MIC 329MIC 329The Gram-positive World*The Gram-positive World*

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

The changing definition of The changing definition of BacillusBacillus::

Any rod-shaped bacteriumAny rod-shaped bacteriumthenthen

Gram-positiveGram-positive rods rodsthenthen

AerobicAerobic Gram (+) rods Gram (+) rodsthen along came 16S sequences…...then along came 16S sequences…...

EnterococcusEnterococcusListeriaListeria

SporosarcinaSporosarcina

SporolactobacillusSporolactobacillus

The ongoing schism of the genus The ongoing schism of the genus BacillusBacillus

• GeobacillusGeobacillus

• ThermobacillusThermobacillus

• VirgibacillusVirgibacillus

• SalibacillusSalibacillus

• PaenibacillusPaenibacillus

• GracibacillusGracibacillus

Why study Why study Bacillus subtilisBacillus subtilis??

• Best-characterized Gram-positive bacteriumBest-characterized Gram-positive bacterium– Biochem., metabolism well-studiedBiochem., metabolism well-studied

• Good genetic system (transformation, transduction)Good genetic system (transformation, transduction)

• Advanced molecular biology techniquesAdvanced molecular biology techniques

• Entire genome sequenced / annotatedEntire genome sequenced / annotated

• Easy to grow, manipulate in cultureEasy to grow, manipulate in culture

• Widely used in industry, agricultureWidely used in industry, agriculture

• ““Simple” model for cellular differentiationSimple” model for cellular differentiation

Bacillus subtilisBacillus subtilis differentiation cycle differentiation cycle

Germination and Outgrowth

Exponential Growth

Postexponential PhaseSporulation

Dormancy

II

III

IV

V*Protection*Protection*Photochemistry*Photochemistry

*Repair*Repair

B. subtilisB. subtilis spore anatomy spore anatomy

outer coatouter coatinner coatinner coatcortexcortexmembranesmembranescorecorenucleoidnucleoid

ococ

icic

Endospores are resistant to:Endospores are resistant to:

• Heat (both wet and dry)Heat (both wet and dry)

• Ultraviolet (UV) radiationUltraviolet (UV) radiation

• Extreme desiccation (including vacuum)Extreme desiccation (including vacuum)

• LysozymeLysozyme

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

SporeSporeResistanceResistance

EnvironmentalEnvironmentalFactorsFactors

SporeSporeProtectiveProtective

MechanismsMechanisms

Repair ofRepair ofDamageDamage

Sporulation/Sporulation/GerminationGerminationPhysiologyPhysiology

GeneticGeneticFactorsFactors

Abundance of spores in extreme localesAbundance of spores in extreme localesSample site Location

(Date)Number of

spores

3 desert soils Tucson, AZ(1996-7) ~1 x 108 / g

Near-subsurface

granite

CatalinaMtns., AZ

(2000) ~ 5 x 102 / g

Painted metalsurface

(unshaded)

Rooftop, Ft.Worth, TX

(1995)~ 2 x 103 / m2

• Solar UV:Solar UV:– ~10 J /m~10 J /m22 sec UV-B (noon) sec UV-B (noon)– ~25 J/m~25 J/m22 sec UV-A (noon) sec UV-A (noon)

• Temperature extremes:Temperature extremes:– Avg. -7 to +46 ˚C (air)Avg. -7 to +46 ˚C (air)– ~70-80 ˚C (surfaces)~70-80 ˚C (surfaces)

• Desiccation:Desiccation:– Avg. 13%-30% RHAvg. 13%-30% RH– Avg. 28 cm rainfall / yearAvg. 28 cm rainfall / year

Sonoran Desert Environment:Sonoran Desert Environment:

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

0 10 20 30 40 50 60 70 80 90

0.01

0.1

1

10

100

UV dose (Joules / square meter)

Per

cen

t su

rviv

al

Spores

Vegetative cells

LD90

(254-nm(254-nmUV-C)UV-C)

wavelength (nm) 200 600 1000 1400 1800 2200

100

50

Rel

ativ

e fl

ux

UV vis IR

200 nm 300 nm 400 nm

UV-C UV- B

UV-A

254 nm used in the lab

>290 nm reaches the Earth's surface

in space on Earth's surface

Solar UVSolar UVSpectrumSpectrum

vs.vs.

LaboratoryLaboratoryUVUV

DNA Protective Factors in SporesDNA Protective Factors in Spores

• Spore coat proteinsSpore coat proteins

• Spore pigment in coatSpore pigment in coat

• Dipicolinic acid in coreDipicolinic acid in core

• SASP in coreSASP in core

UV-C UV-B UV-B+A sun

UV-A sun

0

100

w.t.cotE gerE

Treatment

LD

(

% o

f w

ild

typ

e)90

*

* *

Riesenman and Nicholson. AEM 66: 620. 2000.Riesenman and Nicholson. AEM 66: 620. 2000.

The spore coat layers protect spores fromThe spore coat layers protect spores fromsolar UV wavelengthssolar UV wavelengths

Spore pigment offers significant protection Spore pigment offers significant protection against environmentally-relevant UV wavelengthsagainst environmentally-relevant UV wavelengths

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

Wild-type (+) CuSOWild-type (+) CuSO44

Wild-type (-) CuSOWild-type (-) CuSO44

cotAcotA (+) CuSO (+) CuSO44

N

C

C

C

C

CCC

OO

-OO- Ca + +

DPA

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

• Unique to spore coreUnique to spore core

• Exists as CaExists as Ca+2+2-chelate-chelate

• Abundant (up to 10% of dry Abundant (up to 10% of dry weightweight

• Important in heat resistanceImportant in heat resistance

UV-C UV-BFull sun

UV-Asun

FB108

FB72

PS832

0

5

10

15

20

25

UV resistance

relative to FB108

DPA is especially important for spore DPA is especially important for spore resistance to UV-B radiationresistance to UV-B radiation

Slieman and Nicholson. AEM 67: 1274. 2001.Slieman and Nicholson. AEM 67: 1274. 2001.

Spore photochemistry is due to Spore photochemistry is due to SASP-DNA interactionSASP-DNA interaction

• SASP are Small, Acid-SASP are Small, Acid-soluble Spore Proteinssoluble Spore Proteins

• SASP are synthesized at SASP are synthesized at Stage III of sporulationStage III of sporulation

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

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

Bacillus subtilisBacillus subtilis differentiation cycle differentiation cycle

Germination and Outgrowth

Exponential Growth

Postexponential PhaseSporulation

Dormancy

II

III

IV

V

SASP productionSASP production

SP RepairSP Repair

UV-->UV-->SP producedSP producedin DNAin DNA

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

cis-syn cyclobutyl thymine dimer (csTT)

C

C

CC

C N

6 54

21

3H

ON

H

HO

SUGAR

PHOSPHATE

SUGAR

3

3

O H

H

N O

H3

1 245

NC

C C

C

C6

spore photoproduct (SP)

C

C

CC

C N

6 54

21

H

ON

H

HO

SUGAR

PHOSPHATE

SUGAR

3

3

O H

H

N O

H3

1 24

5

NC

C C

C

C6

H

2

adjacent thymines

6 C

C

CC

C N

54

21

3H

ON

H

HO

3

3

SUGAR

PHOSPHATE

SUGAR

O H

H

N O

H3

12

456

NC

C C

C

C

+UV+UV

vegetative cells DNA in solution (B-DNA)

spores dehydrated DNA (A-DNA)

3210-10

10

20

30

Hours relative to solar noon

60

70

80 Temperature (Þ

C)

UV

flu

x (J

/ m

s

ec)

2

A. Filter lidA. Filter lidB. 3x dried spore spotsB. 3x dried spore spotsC. Microscope slideC. Microscope slideD. PlatformD. PlatformE. BoxE. Box

Saran WrapSaran Wrap

1/2”1/2”PlatePlateglassglass

Poly-Poly-styrenestyrene

3210.001

.01

.1

1

10

100

%Sw.t.uvrB42

splB1

uvrB42, splB1

same strains, shielded

Solar UV-A dose (MJ/m )2

Solar UV, not heat or desiccation, determines spore survivalSolar UV, not heat or desiccation, determines spore survival

30020010000.1

1

10

100

wild-typeuvrB42splB1uvrB42,splB1

% S

urv

ival

UV dose ( J / m )2

SP is repaired in germinating SP is repaired in germinating spores by SP lyase and NER spores by SP lyase and NER

vegetative cellsvegetative cells

(254-nm(254-nmUV-C)UV-C)

LDLD9090

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

Yaming XueYaming Xue

w.t.w.t.uvrB42uvrB42splB1splB1

Appl. Environ. Microbiol.Appl. Environ. Microbiol.6262: 2221-2227. 1996.: 2221-2227. 1996.

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

cis-syn cyclobutyl thymine dimer (csTT)

C

C

CC

C N

6 54

21

3H

ON

H

HO

SUGAR

PHOSPHATE

SUGAR

3

3

O H

H

N O

H3

1 245

NC

C C

C

C6

spore photoproduct (SP)

C

C

CC

C N

6 54

21

H

ON

H

HO

SUGAR

PHOSPHATE

SUGAR

3

3

O H

H

N O

H3

1 24

5

NC

C C

C

C6

H

2

adjacent thymines

6 C

C

CC

C N

54

21

3H

ON

H

HO

3

3

SUGAR

PHOSPHATE

SUGAR

O H

H

N O

H3

12

456

NC

C C

C

C

+UV+UV

vegetative cells DNA in solution (B-DNA)

spores dehydrated DNA (A-DNA)

Probing DNA damages with EndoV and alkaliProbing DNA damages with EndoV and alkaliDNA

UV

T<>T

10 kb

ss break ds break CPD

10 kb 10 kb4 kb 6 kb

Endo V

T<>T

10 kb

High pH High pH

High pH

10 kb 10 kb4 kb

4 kb

6 kb

6 kb6 kb

4 kb4 kb

6 kb

B. subtilisB. subtilis spore DNA spore DNAexposed to sunlight exposed to sunlight accumulates ss breaks,accumulates ss breaks,ds breaks and cyclobutane ds breaks and cyclobutane dimers in addition to SP.dimers in addition to SP.

0.8% neutral agarose0.8% neutral agarose

0.8% alkaline agarose0.8% alkaline agarose

UV wavelength (nm)300 350 400250

1 23

4

UV-C UV-B UV-A

UV treatment: 1 2 3 4 SP* + + + + Py<>Py + + + - SS + - + + DS + - +/- - AP - - - -

A

B

Tony SliemanTony SliemanAppl. Environ. Microbiol. 66:199-205. 2000.Appl. Environ. Microbiol. 66:199-205. 2000.

Summary of DNA Summary of DNA Damage in solar Damage in solar UV-irradiated UV-irradiated spores.spores.

30020010000.1

1

10

100

wild-typeuvrB42splB1uvrB42,splB1

% S

urv

ival

UV dose ( J / m )2

SP is repaired in germinating SP is repaired in germinating spores by SP lyase and NER spores by SP lyase and NER

vegetative cellsvegetative cells

(254-nm(254-nmUV-C)UV-C)

LDLD9090

SP lyase-mediated DNA repair in SP lyase-mediated DNA repair in B. subtilisB. subtilis

• Encoded by Encoded by splBsplB gene. gene.

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

• Active during spore germination.Active during spore germination.

• Direct reversal of SP to thymines Direct reversal of SP to thymines in situ.in situ.

• ““Dark repair” process.Dark repair” process.

Organization and expression of Organization and expression of the the splABsplAB operon in operon in B. subtilisB. subtilis

ptsI splA splB

Enz I of PTS

UUUUUUUU

UUUUP1--EsigG

P3--EsigG

SplA 9.2 kD

TRAP-likeSplB

40 kD SP lyase

?

Patricia FajardoPatricia Fajardo

Mario PedrazaMario PedrazaJ. Bact. J. Bact. 175:175:1735.1993. Curr. Micro.1735.1993. Curr. Micro.34:34:133.1997.133.1997.J.Bact. J.Bact. 176176: 3983.1994. MGG : 3983.1994. MGG 255255:587.1997.:587.1997.J.Bact.J.Bact.177:177: 4402. 1995. J.Bact. 4402. 1995. J.Bact. 182:182:555.2000.555.2000.

““Radical SAM” Model for SP repairRadical SAM” Model for SP repair

TT5' 3'

TT5' 3'

Sp lB Sp lB

Sp lBSp lB

4 Fe4 S

1

2

Sp lB Sp lB

TT

5'

3'

3

S-AdoMet

5'-Ade

met

4

Sp lB Sp lB

1. SplB dimerizes via a [4Fe-4S] center.1. SplB dimerizes via a [4Fe-4S] center.2. Specific binding to SP in DNA.2. Specific binding to SP in DNA.3. SAM split by electron donation from Fe-S 3. SAM split by electron donation from Fe-S center, producing 5’-adenosyl radical.center, producing 5’-adenosyl radical.4. Radical abstracts proton from C-6 of SP, 4. Radical abstracts proton from C-6 of SP, reverses SP back to 2 T’s.reverses SP back to 2 T’s.

Roberto RebeilRoberto Rebeil Tony SliemanTony Slieman

J.Bact. J.Bact. 180:180:4879. 1998. PNAS 4879. 1998. PNAS 9898: 9038. 2001.: 9038. 2001.J.Bact. J.Bact. 182182: 6412. 2000.: 6412. 2000.

CONCLUSIONSCONCLUSIONS• In the laboratory:In the laboratory:

– Spores are highly UV resistant.Spores are highly UV resistant.

– SP is the major DNA damage.SP is the major DNA damage.

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

– SP lyase > NER during germination.SP lyase > NER during germination.

• In the environment:In the environment:– Spores are highly UV resistant.Spores are highly UV resistant.– SP is still the major DNA damage.SP is still the major DNA damage.– CPD, ss, ds breaks CPD, ss, ds breaks areare significant at biol. relevant UV doses. significant at biol. relevant UV doses.– SP lyase = NER during germination.SP lyase = NER during germination.– Heat not a significant lethal component of sunlight.Heat not a significant lethal component of sunlight.

Survival and persistence of Survival and persistence of bacterial endospores in extreme bacterial endospores in extreme

environmentsenvironmentsPatricia FajardoPatricia Fajardo Mario Pedraza Mario Pedraza

Lilian ChoobackLilian Chooback Roberto Rebeil Roberto Rebeil

Heather GlanzbergHeather Glanzberg Paul Riesenman Paul Riesenman

Jocelyn LawJocelyn Law Tony Slieman Tony Slieman

Rachel MastrapaRachel Mastrapa Yubo Sun Yubo Sun

Heather MaughanHeather Maughan Yaming Xue Yaming Xue