftf expression, biofilm formation and genetic competence development. J. Bacteriol. 2005, 187: 4064-4076
[51]Davies, J. Origins and evolution of antibiotic resistance.microbiologia. 1996, 12: 9-16
[52] Havarstein L. S. et al. Natural competence in the genus Streptococcus: evidence that streptococci can change pherotype by interspecies recombinational exchanges. J. Bacteriol. 1997, 179: 6589-6594
[53] Havarstein L. S. and D. A. Morrison. Quorum sensing and peptide pheromones in streptococcal competence for genetic transformation. In Cell-Cell signaling in Bacteria. Dunny, G. M. and Winans, S, C.,eds. ASM Press,1999, pp. 9-26
[54]Magnuson, R et al. Biochemical and genetic characterization of a competence pheromone from Bacillus subtilis. Cell. 1994, 77: 207-216
[55]Kleerebezem, M. et al. Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria. Mol. Microbiol. 1997, 24: 895-904
[56]Perry, D. and H. K. Kuramitsu. Genetic transformation of Streptococcus mutans. Infect Immun. 1981, 32: 1295-1297
[57]Kuramitsu, H. K. and C. M. Long. Plasmid-mediated transformation of Streptococcus mutans. Infect Immun. 1982, 36: 435-436
[58]Dowson, C. G. et al. Horizontal gene transfer and the evolution of resistance and virulence determinants in Streptococcus. Sco. Appl. Baxteriol. Symp. Ser. 1997, 26: 42S-51S
[59]Mercer, D. K. et al. Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DLI by plasmid DNA in human saliva. Appl. Environ. Microbiol. 1999, 65: 6-10
[60]Cvitkovitch, D. G. Genetic competence and transformation on oral streptococci. Crit. Rev. Oral. Biol. Med. 2001, 12: 217-243
[61] Cvitkovitch, D. G. Genetic exchange in biofilms. Microbial Biofilms. M. Ghannoum and G. A.O'Toole eds. ASM press. Washington, D. C. 2004, p. 192-205
[62]Marsh, P. D. Oral ecology and its impact on oral microbial diversity. Chapter 1 in Oral Bacterial Ecology: The Molecular Basis. H. Kuramitsu and R. P. Ellen, eds. Wymondham, Norfolk, UK: Horizon Scientific Press.
[63]Edgar, W. M. and S. M. Higham. Saliva and the control of plaque pH, p.81-94. In W. M. Edgar and D. M. O'Mullane(ed.), Saliva and oral health, 2~(nd)ed. British Dental Association, London, United Kingdom.
[64]Bender, G. R. et al. Acid tolerance, proton permeabilities, and membrane ATPases of oral streptococci. Infect. Immun. 1986, 53: 331-338
[65]Hamilton, I. R. and N. D. Bucklry. Adaptation by Streptococcus mutans to acid tolerance. Oral Microbiol. Immunol. 1991, 6: 65-71
[66]Belli, W. A. and R. E. Marquis. Adaptation of Streptococcus mutans and enterococcus hirae to acid stress in continuous culture. Appl. Environ. Microbiol. 1991,57:1134-1138
[67]Hahn, K. et al. Induction of an AP endonuclease activity in Streptococcus mutans during growth at low Ph. Mol. Microbiol. 1999, 31: 1489-1498
[68]Svensater G, et al. Acid tolerance response and survival by oral bacteria. Oral. Microbiol. Immunol. 1997, 12: 266-273
[69]Verhamme, D. et al. Investigation of in vivo cross-talk between key two-component systems of Escherichia coli. Microbiol. 2002, 148: 69-78
[70]Stoodley, P. et al. Biofilms as complex differentiated communities. Annu. Rev. Microbiol. 2002, 56: 187-209
[71]Loo, C. et al. Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes. J. Bacteriol. 2000, 182: 1374-1382
[72]Bhagwat, S. et al. Effects of mutating putative two component systems on biofilm formation by Streptococcus mutans UA159. FEMS Microbiol. Lett. 2001, 205: 225-230
[73]Havarstein, L. S. et al. An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA. 1995, 92: 11140-11144
[74]Lunsford, R. D. and J. London. DNA-binding activities in Streptococcus gordonii: identification of a receptor-nickase and a histone like protein. Curr. Microbiol. 1996, 32:95-100
[75]Havarstein, L. S. et al. The leader peptide of colicin V shares consensus sequences with leader peptides that are common among peptide bacteriocins produced by gram-positive bacteria. Microbiology. 1994, 140: 2383-2389
[76]Oggioni, M. R. et al. Antibacteral activity of a competence-stimulating peptide in experimental sepsis caused by Streptococcus pneumoniae. Antimicrob. Agents Chemother. 2004, 48: 4725-4732
[77]Qi, F. et al. Signal peptide pheromone induced cell death of Streptococcus mutans. Manuscript submitted. 2005.
[78] Galperin M.Y.(2004). Bacterial signal transduction network in a genomic perspective. Env microbiol 6, 552-567
[79] R(o|¨)mling U., Gomelsky M. and Galperin M.Y.(2005). C-di-GMP: the dawning of a novel bacterial signaling system. Mol microbiol 57: 629-639
[80] Ryjenkov D.A., Tarutina M., Moskvin O.V., and Gomelsky M. (2005).Cyclic Diguanylate Is a Ubiquitous Signaling Molecule in Bacteria: Insights into Biochemistry of the GGDEF Protein Domain. J Bacteriol 187, 1792-1798
[81]Tal R., Wong H.C., Calhoon R., Gelfand D., Fear A.L., Volman G., Mayer R., Ross P., Amikam D., Weinhouse H. et al(1998). Three cdg operons control cellular turnover of cyclic di-GMP in Acetobacter xylinum: genetic organization and occurrence of conserved domains in isoenzymes. J Bacteriol 180,4416-4425.
[82] R(o|¨)mling, U. (2005) Characterization of the rdar morphotype, a multicellular behaviour in Enterobacteriaceae. CLMS 62, 1-13.
[83] Tischler A. D. and Camilli A.(2004). Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation. Mol microbiol 53, 857-869
[84] Karaolis D., Rashid M., Chythanya R., Luo W., Hyodo M., and Hayakawa Y.(2005). c-di-GMP (3'-5'-Cyclic diguanylic acid) inhibits Staphylococcus aureus cell-cell interactions and biofilm formation. Antimicrob agents chemother 49, 1029-1038
[85] D'Argenio D. A. and Miller S. I.(2004) Cyclic di-GMP as a bacterial second messenger. Microbiol 150, 2497-2502.
[86] Aldridge, P., R. Paul, P. Goymer, P. Rainey, and U. Jenal.(2003). Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus. Mol. Microbiol. 47,1695-1708.
[87] Tischler A.D., Camilli A.(2005). Cyclic diguanylate regulates Vibrio cholerae virulence gene expression. Infect Immun.73, 5873-5882.
[88] Galperin M Y (2005).A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts. BMC Microbiol. 14,35
[89] Mendez-Ortiz MM, Hyodo M, Hayakawa Y, Membrillo-Hemandez J (2006). Genome wide transcriptional profile of Escherichia coli in response to high levels of the second messenger c-di-GMP.J Biol Chem. Jan 17; [Epub ahead of print]
[90]马建岗主编．基因工程学原理．西安：西安交通大学出版社，2001．148-155．
[91]白雪源，陈香美．基因打靶技术及其在生物医学中的应用．中华肾脏病杂志，2002；16(6)：403-405．
[92]陈效友，李传友，马玛，刘冲，王敬慧，张雪峰，昌增益．卡介苗菌MDP1基因敲除技术的研究．中华结核和呼吸杂志，2004；27：183-187．
[93]Bomchil.N et al Identification and Characterization of a Vibrio cholerae GenembaA Involved in Maintenance of Biofilm Architecture. J Bacteriol. 2003 February;185(4): 1384-1390.
[94]Tao L. Streptococcal integration vectors for gene inactivation and cloning.Methods Cell Sci 1998,20: 59-64.
[95] Hinds J, Mahenthiralingam E, Kempsell KE, Duncan K, Stokes RW, Parish T,Stoker NG. Enhanced gene replacement in mycobacteria. Microbiology. 1999,145(Pt 3): 519-527.
[96] Nelson DL, CoxMM. Lehninger principles of biochemistry. 3th ed. New York:Worth Publishers, 2000.931-977.
[97]Yim HH, Rubens CE. Site-specific homologous recombination mutanenesis ingroup B streptococci. Methods Cell Sci. 1998,20:13-20.
[98]Suzuki T, Tagami J and Hanada N. Role of F_1F_0-ATPase in the growth ofStreptococcus mutans GS5. J. Applied Microbiol. 2000,88: 555-562
[99] Ann SJ, Lemos JA, Burne RA. Role of HtrA in growth and competence ofStreptococcus mutans UA159. J Bacteriol.2005,187(9): 3028-38.
[100]Gainesa S, Jamesa TC, Folanc M, Bairdd AW, O'Farrelly C. A novel??spectrofluorometric microassay for Streptococcus mutans adherence to hydroxylapatite. J Microbiol Methods 2003,54: 315-323.
[101] Hao YQ, Zhou XD, Xiao XR, Lu JJ, Zhang FC, Hu T, Wu HK, Chen XM. Effects of cecropin-XJ on growth and adherence of oral cariogenic bacteria in vitro.Chin Med J(Engl). 2005,118(2): 155-60.
[115]周学东 主编．口腔生物化学．成都：四川大学出版社，2002．84-88
[116] Clark WB, Lane MD, Beem JE, Bragg SL, Wheeler TT. Relative hydrophobicities of Actinomyces viscosus and Actinomyces naeslundii strains and their adsorption to saliva-treated hydroxyapatite. Infect Immun. 1985; 47(3): 730-6.
[2] World Health Organization (2003): The World Oral Health Report 2003. Geneva:WHO.
[3] WHO releases new report on global problem of oral diseases:http://www.who.int /mediacentre/news /releases/2004/pr15/en/print.html
[4]全国牙病防治指导组．第二次全国口腔健康流行病学抽样调查．第1版，北京：人民卫生出版社．1999．
[5]赵连香．变形链球菌毒力因子的研究职业与健康，2003,19(11)：45-47．
[6]Loesche WI. Role of Streptococcus mutants in human dental decay.Microbiol Rev.1986; 50:53-380
[7] Howard K. Kuramitsu. Virulence properties of oral bacteria: impact of molecular biology. Curr Issues Mol Biol. 2001,3: 35-36.
[8]刘筱娣．变形链球菌生物膜形成相关基因的研究进展 牙体牙髓牙周病杂志2006；16(9)：532-535
[9]Fuqua WC et al. Annu Rev Genet,2001,35: 439-468
[10]Martin CA.Therapeutic frontiers: preventing and treating infectious diseases byinhibiting bacterial quorum sensing. Eur J Clin Microbiol Infect Dis. 2008 Mar 6
[11]KleerebezemM et al. Mol Microbiol, 1997, 24(5): 895-904
[12] Deziel E etal. Proc Nail Acad Sci USA, 2004, 101(5): 1339-1344
[13]MashburnLM et al. Nature, 2005, 437: 422-425
[14]Xavier KB et al. Nature, 2005, 437(7059): 750-753
[15]宋水山等．生物技术，2005，15(1)：7-10
[16]Manefield M et al. Microbiology, 2002, 148(Pt4): 1119-1127
[17]Chun CK et al. Proc Natl Acad Sci USA , 2004, 101(10): 3587-3590
[18]宋水山等．细菌小分子的信号转导．生命的化学，2007，27(5)：396-398
[19]Moore. W. E. C et al. The bacteria of periodontal diseases.Periodontol.20005:66-67
[20]Socransky, S.S et al. Evidence of bacterial etiology:a historicalperspective.Periodontol.2000 5:7-25
[21]Facklam R et al. What happened to the streptococci: overview of taxonomic andNomenclature changes. Clin. Microbiol. Rev. 2002,15: 613-630
[22]Kolenbrander. P. E. Oral microbial communities:biofilms,interactions,andgenetic systems. Annu. Rev. Microbiol. 2000,54: 413-437
[23]Marsh, P. D. Dental plaque as a microbial biofilm. Caries Res. 2004,38:204-211
[24]Whittaker, C.J. et al. Mechanisms of adhesion by oral bacteria. Annu. Rev.Microbiol. 1996,50: 513-552
[25]Weerkamp, A et al. Antagonistic substances produced by streptococci fromhuman dental plaque and their significance in plaque ecikogy. Caries Res. 1977b, 11:245-56
[26]Weerkamp, A. et al. Bacteriocins as factors in the in vitro interaction betweenoral streptococci in plaque. Infect.Immun. 1977a, 16: 773-780
[27]Bassler, B.L Cell-to-cell communication in bacteria. Cell. 1999,109:421-424
[28]Shapiro, J. A.Thinking about bacterial populations as multicellular organisms.Annu. Rev.Microbiol. 1998,52: 81-104
[29]Nealson, K.H. et al. Cellular control of the synthesis and activity of the bacterial luninescent system.J. Bacteriol. 1970, 104:313-322
[30]Davies, D.G. et al. The involvemont of cell-to cell signals in the development of a bacterial biofilm. Science. 1998, 280:295-298
[31]Givskov, M. et al. Eukaryotic interference with homoserine lactone-mediaterd prokaryotic signaling. J.Bacteriol. 1996, 178:6618-6622
[32]Lee, M.S. and D.A. Morrison. Identification of a new regulator in Streptococcus pneumoniae linking quorum sensing to competence for genetic transformation. J.Baceriol. 1999, 181:5004-5016
[33]Li,Y-H. et a.l Natural genetic transformation of Streptococcus mutans growing in biofilms. J.Bacteriol. 2001b, 183:879-908
[34]Ji, G. et al. Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. Proc. Natl. Acad. Sci. USA. 1995, 92:12055-12059
[35]Bassler, B. L. Small talk: Cell-to-cell communication in bacteria. Cell. 2002, 109:421-424
[36]Podbielski, A and B. Kreikemeyer. Cell density-dependent regulation:basic principles and effects on the virulence of Gram-positive cocci. Int. J. Infect. Dis.2004, 8: 81-95
[37]Sturme, M. et al. Cell to cell communication by autoinducing peptides in grampositive bacteria. Antonie van Leeuwenhoek. 2002, 81: 233-243
[38]Bassler, B. L. et al. Multiple signaling systems controlling expression of luminescence in Vibro harveyi: sequence and function of genes encoding a second sensory pathway. Mol. Microbiol. 1994,13: 273-286
[39]Merritt, J. et al. Mutation of luxS affects biofilm formation in Streptococcrs mutans. Infec. Immun. 2003, 71: 1972-1979
[40]Yoshida, A. et al. LuxS-based signaling affects Streptococcus mutans biofilmformation. Appli. Environ. Microbiol. 2005,71: 2372-2380
[41]Wen, Z. T. and R. A. Burne. LuxS- mediated signaling in Streptococcus mutansis involved in regulation of acid and oxidative stress tolerance and biofilm formation.J. Bacteriol. 2004,186: 2862-2691
[42]Jayaraman, G C. et al. Transcriptional analysis of the Streptococcus mutanshrcA, grpE and dnakK genes and regulation of expression in response to heat shockand environmental acidification. Mol. Microbiol. 1997,25: 329-341
[43]Li, Y. et al. Cell density modulates acid adaptation in Streptococcus mutans:implications for survival in biofilms. J. Bacteriol. 2001a, 183: 6875-6884
[44]Li, Y. et al. A quorum-sensing signaling system essential for genetic competencein Streptococcus mutans is involved in biofilm formation. J. Bacteriol. 2002b, 184:2699-2708
[45]Wang, B. Y. and H. K. Kuramitsu. Interactions between oral bacteria: inhibitionof Streptococcus mutans bacteriocin production by Streptococcus gordonii. Appl.Environ. Microbiol. 2005,71: 345-362
[46]Li, Y. et al. Novel two-component regulatory system involved in biofilmformation and acid resistance in Streptococcus mutans. J. Bacteriol. 2002a, 184:6333-6342
[47]Luo, P. et al. CotnX is a unique link between multiple quorum sensing outputsand competence in Streptococcus pneumoniae. Mol. Microbiol. 2003,50: 623-633
[48] Luo, P.and D, A, Morrison. Transient association of an alternative sigma factor,comX, with RNA polymerase during the period of competence for genetictransformation in Streptococcus pneumoniae. J. Bacteriol. 2003,185: 349-358
[49]Ajdic, D. et al. Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc. Natl. Acad. Sci. USA. 2002, 22: 14434-14439
[50]Senadheera, M. D. et al. A VicR/K signal transduction system in Streptococcus mutans affects gtfB, gtfC and ftf expression, biofilm formation and genetic competence development. J. Bacteriol. 2005, 187: 4064-4076
[51]Davies, J. Origins and evolution of antibiotic resistance.microbiologia. 1996, 12: 9-16
[52] Havarstein L. S. et al. Natural competence in the genus Streptococcus: evidence that streptococci can change pherotype by interspecies recombinational exchanges. J. Bacteriol. 1997, 179: 6589-6594
[53] Havarstein L. S. and D. A. Morrison. Quorum sensing and peptide pheromones in streptococcal competence for genetic transformation. In Cell-Cell signaling in Bacteria. Dunny, G. M. and Winans, S, C.,eds. ASM Press,1999, pp. 9-26
[54]Magnuson, R et al. Biochemical and genetic characterization of a competence pheromone from Bacillus subtilis. Cell. 1994, 77: 207-216
[55]Kleerebezem, M. et al. Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria. Mol. Microbiol. 1997, 24: 895-904
[56]Perry, D. and H. K. Kuramitsu. Genetic transformation of Streptococcus mutans. Infect Immun. 1981, 32: 1295-1297
[57]Kuramitsu, H. K. and C. M. Long. Plasmid-mediated transformation of Streptococcus mutans. Infect Immun. 1982, 36: 435-436
[58]Dowson, C. G. et al. Horizontal gene transfer and the evolution of resistance and virulence determinants in Streptococcus. Sco. Appl. Baxteriol. Symp. Ser. 1997, 26: 42S-51S
[59]Mercer, D. K. et al. Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DLI by plasmid DNA in human saliva. Appl. Environ. Microbiol. 1999, 65: 6-10
[60]Cvitkovitch, D. G. Genetic competence and transformation on oral streptococci. Crit. Rev. Oral. Biol. Med. 2001, 12: 217-243
[61] Cvitkovitch, D. G. Genetic exchange in biofilms. Microbial Biofilms. M. Ghannoum and G. A.O'Toole eds. ASM press. Washington, D. C. 2004, p. 192-205
[62]Marsh, P. D. Oral ecology and its impact on oral microbial diversity. Chapter 1 in Oral Bacterial Ecology: The Molecular Basis. H. Kuramitsu and R. P. Ellen, eds. Wymondham, Norfolk, UK: Horizon Scientific Press.
[63]Edgar, W. M. and S. M. Higham. Saliva and the control of plaque pH, p.81-94. In W. M. Edgar and D. M. O'Mullane(ed.), Saliva and oral health, 2~(nd)ed. British Dental Association, London, United Kingdom.
[64]Bender, G. R. et al. Acid tolerance, proton permeabilities, and membrane ATPases of oral streptococci. Infect. Immun. 1986, 53: 331-338
[65]Hamilton, I. R. and N. D. Bucklry. Adaptation by Streptococcus mutans to acid tolerance. Oral Microbiol. Immunol. 1991, 6: 65-71
[66]Belli, W. A. and R. E. Marquis. Adaptation of Streptococcus mutans and enterococcus hirae to acid stress in continuous culture. Appl. Environ. Microbiol. 1991,57:1134-1138
[67]Hahn, K. et al. Induction of an AP endonuclease activity in Streptococcus mutans during growth at low Ph. Mol. Microbiol. 1999, 31: 1489-1498
[68]Svensater G, et al. Acid tolerance response and survival by oral bacteria. Oral. Microbiol. Immunol. 1997, 12: 266-273
[69]Verhamme, D. et al. Investigation of in vivo cross-talk between key two-component systems of Escherichia coli. Microbiol. 2002, 148: 69-78
[70]Stoodley, P. et al. Biofilms as complex differentiated communities. Annu. Rev. Microbiol. 2002, 56: 187-209
[71]Loo, C. et al. Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes. J. Bacteriol. 2000, 182: 1374-1382
[72]Bhagwat, S. et al. Effects of mutating putative two component systems on biofilm formation by Streptococcus mutans UA159. FEMS Microbiol. Lett. 2001, 205: 225-230
[73]Havarstein, L. S. et al. An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA. 1995, 92: 11140-11144
[74]Lunsford, R. D. and J. London. DNA-binding activities in Streptococcus gordonii: identification of a receptor-nickase and a histone like protein. Curr. Microbiol. 1996, 32:95-100
[75]Havarstein, L. S. et al. The leader peptide of colicin V shares consensus sequences with leader peptides that are common among peptide bacteriocins produced by gram-positive bacteria. Microbiology. 1994, 140: 2383-2389
[76]Oggioni, M. R. et al. Antibacteral activity of a competence-stimulating peptide in experimental sepsis caused by Streptococcus pneumoniae. Antimicrob. Agents Chemother. 2004, 48: 4725-4732
[77]Qi, F. et al. Signal peptide pheromone induced cell death of Streptococcus mutans. Manuscript submitted. 2005.
[78] Galperin M.Y.(2004). Bacterial signal transduction network in a genomic perspective. Env microbiol 6, 552-567
[79] R(o|¨)mling U., Gomelsky M. and Galperin M.Y.(2005). C-di-GMP: the dawning of a novel bacterial signaling system. Mol microbiol 57: 629-639
[80] Ryjenkov D.A., Tarutina M., Moskvin O.V., and Gomelsky M. (2005).Cyclic Diguanylate Is a Ubiquitous Signaling Molecule in Bacteria: Insights into Biochemistry of the GGDEF Protein Domain. J Bacteriol 187, 1792-1798
[81]Tal R., Wong H.C., Calhoon R., Gelfand D., Fear A.L., Volman G., Mayer R., Ross P., Amikam D., Weinhouse H. et al(1998). Three cdg operons control cellular turnover of cyclic di-GMP in Acetobacter xylinum: genetic organization and occurrence of conserved domains in isoenzymes. J Bacteriol 180,4416-4425.
[82] R(o|¨)mling, U. (2005) Characterization of the rdar morphotype, a multicellular behaviour in Enterobacteriaceae. CLMS 62, 1-13.
[83] Tischler A. D. and Camilli A.(2004). Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation. Mol microbiol 53, 857-869
[84] Karaolis D., Rashid M., Chythanya R., Luo W., Hyodo M., and Hayakawa Y.(2005). c-di-GMP (3'-5'-Cyclic diguanylic acid) inhibits Staphylococcus aureus cell-cell interactions and biofilm formation. Antimicrob agents chemother 49, 1029-1038
[85] D'Argenio D. A. and Miller S. I.(2004) Cyclic di-GMP as a bacterial second messenger. Microbiol 150, 2497-2502.
[86] Aldridge, P., R. Paul, P. Goymer, P. Rainey, and U. Jenal.(2003). Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus. Mol. Microbiol. 47,1695-1708.
[87] Tischler A.D., Camilli A.(2005). Cyclic diguanylate regulates Vibrio cholerae virulence gene expression. Infect Immun.73, 5873-5882.
[88] Galperin M Y (2005).A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts. BMC Microbiol. 14,35
[89] Mendez-Ortiz MM, Hyodo M, Hayakawa Y, Membrillo-Hemandez J (2006). Genome wide transcriptional profile of Escherichia coli in response to high levels of the second messenger c-di-GMP.J Biol Chem. Jan 17; [Epub ahead of print]
[90]马建岗主编．基因工程学原理．西安：西安交通大学出版社，2001．148-155．
[91]白雪源，陈香美．基因打靶技术及其在生物医学中的应用．中华肾脏病杂志，2002；16(6)：403-405．
[92]陈效友，李传友，马玛，刘冲，王敬慧，张雪峰，昌增益．卡介苗菌MDP1基因敲除技术的研究．中华结核和呼吸杂志，2004；27：183-187．
[93]Bomchil.N et al Identification and Characterization of a Vibrio cholerae GenembaA Involved in Maintenance of Biofilm Architecture. J Bacteriol. 2003 February;185(4): 1384-1390.
[94]Tao L. Streptococcal integration vectors for gene inactivation and cloning.Methods Cell Sci 1998,20: 59-64.
[95] Hinds J, Mahenthiralingam E, Kempsell KE, Duncan K, Stokes RW, Parish T,Stoker NG. Enhanced gene replacement in mycobacteria. Microbiology. 1999,145(Pt 3): 519-527.
[96] Nelson DL, CoxMM. Lehninger principles of biochemistry. 3th ed. New York:Worth Publishers, 2000.931-977.
[97]Yim HH, Rubens CE. Site-specific homologous recombination mutanenesis ingroup B streptococci. Methods Cell Sci. 1998,20:13-20.
[98]Suzuki T, Tagami J and Hanada N. Role of F_1F_0-ATPase in the growth ofStreptococcus mutans GS5. J. Applied Microbiol. 2000,88: 555-562
[99] Ann SJ, Lemos JA, Burne RA. Role of HtrA in growth and competence ofStreptococcus mutans UA159. J Bacteriol.2005,187(9): 3028-38.
[100]Gainesa S, Jamesa TC, Folanc M, Bairdd AW, O'Farrelly C. A novel??spectrofluorometric microassay for Streptococcus mutans adherence to hydroxylapatite. J Microbiol Methods 2003,54: 315-323.
[101] Hao YQ, Zhou XD, Xiao XR, Lu JJ, Zhang FC, Hu T, Wu HK, Chen XM. Effects of cecropin-XJ on growth and adherence of oral cariogenic bacteria in vitro.Chin Med J(Engl). 2005,118(2): 155-60.
[115]周学东 主编．口腔生物化学．成都：四川大学出版社，2002．84-88
[116] Clark WB, Lane MD, Beem JE, Bragg SL, Wheeler TT. Relative hydrophobicities of Actinomyces viscosus and Actinomyces naeslundii strains and their adsorption to saliva-treated hydroxyapatite. Infect Immun. 1985; 47(3): 730-6.
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