ClosTron-Mediated Engineering of Clostridium

Kuehne, Sarah A.; Heap, John; Cooksley, Clare; Cartman, Stephen T.; Minton, Nigel P.

doi:10.1007/978-1-61779-197-0_23

Cited by 47 publications

(36 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mutations in the genes sleC, spo0A, fliC, and luxS were generated using the insertional inactivation system, ClosTron, as previously described (27)(28)(29)(30). Construction of the fliC and luxS mutants is described in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

Multiple Factors Modulate Biofilm Formation by the Anaerobic Pathogen Clostridium difficile

Đapa

Leuzzi

et al. 2012

Journal of Bacteriology

246

252

View full text Add to dashboard Cite

bBacteria within biofilms are protected from multiple stresses, including immune responses and antimicrobial agents. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Although biofilms have been well studied for several gut pathogens, little is known about biofilm formation by anaerobic gut species. The obligate anaerobe Clostridium difficile causes C. difficile infection (CDI), a major health care-associated problem primarily due to the high incidence of recurring infections. C. difficile colonizes the gut when the normal intestinal microflora is disrupted by antimicrobial agents; however, the factors or processes involved in gut colonization during infection remain unclear. We demonstrate that clinical C. difficile strains, i.e., strain 630 and the hypervirulent strain R20291, form structured biofilms in vitro, with R20291 accumulating substantially more biofilm. Microscopic and biochemical analyses show multiple layers of bacteria encased in a biofilm matrix containing proteins, DNA, and polysaccharide. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella, and a putative quorum-sensing regulator, LuxS, are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in Spo0A, a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

show abstract

Section: Methodsmentioning

confidence: 99%

Multiple Factors Modulate Biofilm Formation by the Anaerobic Pathogen Clostridium difficile

Đapa

Leuzzi

et al. 2012

Journal of Bacteriology

246

252

View full text Add to dashboard Cite

show abstract

“…Over the past two decades genetic techniques for clostridia, such as antisense RNA strategies [200], reporter gene systems [201][202][203][204], inducible promoter-repressor systems [205,206], and several double-crossover homologous recombination strategies [207][208][209][210][211][212], have been developed. More recently, integration based techniques such as ClosTron [213][214][215], as well as marker-less integration methods [216], have been applied to C. acetobutylicum and other species such as the cellulolytic C. thermocellum. Several review articles have been published recently that give a detailed overview of the developed tools [92,171,217].…”

Section: Strain Improvementmentioning

confidence: 99%

Commercial Biomass Syngas Fermentation

2012

View full text Add to dashboard Cite

Abstract:The use of gas fermentation for the production of low carbon biofuels such as ethanol or butanol from lignocellulosic biomass is an area currently undergoing intensive research and development, with the first commercial units expected to commence operation in the near future. In this process, biomass is first converted into carbon monoxide (CO) and hydrogen (H 2 )-rich synthesis gas (syngas) via gasification, and subsequently fermented to hydrocarbons by acetogenic bacteria. Several studies have been performed over the last few years to optimise both biomass gasification and syngas fermentation with significant progress being reported in both areas. While challenges associated with the scale-up and operation of this novel process remain, this strategy offers numerous advantages compared with established fermentation and purely thermochemical approaches to biofuel production in terms of feedstock flexibility and production cost. In recent times, metabolic engineering and synthetic biology techniques have been applied to gas fermenting organisms, paving the way for gases to be used as the feedstock for the commercial production of increasingly energy dense fuels and more valuable chemicals.

show abstract

“…Uracil-auxotrophic mutants of M. thermoacetica [41 ] and C. thermocellum [43] were constructed allowing for both positive-and counter-selection of desired recombinants. Moreover, methods employing bacterial group II intron for gene deletion [44,45,46 ] and a Bacillus subtilis resolvase [47-49] were developed as universal genetic tools for Clostridium species. Genetic systems were also established based on replicative plasmids capable of double crossover chromosomal integration [50], inducible counter-selection for markerless gene deletions/insertions at any desired genomic loci [51], coupled expression of heterologous selectable markers to a chromosomal promoter to select for double crossover events [52 ], and antisense RNA for protein down-regulation [53].…”

Section: Advances In Genetic Manipulation Toolsmentioning

confidence: 99%