Research Article

The essential gene set of a photosynthetic organism

Benjamin E. Rubin, Kelly M. Wetmore, Morgan N. Price, Spencer Diamond, Ryan K. Shultzaberger, Laura C. Lowe, Genevieve Curtin, Adam P. Arkin, Adam Deutschbauer, and Susan S. Golden
PNAS December 1, 2015 112 (48) E6634-E6643; first published October 27, 2015; https://doi.org/10.1073/pnas.1519220112

  1. Contributed by Susan S. Golden, September 29, 2015 (sent for review July 16, 2015; reviewed by Caroline S. Harwood and William B. Whitman)

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Article Figures & SI

Figures

  • Fig. 1.
    Fig. 1.

    The distribution of transposon mutations in the library overlaid across the S. elongatus main chromosome and two plasmids. Upper, the number of transposons (in 1,000-bp bins for the main chromosome and 100-bp bins for the plasmids) is represented by the length of the green bars in the outer circles. The locations of essential genes are shown in red in the inner circles. Lower shows a blown-up view of a region with underrepresentation of transposon insertions that encodes subunits of RNA polymerase. Lengths of black vertical bars represent numbers of sequence reads, and green bars indicate positions of insertions. Essential genes are in red, and nonessential genes are in blue (numbers represent SynPCC7942 gene numbers from the Joint Genome Institute annotation).

  • Fig. S1.
    Fig. S1.

    Divergence of antibiotic and no antibiotic library outgrowths. Each gene’s fitness score is plotted after outgrowth with kanamycin (y axis) or without kanamycin (x axis) for seven generations. Negative values represent genes that had negative effects when mutated (beneficial genes), and positive values represent genes that had positive effects when mutated (disadvantageous genes).

  • Fig. S2.
    Fig. S2.

    Lack of sequence bias around insertion locations. The y axis shows bit score, a measure of sequence conservation, with a perfectly conserved base denoted by a score of two (24). The insertion location is between −1 and 0 bp.

  • Fig. S3.
    Fig. S3.

    Normalizing for GC bias of insertion density. A, Upper shows the insertion density (y axis) for each gene compared with its GC% (x axis) before normalization. These values were normalized by dividing each insertion density by the trend line value for its GC%. A, Lower shows the insertion density normalized for GC% (insertion index). B, Upper shows the insertion density for each of the recently annotated ncRNAs (45) compared with its GC% before normalization. B, Lower shows the insertion density normalized for GC% (insertion index).

  • Fig. 2.
    Fig. 2.

    The determination of gene essentiality. (A) The distribution of insertion indexes of all analyzed genes immediately after creation of the library, which was used to determine gene essentiality. The y axis indicates the number of genes, with the insertion index shown on the x axis. (B) The distribution of fitness for each gene after six generations used to refine the essentiality measurements and assign genes that are beneficial (growth defect when mutated). The y axis indicates the number of genes, with the fitness score shown on the x axis. The cutoff for beneficial genes that have significant growth defects when mutated is denoted by a dotted vertical line. Each gene’s fitness is averaged from four growth samples in control conditions and normalized to zero, which represents a neutral fitness contribution. (C) The number of genes in the genome that are nonessential, essential, beneficial, ambiguous, or not analyzed.

  • Fig. 3.
    Fig. 3.

    Comparing the essential gene set with other predictors of gene importance. (A) The numbers of essential, conserved (26), and hypothetical genes that are overlapping and unique. (B) Fold enrichment for functional categories (TIGR function roles) that were significantly enriched or underrepresented in the essential gene sets of E. coli (29) (black bars) and S. elongatus (white bars).

  • Fig. 4.
    Fig. 4.

    Gene essentiality in central metabolism. (A) The number of genes that are conserved and nonredundant members of the Calvin–Benson cycle (CBC), the TCA cycle, glycolysis (Gly), and the pentose phosphate pathway (PPP). In the disagreements column, conserved nonredundant members of these pathways that are not essential are shown. (B) Genotypic characterization of the recreated fumC mutant. Lane 1, standard 1-kb ladder (New England BioLabs); lane 2, amplification of WT DNA with primers surrounding the fumC gene; lane 3, amplification of fumC mutant (8S42-O6), in which a 1.3-kb insertion is present, with the same primers. Each band is representative of three colonies tested. (C) Growth curves of the WT and fumC mutant strain. The error bars indicate the SDs for three independent replicates. (D) Essentiality in the TCA cycle. For enzymes that are present in S. elongatus, their names are shown: acnB (SynPCC7942_0903), icd (SynPCC7942_1719), sdhB (SynPCC7942_1533), fumC (SynPCC7942_1007), gltA (SynPCC7942_0612), maeA (SynPCC7942_1297), and ppc (SynPCC7942_2252). Abbreviations for enzymes that are missing are shown in white boxes: MDH, malate dehydrogenase; MQO, malate:quinone oxidoreductase; 2-OGDH, 2-oxoglutarate dehydrogenase; STK, succinate thiokinase.

  • Fig. S4.
    Fig. S4.

    Genotypic characterization of the recreated tal mutant. Lane 1, standard 1-kb ladder (New England BioLabs); lane 2, amplification of WT DNA with primers surrounding the tal gene; lane 3, amplification of tal mutant (UGS-3-C-11), which carries a 1.3-kb insertion, with the same primers. Each band is representative of three colonies tested.

  • Fig. 5.
    Fig. 5.

    Essentiality of ncRNAs. (A) The distribution of insertion indexes for the recently discovered ncRNAs (45). Axes are the same as in Fig. 2A. (B) The insertion distribution in and around the group I intron: ncRNA136. Lengths of black vertical bars represent numbers of sequence reads, and green bars indicate positions of insertions. The nonessential genes surrounding the essential ncRNA136 (red arrow) are shown as blue arrows. Black triangles indicate the locations of insertion mutations used to support the essentiality of ncRNA136. (C) Genotypic characterization of the failure to create a mutant of ncRNA136. Lane 1, standard 1-kb ladder (New England BioLabs); lane 2, amplification of WT DNA with primers surrounding ncRNA136 and both flanking genes; lane 3, amplification with the same primers of the region, in which the gene that flanks the ncRNA136 on the left, SynPCC7942_0413 (2E11-E-C4), carries a 1.3-kb insertion; lane 4, amplification with the same primers of a putative transformant, in which interruption of ncRNA136 (2E11-E-N7) was attempted, but the 1.3-kb insertion is absent; lane 5, amplification with the same primers of the region, in which the gene that flanks the ncRNA136 on the right, SynPCC7942_0414 (2E11-E-N11), carries a 1.3-kb insertion. Each band is representative of genotyping of three colonies.

  • Fig. 6.
    Fig. 6.

    Essential regulatory regions. (A) Transposon insertion density (insertions per base pair) on the y axis is plotted against the distance upstream from the translational start sites of essential genes (red) and nonessential genes (blue). Shading around the solid lines represents 95% confidence intervals (Poisson distribution). (B) For 557 essential genes that had an insertion before the nearest upstream gene and within 200 bp of the translation start site, the location of the closest insertion relative to the start site is shown.

Tables

  • Table S1.

    The major variations of techniques used for conjugation

    Conjugation light intensity (μmol·m−2⋅s−1)Ratio (mL:mL) of Synechococcus elongatus:Escherichia coliConjugation mediumConjugation time (h)Efficiency*
    10–405:1Filter (directly transferred to selective plate)6–81.1 × 10−8 (n = 1)
    10–402:1Filter6–81.5 × 10−8 (n = 1)
    10–4010:1Filter242.2 × 10−8 (n = 1)
    10–4010:1Filter6–83.3 × 10−8 (n = 1)
    10–4010:1Agar transferred by wash to selective plate244.4 × 10−8 (n = 2)
    10–4010:1Agar transferred by wash to selective plate6–84.7 × 10−8 (n = 2)
    100–1405:1Filter242.1 × 10−7 (n = 1)
    100–1405:1Filter6–82.1 × 10−7 (n = 1)
    100–1402:1Filter242.8 × 10−7 (n = 1)
    10–401:1Filter6–83.0 × 10−7 (n = 2)
    100–1402:1Filter6–83.3 × 10−7 (n = 1)
    10–401:1Filter245.0 × 10−7 (n = 2)
    10–401:1Agar with selective agent underlayed247.8 × 10−7 (n = 1)
    10–401:1Filter481.1 × 10−6 (n = 1)
    100–1401:1Filter242.0 × 10−6 (n = 1)
    100–1401:1Filter6–83.9 × 10−6 (n = 4)†

    • ↵* Colony forming units (CFU) of conjugated mutants per CFUs of S. elongatus culture.

    • ↵† The conditions used for generation of the RB-TnSeq library.

  • Table S2.

    Essential genes in central carbon metabolism

    Synechococcus elongatus identificationNamePathway*EssentialityConserved†Redundant‡
    SynPCC7942_2297talPentose phosphate pathwayNonessential10
    SynPCC7942_0538tktAPentose phosphate pathwayEssential10
    SynPCC7942_0604cbbEPentose phosphate pathwayEssential10
    SynPCC7942_0584rpiAPentose phosphate pathwayEssential10
    SynPCC7942_0529pglPentose phosphate pathwayNonessential10
    SynPCC7942_2334zwfPentose phosphate pathwayNonessential10
    SynPCC7942_0039gndPentose phosphate pathwayNonessential10
    SynPCC7942_0098pykGlycolysisEssential10
    SynPCC7942_0639enoGlycolysisEssential10
    SynPCC7942_0469pgmMGlycolysisEssential11
    SynPCC7942_1116pgkGlycolysisEssential10
    SynPCC7942_1939gap3GlycolysisNonessential01
    SynPCC7942_0245gap1GlycolysisNonessential01
    SynPCC7942_1261tpiAGlycolysisEssential10
    SynPCC7942_1443cbbAGlycolysisEssential10
    SynPCC7942_0592pfkAGlycolysisNonessential00
    SynPCC7942_2029pgiGlycolysisEssential10
    SynPCC7942_0781ppsAGlycolysisNonessential00
    SynPCC7942_2335fbpGlycolysisNonessential01
    SynPCC7942_0505fbpIGlycolysisEssential11
    SynPCC7942_0485pgam1GlycolysisEssential11
    SynPCC7942_1516pgam2GlycolysisEssential01
    SynPCC7942_2078pgam3GlycolysisNonessential01
    SynPCC7942_0612gltATCA cycleEssential10
    SynPCC7942_0903acnBTCA cycleEssential00
    SynPCC7942_1007fumCTCA cycleNonessential10
    SynPCC7942_1719icdTCA cycleEssential00
    SynPCC7942_1533sdhBTCA cycleNonessential00
    SynPCC7942_1427cbbSCalvin–Benson–Bassham cycleEssential10
    SynPCC7942_1426cbbLCalvin–Benson–Bassham cycleEssential10
    SynPCC7942_1116pgkCalvin–Benson–Bassham cycleEssential10
    SynPCC7942_1742gap2Calvin–Benson–Bassham cycleEssential10
    SynPCC7942_1261tpiACalvin–Benson–Bassham cycleEssential10
    SynPCC7942_1443cbbACalvin–Benson–Bassham cycleEssential10
    SynPCC7942_2335fbpCalvin–Benson–Bassham cycleNonessential01
    SynPCC7942_0538tktACalvin–Benson–Bassham cycleEssential10
    SynPCC7942_0505fbpICalvin–Benson–Bassham cycleEssential11
    SynPCC7942_0604cbbECalvin–Benson–Bassham cycleEssential10
    SynPCC7942_0977prkCalvin–Benson–Bassham cycleEssential00
    SynPCC7942_0584rpiACalvin–Benson–Bassham cycleEssential10

    • ↵* Metabolic pathway as taken from the BioCyc database (31).

    • ↵† Refers to conservation among all 13 of the cyanobacterial genomes studied in the work by Shi and Falkowski (26).

    • ↵‡ Functional redundancy as shown in the BioCyc database (31).

  • Table S3.

    Essential genes for the photosynthetic lifestyle

    Synechococcus elongatus identificationNameDescriptionCyano conserved*
    SynPCC7942_0027bioF8-Amino-7-oxononanoate synthaseNo
    SynPCC7942_0203ribD5-Amino-6-(5-phosphoribosylamino)uracil reductase…Yes
    SynPCC7942_0222Hypothetical proteinNo
    SynPCC7942_0226Sec-independent protein translocase TatANo
    SynPCC7942_0257Protein of unknown function DUF92, transmembraneYes
    SynPCC7942_0297ftsHFtsH peptidase homolog, chloroplast; metallopeptidaseNo
    SynPCC7942_0320galEUDP-galactose 4-epimeraseNo
    SynPCC7942_0322ycf44c-Type cytochrome biogenesis proteinYes
    SynPCC7942_0323ccdACytochrome c biogenesis protein-likeYes
    SynPCC7942_0330atp1Hypothetical proteinYes
    SynPCC7942_0333atpGF0F1 ATP synthase subunit B′Yes
    SynPCC7942_0439chlMMg-protoporphyrin IX methyl transferaseYes
    SynPCC7942_0450nifUPutative NifU-like proteinYes
    SynPCC7942_0485gpmBPhosphoglycerate mutaseYes
    SynPCC7942_0492ribFBifunctional riboflavin kinase/FMN adenylyltransferaseYes
    SynPCC7942_0537fabF3-Oxoacyl-(acyl carrier protein) synthase IIYes
    SynPCC7942_0560ATPaseNo
    SynPCC7942_0584rpiARibose-5-phosphate isomerase AYes
    SynPCC7942_0632rplJ50S ribosomal protein L10Yes
    SynPCC7942_0684fabG3-oxoacyl-[acyl-carrier-protein] reductaseYes
    SynPCC7942_0694rps130S ribosomal protein S1Yes
    SynPCC7942_0775Hypothetical proteinNo
    SynPCC7942_0853dapL1l,l-diaminopimelate aminotransferaseNo
    SynPCC7942_0884EF-TuElongation factor TuYes
    SynPCC7942_0894aroKShikimate kinaseYes
    SynPCC7942_0897minECell division topological specificity factor MinEYes
    SynPCC7942_0912dnaXDNA polymerase III, τ-subunitYes
    SynPCC7942_0928Outer envelope membrane proteinNo
    SynPCC7942_0942ftsHFtsH peptidase homolog, chloroplast, metallo peptidaseNo
    SynPCC7942_0959obgEGTPase ObgEYes
    SynPCC7942_0977prkPhosphoribulokinaseNo
    SynPCC7942_0978petHFerredoxin-NADP oxidoreductaseYes
    SynPCC7942_0988ycf54Conserved hypothetical protein YCF54No
    SynPCC7942_1002psaDPhotosystem I reaction center subunit IIYes
    SynPCC7942_1068pdhCBranched-chain α-keto acid dehydrogenase subunit E2Yes
    SynPCC7942_1083Probable glycosyltransferaseYes
    SynPCC7942_1086hemEUroporphyrinogen decarboxylaseYes
    SynPCC7942_1198phdDDihydrolipoamide dehydrogenaseYes
    SynPCC7942_1232petCCytochrome b6-f complex iron-sulfur subunitYes
    SynPCC7942_1274ycf37TPR repeatYes
    SynPCC7942_1351HAD-superfamily hydrolase subfamily IA, variant 3No
    SynPCC7942_1359Coenzyme F420 hydrogenaseNo
    SynPCC7942_1427rbcSRibulose 1,5-bisphosphate carboxylase small subunitYes
    SynPCC7942_1457plsC1-Acyl-sn-glycerol-3-phosphate acyltransferaseYes
    SynPCC7942_1497Hypothetical proteinNo
    SynPCC7942_1499petFFerredoxin (2Fe-2S)No
    SynPCC7942_1520rpsT30S ribosomal protein S20Yes
    SynPCC7942_1590Hypothetical proteinNo
    SynPCC7942_1673Hypothetical proteinYes
    SynPCC7942_1734ftrCFerredoxin-thioredoxin reductase catalytic chainNo
    SynPCC7942_1737sufDIron-regulated ABC transporter permease protein SufDYes
    SynPCC7942_1830trxAThioredoxinYes
    SynPCC7942_1883ycf53Conserved hypothetical protein YCF53No
    SynPCC7942_1907acsFMg-protoporphyrin IX monomethyl ester (oxidative) cyclaseNo
    SynPCC7942_1959proSProlyl-tRNA synthetaseYes
    SynPCC7942_1983pdsζ-Carotene desaturase/three-step phytoene desaturaseNo
    SynPCC7942_2017Hypothetical proteinYes
    SynPCC7942_2062crtLLycopene cyclase (CrtL-type)No
    SynPCC7942_2084chlGBacteriochlorophyll/chlorophyll a synthaseYes
    SynPCC7942_2089Thioredoxin domain 2Yes
    SynPCC7942_2113kprSRibose-phosphate pyrophosphokinaseYes
    SynPCC7942_2117gatAAspartyl/glutamyl-tRNA amidotransferase subunit AYes
    SynPCC7942_2136dapBDihydrodipicolinate reductaseYes
    SynPCC7942_2274chlDProtoporphyrin IX magnesium-chelataseYes
    SynPCC7942_2312murGUDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)…Yes
    SynPCC7942_2330ctpAC-terminal processing peptidase-2; serine peptidaseNo
    SynPCC7942_2359nha3Na+/H+ antiporterNo
    SynPCC7942_2415lysSLysyl-tRNA synthetaseYes
    SynPCC7942_2503porProtochlorophyllide oxidoreductaseYes
    SynPCC7942_2524tigTrigger factorYes
    SynPCC7942_2531tsfElongation factor TsYes
    SynPCC7942_2537clpPIIIATP-dependent Clp protease proteolytic subunitYes
    SynPCC7942_2538clpRATP-dependent Clp protease-like proteinYes
    SynPCC7942_2581petFFerredoxin (2Fe-2S)Yes

    • S. elongatus essential genes conserved in the green lineage (plants and green algae) but not in nonphotosynthetic organisms as determined using the greencut2 dataset (30). GTPase, guanosine triphosphate hydrolase.

    • ↵* Refers to conservation among all 13 of the cyanobacterial genomes studied in the work by Shi and Falkowski (26).

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Photosynthetic essential gene set
Benjamin E. Rubin, Kelly M. Wetmore, Morgan N. Price, Spencer Diamond, Ryan K. Shultzaberger, Laura C. Lowe, Genevieve Curtin, Adam P. Arkin, Adam Deutschbauer, Susan S. Golden
Proceedings of the National Academy of Sciences Dec 2015, 112 (48) E6634-E6643; DOI: 10.1073/pnas.1519220112
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Proceedings of the National Academy of Sciences: 112 (48)
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