The complete genome sequence of Mycobacterium avium subspecies paratuberculosis 27644 Comparative Microbial Genomics

1. The complete genome sequence of Mycobacteriumavium subspecies paratuberculosis27644 Comparative Microbial Genomics Mette Herold, s001788 Bent Petersen, s991687 Martin Bau Clausen, s011398

2. Outline Introduction Characteristics of the Map genome Virulence factors and diagnostics Summary

3. Artiklens navn. Forfattere. Hvor den er udgivet. �rstalArtiklens navn. Forfattere. Hvor den er udgivet. �rstal

4. Mycobacterium avium subspecies paratuberculosis (Map) Taxgroup Actinobacteria Rod shaped Gram positive, but are hard to stain because of very high lipid content of the cell wall Resistant to both chemical and environmental changes High G-C content Extremely slow growing Can not produce mycobactin Rod shaped Gram positive, but are hard to stain because of very high lipid content of the cell wall (martin will tell about the cell wall later) High G-C content (karakteristika for this species) Extremely slow growing (up to 16 weeks) Resistant to both chemical and environmental changes (because of the high lipidlayer of the cell wall) Can not produce mycobactin (mycobactin will be descibed later by bent)Rod shaped Gram positive, but are hard to stain because of very high lipid content of the cell wall (martin will tell about the cell wall later) High G-C content (karakteristika for this species) Extremely slow growing (up to 16 weeks) Resistant to both chemical and environmental changes (because of the high lipidlayer of the cell wall) Can not produce mycobactin (mycobactin will be descibed later by bent)

5. Johne�s disease Chronic inflammation of the intestine Infect all animals especially live stock Costs 1,5 billion dollars per year in the USA Three stages � in 2. and 3. bacteria are shed in feces and the animal eventually dies Contaminates through feces and oral Difficult to treat because of the high lipid content and complexity of the cell wall; can survive within macrophages Treatment long and expensive � not feasible in livestock Has been found in humans with Crohn�s disease chronic inflammation of the intestine Infect all animals especially live stock (because are in close kantact with each other) Costs 1,5 billion dollars per year in the USA Three stages � in 2. and 3. bacteria are shed in feces and the animal eventually dies Contaminates through feces and oral ASS EALIER MEANTIONED THE CELL WALL ARE : Difficult to treat because of the high lipid content and complexity of the cell wall; can survive within macrophages THIS OF COUSE MEANS THAT Treatment long and expensive � not feasible in livestock Has been found in humans with crohn�s disease BUT ARE NOT SURE THAT THIS ARE THE CAUSE chronic inflammation of the intestine Infect all animals especially live stock (because are in close kantact with each other) Costs 1,5 billion dollars per year in the USA Three stages � in 2. and 3. bacteria are shed in feces and the animal eventually dies Contaminates through feces and oral ASS EALIER MEANTIONED THE CELL WALL ARE : Difficult to treat because of the high lipid content and complexity of the cell wall; can survive within macrophages THIS OF COUSE MEANS THAT Treatment long and expensive � not feasible in livestock Has been found in humans with crohn�s disease BUT ARE NOT SURE THAT THIS ARE THE CAUSE

6. Characteristics of the Map genome 4,829,781 base pairs 1 rRNA operon 4,350 ORFs 91.30 % codes for proteins G-C content of 69.3 % Relatively constant G-C content Sidste punkt skal bruges til at herf�re til n�ste billede. Sig according to the tekst of the articleSidste punkt skal bruges til at herf�re til n�ste billede. Sig according to the tekst of the article

7. D�rlig figur. The dark purple histogram represents the gc-content. It lacks to state what are more and less side, and how much the deviation is. Not really used in the text Seems to be made because they couldD�rlig figur. The dark purple histogram represents the gc-content. It lacks to state what are more and less side, and how much the deviation is. Not really used in the text Seems to be made because they could

8. All looks like each other. Not really used. They forgot to tell where their data are from � can we trust the comparison. Are the results based on the same criteria Mycobacterium tyberculosis og myAll looks like each other. Not really used. They forgot to tell where their data are from � can we trust the comparison. Are the results based on the same criteria Mycobacterium tyberculosis og my

9. MTB OG M. BOVIS LIGNER HINANDEN MEGET MTB OG M. BOVIS LIGNER HINANDEN MEGET

10. Homologous Proteins 60 % of putative proteins has homologs to other microbial proteins with known functions. 25 % were homologs to hypothetical proteins 39 predicted genes are unique to Map no identical homologous in current databases I�m going to talk about the proteins, genes and repeats in Map, compared to other Mycobacterium species. The authors have found, that a total of 60 % of the putative proteins (possibly but not proven) in Map, has homologs to other microbial proteins, that have a known function. and 25 % were homologous to hyppthetical proteins. And actually 39 predicted genes are unique to Map � with no identical homologous in current databasesI�m going to talk about the proteins, genes and repeats in Map, compared to other Mycobacterium species. The authors have found, that a total of 60 % of the putative proteins (possibly but not proven) in Map, has homologs to other microbial proteins, that have a known function. and 25 % were homologous to hyppthetical proteins. And actually 39 predicted genes are unique to Map � with no identical homologous in current databases

11. Repeats 1.5 % of Map DNA is repeats (72,7 kb) Insertion Sequences (IS) Duplicated householding genes 1.5 % of the DNA in Map consist of repeats, which in Map is Insertion Sequences and Duplicated Householding genes. Insertion Sequences: a small bacterial transposon, approximately 1000 bases long, with a short run of inverted repeated sequences at its termini, it causes duplication of the recipient DNA site into which it inserts, one copy of the recipient DNA flanking it on each side. 1.5 % of the DNA in Map consist of repeats, which in Map is Insertion Sequences and Duplicated Householding genes. Insertion Sequences: a small bacterial transposon, approximately 1000 bases long, with a short run of inverted repeated sequences at its termini, it causes duplication of the recipient DNA site into which it inserts, one copy of the recipient DNA flanking it on each side.

12. As we can see, Mtb and M. Bovis is very alike in their repeats, and Map is far away when comparing�. As we can see, Mtb and M. Bovis is very alike in their repeats, and Map is far away when comparing�.

13. Insertion Sequences (IS) 17 copies of IS900 - hypothetical protein IS_MAP02 � 6 copies � 28 % identity with a transporase in Legionella pneumophila Some IS are homologous with IS in Mtb, Mav, M. bovis and M. marinum., but there are also IS with no identifiable homologs in other mycobacteria. Insertion Sequences with no homology in other mycobacteria is of interest, due to their possible usage as diagnostic targets. Map has some interesting Insertion sequences. It has 17 copies of an Insertion sequence, which is called IS900. Hypothetical protein. Of these IS with no homolog in other mycobacteria are the two examples IS_MAP02 and IS_MAP04. IS_MAP02 has 6 copies in the Map genome, is not present in any other mycobacteria and has a very low level of identity with a transporase in Legionella pheumophila. Talk about Legionellas ability to survive within macrophages.This is interesting because Map has strategies to survive in macrophages. Martin will talk about that later. transporase: the transposase apparently recognizes, cuts and eventually ligates the dna duing tranposition Legionella pheumophila can be found in water, and is a parasite of macrophages. possessing a mechanism that prevents them being destroyed after they have been ingested into the cell. Multiplication takes place within the macrophage, which eventually ruptures, releasing the flu-like, with malaise, headache, stiffness and fever. Some of the Insertion sequences are homologs with insertion sequences in other mycobacterium species, but there are also some that has no identifiable homologs in other mycobacteria� and these are interesting�. Mtb: Mycobacterium tuberculosis Mav: Mycobacterium avium M. Bovis: Mycobacterium bovis M. Marinum: Mycobacterium marinum It is just notable to say, that insertion sequences with no homology in other mycobacteria, is of interest, due to their possible usage as diagnostic targets.Map has some interesting Insertion sequences. It has 17 copies of an Insertion sequence, which is called IS900. Hypothetical protein. Of these IS with no homolog in other mycobacteria are the two examples IS_MAP02 and IS_MAP04. IS_MAP02 has 6 copies in the Map genome, is not present in any other mycobacteria and has a very low level of identity with a transporase in Legionella pheumophila. Talk about Legionellas ability to survive within macrophages.This is interesting because Map has strategies to survive in macrophages. Martin will talk about that later. transporase: the transposase apparently recognizes, cuts and eventually ligates the dna duing tranposition Legionella pheumophila can be found in water, and is a parasite of macrophages. possessing a mechanism that prevents them being destroyed after they have been ingested into the cell. Multiplication takes place within the macrophage, which eventually ruptures, releasing the flu-like, with malaise, headache, stiffness and fever. Some of the Insertion sequences are homologs with insertion sequences in other mycobacterium species, but there are also some that has no identifiable homologs in other mycobacteria� and these are interesting�. Mtb: Mycobacterium tuberculosis Mav: Mycobacterium avium M. Bovis: Mycobacterium bovis M. Marinum: Mycobacterium marinum It is just notable to say, that insertion sequences with no homology in other mycobacteria, is of interest, due to their possible usage as diagnostic targets.

14. Mycobactin production Map cannot produce Mycobactin in laboratory cultures Mycobactin is an extracelluary molecule that binds very tightly to iron and transports it into cells. Map is also different on another way. It is not able to produce Mycobactin in laboratory cultures. To those that is not aware of what Mycobactin is, it is an extracellulary molecule that binds very tightly to iron and transports it into cells. Iron is very inportant to a cell.Map is also different on another way. It is not able to produce Mycobactin in laboratory cultures. To those that is not aware of what Mycobactin is, it is an extracellulary molecule that binds very tightly to iron and transports it into cells. Iron is very inportant to a cell.

15. Mycobactin production Mtb has a cluster of 10 genes, (mbtA-J),which is responsible for mycobactin production has been found. Map has a homolog, but it is different in structure It is known that Mycobacterium tuberculosis has a cluster of 10 genes, that is responsible for mycobactin production. Map has a homolog, but it is a bit different in it�s structure�.It is known that Mycobacterium tuberculosis has a cluster of 10 genes, that is responsible for mycobactin production. Map has a homolog, but it is a bit different in it�s structure�.

16. Mycobactin production Here we can see the cluster of 10 genes, that are responsible for the mycobactin production in Mtb.. We can see that the homolog cluster for mav and map. We can see that the genes of Mtb mtbI(trpE2) and mbtJ(lipK) are adjacent to each other and immediately downstream of the mycobactin biosynthesis gene cluster. However in Mav and Map there is a gab between the two genes, plus the 19,3 kb gab and the 197,3 kb gab. But, because Mav successfully can produce Mycobactin, the distance is not likely the limiting factor. Here we can see the cluster of 10 genes, that are responsible for the mycobactin production in Mtb.. We can see that the homolog cluster for mav and map. We can see that the genes of Mtb mtbI(trpE2) and mbtJ(lipK) are adjacent to each other and immediately downstream of the mycobactin biosynthesis gene cluster. However in Mav and Map there is a gab between the two genes, plus the 19,3 kb gab and the 197,3 kb gab. But, because Mav successfully can produce Mycobactin, the distance is not likely the limiting factor.

17. Mycobactin production If we look at the cluster again, we find another difference between the species. The major difference is the mtbA gene. Gene mtbA is shorter in Map than in the two others. Because mbtA is thought to initiate Mycobactin production, the truncation observed suggests that the entire cascade leading to mycobactin production may be attenuated in Map. (34) It is still not tested if that is the reason. If we look at the cluster again, we find another difference between the species. The major difference is the mtbA gene. Gene mtbA is shorter in Map than in the two others. Because mbtA is thought to initiate Mycobactin production, the truncation observed suggests that the entire cascade leading to mycobactin production may be attenuated in Map. (34) It is still not tested if that is the reason.

18. The virulence of M. avium ssp. paratuberculosis Lige en note om noterne: I mange af mine slides siger jeg ikke v�sentligt mere end det der st�r p� sk�rmen. S� de slides har jeg ikke skrevet noter til�Lige en note om noterne: I mange af mine slides siger jeg ikke v�sentligt mere end det der st�r p� sk�rmen. S� de slides har jeg ikke skrevet noter til�

19. The virulence of Map Factors that are assumed to be important for the virulence of Map: The PE/PPE protein family Membrane lipids The mce (Mammalian Cell Entry) gene

20. The PE/PPE protein family PE/PPE genes comprise 10% of the Mtb genome. In Map, it is only 1 %. Theory: PE/PPE proteins are there to create antigenic variation

21. Effects of membrane lipids Mycobacteria have a lipid-rich, hydrophobic membrane. This helps the bacteria to survive in a macrophage Phagosomes containing large hydrophobic granules have been shown to not merge with lysosomes Her skal jeg tegne lidt p� tavlen ved siden af, hvor jeg viser, hvordan bakterien bliver trukket ind i et fagosom. (Lysosomer er de organeller, som indeholder de enzymer, der kan nakke bakteriet)Her skal jeg tegne lidt p� tavlen ved siden af, hvor jeg viser, hvordan bakterien bliver trukket ind i et fagosom. (Lysosomer er de organeller, som indeholder de enzymer, der kan nakke bakteriet)

22. How to diagnose an M. avium ssp. Paratuberculosis?

23. Old method: IS900 Previously, Map was diagnosed using a PCR method, where primers specific to the insertion sequence IS900 were used But this method was shown to give false positives, as it also detected some other mycobacteria

24. Unique sequences 161 unique sequences have been found in Map A method has been developed, in which PCR is used to target a unique sequence How specific is the PCR method? The authors say it�s highly specific, but we think they might have patented it. So they might not be objective. There�s no guarantee that some of these �unique� sequences won�t pop in other genomes that we haven�t sequenced yet.How specific is the PCR method? The authors say it�s highly specific, but we think they might have patented it. So they might not be objective. There�s no guarantee that some of these �unique� sequences won�t pop in other genomes that we haven�t sequenced yet.

25. Summary Map is the causing agent of Johne�s disease, which costs USD$1.5 billion per year in the USA Map is unable to produce mycobactin due to a smaller mbtA gene PE/PPE-proteins, the mce gene and membrane lipids all contribute to the virulence of Map Unique sequences found in Map can be used to more easily diagnose the bacteria Parts of the article indicates sloppy work from the authors