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)