Identification of genes involved in gliding motility and proteomic analysis of spore inner membrane proteins in Clostridium perfringens

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Virginia Tech

Clostridium perfringens is a Gram-positive anaerobic pathogen of humans and animals. While lacking flagella, C. perfringens cells can still migrate across surfaces using a type of gilding motility that involves the formation of filaments of bacteria lined up in an end to end conformation.

To discover the gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. Twenty-four mutants with deficiency in gliding motility were identified and one gene, which encodes a homolog of the SagA cell wall-dependent endopeptidase, was further characterized.

We also isolated and characterized two hypermotile variants of strain SM101. Compared to wide type cells, the hypermotile cells are longer and video microscopy of their gliding motility suggests they form long, thin filaments that move rapidly away from a colony, analogous to swarmer cells in bacteria with flagella. Whole genome sequencing analysis showed that both mutants have mutations in cell division genes. Complementation of these mutations with wild-type copies of each gene restored the normal motility phenotype. A model is presented explaining the principles underlying the hypermotility phenotype.

Heat resistant spores are the major route for disease transmission for C. perfringens, which cause food poisoning. To elucidate the molecular mechanisms involved in spore germination as well as to identify attractive targets for development of germination inhibitors to kill spores, we combined 1D-SDS-PAGE and MALDI-TOF-MS/MS to map the whole spore inner membrane proteome, both from dormant and germinated spores. As the first comprehensive spore inner membrane proteome study, we identified 494 proteins in total and 119 are predicted to be membrane-associated proteins. Among those membrane-associated proteins, 71 changed at least two-fold in abundance after germination. This study provides the first comprehensive list of the spore inner membrane proteins that may be involved in germination of the C. perfringens spore and their relative levels during germination.

mutagenesis, transposon, motility, spore, proteome, membrane