Supplementary Materials for

Identification of Fic-1 as an enzyme that inhibits bacterial DNA

replication by AMPylating GyrB, promoting filament formation

Canhua Lu, Ernesto S. Nakayasu, Li-Qun Zhang,* Zhao-Qing Luo*

*Corresponding author. E-mail: zhanglq@cau.edu.cn (L.-Q.Z.); luoz@purdue.edu (Z.-Q.L.)

Published 26 January 2016, Sci. Signal. 9, ra11 (2016)

DOI: 10.1126/scisignal.aad0446

The PDF file includes:

Fig. S1. Diagrams of the three fic genes present in P. fluorescens strain 2P24.

Fig. S2. Alignment of Fic proteins similar to Fic-1 from other bacteria.

Fig. S3. Fic-1 affects the DNA yield of plasmids from different incompatibility

groups.

Fig. S4. Mutants of Fic-2 and Fic-3 defective in the intramolecular inhibitory motif

did not affect plasmid DNA yield.

Fig. S5. Fic-1 inhibits the growth of both E. coli and P. fluorescens.

Fig. S6. Fic-1 AMPylates the N-terminal domain of GyrB (1–200).

Fig. S7. Fic-1 AMPylates GyrB from P. fluorescens on Tyr111.

Fig. S8. The Fic-1Y5A mutant is defective in self-AMPylation and has low activity

against GyrB.

Fig. S9. AntF inhibits the activity of Fic-1 by direct interactions.

Table S1. Proteins analyzed for interactions with Fic-1.

Table S2. Bacterial strains, plasmids, and primers used in the study.

Reference (60)

www.sciencesignaling.org/cgi/content/full/9/412/ra11/DC1

Fig. S1. Diagrams of the three fic genes present in P. fluorescens strain 2P24. The lengths of the proteins are indicated

by numbers and the blowouts in light yellow background indicate the sequences of the predicted Fic domain. The positions

and sequences of the putative inhibitory domains present in a separate gene (for fic-1) or within the Fic protein are shown in

light blue background.

Fig. S2. Alignment of Fic proteins similar to Fic-1 from other bacteria. Ten Fic proteins from the indicated bacteria

identified by PSI-BLAST searches were aligned by the Clustal W 2.0 program. The Fic domain starting with the catalytic

histidine is highlighted in red. Note the diversity of the bacteria and the sequence disparity in regions beyond the Fic

domain. Predicted Fic proteins from indicated microorganisms were aligned with CLUSTAL 2.1. The proteins appear to

have two domains with the catalytic Fic domain localized to their C-termini. Identical residues are indicated by letters in

black background with the Histidine residue critical for catalysis marked in red background. Conserved residues are

highlighted in yellow background. The positions of the residues in these proteins and the number of residues omitted in the

alignments are indicated by numbers. The proteins included are as follows: Pseudomonas fluorescens strain 2P24

(gi|KT020755); Azotobacter chroococcum (gi|749168471); Vibrio parahaemolyticus (gi|646572781); Escherichia coli

(gi|1789761); Salmonella enterica (gi|538466839); Bartonella queenslandensis (gi|748190205); Streptococcus pneumoniae

(gi|321157304); Mycobacterium tuberculosis (gi|13883608); Staphylococcus aureus (gi|678252119); Neisseria meningitidis

(gi|81707273).

Fig. S3. Fic-1 affects the DNA yield of plasmids from different incompatibility groups. DNA fragments harboring fic-1

or fic-1H135A were inserted into plasmid pHSG399 and the resulting constructs were co-transformed with pRK415 (IncP)

(1), pDSK519 (IncQ) (1) and pBBR1MCS-2 (unknown Inc) (2), respectively. The resulting bacterial strains were used for

plasmid DNA purification. Note that compared to the vector or plasmids containing the fic-1H135A mutant, the DNA yield of

the plasmids co-existing with the one harboring wild-type fic-1 is lower.

Fig. S4. Mutants of Fic-2 and Fic-3 defective in the intramolecular inhibitory motif did not affect plasmid DNA

yield. Derivatives of pHSG399 carrying the wild type or the indicated mutants of Fic-2 (upper panel) and Fic-3 (lower

panel) were isolated from identical amounts of E. coli. DNA digested with HindIII and XbaI was separated by agarose gels

for imaging. An identical set of samples were probed for isocitrate dehydrogenase (ICDH) as a loading control.

Fig. S5. Fic-1 inhibits the growth of both E. coli and P. fluorescens. A. E. coli strain BL21(DE3) was transformed with

pET-Sumo (a), its derivatives harboring fic-1(b) and fic-1H135A (c), respectively. After spreading transformed cells onto

medium with appropriate antibiotic, the plate was incubated at 37oC for 16 h before imaging. B. Single colonies from the

plate in A were patched onto LB agar and cells suspended in PBS were diluted into LB broth to identical densities (OD600).

Growth was monitored by measuring OD600 (left panel) or by determining colony forming units (right panel) at 2 h

intervals. C. P. fluorescens strains harboring plasmids carrying the indicated genes were grown to saturation and the cells

were diluted 1:100 in ABM medium. Arabinose was added to 0.2% and growth of the cells was monitored by measuring

OD600 and colony forming units at the indicated time points. Note that very little growth was detected in the first 28 h in the

strain expressing the wild type Fic-1.

Fig. S6. Fic-1 AMPylates the N-terminal domain of GyrB (1–200). Recombinant GyrB, GyrB1-200 and GyrB201-804 were

each incubated with Fic-1 or the Fic-1H135A mutant proteins and 32P-α-ATP. Samples resolved by SDS-PAGE were

subjected to autoradiograph (left panel) or stain with Coomassie blue (right panel). Note that AMPylation signals were

detected only in samples containing the wild type Fic-1 and full-length GyrB or GyrB1-200.

Fig. S7. Fic-1 AMPylates GyrB from P. fluorescens on Tyr111. Reactions were established with 32P-α-ATP, Fic-1, the

Fic-1H135A mutant, GyrB and its Y111A mutant as indicated. After incubation at 35oC for 30 min, samples were resolved by

SDS-PAGE. The gels were dried and 32P-α-AMP-labeled proteins were detected by autoradiograph for 2 min (upper panel).

The same gel was stained with Coomassie blue to detect the levels of proteins used in the reactions (lower panel). The 1st

lane contain protein size markers (KDa). Note that residues H135 in Fic-1 and Tyr111 in GyrB are essential for the

modification.

Fig. S8. The Fic-1Y5A mutant is defective in self-AMPylation and has low activity against GyrB. The indicated

amounts of Fic-1 or Fic-1Y5A were incubated with GyrB or by alone in the presence of 32P-α-ATP for 30 min at 35oC. Self-

AMPylation or AMPylation of GyrB was detected by autoradiography (upper panel). Total protein in the reactions was

detected by Coomassie staining (lower panel). Note that self-AMPylation was readily detectable in reactions containing 22

pmol Fic-1 but was not detected in those containing 440 pmol of Fic-1Y5A.

Fig. S9. AntF inhibits the activity of Fic-1 by direct interactions. A. Plasmid DNA was isolated from equal amounts of

E. coli strains harboring the indicated plasmids. Equal amounts of DNA samples digested with restriction enzymes BamHI

and SalI were resolved by agarose gel electrophoresis. Note that the amount of DNA from cells harboring both fic-1 and the

α-inhibitor was comparable to that from the mutant, and each was higher than that from cells carrying the wild type gene.

B. Fic-1 and the α-inhibitor were reciprocally fused to the T18 or the T25 fragment of adenylate cyclase and the resulting

plasmids were co-transformed into E. coli strain BTH101. A strain harboring plasmids expressing the Zipper fusions was

used as a positive control whereas a strain that contains T18::Fic-1 and the control vector pUT18C, served as controls. Cells

diluted from 100-l of saturated cultures were used for-galactosidase assay. The results shown are from one

representative experiment done in triplicate. Similar results were obtained in at least three independent experiments.

Table S1. Proteins analyzed for interactions with Fic-1

EcoGene

Accession Number Gene Description

EG10235 dnaA DNA synthesis initiator and global transcription regulator

EG10236 dnaB Replicative DNA helicase

EG10237 dnaC DNA biosynthesis, helicase DnaB loader

EG10238 dnaE DNA polymerase III, alpha subunit

EG10239 dnaG Primase for DNA replication

EG10240 dnaJ DnaK co-chaperone HSP40

EG10241 dnaK Hsp70 molecular chaperone, heat-inducible

EG10242 dnaN DNA polymerase III sliding clamp beta subunit

EG10243 dnaQ DNA polymerase III epsilon subunit

EG10244 dnaT Primasomal protein i

EG10245 dnaX DNA polymerase III holoenzyme, tau and gamma ATPase subunits

EG10317 fis Transcriptional activator for rRNA operons, bends DNA

EG10423 gyrA DNA gyrase, subunit A

EG10424 gyrB DNA gyrase, subunit B

EG10440 ihfA Integration Host Factor (IHF), alpha subunit

EG10441 ihfB Integration Host Factor (IHF), beta subunit

EG10466 hupA Histone-like protein HU-alpha, HU-2

EG10467 hupB Histone-like protein HU-beta, HU-1

EG10490 argP Inhibitor of chromosome initiation, transcriptional activator

EG10510 rpoS RNA polymerase subunit, stress and stationary phase sigma S

EG10534 ligA DNA ligase A, NAD(+)-dependent

EG10686 parC Topoisomerase IV, subunit A, ATP-dependent, type II

EG10687 parE Topoisomerase IV, subunit B, ATP-dependent, type II

EG10746 polA DNA polymerase I

EG10746 polA DNA polymerase I

EG10747 polB DNA polymerase II, capable of translesion synthesis

EG10763 priA Primosome factor Y, also called protein n'

EG10764 priB Primosomal protein n

EG10765 priC Primosomal protein n

EG10774 prs Phosphoribosylpyrophosphate synthase

EG10823 recA Multifunctional DNA recombination and repair protein

EG10828 recF Recombination and repair

EG10837 rep

ATP-dependent DNA helicase Rep, involved in DNA replication

Table S1 continued

EG10860 rnhA RNase HI

EG10860 rnhA RNase HI

EG10861 rnhB Degrades RNA of DNA-RNA hybrids

EG10893 rpoA RNA polymerase, alpha subunit

EG10894 rpoB RNA polymerase, beta subunit

EG10895 rpoC RNA polymerase, beta' subunit

EG10896 rpoD RNA polymerase subunit, sigma 70, initiates transcription

EG10897 rpoH RNA polymerase subunit, sigma 32, heat shock transcription

EG10898 rpoN RNA polymerase subunit, sigma 54

EG10899 rpoZ RNA polymerase subunit, omega subunit

EG10899 rpoZ RNA polymerase subunit, omega subunit

EG10951 ytjB SMP_2 family predicted membrane-anchored periplasmic protein

EG10976 ssb Single-stranded DNA-binding protein

EG11013 topA Topoisomerase I

EG11014 topB Topoisomerase III

EG11033 tsf Translation_elongation_factor_EF-Ts.seq

EG11036 tufA Translation elongation factor EF-Tu 1

EG11037 tufB Translation_elongation_factor_EF-Tu_2.seq

EG11083 rapA RNA polymerase-associated, ATP-dependent RNA translocase

EG11128 yciH Mimics some initiation factor functions, binds 30S subunit

EG11412 holA DNA polymerase III, delta subunit

EG11413 holC DNA polymerase III, chi subunit

EG11414 holD DNA polymerase, psi subunit, clamp loader complex subunit

EG11415 dps Stress-induced Fe-binding and storage protein

EG11500 holB DNA polymerase III, delta' subunit

EG11505 holE DNA polymerase III, theta subunit

EG11507 rlmE 23S rRNA U2552 2'-O-ribose methyltransferase, SAM-dependent

EG11608 clsA Cardiolipin synthase 1

EG11892 sbmC DNA gyrase inhibitor

EG12080 recX Blocks RecA filament extension

EG12164 ytjC Phosphatase, function unknown

EG12197 seqA Multi-faceted genome stability factor

EG12216 tusA Sulfurtransferase, 2-thiolation of mnm(5)s(2)U34-tRNA

EG12314 yacG DNA gyrase inhibitor

EG12780 diaA DnaA-binding protein

EG12795 obgE DNA-binding GTPase involved in cell partioning and DNA repair

EG12855 matP Ter macrodomain organizer matS-binding protein

Table S1 continued

EG13141 dinB DNA polymerase IV, capable of translesion synthesis

EG13671 clsB Cardiolipin synthase 2

EG13875 clsC Cardiolipin synthase 3, active in stationary phase

EG13992 cedA DNA-binding protein that modulates cell division

EG14201 hda Required for regulatory inactivation of DnaA

EG14207 der Multicopy suppressor of ftsJ, GTPase, ribosome biogenesis

EG14398 ytjA Function unknown

EG20005 oriC Origin of chromosomal DNA replication, bidirectional

Table S2. Bacterial strains, plasmids, and primers used in the study

Name Relevant characteristics References or sources

Pseudomonas fluorescens

2P24 Apr; Wide type 33

PM933 Apr; fic-1 gene in-frame deletion in 2P24 This study

PM936 Apr; antF gene in-frame deletion in 2P24 This study

PM937 Apr; antF and fic-1 double genes in-frame deletion in 2P24 This study

PM938 Apr; sulA gene in-frame deletion in 2P24 This study

Escherichia coli

DH5α F-，φ80dlacZΔM15，Δ(lacZYA-argF)U169，deoR，recA1，endA1，hsdR17(rk-，mk+)，phoA

，supE44，λ-，thi-1，gyrA96，relA1. Our collection

BTH101 F-, cya-99, araD139, galE15, galK16, rpsL1 (Str r), hsdR2, mcrA1, mcrB1. 34

BL21(DE) F-，ompT， hsdS（rBB-mB－），gal， dcm（DE3）. Novagen

S171 (λ-π) Donor strain for biparental mating Our collection

MG1655 Wide type Dr. Laszlo Csonka (Purdue University)

HB32 F+, sulA1 The Coli Genetic Stock Center

WU38 F-, lon-11, sulA1, tyrA14, recA1 The Coli Genetic Stock Center

Plasmid

pBAD22 Tight regulation of protein expression by vector containing the arabinose PBAD promoter 53

pBBR1MCS-2 Kmr; pBBR1MCS-2 containing pBluescript II KS-lacZα; the broad host range cloning vector 54

pCL008 Kmr; pBBR1MCS-2 containing the arabinose PBAD promoter from pBAD22 vector This study

pDSK519 Kmr; broad host range vector 60

pET22b(+) Apr; expression vector Novagen

pETSUMO Kmr; expression vector Champion

pGEX-6P1 Amr; expression of GST-fusion protein

pHSG399 Cmr; ColE1 origin TaKaRa

pRK415 Tcr; IncP1 replicon, polylinker of pUC19, Mob+ 60

pEX18-KCL Kmr; suicide vector for inframe-deletion gene This study

Note: Apr, Kmr, Cmr , and Tcr indicate resistance to ampicillin, kanamycin, chloramphenicol and tetracycline, respectively.

Primers used

Name Sequence Purpose

Ec_argP-Xba I-F ACTCTAGAGAAACGCCCGGACTACAGAAC 5' primer for cloning argP gene onto pKT25

Ec_argP-EcR I-R ATGAATTCTTAATCCTGACGAAGGACTTTG 3’ primer for cloning argP gene onto pKT25

Ec_cedA-Xba I-F ACTCTAGAGAAGAAACCGCTCCGCCAGC 5’ primer for cloning cedA gene onto pKT25

Ec_cedA-EcR I-R ATGAATTCTTACTCAGTCACTTCCCCTTC 3’ primer for cloning cedA gene onto pKT25

Ec_clsA-Xba I-F ATTCTAGAGACAACCGTTTATACGTTGGTG 5’ primer for cloning clsA gene onto pKT25

Ec_clsA-Kpn I-R ATTGGTACCTTACAGCAACGGACTGAAGAAG 3’ primer for cloning clsA gene onto pKT25

Ec_clsB-Xba I-F ACTCTAGAGAAATGTAGCTGGCGCGAAGG 5’ primer for cloning clsB gene onto pKT25

Ec_clsB-EcR I-R ATGAATTCAGGGTTTTACCCCCGTG 3’ primer for cloning clsB gene onto pKT25

Ec_clsC-Xba I-F ACTCTAGAGCCCCGGCTGGCGAGCGC 5’ primer for cloning clsC gene onto pKT25

Ec_clsC-EcR I-R ATGAATTCTTACAATAACCATTCCACGGGC 3’ primer for cloning clsC gene onto pKT25

Ec_der-Xba I-F ACTCTAGAGGTACCTGTGGTCGCGCTTG 5’ primer for cloning der gene onto pKT25

Ec_der-EcR I-R ATGAATTCTTATTTATTTTTCTTGATGTGCTTC 3’ primer for cloning der gene onto pKT25

Ec_diaA-Xba I-F ACTCTAGAGCAAGAAAGAATTAAAGCTTGCTTC 5’ primer for cloning diaA gene onto pKT25

Ec_diaA-EcR I-R ATGAATTCTTAATCATCCTGGTGAGGGAAAAG 3’ primer for cloning diaA gene onto pKT25

Ec_dinB-Xba I-F ACTCTAGAGCGTAAAATCATTCATGTGG 5’ primer for cloning dinB gene onto pKT25

Ec_dinB-EcR I-R TAGAATTCATAATCCCAGCACCAGTTG 3’ primer for cloning dinB gene onto pKT25

Ec_dnaA-Xba I-F ATTCTAGAGTCACTTTCGCTTTGGCAGCAG 5’ primer for cloning dnaA gene onto pKT25

Ec_dnaA-Kpn I-R ATTGGTACCTTACGATGACAATGTTCTG 3’ primer for cloning dnaA gene onto pKT25

Ec_dnaB-Xba I-F ATTCTAGAGGCAGGAAATAAACCCTTCAAC 5’ primer for cloning dnaB gene onto pKT25

Ec_dnaB-Kpn I-R ATTGGTACCTTATTCGTCGTCGTACTGCGGC 3’ primer for cloning dnaB gene onto pKT25

Ec_dnaC-Xba I-F ATTCTAGAGAAAAACGTTGGCGACCTGATGC 5’ primer for cloning dnaC gene onto pKT25

Ec_dnaC-EcR I-R ATTGAATTCTTAATACTCTTTACCTGTTACCCG 3’ primer for cloning dnaC gene onto pKT25

Ec_dnaE-Xba I-F ATTCTAGAGTCTGAACCACGTTTCGTACACC 5’ primer for cloning dnaE gene onto pKT25

Ec_dnaE-Kpn I-R ATTGGTACCTTAGTCAAACTCCAGTTCCACC 3’ primer for cloning dnaE gene onto pKT25

Ec_dnaG-Xba I-F ATTCTAGAGGCTGGACGAATCCCACGCG 5’ primer for cloning dnaG gene onto pKT25

Table S2 continued

Ec_dnaG-Kpn I-R ATTGGTACCTCACTTTTTCGCCAGCTCCTG 3’ primer for cloning dnaG gene onto pKT25

Ec_dnaN-Xbal I-F ACTCTAGAGAAATTTACCGTAGAACGTGAG 5’ primer for cloning dnaN gene onto pKT25

Ec_dnaN-EcR I-R ATGAATTCTTACAGTCTCATTGGCATGAC 3’ primer for cloning dnaN gene onto pKT25

Ec_dnaJ-Xba I-F ACTCTAGAGGCTAAGCAAGATTATTACGAG 5’ primer for cloning dnaJ gene onto pKT25

Ec_dnaJ-EcR I-R ATGAATTCTTAGCGGGTCAGGTCGTCAA 3’ primer for cloning dnaJ gene onto pKT25

Ec_dnaK-Xba I-F ACTCTAGAGGGTAAAATAATTGGTATCGACC 5’ primer for cloning dnaK gene onto pKT25

Ec_dnaK-Kpn I-R ATTGGTACCTTATTTTTTGTCTTTGACTTCTTC 3’ primer for cloning dnaK gene onto pKT25

Ec_dnaQ-Xba I-F ACTCTAGAGAGCACTGCAATTACACGCC 5’ primer for cloning dnaQ gene onto pKT25

Ec_dnaQ-EcR I-R ATGAATTCTTATGCTCGCCAGAGGCAAC 3’ primer for cloning dnaQ gene onto pKT25

Ec_dnaT-Xba I-F ATTCTAGAGTCTTCCAGAGTTTTGACCCCG 5’ primer for cloning dnaT gene onto pKT25

Ec_dnaT-Kpn I-R ATTGGTACCTTACCCTCTGAATCCTGGTGG 3’ primer for cloning dnaT gene onto pKT25

Ec_dnaX-Xba I-F ATTCTAGAGAGTTATCAGGTCTTAGCCC 5’ primer for cloning dnaX gene onto pKT25

Ec_dnaX-EcR I-R ATGAATTCAAATGGGGCGGATACTTTC 3’ primer for cloning dnaX gene onto pKT25

Ec_dps-Xba I-F ACTCTAGAGAGTACCGCTAAATTAGTTAAATC 5’ primer for cloning dps gene onto pKT25

Ec_dps-EcR I-R ATGAATTCTTATTCGATGTTAGACTCGATAAAC 3’ primer for cloning dps gene onto pKT25

Ec_fis-Xba I-F ACTCTAGAGTTCGAACAACGCGTAAATTCTG 5’ primer for cloning fis gene onto pKT25

Ec_fis-EcR I-R ATGAATTCTTAGTTCATGCCGTATTTTTTC 3’ primer for cloning fis gene onto pKT25

Ec_gltD-Xba I-F ATTCTAGAGAGTCAGAATGTTTATCAATTTATC 5’ primer for cloning gltD gene onto pKT25

Ec_gltD-Kpn I-R ATTGGTACCTTAAACTTCCAGCCAGTTCATAA 3’ primer for cloning gltD gene onto pKT25

Ec_gyrA-Xba I-F ACTCTAGAGAGCGACCTTGCGAGAGAAAT 5’ primer for cloning gyrA gene onto pKT25

Ec_gyrA-EcR I-R ATGAATTCTTATTCTTCTTCTGGCTCGTCG 3’ primer for cloning gyrA gene onto pKT25

Ec_gyrB-Xba I-F ACTCTAGAGTCGAATTCTTATGACTCCTCC 5’ primer for cloning gyrB gene onto pKT25

Ec_gyrB- Kpn I-R ATGGTACCTTAAATATCGATATTCGCCGCTTT 3’ primer for cloning gyrB gene onto pKT25

Ec_hda-Xba I-F ACTCTAGAGAACACACCGGCACAGCTCTC 5’ primer for cloning hda gene onto pKT25

Ec_hda-Kpn I-R ATTGGTACCTACAACTTCAGAATTTCTTTCAC 3’ primer for cloning hda gene onto pKT25

Ec_holA-Xba I-F ACTCTAGAGATTCGGTTGTACCCGGAACA 5’ primer for cloning holA gene onto pKT25

Table S2 continued

Ec_holA-EcR I-R ATGAATTCAACCGTCGATAAATACGTC 3’ primer for cloning holA gene onto pKT25

Ec_holC -Xba I-F ACTCTAGAGAAAAACGCGACGTTCTACCTTC 5’ primer for cloning holC gene onto pKT25

Ec_holC-EcR I-R ATGAATTCTTATTTCCAGGTTGCCGTATTC 3’ primer for cloning holC gene onto pKT25

Ec_holD-Xba I-F ACTCTAGAGACATCCCGACGAGACTGGC 5’ primer for cloning holD gene onto pKT25

Ec_holD-EcR I-R ATGAATTCAGTCGTTTCGAGGGAAGAA 3’ primer for cloning holD gene onto pKT25

Ec_holB-Xba I-F ACTCTAGAGAGATGGTATCCATGGTTACGAC 5’ primer for cloning holB gene onto pKT25

Ec_holB-EcR I-R ATGAATTCTTAAAGATGAGGAACCGGTAGC 3’ primer for cloning holB gene onto pKT25

Ec_holE-Xba I-F ACTCTAGAGCTGAAGAATCTGGCTAAACTGG 5’ primer for cloning holE gene onto pKT25

Ec_holE-Kpn I-R ATTGGTACCTTATTTAAGTTTGGGCTCGTAAG 3’ primer for cloning holE gene onto pKT25

Ec_hupA-Xba I-F ACTCTAGAGAACAAGACTCAACTGATTGATG 5’ primer for cloning hupA gene onto pKT25

Ec_hupA-EcR I-R ATGAATTCTTACTTAACTGCGTCTTTCAGTG 3’ primer for cloning hupA gene onto pKT25

Ec_hupB-Xba I-F ACTCTAGAGAATAAATCTCAATTGATCGAC 5’ primer for cloning hupB gene onto pKT25

Ec_hupB-EcR I-R ATGAATTCTTAGTTTACCGCGTCTTTCAGT 3’ primer for cloning hupB gene onto pKT25

Ec_ihfA-Xba I-F ACTCTAGAGGCGCTTACAAAAGCTGAAATG 5’ primer for cloning ihfA gene onto pKT25

Ec_ihfA-EcR I-R ATGAATTCTTACTCGTCTTTGGGCGAAGC 3’ primer for cloning ihfA gene onto pKT25

Ec_ihfB-Xba I-F ACTCTAGAGACCAAGTCAGAATTGATAGAAA 5’ primer for cloning ihfB gene onto pKT25

Ec_ihfB-EcR I-R ATGAATTCTTAACCGTAAATATTGGCGCG 3’ primer for cloning ihfB gene onto pKT25

Ec_ligA-Xba I-F ACTCTAGAGGAATCAATCGAACAACAACTGAC 5’ primer for cloning ligA gene onto pKT25

Ec_ligA-EcR I-R ATGAATTCAGCTACCCAGCAAACGC 3’ primer for cloning ligA gene onto pKT25

Ec_matP-Xba I-F ACTCTAGAGAAATATCAACAACTTGAAAATCTTG 5’ primer for cloning matP gene onto pKT25

Ec_matP-EcR I-R ATGAATTCTTATTCCTTACCCAGCAATGC 3’ primer for cloning matP gene onto pKT25

Ec_obgE-Xba I-F ACTCTAGAGAAGTTTGTTGATGAAGCATCGAT 5’ primer for cloning obgE gene onto pKT25

Ec_obgE-Kpn I-R ATTGGTACCTTAACGCTTGTAAATGAACTCAAC 3’ primer for cloning obgE gene onto pKT25

Ec_parC-Xba I-F ACTCTAGAGAGCGATATGGCAGAGCGC 5’ primer for cloning parC gene onto pKT25

Ec_parC-EcR I-R ATGAATTCTTACTCTTCGCTATCACCGC 3’ primer for cloning parC gene onto pKT25

Ec_parE-Xba I-F ACTCTAGAGACGCAAACTTATAACGCTGATG 5’ primer for cloning parE gene onto pKT25

Table S2 continued

Ec_parE-EcR I-R ATGAATTCTTAAACCTCAATCTCCGCCATG 3’ primer for cloning parE gene onto pKT25

Ec_polA-Xba I-F ACTCTAGAGGTTCAGATCCCCCAAAATCC 5’ primer for cloning polA gene onto pKT25

Ec_polA-EcR I-R ATGAATTCTTAGTGCGCCTGATCCCAG 3’ primer for cloning polA gene onto pKT25

Ec_polB-Xba I-F ACTCTAGAGGCGCAGGCAGGTTTTATCTTAAC 5’ primer for cloning polB gene onto pKT25

Ec_polB-EcR I-R ATGAATTCAAAATAGCCCAAGTTGCCC 3’ primer for cloning polB gene onto pKT25

Ec_priA-Xba I-F ACTCTAGAGCCCGTTGCCCACGTTGCC 5’ primer for cloning priA gene onto pKT25

Ec_priA-EcR I-R ATGAATTCTTAACCCTCAATCGGATCAAC 3’ primer for cloning priA gene onto pKT25

Ec_priB-Xba I-F ACTCTAGAGACCAACCGTCTGGTGTTGTC 5’ primer for cloning priB gene onto pKT25

Ec_priB-Kpn I-R ATGGTACCTAGTCTCCAGAATCTATCAATTC 3’ primer for cloning priB gene onto pKT25

Ec_priC-Xba I-F ACTCTAGAGAAAACCGCCCTGCTGCTGG 5’ primer for cloning priC gene onto pKT25

Ec_priC-EcR I-R ATGAATTCTAGCGGGTTAAACGCGCTA 3’ primer for cloning priC gene onto pKT25

Ec_prs-Xba I-F TATCTAGAGCCTGATATGAAGCTTTTTGCTG 5’ primer for cloning prs gene onto pKT25

Ec_prs-EcR I-R ATGAATTCTTAGTGTTCGAACATGGCAGAG 3’ primer for cloning prs gene onto pKT25

Ec_rapA-Xba I-F ACTCTAGAGCCTTTTACACTTGGTCAACGC 5’ primer for cloning rapA gene onto pKT25

Ec_rapA-EcR I-R ATGAATTCTTACTGATGCGTTACAACGATC 3’ primer for cloning rapA gene onto pKT25

Ec_recA-Xba I-F ATTCTAGAGGCTATCGACGAAAACAAACAG 5’ primer for cloning recA gene onto pKT25

Ec_recA-Kpn I-R ATTGGTACCTTAAAAATCTTCGTTAGTTTCTGC 3’ primer for cloning recA gene onto pKT25

Ec_recF-Xba I-F ATTCTAGAGTCCCTCACCCGCTTGTTGATC 5’ primer for cloning recF gene onto pKT25

Ec_recF-EcR I-R ATGAATTCTTAATCCGTTATTTTACCCTTTTCC 3’ primer for cloning recF gene onto pKT25

Ec_recX-Xba I-F ATTCTAGAGACAGAATCAACATCCCGTCG 5’ primer for cloning recX gene onto pKT25

Ec_recX-EcR I-R ATGAATTCAGTCGGCAAAATTTCGCC 3’ primer for cloning recX gene onto pKT25

Ec_rep-Xba I-F ACTCTAGAGCGTCTAAACCCCGGCCAAC 5’ primer for cloning rep gene onto pKT25

Ec_rep-Kpn I-R TATGGTACCTTATTTCCCTCGTTTTGCCGC 3’ primer for cloning rep gene onto pKT25

Ec_rlmE-Xba I-F ACTCTAGAGACAGGTAAGAAGCGTTCTGC 5’ primer for cloning rlmE gene onto pKT25

Ec_rlmE-Kpn I-R ATTGGTACCTTAGGGTTTACGCCCGGTC 3’ primer for cloning rlmE gene onto pKT25

Ec_rnhA-Xbal I-F ATTCTAGAGCTTAAACAGGTAGAAATTTTCACC 5’ primer for cloning rnhA gene onto pKT25

Table S2 continued

Ec_rnhA-EcR I-R ATGAATTCTTAAACTTCAACTTGGTAGCCTG 3’ primer for cloning rnhA gene onto pKT25

Ec_rnhB-Xba I-F ACTCTAGAGATCGAATTTGTTTATCCGCACAC 5’ primer for cloning rnhB gene onto pKT25

Ec_rnhB-EcR I-R ATGAATTCAGGACGCAAGTCCCAGTG 3’ primer for cloning rnhB gene onto pKT25

Ec_rpoA-Xba I-F ATTCTAGAGCAGGGTTCTGTGACAGAGTTTC 5’ primer for cloning rpoA gene onto pKT25

Ec_rpoA-Kpn I-R ATTGGTACCTTACTCGTCAGCGATGCTTGC 3’ primer for cloning rpoA gene onto pKT25

Ec_rpoB-Pst I-F ATTCTGCAGGGTTTACTCCTATACCGAG 5’ primer for cloning rpoB gene onto pKT25

Ec_rpoB-Xba I-R ATTCTAGATTACTCGTCTTCCAGTTCG 3’ primer for cloning rpoB gene onto pKT25

Ec_rpoC-Xba I-F ATTCTAGAGAAAGATTTATTAAAGTTTCTGAAAGC 5’ primer for cloning rpoC gene onto pKT25

Ec_rpoC-Sac I-R ATGAGCTCTTACTCGTTATCAGAACCGCC 3’ primer for cloning rpoC gene onto pKT25

Ec_rpoD-Xba I-F ACTCTAGAGGAGCAAAACCCGCAGTCACAG 5’ primer for cloning rpoD gene onto pKT25

Ec_rpoD-EcR I-R ATGAATTCTTAATCGTCCAGGAAGCTACG 3’ primer for cloning rpoD gene onto pKT25

Ec_rpoH-Xba I-F ACTCTAGAGACTGACAAAATGCAAAGTTTAGC 5’ primer for cloning rpoH gene onto pKT25

Ec_rpoH-EcR I-R ATGAATTCTTACGCTTCAATGGCAGCACG 3’ primer for cloning rpoH gene onto pKT25

Ec_rpoN-Xba I-F ACTCTAGAGAAGCAAGGTTTGCAACTCAGG 5’ primer for cloning rpoN gene onto pKT25

Ec_rpoN-EcR I-R ATGAATTCAAACGAGTTGTTTACGCTGG 3’ primer for cloning rpoN gene onto pKT25

Ec_rpoS-Xba I-F ACTCTAGAGAGTCAGAATACGCTGAAAGTT 5’ primer for cloning rpoS gene onto pKT25

Ec_rpoS-EcR I-R ATGAATTCTTACTCGCGGAACAGCGCTTC 3’ primer for cloning rpoS gene onto pKT25

Ec_rpoZ-Xba I-F ATTCTAGAGGCACGCGTAACTGTTCAGG 5’ primer for cloning rpoZ gene onto pKT25

Ec_rpoZ-EcR I-R ATTGAATTCTTAACGACGACCTTCAGCAATAGC 3’ primer for cloning rpoZ gene onto pKT25

Ec_sbmC-Xba I-F ACTCTAGAGAACTACGAGATTAAGCAGGAAG 5’ primer for cloning sbmC gene onto pKT25

Ec_sbmC-EcR I-R ATGAATTCTTAGTGATGTTTTGGCTGCAC 3’ primer for cloning sbmC gene onto pKT25

Ec_seqA-Xba I-F ACTCTAGAGAAAACGATTGAAGTTGATGATGA 5’ primer for cloning seqA gene onto pKT25

Ec_seqA-EcR I-R ATGAATTCTTAGATAGTTCCGCAAACCTTC 3’ primer for cloning seqA gene onto pKT25

Ec_ssb-Xba I-F ACTCTAGAGGCCAGCAGAGGCGTAAACA 5’ primer for cloning ssb gene onto pKT25

Ec_ssb-EcR I-R ATGAATTCAGAACGGAATGTCATCATC 3’ primer for cloning ssb gene onto pKT25

Ec_topA-Xba I-F ACTCTAGAGGGTAAAGCTCTTGTCATCGTTG 5’ primer for cloning topA gene onto pKT25

Table S2 continued

Ec_topA-Kpn I-R ATGGTACCTTATTTTTTTCCTTCAACCCATTTG 3’ primer for cloning topA gene onto pKT25

Ec_topB-Xba I-F ACTCTAGAGCGGTTGTTTATTGCCGAAAAAC 5’ primer for cloning topB gene onto pKT25

Ec_topB-EcR I-R ATGAATTCTTACGCTATCGCCCCGCTTC 3’ primer for cloning topB gene onto pKT25

Ec_yacG-Xba I-F ACTCTAGAGTCAGAAACTATTACGGTGAATTG 5’ primer for cloning yacG gene onto pKT25

Ec_yacG-Kpn I-R ATTGGTACCTCACTGCTTTGGTTCTTCGC 3’ primer for cloning yacG gene onto pKT25

Ec_ycaC-Xba I-F ATTCTAGAGACCAAACCGTATGTTCGTC 5’ primer for cloning ycaC gene onto pKT25

Ec_ycaC-Kpn I-R ATTGGTACCTTATTTCTGCTTCGTTAACGTG 3’ primer for cloning ycaC gene onto pKT25

Ec_yciH-Xba I-F ACTCTAGAGAGTGATTCCAACAGCCGTCTG 5’ primer for cloning yciH gene onto pKT25

Ec_yciH-EcR I-R ATGAATTCTTAACCGCCTGCGAGTTTTAC 3’ primer for cloning yciH gene onto pKT25

Ec_ytjA-Xba I-F ACTCTAGAGTTTCGTTGGGGCATCATATTTC 5’ primer for cloning ytjA gene onto pKT25

Ec_ytjA-EcR I-R ATGAATTCTAGGGTCGTTTTCGGCCC 3’ primer for cloning ytjA gene onto pKT25

Ec_ytjB-Xba I-F ACTCTAGAGGCTCGCACAAAACTGAAATTC 5’ primer for cloning ytjB gene onto pKT25

Ec_ytjB-Kpn I-R ATTGGTACCTCACTCTTTTTTCTCGCTTTCT 3’ primer for cloning ytjB gene onto pKT25

Ec_ytjC-Xba I-F ACTCTAGAGTTACAGGTATACCTAGTCCGC 5’ primer for cloning ytjC gene onto pKT25

Ec_ytjC-EcR I-R ATGAATTCTTAACGCTGCAGCTCATCTAATG 3’ primer for cloning ytjC gene onto pKT25

Ec_tusA-Xba I-F ATTCTAGAGATGACCGATCTCTTTTCCAG 5’ primer for cloning tusA gene onto pKT25

Ec_tusA-EcR I-R ATTGAATTCTCAACCGCCTTTACGAATCAAA 3’ primer for cloning tusA gene onto pKT25

Ec_tufA-Xbal I-F ACTCTAGAGTCTAAAGAAAAATTTGAACGTAC 5’ primer for cloning tufA gene onto pKT25

Ec_tufA-EcR I-R ATGAATTCTTAGCCCAGAACTTTAGCAAC 3’ primer for cloning tufA gene onto pKT25

Ec_tufB-Xbal I-F ACTCTAGAGTCTAAAGAAAAGTTTGAACGTAC 5’ primer for cloning tufB gene onto pKT25

Ec_tufB-EcR I-R ATGAATTCTTAGCTCAGAACTTTTGCTACAAC 3’ primer for cloning tufB gene onto pKT25

Ec_tsf-Xbal I-F ACTCTAGAGGCTGAAATTACCGCATCCC 5’ primer for cloning tsf gene onto pKT25

Ec_tsf-Kpn I-R ATGGTACCTTAAGACTGCTTGGACATCGC 3’ primer for cloning tsf gene onto pKT25

Pf_fic-1- Sal I ATGTCGACAGTGACCTTCGCGGACGG 5’ primer for cloning fic-1 gene onto pHSG399

Pf_fic-1-BamH I ATTGGATCCGTAAGACGGTGAGCTTGG 3’ primer for cloning fic-1 gene onto pHSG399

Pf_Δfic-1-u-Xba I ATTCTAGAATCACCTGCGTGCTCTGC Primer for fic-1 gene knock out

Table S2 continued

Pf_Δfic-1-u-BamH I ATGGATCCGGGCTGAATTCGACGTTG Primer for fic-1 gene knock out

Pf_Δfic-1-d-BamH I ATGGATCCGCGGCTTATAACGGCGTC Primer for fic-1 gene knock out

Pf_Δfic-1-d-Kpn I ATTGGTACCAGTGATAGCGTCCTTGGC Primer for fic-1 gene knock out

Pf_fic-1-H135A-F GGGGCTACGACGTTGATGTCGG Primer for Fic-1 H135A mutation

Pf_fic-1-H135A-R CTTCCGCGAAGGCAATGGCC Primer for Fic-1 H135A mutation

Pf_antF-Fic-1-Sal I-F ATTGTCGACGAGTCGCCAGGCCACGATG 5’ primer for cloning operon of antF and fic-1

Pf_fic-1-BamH I-R ATTGGATCCTCAAGCTTGAATCGCCTGCC 3’ primer for cloning operon of antF and fic-1

Pf_fic-1-Tag-Nde I-F ATTCATATGCCTGACAAATATGGGGTCG 5’ primer for cloning fic-1 gene onto pET22b(+)

Pf_fic-1-Tag-Sal I-R ATTGTCGACAGCTTGAATCGCCTGCCCGA 3’ primer for cloning fic-1 gene onto pET22b(+)

Pf_fic-1-Xba I-F ATTCTAGAGCCTGACAAATATGGGGTCG 5’ primer for cloning fic-1 gene onto pKT25 or pUT18C

Pf_fic-1-Kpn I-R ATTGGTACCTCAAGCTTGAATCGCCTGCCC 3’ primer for cloning fic-1 gene onto pKT25 or pUT18C

Pf_fic-1-His-Xba I-F ATTCTAGAGCCTGACAAATATGGGGTCG 5’ primer for cloning fic-1 gene onto pDB-His

Pf_fic-1-His-Hind III-R ATTAAGCTTAATCGCCTGCCCGATACAC 3’ primer for cloning fic-1 gene onto pDB-His

Pf_fic-2-BamH I-F ATGGATCCAAGCCTTGCCCAATCTGATG 5’ primer for cloning fic-2 gene onto pHSG399

Pf_fic-2-Xba I-R ATTCTAGAAGCTCAACTCGCCACAACC 3’ primer for cloning fic-2 gene onto pHSG399

Pf_fic-3-Hind III-F ATTAAGCTTCTGCCGCCAGGTCGCCC 5’ primer for cloning fic-3 gene onto pHSG399

Pf_fic-3- Xba I ATTCTAGACAGGTTCGTTCTGTTCAGTCG 3’ primer for cloning fic-3 gene onto pHSG399

Pf_antF-Tag-Nde I-F ATTCATATGGGCAATGTCAGCCTTGAA 5’ primer for cloning antF gene onto pET22b(+)

Pf_antF-Tag-Sal I-D ATTGTCGACGGTTCGGGTCTGGGTGAAGG 3’ primer for cloning antF gene onto pET22b(+)

Pf_antF-BamH I-F ATGGATCCCGGCAATGTCAGCCTTGAAACC 5’ primer for cloning antF gene onto pKT25

Pf_antF-EcR I-R ATGAATTCAGGTTCGGGTCTGGGTGAAG 3’ primer for cloning antF gene onto pKT25

Pf_antF-Fic-1-KpnI-F ATTGGTACCGGGCAATGTCAGCCTTGAAACC 5’ primer for cloning the operon containing antF and fic-1 onto

pBAD22

Pf_fic-1-Tag-Sal I-R ATTGTCGACAGCTTGAATCGCCTGCCCGA 3’ primer for cloning the operon containing antF and fic-1 onto

pBAD22

Ec_recX-BamH I-F ATTGGATCCATGACAGAATCAACATCCCGT 5’ primer for cloning recX gene onto pETSUMO

Table S2 continued

Ec_recX-Sal I-R ATTGTCGACTCAGTCGGCAAAATTTCGCC 3’ primer for cloning recX gene onto pETSUMO

Ec_priA-BamH I-F ATTGGATCCATGCCCGTTGCCCACGTTG 5’ primer for cloning N-terminus of priA gene onto pETSUMO

Ec_priA-Age I-R CGGAACCGGTAACGCCCGC 3’ primer for cloning N-terminus of priA gene onto pETSUMO

Ec_priA-Age I-F GGCGTTACCGGTTCCGGT 5’ primer for cloning C-terminus of priA gene onto pETSUMO

Ec_priA-Sal I-R ATTGTCGACTTAACCCTCAATCGGATCAAC 3’ primer for cloning C-terminus of priA gene onto pETSUMO

Ec_polB-BamH I-F ATTGGATCCGTGGCGCAGGCAGGTTTTATC 5’ primer for cloning polB gene onto pETSUMO

Ec_polB-Sal I-R ATTGTCGACTCAAAATAGCCCAAGTTGCC 3’ primer for cloning polB gene onto pETSUMO

Ec_parE-Bgl II-F ATTAGATCTATGACGCAAACTTATAACGCT 5’ primer for cloning parE gene onto pETSUMO

Ec_parE-Sal I-R ATTGTCGACTTAAACCTCAATCTCCGCCATG 3’ primer for cloning parE gene onto pETSUMO

Ec_ligA-BamH I-F ATTGGATCCATGGAATCAATCGAACAACAAC 5’ primer for cloning ligA gene onto pETSUMO

Ec_ligA-Sal I-R ATTGTCGACTCAGCTACCCAGCAAACGC 3’ primer for cloning ligA gene onto pETSUMO

Ec_gyrA-BamH I-F ATTGGATCCATGAGCGACCTTGCGAGAG 5’ primer for cloning gyrA gene onto pETSUMO

Ec_gyrA-Sal I-R ATTGTCGACTTATTCTTCTTCTGGCTCGTC 3’ primer for cloning gyrA gene onto pETSUMO

Ec_gyrB-BamH I-F ATTGGATCCATGTCGAATTCTTATGACTCC 5’ primer for cloning gyrB gene onto pETSUMO

Ec_gyrB-Xho I-R ATTCTCGAGTTAAATATCGATATTCGCCGC 3’ primer for cloning gyrB gene onto pETSUMO

Pf_gyrB-U-BamH I-F ATTGGATCCTTGAGCGAAGAAAATACGTACG 5’ primer for cloning N-terminus of gyrB gene onto pETSUMO

Pf_gyrB-U-Age I-R GCATCGTCACCGGTGGTGG 3’ primer for cloning N-terminus of gyrB gene onto pETSUMO

Pf_gyrB-D-Age I-F CCACCACCGGTGACGATGC 5’ primer for cloning C-terminus of gyrB gene onto pETSUMO

Pf_gyrB-D-Xho I-R ATaCTCGAGTCAGAAATCCAGGTTGGACAC 3’ primer for cloning C-terminus of gyrB gene onto pETSUMO

Pf_gyrB-Y111A-F ACCGCCGGAGACCTTGGCGGAGTTATCGTCGAAC Primer for GyrB Y111A mutation of P. fluorescens 2P24

Pf_gyrB-Y111A-R GTTCGACGATAACTCCGCCAAGGTCTCCGGCGGT Primer for GyrB Y111A mutation of P. fluorescens 2P24

Pf_parE-U-Bgl II-F ATTAGATCTATGGCCACTCCCAGCGCTAGC 5’ primer for cloning N-terminus of parE gene onto pETSUMO

Pf_parE-U-Age I-R CAGGGCCAAACCGGTTTCC 3’ primer for cloning N-terminus of parE gene onto pETSUMO

Pf_parE-D-Age I-F GGAAACCGGTTTGGCCCTG 5’ primer for cloning C-terminus of parE gene onto pETSUMO

Pf_parE-D-Xho I-R ATTCTCGAGTCAGGCCAGCACCTCAGC 3’ primer for cloning C-terminus of parE gene onto pETSUMO

Pf_parE-Y109A-F CAGACCGCCGGAGAACTGGGCGTTCTTGTTGGAAAACTTG Primer for ParE Y109A mutation of P. fluorescens 2P24

Table S2 continued

Pf_parE-Y109A-R CAAGTTTTCCAACAAGAACGCCCAGTTCTCCGGCGGTCTG Primer for ParE Y109A mutation of P. fluorescens 2P24

Ec_gyrB-N-Sal I-R ATTGTCGACTCAGCCTTCATAGTGGAAGTGG 3’ primer for cloning N-terminus of gyrB gene onto pETSUMO

Ec_gyrB-C-BamH I-F ATTGGATCCGGCATCAAGGCGTTCGTTG 5’ primer for cloning C-terminus of gyrB gene onto pETSUMO

Ec_gyrB-Y109F-F ATTTGACGATAACTCCTTTAAAGTGTCCGGCGGTC Primer for GyrB Y109F mutation of E. coli strain DH5α

Ec_gyrB-Y109F-R GACCGCCGGACACTTTAAAGGAGTTATCGTCAAAT Primer for GyrB Y109F mutation of E. coli strain DH5α