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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