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MIC 329 The Gram-positive World*






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MIC 329 The Gram-positive World*. *(Well, really a talk about my favorite Gram (+) organism, Bacillus subtilis ). The changing definition of Bacillus : Any rod-shaped bacterium then Gram-positive rods then Aerobic Gram (+) rods then along came 16S sequences…. Sporosarcina.
MIC 329 The Gram-positive World*

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Mic 329 the gram positive world l.jpgSlide 1

MIC 329The Gram-positive World*

*(Well, really a talk about my favorite Gram (+) organism, Bacillus subtilis)

Slide4 l.jpgSlide 4

The changing definition of Bacillus:

Any rod-shaped bacterium

then

Gram-positive rods

then

Aerobic Gram (+) rods

then along came 16S sequences…...

Slide5 l.jpgSlide 5

Sporosarcina

Listeria

Enterococcus

Sporolactobacillus

The ongoing schism of the genus bacillus l.jpgSlide 6

The ongoing schism of the genus Bacillus

  • Geobacillus

  • Thermobacillus

  • Virgibacillus

  • Salibacillus

  • Paenibacillus

  • Gracibacillus

Why study bacillus subtilis l.jpgSlide 7

Why study Bacillus subtilis?

  • Best-characterized Gram-positive bacterium

    • Biochem., metabolism well-studied

  • Good genetic system (transformation, transduction)

  • Advanced molecular biology techniques

  • Entire genome sequenced / annotated

  • Easy to grow, manipulate in culture

  • Widely used in industry, agriculture

  • “Simple” model for cellular differentiation

Slide8 l.jpgSlide 8

Bacillus subtilis differentiation cycle

*Repair

*Protection

*Photochemistry

B subtilis spore anatomy l.jpgSlide 9

B. subtilis spore anatomy

outer coat

inner coat

cortex

membranes

core

nucleoid

oc

ic

Endospores are resistant to l.jpgSlide 10

Endospores are resistant to:

  • Heat (both wet and dry)

  • Ultraviolet (UV) radiation

  • Extreme desiccation (including vacuum)

  • Lysozyme

  • Chemicals (organic solvents, oxidizing agents, etc.)

Slide11 l.jpgSlide 11

Spore

Protective

Mechanisms

Environmental

Factors

Spore

Resistance

Repair of

Damage

Genetic

Factors

Sporulation/

Germination

Physiology

Abundance of spores in extreme locales l.jpgSlide 12

Abundance of spores in extreme locales

Slide13 l.jpgSlide 13

Sonoran Desert Environment:

  • Solar UV:

    • ~10 J /m2 sec UV-B (noon)

    • ~25 J/m2 sec UV-A (noon)

  • Temperature extremes:

    • Avg. -7 to +46 ˚C (air)

    • ~70-80 ˚C (surfaces)

  • Desiccation:

    • Avg. 13%-30% RH

    • Avg. 28 cm rainfall / year

Spores are 1 2 orders of magnitude more uv resistant than vegetative cells l.jpgSlide 14

Spores are 1-2 orders of magnitude more UV resistant than vegetative cells

(254-nm

UV-C)

Slide15 l.jpgSlide 15

Solar UV

Spectrum

vs.

Laboratory

UV

Dna protective factors in spores l.jpgSlide 16

DNA Protective Factors in Spores

  • Spore coat proteins

  • Spore pigment in coat

  • Dipicolinic acid in core

  • SASP in core

Slide17 l.jpgSlide 17

The spore coat layers protect spores from

solar UV wavelengths

Riesenman and Nicholson. AEM 66: 620. 2000.

Slide18 l.jpgSlide 18

Spore pigment offers significant protection

against environmentally-relevant UV wavelengths

Wild-type (+) CuSO4

Wild-type (-) CuSO4

DcotA (+) CuSO4

Hullo, et al. J. Bacteriol. 183: 5426. 2001.

Slide19 l.jpgSlide 19

Dipicolinic acid (pyridine-2,6-dicarboxylate)

  • Unique to spore core

  • Exists as Ca+2-chelate

  • Abundant (up to 10% of dry weight

  • Important in heat resistance

Slide20 l.jpgSlide 20

DPA is especially important for spore

resistance to UV-B radiation

Slieman and Nicholson. AEM 67: 1274. 2001.

Spore photochemistry is due to sasp dna interaction l.jpgSlide 21

Spore photochemistry is due to SASP-DNA interaction

  • SASP are Small, Acid-soluble Spore Proteins

  • SASP are synthesized at Stage III of sporulation

  • SASP bind to DNA and shift its conformation from B to A

  • UV irradiation of SASP-DNA complexes results in formation of SP and not T<>T

Slide22 l.jpgSlide 22

Bacillus subtilis differentiation cycle

SP Repair

UV-->

SP produced

in DNA

SASP production

The uv photochemistry of dna in vegetative cells and spores is different l.jpgSlide 23

The UV photochemistry of DNA in vegetative cells and spores is different

Slide24 l.jpgSlide 24

Saran Wrap

1/2”

Plate

glass

Poly-

styrene

A. Filter lid

B. 3x dried spore spots

C. Microscope slide

D. Platform

E. Box

Slide25 l.jpgSlide 25

Solar UV, not heat or desiccation, determines spore survival

Sp is repaired in germinating spores by sp lyase and ner l.jpgSlide 26

SP is repaired in germinating spores by SP lyase and NER

LD90

(254-nm

UV-C)

vegetative cells

Spores of b subtilis dna repair mutants respond differently to lab uv and solar uv l.jpgSlide 27

Spores of B. subtilis DNA repair mutants respond differently to lab UV and Solar UV

w.t.

uvrB42

splB1

Yaming Xue

Appl. Environ. Microbiol.

62: 2221-2227. 1996.

Do spores exposed to solar uv accumulate different types of dna damage s l.jpgSlide 28

Do spores exposed to solar UV accumulate different types of DNA damage(s)?

Probing dna damages with endov and alkali l.jpgSlide 29

Probing DNA damages with EndoV and alkali

Slide30 l.jpgSlide 30

B. subtilis spore DNA

exposed to sunlight

accumulates ss breaks,

ds breaks and cyclobutane

dimers in addition to SP.

0.8% neutral agarose

0.8% alkaline agarose

Slide31 l.jpgSlide 31

Summary of DNA

Damage in solar

UV-irradiated

spores.

Tony Slieman

Appl. Environ. Microbiol. 66:199-205. 2000.

Sp is repaired in germinating spores by sp lyase and ner32 l.jpgSlide 32

SP is repaired in germinating spores by SP lyase and NER

LD90

(254-nm

UV-C)

vegetative cells

Sp lyase mediated dna repair in b subtilis l.jpgSlide 33

SP lyase-mediated DNA repair in B. subtilis

  • Encoded by splB gene.

  • Synthesized at Stage III of sporulation, packaged in the dormant spore.

  • Active during spore germination.

  • Direct reversal of SP to thymines in situ.

  • “Dark repair” process.

Organization and expression of the splab operon in b subtilis l.jpgSlide 34

Organization and expression of the splAB operon in B. subtilis

Patricia Fajardo

J. Bact. 175:1735.1993. Curr. Micro.34:133.1997.

J.Bact. 176: 3983.1994. MGG 255:587.1997.

J.Bact.177: 4402. 1995. J.Bact. 182:555.2000.

Mario Pedraza

Radical sam model for sp repair l.jpgSlide 35

“Radical SAM” Model for SP repair

1. SplB dimerizes via a [4Fe-4S] center.

2. Specific binding to SP in DNA.

3. SAM split by electron donation from Fe-S center, producing 5’-adenosyl radical.

4. Radical abstracts proton from C-6 of SP, reverses SP back to 2 T’s.

Tony Slieman

Roberto Rebeil

J.Bact. 180:4879. 1998. PNAS 98: 9038. 2001.

J.Bact. 182: 6412. 2000.

Conclusions l.jpgSlide 36

In the laboratory:

Spores are highly UV resistant.

SP is the major DNA damage.

CPD, ss, ds breaks negligible at biol. relevant UV doses.

SP lyase > NER during germination.

CONCLUSIONS

  • In the environment:

    • Spores are highly UV resistant.

    • SP is still the major DNA damage.

    • CPD, ss, ds breaks are significant at biol. relevant UV doses.

    • SP lyase = NER during germination.

    • Heat not a significant lethal component of sunlight.

Survival and persistence of bacterial endospores in extreme environments l.jpgSlide 37

Survival and persistence of bacterial endospores in extreme environments

Patricia Fajardo Mario Pedraza

Lilian Chooback Roberto Rebeil

Heather Glanzberg Paul Riesenman

Jocelyn Law Tony Slieman

Rachel Mastrapa Yubo Sun

Heather Maughan Yaming Xue


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