Article Figures & Data
Figures
- FIG 1
Tn-Seq is reproducible between library replicates and between experimental replicates in Geobacter sulfurreducens. (A) Comparison of two subsamples of the same library grown in two independent replicates with fumarate. The number of reads mapped to a gene (normalized for read depth) is plotted against reads mapped to the same gene for two experiments prepared and sequenced separately. (B) Comparison of two cultures inoculated separately in reactors a poised electrode as a terminal electron acceptor, recovered and sequenced separately. Both fumarate and poised electrode libraries have a Pearson correlation coefficient of 0.98.
- FIG 2
Estimation of essential genes based on low insertion densities and use of in silico model data to verify essentiality predictions. (A) The frequency distribution of insertions is centered around 10 insertions/kb, with enrichment below 4 insertions per kb (left of the dashed line). Genes with few insertions are predicted to be essential under these conditions. (B) Most genes labeled as essential in in silico modeling also contained fewer than 4 insertion sites per kb and had fewer than 300 mapped reads/gene (27).
- FIG 3
Replicate G. sulfurreducens Tn-Seq library biofilms grow at similar rates with similar current density. Replicate (n = 2) Tn-Seq libraries were inoculated independently into 3-electrode bioreactors, with anodes poised to mimic Fe(III) oxides (−0.1 V versus SHE). Biofilms were harvested after the equivalent of 6 doublings in biofilm growth mode. Error bars represent standard deviations.
- FIG 4
Genes essential for growth with electrodes are not required for Fe(III) reduction. (A and B) Growth of scarless deletion mutants of chemosensory genes esnA, esnB, esnC, and esnD, and extracellular conduit clusters extABCD, omcBC, extEFG, and extHIJKL with poised electrodes (A) or insoluble Fe(III) oxides (B). All incubations or reactor experiments were performed for each strain in triplicate. A mutant lacking both inner membrane cytochromes important for using extracellular acceptors (ΔcbcL ΔimcH) was used as a negative control. Representative curves are from replicates in all growth experiments. Error bars represent standard deviations.
- FIG 5
Genes involved in electrode reduction fall into four main categories. Of all genes identified under Tn-Seq conditions as causing a >50% reduction in growth rate on electrodes, most appeared to be involved in modifying sugars on the external surface of the cell, producing the pilus secretion apparatus, or modifying protein translation inside the cell. At −0.1 V versus SHE, only the CbcL inner membrane cytochrome and the ExtABCD porin-cytochrome conduit were essential. The only two regulatory systems identified involved cyclic di-GMP signaling and a previously unstudied MarR family DNA-binding protein.
Tables
- TABLE 1
Tn-Seq mutations which after growth on an electrode showed a decrease in reads mapped by at least a log2 ratio of −2, equivalent to a predicted 50% reduction in growth rate
Locus Gene symbol and/or product description Log2 ratio Cytochrome GSU0274 cbcL, c- and b-type cytochrome −3.3 GSU2643 extC, lipoprotein cytochrome c −2.0 GSU2645 extA, cytochrome c −2.5 Metabolism and protein processing GSU0140 Phosphoribosylaminoimidazole carboxylase-like protein −2.2 GSU0503 crcB integral membrane protein, putative transporter −2.2 GSU0536 Adenosine nucleotide alpha-hydrolase superfamily protein −2.6 GSU0994 fumB, fumarate hydratase −8.0 GSU1105 Prolidase family protein −2.0 GSU1279 nikMN, nickel ABC transporter membrane protein NikMN −2.0 GSU1752 efp-2, elongation factor P −2.5 GSU1753 genX, translation elongation factor P-lysine lysyltransferase −2.9 GSU1754 yjeK, translation elongation factor P-lysyl-lysine 2,3-aminomutase −2.8 GSU3278 Outer membrane TPR-containing proteina −2.0 Signaling and regulation GSU0013 MarR family winged helix-turn-helix transcriptional regulator −2.1 GSU1704 esnA, GAF sensor methyl-accepting chemotaxis sensory transducer, class 40H −2.4 GSU2220 esnB, scaffold protein CheW associated with MCPs of class 40H −2.4 GSU2221 ATPase −2.1 GSU2222 esnC, sensor histidine kinase CheA associated with MCPs of class 40H −2.8 Extracellular structures GSU1114 Lipoprotein −2.1 GSU1493 pilC, type IV pilus inner membrane protein PilC −2.2 GSU1494 pilS, sensor histidine kinase PilS, PAS domain containing −2.2 GSU1501 xapD, ABC transporter ATP-binding protein −2.0 GSU1816 ugd, UDP-glucose 6-dehydrogenase −2.0 GSU1889 lptA, lipopolysaccharide ABC transporter periplasmic protein LptA −6.5 GSU1976 YqgM-like family glycosyltransferase −4.9 GSU2028 pilQ, type IV pilus secretin lipoprotein PilQ −2.5 GSU2029 pilP, type IV pilus assembly lipoprotein PilP −3.3 GSU2030 pilO, type IV pilus biogenesis protein PilO −2.5 GSU2032 pilM, type IV pilus biogenesis ATPase PilM −2.4 GSU2085 hldE, d-glycero-d-mannoheptose-7-phosphate kinase and d-glycero-d-mannoheptose-1-phosphate adenylyltransferase −3.1 GSU2086 Hypothetical cytoplasmic protein in sugar biosynthesis operon −3.2 GSU2087 gmhA, phosphoheptose isomerase −3.7 GSU2257 Hypothetical cytoplasmic protein in LPS biosynthesis operon −2.4 GSU2973 Lipoprotein −2.7 GSU3321 Phosphoglucomutase/phosphomannomutase family protein −2.2 Hypothetical GSU0141 Hypothetical cytoplasmic protein −2.3 GSU0959 Hypothetical protein −2.3 GSU2048 Hypothetical protein −2.1 GSU2713 Hypothetical protein (chaperone-like protein) −2.9 ↵a TPR, tetratricopeptide repeat.
- TABLE 2
Evidence for EsnABC association via two-hybrid analysisa
Coexpression with pEsnA bait LacZ activity (ΔOD420 min−1 · OD600−1) pSR658 (negative control) 1,378.5 ± 131.8 pEsnA prey (EsnA-EsnA) 287.9 ± 15.6 pEsnB prey (EsnA-EsnB) 473.6 ± 44.2 pEsnC prey (EsnA-EsnC) 448.3 ± 46.5 ↵a LacZ activity is calculated based on the results from biological triplicates ± standard deviations. Lower LacZ activity indicates stronger protein-protein associations, causing stronger repression.
- TABLE 3
Strains and plasmids used in this work
Strain or plasmid Description or relevant genotype Source or reference Strains G. sulfurreducens DB790 ΔcbcL ΔimcH (ΔGSU0274 ΔGSU3259) This study DB823 ΔesnA (ΔGSU1704) This study DB836 ΔesnB (ΔGSU2220) This study DB824 ΔesnC (ΔGSU2222) This study DB1130 ΔesnD (ΔGSU3376) This study DB1280 ΔextABCD (ΔGSU2645 to GSU2642) This study DB1282 ΔextEFG (ΔGSU2726 to GSU2724) This study DB1279 ΔomcBC cluster (ΔGSU2739 to GSU2731) This study DB1281 ΔextHIJKL (ΔGSU2940 to GSU2936) This study E. coli S17-1 recA pro hsdR RP4-2-Tc::Mu-Km::Tn7 72 SU202 lexA71::Tn5(Def)sulA211 Δ(laclPOZYA) 169/F′ lacl q lacZΔM15::Tn9 71 BW29427 (WM3064) thrB1004 pro thi rpsL hsdS lacZΔM15 RP4-1360 Δ(araBAD)567 ΔdapA1341::[erm pir] K. Datsenko and B. L. Wanner Plasmids pEB001 Plasmid carrying mini-Himar RB1 transposon with engineered MmeI restriction sites 24 pK18mobsacB SacB-encoding scarless deletion vector 72 pDGSU0274 Flanking regions of GSU0274 in pK18mobsacB 9 pDGSU3259 Flanking regions of GSU3259 in pK18mobsacB 66 pDGSU1704 Flanking regions of GSU1704 in pK18mobsacB This study pDGSU2220 Flanking regions of GSU2220 in pK18mobsacB This study pDGSU2222 Flanking regions of GSU2222 in pK18mobsacB This study pDGSU3376 Flanking regions of GSU3376 in pK18mobsacB This study pDGSU2645-2642 Flanking regions of GSU2645 to GSU2642 in pK18mobsacB This study pDGSU2726-2724 Flanking regions of GSU2726 to GSU2724 in pK18mobsacB This study pDGSU2739-2731 Flanking regions of GSU2739 to GSU2731 in pK18mobsacB This study pDGSU2940-2936 Flanking regions of GSU2940 to GSU2936 in pK18mobsacB This study pSR658 Bacterial two-hybrid vector, WT LexA DNA-binding domain 71 pSR659 Bacterial two-hybrid vector, variant LexA DNA-binding domain 71 pEsnA-4 (bait) pSR659 expressing the soluble domain of EsnA fused to variant LexA DNA-binding domain This study pEsnA-6 (prey) pSR658 expressing the soluble domain of EsnA fused to WT LexA DNA-binding domain This study pEsnB-2 (prey) pSR658 expressing EsnB fused to WT LexA DNA-binding domain This study pEsnC-2 (prey) pSR658 expressing EsnC fused to WT LexA DNA-binding domain This study
Supplemental material
- Supplemental file 1 -
Table S1 (Primers)
XLSX, 12K
- Supplemental file 2 -
Table S2 (Raw data)
XLSX, 1005K
- Supplemental file 3 -
Table S3 (Essential genes predicted in this study versus the in silico model of Mahadevan et al.)
XLSX, 52K
- Supplemental file 4 -
Table S4 (Genes found to be essential in the acetate/fumarate outgrowth deep sequencing library)
XLSX, 61K
- Supplemental file 5 -
Table S5 (Reactions associated with central metabolism)
XLSX, 13K
- Supplemental file 6 -
Table S6 (Functional and sequence homologs in the G. sulfurreducens genome)
XLSX, 30K
- Supplemental file 7 -
File S7 (Supplemental text)
PDF, 67K
- Supplemental file 1 -
