Assessing the diverstiy of the monomorphic subspecies Bifidobacterium animalis subsp. lactis

Open Access
Author:
Loquasto, Joseph Robert
Graduate Program:
Food Science
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
August 15, 2013
Committee Members:
  • Robert F Roberts, Committee Chair
  • Edward G Dudley, Committee Member
  • Rodolphe Barrangou, Committee Member
  • Eric Thomas Harvill, Committee Member
Keywords:
  • bifidobacteria
  • probiotics
  • genomics
  • dairy products
Abstract:
Bifidobacterium animalis subsp. lactis is a widely consumed probiotic microorganism commonly added to fermented dairy products such as yogurt. Health benefits associated with this subspecies are considered to be strain-specific, thus proper identification of these strains is critical. However, the identification and differentiation of strains has remained difficult using both phenotypic and molecular methods. Prior to this work very little genomic sequence existed for strains of this subspecies and no completed genomes were publically available. To develop a better understanding of the subspecies B. animalis subsp. lactis, the genome of a commercial strain Bl-04 and the Type strain DSM 10140 were sequenced and compared. Comparison of the DSM 10140 and Bl-04 genome sequences (99.975% identical) revealed a high degree of identity and synteny as well as a total of 47 single nucleotide polymorphism (SNPs) and 4 insertion/deletions (INDELs). One non-synonymous SNP was identified in a putative glucose uptake protein (glcU). The two strains were shown to have differential ability to grow on glucose as the sole carbohydrate source. The high degree of similarity observed between these two strains explains the difficulties encountered in differentiation of strains of this subspecies. A collection of 24 strains of B. animalis subsp. lactis were screened using the SNPs/INDELs identified between DSM 10140 and Bl-04 as possible targets. Results obtained from this analysis revealed a combination of nine SNPs/INDELs could be used to differentiate strains into 14 distinct genotypic groups. The method reported here is the first available for B. animalis subsp. lactis and can be used in clinical, regulatory, and commercial applications requiring identification of B. animalis subsp. lactis at the strain level. The genome of the closely related B. animalis subsp. animalis ATCC 25527 was sequenced in an effort to understand what genomic features differentiate the two subspecies. Comparative genomic analysis revealed 156 and 182 genes that were unique to and absent in the B. animalis subsp. animalis genome when compared to B. animalis subsp. lactis genome, respectively. Among the differential content was a set of unique clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes and a novel CRISPR locus containing 30 spacers in the genome of B. animalis subsp. animalis. Although, previous research had suggested the ability to grow in milk was a differential phenotype between these two subspecies. Analysis revealed there is no significant difference between the subspecies in their ability to grow in milk and no differential gene content that would lead to such a result. Supplementation with a protein hydrolysate allowed growth in milk by both subspecies suggesting lack of the ability to utilize milk proteins as the factor limiting growth in milk. Subsequent to publication of the complete genome of B. animalis subsp. lactis DSM 10140 and Bl-04, seven additional strains of the subspecies have been reported and deposited in GenBank. These genomes reveal remarkably little diversity leading to the term “monomorphic” being applied to this group. B. animalis subsp. lactis ATCC 27673 was selected for sequencing based on the report of a unique MLST profile when compared to other strains of the subspecies. The complete genome of ATCC 27673 was 1,963,012 bp and contained 1,616 genes, 4 rRNA operons, and had a G+C content of 61.55%. Comparative analyses of the typical B. animalis subsp. lactis revealed the genome of ATCC 27673 contained six distinct genomic islands, encoding 83 ORFs that are absent from other strains of the same subspecies. Phage or mobile genetic elements were identified in four of these genomic islands. In island 6, the largest island, a novel type I-E CRISPR-cas system was identified, which contains 81 novel spacers. It is noteworthy that all other sequenced strains of this subspecies contain type I-C systems. This study revealed ATCC 27673 is a strain of B. animalis subsp. lactis with novel genetic content, suggesting the lack of genetic variability observed to date is potentially due to the repeated sequencing of a limited number of widely distributed commercial strains. In conclusion, the 47 SNPs and 4 INDELs identified between the sequenced strains DSM 10140 and Bl-04 separated the collection into 14 distinct genomic clusters. B. animalis subsp. animalis ATCC 25527 was sequenced and compared to sequenced strains of B. animalis subsp. lactis and the previously identified defining phenotype that separates the two subspecies was examined and no differences were observed when both subspecies were grown in milk. B. animalis subsp. lactis ATCC 27673 was identified as a potentially novel strain of the subspecies and was sequenced. Six genomic islands containing novel lactis genomic sequence were identified. The identification of this unique strain suggests more unique strains exist and the full diversity of the subspecies has not yet been explored.