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Use of a mariner-based transposon mutagenesis system to isolate Clostridium perfringens mutants deficient in gliding
Hualan Liu1, Laurent Bouillaut, Abraham L Sonenshein
1Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, USA.
Abstract:
Clostridium perfringens is an anaerobic Gram-positive pathogen that causes many human and animal diseases, including food poisoning and gas gangrene. C. perfringens lacks flagella but possesses type IV pili (TFP). We have previously shown that C. perfringens can glide across an agar surface in long filaments composed of individual bacteria attached end to end and that two TFP-associated proteins, PilT and PilC, are needed for this. To discover additional gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. More than 10,000 clones were screened for mutants that lacked the ability to move away from the edge of a colony. Twenty-four mutants (0.24%) were identified that fit the criteria. The genes containing insertions that affected gliding motility fell into nine different categories. One gene, CPE0278, which encodes a homolog of the SagA cell wall-dependent endopeptidase, acquired distinct transposon insertions in two independent mutants. sagA mutants were unable to form filaments due to a complete lack of end-to-end connections essential for gliding motility. Complementation of the sagA mutants with a wild-type copy of the gene restored gliding motility. We constructed an in-frame deletion mutation in the sagA gene and found that this mutant had a phenotype similar to those of the transposon mutants. We hypothesize that the sagA mutant strains are unable to form the molecular complexes which are needed to keep the cells in an end-to-end orientation, leading to separation of daughter cells and the inability to carry out gliding motility.
Insights
Clostridium perfringens gliding motility requires end-to-end bacterial connections. A new study identifies the SagA endopeptidase as crucial for maintaining these connections, essential for movement.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Clostridium perfringens is an anaerobic Gram-positive pathogen causing significant human and animal diseases.
- This bacterium lacks flagella but utilizes type IV pili (TFP) for motility.
- Previous research identified TFP-associated proteins PilT and PilC as necessary for C. perfringens gliding motility.
Purpose of the Study:
- To identify novel gene products involved in Clostridium perfringens gliding motility.
- To develop and utilize a plasmid-based mariner transposon mutagenesis system for genetic screening in C. perfringens.
Main Methods:
- A mariner transposon mutagenesis system was employed to screen over 10,000 C. perfringens clones.
- Mutants exhibiting impaired colony edge motility were isolated and analyzed.
- Genes disrupted by transposon insertions affecting gliding were identified and categorized.
- sagA gene function was further investigated through complementation and in-frame deletion studies.
Main Results:
- Twenty-four mutants (0.24%) with defects in gliding motility were identified.
- Transposon insertions in the CPE0278 gene, encoding a SagA homolog, were found in two independent mutants.
- sagA mutants demonstrated an inability to form filaments due to a lack of end-to-end bacterial connections.
- Complementation and deletion analyses confirmed the essential role of SagA in maintaining cell-to-cell adhesion for gliding.
Conclusions:
- The SagA endopeptidase is critical for maintaining end-to-end bacterial connections in Clostridium perfringens.
- Disruption of SagA function leads to cell separation and loss of gliding motility.
- These findings elucidate a novel mechanism essential for the gliding motility of this important pathogen.
