GAF competence
Ontology highlight
ABSTRACT:
ORGANISM(S): Streptococcus pneumoniae R6 Streptococcus pneumoniae
PROVIDER: GSE8362 | GEO | 2007/09/05
SECONDARY ACCESSION(S): PRJNA101385
REPOSITORIES: GEO
Similar Datasets
Project description:We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner-transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counter-selection of mutants in 21 different genes during the challenge. Eight of the counter-selected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants in which seven of the eight remaining genes, i.e., spr0459/spr0460, spr0777, spr0838, spr1259/spr1260, and spr1357, were deleted, displayed reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation of chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and DNA damage repair in S. pneumoniae. Keywords: GAF competence Selection for genes essential for transformation. Aliquots of a marinerT7 transposon library generated in S. pneumoniae R6 containing approximately 40,000 independent mutants were transformed with an antibiotic resistance marker and grown without (initial library) or with (challenged library) antibiotics for 25 generation until DNA extraction.
2007-09-04 | E-GEOD-8362 | biostudies-arrayexpress
Project description:Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra- and interspecies competition within the human host. What triggers pneumocin expression is poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39 and show that several antibiotics activate the blp-genes. Real-time gene expression measurements showed that while the promoter driving expression of the two-component regulatory system blpS/H was constitutive, the remaining blp-promoters (e.g. controlling pneumocin expression, immunity and the inducer peptide BlpC) was pH-dependent, induced in the late exponential phase and occurred in all cells. Intriguingly, competence for genetic transformation, mediated by the ComD/E two-component quorum system, was induced by the same stimuli. To test for regulatory interplay, we utilized synthetic BlpC and competence-stimulating peptide (CSP). Strikingly, immediately upon addition of CSP, the blp-promoters were activated in a comD/E dependent manner. After a delay, blp-expression was highly induced but strictly dependent on the presence of blpRH and blpC. This raised the question how BlpC is exported since phylogenetic analysis showed that the putative exporter for BlpC, blpAB, is not intact in strain D39 and most other strains. However, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. In fact, we show that high expression of the blp-genes requires comAB. Together, we demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might promote survival under antibiotic stress by killing and liberating DNA from neighboring (antibiotic-resistant) bacteria, which can then be used for transformation or repair. Pairwise comparison of untreated and antibiotic-treated cells (either DNA-seq or RNA-seq). It was performed with deep sequencing, using an Illumina HiSeq 2000 machine with 100 nt paired-end reads. Control and HPUra-treated samples were analysed from duplicate samples, while other conditions were from single samples.
2016-01-08 | E-GEOD-69729 | biostudies-arrayexpress
Project description:Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra- and interspecies competition within the human host. What triggers pneumocin expression is poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39 and show that several antibiotics activate the blp-genes. Real-time gene expression measurements showed that while the promoter driving expression of the two-component regulatory system blpS/H was constitutive, the remaining blp-promoters (e.g. controlling pneumocin expression, immunity and the inducer peptide BlpC) was pH-dependent, induced in the late exponential phase and occurred in all cells. Intriguingly, competence for genetic transformation, mediated by the ComD/E two-component quorum system, was induced by the same stimuli. To test for regulatory interplay, we utilized synthetic BlpC and competence-stimulating peptide (CSP). Strikingly, immediately upon addition of CSP, the blp-promoters were activated in a comD/E dependent manner. After a delay, blp-expression was highly induced but strictly dependent on the presence of blpRH and blpC. This raised the question how BlpC is exported since phylogenetic analysis showed that the putative exporter for BlpC, blpAB, is not intact in strain D39 and most other strains. However, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. In fact, we show that high expression of the blp-genes requires comAB. Together, we demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might promote survival under antibiotic stress by killing and liberating DNA from neighboring (antibiotic-resistant) bacteria, which can then be used for transformation or repair.
2016-01-08 | GSE69729 | GEO
Project description:Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other inflammatory factors. We identified a novel peptide in the Klebsiella pneumoniae secretome, which enters Streptococcus pneumoniae via its AmiA-AliA/AliB permease. Subsequent downregulation of genes for amino acid biosynthesis and peptide uptake was associated with reduction of pneumococcal growth in defined medium and human cerebrospinal fluid, irregular cell shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was specific to S. pneumoniae and Streptococcus pseudopneumoniae with no effect on Streptococcus mitis, Haemophilus influenzae, Staphylococcus aureus or K. pneumoniae. Peptide sequence and length were crucial to growth suppression. The peptide reduced pneumococcal adherence to primary human airway epithelial cell cultures and colonization of rat nasopharynx, without toxicity. We also analysed the effect of peptide on the proteome of S. pneumoniae. We found alteration of the proteome by the peptide with some proteins turned on or off in line with the transcriptomic changes. We therefore identified a peptide with potential as a therapeutic for pneumococcal diseases suppressing growth of multiple clinical isolates, including antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis.
2024-04-08 | PXD050408 | Pride
Project description:Streptococcus pneumoniae D39 AdcR (adhesion competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling of a ∆adcR strain grown in liquid culture under microaerobic conditions revealed that transcript amounts for 13 genes were up-regulated relative to the wild-type strain, among them adcR, adcCBA, encoding a high affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (pht) and adcII (lmb, laminin binding) proteins. Down-regulated transcripts included those encoding two putative zinc-containing alcohol dehydrogenases.
2010-10-01 | GSE21506 | GEO
Project description:We applied genomic array footprinting (GAF) in the search for genes of S. pneumoniae essential during the establishment and progression of experimental meningitis. Four libraries with a total of 6,000 independent TIGR4 marinerT7 transposon mutants were injected intra-cisternally in rabbits, and cerebrospinal fluid (CSF) was collected after three, nine, and fifteen hours. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 genes of which mutants had become attenuated and eleven genes of which mutants had become enriched during infection. Screening results particularly point to an essential role for capsular polysaccharides (cps), nutrient uptake, and metabolism in meningitis. Detailed study on directed mutants of a subset of sixteen GAF targets in an experimental model of rat meningitis revealed that ten were significantly attenuated or enriched during competitive infection. Furthermore, we showed that mutants of adenylosuccinate synthetase (purA), flavodoxin (fld), and the substrate binding protein (livJ) of a branched chain amino acid ABC-transporter were essential for full blown meningitis in a mono-infection setup of rat meningitis. Overall, the GAF screen revealed the general restraint on nutrients encountered by the pneumococcus in CSF, while, except for cps genes, no ‘classic’ virulence factors appeared to be required for infection. This knowledge will contribute to a better understanding of the molecular pathogenesis of pneumococcal meningitis.
2010-11-16 | GSE21729 | GEO
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.
2011-04-21 | E-MEXP-3170 | biostudies-arrayexpress
Project description:We applied genomic array footprinting (GAF) in the search for genes of S. pneumoniae essential during the establishment and progression of experimental meningitis. Four libraries with a total of 6,000 independent TIGR4 marinerT7 transposon mutants were injected intra-cisternally in rabbits, and cerebrospinal fluid (CSF) was collected after three, nine, and fifteen hours. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 genes of which mutants had become attenuated and eleven genes of which mutants had become enriched during infection. Screening results particularly point to an essential role for capsular polysaccharides (cps), nutrient uptake, and metabolism in meningitis. Detailed study on directed mutants of a subset of sixteen GAF targets in an experimental model of rat meningitis revealed that ten were significantly attenuated or enriched during competitive infection. Furthermore, we showed that mutants of adenylosuccinate synthetase (purA), flavodoxin (fld), and the substrate binding protein (livJ) of a branched chain amino acid ABC-transporter were essential for full blown meningitis in a mono-infection setup of rat meningitis. Overall, the GAF screen revealed the general restraint on nutrients encountered by the pneumococcus in CSF, while, except for cps genes, no ‘classic’ virulence factors appeared to be required for infection. This knowledge will contribute to a better understanding of the molecular pathogenesis of pneumococcal meningitis. The rabbit meningitis model was performed as previously described by Østergaard (PMID: 9661008 ). Briefly, 10E6 CFU of a S. pneumoniae mutant library was suspended in 20 µl beef-broth and injected into cisterna magna of four groups of four outbred New Zealand White rabbits of app. 2.5 kg (in one group one rabbit died). CSF (0.3 ml) samples were repeatedly obtained from the same rabbit by aspiration from indwelling spinal- and arterial cannules. Samples were collected before pleocytosis developed (three hrs), at the time of maximal pleocytosis (nine hrs), and at a time of slowing bacterial growth and abating pleocytosis (fifteen hrs ). Until further processing in the GAF protocol, CSF and inocula were stored with 15% glycerol at -80ºC. The GAF technology was performed as described previously (PMID: 17261526 ). Briefly, stored inocula and CSF obtained during the experimental rabbit meningitis experiments were defrosted, diluted in GM17 medium supplemented with spectinomycin, and grown to mid-log phase. Chromosomal DNA was isolated, digested with AluI endonuclease, and used as a template for in vitro transcription initiated from the T7 promoters of the marinerT7 transposon that is present in each mutant. After removal of template DNA by DNAseI treatment, mutant-specific T7 RNA was reverse transcribed in the presence of fluorescent Cy3/Cy5-labeled dUTP nucleotides. Labeled Cy3/Cy5-cDNA from the CSF samples and oppositely labeled Cy5/Cy3 cDNA from the inocula were combined, purified, and washed by ultrafiltration. The cDNA was hybridized to pneumococcal microarrays containing, 2,087 ORFs of Streptococcus pneumoniae TIGR4 and 70-mer oligo's specific for the non-homologous ORFs in the R6, D39, 23F, INV104B, INV200, OXC141, and G54 strains, all spotted in duplicate (PMID: 18174343) . Dual channel array images were acquired on a GenePix 4200AL microarray scanner (Axon Instruments, Union City, CA). Microarray image files were analyzed with GenePix Pro software (Axon Instruments, Union City, CA). Spots were screened visually to identify those of low quality and removed from the data set prior to analysis. A net mean intensity filter based on hybridization signals obtained with R6-specific spots was applied in all experiments. Slide data were processed and normalized using MicroPreP (PMID: 15907200). Further analysis was performed using a CyberT implementation of the Student’s t test (cybert.microarray.ics.uci.edu). This web-based program lists the ratios of all intra-replicates (duplicate spots) and inter-replicates (different slides), the mean ratios per gene, and standard deviations and (Bayesian) p values assigned to the mean ratios. For identification of conditionally essential genes, only genes with a minimum of 6/8 (for groups of four rabbits) or 5/6 for the group of three rabbits) reliable measurements and a Bayesian p-value < 0.001 were included. Further selection criteria were applied, as only mutants displaying an average fold-change of >2.5 at a minimum of two time-points, or >4.0 at one time-point were accepted as significantly attenuated or enriched. The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo/) under GEO Series accession number ...
2010-11-16 | E-GEOD-21729 | biostudies-arrayexpress
Project description:The aim of this study is to identify the rapid (RR) and adaptative (AR) response of S. pneumoniae to aztreonam and clavulanic acid stress on transcriptional level Streptococcus pneumoniae is able to acquire antibiotic resistance by activation of competence and subsequent DNA uptake. Several antibiotics induce competence by disrupting protein-quality control or perturbing DNA replication. Here, we demonstrate that aztreonam (AZT) and clavulanic acid (CLA) also promote competence. We show that AZT and CLA induce cell chain formation by targeting the D,D-carboxypeptidase PBP3. In support of the hypothesis that chain forming promotes competence, we demonstrate that an autolysin mutant ( lytB) is hypercompetent. As competence is initiated by the binding of a small extracellular peptide (CSP) to a membrane-anchored receptor (ComD), we wondered if chain formation alters CSP diffusion and thereby sensing by ComD. Indeed, the presence of AZT or CLA affects competence synchronization. Artificially reducing CSP diffusion by increasing culture medium viscosity also triggers competence. Together, our data show that AZT and CLA effectively switch CSP-based quorum sensing to autocrine-like signalling, as CSP is now retained to chained cells and no longer shared in a common pool.
2018-03-09 | GSE111562 | GEO