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Lectures 7 BL 246 Bacterial sporulation resistance

Lecture 7 BL246 Bacterial sporulation (page 16). Type of cell divisionintegrated sequence of new biochemical pathways'NEW enzymes

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Lectures 7 BL 246 Bacterial sporulation resistance

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    1. Lectures 7 BL 246 Bacterial sporulation & resistance Review at front Setlow, P (2000) ‘Resistance of bacterial spores’.pp 217-230. In ‘Bacterial stress responses’ Eds. G. Storz & R.Hengge-Aronis. ASM Press, Washington DC. Overview of sporulation & resistance

    2. Lecture 7 BL246 Bacterial sporulation (page 16) Type of cell division ‘integrated sequence of new biochemical pathways’ NEW enzymes & polymers & structures DIFFERENTATION

    3. But who sporulates? 20 different genera: *Bacillus, *Clostridium (*most studied) Sporosarcina, Sporolactobacillus etc model sporulator - B.subtilis

    4. Endospores properties (page 16) 1. Refractile (slide) 2. Cannot stain with basic dyes 3. Dormant - long periods 4. Heat resistant -1200C/20p.s.i./20min to kill all spores 5. Resistant to short range radiation & chemicals

    5. Dormant spore structure (p 16 & 18) (slides) Thick coats (inner & outer) TEM of dormant spore – electron transparent cortex Germ cell wall Spore core (germ cell)

    6. 250 million years old spore??? Nature 407; 897-900 (2000) ‘Alarm bells ring’…sceptics question the work…. Contamination of sample? Through cracks in salt cystal strain 2-9-3 ~Bacillus marismortui found in the dead sea

    7. Sporulation stages-stage I-axial filament formation (p 17) 1. End of log phase 2. Carbon ? pH ? etc… 3. DNA synthesis stops 4 Spo genes activated (? factors) 5. Reversible 6. DNA attached to membrane at poles

    8. Sporulation stages II & III Forespore development 1. Outer forespore membrane Inner forespore membrane 2. New membrane synthesis 3. DNA trapped in forespore 4. FtsZ ring forms at a polar site 5. reversible

    9. Sporulation stage IV- cortex development 1. Cortex forms between ifm & ofm 2. ATP levels ? 3. 3 Phosphoglyceric acid ? (3PGA) 4. Refractility develops 5. Dipicolinic acid (DPA) & calcium ??? in forespore 6. Committed

    10. Sporulation Stage V-coat formation 1. Refractility complete 2. Coat proteins made in mother cell 3. Coats discontinuous & join up Stage VI Release of spore - autolysis of mother cell

    11. Sporulation (cont) Time taken for population to sporulate - 6h (page 19 ) 50-75% cells sporulate polarity of membranes directs biosynthesis (page 20) ifm ? new cell wall ofm ? cortex

    12. Lecture 8 BL246 Bacterial endospores Today :- Spore specific components Resistance Gemination

    13. Spore specific polymers & sporulation Polarity of membranes during sporulation (page 20) Ifm & germ cell DNA ? germ cell wall biosynthesis Ofm & mother cell DNA ? cortex & coat biosynthesis, and DPA synthesis

    14. Cortex composition? peptidoglycan = PG (no teichoic acid) thick ‘cell wall like layer’ lightly x linked (7%) maintenance of dormancy?

    15. Accumulation of spore specific components Calcium accumulation 2% dry wt in mature spore Facilitated diffusion Dipicolinic acid synthesis (DPA) DPA made in mother cell pyridine-2-6-dicarboxylic acid

    16. Spore specific components (cont) Ca : DPA chelate Ca : DPA present in a 1:1 molar ratio low free Ca Ca linked to ? wet heat resistance (mechanism unknown) but NOT DPA function of DPA unknown

    17. Spore coat composition 50% spore volume (slides) 40-60% total dry weight Protein (~60 polypeptides) resistant to chemicals eg 8M urea, 80% phenol stable disulphide rich proteins

    18. Maintenance of dormancy Hypothesis:- Cortex exerts an osmotic pressure on the spore core ? dehydrating it cortex has an osmoregulatory function partially dehydrated core (25% water) in equilibrium with a wet cortex (75% water).

    19. Small acid soluble proteins (SASPS) Provides resistance to dry & wet heat, UV, ?/? SASPS made in developing forespore 3-8% spore protein DNA binding proteins (DS DNA) changes DNA structure

    20. Spore specific components (summary) 1. Inner & outer coats (protection) 2. Cortex (dehydrates the core) 3. Ca : DPA (Ca wet heat resistance) 4. SASPS (multifunctional) binding to DNA ? wet & dry ht & UV resistance

    21. Germination of spores Process of degradation Activation (reversible) germination (irreversible) outgrowth into a new vegetative cell

    22. Activation Conditioning spores to germinate Heat activation (eg 5min at 600C) Ageing Activated spores ? no endogenous metabolism

    23. Germination (page 21A) Resistant dormant spore ? sensitive metabolically active spore trigger reaction 150 genes in sporulation (spo) & germination (ger) slides to show sections of germinating spores

    24. Inititiation of germination (page 22 top etc) Sugars - glu, fru, amino-acids - ala, leu, pro purines and others population takes 40-100min one spore takes <1 min.

    25. Germination - a series of degradative events 1. Commitment 2. Loss of heat resistance & Ca & DPA 3. Loss of cortex & refractility 4. Protein hydrolysis 1-4 NO ATP

    26. Germination cont ATP dependent from now on… 5. Metabolism - O2 , glycolysis, TCA cycle etc 6. Macromolecular synthesis - RNA & protein, DNA, cell wall….. NEW CELL HAS EMERGED! Yippee

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