Clostridium perfringens-Escherichia coli Shuttle Vectors That Carry Single Antibiotic Resistance Determinants

doi:10.1006/plas.1993.1025

Plasmid

Volume 29, Issue 3, May 1993, Pages 233-235

https://doi.org/10.1006/plas.1993.1025 Get rights and content

Abstract

Two versatile Clostridium perfringens-Escherichia coli shuttle vectors were constructed. Each plasmid carried a single antibiotic resistance gene which was expressed in both organisms. The plasmid pJIR750 encoded resistance to chloramphenicol and pJIR751 encoded resistance to erythromycin. Each plasmid contained the pUC18-derived multiple cloning site and the lacZ′ gene which enabled direct screening for recombinants in E. coli . These plasmids should prove invaluable for the genetic manipulation of C. perfringens.

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Cited by (118)

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The gut microbiota plays a significant role in influencing human immunity, metabolism, development, and behavior by producing a wide range of metabolites. While there is accumulating data on several microbiota-derived small molecules that contribute to host health and disease, our knowledge regarding the molecular mechanisms underlying metabolite-mediated microbe-host interactions remains limited. This is primarily due to the lack of efficient genetic tools for most commensal bacteria, especially those belonging to the dominant phyla Bacteroides spp. and Clostridium spp., which hinders the application of synthetic biology to these gut commensal bacteria. In this review, we provide an overview of recent advances in synthetic biology tools developed for the two dominant genera, as well as their applications in deciphering the mechanisms of microbe-host interactions mediated by microbiota-derived small molecules. We also discuss the potential biomedical applications of engineering commensal bacteria using these toolboxes. Finally, we share our perspective on the future development of synthetic biology tools for a better understanding of small molecule-mediated microbe-host interactions and their engineering for biomedical purposes.
The serine proteases CspA and CspC are essential for germination of spores of Clostridium perfringens SM101 through activating SleC and cortex hydrolysis
2020, Food Microbiology
Citation Excerpt :
To construct the cspA-cspC operon complementation plasmid, a ~4.1-kb DNA fragment carrying the 571-bp upstream region of the cspA, the cspA-ORF, the cspC ORF and the 180-bp cspC downstream region was PCR amplified using primer pairs CPP1160/CPP1159 (forward and reverse primers had KpnI and SalI sites, respectively, at their 5’ ends). These ~2.2 and 4.1-kb PCR fragments were digested with KpnI and SalI and cloned into the KpnI-SalI sites of C. perfringens-E. coli shuttle multicopy vector pJIR750 (Bannam and Rood, 1993), yielding plasmids pPKT108 and pPKT110, respectively. To construct the cspA mutant complemented with wild-type cspA or cspA-cspC operon, plasmids pPKT108 and pPKT110 were introduced into the C. perfringens cspA mutant strain PKT133 by electroporation, and Emr Cmr transformants were selected, generating strains PKT133(pPKT108) (cspA mutant carrying wild-type cspA) and PKT133(pPKT110) (cspA mutant carrying wild-type cspA-cspC), respectively.
Clostridium perfringens SM101 genome encodes three serine proteases (CspA, CspB, and CspC), and genetic evidence indicates that CspB is required for processing of pro-SleC into active SleC, an enzyme essential for degradation of the peptidoglycan cortex during spore germination. In this study, the expression of cspA and cspC, as well as the germination and colony formation by spores of cspAC and cspC mutants of strain SM101, were assessed. We demonstrated that 1) the cspA and cspC genes were expressed as a bicistronic operon only during sporulation in the mother cell compartment of SM101; 2) both cspAC and cspC mutant spores were unable to germinate significantly with either KCl, l-glutamine, brain heart infusion (BHI) broth, or a 1:1 chelate of Ca²⁺ and dipicolinic acid (DPA); 3) consistent with germination results, both cspAC and cspC mutant spores were defective in normal DPA release; 4) the colony formation by cspAC and cspC mutant spores was ~10⁶-fold lower than that of wild-type spores, although decoated mutant spores yielded wild-type level colony formation on plates containing lysozyme; 5) no processing of inactive pro-SleC into active SleC was observed in cspAC and cspC mutant spores during germination; and finally, 6) the defects in germination, DPA release, colony formation and SleC processing in cspAC and cspC mutant spores were complemented by the wild-type cspA-cspC operon. Collectively, these results indicate that both CspA and CspC are essential for C. perfringens spore germination through activating SleC and inducing cortex hydrolysis.
Phosphorothioation of foreign DNA influences the transformation efficiency in Clostridium perfringens NCTC8239
2020, Anaerobe
Major advances in Clostridium perfringens genetics have been achieved through the development of electroporation-induced transformation; however, highly transformable strains are still limited. SM101 is the only useful strain for genetic manipulation via transformation in C. perfringens causing foodborne illness (FBI). We focused on the FBI strain NCTC8239, which is transformed at a low frequency, because it has a gene cassette that is predicted to encode enzymes involved in DNA phosphorothioation (PT). The oxidant-dependent degradation of NCTC8239 genomic DNA suggested that the genome is PT-modified. When foreign DNA was PT-modified using a plasmid expressing Salmonella enterica PT modification enzymes, the transformation efficiency of NCTC8239 was significantly higher than that using an unmodified plasmid. We then attempted to establish a highly transformable derivative of NCTC8239, and focused on DptFGH, which are predicted to be PT restriction enzymes. A dptG-null mutant exhibited significantly higher transformation efficiency with unmodified foreign DNA than did the wild-type strain. Furthermore, the mutant was transformed with the unmodified plasmid as efficiently as with a PT-modified plasmid, implying that DptG is involved in PT-dependent restriction. Thus, the present results revealed that PT modifications of foreign DNA influence the transformation frequency of NCTC8239 and suggest that PT is a factor contributing to transformation efficiency in NCTC8239.
The Tcp plasmids of Clostridium perfringens require the resP gene to ensure stable inheritance
2020, Plasmid
Citation Excerpt :
The results showed that the pCW3ΔresP plasmids were less stable than wild-type pCW3, with an average of 67.5 ± 6% of the population retaining the plasmid following six days passage, in comparison to 93.3 ± 1% for wild-type pCW3 (Fig. 2A). Complementation of the ΔresP1 mutant in trans with the wild-type resP gene on the multicopy shuttle vector, pJIR750 (Bannam and Rood, 1993), restored plasmid stability to wild-type levels (95.3 ± 1%) (Fig. 2B). These results showed that resP is required for the stable inheritance of pCW3 under these conditions.
Many of the disease-causing toxins of the pathogenic bacterium Clostridium perfringens are harboured on large, highly stable, conjugative plasmids. Previous work has established the requirement of a ParMRC-like partitioning system for plasmid maintenance, but little is known about other mechanisms used to ensure stable plasmid inheritance. The archetypal 47 kb Tcp plasmid, pCW3, encodes a gene, resP, whose putative product has sequence similarity to members of the serine recombinase family of site-specific recombinases. ResP is therefore likely to function to resolve plasmid multimers. Sequence analysis identified that resP genes are present on all C. perfringens plasmid families, suggesting a conserved function in these plasmids. To assess the requirement of resP for the stability of pCW3, deletion mutants were constructed. Deletion of resP from pCW3 resulted in a marked instability phenotype that was rescued upon complementation with the wild-type resP gene. Complementation with resP genes from two different C. perfringens plasmids demonstrated that only closely related resP genes can complement the mutation on pCW3. The function of ResP in vivo was examined using an Escherichia coli model system, which determined that two directly repeated res sites were required for the resolution of DNA and that ResP could resolve multimeric plasmid forms into monomeric units. Based on these findings we concluded that ResP could catalyse the resolution of plasmid multimers and was required for the maintenance of Tcp plasmids within C. perfringens. Overall, the results of this study have significant implications for our understanding of the maintenance of toxin-encoding plasmids within C. perfringens.
A chemical and biological toolbox for Type Vd secretion: Characterization of the phospholipase A1 autotransporter FplA from Fusobacterium nucleatum
2017, Journal of Biological Chemistry
Citation Excerpt :
Genetic manipulation of Fusobacterium spp. is technically challenging, and of the seven strains used for analysis in this paper, only F. nucleatum 23726 and 10953 have been successfully mutated by gene deletion (45). A single homologous crossover plasmid (pDJSVT100; supplemental Table S2) that we developed from a Clostridium shuttle vector (46) using a recombination method previously established for F. nucleatum (45) was used to create a ΔfplA strain (gene HMPREF0397_1968) (strain DJSVT01; supplemental Table S1) marked with chloramphenicol resistance (Fig. 7, A and B). We verified by PCR that the fplA gene was disrupted by the chromosomally inserted plasmid and further showed expression of the protein had been abolished by a fluorescent probe and Western blots probed with an anti-FplA antibody (Fig. 7C).
Fusobacterium nucleatum is an oral pathogen that is linked to multiple human infections and colorectal cancer. Strikingly, F. nucleatum achieves virulence in the absence of large, multiprotein secretion systems (Types I, II, III, IV, and VI), which are widely used by Gram-negative bacteria for pathogenesis. By contrast, F. nucleatum strains contain genomic expansions of Type V secreted effectors (autotransporters) that are critical for host cell adherence, invasion, and biofilm formation. Here, we present the first characterization of an F. nucleatum Type Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby rename Fusobacterium phospholipase autotransporter (FplA). Biochemical analysis of multiple Fusobacterium strains revealed that FplA is expressed as a full-length 85-kDa outer membrane–embedded protein or as a truncated phospholipase domain that remains associated with the outer membrane. Whereas the role of Type Vd secretion in bacteria remains unidentified, we show that FplA binds with high affinity to host phosphoinositide-signaling lipids, revealing a potential role for this enzyme in establishing an F. nucleatum intracellular niche. To further analyze the role of FplA, we developed an fplA gene knock-out strain, which will guide future in vivo studies to determine its potential role in F. nucleatum pathogenesis. In summary, using recombinant FplA constructs, we have identified a biochemical toolbox that includes lipid substrates for enzymatic assays, potent inhibitors, and chemical probes to detect, track, and characterize the role of Type Vd secreted phospholipases in Gram-negative bacteria.
Identification of orphan histidine kinases that impact sporulation and enterotoxin production by Clostridium perfringens type F strain SM101 in a pathophysiologically-relevant ex vivo mouse intestinal contents model
2023, PLoS Pathogens
When causing food poisoning or antibiotic-associated diarrhea, Clostridium perfringens type F strains must sporulate to produce C. perfringens enterotoxin (CPE) in the intestines. C. perfringens is thought to use some of its seven annotated orphan histidine kinases to phosphorylate Spo0A and initiate sporulation and CPE production. We previously demonstrated the CPR0195 orphan kinase, but not the putative CPR1055 orphan kinase, is important when type F strain SM101 initiates sporulation and CPE production in modified Duncan-Strong (MDS) sporulation medium. Since there is no small animal model for C. perfringens sporulation, the current study used diluted mouse intestinal contents (MIC) to develop an ex vivo sporulation model and employed this model to test sporulation and CPE production by SM101 CPR0195 and CPR1055 null mutants in a pathophysiologically-relevant context. Surprisingly, both mutants still sporulated and produced CPE at wild-type levels in MIC. Therefore, five single null mutants were constructed that cannot produce one of the previously-unstudied putative orphan kinases of SM101. Those mutants implicated CPR1316, CPR1493, CPR1953 and CPR1954 in sporulation and CPE production by SM101 MDS cultures. Phosphorylation activity was necessary for CPR1316, CPR1493, CPR1953 and CPR1954 to affect sporulation in those MDS cultures, supporting their identity as kinases. Importantly, only the CPR1953 or CPR1954 null mutants exhibited significantly reduced levels of sporulation and CPE production in MIC cultures. These phenotypes were reversible by complementation. Characterization studies suggested that, in MDS or MIC, the CPR1953 and CPR1954 mutants produce less Spo0A than wild-type SM101. In addition, the CPR1954 mutant exhibited little or no Spo0A phosphorylation in MDS cultures. These studies, i) highlight the importance of using pathophysiologically-relevant models to investigate C. perfringens sporulation and CPE production in a disease context and ii) link the CPR1953 and CPR1954 kinases to C. perfringens sporulation and CPE production in disease-relevant conditions.

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Short CommunicationsClostridium perfringens-Escherichia coli Shuttle Vectors That Carry Single Antibiotic Resistance Determinants

Abstract

Short Communications
Clostridium perfringens-Escherichia coli Shuttle Vectors That Carry Single Antibiotic Resistance Determinants