Quasi-irreversible inhibition of enolase of Streptococcus mutans by flouride

Abstract Fluoride at concentrations greater than 0.01 mM was found to be a quasi-irreversible inhibitor of enolase of permeabilized cells of Streptococcus mutans GS-5 and also of isolated yeast enolase. The inhibition appeared to be of the type that has been described for P-ATPases, but was not dependent on added Al³⁺ or Be²⁺ ions. Fluoride inhibition of enolase was not reversed by repeatedly washing the permeabilized cells in chilled fluoride-free medium but could be reversed by the product, phosphoenolpyruvate, or by very high levels of the substrate, 2-phosphoglycerate. Irreversible inhibition of glycolysis was not evident after fluoride treatment of intact cells, washing to remove unbound or loosely bound fluoride and addition of glucose, presumably because intracellular levels of phosphoenolpyruvate were sufficiently high to preclude irreversible fluoride inhibition of enolase.     

变异链球菌和血链球菌全代谢途径比对分析

为了比较变异链球菌和血链球菌全代谢途径,依据KEGG数据库(http://www.genome.ad.jp/kegg)对变异链球菌和血链球菌的全部代谢途径作逐项比对。结果显示,二者参与了85个代谢途径,包括多数以相同的酶参与的中央代谢途径,即糖酵解、三羧酸循环、磷酸戊糖途径等,和多数以不同的酶参与的双组分感应系统等。通过变异链球菌和血链球菌整体代谢网络对比,了解了变异链球菌和血链球菌理论上的全部代谢途径,为全面揭示二者代谢交流研究奠定了基础。     

Streptococcus mutans glucan-binding protein-A affects Streptococcus gordonii biofilm architecture

The glucan-binding protein-A (GbpA) of Streptococcus mutans has been shown to contribute to the architecture of glucan-dependent biofilms formed by this species and influence virulence in a rat model. As S. mutans synthesizes multiple glucosyltransferases and nonglucosyltransferase glucan-binding proteins (GBPs), it is possible that there is functional redundancy that overshadows the full extent of GbpA contributions to S. mutans biology. Glucan-associated properties such as adhesion, aggregation, and biofilm formation were examined independently of other S. mutans GBPs by cloning the gbpA gene into a heterologous host, Streptococcus gordonii, and derivatives with altered or diminished glucosyltransferase activity. The presence of GbpA did not alter dextran-dependent aggregation nor the initial sucrose-dependent adhesion of S. gordonii. However, expression of GbpA altered the biofilm formed by wild-type S. gordonii as well as the biofilm formed by strain CH107 that produced primarily alpha-1,6-linked glucan. Expression of gbpA did not alter the biofilm formed by strain DS512, which produced significantly lower quantities of parental glucan. These data are consistent with a role for GbpA in facilitating the development of biofilms that harbor taller microcolonies via binding to alpha-1,6-linkages within glucan. The magnitude of the GbpA effect appears to be dependent on the quantity and linkage of available glucan.     

Carboxyl-terminal deletion analysis of the Streptococcus mutans glucosyltransferase-I enzyme

Sequential deletion of the carboxyl-terminal amino acids (including the six direct repeating units) of the glucosyltransferase-I (GTF-I) enzyme of Streptococcus mutans revealed differential effects on sucrase and GTF activities. Removal of all but one repeating unit resulted in a truncated enzyme with significant sucrase activity but no detectable GTF activity. These results are compatible with the presence of two functional domains in the enzyme.     

电转化法构建变形链球菌UA159 comD缺陷菌株

目的构建变形链球菌UAl59密度感应相关的comD基因缺陷菌株,为进一步研究该基因功能做准备。方法根据同源重组原理,利用变形链球菌UAl59comD基因同源重组DNA片段,采用电击转化方法获取转化菌落,通过形态学观察、生化特性检测、PCR及测序、RT-PCR对缺陷菌进行鉴定。结果在含有红霉素（10μg/mL）的琼脂平板上出现转化菌落,鉴定结果均显示转化菌为comD缺陷菌。结论成功构建了变形链球菌UA159comD基因缺陷菌株。     

Thermophysiology of Streptococcus mutans and related lactic-acid bacteria

The temperature ranges for growth of Streptococcus mutans GS-5 and S. sobrinus 6715 were found to be very narrow, from about 30 to 47 °C, with optimal growth around 37 °C. Thus, the organisms showed little potential to grow in the environment outside of the animal host. In contrast wider ranges were found for Enterococcus hirae, S. rattus and S. sanguis. Detailed study of S. mutans GS-5 showed that energetic coupling, reflected in yields of biomass per mol of glucose utilized, were not greatly affected by changes in temperature within the growth range. However, since glycolysis occurred over a wider temperature range (about 10 to 52 °C) than growth, yield values dropped to zero at temperatures above or below the growth range. The temperature range for glycolysis could be related to temperature sensitivity of the phosphoenolpyruvate: sugar phosphotransferase system for sugar uptake. F-ATPases were active over a similar range of temperatures, but with a broad optimal range from about 30 to 50 °C. Proton permeability of S. mutans increased steadily with temperature in a manner similar to that of other mesophilic bacteria, such as Escherichia coli. Growth of the bacteria in media supplemented with various fatty acids had major effects on proton permeabilities but the effects were not well reflected by changes in growth or glycolysis of the bacteria. The overall conclusions were that S. mutans is a typical mesophile in relation to membrane and catabolic functions but its narrow temperature range for growth is related to temperature sensitivities of anabolic systems.     

变形链球菌电击转化条件的优化研究

目的为增强对变形链球菌的电击转化效率,探索常用的胞壁弱化剂甘氨酸在电击转化中的加入模式。方法以氨苄青霉素抗性的pGL3 basic质粒作为外源DNA,通过电击转化导入变形链球菌参考株UA159内,并在选择性培养基上筛选阳性转化克隆,以优化筛选出最佳的甘氨酸加入浓度与模式,同时比较了不同电击方案转化效率的差异。结果在变形链球菌对数生长期后加入终浓度为10%的甘氨酸可有效地增强电击转化的效率;而不同的电转电压的选择对于转化效率的影响,在本实验中差异未见显著性。结论研究证实了甘氨酸作为胞壁弱化剂可增强对变形链球菌的转化效率,并优化了对变形链球菌的电击转化方案。     

Enhancement of fluoride activity against Streptococcus mutans biofilms by a substance separated from Polygonum cuspidatum

Polygonum cuspidatum is a plant with spreading rhizomes and numerous reddish-brown stems that has been used in Korean folk medicine to improve oral hygiene. Nevertheless, there are no reports related to its possible effect on the virulence of dental biofilms. In this study, the ability of a fraction (F1) separated from P. cuspidatum, alone or in combination with fluoride, to disrupt virulence factors and the composition of Streptococcus mutans biofilms was examined. F1 was mainly composed of resveratrol, emodin and physcion (approximately 16.2%, 18.9% and 2.07% of the weight of F1, respectively). F1 showed inhibitory effects on acid production and F-ATPase activity of S. mutans in biofilms, and could enhance fluoride activity against acid production and acid tolerance of S. mutans in biofilms. When S. mutans biofilms were briefly treated with F1 (10 min, a total of five times), the biomass accumulation, water-insoluble polysaccharides and intracellular iodophilic polysaccharides were reduced. Furthermore, the fluoride activity against biomass accumulation was enhanced by F1. These results suggest that F1 may be useful in the control of dental biofilms and in improving the cariostatic properties of fluoride without increasing its exposure.     

变形链球菌UA159及其基因缺陷菌生长特性变化的初步研究

目的探讨变形链球菌密度信号系统相关基因缺陷后其生长特性的变化情况。方法分别配制BHI液体培养基,含2%葡萄糖的BHI液体培养基,含2%蔗糖的BHI液体培养基,然后将细菌接种于上述3个营养环境中生长,采用722S型可见光分光光度计,进行细菌生长曲线的测定。结果在3种不同的营养环境中,变形链球菌UA159△ComD基因缺陷菌株的生长速度最快,变形链球菌UA159菌株变形链球菌UA159△LuxS基因变异株次之,变形链球菌UA159野生菌株最慢。结论在本实验中,无论那种密度感应信号系统被阻断后,均会影响变形链球菌的生长。     

Glucan-binding proteins are essential for shaping Streptococcus mutans biofilm architecture

Glucan plays a central role in sucrose-dependent biofilm formation by the dental pathogen Streptococcus mutans. This organism synthesizes several proteins capable of binding glucan. These are divided into the glucosyltransferases that catalyze the synthesis of glucan and the nonglucosyltransferase glucan-binding proteins (Gbps). The biological significance of the Gbps has not been thoroughly defined, but studies suggest that these proteins influence virulence and play a role in maintaining biofilm architecture by linking bacteria and extracellular molecules of glucan. We engineered a panel of Gbp mutants, targeting GbpA, GbpC, and GbpD, in which each gene encoding a Gbp was deleted individually and in combination. These strains were then analyzed by confocal microscopy and the biofilm properties were quantified by the biofilm quantification software comstat. All biofilms produced by mutant strains lost significant depth, but the basis for the reduction in height depended on which particular Gbp was missing. The loss of the cell-bound GbpC appeared dominant as might be expected based on losing the principal receptor for glucan. The loss of an extracellular Gbp, either GbpA or GbpD, also profoundly changed the biofilm architecture, each in a unique manner.     

Genome sequence of Streptococcus mutans bacteriophage M102

Bacteriophage M102 is a lytic phage specific for serotype c strains of Streptococcus mutans, a causative agent of dental caries. In this study, the complete genome sequence of M102 was determined. The genome is 31,147 bp in size and contains 41 ORFs. Most of the ORFs encoding putative phage structural proteins show similarity to those from bacteriophages from Streptococcus thermophilus. Bioinformatic analysis indicated that the M102 genome contains an unusual lysis cassette, which encodes a holin and two lytic enzymes.     

The regulation of Streptococcus mutans glucan-binding protein A expression

The S. mutans GBP-A is hypothesized to be constitutively expressed and to contribute to the sucrose-dependent colonization of S. mutans. To investigate GBP-A expression, a reporter gene encoding chloramphenicol acetyltransferase (CAT) was placed downstream of the gbpA promoter and CAT activity was measured under conditions that would be associated with the sucrose-dependent colonization of S. mutans. Expression of GBP-A was optimal under anaerobiosis and neutral pH conditions, and correlated with optimal growth. The addition of sucrose to the growth medium did not elevate the expression of GBP-A.     

Tea extracts differentially inhibit Streptococcus mutans and Streptococcus sobrinus biofilm colonization depending on the steeping temperature

Abstract

This study aimed to evaluate the effects of tea extracts on oral biofilm colonization depending on steeping temperature. S. mutans and S. sobrinus were cultured and treated with green or black tea extracts prepared under different steeping conditions. Biofilm formation, glucosyltransferase (GTF) levels, bacterial growth, and acidogenicity were evaluated. Biofilms were also assessed by gas chromatography-mass spectrometry and confocal laser scanning microscopy. All extracts with hot steeping showed higher inhibitory effects on biofilm formation and cell viability and lower GTF levels compared with those with cold steeping (p?<?0.05). Hot steeping significantly reduced bacterial growth (p?<?0.05) and maintained the pH. Catechins were only identified from hot steeping extracts. Within the limits of this study, extracts with cold steeping showed lower inhibitory effects on oral biofilms. The different effects between steeping extracts may be attributed to the difference in catechins released from tea extracts under the different steep conditions.     

Acid tolerance response of biofilm cells of Streptococcus mutans

Streptococcus mutans, a major etiological agent of dental caries, is a component of the dental plaque biofilm and functions during caries progression in acidic lesions that may be at or below pH 4. In this study, we were interested in determining the acid tolerance of 1-7-day chemostat-grown biofilm cells of S. mutans BM71 growing in a semi-defined medium at a rate consistent with that of cells in dental plaque (dilution rate=0.1 h(-1)), as well as, assessing the capacity of 2- and 5-day biofilms to induce an acid tolerance response that would enhance survival at a killing pH (3.5). As expected, biofilm cell growth increased (2.5-fold) from day 1 to day 7 (10.6-25.7 x 10(6) cells cm(-)(2)) with the percentage live cells over that period averaging 79.4%, slightly higher than that of planktonic cells (77.4%). Biofilms were highly resistant to acid killing at pH 3.5 for 2 h with survival ranging from 41.8 (1 day) to 63.9% (7 day), while the percentage of live cells averaged 43.4%. Planktonic and dispersed biofilm cells were very acid-sensitive with only 0.0009%- and 0.0002-0.2% survivors, respectively. Unlike the planktonic cells, the incubation of 2- and 5-day biofilms at pH 5.5 for periods of up to 6 h induced strong acid tolerance responses that enhanced survival during a subsequent exposure to acid killing at pH 3.5.     

氟对正畸儿童牙面菌斑变形链球菌的影响

目的:观察和探讨2%氟化钠对正畸患者牙面菌斑内细菌总数及变形链球菌数的影响.方法:选择34例正畸儿童,分为两组,试验组涂布2%氟化钠;对照组不做处理.分别于戴用矫治器前,戴入后1月采集上颌牙唇颊面菌斑,测定菌斑中细菌总数及变形链球菌数.结果:对照组戴用后1月,细菌总数及变形链球菌数较戴用前明显增加(P＜0.01).对照组与试验组戴用后1月相比,试验组细菌总数及变形链球菌数明显少于对照组(P＜0.05).结论:戴用固定矫治器后,牙面菌斑内细菌总数及变形链球菌数较戴用前增加,应用2%氟化钠可明显抑制正畸患者口腔内变形链球菌数,减少龋坏发生.     

Differential localization of the Streptococcus mutans GS-5 fructan hydrolase enzyme, FruA

Abstract Streptococcus mutans GS-5 synthesizes an exo-β-d-fructosidase, FruA, capable of degrading levans, inulins, sucrose and raffinose, with the greatest activity on levans. A previous analysis of the deduced amino acid sequence of the FruA protein revealed the presence of a C-terminus with an LPXTGX membrane sorting sequence and membrane spanning domain, characteristic of many Gram-positive cocci surface proteins. Here it is demonstrated that FruA, which had been previously shown to exist almost exclusively as an extracellular enzyme, can be detected in significant proportions at the surface of S. mutans cells. Moreover, growth of S. mutans GS-5 at steady state in continuous culture at pH values of 7.0, 6.0, or 5.0 revealed that the amount of cell-associated enzyme increased with decreasing pH values, such that roughly 50% of the total fructanase activity of pH 5.0-grown organisms was cell-associated. This result was confirmed using anti-recombinant-FruA antisera in Western blotting of culture supernate and cell-associated enzyme preparations from chemostat-grown cells. Incubation of S. mutans at pH values of 5.0, 6.0 or 7.0 in buffered media yielded results similar to those observed in the chemostat experiments. The release of FruA from S. mutans was also shown to be inhibitable by copper, which is known to interfere with the release of the surface adhesin, P1, from intact cells and protoplasts of S. mutans . These data provide evidence for a unique post-translational mechanism for the regulation of the catabolism of polysaccharides by bacteria. The control of degradation of plaque fructans by modulation of the release of the fructanase enzyme from S. mutans may play a critical role in the temporal and spatial separation of the synthesis and degradation of dental plaque fructans.     

Complete nucleotide sequence of the sr gene from Streptococcus mutans OMZ 175

The nucleotide sequence encoding the SR protein of Streptococcus mutans OMZ 175 (serotype f) has been determined. The sr gene consists of 4667 bp and codes for a 171177 Da protein. Comparison of the inferred amino acid sequence with the one of PAc antigen from S. mutans MT 8148 (serotype c) indicates a 88% conservation of amino acid residues which reflects the close relatedness of both proteins. Major differences in amino acid composition are located at the C-terminal part of the sequence where only 298 amino acids of the terminal 420 are conserved.     

DnaK expression in response to heat shock of Streptococcus mutans

Abstract The oral pathogen, Streptococcus mutans , persistently colonizes human hosts and initiates oral disease despite extreme variations in environmental conditions. To begin to investigate the role of the stress protein, DnaK (Hsp70), in environmental stress responses by S. mutans , pulse—chase experiments were initially used to establish that a functional heat shock response existed in this organism. A C-terminal fragment of the S. mutans dnaK gene was cloned and engineered to be expressed with a histidine tag. Using the recombinant DnaK protein that had been purified by nickel affinity chromatography, an antibody specific for the S. mutans DnaK protein was generated to analyse DnaK expression under homeostatic and heat shock conditions. Western blot analysis indicated that the anti-recombinant DnaK antibody specifically recognized a protein (molecular mass approx. 68 kDa) which was induced in response to thermal stress. Elucidating the role of DnaK in responses by S. mutans to various environmental Stressors will provide a better understanding of how DnaK is involved in survival of extreme environments and the contribution of the DnaK protein to the virulence of S. mutans .     

Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ?lytS and ?lytT; LTA – ?dltA and ?dltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.     

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis

Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.