ABSTRACT: Investigation of whole genome gene expression level changes in S. pneumoniae KCTC 5080T, S. mitis KCTC 3556T, S. oralis KCTC 13048T, and S. pseudopneumoniae CCUG 49455T. This proves that transcriptional profiling can facilitate in elucidating the genetic distance between closely related strains. A one chip study using total RNA recovered from S. pseudopneumoniae CCUG 49455T with three strain. For the the transcriptome of S. pseudopneumoniae CCUG 49455T was analyzed using the S. pneumoniae R6 microarray platform and compared with those of S. pneumoniae KCTC 5080T, S. mitis KCTC 3556T, and S. oralis KCTC 13048T strains.
Project description:In order to appreciate the presence of surface protein gene homologues in commensal species S. mitis and S. oralis, comparative genomic hybridization studies using DNA microarrays were performed with 8 S. mitis and 11 S. oralis from different geographic locations. The oligonucleotide microarray was designed based on the genomes of S. pneumoniae R6 and TIGR4 as well as S. mitis B6 to include genes of 63 cell surface proteins. The denatured genomic DNA of the S. mitis and S. oralis strains was labeled with Cy3-dCTP and control S. mitis B6 DNA was labeled with Cy5-dCTP. Hybridization was performed following the manufacturers recommendations using an hybridization temperature of 40C for 16 h. For data processing, microarrays were scanned on the laser scanner Pro Scan Array GX (PerkinElmer) with the low resolution of 50 µm using ScanArrayExpress Software version 4.0. Photomultiplier tube was adjusted to balance the two fluorescence channels and biochips were scanned with a resolution of 10 µm. After elimination of background values fluorescence intensity was determined. Signals that showed an intensity ratio of 0.3 and above were considered to be positive.
Project description:Antibiotic resistance in Streptococcus pneumoniae is often the result of horizontal gene transfer events involving closely related streptococcal species. Laboratory experiments confirmed that S. mitis DNA functions as donor in transformation experiments, using the laboratory strain S. pneumoniae R6 as recipient and chromosomal DNA of a high level penicillin resistant S. mitis B6 strain. After four transformation steps, alterations in five penicillin-binding proteins (PBP) were observed, and sequence analysis confirmed recombination events in the corresponding PBP genes. In order to detect regions where recombination with S. mitis DNA has occurred we analyzed the S. pneumoniae transformants by microarray analyses, using oligonucleotide microarrays designed for the S. pneumoniae genome and the S. mitis B6 genome as well.
Project description:Seven streptococcal isolates from the Mitis group were analysed for the presence of pneumococcal gene homologues using comparative genomic hybridization studies with microarrays based on open reading frames from the genomes of Streptococcus pneumoniae TIGR4 and R6. The diversity of pneumolysin (ply) and neuramindase A (nanA) gene sequences was explored in more detail in a collection of 14 S. pseudopneumoniae and 29 Mitis group isolates respectively. The Mitis group isolates used in the microarray experiments included a type strain (NCTC 12261), 2 S. mitis isolates from the nasopharynx of children, one S.mitis from infective endocarditis, one S. mitis isolate from a dental abscess, plus one S. oralis isolate and one S. pseudopneumoniae isolate from the nasopharynx of children. The results of the microarray study showed that the 5 S. mitis isolates had homologues to between 67-82% of pneumococcal virulence genes, S. oralis hybridised to 83% and S. pseudopneumoniae to 92% of identified pneumococcal virulence genes. Comparison of the pneumolysin, mitilysin (mly) and newly identified pseudopneumolysin (pply) gene sequences revealed that mly and pply genes are more closely related to each other than either is to ply. In contrast, the nanA gene sequences in the pneumococcus and streptococci from the Mitis group are closely clustered together sharing 99.4-99.7% sequence identity with pneumococcal nanA alleles. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-82
Project description:We performed a comparative study of the two control sample options with a Streptococcus pneumoniae microarray designed with three fully sequenced strains. We hybridized two of these strains (R6 and G54) as test samples using the third strain alone (TIGR4) or a mix of the three strains as the control sample.
Project description:Investigation of whole genome gene expression level changes in Lactococcus lactis KCTC 3769T,L. raffinolactis DSM 20443T, L. plantarum DSM 20686T, L. fujiensis JSM 16395T, L. garvieae KCTC 3772T, L. piscium DSM 6634T and L. chungangensis CAU 28T . This proves that transcriptional profiling can facilitate in elucidating the genetic distance between closely related strains. A one chip study using total RNA recovered from of L. raffinolactis DSM 20443T, L. plantarum DSM 20686T, L. fujiensis JSM 16395T, L. garvieae KCTC 3772T, L. piscium DSM 6634T and L. chungangensis CAU 28T . For the the transcriptome of of L. raffinolactis DSM 20443T, L. plantarum DSM 20686T, L. fujiensis JSM 16395T, L. garvieae KCTC 3772T, L. piscium DSM 6634T and L. chungangensis CAU 28T was analyzed using the Lactococcus lactis KCTC 3769T microarray platform
Project description:Oral streptococci metabolize carbohydrate to produce organic acids, not only decrease the environmental pH, but also increase osmolality of dental plaque fluid due to tooth demineralization and consequent calcium and phosphate accumulation. Thus, to persevere in the dental plaque, acidogenic bacteria should evolve sophisticated molecular machineries to counter the detrimental effect of elevated osmolality. This study was aimed to obtain a global view on strategies taken by streptococcus mutans to deal with physiologically relevant elevated osmolality, and preserves within a cariogenic dental plaque. We investigated phenotypic change of S. mutans biofilm upon sub-lethal level of hyperosmotic challenge. We found that hyperosmotic condition was able to initiate S. mutans biofilm dispersal by reducing both microbial content and extracellular polysaccharides matrix. We then used DNA microarray with qPCR validation to systemically investigate the underlying molecular machinery of this bacteria in response to hyperosmotic stimuli. Among those identified 50 differentially regulated genes, down-regulation of gtfB and comC were believed to be responsible for the observed biofilm dispersal. Further analysis of microarray data showed significant up-regulation of genes and pathways involved in carbohydrates metabolism. Specific genes involved in heat shock response and acid tolerance were also upregulated, indicating potential cross-talk between hyperosmotic and other environmental stress. Based on the data obtained in this study, we believe that although hyperosmotic condition may induce significant stress response on S. mutans, this cariogenic bacterium has evolved sophisticated molecular machineries to counter those elicited detrimental effects. In the meantime, it will take full advantage of these environmental stimuli to better fit the fluctuating environments within oral cavity, and thus emerge as numeric-predominant bacteria under cariogenic conditions. A six-chip study using total RNA recovered from mid-logarithmic phase of S. mutans UA159 from three separate cultures of strains submitted for 15 minutes to hyperosmotic stimuli (0.4M NaCl) and three separate cultures of strains kept under no stress condition.
Project description:PcsB is a protein of unknown function that plays a critical role in cell division in Streptococcus pneumoniae and other ovococcus species of Streptococcus. We constructed isogenic sets of mutants expressing different amounts of PcsB in laboratory strain R6 and virulent serotype 2 strain D39 to evaluate its cellular roles. Insertion mutagenesis in parent and pcsB+ merodiploid strains indicated that pcsB is essential in serotype 2 S. pneumoniae. Quantitative Western blotting of wild-type and epitope-tagged PcsB showed that all PcsB was processed into cell-associated and secreted forms of the same molecular mass. These analyses showed that PcsB bound to cells is present in relatively low amounts of only ˜ 300 molecules per cell. Controlled expression and complementation experiments indicated that there was a causative relationship between the severity of defects in cell division and decreasing PcsB amount. These experiments also indicated that perturbations of expression of the upstream mreCD genes did not contribute to the cell division defects of pcsB mutants and that mreCD could readily be deleted in these strains. Unexpectedly, the defects in cell division and cell shape in pcsB mutants or other mutants defective in cell wall biosynthesis, such as dacA, were strongly influenced by capsule. Underexpression of PcsB did not result in changes in the amounts or composition of lactoyl-muropeptides, which were markedly different in the R6 and D39 strains, and there was no correlation between decreased PcsB amount and sensitivity to penicillin. Finally, microarray analyses indicated that underexpression of PcsB may generate a signal that increases expression of the VicRK regulon, which includes pcsB. Overall design: Four independent hybridizations using independent RNA preparations from the strain IU1533 (reference strain) and strain IU1979 (a strain with decreased expression of PcsB protein) were performed. Dye swap was performed with the reference strain labeled with Cy3 in two hybridizations and Cy5 in the other two hybridizations.
Project description:The objective of the study is to obtain a high resolution transcription map of S. pneumoniae genome and make it available through a genome browser. The study also help to identify gene expression pattern, presence of small RNAs, Antisense expression and operon structure. S. pneumoniae strain TIGR4 (ref) was grown in THYmedium, Todd-Hewitt broth supplemented with 0.5% yeast extract. Cells were harvested from duplicate cultures from mid-log phase (OD600 nm, 0.4–0.6) of growth by centrifugation. The harvested pellets were washed twice in sterile PBS (pH 7.0) and stored at -80°C. RNA was purified from frozen bacterial pellets using Qiagen RNeasy kit by following the manufacturer’s protocol. Isolated RNA was treated with DNase to remove genomic DNA contamination, and purity was checked by performing a one-step RT-PCR using primers specific for 16S rRNA in presence or absence of reverse transcriptase. RNA concentration and quality were determined by using Agilent Bioanalyzer. Purified RNA was stored in nuclease free water at -80°C. One microgram of total RNA was used by Nimblegen systems for labeling and hybridization. 2 biological replicates (single colored) were used in the analysis. 20,000 random probes were designed on the chip as a negative controls to measure non-specific hybridization. 35 genes identified through proteomics of same sample were used as positive controls.