The Signal Sequence of the Abundant Extracellular Metalloprotease PPEP-1 Can Be Used to Secrete Synthetic Reporter Proteins in <i>Clostridium difficile</i>

Paiva, Ana Mafalda; Friggen, Annemieke H.; Hossein-Javaheri, S.H.J.; Smits, Wiep Klaas

doi:10.1021/acssynbio.6b00104

Cited by 37 publications

(42 citation statements)

References 33 publications

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“…All the constructs were placed in a modular vector under the control of the anhydrotetracycline (ATc) -inducible promoter P tet (28). As observed in other bioluminescence assays (29, 30) a background signal is detected from non-induced cells (Fig. 3A, T0), which is comparable to that of a medium only control (17872.8 ± 4397.7 RLU/OD; data not shown).…”

Section: Resultssupporting

confidence: 84%

“…Our experiments do not allow us to determine the possible effects of replacing the serine and cysteine residues on the structural stability of TcdC and/or its interactions with binding partners (37). Nevertheless, our results are confirmed in independent experiments using the HiBiT opt system, which to our knowledge, is applied for the first time in C. difficile here and extends our existing luciferase toolkit (29, 30).…”

Section: Discussionsupporting

confidence: 77%

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The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

Paiva

Jong

Friggen

et al. 2019

Preprint

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21Clostridioides difficile is an anaerobic gram-positive bacterium that can can produce the large 22 clostridial toxins, Toxin A and Toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also 23 encodes the sigma factor TcdR, that positively regulates toxin gene expression, and TcdC, a putative 24 negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor, however, several 25 studies failed to show an association between tcdC genotype and toxin production. Consequently, TcdC 26 function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated 27 protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is 28 mediated through the OB-fold domain present at the C-terminus of the protein. This domain was 29 previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating 30 a cytoplasmic localization of the C-terminal OB-fold. 31In this study we aimed to obtain topological information on the C-terminus of TcdC. Using Scanning 32 Cysteine Accessibility Mutagenesis and a HiBiT-based system, we demonstrate that the C-terminus of 33 TcdC is located extracellularly. The extracellular location of TcdC is not compatible with direct binding of 34 the OB-fold domain to intracellular nucleic acid or protein targets, and suggests a mechanism of action 35 that is different from characterized anti-sigma factors. 363 Importance 37Transcription of the C. difficile large clostrididial toxins (TcdA and TcdB) is directed by the sigma 38 factor TcdR. TcdC has been implicated as a negative regulator, possible acting as an anti-sigma factor.

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Section: Resultssupporting

confidence: 84%

Section: Discussionsupporting

confidence: 77%

The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

Paiva

Jong

Friggen

et al. 2019

Preprint

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“…We compared transcriptome data from strain IB58 (sigB::CT Ptet-sigB) to that of strain IB61 (sigB::CT Ptet-sluc opt ). IB61 harbors a vector for the inducible expression of a luciferase gene which does not lead to any toxicity and growth phenotype (24) We expected no or a limited number of genes to be differentially expressed (log2 foldchange (log2FC) of ≤ -1.5 or ≥1.5, and adjusted p-value of <0.05) under non-inducing conditions. Indeed, we found only five differentially expressed genes in the Ptet-sigB strain (IB58) compared to the Ptet-sluc opt control (IB61) strain (hybridizations 1 and 3) (Dataset S1).…”

Section: σ B Primarily Activates Genes Relating To Oxidative/nitrosatmentioning

confidence: 99%

Redefining theClostridioides difficileσ^Bregulon: σ^Bactivates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

Boekhoud

Michel

Corver

et al. 2020

Preprint

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In many gram-positive bacteria the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile σB has been implicated in protection against stressors such as reactive oxygen species and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allowed for the refinement of the previously proposed σB regulon. At least 32% the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σB was demonstrated through in vitro run-off transcription of specific target genes (cd0350, cd3614, cd3605, cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to σB-dependent gene activation.ImportanceSigma B is the alternative sigma factor governing stress response in many gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together our data suggest that σB is a key player in dealing with toxic radicals.

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“…has been used in C. acetobutylicum 65 as a reporter to study the phenomenon of strain degeneration (loss of solventogenesis) which is often caused by loss of the pSOL1 megaplasmid on which amyP is located. A codon-optimized amylase (AmyE opt ) has been used successfully as a secreted reporter in C. difficile by addition of a zinc metalloprotease PPEP-1 signal sequence 66…”

Section: Carbohydrate Hydrolases: -Glucuronidase -Galactosidase Amymentioning

confidence: 99%

“…This assay has the lowest background signal level but the requirement for live cell exposure to oxygen may introduce variability. A codon-optimized luciferase (sLuc opt ) was also successfully secreted in C. difficile using the aforementioned zinc metalloprotease signal peptide 66 . While there are many reporter choices available to clostridial researchers, we would argue that the multiplicity of reporters used has not helped ease the comparison of data obtained by different laboratories.…”

Section: Luciferasementioning

confidence: 99%

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

et al. 2017

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The solventogenic Clostridia are of interest to the chemical industry because of their natural ability to produce chemicals such as butanol, acetone and ethanol from diverse feedstocks. Their use as whole cell factories presents multiple metabolic engineering targets that could lead to improved sustainability and profitability of Clostridium industrial processes. However, engineering efforts have been held back by the scarcity of genetic and synthetic biology tools. Over the past decade, genetic tools to enable transformation and chromosomal modifications have been developed, but the lack of a broad palette of synthetic biology parts remains one of the last obstacles to the rapid engineered improvement of these species for bioproduction. We have systematically reviewed existing parts that have been used in the modification of solventogenic Clostridia, revealing a narrow range of empirically chosen and nonengineered parts that are in current use. The analysis uncovers elements, such as promoters, transcriptional terminators and ribosome binding sites where increased fundamental knowledge is needed for their reliable use in different applications. Together, the review provides the most comprehensive list of parts used and also presents areas where an improved toolbox is needed for full exploitation of these industrially important bacteria.

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The Signal Sequence of the Abundant Extracellular Metalloprotease PPEP-1 Can Be Used to Secrete Synthetic Reporter Proteins in Clostridium difficile

Cited by 37 publications

References 33 publications

The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

Redefining theClostridioides difficileσ^Bregulon: σ^Bactivates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

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The Signal Sequence of the Abundant Extracellular Metalloprotease PPEP-1 Can Be Used to Secrete Synthetic Reporter Proteins in Clostridium difficile

Cited by 37 publications

References 33 publications

The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

The C-terminal domain of Clostridioides difficile TcdC is exposed on the bacterial cell surface

Redefining theClostridioides difficileσBregulon: σBactivates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

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Redefining theClostridioides difficileσ^Bregulon: σ^Bactivates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide