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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.

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Mic 329 the gram positive world l.jpg

MIC 329The Gram-positive World*

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


Slide4 l.jpg

The changing definition of Bacillus:

Any rod-shaped bacterium

then

Gram-positive rods

then

Aerobic Gram (+) rods

then along came 16S sequences…...


Slide5 l.jpg

Sporosarcina

Listeria

Enterococcus

Sporolactobacillus


The ongoing schism of the genus bacillus l.jpg
The ongoing schism of the genus Bacillus

  • Geobacillus

  • Thermobacillus

  • Virgibacillus

  • Salibacillus

  • Paenibacillus

  • Gracibacillus


Why study bacillus subtilis l.jpg
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.jpg

Bacillus subtilis differentiation cycle

*Repair

*Protection

*Photochemistry


B subtilis spore anatomy l.jpg
B. subtilis spore anatomy

outer coat

inner coat

cortex

membranes

core

nucleoid

oc

ic


Endospores are resistant to l.jpg
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.jpg

Spore

Protective

Mechanisms

Environmental

Factors

Spore

Resistance

Repair of

Damage

Genetic

Factors

Sporulation/

Germination

Physiology



Slide13 l.jpg

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



Slide15 l.jpg

Solar UV vegetative cells

Spectrum

vs.

Laboratory

UV


Dna protective factors in spores l.jpg
DNA Protective Factors in Spores vegetative cells

  • Spore coat proteins

  • Spore pigment in coat

  • Dipicolinic acid in core

  • SASP in core


Slide17 l.jpg

The spore coat layers protect spores from vegetative cells

solar UV wavelengths

Riesenman and Nicholson. AEM 66: 620. 2000.


Slide18 l.jpg

Spore pigment offers significant protection vegetative cells

against environmentally-relevant UV wavelengths

Wild-type (+) CuSO4

Wild-type (-) CuSO4

DcotA (+) CuSO4

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


Slide19 l.jpg

Dipicolinic acid (pyridine-2,6-dicarboxylate) vegetative cells

  • Unique to spore core

  • Exists as Ca+2-chelate

  • Abundant (up to 10% of dry weight

  • Important in heat resistance


Slide20 l.jpg

DPA is especially important for spore vegetative cells

resistance to UV-B radiation

Slieman and Nicholson. AEM 67: 1274. 2001.


Spore photochemistry is due to sasp dna interaction l.jpg
Spore photochemistry is due to SASP-DNA interaction vegetative cells

  • 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.jpg

Bacillus subtilis vegetative cells differentiation cycle

SP Repair

UV-->

SP produced

in DNA

SASP production



Slide24 l.jpg

Saran Wrap is different

1/2”

Plate

glass

Poly-

styrene

A. Filter lid

B. 3x dried spore spots

C. Microscope slide

D. Platform

E. Box



Sp is repaired in germinating spores by sp lyase and ner l.jpg
SP is repaired in germinating spores by SP lyase and NER is different

LD90

(254-nm

UV-C)

vegetative cells


Spores of b subtilis dna repair mutants respond differently to lab uv and solar uv l.jpg
Spores of is differentB. 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.




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B. subtilis DNA damage(s)? 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.jpg

Summary of DNA DNA damage(s)?

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.jpg
SP is repaired in germinating spores by SP lyase and NER DNA damage(s)?

LD90

(254-nm

UV-C)

vegetative cells


Sp lyase mediated dna repair in b subtilis l.jpg
SP lyase-mediated DNA repair in DNA damage(s)?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.jpg
Organization and expression of the DNA damage(s)?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.jpg
“Radical SAM” Model for SP repair DNA damage(s)?

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.jpg

In the laboratory: DNA damage(s)?

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.jpg

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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