lactic acid bacteria whole genome sequencing

Genome Sequencing of Lactic Acid

Bacteria: Lessons to be LearntDiwas Pradhan

Dairy Microbiology

ICAR-NDRI, Karnal

Oct., 2011

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.

DNA Sequencing TechnologiesTypes 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 GENERATIONSEQUENCING(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

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


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


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 --


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 --



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

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 --



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 --



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 freudenreichiiCIRM-BIA1T

2.7 67



Genomes in progress

GENUS NO. OF STRAINS

Lactobacilli 141

Bifidobacteria 29

Lactococci 5

Leuconostoc 10

Oenococci 4

Pediococci 3



Indian scenario

ORGANISM CENTER

Lactobacillus helveticus MTCC 5463

SMC College of Dairy Science, Anand

Anand Agricultural University

Lactobacillus rhamnosus MTCC 5462



Genome Sequences Of Commercial Probiotic

BacteriaSpecies Strain Genome

size (Mb)

Company Reference

Bifidobacterium animalis spp.

lactis

BB-12 2.0 Chr. Hansen, Denmark [email protected]

Bifidobacterium breve Yakult 2.35 Yakult, Japan [email protected]

Bifidobacterium breve M-16V 2.3 Morinaga Milk, Japan [email protected]

Bifidobacterium longum biot

infantis

M-63 2.8 Morinaga Milk, Japan [email protected]

Bifidobacterium longum BB536 2.5 Morinaga Milk, Japan [email protected]

Bifidobacterium lactis 1.94 Danone, France [email protected]

Lactobacillus brevis KB290 2.49 Kagome, Japan [email protected]

Lactobacillus casei Shirota 3.03 Yakult, Japan [email protected]

Lactobacillus casei 3.14 Danone, France [email protected]

Lactobacillus reuteri ATCC55

730

2.0 SLU, Sweden [email protected]

Roland J. Siezen and Greer Wilson, 2010

Bioinformatics Toolbox

Roland J Siezen et al., 2004

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

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

Lactobacillus plantarum WCFS1

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

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

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

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)

Flavour Formation

(Mengjin Liu et al., 2008)

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

• 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

• 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)

Factors contributing to the optimal functioning of probiotic bacteria

Adaptation Factors Probiotic Factors

Stress resistance

Adherence

Adapted metabolism

Microbe-microbe interaction

Epithelial barrier protection

Immunomodulation

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

Identification of pili in L. rhamnosus GG by immunogold high-resolution

electron micrography.

Multiple pili are shown with gold-labelled SpaC proteins.

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).

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

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)

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

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

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.

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

lactic acid bacteria whole genome sequencing

Health & Medicine