subtilis, where pckA was shown to be under indirect control of CcpA [32]. The pentose phosphate pathway, selleck compound an alternative glucose degradation pathway in S. aureus [30], provides the cell with NADPH and precursors for biomass, which are needed in many anabolic reactions. gntRKP was the only operon of the pentose phosphate pathway we found to be regulated at least partially by CcpA (Table 3). When glucose is depleted from the medium, S. aureus reintroduces products of carbon overflow, such as acetate or acetoin, into central metabolism [33, 34]. The genes for acetolactate

synthase (alsS) and acetolactate decarboxylase (alsD), both involved in acetoin production, were up-regulated by glucose (Table 3). Although up-regulation was found in wild-type and ΔccpA mutant, it was three times higher in the wild-type, indicating a substantial contribution of CcpA in alsD and alsS transcription in response to glucose. ROCK inhibitor While the amount of acetate in the medium increased upon glucose addition in

both, wild-type and mutant (Fig. 1), we neither observed an increase in transcription of genes encoding proteins being involved in acetate formation (i.e. phosphotransacetylase [pta] and acetate RG7112 chemical structure kinase [ackA]), nor of genes with products responsible for acetate and acetoin utilization (i.e. acetyl-CoA synthetase [acsA], acetoin dehydrogenase [acuA], and the acetoin utilization protein [acuC]). In the presence of glucose, CcpA repressed several genes of the TCA cycle, including aconitate hydratase (citB), isocitrate dehydrogenase (citC), and citrate synthase (citZ), Fossariinae confirming previous findings [23]. Also succinate dehydrogenase (sdhB), succinyl-CoA synthetase (sucCD), and 2-oxoglutarate dehydrogenase (odhAB) were repressed by glucose

in a CcpA-dependent manner (Fig. 4, Additional file 3: CcpA-dependent down-regulation by glucose). The majority of promoter regions of these genes contained a putative cre-site (see Additional file 3: CcpA-dependent down-regulation by glucose), indicating that the TCA cycle is under direct control of CcpA. The pdhABCD operon, coding for the pyruvate dehydrogenase complex, which links glycolysis to the TCA cycle by converting pyruvate to acetyl-CoA, was not found to be regulated by CcpA in S. aureus. S. aureus is able to use amino acids as secondary carbon sources. However, this is not necessary in the presence of high amounts of glucose. Accordingly, we found that several genes coding for enzymes of amino acid degradation (rocA, arg, rocD, glnA, hutI, hutU, aldA, ald, gudB, SA1365, SA1366, SA1367) were repressed by glucose in a CcpA-dependent fashion (see Additional file 3: CcpA-dependent down-regulation by glucose).

Finally, the cells, wells, and membranes were washed with PBS. For FACS analysis, the cells were fixed with 2% p-formaldehyde. Then absorbance at 450 nm (ELISA), chemiluminescence (dot-blotting analysis), or fluorescence (FACS; Excalibur, Beckton Dickinson) were detected. Biofilm formation Homotypic biofilm formation by P. gingivalis was performed as described by others [50]. Briefly, P. gingivalis cells were grown on ABA plates, then in BM supplemented with hemin or dipyridyl to OD660 = 1.0 and used to inoculate fresh LY2606368 cultures to OD660 = 0.1. Cells in the appropriate medium were transferred (200

μl) into sterile round-bottom microtiter plates (Sarstedt) and incubated under anaerobic conditions at 37°C for 24 or 48 h. The resulting biofilms were washed with PBS, stained with I-BET151 1% crystal violet, washed with PBS, and de-stained with 96% ethanol. Absorbance (A) was determined at 570 nm using a Multiskan Ascent microplate reader. The assays were repeated at least three times with each strain ZD1839 purchase grown in eight wells. To confirm that the P. gingivalis cells were viable, the biofilm cells were scrapped into the respective medium and the OD at 660 nm and colony-forming

unit (CFU) values were evaluated after 24 and 48 h (see Additional file 3). In parallel, bacteria were grown in planktonic form and the OD at 660 nm and CFU values were measured after 24 and 48 h. Growth and biofilm inhibition studies Bacteria were grown overnight on ABA plates and then in BM supplemented with hemin or dipyridyl to OD660 = 1.0. After centrifugation, the bacteria were washed and suspended in PBS to OD660 = 0.1. Then

5 ml of the bacterial suspension was centrifuged and the bacteria were incubated in 200 μl of PBS for 1 h at 37°C with the IgG fraction purified from pre-immune or immune anti-HmuY rabbit serum (200 ng). After addition of 5 ml of the appropriate medium, planktonic bacterial growth was monitored by measuring the OD at 660 nm or biofilm formed as described above. Assays were performed three times in duplicate. AZD9291 supplier Statistical analysis Data are expressed as means values ± standard deviations (mean ± SD). Statistical analysis was performed using unpaired Student’s t test (GraphPad Prism 5). Values of p < 0.05 were considered statistically significant. Acknowledgements This work was supported in part by grant nos. N401 029 32/0742, N N303 406136, and N N303 518438 from the Ministry of Science and Higher Education, and by Wroclaw Research Center EIT+ under the project “”Biotechnologies and advanced medical technologies – BioMed”" (POIG 01.01.02-02-003/08/00) financed from the European Regional Development Fund (Operational Program Innovative Economy, 1.1.2) (TO) and the European Social Fund (Human Capital Program, 8.2.

Analysis of the spectra using the CDSSTR variable selection method gave secondary structure estimates of 58% helix, 8% strand, 16% turns and 18% unordered structure. The normalized check details root mean standard deviation (NRMSD) for the estimates provides a goodness-of-fit measure of the correspondence between the experimental and calculated spectra (Fig. 7A); we obtained a NRMSD value of 0.011, which suggests a very accurate prediction of the secondary

structure. However, this prediction depends ultimately on how closely the reference dataset proteins used to derive the calculated spectra share structural similarity to Pam [13]. Figure 7 Structural properties of Pam. (A) Graphical output of far-UV CD data for Pam reveals that experimental data (green crosses) Pevonedistat cost and calculated spectrum (blue boxes), derived from the calculated output secondary structure, show agreement. The difference spectrum (purple lines) is very close to zero throughout the wavelength range,

indicating the goodness of fit of the structural predictions. The CD data indicate that Pam is largely helical (58%), with only a small fraction of residues forming β-strands. (B) Thermal stability of Pam measured by differential scanning Selleck PD0332991 calorimetry. The normalised thermal transition curve (red line) shows energy uptake by Pam reached a peak (Tm) at 77.4°C, representing the temperature at which 50% of the protein molecules are unfolded. This was almost identical after cooling the sample and repeating (black line). The temperature stability of Pam was measured using DSC. Energy changes in purified recombinant protein were recorded as the sample was heated at a constant rate from 20°C to 95°C. The sample was then allowed to cool before the analysis was repeated. The thermal transition curve measured for Pam reveals two things: firstly, the Methocarbamol protein is relatively thermostable, not undergoing a change in enthalpy until the

temperature of the system was above 60°C, and reaching a transition midpoint at 77.4°C. Above this midpoint, energy is released and the thermal profile drops toward the baseline (Fig. 7B). Secondly, upon reheating Pam follows a similar profile, except for a slight shoulder between approximately 60°C and 70°C. This shoulder is indicative of misfolding, with the protein not making all of its native contacts, but its magnitude suggests that the protein was largely able to refold to its original conformation and unfold at a rate identical to that measured in the first scan. Discussion We have studied a previously identified protein (Plu1537, here renamed Pam) which in P. asymbiotica ATCC43949 is secreted in a temperature-dependent manner, suggestive of a host-specific role in insects. In the closely related insect-only pathogen P.

Furthermore, significant changes in the organic carbon LGK 974 can also be one of the important soil factors to cause temporal shifts in the actinomycetal community, since changes in the microbial community are correlated with organic carbon content [45]. Changes in the other soil variables (mineral-N, K2O, S, Zn, Fe, Mn and soil pH) with respect to plant-age [54], can also have significant role in the maintenance of the rhizospheric microbial community. The present study also supports the view that the extent of

genetic modification depends on the plant type, transgenes, and the conditions prevailing [23]. Irrespective of the crop type, flowering stage harbours more diverse actinomycetes compared to others. Some studies suggested that the structure and function of rhizospheric microflora was affected by physiological activities of plant [18, 55, 56]. Therefore, flowering stage may be the favourable one for microbial proliferation due to the active release of root exudates [52, 57]. Observations in the present study are in agreement with the fact that the natural factors PXD101 other than genetic modification have strong bearing on temporal shifting of the microbial

community including the actinomycetes [36]. We now can summarize that changes in the actinomycetal community structure are closely associated with environmental factors such as soil variables that may favour the optimal proliferation of actinomycetal community [30]. The Cry1Ac

gene induced effect has the potential in shifting of the actinomycetal community although it is transient compared to the plant-age effect in the transgenic brinjal agroecosystem. Conclusions Changes in the organic carbon content between the non-Bt and Bt planted soil can be attributed to alterations in the quality and composition of root exudates that could be regulated by the genetic modifications in the crop. Alteration in the organic carbon between the soils of non-Bt and Bt brinjal could be one of the possible reasons for the minor Racecadotril fluctuations in the actinomycetes population density and diversity, although the dominant groups (Micrococaceaea and Nocardiodaceae) were more prominent than the exclusive groups as detected in non-Bt and Bt brinjal planted soil NVP-BSK805 nmr during the crop duration. Since, the present study is confined to small scale field experiments that are not sensitive to detect anything other than large and obvious effects, the assessment of risks to biological diversity has to be conducted on a long-term and large-scale basis. Therefore, to assess the behaviour of transgenic line, there is need to include natural cultivar deployed by the local farmer, in addition to Bt and its near-isogenic Bt crop.

We refer to these sequences as probable unique sequences, because there are nearly no identical sequences found in other organisms (Figure 1). Figure 1 Pictorial representation of the bioinformatics strategy employed to churn out the unique genic regions from Las genome. The input and output of each step are shown in oval or square boxes. Actions taken are noted to the left side of the arrow mark, while the information used is indicated to the right side of the arrow. We performed the sequence similarity searches first by using stringent E-value of ≤ 1 × 10-3 against nt database (Figure 1). This search resulted in ~200 sequences that are unique to Las. This set of sequences is relatively high to validate experimentally;

therefore, to PU-H71 further reduce the number VX-680 of unique sequences, we performed the second sequence similarity search with a relaxed E-value of ≤ 1. This search resulted in 38 unique sequences. The E-value of ≤ 1 excludes the sequences with even little similarity to other organisms. Therefore, the resulting 38 unique sequences are

considered unique to Las and constitute the promising candidates for qRT-PCR based detection (Figure 1). We further searched the 38 unique sequences of Las against the phylogenetically closely related Lso, Lam, and Lcr. Because these find more organisms are closely related, we used the stringent E-value threshold of ≤ 1 × 10-3 for this similarity search. In order to achieve this E-value, the sequences need to be highly similar between the Las,

Lso, Lam, and Lcr. Therefore, this close species filter procedure potentially eliminates all the Las sequence targets that could lead to false positive results in qRT-PCR based molecular diagnostic assays. Consequently, we further ADP ribosylation factor eliminated four conserved sequences from the list of 38 unique sequences, resulting in a total of 34 potential sequence signatures. We could not apply this close species filter step against Laf genome as its genome is yet to be sequenced. Five (~15%) of the 34 unique gene sequences namely CLIBASIA_05545, CLIBASIA_05555, CLIBASIA_05560, CLIBASIA_05575 and CLIBASIA_05605 are in the prophage region of the Las genome. All these five unique sequences are located upstream of the genomic locus CLIBASIA_05610 encoding a phage terminase. There are possibly 30 genes that represent the complete prophage genome within the Las genome [25, 44], of which 16 open reading frames (ORFs) are upstream of the phage terminase, while the remaining 13 ORFs are downstream. The prophage genes CLIBASIA_05610 (primer pair 766 F and 766R) and CLIBASIA_05538 (primer pair LJ900F and LJ900R) have been targeted in previous studies by both conventional as well as qRT-PCR based assays [25, 44]. We further analyzed the genomic orientation of the 34 unique genes. This analysis revealed that 15 (~44%) of them are oriented on the sense strand, while the remaining 19 (~56%) were present on the anti-sense strand (Additional file 3: Figure S1).

J Bacteriol 2002,184(3):806–811.PubMedCrossRef www.selleckchem.com/products/arn-509.html 44. Levert M, Zamfir O, Clermont O, Bouvet O, Lespinats S, Hipeaux MC, Branger C, Picard B, Saint-Ruf C, Norel F, Balliau T, Zivy M, Le Nagard

H, Cruveiller S, Chane-Woon-Ming B, Nilsson S, Gudelj I, Phan K, Ferenci T, Tenaillon O, Denamur E: Molecular and buy CRT0066101 evolutionary bases of within-patient genotypic and phenotypic diversity in Escherichia coli extraintestinal infections. PLoS Pathog 2010,6(9):e1001125.PubMedCrossRef 45. Roos V, Klemm P: Global gene expression profiling of the asymptomatic bacteriuria Escherichia coli strain 83972 in the human urinary tract. Infect Immun 2006,74(6):3565–3575.PubMedCrossRef 46. Alexeeva S, de Kort B, Sawers G, Hellingwerf KJ, de Mattos MJ: Effects of limited aeration and of the ArcAB system on intermediary pyruvate catabolism in Escherichia coli. J

Bacteriol 2000,182(17):4934–4940.PubMedCrossRef 47. Korshunov S, Imlay JA: Two sources of endogenous hydrogen peroxide in Escherichia coli. Mol Microbiol 2010,75(6):1389–1401.PubMedCrossRef 48. Sabarly V, Bouvet O, Glodt J, Clermont O, Skurnik D, Diancourt L, de Vienne D, Denamur E, find more Dillmann C: The decoupling between genetic structure and metabolic phenotypes in Escherichia coli leads to continuous phenotypic diversity. J Evol Biol 2011,24(7):1559–1571.PubMedCrossRef 49. Hancock V, Vejborg RM, Klemm P: Functional genomics of probiotic Escherichia coli Nissle 1917 and 83972, and UPEC strain CFT073: comparison of transcriptomes, growth and biofilm formation. Mol Genet Genomics 2010,284(6):437–454.PubMedCrossRef 50. Vejborg RM, Hancock V, Schembri MA, Klemm P: Comparative genomics of Escherichia coli strains causing urinary tract infections. Appl Environ Microbiol 2011,77(10):3268–3278.PubMedCrossRef 51. Roos V, Nielsen EM, Klemm P: Asymptomatic bacteriuria Escherichia coli strains: adhesins, growth and

competition. FEMS Microbiol Lett 2006,262(1):22–30.PubMedCrossRef 52. Hancock V, Seshasayee AS, Ussery DW, Luscombe NM, Klemm P: Transcriptomics and adaptive genomics of the asymptomatic bacteriuria Escherichia coli strain 83972. Mol Genet Genomics 2008,279(5):523–534.PubMedCrossRef 53. Johnson JR, Clabots C, Rosen Succinyl-CoA H: Effect of inactivation of the global oxidative stress regulator oxyR on the colonization ability of Escherichia coli O1:K1:H7 in a mouse model of ascending urinary tract infection. Infect Immun 2006,74(1):461–468.PubMedCrossRef 54. Klemm P, Hancock V, Schembri MA: Mellowing out: adaptation to commensalism by Escherichia coli asymptomatic bacteriuria strain 83972. Infect Immun 2007,75(8):3688–3695.PubMedCrossRef 55. Boles BR, Singh PK: Endogenous oxidative stress produces diversity and adaptability in biofilm communities. Proc Natl Acad Sci USA 2008,105(34):12503–12508.PubMedCrossRef 56.

In this work we observed that the adherence of different T3SS mutants to host cell tissue was not altered. Studies in several pathogenic bacteria, such as Salmonella typhimurium[35], E. coli[36, 37] and the plant pathogen P. syringae[38] revealed that mutants unable to produce T3SS appendages become affected in their interactions with host cells. However, in the phytopathogen Ralstonia solanacearum, it has been shown that the lack of a T3SS pilus does not affect attachment to plant cells [39], and this is consistent with our observation that adherence of X. citri to the host tissue was not affected by the absence of a functional T3SS. In addition, we determined that T3SS is required for X. citri

survival on citrus leaves and that T3SS genes are expressed while bacteria reside on the plant surface. Expression of T3SS genes on the leaf surface was also detected in Xanthomonas euvesicatoria cells suggesting a role for T3SS in epiphytic survival of the bacteria [40]. Selleckchem AZD5582 In a recent report, it was revealed that Nutlin-3a in vitro the survival of Pseudomonas syringae T3SS-deficient strains on leaf surfaces is reduced, supporting a role of T3SS and effector proteins in the promotion of epiphytic bacterial survival

[41]. Our results suggest that T3SS plays a role in X. citri leaf-associated survival on the leaf surface by enabling biofilm formation. The proteomic study revealed differentially expressed proteins between X. citri and the hrpB − mutant strain and GO analysis detected enrichment of up-regulated proteins in different metabolic processes and generation of energy in the hrpB − mutant. Similarly, in a previous proteomic study, these categories were also enriched with up-regulated proteins in X. citri planktonic cells compared to biofilm, suggesting a slower metabolism and reduction in aerobic respiration in the X. citri biofilm [42]. Therefore, the higher expression of proteins involved in these processes in the hrpB − mutant compared to X. citri may be caused by the lack of biofilm formation of the mutant. It is remarkable that among the differentially Thiamet G expressed proteins between the mutant and

the wild type strain, some have been previously characterized as involved in biofilm formation in X. citri or in other pathogenic bacteria. Such is the case of DNA-directed RNA polymerase subunit β [32], tryptophan synthase [43], GroEL [44, 45], FadL [32, 42, 46] and several TBDTs [42, 47]. Interestingly, high intracellular L-tryptophan concentration prevents biofilm formation and triggers degradation of mature biofilm in E. coli[43]. The proteomic assay showed that tryptophan synthase (XAC2717) was up-regulated, while the tryptophan repressor binding protein (XAC3709) was down-regulated in hrpB − strain suggesting a link also between tryptophan metabolism and biofilm formation in X. citri. Another example is the outer membrane protein XAC0019 that displays high homology to the fatty acid transport porin FadL.

After the first 90 min, single Rt24.2 cells were visible on the surface of root hairs (Figure 10A). After 24 h, attachment of numerous check details Rt24.2 cells to root hairs was seen. Bacteria were located mainly on root hair tops, forming caps and rhizobial clouds (Figure 10B). In the zone of growing root hairs, the majority of the root hairs were coated with Rt24.2 cells (Figure 10C). After 6 days post infection (dpi), infection

threads inside some of the root hairs were initiated or already extended and reached root epidermal cells (Figure 10D). In contrast, Rt2472 cells were seen on the root surface but were attached to the root hairs only sporadically demonstrating a much weaker attachment ability (Figure 10E). The caps formed CYT387 by rosR cells on the top of root hairs were detected very rarely (Figure 10F). In addition, several root hairs had an atypical, expanded shape resembling ginger roots (Figure 10G) in contrast to the typical curled root hairs in clover inoculated with the wild type. In the case of rosR mutant-inoculated plants, infection threads inside root hairs were observed sporadically, and their elongation was frequently interrupted (Figure 10H). Figure 10 Root attachment and infection of clover roots by the rosR mutant and the wild type. Fluorescence microscopy analyses of clover root colonization and invasion by GFP-expressing cells of R. leguminosarum bv. trifolii wild type (A-D) and the rosR mutant (Rt2472)

(E-H). The Rt24.2 cells attached very fast and effectively to root hairs (A-B), and formed caps on the top of root hairs (C). (D) Curled root hairs with an extended infection thread filled with the wild type cells. The infection thread started from the Shepherd’s ifenprodil crook of the curled root hair and reached the base of root hair. The ability of root attachment and root cap formation of the rosR mutant was substantially decreased (E-F). Only individual cells of the Rt2472 rosR mutant attached to root hairs (E) and root caps were formed sporadically (F). Several root hairs showed abnormal deformation (G). The root hair colonized by the rosR mutant, which had developed an aborted

infection thread (H). (I) Attachment to clover roots 0.5 h and 48 h post inoculation with the wild type, and the Rt2472 and Rt2441 rosR mutants, and their derivatives complemented with pRC24. For each strain, ten roots were examined. Data shown are the means of two replicates ± SD. (J) Kinetics of curled root hair (CRH) formation, infection thread (IT) initiation and extension on clover plants inoculated with the wild type and the rosR mutant (Rt2472). For each strain, 25 plants were used. Data shown are the means of two experiments. To quantitatively determine the attachment ability to the surface of clover roots, Rt24.2 wild type, Rt2472 and Rt2441 rosR mutants, and their derivatives bearing plasmid pRC24 were incubated with clover roots for 0.5 h and 48 h.

The organisms were chosen from IMG based on their possession of multiple nifH gene homologs in their genome except for Klebsiella pneumoniae 342. The number of nifH gene homologs from each

species are; five from Methanosarcina acetivorans C2A (blue bullets), six from Anabaena variabilis ATCC 29413 (green bullets), a total of nine from Firmicutes (red bullets); four from D. hafniense DCB-2 and five from Clostridium kluyveri DSM 555, and a total of eight from Proteobacteria (black bullets), including four from Rhodobacter sphaeroides ATCC 17025, one from K. pneumoniae 342, and three from Geobacter sp. FRC-32. The tree shows that the NifH encoded by Dhaf_1049 belongs to a more conserved NifH cluster and is distant from other NifH homologs of D. hafniense DCB-2. Oxidative stresses Although classified as an obligatory GDC-0973 ic50 anaerobe, D. hafniense DCB-2 can tolerate considerable oxygen in

liquid culture and can resume its anaerobic growth after 24 hours’ exposure to oxygen [4]. Most Clostridium species can accept microoxic conditions and are considered to possess systems to metabolize oxygen as well as to scavenge reactive oxygen species (ROS)[62–64]. NoxA, a H2O-forming NADH oxidase, has been implicated in oxygen consumption in Clostridium aminovalericum [64]. Our total genome microarray study PI3K inhibitor cancer revealed that among four noxA homologous genes identified in the DCB-2 genome, a gene encoded by Dhaf_1505, which MG132 also showed the lowest E-value of 1e-43, was significantly upregulated upon oxygen exposure (~5 fold). Cytochrome bd quinol oxidase (CydA, B), a respiratory cytochrome oxidase unusual for strict anaerobes, was reported to catalyze reduction of low levels of oxygen in the strict anaerobe, Moorella thermoacetica [65]. A complete cyd operon (cydA, B, C, D) was also identified in DCB-2 (Dhaf_1310-1313). However, the operon was not induced under the microoxic conditions that we tested. Under the same conditions, Dhaf_2096 encoding a putative bifunctional catalase/peroxidase

was highly upregulated (~12 fold) and the expression of heme catalase-encoding Dhaf_1029 was also considerably induced (~3 fold). No significant induction was observed for three other catalase-encoding genes (Dhaf_1329, Dhaf_1481, and Dhaf_1646) and two Fe/Mn-type superoxide dismutase genes (SOD genes; Dhaf_1236 and Dhaf_2597), although a gel-based cDNA detection study indicated that the Dhaf_1236 SOD gene was expressed constitutively. Other oxygen responsive genes include those for thioredoxin (Dhaf_1227 and Dhaf_3584), thioredoxin reductase (Dhaf_0850), and rubrerythrin (Dhaf_4567). These results suggest that D. hafniense DCB-2 is equipped with and can operate defensive machinery against oxygen, which includes ROS scavenging, oxygen metabolism, and other oxygen-responsive reductive activities. Sporulation and germination Of the 12 Desulfitobacterium strains that have been examined, seven strains including D. hafniense DCB-2 were observed to sporulate [1].

actinomycetemcomitans JCM8577, A. actinomycetemcomitans SUNYaB67, A. actinomycetemcomitans SUNYaB75, Aggregatibacter naeslundii JCM8350, Prevotella loescheii JCM8530, Prevotella denticola JCM8525, Prevotella bivia JCM6331, Prevotella pallens JCM1140, Prevotella veroralis JCM6290, and Prevotella oralis ATCC

33322. Trichostatin A price Ethics statement All patients were treated in accordance with the Helsinki Declaration regarding the participation of human subjects in medical research. Ethics clearance for the study was obtained from the Ethics Committee of Kyushu Dental University Hospital (reference number 11–40). The parents of participants were fully informed about the study and signed informed consent forms. Study subjects and oral specimen sampling Twenty-one subjects ranging in age from 3 to 10 years and who had dental caries were included in the caries group (mean age ± S.D. = 7.86 ± 0.43 years; 11 males and 10 females). A healthy (completely caries-free) control group consisted of 24 subjects (ages 3 to 12 years; mean age ± S.D. = 7.29 ± 0.56 years; 13 males and 11 females). The carious dentin Ku-0059436 in vitro was

excavated from cavitated lesions. Before excavation of the carious dentin, the plaque on the surfaces of cavitated lesions was swiped. The dental plaque samples from healthy subjects were collected from the buccal or lingual surface of the second primary molar. Collected carious dentin and dental plaque were placed in 200 μl of PBS in a sterile 1.5-ml microcentrifuge tube. These samples were washed and placed in PBS solution adjusted to 1 mg per 100 μl. Saliva was collected

from both the caries and healthy control groups. Fifty microliters Phospholipase D1 of saliva was washed with PBS and used for analysis. Bacterial counting from oral specimens on an agar plate Serially diluted carious dentin or dental plaque was plated on a Mitis-Salivarius agar plate (Becton Dickinson, Franklin Lakes, NJ) supplemented with 150 g/l sucrose and 200 U/l bacitracin for selection of mutans streptococci (MSB agar). Bacterial counting was performed using a magnifying loupe. Propidium monoazide treatment For only viable cell quantification, PMA (3-amino-8-azido-5-[3-(diethylmethylammonio)propyl]-6-phenyl dichloride; Wako Pure Chemical, Osaka, Japan) treatment was performed for bacterial cells prior to DNA extraction, as previously described [19]. Briefly, PMA was dissolved in 20% DMSO to produce a 25-mM stock solution. Following incubation with the dye for 5 min in the dark, similarly prepared cells were exposed for 5 min to a 500-W halogen light placed 15 cm above 500-μl samples in open microcentrifuge tubes on ice. The toxicity of PMA at 2.5–250 μM to S. mutans and S. sobrinus was analyzed at 37°C. In the present study, 25 μM PMA was employed for the analysis. All data presented are from triplicate independent cultures and/or biofilms.