CLCF, Not CrcB, Protects Pseudomonas putida ATCC 12633 During Intense Fluoride Stress.

Figure S2: Creation of the pCLC^F plasmids and verification of co-expression with the Delftia fluoroacetate dehalogenase. (A) Map of the constitutive mNeonGreen expression plasmids. The low (pSW002) or higher copy (pSW003) versions diEer only by the A246V mutation in the pSW003 RepA protein. (B) Plasmid map for constitutive expression of the Pseudomonas syringae CLC^F from the P_psbA promoter created by inserting the CLC^F gene in place of the mNeonGreen gene (C) SDS-PAGE to verify co-expression of CLC^F and Delftia fluoroacetate dehalogenase (DEF1) in P. putida ATCC 12633 + pBBR1-P_T5-DEF1 + pCLC^F-low after growth on 40 mM a-fluorophenylacetic acid as sole carbon. Specific his-tag staining and fluorescent imaging were done as described in the Methods. The two samples shown (total cell protein and protein size standards) were run on the same gel and the original image was spliced to create the image shown above.

Figure S3: Creation of inducible expression vectors for Pseudomonas spp. (A) Template for the PCR to amplify a fragment containing the arabinose operon, MCS and terminators. The grey arrow in the map interior are the annealing sites for the PCR primers (Table S1). (B) pBBR1MCS-2 was linearized at the indicated restrictions sites for insertion of the PCR fragment. (C) Assembled plasmid with the BBR1 origin for replication in Pseudomonas and the inducible arabinose promoter and regulatory elements from pBAD18. (D) Plasmid for inducible expression of mNeonGreen. (E) Plasmid for inducible expression of wild-type CrcB from P. putida ATCC 12633.

Figure S4: (A) Suicide vector that was linearized at the indicated restriction sites for insertion of mutagenic fragments. (B) Mutagenic plasmid with a fragment amplified from NaF-adapted strains containing the crcB with a point mutation plus upstream and downstream flanking regions to facilitate homologous recombination. (C) Mutagenic plasmid assembled from PCR fragments amplified from wild-type P. putida 12633 to create an internal in-frame deletion in crcB.

Figure S5: OD600_max measured during adaption of the (A) +CLCF-low strain or the (B) +CLCF-high strain to increasing concentrations of NaF in LB. Error bars in panel A represent the standard deviation of three replicates. Error bars in panel B represent standard deviation of three replicates from 25 to 400mM, two replicates from 425 to 500mM, or single cultures from 525 to 550mM.

Figure S6: (A) Alignment (Clustal Omega) of P_psbA promoter sequences on CLC^F expression plasmids isolated from P. putida ATCC 12633 strains that were unadapted or adapted to growth in LB + 425mM NaF. The single nt change (lower case, bold) was found on both pCLC^F-low or pCLC^F-high plasmids isolated from the adapted strains. (B) Growth curves and (C) specific fluorescence of P. putida cells expressing mNeonGreen from the constitutive wild-type (WT) or mutated (Mut) constitutive P_psbA promoter. Cells were grown in LB + Tc in 96-well plates.

Figure S7: Normalized mNeonGreen fluorescence of P. putida ATCC 12633 + pBBR1-P_araBAmNeonGreen growing in LB with 0.0%–1.0% L-arabinose. Error bars represent the standard deviations of three replicates.

Figure S8: Growth curves of strain P. putida ATCC 12633 crcB(?nt 55–363) + pBBR1-P_araBAD–crcB in LB + Km and 0, 5 or 50mM NaF. The cloned crcB gene was induced with 0.0% to 0.5% L-arabinose, which was added to the medium before inoculation. Error bars represent the standard deviations of three replicates.

Figure S9: Initial growth of (A) wild-type P. putida ATCC 12633 or (B) P. putida ATCC 12633 + pCLC^F-low in shake flasks with 0–200mM NaF in MSB with 20 ug/mL Tc, 50 ug/mL Km and 20mM (RS)-mandelic acid as the sole carbon source.

Figure S10: Growth of P. putida ATCC12633 + pCLC^F-low + pBBR1-P_T5-DEF1 in normal MSB (40mM phosphate buffer) and in MSB with the phosphate buffer concentration increased as indicated. Assays were conducted in a 96-well plate in 200 mL aliquots of media with antibiotics (Tc and Km) and 20mM D-lactic acid as the sole carbon source. Error bars represent the standard deviations of three replicates.

Table S1: Oligonucleotides used in this study (UPPER CASE = annealing, lower case = complementary sequence for cloning via Gibson-type assembly).

Table S2: Genomic sequencing coverage ratios: sequencing coverage of CLC^F plasmid sequences relative to sequencing coverage of the chromosome.

Table S3: Mutations identified in the genomic sequences of the PCLC^F-low or pCLC^F-high strains adapted to growth in LB + 425mM NaF Using the unadapted strain sequences and the P. putida ATCC 12633 genome in GenBank (NCBI RefSeq assembly GCF_024508115) as the reference. Chromosomal mutations were identified using BreSeq v0.35.0. Mutations in the plasmids were identified by BLAST alignments of the resolved plasmid contigs in the genomic sequences with the sequences of plasmids purified from the unadapted strains.