ChIP-seq assay for RocS binding to the Streptococcus pneumoniae genome
Ontology highlight
ABSTRACT:
ORGANISM(S): Streptococcus pneumoniae
PROVIDER: GSE129717 | GEO | 2019/04/14
REPOSITORIES: GEO
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Project description:The most basic level of transcription regulation in Streptococcus pneumoniae is the organization of its chromosome in topological domains. In response to drugs that caused DNA-relaxation, a global transcriptional response was observed. Separate domains were identified depending of the transcription of their genes: up-regulated (U), down-regulated (D), non-regulated (N), and flanking (F). We show here that these distinct domains have different expression and conservation tendencies. Microarray fluorescence units under non-relaxation conditions, taken as a measure of gene transcription level, were significantly lower in F genes than in the other domains in the same range of AT content. Transcription level categorization of the domains was D>U>F. In addition, a comparison of 12 S. pneumoniae genome sequences evidenced conservation of gene composition in the U and D domains and extensive gene interchange in F domains. We tested domain organization by measuring the relaxation-mediated transcription of eight insertions of a heterologous Ptccat cassette, two in each type of domain, showing that transcription depended on their chromosomal location. Moreover, transcription from the four promoters directing the five genes involved in supercoiling homeostasis, located either in U (gyrB), D (topA), or N (gyrA and parEC) domains was analyzed both in their chromosomal locations and in a replicating plasmid. Although expression from the chromosomal PgyrB and PtopA showed the expected domain regulation, their expression was down-regulated in the plasmid, which behaved as a D domain. However, both PparE and PgyrA carried their own regulatory signals, their topology-dependent expression being equivalent in the plasmid or in the chromosome. In PgyrA a DNA bend acted as a DNA supercoiling sensor. These results revealed that DNA topology works as a general transcriptional regulator, superimposed to other kind of more specific regulatory mechanisms.
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Project description:Real-time PCR targeting lytA (the major autolysin gene) and piaB (permease gene of the pia ABC transporter) are currently used as the gold-standard culture-independent assays for Streptococcus pneumoniae identification. We evaluated the performance of a new real-time PCR assay - targeting SP2020 (putative transcriptional regulator gene) - and compared its performance with the assays previously described. A collection of 150 pneumococci, 433 non-pneumococci and 240 polymicrobial samples (obtained from nasopharynx, oropharynx, and saliva; 80 from each site) was tested. SP2020 and lytA-CDC assays had the best performance (sensitivity of 100% for each compared to 95.3% for piaB). The specificity for lytA and piaB was 99.5% and for SP2020 was 99.8%. Misidentifications occurred for the three genes: lytA, piaB and SP2020 were found in non-pneumococcal strains; piaB was absent in some pneumococci including a serotype 6B strain. Combining lytA and SP2020 assays resulted in no misidentifications. Most polymicrobial samples (88.8%) yielded concordant results for the three molecular targets. The remaining samples seemed to contain non-typeable pneumococci (0.8%), and non-pneumococci positive for lytA (1.7%) or SP2020 (8.7%). We propose that combined detection of both lytA-CDC and SP2020 is a powerful strategy for the identification of pneumococcus either in pure cultures or in polymicrobial samples.
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Project description:Purpose: The goal of this study is to identify genes selectively associated with C. elegans CEBP-1. Methods: We generated transgenic animals expressing FLAG-tagged CEBP-1 in a cebp-1(tm2807) mutant background (cebp-1(0); juIs418 [Pcebp-1::FLAG::CEBP-1::cebp-1 3’UTR]) and then immunoprecipitated FLAG-CEBP-1-associated DNA fragments using anti-FLAG antibodies (M2 anti-FLAG magnetic beads; Sigma). We collected mixed stage worms grown at 20˚C on NGM plates followed by 2% formaldehyde and sonicated the samples as described (Mukhopadhyay et al., 2008). We next generated ChIP-seq DNA libraries. Briefly, both ChIPed DNA and input genomic DNA were ligated to specific adaptors and amplified by barcode primers followed by sequencing on the Illumina HiSeq-2000 platform. We performed two independent ChIP-seq experiments, with parallel genomic DNA controls prepared from the same strain. We conducted peak-calling using CLC genomics workbench 6.0 (CLCbio). To define genes associated with the peaks, we used the annotation of transcription start site (TSS) and transcription end site (TES) from WS220 and annotated the peak if it overlapped the gene or the 3 kb upstream of the TSS. We then manually confirmed the peaks and associated genes using UCSC browser and update to WS252. Results: We found 209 CEBP-1 ChIP-seq peaks in the genome that were associated with 212 coding genes. Through motif discovery tools, we also found that CEBP-1 binds conserved DNA motifs. Conclusions: The conservation of C.elegans CEBP-1 binding motif supports functional parallels between C. elegans CEBP-1 and vertebrate C/EBPs.
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Project description:A semiautomated method for the determination of five serotypes and three serogroups in Streptococcus pneumoniae was developed. Primers specific for serotypes 1, 3, 14, 19F, and 23F and serogroups 6, 19, and 23 were combined in three multiplex PCRs. Products were separated by capillary electrophoresis with a 7-min run time, and a serotype or serogroup was assigned on the basis of fragment size. The method was used to test 93 clinical isolates, and all isolates of the serotypes concerned were correctly detected. The strategy would allow the detection of multiple serotypes in a single sample. Detection of additional serotypes could be included as capsule locus sequences become available.
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Project description:BackgroundPrecise serotyping of pneumococci is essential for vaccine development, to better understand the pathogenicity and trends of drug resistance. Currently used conventional and molecular methods of serotyping are expensive and time-consuming, with limited coverage of serotypes. An accurate and rapid serotyping method with complete coverage of serotypes is an urgent necessity. This study describes the development and application of a novel technology that addresses this need.MethodsPolymerase chain reaction (PCR) was performed, targeting 1061 bp cpsB region, and the amplicon was subjected to sequencing. The sequence data was analyzed using the National Centre for Biotechnology Information database. For homologous strains, a second round of PCR, sequencing, and data analysis was performed targeting 10 group-specific genes located in the capsular polysaccharide region. Ninety-one pneumococcal reference strains were analyzed with PCRSeqTyping and compared with Quellung reaction using Pneumotest Kit (SSI, Denmark).ResultsA 100% correlation of PCRSeqTyping results was observed with Pneumotest results. Fifty-nine reference strains were uniquely identified in the first step of PCRSeqTyping. The remaining 32 homologous strains out of 91 were also uniquely identified in the second step.ConclusionThis study describes a PCRSeqTyping assay that is accurate and rapid, with high reproducibility. This assay is amenable for clinical testing and does not require culturing of the samples. It is a significant improvement over other methods because it covers all pneumococcal serotypes, and it has the potential for use in diagnostic laboratories and surveillance studies.
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