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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*

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MIC 329The 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

Listeria

Enterococcus

Sporolactobacillus


The ongoing schism of the genus Bacillus

  • Geobacillus

  • Thermobacillus

  • Virgibacillus

  • Salibacillus

  • Paenibacillus

  • Gracibacillus


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


Bacillus subtilis differentiation cycle

*Repair

*Protection

*Photochemistry


B. subtilis spore anatomy

outer coat

inner coat

cortex

membranes

core

nucleoid

oc

ic


Endospores are resistant to:

  • Heat (both wet and dry)

  • Ultraviolet (UV) radiation

  • Extreme desiccation (including vacuum)

  • Lysozyme

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


Spore

Protective

Mechanisms

Environmental

Factors

Spore

Resistance

Repair of

Damage

Genetic

Factors

Sporulation/

Germination

Physiology


Abundance of spores in extreme locales


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

(254-nm

UV-C)


Solar UV

Spectrum

vs.

Laboratory

UV


DNA Protective Factors in Spores

  • Spore coat proteins

  • Spore pigment in coat

  • Dipicolinic acid in core

  • SASP in core


The spore coat layers protect spores from

solar UV wavelengths

Riesenman and Nicholson. AEM 66: 620. 2000.


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.


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


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

  • 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


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


Saran Wrap

1/2”

Plate

glass

Poly-

styrene

A. Filter lid

B. 3x dried spore spots

C. Microscope slide

D. Platform

E. Box


Solar UV, not heat or desiccation, determines spore survival


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

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)?


Probing DNA damages with EndoV and alkali


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


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 NER

LD90

(254-nm

UV-C)

vegetative cells


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

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

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.


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

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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