Joseph Loquasto Department of Food Science The Pennsylvania State University Comparison of the complete genomes of Bifidobacterium animalis subsp. animalis.

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Joseph Loquasto Department of Food Science The Pennsylvania State University Comparison of the complete genomes of Bifidobacterium animalis subsp. animalis and Bifidobacterium animalis subsp. lactis

Probiotics “Live microorganisms which when administered in adequate amounts confer a health benefit on the host” -(FAO/WHO 2002)

Potential Probiotic Organisms Bifidobacterium B. animalis subsp. lactis B. longum subsp. longum B. longum subsp. infantis B. adolescentis B. breve B. bifidum Lactobacillus L. rhamnosus L. acidophilus L. casei L. paracasei L. johnsonii L. reuterii

Mechanism of Probiotics Sherman et al. 2009

Bifidobacterium animalis ssp. Gram positive Non-spore forming Non-motile Obligate anaerobe

Bifidobacterium animalis subsp. lactis First isolated and identified as a new species in 1997 (Meile et al. 1997) Most common (sub)species of bifidobacteria isolated from dairy products (Fasoli et al. 2003) Important technological advantages  Oxygen-tolerant  Acid-resistant  Bile-tolerant (Jayamanne and Adams 2006)  Growth observed in milk and milk based media (Masco et al. 2004)

Bifidobacterium animalis subsp. lactis Comparison of the complete genome sequences of Bifidobacterium animalis subsp. lactis DSM and Bl- 04. Barrangou R, Briczinski EP, Traeger LL, Loquasto JR, Richards M, Horvath P, Coûté-Monvoisin AC, Leyer G, Rendulic S, Steele JL, Broadbent JR, Oberg T, Dudley EG, Schuster S, Romero DA, Roberts RF. J Bacteriol Jul;191(13): Genome sequence of the probiotic bacterium Bifidobacterium animalis subsp. lactis AD011. Kim JF, Jeong H, Yu DS, Choi SH, Hur CG, Park MS, Yoon SH, Kim DW, Ji GE, Park HS, Oh TK.J Bacteriol Jan;191(2): Complete genome sequence of Bifidobacterium animalis subsp. lactis BB-12, a widely consumed probiotic strain. Garrigues C, Johansen E, Pedersen MB.J Bacteriol May;192(9):2467-8

Bifidobacterium animalis subsp. animalis First described in 1969 by Mitsuoka. Isolated from the feces of rat Believed to: Lack the ability to grow in milk (Meile et al. 1997) Reduced oxygen-tolerance (when compared to BAL) Reduced ability to survive in acid and bile (when compared to BAL) Little evidence to support probiotic status Potentially pathogenic-Caused colonic inflammation in rats (Moran et al. 2009)

Bifidobacterium animalis subsp. animalis Previous reports have suggested the level of homology between B. animalis subsp. animalis and B. animalis subsp. lactis to be around 85-95% homologous by DNA-DNA hybridization (Meile et al. 1997). No complete genome existed for any strain of B. animalis subsp. animalis. Despite obvious phenotypic differences there exists a high level of similarity between the genomes of the two organisms.

Objectives 1a.To determine the complete genome sequence of B. animalis subsp. animalis ATCC T. 1b.. To conduct a comparative analysis between the type strains of B. animalis subsp. animalis (ATCC T) and B. animalis subsp. lactis (DSMZ T ). 1c. Use the Ka/Ks (dN/dS) ratio to determine if there is a positive selection for genes related to growth in milk 2. Investigate the phylogeny between B. animalis subsp. animalis and B. animalis subsp. lactis. 3. Examine interesting genetic differences observed in Objective 1.

Objective 1 Genomic DNA was isolated from cells grown overnight in MRS broth Isolated DNA was submitted to the lab of Dr. Stephan Schuster at Penn State for 454 DNA sequencing 30 contigs were generated with 146X coverage (a total of 295,919,203 bases were generated) Contigs were aligned using PGAP

BAL Scaffold Bifidobacterium animalis subsp. lactis DSMZ Complete Genome Bifidobacterium animalis subsp. animalis ATCC Unassembled Contigs Bifidobacterium animalis subsp. animalis ATCC 25527Assembled Contigs for closing

Closing Reactions Bifidobacterium animalis subsp. lactis DSMZ Complete Genome Bifidobacterium animalis subsp. animalis ATCC 25527Assembled Contigs for closing Primers were designed on the 5’ and 3’ ends of each contig. PCR was conducted on primers of matching ends. PCR products were sequenced to “fill in the gaps” and assembled to the existing contigs.

Bifidobacterium animalis subsp. animalis genome 1,932,693 bases in length encoding 1,595 genes and 60.4% G+C The B. animalis subsp. lactis genome is 1,938,482 bases in length encoding 1,631 genes and 60.5% G+C Of all 12 species with at least draft genomes publicly available, B. animalis spp. are the smallest. All other genomes are greater than 2 MB in size. Other genomes of this genus have a G+C content between %

Objective 1 BLAST Dot Plot of B. animalis subsp. animalis ATCC vs. B. animalis subsp. lactis DSMZ 10140

Objective 1 B. animalis subsp. animalis ATCC B. animalis subsp. lactis DSMZ WebACT Comparison of B. animalis subsp. animalis ATCC and B. animalis subsp. lactis DSMZ 10140

Objective 1 Optical Map Comparison of B. animalis subsp. animalis ATCC (with KpnI) and B. animalis subsp. lactis DSMZ (in silico, KpnI)

Positive Selection? Beta-galactosidase

Positive Selection? Beta-galactosidase Lactose Transporter Aminopeptidase Endopeptidase

Objective 1- Summary The genomes of B. animalis subsp. animalis ATCC T and of B. animalis subsp. lactis DSMZ T revealed: Similar size, similar number of genes, similar G+C content High levels of synteny (Dot Plot) High levels of similarity in gene content (WebACT) There are 126 and 155 genes in the animalis subspecies that are absent and unique, respectively, as compared to the lactis subspecies Differential base content (Optical Maps) Does not appear to be positive selection on lacZ and select peptidases

Objective 2 2. Investigate the phylogeny between B. animalis subsp. animalis and B. animalis subsp. lactis. Our lab has a collection of 24 strains of B. animalis subsp. lactis (Mostly commercial strains, and some from culture collections). We would like to identify a parental strain of B. animalis subsp. lactis to better understand the evolution between the two subspecies. The work of Briczinski et al. (2009) identified differences between highly related strains and gives some indication about the relatedness of our collection strains.

DSMZ (1) RB 5251 (9) RB 1573 (3) RB 5851 (5) RB 1280 (2) RB 4052 (3) RB 4825 (9) RB 4536 (3) Bl-04 (9) RB 8613 (12) RB 9321 (3) RB 9632 (12) RB 0171 (13) RB 7239 (4) RB 7339 (3) RB 5422 (11) RB 5859 (10) RB 3046 (11) RB 5733 (12) RB 1281 (12) RB 4753 (6) RB 1791 (7) HN019 (8) ATCC (14)

Objective 3 3. Examine interesting genetic differences observed in Objective 1 Comparative genomic analysis revealed ~250 gene differences, however most were classified as “hypothetical protein” No genes have yet to be identified that would explain differences seen in oxygen tolerance, bile resistance, or acid tolerance.

Objective 3 However, a difference was observed in the yafQ-dinJ toxin-antitoxin module May be responsible for the pathogenic effect of ATCC seen by Moran et al. (2009) B. animalis subsp. animalis ATCC B. animalis subsp. lactis DSMZ Truncated version of yafQ in the yafQ and DnaJ Toxin-antitoxin module Schematic of the comparison toxin-antitoxin modules

Objective 3 Approach for Objective 3 Use RT-PCR to detect the transcript of yafQ gene in ATCC Use Western Blot to detect protein of the yafQ gene Construct a knockout of the yafQ gene in ATCC Test ATCC and ATCC  yafQ mutant for pathogenicity

Summary The genome of B. animalis subsp. animalis ATCC was determined and compared to the genome of B. animalis subsp. lactis DSMZ A putative toxin-antitoxin module was identified in the genome of B. animalis subsp. animalis ATCC that has the potential to explain pathogenic effects observed by Moran et al. (2009). A truncated version of the toxin was identified in the genome of B. animalis subsp. lactis DSMZ which may explain it long history of safe use.

XYPLOT Beta-galactosidase