The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533

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• The genome sequence of the probiotic intestinal
  bacterium Lactobacillus johnsonii NCC 533
• Group 5
• Steinunn Maria Stefánsdóttir
• Subhasree Dash
• Wan Peng

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Information

• 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

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Outline

• Introduction
• Genome Characteristics
• Limited biosynthetic capabilities
• Transport,metabolism and regulation
• Uncommon genes of Lactobacillus johnsonii
• Importance of NCC 533
• Summary

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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 (gt12) 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 gt95 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

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Introduction

• Probiotic
• They can be found in products such as yogurt
• Examples of probiotics are
• Lactobacillus johnsonii
• Lactobacillus acidophilus
• Are to assist the bodys naturally occurring
  flora within the gastrointestinal tract to
  reestablish themselves

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Introduction
A research Nestlé on milk products containing
Lactobacillus johsonii

• 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

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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 gt500 species of
  bacteria that produce nutrients for their host
  providing up to 15 of total caloric intake.

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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

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Taxonomy of Lactobacillus johnsonii NCC 533

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

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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

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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

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Transport

• Among the highly expressed genes in NCC 533 are
  transporter genes (15)
• L.johnsonii has gt 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

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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 gt 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

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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

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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 ORFS were absent from the L.gasseri
  draft genome
• gt95 of these ORFs were confirmed as NCC 533
  specific. By hybridizing whole genome micro
  arrays with labelled L.gasseri DNA

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Example of specific genomes in NCC 533

• LJ0854-LJ0858 is a gene cluster that confers on
  NCC 533 the ability to use uncommon
  ß-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

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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 BSHs which is a very large
  number found in bacterial genome
  
• Adjacent to one of its BSH encoding genes NCC 533
  has 2 ORFs 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

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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 BSHs and the unique bile transporters