Occurrence of genes encoding spore germination in Clostridium species that cause meat spoilage

Abstract

Members of the genus Clostridium are frequently associated with meat spoilage. The ability for low numbers of spores of certain Clostridium species to germinate in cold-stored vacuum-packed meat can result in blown pack spoilage. However, little is known about the germination process of these clostridia, despite this characteristic being important for their ability to cause spoilage. This study sought to determine the genomic conditions for germination of 37 representative Clostridium strains from seven species (C. estertheticum, C. tagluense, C. frigoris, C. gasigenes, C. putrefaciens, C. aligidicarnis and C. frigdicarnis) by comparison with previously characterized germination genes from C. perfringens, C. sporogenes and C. botulinum. All the genomes analysed contained at least one gerX operon. Seven different gerX operon configuration types were identified across genomes from C. estertheticum, C. tagluense and C. gasigenes. Differences arose between the C. gasigenes genomes and those belonging to C. tagluense/C. estertheticum in the number and type of genes coding for cortex lytic enzymes, suggesting the germination pathway of C. gasigenes is different. However, the core components of the germination pathway were conserved in all the Clostridium genomes analysed, suggesting that these species undergo the same major steps as Bacillus subtilis for germination to occur.

Keywords: Clostridium; meat spoilage; spore germination.

Fig. 1.

Core genome comparison of 40 Clostridium strains and one Bacillus subtilis strain. A phylogenetic network was constructed in SplitsTree using a core-genome alignment generated by Roary (v.3.8.2). Bar, 0.01 nucleotide changes per position.

Fig. 2.

Conservation of selected germination genes in Clostridium species associated with meat spoilage. This heat-map shows 20 genes involved in spore germination (a). The presence of a homologue was determined by using the OrthoMCL algorithm in the GET_HOMOLOGUES tool. No homologues were identified in the Clostridium genomes of the additional representative genes from B. subtilis: gerPA, gerPB, gerPC, gerPD, gerPE, gerPF, yfkQ, yfkR, yfkT. (b) Three potential pathways as represented by each colour. (c) Those genes that are linked.

Fig. 3.

Germination receptor gerX operon configurations in selected strains of Clostridium associated with meat spoilage. The gerX operon configurations (a) were designated types 1–7 based on the classifications defined previously [59]. Maximum-likelihood phylogenetic trees of the GerA (b), GerB (c) and GerC (d) amino acid sequences from the C. estertheticum strains DSM 14864^T and CF003, the C. tagluense strains A121, FP1 and FP2, the C. gasigenes strain CGAS001, the New Zealand strains FP3, FP4 and M14, as well as the reference strains C. botulinum ATCC 3502^T, BKT015925, Loch Maree, Ekland 17-B, C. sporogenes NCIMB10696 and B. subtilis 168. The GerX type is denoted by the number after the X and the colour block represents each different gerX configuration. The scale bar is the number of substitutions per site.

Fig. 4.

Schematic depiction for the proposed pathways during the germination of spores of Clostridium species associated with meat spoilage.