Search

Background: There is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol (ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (< 1 g/L). However, despite its relative ancient discovery, few genomic details have been described for this strain. Research efforts including omics and genetic engineering approaches are therefore needed to enable the use of C. beijerinckii as a microbial cell factory for production of isopropanol. Results: The complete genome sequence and a first transcriptome analysis of C. beijerinckii DSM 6423 are described in this manuscript. The combination of MiSeq and de novo PacBio sequencing revealed a 6.38 Mbp chromosome containing 6254 genomic objects. Three Mobile Genetic Elements (MGE) were also detected: a linear double stranded DNA bacteriophage (ϕ6423) and two plasmids (pNF1 and pNF2) highlighting the genomic complexity of this strain. A first RNA-seq transcriptomic study was then performed on 3 independent glucose fermentations. Clustering analysis allowed us to detect some key gene clusters involved in the main life cycle steps (acidogenesis, solvantogenesis and sporulation) and differentially regulated among the fermentation. These putative clusters included some putative metabolic operons comparable to those found in other reference strains such as C. beijerinckii NCIMB 8052 or C. acetobutylicum ATCC 824. Interestingly, only one gene was encoding for an alcohol dehydrogenase converting acetone into isopropanol, suggesting a single genomic event occurred on this strain to produce isopropanol. Conclusions: We present the full genome sequence of Clostridium beijerinckii DSM 6423, providing a complete genetic background of this strain. This offer a great opportunity for the development of dedicated genetic tools currently lacking for this strain. Moreover, a first RNA-seq analysis allow us to better understand the global metabolism of this natural isopropanol producer, opening the door to future targeted engineering approaches. [ABSTRACT FROM AUTHOR]

InClostridium difficile,erm(B) genes are located on mobile elements like Tn5398and Tn6215. In previous studies, some of these elements were transferred by conjugation-like mechanisms, mobilizedin transby helper conjugative systems. In this study, we analyzed the genomes of several recipient strains that acquired either Tn5398or Tn6215-like elements. We demonstrated that the integration of the transposons in the genome of the recipient cell was always due to homologous recombination events, involving exchange of large chromosomal segments. We did not observed transposon transfer to aC. difficilestrain in presence of DNAse, suggesting that a possible transformation-like mechanism occurred in this recipient. [ABSTRACT FROM PUBLISHER]

Resistance to the macrolide-lincosamide-streptogramin B group of antibiotics in Clostridium difficile is generally due to erm(B) genes. Tn 6194, a conjugative transposon initially detected in PCR-ribotype 027 isolates, is an erm(B)-containing element also detected in other relevant C. difficile PCR-ribotypes. In this study, the genome of a C. difficile PCR-ribotype 001 strain was sequenced, and an element with two nucleotidic changes compared to Tn 6194 was detected. This element was transferred by filter mating assays to recipient strains of C. difficile belonging to PCR-ribotype 009 and 027 and to a recipient strain of Enterococcus faecalis. Transconjugants were characterized by Southern blotting and genome sequencing, and integration sites in all transconjugants were identified. The element integrated the genome of C. difficile at different sites and the genome of E. faecalis at a unique site. This study is the first molecular characterization of an erm(B)-containing conjugative transposon in C. difficile and provides additional evidence of the antibiotic resistance transmission risk among pathogenic bacteria occupying the same human intestinal niche. [ABSTRACT FROM AUTHOR]

Molecular analysis is an important tool to investigate Clostridium difficile resistance to macrolide-lincosamide-streptogramin B (MLSB). In particular, the protocols described in this chapter have been designed to investigate the genetic organization of erm(B)-containing elements and to evaluate the capability of these elements to transfer in C. difficile recipient strains using filter mating assay.

Resistance to the macrolide-lincosamide-streptogramin B group of antibiotics in Clostridium difficile is generally due to erm(B) genes. Tn6194, a conjugative transposon initially detected in PCR-ribotype 027 isolates, is an erm(B)-containing element also detected in other relevant C. difficile PCR-ribotypes. In this study, the genome of a C. difficile PCR-ribotype 001 strain was sequenced, and an element with two nucleotidic changes compared to Tn6194 was detected. This element was transferred by filter mating assays to recipient strains of C. difficile belonging to PCR-ribotype 009 and 027 and to a recipient strain of Enterococcus faecalis. Transconjugants were characterized by Southern blotting and genome sequencing, and integration sites in all transconjugants were identified. The element integrated the genome of C. difficile at different sites and the genome of E. faecalis at a unique site. This study is the first molecular characterization of an erm(B)-containing conjugative transposon in C. difficile and provides additional evidence of the antibiotic resistance transmission risk among pathogenic bacteria occupying the same human intestinal niche.