Genomic approach to studying nutritional requirements of Clostridium tyrobutyricum and other Clostridia causing late blowing defects

Abstract

Clostridium tyrobutyricum is the main microorganism responsible for the late blowing defect in hard and semi-hard cheeses, causing considerable economic losses to the cheese industry. Deeper knowledge of the metabolic requirements of this microorganism can lead to the development of more effective control approaches. In this work, the amino acids and B vitamins essential for sustaining the growth of C. tyrobutyricum were investigated using a genomic approach. As the first step, the genomes of four C. tyrobutyricum strains were analyzed for the presence of genes putatively involved in the biosynthesis of amino acids and B vitamins. Metabolic pathways could be reconstructed for all amino acids and B vitamins with the exception of biotin (vitamin B7) and folate (vitamin B9). The biotin pathway was missing the enzyme amino-7-oxononanoate synthase that catalyzes the condensation of pimeloyl-ACP and l-alanine to 8-amino-7-oxononanoate. In the folate pathway, the missing genes were those coding for para-aminobenzoate synthase and aminodeoxychorismate lyase enzymes. These enzymes are responsible for the conversion of chorismate into para-aminobenzoate (PABA). Two C. tyrobutyircum strains whose genome was analyzed in silico as well as other 10 strains isolated from cheese were tested in liquid media to confirm these observations. 11 strains showed growth in a defined liquid medium containing biotin and PABA after 6-8 days of incubation. No strain showed growth when only one or none of these compounds were added, confirming the observations obtained in silico. Furthermore, the genome analysis was extended to genomes of single strains of other Clostridium species potentially causing late blowing, namely Clostridium beijerinckii, Clostridium sporogenes and Clostridium butyricum. Only the biotin biosynthesis pathway was incomplete for C. butyricum and C. beijerincki. In contrast, C. sporogenes showed missing enzymes in biosynthesis pathways of several amino acids as well as biotin, folate, and cobalamin (vitamin B12). These observations agree with the results of growth experiments of these species in liquid media reported in the literature. The results of this study suggest that biotin and folate are potential targets for reducing late blowing in cheese and highlight the usefulness of genomic analysis for identifying essential nutrients in bacteria.

The late blowing defect of hard cheeses: Behaviour of cells and spores of Clostridium tyrobutyricum throughout the cheese manufacturing and ripening

“…Furthermore, none of the FAA was depleted, indicating that availability of FAA was not a limiting factor for cell growth itself. This observation is supported by findings of Storari et al, (2016), who have recently reconstructed the presence of genes putatively involved in the biosynthesis of 19 amino acids in the genomes of four C. tyrobutyricum strains isolated from hard and semi-hard cheeses, including UC7086 used in this study. Alanine had an interesting behaviour because its content was always lower in the S-tubes.…”

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

“…Although the core genome of the analyzed C. tyrobutyricum strains represented ~40% of the average number of genes per genome, when we consider it as a fraction of pangenome size, it is found to be relatively small (12.1%). By comparison, previous studies had found the C. tyrobutyricum core genome to contain 2,409–2,764 genes ( Storari et al, 2016 ; Liu et al, 2020 ) representing roughly 80–96% of the total C. tyrobutyricum pangenome. This discrepancy is most probably due to the different number of genomes previously used (3–4) and the different methods employed to calculate core genes: in contrast to previous work, we have performed clustering of the 28 genomes and identified strain-specific genes within the merged dataset (see “Materials and Methods” section).…”

“…For instance, whole-genome sequencing of C. tyrobutyricum has uncovered the genomic basis for their potential application in industry and human health ( Liu et al, 2020 ). Based on comparative analysis within C. tyrobutyricum and within Clostridium spp., Storari et al (2016) provided significant information about nutritional requirements. However, research to date is limited to a small number of C. tyrobutyricum strains (18 strains).…”

Transcriptional analysis of amino acid, metal ion, vitamin and carbohydrate uptake in butanol-producing Clostridium beijerinckii NRRL B-598

“…According to the latest in silico analysis, genes encoding all essential enzymes for proteinogenic amino acid synthesis are present in the C . beijerinckii G117 genome [37]. However, uptake of free amino acids from the culture medium is energetically more advantageous for the cell than their biosynthesis [38].…”