Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains

Bhattacharjee, Disha; Sorg, Joseph A.

doi:10.1128/msphere.00941-19

Cited by 9 publications

(8 citation statements)

References 50 publications

(21 reference statements)

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“…Similar numbers of colonies were observed at DNA concentrations from 1 to 6 μg, supporting that DNA uptake does not limit the transformation efficiency (Figure C). Glycine improves electrotransformation in other Clostridia by incorporating into the cell wall in place of d -alanine, weakening the cell wall by reducing peptidoglycan linkages. , Supplementing GS2 medium with >100 mM glycine significantly reduced the growth rate, but 50, 100, or 150 mM glycine supplementation did not affect the transformation efficiency (data not shown). We also found that electrocompetent C.…”

Section: Resultsmentioning

confidence: 95%

“…Glycine improves electrotransformation in other Clostridia by incorporating into the cell wall in place of D-alanine, weakening the cell wall by reducing peptidoglycan linkages. 32,33 Supplementing GS2 medium with >100 mM glycine significantly reduced the growth rate, but 50, 100, or 150 mM glycine supplementation did not affect the transformation efficiency (data not shown). We also found that electrocompetent C. phytofermentans cells can be stored frozen at −80 °C without loss of transformation efficiency, even when the cells were frozen for 12 weeks (Figure 2D).…”

Section: Restriction Modification Systems In C Phytofermentansmentioning

confidence: 92%

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Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi

et al. 2022

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Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.

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Section: Resultsmentioning

confidence: 95%

Section: Restriction Modification Systems In C Phytofermentansmentioning

confidence: 92%

Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi

et al. 2022

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“…To construct the CRISPR-Cas9 spo0A deletion mutant, the gBlock for spo0A targeting sgRNA, CRISPR_spo0A_2, was introduced by Gibson assembly into the Kpn I and Mlu I restriction sites in pKM197 (49) and transformed into E. coli DH5α resulting in pKM213. The homology arms to be used for homology directed repair was PCR amplified from C. difficile R20291 genomic DNA using primers 5’spo0A_UP and 3’spo0A_UP for the 500 bp upstream arm and primers 5’spo0A_DN and 3’spo0A_DN for the 500 bp downstream arm.…”

Section: Methodsmentioning

confidence: 99%

The selenophosphate synthetase, selD, is important for Clostridioides difficile physiology

McAllister

Aguirre

Sorg

2021

Preprint

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The endospore-forming pathogen, Clostridioides difficile, is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and healthcare. C. difficile, like all forms of life, incorporates selenium into proteins through a selenocysteine synthesis pathway. The known selenoproteins in C. difficile are involved in a metabolic process that uses amino acids as the sole carbon and nitrogen source (Stickland metabolism). The Stickland metabolic pathway requires the use of two selenium-containing reductases. In this study, we built upon our initial characterization of the CRISPR-Cas9-generated selD mutant by creating a CRISPR-Cas9-mediated restoration of the selD gene at the native locus. Here, we use these CRISPR-generated strains to analyze the importance of selenium-containing proteins on C. difficile physiology. SelD is the first enzyme in the pathway for selenoprotein synthesis and we found that multiple aspects of C. difficile physiology were affected (e.g., growth, sporulation, and outgrowth of a vegetative cell post-spore germination). Using RNAseq, we identified multiple candidate genes which likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Our results suggest that the absence of selenophosphate (i.e., selenoprotein synthesis) leads to alterations to C. difficile physiology so that NAD+ can be regenerated by other pathways.ImportanceC. difficile is a Gram-positive, anaerobic gut pathogen which infects thousands of individuals each year. In order to stop the C. difficile lifecycle, other non-antibiotic treatment options are in urgent need of development. Towards this goal, we find that a metabolic process used by only a small fraction of the microbiota is important for C. difficile physiology – Stickland metabolism. Here, we use our CRISPR-Cas9 system to ‘knock in’ a copy of the selD gene into the deletion strain to restore selD at its native locus. Our findings support the hypothesis that selenium-containing proteins are important for several aspects of C. difficile physiology – from vegetative growth to spore formation and outgrowth post-germination.

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“…In this concept, microwaves at sub-lethal temperatures induce the formation of pores in a cellular membrane due to their interaction with polar molecules. Although this is yet to be fully understood, current theories suggest that the polar molecules in the cell membrane rotate and create reversible pores; once the microwave is removed the pores close and return to the original structure [ 23 , 24 , 25 , 26 ]. These pores allow the cellular contents to leak outside, including substances such as DNA that are not normally able to cross the cell membrane.…”

Section: Non-thermal Interactions With Bacteriamentioning

confidence: 99%

“…These pores allow the cellular contents to leak outside, including substances such as DNA that are not normally able to cross the cell membrane. These released components from the cell are fully intact at sub-lethal temperatures and once purified can be used for further research [ 26 ]. Cells have shown to initially shrink after non-lethal microwave exposure, however, once the pores close, within 30 s the cell has been observed to return to its original size [ 22 , 27 ].…”

Section: Non-thermal Interactions With Bacteriamentioning

confidence: 99%

Applications of Microwave Energy in Medicine

Gartshore

Kidd²,

Joshi

2021

Biosensors

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Microwaves are a highly utilized electromagnetic wave, used across a range of industries including food processing, communications, in the development of novel medical treatments and biosensor diagnostics. Microwaves have known thermal interactions and theorized non-thermal interactions with living matter; however, there is significant debate as to the mechanisms of action behind these interactions and the potential benefits and limitations of their use. This review summarizes the current knowledge surrounding the implementation of microwave technologies within the medical industry.

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Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains

Cited by 9 publications

References 50 publications

Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi

Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi

The selenophosphate synthetase, selD, is important for Clostridioides difficile physiology

Applications of Microwave Energy in Medicine

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