Establishing a genetic system for ecological studies of Streptococcus oligofermentans

Streptococcus oligofermentans is a newly characterized species belonging to the mitis group of oral streptococci. So far no correlation has been demonstrated between S. oligofermentans and dental caries. Furthermore, a reverse correlation has been observed between the number of S. oligofermentans and the number of Streptococcus mutans, a major cariogenic pathogen, in the oral cavity. These properties suggest that S. oligofermentans may have a potential to be used as a 'probiotics' for caries prevention. In this study, we aim to establish a genetic system in S. oligofermentans to further study the biology of this new species. Using homologous regions of the comCDE locus in other streptococci, the comC gene was isolated and sequenced. A synthetic competence-stimulating peptide (CSP) was synthesized and shown to be able to effectively induce competence in S. oligofermentans. This CSP-induced transformation system in S. oligofermentans was used to construct green fluorescent protein (gfp) and luciferase (luc) reporter systems, both of which are driven by the lactate dehydrogenase (ldh) promoter. These reporter systems were further shown to be highly expressed in planktonic and biofilm cells, suggesting that these reporter systems can be used in future ecological studies of S. oligofermentans.     

Cell Density Modulates Acid Adaptation in Streptococcus mutans: Implications for Survival in Biofilms

Streptococcus mutans normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates. The ability of S. mutans to survive at low pH is an important virulence factor in the pathogenesis of dental caries. Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of S. mutans growing in high-cell-density biofilms. It is unknown whether biofilm growth mode or high cell density affects acid adaptation by S. mutans. This study was initiated to examine the acid tolerance response (ATR) of S. mutans biofilm cells and to determine the effect of cell density on the induction of acid adaptation. S. mutans BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation. Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, followed by a pH 3.5 acid shock for 3 h. Both biofilm and planktonic cells were removed to assay percentages of survival. The results showed that S. mutans BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation. Cell density was found to modulate acid adaptation in S. mutans log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation in S. mutans. Heat or proteinase treatment abolished the induction by the culture filtrate. The results also showed that mutants defective in the comC, -D, or -E genes, which encode a quorum sensing system essential for cell density-dependent induction of genetic competence, had a diminished log-phase ATR. Addition of synthetic competence stimulating peptide (CSP) to the comC mutant restored the ATR. This study demonstrated that cell density and biofilm growth mode modulated acid adaptation in S. mutans, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication.     

Natural genetic transformation of Streptococcus mutans growing in biofilms

Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm "lifestyle" for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.     

Firefly luciferase as a reporter to study gene expression in Streptococcus mutans.

The utility of firefly luciferase as a reporter was tested in Streptococcus mutans. Under control of an endogenous promoter, the luciferase coding sequence was strongly expressed, while a promoterless version was indistinguishable from the background. Luciferase activity was easily extracted and the assay rapid and reproducible. In addition, the half-life of luciferase activity was found to be comparable to those of other frequently used reporters. Thus, firefly luciferase can readily be used as a reporter in S. mutans, a useful alternative to methods requiring radioactive isotopes.     

Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa.

Reporter gene technology was employed to detect the activity of an alginate promoter of Pseudomonas aeruginosa when the organism was grown as a biofilm on a Teflon mesh substratum and as planktonic cells in liquid medium. Alginate biosynthetic activity was determined with a mucoid cell line derived from a cystic fibrosis isolate and containing an alginate algC promoter fused to a lacZ reporter gene. Reporter activity was demonstrated with chromogenic and fluorogenic substrates for beta-galactosidase. Expression of algC was shown to be upregulated in biofilm cells compared with planktonic cells in liquid medium. Gene up-expression correlated with alginate biosynthesis as measured by Fourier transform infrared spectroscopy, uronic acid accumulation, and alginate-specific enzyme-linked immunosorbent assay. The algC promoter was shown to have maximum activity in planktonic cultures during the late lag and early log phases of the cell growth cycle. During a time course experiment, biofilm algC activity exceeded planktonic activity except during the period immediately following inoculation into fresh medium. In continuous-culture experiments, conversion of lacZ substrate was demonstrated microscopically in individual cells by epifluorescence microscopy.     

An in vitro reporter gene assay method incorporating metabolic activation with human and rat S9 or liver microsomes

A metabolic activation system with an S9 fraction or liver microsomes was applied to a reporter gene assay in vitro for the screening of estrogenicity of chemicals. The endpoint (luciferase) was luciferase induction in cells transfected with a reporter plasmid containing an estrogen-responsive element linked to the luciferase gene. Compounds were applied to the reporter gene assay system after pretreatment or simultaneous treatment with an S9 fraction or liver microsomes. Both trans-stilbene and methoxychlor themselves showed no or little estrogenicity, but when they were treated with an S9 fraction or liver microsomes, they demonstrated strong effects, indicating their metabolites to be estrogenic. When four pyrethroid insecticides were subjected to this assay system, however, they showed no estrogenicity even with liver microsome or S9 mix treatment.     

Functional genomics approach to identifying genes required for biofilm development by Streptococcus mutans

Streptococcus mutans, the primary etiological agent of human dental caries, is an obligate biofilm-forming bacterium. The goals of this study were to identify the gene(s) required for biofilm formation by this organism and to elucidate the role(s) that some of the known global regulators of gene expression play in controlling biofilm formation. In S. mutans UA159, the brpA gene (for biofilm regulatory protein) was found to encode a novel protein of 406 amino acid residues. A strain carrying an insertionally inactivated copy of brpA formed longer chains than did the parental strain, aggregated in liquid culture, and was unable to form biofilms as shown by an in vitro biofilm assay. A putative homologue of the enzyme responsible for synthesis of autoinducer II (AI-2) of the bacterial quorum-sensing system was also identified in S. mutans UA159, but insertional inactivation of the gene (luxS(Sm)) did not alter colony or cell morphology or diminish the capacity of S. mutans to form biofilms. We also examined the role of the homologue of the Bacillus subtilis catabolite control protein CcpA in S. mutans in biofilm formation, and the results showed that loss of CcpA resulted in about a 60% decrease in the ability to form biofilms on an abiotic surface. From these data, we conclude that CcpA and BrpA may regulate genes that are required for stable biofilm formation by S. mutans.     

Acid tolerance of biofilm cells of Streptococcus mutans

Streptococcus mutans, a member of the dental plaque community, has been shown to be involved in the carious process. Cells of S. mutans induce an acid tolerance response (ATR) when exposed to sublethal pH values that enhances their survival at a lower pH. Mature biofilm cells are more resistant to acid stress than planktonic cells. We were interested in studying the acid tolerance and ATR-inducing ability of newly adhered biofilm cells of S. mutans. All experiments were carried out using flow-cell systems, with acid tolerance tested by exposing 3-h biofilm cells to pH 3.0 for 2 h and counting the number of survivors by plating on blood agar. Acid adaptability experiments were conducted by exposing biofilm cells to pH 5.5 for 3 h and then lowering the pH to 3.5 for 30 min. The viability of the cells was assessed by staining the cells with LIVE/DEAD BacLight viability stain. Three-hour biofilm cells of three different strains of S. mutans were between 820- and 70,000-fold more acid tolerant than corresponding planktonic cells. These strains also induced an ATR that enhanced the viability at pH 3.5. The presence of fluoride (0.5 M) inhibited the induction of an ATR, with 77% fewer viable cells at pH 3.5 as a consequence. Our data suggest that adhesion to a surface is an important step in the development of acid tolerance in biofilm cells and that different strains of S. mutans possess different degrees of acid tolerance and ability to induce an ATR.     

Competence-dependent endogenous DNA rearrangement and uptake of extracellular DNA give a natural variant of Streptococcus mutans without biofilm formation

The production of water-insoluble glucan (WIG) enables Streptococcus mutans to survive and persist in the oral niche. WIG is produced from sucrose by glucosyltransferase encoded tandemly by the highly homologous gtfB and gtfC genes. Conversely, a single hybrid gene from the endogenous recombination of gtfB and gtfC is easily generated using RecA, resulting in S. mutans UA159 WIG- (rate of ～1.0×10(-3)). The pneumococcus recA gene is regulated as a late competence gene. comX gene mutations did not lead to the appearance of WIG- cells. The biofilm collected from the flow cell had more WIG- cells than among the planktonic cells. Among the planktonic cells, WIG- cells appeared after 16 h and increased ～10-fold after 32 h of cultivation, suggesting an increase in planktonic WIG- cells after longer culture. The strain may be derived from the biofilm environment. In coculture with donor WIG+ and recipient WIG- cells, the recipient cells reverted to WIG+ and acquired an intact gtfBC region from the environment, indicating that the uptake of extracellular DNA resulted in the phenotypic change. Here we demonstrate that endogenous DNA rearrangement and uptake of extracellular DNA generate WIG- cells and that both are induced by the same signal transducer, the com system. Our findings may help in understanding how S. mutans can adapt to the oral environment and may explain the evolution of S. mutans.     

表达萤光素酶的HIV药物筛选细胞模型的建立

目的:构建基于萤光素酶的单次复制人免疫缺陷病毒(HIV)细胞模型,用于抗HIV药物的筛选。方法:构建含萤光素酶报告基因的假型慢病毒质粒,将疱疹性口炎病毒外膜糖蛋白(VSV-G)的表达质粒、HIV-1 Rev蛋白表达质粒、HIV Gag-Pol蛋白表达质粒和含萤光素酶报告基因的重组慢病毒质粒共转染HEK 293FT细胞,制备假型慢病毒;在假型慢病毒生产和再感染新鲜HEK 293FT细胞的过程中加入逆转录酶和蛋白酶抑制剂(如AZT),检测再感染的细胞中萤光素酶的表达水平,从而判断药物对HIV的抑制作用。结果:构建了含萤光素酶报告基因的重组慢病毒质粒pLenti-Luc;利用已知抗HIV药物AZT进行测试,发现HIV药物处理组细胞中萤光素酶活性远低于对照组。结论:建立了基于萤光素酶的HIV药物筛选细胞模型,该系统使用单次复制的报告病毒,具有良好的安全性,而使用萤光素酶基因作为报告基因使该系统具备极高的敏感性,该系统适合于进行高通量药物筛选。     

Both lactoferrin and iron influence aggregation and biofilm formation in Streptococcus mutans

Streptococcus mutans, a gram-positive immobile bacterium, is an oral pathogen considered to be the principal etiologic agent of dental caries. Although some researches suggest that trace metals, including iron, can be associated with dental caries, the function of salivary iron and lactoferrin in the human oral cavity remains unclear. The data reported in this study indicates that iron-deprived saliva (Fe3+ < 0.1 microM) increases S. mutans aggregation and biofilm formation in the fluid and adherent phases as compared with saliva (Fe3+ from 0.1 to 1 microM), while iron-loaded saliva (Fe3+ > 1 microM) inhibits both phenomena. Our findings are consistent with the hypothesis that S. mutans aggregation and biofilm formation are negatively iron-modulated as confirmed by the different effect of bovine lactoferrin (bLf), added to saliva at physiological concentration (20 microg/ml) in the apo- or iron-saturated form. Even if saliva itself induces bacterial aggregation, iron binding capability of apo-bLf is responsible for the noticeable increase of bacterial aggregation and biofilm development in the fluid and adherent phases. On the contrary, iron-saturated bLf decreases aggregation and biofilm development by supplying iron to S. mutans. Therefore, the iron-withholding capability of apo-Lf or native Lf is an important signal to which S. mutans counteracts by leaving the planktonic state and entering into a new lifestyle, biofilm, to colonize and persist in the human oral cavity. In addition, another function of bLf, unrelated to its iron binding capability, is responsible for the inhibition of the adhesion of S. mutans free, aggregated or biofilm on abiotic surfaces. Both these activities of lactoferrin, related and unrelated to the iron binding capability, could have a key role in protecting the human oral cavity from S. mutans pathogenicity.     

Yeast mutant with efficient secretion identified by a novel secretory reporter, Cluc

Yeast is an important host for the production of pharmaceutical or industrial proteins by virtue of its genetic information and easy handling. A number of heterologous proteins have been produced and purified from yeast cell cultures as secreted forms. Here, we describe a novel screening system of Saccharomyces cerevisiae and its application to improve the secretion efficiency of yeast. In our system, a natural secretory luciferase from Cypridina noctiluca is used as a reporter enzyme. The accumulation of enzymatically active luciferase in culture medium makes it possible to screen many samples simultaneously in a simple and sensitive assay. Using this system, we have discovered that the deletion mutant of MON2, which encoded a scaffold protein for vesicle formation located at the late Golgi, secreted luciferase highly efficiently to the extracellular space. Thus, we conclude that this new reporter assay is useful for the improvement and screening of yeast secretory strains.     

Protein expression by planktonic and biofilm cells of Streptococcus mutans

Streptococcus mutans, a major causal agent of dental caries, functions in nature as a component of a biofilm on teeth (dental plaque) and yet very little information is available on the physiology of the organism in such surface-associated communities. As a consequence, we undertook to examine the synthesis of proteins by planktonic and biofilm cells growing in a biofilm chemostat at pH 7.5 at a dilution rate of 0.1 h(-1) (mean generation time=7 h). Cells were incubated with (14)C-labelled amino acids, the proteins extracted and separated by two-dimensional electrophoresis followed by autoradiography and computer-assisted image analysis. Of 694 proteins analysed, 57 proteins were enhanced 1.3-fold or greater in biofilm cells compared to planktonic cells with 13 only expressed in sessile cells. Diminished protein expression was observed with 78 proteins, nine of which were not expressed in biofilm cells. The identification of enhanced and diminished proteins by mass spectrometry and computer-assisted protein sequence analysis revealed that, in general, glycolytic enzymes involved in acid formation were repressed in biofilm cells, while biosynthetic processes were enhanced. The results show that biofilm cells possess novel proteins, of as yet unknown function, that are not present in planktonic cells.     

伊犁黑蜂蜂胶对不同状态下变形链球菌乳酸脱氢酶及其相关基因影响的实验研究

目的目的通过新疆伊犁黑蜂蜂胶乙醇提取物(Ethanol Extract of Propolis,EEP)对不同状态下变形链球菌乳酸脱氢酶活性及其相关基因表达影响的作用,研究伊犁黑蜂蜂胶抑制变形链球菌产酸的原因并探讨其可能的防龋机制。方法 (1)分别培养浮游状态与生物膜状态下生长的变形链球菌,根据实验分组用含梯度浓度EEP的BHI培养基、50 mg/L氟化钠的BHI培养基作用18 h,通过还原性辅酶I氧化法测定乳酸脱氢酶活性。(2)分别培养浮游状态与生物膜状态下生长变形链球菌,根据实验分组用含梯度浓度EEP的BHI培养基、含50 mg/L氟化钠的BHI培养基作用18 h,反转录-实时荧光定量PCR(RTq PCR)法测定各组乳酸脱氢酶编码基因ldh表达情况。结果 (1)在浮游状态与生物膜状态下,EEP组和Na F组乳酸脱氢酶活性均有降低,差异具有统计学意义(P0.05)。(2)浮游状态时,实验组组和阳性对照组ldh表达明显受到抑制(P0.05);生物膜状态下,实验组在1 MBEC、1/2 MBEC、1/4 MBEC浓度时ldh表达受到抑制(P0.05),Na F组ldh表达差异没有统计学意义(P0.05)。结论伊犁黑蜂蜂胶能够抑制浮游状态与生物膜状态下变形链球菌乳酸脱氢酶活性及其编码基因ldh表达,来抑制细菌产酸,伊犁黑蜂蜂胶可能是通过此途径抑制变形链球菌产酸,从而达到防龋的效果。     

Inhibition of biofilm formation, quorum sensing and infection in Pseudomonas aeruginosa by natural products-inspired organosulfur compounds

Using a microplate-based screening assay, the effects on Pseudomonas aeruginosa PAO1 biofilm formation of several S-substituted cysteine sulfoxides and their corresponding disulfide derivatives were evaluated. From our library of compounds, S-phenyl-L-cysteine sulfoxide and its breakdown product, diphenyl disulfide, significantly reduced the amount of biofilm formation by P. aeruginosa at levels equivalent to the active concentration of 4-nitropyridine-N-oxide (NPO) (1 mM). Unlike NPO, which is an established inhibitor of bacterial biofilms, our active compounds did not reduce planktonic cell growth and only affected biofilm formation. When used in a Drosophila-based infection model, both S-phenyl-L-cysteine sulfoxide and diphenyl disulfide significantly reduced the P. aeruginosa recovered 18 h post infection (relative to the control), and were non-lethal to the fly hosts. The possibility that the observed biofilm inhibitory effects were related to quorum sensing inhibition (QSI) was investigated using Escherichia coli-based reporters expressing P. aeruginosa lasR or rhIR response proteins, as well as an endogenous P. aeruginosa reporter from the lasI/lasR QS system. Inhibition of quorum sensing by S-phenyl-L-cysteine sulfoxide was observed in all of the reporter systems tested, whereas diphenyl disulfide did not exhibit QSI in either of the E. coli reporters, and showed very limited inhibition in the P. aeruginosa reporter. Since both compounds inhibit biofilm formation but do not show similar QSI activity, it is concluded that they may be functioning by different pathways. The hypothesis that biofilm inhibition by the two active compounds discovered in this work occurs through QSI is discussed.     

Bioluminescent Mycobacterium aurum expressing firefly luciferase for rapid and high throughput screening of antimycobacterial drugs in vitro and in infected macrophages

The slow growth and highly infectious nature of Mycobacterium tuberculosis is a limiting factor in its use as test organism in high throughput screening for inhibitory compounds. To overcome these problems, use of surrogate strains and reporter genes have been considered. In this study, we have investigated the application of a fast growing nonpathogenic M. aurum expressing firefly luciferase in rapid screening of antituberculosis compounds in vitro and in infected macrophages using bioluminescence assay. The assay is based on luminescence determination using luciferin as substrate. Inhibition of bioluminescence was obtained with frontline antimycobacterial drugs like streptomycin, rifampicin, isoniazid, ethambutol, ofloxacin, and sparfloxacin at their reported MICs. Inhibition could be observed as early as 2 h in vitro and within 24 h in infected macrophages. The system can reliably be used in high throughput screening.     

Systemic inactivation and phenotypic characterization of two-component systems in expression of Streptococcus mutans virulence properties

AIM: To assess potential function of each two-component signal transduction system in the expression of Streptococcus mutans virulence properties. METHODS AND RESULTS: For each two-component system (TCS), the histidine kinase-encoding gene was inactivated by a polymerase chain reaction (PCR)-based deletion strategy and the effects of gene disruption on the cell's ability to form biofilms, become competent, and tolerate acid, osmotic, and oxidative stress conditions were tested. Our results demonstrated that none of the mutations were lethal for S. mutans. The TCS-2 (CiaRH) is involved in biofilm formation and tolerance to environmental stresses, the TCS-3 (ScnRK-like) participates in the survival of cells at acidic pH, and the TCS-9 affects the acid tolerance response and the process of streptococcal competence development. CONCLUSIONS: Our results confirmed the physiological role of the TCS in S. mutans cellular function, in particular the SncRK-like TCS and TCS-9 as they may represent new regulatory systems than can be involved in S. mutans pathogenesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiple TCS govern important biological parameters of S. mutans enabling its survival and persistence in the biofilm community.     

Evaluating the Metal Tolerance Capacity of Microbial Communities Isolated from Alberta Oil Sands Process Water

Anthropogenic activities have resulted in the intensified use of water resources. For example, open pit bitumen extraction by Canada’s oil sands operations uses an estimated volume of three barrels of water for every barrel of oil produced. The waste tailings–oil sands process water (OSPW)–are stored in holding ponds, and present an environmental concern as they are comprised of residual hydrocarbons and metals. Following the hypothesis that endogenous OSPW microbial communities have an enhanced tolerance to heavy metals, we tested the capacity of planktonic and biofilm populations from OSPW to withstand metal ion challenges, using Cupriavidus metallidurans, a known metal-resistant organism, for comparison. The toxicity of the metals toward biofilm and planktonic bacterial populations was determined by measuring the minimum biofilm inhibitory concentrations (MBICs) and planktonic minimum inhibitory concentrations (MICs) using the MBEC ™ assay. We observed that the OSPW community and C. metallidurans had similar tolerances to 22 different metals. While thiophillic elements (Te, Ag, Cd, Ni) were found to be most toxic, the OSPW consortia demonstrated higher tolerance to metals reported in tailings ponds (Al, Fe, Mo, Pb). Metal toxicity correlated with a number of physicochemical characteristics of the metals. Parameters reflecting metal-ligand affinities showed fewer and weaker correlations for the community compared to C. metallidurans, suggesting that the OSPW consortia may have developed tolerance mechanisms toward metals present in their environment.