The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533 Group 5 Steinunn Maria

1. The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533 Group 5 Steinunn Maria Stef�nsd�ttir Subhasree Dash Wan Peng

2. Information

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

4. 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

5. 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

6. 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

7. 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.

8. 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

9. 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

10. 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

18. 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 No complete or even partial amoino acid biosynthetic pathways were indentified from the predicted ORF�s so it can�t synthesis most amino acids from start In the article the think it may be able to synthesize some but in general it has to depend on amino acids or peptides that come from outside to fuel protein synthesis and will depend on enhanced transport capabilities to acquire that L.johnsonii can�t either absorb sulfur because it lacks homologs of the enzyme that synthesize many cofactors Additionally the gene for de novo purine synthesizes is missing and L.johnsonii needs them for growth so it must obtain them or ther precursors from the enviroment Because of this L.johnsonii must be restricted to enviroment that is rich in such substrates A biochemical substance, such as an intermediate compound in a chain of enzymatic reactions, that gives rise to a more stable or definitive product Having the same morphology and linear sequence of gene loci as another chromosome. No complete or even partial amoino acid biosynthetic pathways were indentified from the predicted ORF�s so it can�t synthesis most amino acids from start In the article the think it may be able to synthesize some but in general it has to depend on amino acids or peptides that come from outside to fuel protein synthesis and will depend on enhanced transport capabilities to acquire that L.johnsonii can�t either absorb sulfur because it lacks homologs of the enzyme that synthesize many cofactors Additionally the gene for de novo purine synthesizes is missing and L.johnsonii needs them for growth so it must obtain them or ther precursors from the enviroment Because of this L.johnsonii must be restricted to enviroment that is rich in such substrates A biochemical substance, such as an intermediate compound in a chain of enzymatic reactions, that gives rise to a more stable or definitive product Having the same morphology and linear sequence of gene loci as another chromosome.

19. 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 And because of that transporter gene are among the more highly expressed genes of NCC 533. Although the percent age of ORFs involved in transport (15%) is not too much higher than in other lactobacilli, the overabundance of certain types of less common transporters Like L. johnsonii has 20 AA-permease type transporters (PFAM )( An enzyme that promotes the passage of a substance across a cell membrane), which is more than twice the number found in most other lactic acid bacteria (LAB). Also its genome also encodes 16 phosphotransferase type transporter systems (PTS) (Phosphotransferase is a category of enzymes with the quality of catalyzing phosphorylation.) ,that is many more than nearly all other microbes with similar sized genomes, Lister ia monocytogenes has more. And because of that transporter gene are among the more highly expressed genes of NCC 533. Although the percent age of ORFs involved in transport (15%) is not too much higher than in other lactobacilli, the overabundance of certain types of less common transporters Like L. johnsonii has 20 AA-permease type transporters (PFAM )( An enzyme that promotes the passage of a substance across a cell membrane), which is more than twice the number found in most other lactic acid bacteria (LAB). Also its genome also encodes 16 phosphotransferase type transporter systems (PTS) (Phosphotransferase is a category of enzymes with the quality of catalyzing phosphorylation.) ,that is many more than nearly all other microbes with similar sized genomes, Lister ia monocytogenes has more.

20. 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 Because NCC 533 is totally dependent on exogenous amino acids for growth, it is not surprising that it has an unusually large number and types of proteases, peptide transporters, and peptidases to obtain these from proteinaceous substrates in the GIT. NCC 533 is predicted to have an extra- cellular, cell-wall bound proteinase (LJ1840), three oligopeptidetransporters (one classic ABC-t ype and two di- and tripeptide types), and an extensive collection of 25 cytoplasmicpepti From looking at this portease and peptides it is clear that NCC 533 is programmed for amino acid and peptide transport and utilization from an environment where these must be readily available. In contrast to some other LAB )L.plantarum and Lactococcus lactis) they harbor fewer peptidasesbut encode more amino acid biosynthetic capability that supports their documented ability to grow and survive in environments deficient in amino acids. LJ1840 is critical in other milkfermenting bacteria for degradtion of milk casein to oligopeptides that serve as source of amio acids The role of this protease in NCC 533 is unclear ,but it may enhance a acquisition of amino acid in the competitive GIT environment Because NCC 533 is totally dependent on exogenous amino acids for growth, it is not surprising that it has an unusually large number and types of proteases, peptide transporters, and peptidases to obtain these from proteinaceous substrates in the GIT. NCC 533 is predicted to have an extra- cellular, cell-wall bound proteinase (LJ1840), three oligopeptidetransporters (one classic ABC-t ype and two di- and tripeptide types), and an extensive collection of 25 cytoplasmicpepti From looking at this portease and peptides it is clear that NCC 533 is programmed for amino acid and peptide transport and utilization from an environment where these must be readily available. In contrast to some other LAB )L.plantarum and Lactococcus lactis) they harbor fewer peptidasesbut encode more amino acid biosynthetic capability that supports their documented ability to grow and survive in environments deficient in amino acids. LJ1840 is critical in other milkfermenting bacteria for degradtion of milk casein to oligopeptides that serve as source of amio acids The role of this protease in NCC 533 is unclear ,but it may enhance a acquisition of amino acid in the competitive GIT environment

21. 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 What they found out about the reuglation in NCC 533 is that most of the regulatory protein families were negative regulators. Many of these are located in gene clusters predicted to be involved in sugar acquisition and utilization. And therefore are likely to be involved in regulation of sugar metabolism they also found out that Homologs of the global regulator Fur (ferric upt ake regulator) are absent from genomes of L. johnsonii consistent some other lactobacilli(L gasser), implying that iron availability is not a major physiological concern for these bacteria. The apparent simplicity of the transcriptional regulatory networks in NCC 533 may reflect the richness and relative stability of the GIT environment. What they found out about the reuglation in NCC 533 is that most of the regulatory protein families were negative regulators. Many of these are located in gene clusters predicted to be involved in sugar acquisition and utilization. And therefore are likely to be involved in regulation of sugar metabolism they also found out that Homologs of the global regulator Fur (ferric upt ake regulator) are absent from genomes of L. johnsonii consistent some other lactobacilli(L gasser), implying that iron availability is not a major physiological concern for these bacteria. The apparent simplicity of the transcriptional regulatory networks in NCC 533 may reflect the richness and relative stability of the GIT environment.

22. 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 L.johnsonii and L.gasseri are described as being very closely related . This is supported by comparison of the 16S ribosomal genes (99.6% identical) and the observation that the DNA sequences of many housekeeping genes of these two bacteria also show high degree of similarity. Unfortunately we can�t show any information about L.gasseri the authors don�t show the Blast results and we found no information about the bacteria elsewhere. But we just have to trust the authors when they say the did a Blast anylyzes and found this out Many of this genes are clustered and have deducible function for example L.johnsonii and L.gasseri are described as being very closely related . This is supported by comparison of the 16S ribosomal genes (99.6% identical) and the observation that the DNA sequences of many housekeeping genes of these two bacteria also show high degree of similarity. Unfortunately we can�t show any information about L.gasseri the authors don�t show the Blast results and we found no information about the bacteria elsewhere. But we just have to trust the authors when they say the did a Blast anylyzes and found this out Many of this genes are clustered and have deducible function for example

23. Example of specific genomes in NCC 533 LJ0854-LJ0858 is a gene cluster that confers on NCC 533 the ability to use uncommon �-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 Galactosidase is any of a group of enzymes that catalyze the hydrolysis of a galactoside. Galactosidase is any of a group of enzymes that catalyze the hydrolysis of a galactoside.

24. 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 Another geneset that likely enhances survival in the GIT are the three BSHs and the unique bile transporters of L. johnsonii. IT HAS 3 BSH which is a very larger number found in bacterial genome and it also has a transporter that has been shown to funciton in a uptake of bile. These have been proposed to play a fundamental role in survival because most GIT lactobacilli posses BSH activity and in one case have been shown to be essential for GIT persistence Circumstantial evidence suggests that BSHs facilitate incorporation of cholesterol or bile into the bacterial membrane , changing its fluidity or charge in a way that could affect sensitivity to defensins and other host defense molecules that could strongly affect a select for commensals with BSHs, while disfavoring pathogens or other transients lacking BSHs. Which is consitent with the fact that you can get a helthier flora by consuming milk product that has L.johnsoii in them. Another geneset that likely enhances survival in the GIT are the three BSHs and the unique bile transporters of L. johnsonii. IT HAS 3 BSH which is a very larger number found in bacterial genome and it also has a transporter that has been shown to funciton in a uptake of bile. These have been proposed to play a fundamental role in survival because most GIT lactobacilli posses BSH activity and in one case have been shown to be essential for GIT persistence Circumstantial evidence suggests that BSHs facilitate incorporation of cholesterol or bile into the bacterial membrane , changing its fluidity or charge in a way that could affect sensitivity to defensins and other host defense molecules that could strongly affect a select for commensals with BSHs, while disfavoring pathogens or other transients lacking BSHs. Which is consitent with the fact that you can get a helthier flora by consuming milk product that has L.johnsoii in them.

26. 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