Comparative Genomics Provides Insights Into Genetic Diversity of Clostridium tyrobutyricum and Potential Implications for Late Blowing Defects in Cheese

Abstract

Clostridium tyrobutyricum has been recognized as the main cause of late blowing defects (LBD) in cheese leading to considerable economic losses for the dairy industry. Although differences in spoilage ability among strains of this species have been acknowledged, potential links to the genetic diversity and functional traits remain unknown. In the present study, we aimed to investigate and characterize genomic variation, pan-genomic diversity and key traits of C. tyrobutyricum by comparing the genomes of 28 strains. A comparative genomics analysis revealed an "open" pangenome comprising 9,748 genes and a core genome of 1,179 genes shared by all test strains. Among those core genes, the majority of genes encode proteins related to translation, ribosomal structure and biogenesis, energy production and conversion, and amino acid metabolism. A large part of the accessory genome is composed of sets of unique, strain-specific genes ranging from about 5 to more than 980 genes. Furthermore, functional analysis revealed several strain-specific genes related to replication, recombination and repair, cell wall, membrane and envelope biogenesis, and defense mechanisms that might facilitate survival under stressful environmental conditions. Phylogenomic analysis divided strains into two clades: clade I contained human, mud, and silage isolates, whereas clade II comprised cheese and milk isolates. Notably, these two groups of isolates showed differences in certain hypothetical proteins, transcriptional regulators and ABC transporters involved in resistance to oxidative stress. To the best of our knowledge, this is the first study to provide comparative genomics of C. tyrobutyricum strains related to LBD. Importantly, the findings presented in this study highlight the broad genetic diversity of C. tyrobutyricum, which might help us understand the diversity in spoilage potential of C. tyrobutyricum in cheese and provide some clues for further exploring the gene modules responsible for the spoilage ability of this species.

Keywords: Clostridium tyrobutyricum; cheese; comparative genomics; dairy; pangenome; spoilage.

Figure 1

Heatmap of the average nucleotide identity (ANI) of 28 Clostridium tyrobutyricum strains. ANIm analysis based on MUMmer alignment (Kurtz et al., 2004) of the genome sequences was performed and visualized using pyANI (see Footnote 4). The strain names are colored according to their isolation source, where red, blue, green, gray, and orange denote milk, cheese, silage, human, and mud, respectively. The type strain is indicated by the superscript capital T.

Figure 2

Phylogenetic analysis of 28 Clostridium tyrobutyricum strains. (A) Maximum-likelihood (ML) phylogenetic tree based on 1,179 single-copy gene families using 500 bootstrap replications. (B) Pangenome tree based on presence/absence of gene family in all genomes. The strain names are colored according to their isolation source, where red, blue, green, gray, and orange denote milk, cheese, silage, human, and mud, respectively. The type strain is indicated by the superscript capital T. The phylogenetic trees were visualized using MEGA X v10.1 (Kumar et al., 2018).

Figure 3

Genomic diversity of 28 Clostridium tyrobutyricum strains. (A) Core and strain-specific genes of 28 C. tyrobutyricum genomes. Each oval represents a strain. The number of genes shared by all strains (core genome) is in the center. The number of strain-specific genes is indicated in non-overlapping portions of each oval. The type strain is indicated by the superscript capital T. (B) Increase and decrease in gene families in the pan-(blue) and core (violet) genome, respectively. Gene accumulation curves were plotted as a function of the number of genomes sequentially added (n = 28) of the power-law regression model. The pangenome size is calculated at 9,748 genes. The trajectory of the pangenome shows characteristics of an open pangenome.

Figure 4

Clusters of orthologous groups (COGs) in core- and accessory genome, and strain-specific genes and their associated functions in 28 Clostridium tyrobutyricum genomes. (A) Distribution of functional COGs in each core, accessory, and unique genome. (B) Detailed distribution of COGs with their functions.