Engineering Clostridium Strain to Accept Unmethylated DNA

Dong, Hongjun; Zhang, Yanping; Dai, Zhendong; Li, Yin

doi:10.1371/journal.pone.0009038

Cited by 72 publications

(60 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We implemented SpCRISPR-Cas9 for genome editing in C. acetobutylicum by first targeting the cac1502 gene (encoding the restriction endonuclease Cac824I), which was previously disrupted using group II intron technology (51). The protospacer adjacent motif (PAM) sequence associated with Cas9 binding (i.e.,5=-NGG-3=) (52) was present adjacent to the targeting protospacer within the cac1502 gene (Fig.…”

Section: Resultsmentioning

confidence: 99%

Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

Bruder

Pyne

Moo‐Young

et al. 2016

Appl Environ Microbiol

View full text Add to dashboard Cite

The discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genus Clostridium has lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied the Streptococcus pyogenes type II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing in Clostridium acetobutylicum DSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression in C. acetobutylicum. Subsequently, we targeted the carbon catabolite repression (CCR) system of C. acetobutylicum through transcriptional repression of the hprK gene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated in Clostridium pasteurianum ATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes. IMPORTANCEAfter recognizing the industrial potential of Clostridium for decades, methods for the genetic manipulation of these anaerobic bacteria are still underdeveloped. This study reports the implementation of CRISPR-Cas technology for genome editing and transcriptional regulation in Clostridium acetobutylicum, which is arguably the most common industrial clostridial strain. The developed genetic tools enable simpler, more reliable, and more extensive derivation of C. acetobutylicum mutant strains for industrial purposes. Similar approaches were also demonstrated in Clostridium pasteurianum, another clostridial strain that is capable of utilizing glycerol as the carbon source for butanol fermentation, and therefore can be arguably applied in other clostridial strains.

show abstract

Section: Resultsmentioning

confidence: 99%

Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

Bruder

Pyne

Moo‐Young

et al. 2016

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…To investigate the specific function of the selected gene SMB_G2359 and SMB_G3117, attempts to disrupt these genes in C. acetobutylicum DSM1731 were made using ClosTron pMTL008 vector (Dong et al, 2010;Heap et al, 2010). To disrupt gene SMB_G2359, three targeted intron insertion sites (732/733s, 867/868s, and 1292/1293s) were tested, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…All 12 putative autolysin genes were overexpressed in C. acetobutylicum SMB009 which is capable of accepting unmethylated DNA (Dong et al, 2010) to evaluate whether these genes are involved in autolysis. Considering that constitutive expression of autolysin genes may lead to cellular toxicity, the inducible expression vector pGusA2-2tetO1 that we previously developed (Dong et al, 2012) was used.…”

Section: Preliminary Evaluation Of the Putative Autolysin Genes In Cmentioning

confidence: 99%

See 1 more Smart Citation

Discovery of a novel gene involved in autolysis ofClostridiumcells

et al. 2013

Self Cite

View full text Add to dashboard Cite

Cell a utolysis plays important physiological roles in the life cycle of clostridial cells. Unders tanding the genetic basis of the autolysis phenomenon of pathogenic Clostridium or solvent producing Clostridium cells might provide new insights into this important species. Genes that might be involved in autolysis of Clostridium acetobutylicum, a model clostridial species, were investigated in this study. Twelve putative autolysin genes were predicted in C. acetobutylicum DSM 1731 genome through bioinformatics analysis. Of these 12 genes, gene SMB_G3117 was selected for testing the in tracellular autolysin activity, growth profi le, viable cell numbers, and cellular morphology. We found that overexpression of SMB_G3117 gene led to earlier ceased growth, signifi cantly increased number of dead cells, and clear electrolucent cavities, while disruption of SMB_G3117 gene exhibited remarkably reduced intracellular autolysin activity. These results indicate that SMB_G3117 is a novel gene involved in cellular autolysis of C. acetobutylicum.

show abstract

“…The methylase protects the self DNA from restriction digestion by methylating the nucleotides at specific DNA sequences (i.e., restriction sites), while the foreign DNA, which usually bears a different methylation pattern, would be recognized and degraded by the endonucleases (18). It has been reported that R-M systems can dramatically reduce the DNA transformation efficiency in a variety of bacteria (13,(19)(20)(21)(22)(23)(24), including cyanobacteria (7,(25)(26)(27)(28). For instance, Elhai and coworkers reported that the efficiency of conjugal transfer of shuttle vectors from E. coli into the cyanobacterium Anabaena sp.…”

mentioning

confidence: 99%

Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

Wang

Zhang

et al. 2015

Appl Environ Microbiol

View full text Add to dashboard Cite

cRestriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11-to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5= untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803.

show abstract

Engineering Clostridium Strain to Accept Unmethylated DNA

Cited by 72 publications

References 50 publications

Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

Discovery of a novel gene involved in autolysis ofClostridiumcells

Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

Contact Info

Product

Resources

About