Mycobacteria

2. Mycobacteria • - aerobic • - acid-fast actinomycetes (Ziehl –Neelsen staining) • - slightly curved or straight nonmotile rods (0.2-0.6 x 1.0-10 um) • branching growth or mycelium growth  fragmentation  rods or coccoid • colony color : whitish or cream , bright yellow or orange (species containing carotenoid) • pigmentation : nonpigmented species (ex. Mycobacterium tuberculosis) • photochromogenic species (ex. Mycobacterium marinum) • scotochromogenic species (ex. Mycobacterium gordonae) • - Mycolic acid-containing genera of cell wall chemotype IV • Analysis of the fatty acid ester, identification of menaquinone • Agent of diseases : Tuberculosis ( Mycobacterium tuberculosis) • Leprosy (Mycobacterium leprae)

3. Mycobacteria • Two major groups on the basis of the growth rate • Slow growers : pathogens • ex) Mycobacterium tuberculosis , Mycobacterium leprae, Mycobacterium bovis • Rapid growers : non-pathogens (strictly…No) • ex) Mycobacterium smegmatis

4. menaquinone 2-methyl-3-multiprenyl-1,4-napthoquinone Gram positive bacteria

5. Taxonomic study • In past, morphological character • Now, numerical taxonomy & chemotaxonomic character • chemotaxonomic character  separation of actinomycete genera • Cell wall, mycolic acid analysis • - numerical taxonomy  subgeneric level • DNA-DNA hybridization, 16S rRNA similarity data • Numer of Mycobacterium species : about 110 species in 2004 • 52 species - ~1983 • 6 new species - 1984-1991 • About 4 new species - 1992-2003 • 12 new species - 2004

6. - 16S rRNA gene ideal target for phylogeny & taxonomy - In Mycobacteria Sequence of the variable stretch between position 175 and 238  Can’t be discriminated on the basis of their 16S rRNA gene sequence  change the target to other gene . ex) gyrB , hsp65 genomic separateness of two groups (rapid growers and slow growers) Sequence idiosyncrasy - slow growers : contained long helix between nucleotide positions 451 and 482 - rapid growers : contained a short helix only.

7. 114 species

11. Schematic graph of a neighbor-joining 16S rRNA gene sequence analysis A : Slow growing species red : containing a long helix between position 451 and 482 orange : containing a short helix B : Fast growing species (gray color)

12. A1 are characterized by a long helix between position 451 and 482 of the 16S rRNA sequence A2, there is no long helix between position 451 and 482

13. A2, there is no long helix between position 451 and 482 A3 is interesting from an evolutionary point of view in that this slow growing species without the characteristic helix insert A4, containing helix insert, distinctly separate position

14. B : Fast growing species (blue color) 1 1 and 2, a bridge between slow growers and fast growers. 2 3 3, Different evolutionary rate B3 : Pathogenecity relatives 4 4 and 5 are defined as slow growers 5 B4

15. 1. Chemotaxonomic study • - The genus Mycobacterium was placed within the CNM (Corynebacterium-Nocardia- Mycobacterium) complex. • cell wall chemotype IV • mycolic acid • 2. Numerical taxonomy study • - DNA hybridization of total genomic DNA with specific DNA probes •  relationship between M. paratuberculosis and the MAIS (M. avium–intracellulare–scrofulaceum) complex. • M. scrofulaceum shows little DNA similarity with M. avium and M. intracellulare • - 16S rRNA PCR useful to be differentiation of mycobacteria at the species level • DNA homology study •  M.tuberculosis, M. bovis, M. bovis BCG, M. microti and M. africanum revealed that all strains exhibited more than 90% DNA relatedness •  DNA relatedness between M. tuberculosis and other slowly growing mycobacteria ranged from 9–53%

16. - diverse biotopes : fresh water, salt water, soil, dust • Many mycobacteria has been isolated from environmental samples • Mycobacteria should be mutiply actively in these environments * “contaminating” mycobacteria – isolate from environment Mycobacteria can survive for very long periods under nongrowth condition • ex) Mycobacterium paratuberculosis was reported to survive for 252 days in a soil-water slurry (Kazda, 1983). • Ecology of mycobacteria • 4 groups of mycobacteria on the basis of ecological properties by Kazda • 1. Obligate pathogen : unable to multiply outside living beings • Ex) M. tuberculosis, M.bovis, M. africanum, M. asiaticum.. • 2. Facultative pathogen • Ex) M. leprae, M. paratuberculosis, M. ulcerans • 3. Potential pathogen : can grow in natural biotopes without losing their pathogenic properties • Ex) M. avium, M. chelonae, M. fortuitum, M. intracellulare,M. kansasii, M. marinum.. • 4. Saprophytic mycobacteria : most of the rapidly growing species and some slow growers • Ex) M. gordonae, M. nonchromogenicum, M. triviale, M. terrae , M. gastri..

17. Character of potential pathogen & saprophytic mycobacteria • Found in many environment • Utilize many different growth substrate • Capacity to survive and multply under a wide range of environments From soil M. fortuitum, (40–80%), M.nonchromogenicum, M. terrae. M, aurum, M. smegmatis From hospital dust M. fortuitum(43%), M. nonchromogenicum complex (25%), M.gordonae (18%) From house dust MAIS (M. avium–intracellulare–scrofulaceum )complex (55%), M.nonchromogenicum complex(23%), M. gordonae (11%). From nonmarine water M. gordonae, M. terrae, M.phlei , M. fortuitum,M. chelonae, M. scrofulaceum, M. avium From marine water MAIS complex, M. gordonae, M. terrae

18. Decontamination procedures - selective media have been developed to increase the efficiency of isolation procedures - Most of the decontamination methods were developed for the isolation of mycobacteria from specimens originating from diseased humans or animals and exploit the resistance of the acid-fast mycobacteria to alkaline and acidic conditions. The most commonly applied decontamination procedures  treatment of the samples with NaOH (2–4%) for 15–30 min at room temperature or at 37°C No information is available on the number and type of mycobacteria  loss of samples !  Over-estimate or under-estimate data  no reliable data

19. mycobacteria were isolated from91% of the soil samples with only 4% of the tubes being contaminated

20. Select for a specific mycobacterialspecies. 80% M. moriokaense 20% M. fortuitum High incubation temperature Short incubation time

21. Specific carbon source • 1. Paraffin : enrichment of mycobacteria in soil and water • 2. Ethene : enrichment cultures •  Xanthobacter and Nocardia species may also grow •  isolation of the faster growing mycobacteria • 3. Morpholine (1-oxa-4-azacyclohexane) • degradation by Mycobacterium isolates • All strains isolated on morpholine from activated sludge systems & river water •  mycobacteria • 4. Methanol • Gram-positive methanol-utilizing bacteria isolates • Treatment with antibiotic polymyxinB  M. fortuitum

22. Glycerol, as a sole source of carbon and energy  all cultivable mycobacteria • Liquid cultures of rapid growers : incubated stationary : lag phase • incubated shaken : lag phase  CO2 accumulation in the medium of the stationary culture • Nitrogen sources ex) amino acids, ammonium , nitrate • Most mycobacteria do not require any specific growth factors or vitamins in the growth medium • Exception) 1. M. haemophilum hemin, • 2. M. paratuberculosis mycobactin Media - Basal mineral salt medium + carbon source limited

23. Problem in liquid culture of mycobacteria hydrophobic property of the cell wall  1. Cell aggregation 2. Cell adhesion to the surface of growth vessels Add Tween 80 detergent  reduce clumping and stimulate growth Special fermentor : stainless steel fermentor M. bovis BCG and M. microti : grow with 0.08% Tween 80 in the growth medium and by aerating with air containing 5% carbon dioxide , stirring rate keep low • Chemostat culture (continuous culture without detergent) • - Mycobacterium phlei : grown dispersed in a fermentor without the addition of a detergent • metabolic studies - M. Aurum strain L1 growing on vinyl chloride • - M. Aurum strain E3 growing on ethene

25. Mycobacteria vs other mycolic acid bacteria • Ziehl-Neelsen acid-fast stain (carbol fuchsin-mycolic acid interaction) • : resist decolorization with HCl-alcohol • - Thin layer chromatography • : analysis of mycolic acid methyl ester • - characterization of major menaquinone • Rapid growers vs Slow growers • - forming colony • Rapid growers : form colonies within 7 days • Slow growers : form colonies more than 7 days (usually more than 10 days) • -16S rRNA study - Pigment production by mycobacterium species - Colonial morphology is not useful

26. Probability matrix -slow growers : large pool of collected data of numerical analysis -rapid growers : not yet List of several physiological and biochemical characteristics  Identification of newly isolated rapidly growing species Additional tests : mycolic acid analysis, DNA-hybridization experiment Cell wall & fatty acid analysis : mesodiaminopimelic acid, arabinose, galactose Mycobacterial peptidoglycan residue : N-glycolated (cf. most of N-acrtylated in other prokaryote)

27. Mycobacteria cell envelope : • - lipid-rich thick cell envelope containing very long-chain mycolic acids • Mycolic acids : • - high-molecular-weight (C60 to C90) • - 3-hydroxy fatty acids substituted with an aliphatic side chain at the C2 position • Pyrolysis of the methyl ester of mycobacterial mycolic acids •  long chain meroaldehydes + fatty acid methylesters with chain lengths of 22–26 carbon atoms

28. α-mycolic acids & α′-mycolic acids • They do not contain any oxygen functions • more polar mycolic acids the longer chain is substituted with methoxy, keto, epoxy, carboxy functions • α-mycolic acids : • contain 74–82 carbon atoms • α′-mycolic acids : • Contain 60-68 carbon atoms • Composition of the α-mycolic acids & α′-mycolic acids •  Gas chromatography or mass spectrometry analysis of mycobacterial mycolic acid methyl esters by TLC provides a sensitive and relatively easy method to determine mycolic acid patterns  the technique clearly can be of great use in the identification of mycobacteria Mycobactin -chemotaxonomic marker -mycobactin yields test by TLC, HPLC analysis M.Smegmatis : 1-8 % M.vaccae, M, chelonae strain . Chelonae : no detectable amounts

30. - metabolically versatile - sugars, alcohols, organic acids, large variety of hydrocarbonsincluding branched-chain, unsaturated, aromatic, and cyclic hydrocarbons, methanol, methylamines - degrade polycyclic aromatic hydrocarbons, such as, pyrene and phenanthrene. M. gastri - 3-hexulose-6-phosphate synthase  ribulose-monophosphate pathway M. smegmatis, M. marinum, M. fortuitum - Autotrophic growth on carbondioxide and hydrogen gas Mycobacterium Py1 - ribulose-1,5-bisphosphate carboxylase and membrane-bound hydrogenase  autotrophic growth

31. C2 hydrocarbons ethane, ethene (ethylene) metabolism Mycobacterium E20 Ethane as carbon source - ethane metabolism was via acetate - ethene metabolism via epoxyethane  epoxyethaneacetyl-coenzyme (Co)A and nicotinamide adenine dinucleotide (NAD)-dependent reaction Propane metabolism Mycobacterium vaccae JOB5 - oxidation of both primary and secondary carbon atoms of propane - aceton, acetol, acetate  intermediate in propane metabolism Propene metabolism Mycobacterium Py1 - propene as a carbon source - no growth with propane or acetone - propne initial oxidation  epoxypropane subsequently carboxylate acetoacetate - acetol------oxidatively transform  acetate & formaldehyde

32. Fatty acid synthetase -typeI : eukaryotic system..(exception of M.smegmatis) -typeII: prokaryotic system Fatty acid biosynthesis inM. smegmatis - two fatty acid synthetase(FAS) activities • 1. FAS-I : similar to the typeI FAS of eukaryotes. • stimulated by certain polysaccharides  restrict chain elongation • 2. FAS-II: similar to the typeII FAS found in other prokaryotes • requirement for an acyl carrier protein(ACP) • actually, fatty acids elongating system • no affect from polysaccharides • Another fatty acid elongation system(FES I) • requires acetyl-CoA, ACP-independent • involve in β-oxidation of fatty acids

33. Long-chain fatty acid-elongation system of M. avium - involve in mycolic acid synthesis - requirment of cofactor - sensitivity toward isoniazid affect the 3-oxoacyl-CoA and enoyl-CoA reductase activities inhibit the synthesis of mycolic acid Glycerol-grown mycobacteria contains amounts of triacylglycerol fat body formation Glycogen, trehalose Nitrogrn limited batch culture of M. phlei  increase of glycogen and lipid transfer of these cell to high nitrogen content without carbon media decrease of glycogen and lipid Nitrogrn limited batch culture of M. smegmatis  increase of glycogen transfer of these cell to high nitrogen and carbon content media decrease of glycogen (terehalose level is not change)

34. Major phospholipids present in mycobacteria • Phosphatidylinositol • phosphatidylethanolamine • diphosphatidylglycerol(cardiolipin) • phosphatidylinositol mannosides phospholipid and fatty acid composition  depend on culture condition • Factors of lipid & fatty acid composition • growth temperature • Carbon source • the ratio of carbon and nitrogen sources • culture age Batch grown culture  growth condition & growth rate rarely constant * Chemostat for continuous culture more reliable data

35. Exochelins and mycobactins : siderophores • Exochelins • Two types of exochelins, differing in their solubility in organic solvents • poorly characterized Mycobactins - Very hydrophobic, locate within the cell envelope Recent work focus - regulation of the biosynthesis of the different siderophores and other iron-regulated proteins

36. - genome sizes of mycobacteria : 3 to 5.5 × 109 daltons cf) E. coli 2.5 × 109 daltons. - high G+C content (66-71 mol% ) - presence of extrachromosomal DNA (plasmids) in several species A 173-kb plasmid isolated from a M. scrofulaceum species  mercury and copper resistance pAL5000 : complete nucleotide sequence, a 4,837-bp plasmid from M. fortuitum pLR7 (15.3kb) from M. intracellulare has been mapped - Study of ribosomal RNA coding gene of mycobacteria Slow growers : only one rRNA operon ex) M. smegmatis Fast growers : two rRNA operon ex) M. tuberculosis, M. intracellulae Cf) Seven rRNA operon in E. coli - rRNA gene coding order : 16S-23S-5S

37. Methods to efficiently transfer DNA between mycobacteria and E. coli • construct a vector ! •  replicates as a plasmid in E. coli and as a phage in mycobacteria. - Introduce an E. coli plasmid replicon into a nonessential region of mycobacteriophage TM4, a temperate phage of M. avium  phasmid Construction of plasmids capable of replicating in both E. coli and mycobacteria. hybrid shuttle plasmids : randomly inserting the E. coli plasmid pIJ666, containing an origin of replication and the genes for kanamycin and chloramphenicol resistance in pAL5000 from M. fortuitum. electroporation : useful in the transformation of other Gram-positive bacteria

38. -Hybrid shuttle plasmids by randomly inserting the E. coli plasmid pIJ666, containing an origin of replication and the genes for kanamycin and chloramphenicol resistance in pAL5000 from M. fortuitum. -Construct the 9.2-kb vector pAL8 by combining pAL5000 with an E. coli plasmid and a gene coding for kanamycin resistance Deletion experiment - Small cloning vector  short pAL5000 • pJRD215 • -contains the phage lambda cos site •  cloning of relatively large DNA fragments in E. coli • facilitatE the construction of genomic DNA libraries - kanamycin resistant • pY6002 • - Replication origin of E. coli • homologous recombination  integration of the vector to genomic DNA of host • pyrF gene : uracil auxotroph  selection for uracil prototroph • aph : kanamycin resistance

39. 1. modification of the steroid nucleus of natural or synthetic sterols 2. selective degradation of the side chain of naturally occurring sterols  M. fortuitum or M. parafortuitum Ex) cholesterol and β-sitosterol 3. biocatalytic process : enzyme or whole cell M. neoaurum (ATCC 25795) contain ‘L-specific aminopeptidase’ L- or D-α-methyl-substituted amino acids can be produced by stereoselective hydrolysis of racemic mixtures of the corresponding amides 4. biocatalysis is the production of optically active epoxides from alkenes Optically pure epoxides form versatile starting materials for the chemical synthesis of optically active pharmaceutical compounds.

40. 6. Environmental biotechnology • Removal of traces of the plant hormone ethene from storage facilities for fruit using immobilized ethene-utilizing mycobacteria • Vinyl chloride utilizing Mycobacterium strain L1 to remove the carcinogenic vinyl chloride from industrial waste gases • Bioremediation • contaminated sediments  polycyclic aromatic hydrocarbons degradation • - Disadvantage : low growth rate • low catalytic activity