The genome sequence of the probiotic intestinal bacterium Lactobacillus

johnsonii NCC 533

Group 5Steinunn Maria StefánsdóttirSubhasree DashWan Peng

● Published online before print February 13, 2004– PNAS | February 24, 2004 | vol. 101 | no. 8 | 2512-2517

● Authors– R. David Pridmore , Bernard Berger , Frank Desiere , David

Vilanova , Caroline Barretto , Anne-Cecile Pittet , Marie-Camille Zwahlen , Martine Rouvet , Eric Altermann , Rodolphe Barrangou , Beat Mollet , Annick Mercenier , Todd Klaenhammer, Fabrizio Arigoni and Mark A. Schell

● The authors work for – Department of Nutrition and Health, Nestlé Research, – Department of Food Science, Southeast Dairy Foods

Research Center, North Carolina State University – Department of Microbiology, University of Georgia, Athens

Information

Outline

● Introduction

● Genome Characteristics

● Limited biosynthetic capabilities

● Transport,metabolism and regulation

● Uncommon genes of Lactobacillus johnsonii

● Importance of NCC 533

● Summary

Abstract

Lactobacillus johnsonii NCC 533 is a member of the acidophilus group of intestinal lactobacilli that has been extensively studied for their "probiotic" activities that include, pathogen inhibition, epithelial cell attachment, and immunomodulation. To gain insight into its physiology and identify genes potentially involved in interactions with the host, we sequenced and analyzed the 1.99-Mb genome of L. johnsonii NCC 533. Strikingly, the organism completely lacked genes encoding biosynthetic pathways for amino acids, purine nucleotides, and most cofactors. In apparent compensation, a remarkable number of uncommon and often duplicated amino acid permeases, peptidases, and phosphotransferase-type transporters were discovered, suggesting a strong dependency of NCC 533 on the host or other intestinal microbes to provide simple monomeric nutrients. Genome analysis also predicted an abundance (>12) of large and unusual cell-surface proteins, including fimbrial subunits, which may be involved in adhesion to glycoproteins or other components of mucin, a characteristic expected to affect persistence in the gastrointestinal tract (GIT). Three bile salt hydrolases and two bile acid transporters, proteins apparently critical for GIT survival, were also detected. In silico genome comparisons with the >95% complete genome sequence of the closely related Lactobacillus gasseri revealed extensive synteny punctuated by clear-cut insertions or deletions of single genes or operons. Many of these regions of difference appear to encode metabolic or structural components that could affect the organisms competitiveness or interactions with the GIT ecosystem

Introduction

● Probiotic– They can be found in products such as yogurt– Examples of probiotics are

● Lactobacillus johnsonii ● Lactobacillus acidophilus

– Are to assist the body´s naturally occurring flora within the gastrointestinal tract to reestablish themselves

Introduction

● Probiotic – Maintaining a healthy flora

has an affect on● Digestion ● Better immune system ● Preventing constipation● Reducing insomnia ● Stress related illnesses● Reducing risk of

colorectal cancer

A research Nestlé on milk products containing Lactobacillus johsonii

Introduction

● GIT– The human

gastrointestinal tract– The part of the

digestive system consisting of the stomach, small intestine, and large intestine

– The GIT microbiota consist of >500 species of bacteria that produce nutrients for their host providing up to 15 % of total caloric intake.

Introduction

●Lactobacillus johnsonii NCC 533

● Member of acidophilus group of intestinal lactobacilli

● Extensively studied● Pathogen inhibition● Epithelial cell attachments● Immunomodulation● Found in the

gastrointestinal tract (GIT)● Synonym

● Lactobacillus acidophilus La1

Taxonomy of Lactobacillus johnsonii NCC 533

● Kingdom: Bacteria● Intermediate Rank 1:Firmicutes● Intermediate Rank 2 :Lactobacillales● Intermediate Rank 3 :Lactobacillaceae ● Intermediate Rank 4 :Lactobacillus johnsonii● Genus: Lactobacillus● Species: johnsonii● Strain: NCC 533

Genome Characteristics

● 1,992,676 base pairs● AT content 65.4%● 6 rRNA operons● 79 tRNAs● 1,821 protein-coding genes ● 14 complete IS elements

Predicted Origin of Replication in Lactobacillus johnsonii strain NCC533

Promoter structure of Lactobacillus johnsonii strain NCC533

Limited biosynthetic capabilities

● L.johnsonii incapable of de novo synthesis of most amino acids. It appears it is because it lacks homologs of glutamate dehydrogenase and synthetase

● It may be able to synthesize some but it depends on large amounts of exogenous amino acids and/or peptides and will depend on enhanced transport capabilities to acquire that

● No sulfur assimilation pathways were found. L.johnsonii lacks homologs of the enzyme to synthesize many cofactors

● Additionally the gene for de novo purine synthesizes is missing and L.johnsonii is auxotrophic for it

● That is why L.johnsonii will be restricted to environments that is rich in such substrates

Transport

● Among the highly expressed genes in NCC 533 are transporter genes (15%)

● L.johnsonii has > 20 AA-permease type transporters.– That is more than 2x the number found in most other

lactic acid bacteria(LAB)

● Its genome also encodes 16 phosphotransferase type transporter system. Much more than all other microbes with similar sized genome.– Only one microbes has more

Proteases and peptides

● NCC 533 is predicted to have – An extracellular,cell-wall bound proteinase(LJ1840) – 3 oligopeptide transporters

● 1 classic ABC-type ● 2 di-and tripeptide types

– And an extensive collection of > 25 cytoplasmic peptidases

● From looking at all this proteases and peptides it is clear that NCC 533 is programmed for amino acid and peptide transport and utilization from an environment where these are available

Regulation

● Regulatory protein families in NCC 533 are negative regulators involving sugar metabolism – GntR – 9 members– LacI – 7 members– RpiR – 5 members– ArsR – 3 members

● Absence of homologs of ferric uptake regulator implies that iron availability is not a major physiological concern

● The regulatory networks in NCC 533 is simple may reflect the richness and stability of the GIT environment

Uncommon Genes of L.johnsonii NCC 533

● L.johnsonnii and L.gasseri are described as being very closely related (99.6% identical) but when they did a BLAST analyses – 150 NCC 533 ORF´S were absent from the L.gasseri

draft genome

– >95 % of these ORF´s were confirmed as NCC 533 specific. By hybridizing whole genome micro arrays with labelled L.gasseri DNA

Example of specific genomes in NCC 533

● LJ0854-LJ0858 is a gene cluster that confers on NCC 533 the ability to use uncommon β-galactosidase encountered in the GIT environment

● LJ0730 – LJ0738 is a gene cluster that confers on NCC 533 the ability to acquire sugars from unusual polysaccharides

● LJ0635 a maltose-6-phosphate glucosidase

● LJ0636 a maltose specific IIBC PTS transporter component

● LJ0637 a RpiR type phosphosugar-responsive regulator

● LJ1654 – LJ1661is a gene cluster that may encode utilization of exogenous deoxyriboses

Importance of NCC 533

● Bile salt hydrolase (BSH) is a enzyme that releases taurine or glycine from bile and may impart a selective advantage in the GIT environment

● NCC 533 encodes 3 BSH´s which is a very large number found in bacterial genome

● Adjacent to one of its BSH encoding genes NCC 533 has 2 ORF

´s with 80% sequence identity that had been shown to function in uptake of bile.

● BSH encoding genes and biles transporter multiplicity implies importance of this gene set for GIT survival and persistence

Summary

● Lactobacillus johnsonii NCC 533 is a member of acidophilus group of intestinal lactobacilli

● Exclusively found in human and animal GITs

● It has limited biosynthetic capabilities and therefore depends

on enhanced transport capabilities

● A quite few gene clusters are only present in L.johnsonii

● Most of these clusters seem to enhance the survival in the

GIT

● Found 3 BSH´s and the unique bile transporters