Some of the technological and functional insights provided by the whole genome sequencing of lactic acid bacteria

1. Genome Sequencing of Lactic Acid
Bacteria: Lessons to be Learnt
Diwas Pradhan
Dairy Microbiology
ICAR-NDRI, Karnal
Oct., 2011

2. Introduction
• One of the most industrially important groups of
bacteria.
• Found in a variety of environments, including milk and
dairy products, plants, cereals and meat.
• Used in fermentation, health improvement and as a
cell factory or as vaccine delivery systems.

3. DNA Sequencing Technologies
Types of Sequencing Run time
hr/GB
Cost/Human
genome($ US)
Ease of use
1ST
GENERATION
1. Sanger’s chain
termination method
(1977)
High Expensive Difficult
2. Maxam-Gilbert Method High Expensive Difficult
3. Whole Genome
Shotgun Sequencing
High Expensive Difficult
2ND GENERATION
(Next
Generation)
1. Pyrosequencing 75 1000,000 Difficult
2. SOLiD Sequencing 42 60,000 Difficult
3. Polonator G 007
4. Solexa GA 56 60,000 Difficult
3RD GENERATION
SEQUENCING
(Next-Next
Generation)
1. True Single Molecule
Sequencing (tSMS™)
~12 70,000 Easy
2. FRET based approach
3. SMRT ™ <1 Low Easy
4. Nanopore sequencing 20 Low Easy
5. Transmission Electron
Microscope
~14 Low Easy

5. Lactococci
Sr. No. SPECIES Genome (Mb) % GC
1. Lc. lactis subsp. cremoris MG1363 2.53 35.7
2. Lc. lactis subsp. cremoris NZ9000 2.5 35.8
3. Lc. lactis subsp. cremoris SK11 2.59 35.8
4. Lc. lactis subsp. lactis CV56 2.52 --
5. Lc. lactis subsp. lactis IL1403 2.36 35.3
6. Lc. lactis subsp. lactis KF147 2.638 34.9
http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

7. Sr. No. STRAIN Genome(Mb) %GC
1 Lb. acidophilus 30 SC 2.12 --
2 Lb. acidophilus NCFM 2 34.7
3 Lb. amylovorus GRL 1112 2.16 --
4 Lb. amylovorus GRL 1118 1.98 --
5 Lb. brevis ATCC 367 2.349 46.1
6 Lb. buchneri NRRL B-30929 2.58 --
7 Lb. casei ATCC 334 2.929 46.6
8 Lb. casei BD-II 3.16 --
9 Lb. casei BL-23 3.0792 46.3
10 Lb. casei LC2W 3.04 --
11 Lb. casei Zhang 2.936 40.1
12 Lb. crispatus ST1 2 --
13 Lb. bulgaricus 2038 1.9 --
14 Lb. bulgaricus ATCC 11842 1.865 49.7
15 Lb. bulgaricus ATCC BAA-365 1.85695 49.7
16 Lb. bulgaricus ND02 2.1062 --
17 Lb. fermentum CECT 5716 2.1 --
18 Lb. fermentum IFO 3956 2.1 51.5
19 Lb. gasseri ATCC 33323 1.9 35.3
http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

8. Srl. No. Strain Genome(Mb) %GC
20 Lb. helveticus DPC 4571 2.1 37.1
21 Lb. helveticus H10 2.13 --
22 Lb. johnsonii DPC 6026 2 --
23 Lb. johnsonii FI 9785 1.8295 34.4
24 Lb. johnsonii NCC533 2 34.6
25 Lb. kefironofaciens ZW3 2.34 --
26 Lb. plantarum JDM1 3.2 44.7
27 Lb. plantarum WCFS1 3.3403 44.4
28 Lb. plantarum ST111 3.354 --
29 Lb. reuteri DSM20016 2 38.9
30 Lb. reuteri JCM 1112 2.03941 38.9
31 Lb. reuteri SD 2112 2.35 38.8
32 Lb. rhamnosus GG 3 46.7
33 Lb. rhamnosus GG 3 46.7
34 Lb. rhamnosus Lc705 3.0336 46.7
35 Lb. sakei 23K 1.9 41.3
36 Lb. salivarus CECT5713 2.105 --
37 Lb. salivarus UCC118 2.13111 33
38 Lb. sanfranciscensis TMW 1.38 --
http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

9. Streptococci
Sr. No. STRAIN Genome (Mb) % GC
1 St. thermophilus JIM 8232 1.9 --
2 St. thermophilus CNRZ1066 1.8 39.1
3 St. thermophilus LMD-9 1.864 39.1
4 St. thermophilus LMG 18311 1.8 39.1
5 St. thermophilus ND03 1.8 --
http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

10. Srl.
No.
STRAIN Genome(Mb) %GC
1. Bifidobacterium adolescentis ATCC 15703 2.1 59.2
2. Bifidobacterium animalis subsp. lactis AD011 1.93369 60.5
3 Bifidobacterium animalis subsp. lactis BB-12BLC1 1.9 60.5
4 Bifidobacterium animalis subsp. lactis Bl-04 1.94 60.5
5 Bifidobacterium animalis subsp. lactis CNCM 1.93871 --
6 Bifidobacterium animalis subsp. lactis I-2494 1.9 60.5
7 Bifidobacterium animalis subsp. lactis DSM 10140 1.93871 --
8 Bifidobacterium animalis subsp. lactis V9 1.9 60.5
9 Bifidobacterium bifidum PRL2010 2.2 --
10 Bifidobacterium bifidum S17 2.2 --
11 Bifidobacterium breve ACS-071-V-Sch8b 2.3 --
12 Bifidobacterium breve UCC2003 2.4 --
13 Bifidobacterium dentium Bd1 2.6 58http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi
Bifidobacteria

11. Srl.
No.
STRAIN Genome(Mb) %GC
14 Bifidobacterium longum DJO10A 2.38949 58.5
15 Bifidobacterium longum NCC2705 2.26024 60.2
16 Bifidobacterium longum subsp. infantis 157F 2.41 60.1
17 Bifidobacterium longum subsp. infantis ATCC 15697 2.83275 --
18 Bifidobacterium longum subsp. infantis ATCC 15697 2.8 59.9
19 Bifidobacterium longum subsp. longum BBMN68 2.3 --
20 Bifidobacterium longum subsp. longum F8 2.4 --
21 Bifidobacterium longum subsp. longum JCM 1217 2.4 --
22 Bifidobacterium longum subsp. longum JDM301 2.5 --
23 Bifidobacterium longum subsp. longum KACC 91563 2.41 --
http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

12. Leuconostoc
Srl. No. STRAIN Genome (Mb) % GC
1. Leuconostoc citreum KM20 1.9 38.9
2. Leuconostoc gasicomitatum
LMG 18811
2 --
3. Leuconostoc kimchii
IMSNU 11154
2.0992 37
4. Leuconostoc mesenteroides
subsp. mesenteroides ATCC
8293
2.0754 37.7
5. Leuconostoc sp. C2 1.9 --
http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

13. Other Related Species
Srl.
No.
Strain Genome
(Mb)
%GC
1. Oenococcus oeni PSU-1 1.8 37.9
2. Pediococcus pentosaceus ATTC
25745 1.83239 37.4
3. Propionibacterium freudenreichii
CIRM-BIA1T
2.7 67
http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

14. Genomes in progress
GENUS NO. OF STRAINS
Lactobacilli 141
Bifidobacteria 29
Lactococci 5
Leuconostoc 10
Oenococci 4
Pediococci 3
http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

15. Indian scenario
ORGANISM CENTER
Lactobacillus helveticus MTCC 5463
SMC College of Dairy Science, Anand
Anand Agricultural University
Lactobacillus rhamnosus MTCC 5462
http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

16. Genome Sequences Of Commercial Probiotic
Bacteria
Species Strain Genome
size (Mb)
Company Reference
Bifidobacterium animalis spp.
lactis
BB-12 2.0 Chr. Hansen, Denmark christel.garrigues@dk.chr-hansen.com
Bifidobacterium breve Yakult 2.35 Yakult, Japan yukioshirasawa@yakult.co.jp
Bifidobacterium breve M-16V 2.3 Morinaga Milk, Japan k_nanba@morinagamilk.co.jp
Bifidobacterium longum biot
infantis
M-63 2.8 Morinaga Milk, Japan k_nanba@morinagamilk.co.jp
Bifidobacterium longum BB536 2.5 Morinaga Milk, Japan k_nanba@morinagamilk.co.jp
Bifidobacterium lactis 1.94 Danone, France tamara.smokvina@danone.com
Lactobacillus brevis KB290 2.49 Kagome, Japan masanori_fukao@kagome.co.jp
Lactobacillus casei Shirota 3.03 Yakult, Japan yukioshirasawa@yakult.co.jp
Lactobacillus casei 3.14 Danone, France tamara.smokvina@danone.com
Lactobacillus reuteri ATCC55
730
2.0 SLU, Sweden klara.bath@mikrob.slu.se
Roland J. Siezen and Greer Wilson, 2010

17. Bioinformatics Toolbox
Roland J Siezen et al., 2004

19. Lactococcus lactis subsp. lactis IL1403
• Relatively small genome – 2365kb, 2310 ORFs
• Presence of men and cytABCD operons- aerobic respiration
• Novel gene poxL, encoding pyruvate oxidase
• Many genes required for de novo synthesis of essential
nutrients and the degradation of complex molecules are absent
• Reflects the adaptation in the nutrient-rich milk environment.
Alexander Bolotin et al., 2001

20. Lactobacillus plantarum WCFS1
• L. plantarum encode an exceptionally large number of
phosphotransferase sugar transport systems
 a large proportion of genes involved in sugar utilization clustered
in a specific ‘lifestyle adaptation’ region of its chromosome
 degradation and utilization of complex carbohydrates, including a
variety of glycosyl hydrolases that are required for utilization of
diverse plant-derived dietary fibres or complex carbohydrate
structures produced by the host
• These characteristics represent critical adaptations of these
bacteria to this highly competitive niche
Michiel Kleerebezem et al., 2002

21. Lactobacillus plantarum WCFS1
Nonrandom distribution of genes belonging to specific functional categories in the L.
plantarum chromosome. sugar transport (PTS - black, other transporters - blue), sugar
metabolism (green), and biosynthesis and/or degradation of polysaccharides (red).

22. Lactobacillus helveticus MTCC 5463
(Indian Origin Strain)
• 1911350 bp long single chromosome
• Comparative analysis with L. helveticus DPC
 L. helveticus MTCC 5463 had additional 57 genes
 Indication of diverse carbohydrate utilization pattern for L.
helveticus MTCC 5463.
 The presence of biotin synthesis genes and difference in
cofactors, vitamins, prosthetic groups and pigments
suggest the differential ability of the strain in production of
such bioactive compounds in contrast to the L. helveticus
DPC 4571 strain.
Prajapati et al., 2011

23. Comparative Genomics
• A new scientific discipline as a result of the success of the
genome project
• Comparison of genomes from different species or strains
 predict the function of unknown genes

24. Proteolytic system of LAB
 Comparative genomic analyses of the distribution of components of the
proteolytic system in 22 completely sequenced LAB
 Members of PepE/PepG (endopeptidases) and PepI/PepR/PepL (proline
peptidases) families absent in lactococci and streptococci.
 Many of the peptidases (e.g. aminopeptidases PepC, PepN, and PepM,
and proline peptidases PepX and PepQ) essential for bacterial growth or
survival are encoded in all LAB genomes.
 Lb. acidophilus, Lb. johnsonii, Lb. gasseri, Lb. bulgaricus and Lb.
helveticus strains encode a relatively higher number and variety of
proteolytic system components.
(Liu et al., 2008)

25. Flavour Formation
(Mengjin Liu et al., 2008)

26. Flavour Forming Enzymes
• Aminotransferases
• BcAT ortholog is present in all lactococcal and streptococcal strains
while lacking in lactobacilli such as Lb. johnsonii, Lb. sakei, Lb. reuteri
• araT genes found in all LAB genomes except Lb. sakei and Lb. brevis,
while the aspAT gene was absent in LAB species of the Lb. acidophilus
group
• Glutamate dehydrogenase
– gdh genes found in the genomes of Lb. plantarum, Lb.
salivarius and S. thermophilus strains

27. • α-Ketoacid conversion enzymes
 No orthologs of kdcA were found in the sequenced LAB genomes
 alcohols and carboxylic acids derived from aldehydes detected in many LAB
• Alcohol and aldehyde dehydrogenases
 Most LAB genomes encode multiple AlcDH members, but only a single AldDH
• Esterases
 estA encodes an esterase which catalyzes the biosynthesis of esters
 lactoccoci and streptococci have one estA gene
 absence of estA gene in Lb. acidophilus, Lb. johnsonni, Lb. salivarius and
others

28. • Enzymes for methionine/cysteine metabolism
 Differences in the distribution of the related enzymes, indicating the
presence of the different routes
 Most of these genes present in L. plantarum and S. thermophilus strains
• Most S. thermophilus strains exhibit no absolute amino acids
requirements for growth indicating the presence of all biosynthesis
enzymes
• S. thermophilus, Lactococcus strains and Lb. casei seem to possess
more abundant genes encoding flavor-related enzymes
 presence of flavor-forming enzymes can vary between strains from
the same species
• Many of these enzymes lacking in Lb. gasseri and Lb. johnsonii
• Lb. plantarum genome also encodes a large set of these enzymes
reflecting its flexibility to grow under different conditions
(Mengjin Liu et al., 2008)

29. Factors contributing to the optimal
functioning of probiotic bacteria
Adaptation Factors Probiotic Factors
Stress resistance
Adherence
Adapted
metabolism
Microbe-microbe
interaction
Epithelial barrier
protection
Immunomodulatio
n

30. Adherence
Comparative genomic analysis of L. rhamnosus GG
and L. rhamnosus LC705.
 Presence of cluster of pilus-encoding genes(SpaCBA) in
the genome of Strain GG
 SpaC is a key factor for adhesion- mutation study
 Presence of mucus-binding pili on the surface reveals a
previously undescribed mechanism for the interaction of
selected probiotic lactobacilli with host tissues.
Kankainena et. al., 2009

31. Identification of pili in L. rhamnosus GG by immunogold high-resolution
electron micrography.
Multiple pili are shown with gold-labelled SpaC proteins.

32. Adapted metabolism
 Relatively large proportion (>10%) of the bifidobacterial genome
dedicated to carbohydrate uptake and metabolism, with many
predominantly intracellular, glycosylhydrolases required for the
degradation of complex carbohydrates such as arabinogalactans,
arabinoxylans, starch etc.
 Associated with these glycosylhydrolases, transport systems for the
internalization of structurally diverse carbohydrates were identified
that include docking sites for carbohydrate binding to the bacterial
cell wall, which presumably prevents loss to nearby competitors
(Kleerebezem and Vaughan, 2009).

33. Immunomodulation
Gene encoding potential probiotic effector molecule,
serine protease inhibitor (serpin) was identified in
the genome of B. longum subsp. longum JDM301.
 In eukaryotes, members of the serpin family
regulate various signalling pathways
 some recognized for their ability to suppress
inflammatory responses by inhibiting elastase
activity.
Yan-Xia Wei et al., 2010

34. Niche specific adaptation
• Adaptation to meat environment by L. sakei
 arc operon for arginine catabolism (ADI pathway)
 Heme acquisition
 Harboring of a sodium-dependent symporter to drive the
accumulation of osmo- and cryoprotective solutes (betaine and
carnitine) and genes encoding the putative cold stress proteins
Csp1-4
O. Ludvig Nyquist (2011)

35. Metabolic Pathway Reconstruction
 Begins with gene annotations of a fully sequenced genome
 Enzymes encoded by the genes are assigned to the reactions
in the metabolic networks (Databases: BRENDA and KEGG)
 Stoichiometric models can serve as a predictive model for
phenotype prediction, experimental data interpretation and
metabolic engineering.
Mengjin Liu., 2008

36. Other Potential Functionality
• Plasmids and its role in evolution and adaptation
 Survival of lactococci in the milk fermentation environment
• Proteins associated with biofilms and EPS
 Homologs of eps genes, encoding EPS synthesis proteins
found varying species of Lactococcus, Lactobacillus, and S.
thermophilus
Douglas and Klaenhammer, 2009
• Discovery of bacteriocins and antimicrobial peptides
Makarova et al., 2006

37. Conclusions
• Genomic and comparative genomic analyses are revealing key
gene regions in LAB worthy of continued investigation for their
potential roles in both bioprocessing and health.
• Identification of key enzymes and the prediction on flavor-
forming capacity of various LAB can be exploited for the
production of flavored products.
• Gives key insights into the natural diversity and phylogenetic
relationships.
• Understanding mechanisms of probiotic action - selection of
specific probiotics for specific purposes
• Metabolic and nutrient engineering, and providing platforms to
engineer LAB for delivery of biotherapeutics.

38. Integration of genomics, transcriptomics, proteomics
and metabolomics data backed up by state-of-the art
bioinformatics tools can be used to develop metabolic
models which can provide a full functioning of a
bacterial cell, opening up new horizons in
bioprocessing, human health and food production