SUPPLEMENTAL MATERIAL Table S1: Strains used in this...
Transcript of SUPPLEMENTAL MATERIAL Table S1: Strains used in this...
SUPPLEMENTAL MATERIAL
Table S1: Strains used in this study:
Strain Genotype Reference
PY79 Prototrophic derivative of B. subtilis subsp.
subtilis168
Youngmann
84
3610 Undomesticated wild strain capable of forming
robust biofilms
AE276 amyE::Pldh-lacZ cat this study
AE311 ∆qcrABC::mls this study
AE313 ∆qcrABC::mls, amyE::kinB-GFP this study
AE253 kinA∆PAS1::mls this study
AE254 kinA∆PAS2::mls this study
AE255 kinA∆PAS3::mls this study
AE256 kinAWT::mls this study
AE360 ∆kinA::mlsamyE::kinAHIS this study
AE380 ∆kinA::mls amyE::kinA∆PAS1HIS this study
AE382 ∆kinA::mls amyE::kinA∆PAS3HIS this study
AE325 ∆narGHIJ::mls this study
KG10 ∆cta::spec this study
KG11 ∆cta::spec ∆qcr::mls this study
RL5474 amyE::kinB-gfp::cam Lab stocks
KG13 amyE::kinC-gfp::mls this study
KG14 ∆cta::spec, amyE::kinB-gfp::cam this study
KG15 ∆kinA::mls ∆kinB::kan amyE::kinBC52S::spec this study
KG17 ∆kinA::mls ∆kinB::kan, amyE::kinB::spec this study
RL5426 ∆kinA::mls, amyE::PsinI-lacZ::cam This study
RL5429 ∆kinB::kan amyE::PsinI-lacZ::cam this study
KG19 ∆kinC::mls amyE::PsinI-lacZ::cam this study
KG20 ∆kinD:: tet amyE::PsinI-lacZ::cam this study
RL4523 ∆kinE::mls amyE::PsinI-lacZ::cam this study
KG22 ∆kinA::mls ∆kinB::kan amyE::PsinI-lacZ::cam this study
KG23 ∆kinC::mls ∆kinD::tet amyE::PsinI-lacZ::cam this study
KG24 ΔnarG-I::mls amyE::PtapA-lacZ::spec this study
KG25 ∆cta::spec amyE::PsinI-lacZ::cam This study
KG26 ∆qcr::mls amyE::PsinI-lacz::cam This study
KG27 ∆kinA::tet ∆cta::spec ∆qcr::mls, amyE::PsinI-
lacZ::cam
This study
KG28 ∆kinA::tet ∆kinB::kan ∆cta::spec ∆qcr::ml,
amyE::PsinI-lacZ::cam
This study
KG29 kinB::kan,sacA::PsinI-lacz::cam This study
KG30 kinA::mls kinB::kan,sacA::PsinI-lacZ::cam,
amyE::kinB::spec
This study
KG31 kinA::mls. kinB::kan,sacA::PsinI-lacz::cam,
amyE::kinB-C52S
This study
KG32 kinA::mls kinB::kan,sacA::PsinI-lacZ::cam,
amyE::kinB::TMI-II
This study
KG33 kinA::mls kinB::kan,sacA::PsinI-lacZ::cam
amyE::kinB-TMIII-IV
This study
KG34 kinA::mls kinB::kan sacA::PsinI-lacZ::cam
amyE::kinB-TMV-VI
This study
RL5278 ∆kinA::mls ∆kinB::kan, sacA::PsinI-lux::cam lab stocks
RL5275 ∆kinC::mls ∆kinD::tet sacA::PsinI-lux::cam Lab stocks
RL4582 amyE::PtapA-lacZ::spec lab stocks
RL4585 sinR::spec amyE::PtapA-lacZ::spec Lab stocks
RL4573 ∆kinA::mls, ∆kinB::kan lab stocks
RL5408 ∆kinC::mls, ∆kinD::tet Lab stocks
RL5477 amyE::PtapA-yfp::spec lab stocks
RL4588 amyE::PsinI-lacz::cam Lab stocks
RL5268 sacA::PsinI-lux::cam This study
Table S2: Primers used in this study:
Primer Sequence
ldh-for CGGAATTCaagtgcccggtcagaatcagc
ldh-rev CCCAAGCTTcattaatcatccttgcagggt
qcr-P1 CAC CTG AAT ATG CGG CGG TGC
qcr-P2 CAA TTC GCC CTA TAG TGA GTC GTC TCT TCT CCC CCC
TCT AAG TC
qcr-P3 CCA GCT TTT GTT CCC TTT AGT GAG CGT ATT GTG AAT
TGT CAA ATG
qcr-P4 AGG CTT CCC CGC TCT GCA GC
kinA-P1 ttcagcctagaatcgtcacaagta
kinA-P2 CAAATATATCCTCCTCACTATcgacatatacagattgactttta
kinA-P3 AAATAACAGATTAAAAAAATTATAAaaacacgatgatcatgcaaagga
kinA-P4 cacgatatcatgatattcttc
PAS1-up TAG GGA TTG ATG GCC TGT TTC CGT ATC CTG TTC CAC
AGA ATC
PAS1-down GAT TCT GTG GAA CAG GAT ACG GAA ACA GGC CAT CAA
TCC CTA
PAS2-up CAG GAT GGT TTG GAA TTT TTT ATA TGT AGT CGA TTC
CGG GCT
PAS2-down AGC CCG GAA TCG ACT ACA TAT AAA AAA TTC CAA ACC
ATC CTG
PAS3-up CTT GAC AAT TTC AGA TTC TGT GCT TTT TTG CAG GAT
GGT TTG
PAS3-down CAA ACC ATC CTG CAA AAA AGC ACA GAA TCT GAA ATT
GTC AAG
kinA-comp-up AAACACACAAATTAAAAACTGGTCTGATCGGATCCggaaaacg
ggaacagcggaaac
kinA-comp-do TCGCCAGGGCTGCAGGAATTCttatttttttggaaatgaaattttaaacg
kinA-his-up TGCGCCATGATGATGATGATGATGTGATCCTCTCATagaatccct
cctttgcatgatcatc
kinA-his-down ATGAGAGGATCACATCATCATCATCATCATGGCGCAatggaac
aggatacgcagcatgt
narG-P1 GCA TCG CCC ACG CCC CGT G
narG-P2 CAA TTC GCC CTA TAG TGA GTC GTG AGA GTT CAC TCC
TTC CGA GTC
narG-P3 CCA GCT TTT GTT CCC TTT AGT GAG GCG GGC AGA TCC
TCT GGG GT
narG-P4 GTG GAT AGA GTA AAT TTG ATA C
cta-P1 GAGCCCGCAATAATCAGTGA
cta-P2 CAA TTC GCC CTA TAG TGA GTC GTG AGA GCT TTT AAT
GCT TTA TTC AT
cta-P3 CCA GCT TTT GTT CCC TTT AGT GAG CGT TAT GAA TCC
AGG CAA TCA
cta-P4 AACAGTTATTAAGGTATTGGCTGAAGC
kinB-GFP-F CTG GAATCCATA CGA AAG GAT TTC ACA CGA ATG
kinB-GFP-R CTGAGATCTAAGATCAGCGGGAAG
KinB-start GAA TTC AAG AAT GCT AAA AAA AGG AAA AGT TAT TTT
AGA G
KinB-TM-12-R AT AAA GCA GAT GTG CAG AAG ATA GTC
KinB-TM-12-F GAC TAT CTT CTG CAC ATC TGC TTT ATATA CAA TAC
GGC CTT CAG ATG
KinB-end GGATCC TAG CAA ATC GAT TGG AAC TTT AAT AGA ATA
AAT
KinB-TM-34-R GAC TGT AAG TCC GGA AGC GGT ACT
KinB-TM-34-F AGT ACC GCT TCC GGA CTT ACA GTCTTT GAA CTG CTT
TTT TAC GAA CCT TCC GCA ATG
KinB-TM-56-R AGT GAA AAC AAA CAT TGC GGA AGG TTC
KinB-TM-56-F GAA CCT TCC GCA ATG TTT GTT TTC ACTTCC AGT GTT
CTT CTA TTA AGC ATC TAT ATT
Figure S1. The effect of nitrate on colony morphology is due to anaerobic
respiration. (A) The wild strain 3610 and the ΔnarG-H (AE325) mutant were grown on
solid biofilm-inducing medium in a 5% oxygen atmosphere for 3 days in an
environmental chamber in the absence or presence of nitrate (20 mM). While wrinkling
was reduced in the wild type in the presence of nitrate, the mutant remained wrinkled.
(B) Matrix gene expression was decreased in the presence of nitrate but the decrease
depended on the narG-H genes. Gene expression was monitored using a PtapA-lacZ-
containing strain (RL4582) and its ΔnarG-H derivative (KG24).
- nitrate + nitrate
Figure S2. The NAD+/NADH ratio increases with increasing oxygen concentration.
The wild strain 3610 was grown on solid biofilm-inducing medium containing 50 µM
iron choloride at the indicated oxygen concentrations for 48 hours in an environmental
chamber. Pairs of colonies were harvested and extracted under either acidic or basic
conditions for measurements of NAD+ and NADH, respectively. The NAD
+/NADH
ratios are the average for six colonies.
0
0.4
0.8
1.2
1.6
5 25 35
NA
D+/
NA
DH
rat
io
Oxygen (%)
Figure S3. The ldh transcription is rex dependent.
Shown is expression of Pldh-lacZ in a wild strain carrying the fusion (AE276), and its rex
derivative (AE277). Cells were grown on solid biofilm-inducing medium containing the
indicated concentration of iron for 48 hr. After 48 hr, colonies were harvested, and Pldh
activity was measured. The results are the average for three colonies for each iron
concentration. Error bars represent the standard deviation between three independent
experiments.
0
10
20
30
40
50
60
70
80
90
100
5 50 250
pld
h-l
acz(
MU
)
Iron concentration (µM)
WT
rex
Figure S4. Respiration suppresses matrix gene expression in kinB and cta qcr
dependent manner. The wild strain 3610 was grown on solid biofilm-inducing medium
containing 250 µM iron choloride at the indicated oxygen concentrations for 2 days in an
environmental chamber. The medium contained 20 mM of KNO3 for the colony grown
on nitrate. The figure shows that matrix gene expression decreased with increasing
oxygen concentration in the wild type strain, while not changing dramatically in a kinB
mutant and in a cytochrome double mutant cta qcr. Gene expression was monitored
using a PtapA-lacZ-containing strain (RL4582), and on its dervatives. Colors indicate
oxygen concentration.
0
200
400
600
800
1000
1200
1400
1600
1800
2000
WT kinB kinA kinB cta qcr
Pta
pA
-lac
Z (M
U)
35%
22%
5%
Figure S5. The sinR mutation restores wrinkling to cytochrome mutants. Shown are
top-down images of colonies of the wild type (NCBI 3610) and mutant strains ∆qcrABC
ctaC (KG11), ∆qcrABC sinR (AE431), ∆ctaC sinR (AE430) and ∆qcrABC ctaC sinR
(AE432) grown on solid MSgg medium for three days at 30°C. The colony wrinkliness of
the sinR mutant strains demonstrates that the effect of the qcrABC and ctaC mutations
could be reversed by derepression of matrix gene expression.
Figure S6. The KinA PASA domain is required for NAD+ binding. HPLC
chromatogram of NAD+ associated with wild-type KinA, KinA-PASA and KinA-
PASC. NAD+ was detected associated with wild-type and the KinA-PASC but not
with KinA-PASA.
0
100
200
300
400
500
600
700
11 12 13
t(min)
KinA
KinA-DPAS1
PAS3
KinA
KinA-PASA
KinA-PASC
A2
60 (
mA
U)