Project description:Staphylococcus epidermidis is a significant opportunistic pathogen of humans. Molecular studies in this species have been hampered by the presence of restriction-modification (RM) systems that limit introduction of foreign DNA. Here, we establish the complete genomes and methylomes for seven clinically significant, genetically diverse S. epidermidis isolates and perform the first systematic genomic analyses of the type I RM systems within both S. epidermidis and Staphylococcus aureus Our analyses revealed marked differences in the gene arrangement, chromosomal location, and movement of type I RM systems between the two species. Unlike S. aureus, S. epidermidis type I RM systems demonstrate extensive diversity even within a single genetic lineage. This is contrary to current assumptions and has important implications for approaching the genetic manipulation of S. epidermidis Using Escherichia coli plasmid artificial modification (PAM) to express S. epidermidishsdMS, we readily overcame restriction barriers in S. epidermidis and achieved electroporation efficiencies equivalent to those of modification-deficient mutants. With these functional experiments, we demonstrated how genomic data can be used to predict both the functionality of type I RM systems and the potential for a strain to be electroporation proficient. We outline an efficient approach for the genetic manipulation of S. epidermidis strains from diverse genetic backgrounds, including those that have hitherto been intractable. Additionally, we identified S. epidermidis BPH0736, a naturally restriction-defective, clinically significant, multidrug-resistant ST2 isolate, as an ideal candidate for molecular studies.IMPORTANCEStaphylococcus epidermidis is a major cause of hospital-acquired infections, especially those related to implanted medical devices. Understanding how S. epidermidis causes disease and devising ways to combat these infections have been hindered by an inability to genetically manipulate clinically significant hospital-adapted strains. Here, we provide the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis and demonstrate that these are distinct from those described for S. aureus Using these insights, we demonstrate an efficient approach for the genetic manipulation of S. epidermidis to enable the study of clinical isolates for the first time.
Project description:Bifidobacterium breve represents one of the most abundant (bifido)bacterial species in the gastro-intestinal tract of (breast-fed) infants, where their presence is believed to be beneficial. In the present study whole genome sequencing, employing PacBio’s Single Molecule, Real-Time (SMRT) sequencing platform, combined with comparative genome analysis allowed the most extensive genetic investigation of this taxon. Our findings demonstrate that Restriction/Modification (R/M) systems constitute a substantial part of the B. breve variome. Availing of the methylome data generated by SMRT sequencing, combined with targeted Illumina bisulfite sequencing (BiSeq) and comparative genome analysis, we were able to detect methylation recognition motifs and assign these to identified B. breve R/M systems, where in several cases such assignments were confirmed by restriction analysis. Furthermore, cloning of a selected methyltransferase-encoding gene validated the activity of the corresponding R/M system, and was shown to overcome the barrier they impose to genetic accessibility, thus allowing future genetic manipulation of members of this species.
Project description:The custom-made S. epidermidis GeneChips(Shanghai Biochip Co., Ltd) included qualifiers representing open reading frame (ORF) sequences identified in the genomes of the S. epidermidis strain RP62A, as well as unique ORFs in S. epidermidis strain 12228. The GeneChips were composed of cDNA array containing PCR products of 2316 genes and oligonucleotide array containing 252 genes.Two-component regulatory systems (TCSs) play a pivotal role in bacterial adaptation, survival, and virulence by sensing changes in the external environment and modulating gene expression in response to a variety of stimuli.To investigate the regulatory role of LytSR, one of the TCSs identified in the genomes of S. epidermidis, we used the GeneChips to perform a transcriptional profile analysis of the wild strain and lytSR mutant.
Project description:The custom-made S. epidermidis GeneChips(Shanghai Biochip Co., Ltd) included qualifiers representing open reading frame (ORF) sequences identified in the genomes of the S. epidermidis strain RP62A, as well as unique ORFs in S. epidermidis strain 12228. The GeneChips were composed of cDNA array containing PCR products of 2316 genes and oligonucleotide array containing 252 genes.Two-component regulatory systems (TCSs) play a pivotal role in bacterial adaptation, survival, and virulence by sensing changes in the external environment and modulating gene expression in response to a variety of stimuli.To investigate the regulatory role of LytSR, one of the TCSs identified in the genomes of S. epidermidis, we used the GeneChips to perform a transcriptional profile analysis of the wild strain and lytSR mutant. Wild type untreated in triplicate is compared to lytSR mutant in triplicate for cDNA array and four replicates on the oligo array.
Project description:Autoinducer 2 (AI-2), a widespread by-product of the LuxS-catalyzed S-ribosylhomocysteine cleavage reaction in the activated methyl cycle, has been suggested to serve as an intra- and interspecies signaling molecule, but in many bacteria AI-2 control of gene expression is not completely understood. Particularly, we have a lack of knowledge about AI-2 signaling in the important human pathogens Staphylococcus aureus and S. epidermidis. Here, to determine the role of LuxS and AI-2 in S. epidermidis, we analyzed genome-wide changes in gene expression in an S. epidermidis luxS mutant and after addition of AI-2 synthesized by over-expressed S. epidermidis Pfs and LuxS enzymes. Genes under AI-2 control included mostly genes involved in sugar, nucleotide, amino acid, and nitrogen metabolism, but also virulence-associated genes coding for lipase and bacterial apoptosis proteins. In addition, we demonstrate by liquid chromatography/mass-spectrometry of culture filtrates that the pro-inflammatory phenol-soluble modulin (PSM) peptides, key virulence factors of S. epidermidis, are under luxS/AI-2 control. Our results provide a detailed molecular basis for the role of LuxS in S. epidermidis virulence and suggest a signaling function for AI-2 in this bacterium. Keywords: wild type without glucose control vs luxS mutant vs luxS mutant with auto-inducer II
Project description:Autoinducer 2 (AI-2), a widespread by-product of the LuxS-catalyzed S-ribosylhomocysteine cleavage reaction in the activated methyl cycle, has been suggested to serve as an intra- and interspecies signaling molecule, but in many bacteria AI-2 control of gene expression is not completely understood. Particularly, we have a lack of knowledge about AI-2 signaling in the important human pathogens Staphylococcus aureus and S. epidermidis. Here, to determine the role of LuxS and AI-2 in S. epidermidis, we analyzed genome-wide changes in gene expression in an S. epidermidis luxS mutant and after addition of AI-2 synthesized by over-expressed S. epidermidis Pfs and LuxS enzymes. Genes under AI-2 control included mostly genes involved in sugar, nucleotide, amino acid, and nitrogen metabolism, but also virulence-associated genes coding for lipase and bacterial apoptosis proteins. In addition, we demonstrate by liquid chromatography/mass-spectrometry of culture filtrates that the pro-inflammatory phenol-soluble modulin (PSM) peptides, key virulence factors of S. epidermidis, are under luxS/AI-2 control. Our results provide a detailed molecular basis for the role of LuxS in S. epidermidis virulence and suggest a signaling function for AI-2 in this bacterium. Keywords: wild type without glucose control vs luxS mutant vs luxS mutant with auto-inducer II wild type without glucose control vs luxS mutant vs luxS mutant with auto-inducer II
Project description:We use the zebrafish embryo model to study the innate immune response against Staphylococcus epidermidis. Therefore, we injected S. epidermidis (and three controls groups) into the yolk at 2 hpf and samples at mutiple timepoints. Gene expression profiles were obtained at 6, 30, 54, 78, 102 and 126 hpi by microarrays. The results show that the gram-positive bacterium S. epidermidis induces a late immune response with a strong response at 102 hpi.
Project description:Methylomics and transcriptomics array-based analysis of 36 placentas: 28 with IUGR (Intra-Uterine Growth Restriction) vs. 8 control pregnancies. Methylomics data were obtained using Illumina HumanMethylation-450k microarrays. Transcriptomics data were obtained using Illumina HumanHT-12 v4 Expression BeadChips.
Project description:We sequenced mRNA from three independent biological replicates of Staphylococcus epidermidis biofilms with different proportion of dormant cells. Whole trancriptome analysis of Staphylococcus epidermidis biofilms with prevented and induced dormancy.
Project description:We use the zebrafish embryo model to study the innate immune response against Staphylococcus epidermidis. Therefore, we injected S. epidermidis into the yolk at 2 hpf and took samples at 5 days post injection.