Article Figures & Data
Figures
- FIG. 1.
Schematic circular diagram of the T. denitrificans ATCC 25259 genome. Outer circle, predicted coding regions on the forward strand, color-coded by role categories (dark gray, hypothetical proteins; light gray, conserved hypothetical and unknown function; brown, general function; red, DNA replication and repair; green, energy metabolism; blue, carbon and carbohydrate metabolism; cyan, lipid metabolism; magenta, transcription; yellow, translation; orange, amino acid metabolism; pink, metabolism of cofactors and vitamins; light red, purine and pyrimidine metabolism; lavender, signal transduction; sky blue, cellular processes; pale green, structural RNAs); second circle, predicted coding regions on the reverse strand, color coded as for the outer circle; third and fourth circles, coding regions (on forward and reverse strands) predicted to be involved in denitrification (blue), sulfur-compound oxidation (red), hydrogen oxidation (green), autotrophic carbon assimilation (orange), and metal ion transport-resistance (brown); fifth and sixth circles, coding regions found to have a CXXCH heme-binding motif and therefore potentially encoding c-type cytochromes; seventh circle, deviation from the average G+C; eighth circle, GC skew (olive, positive; purple, negative).
- FIG. 2.
Schematic overview of key genes/enzymes putatively associated with sulfur-compound oxidation in T. denitrificans. Genes in parenthesis have been shown to be lesser expressed paralogs (this study). The biochemical roles of a number of gene products represented in this figure have not been experimentally demonstrated in T. denitrificans and are uncertain. Sulfide:quinone oxidoreductase is not proposed to catalyze the direct oxidation of sulfide to sulfite but rather may participate in an indirect pathway (20). The arrow between thiosulfate and sulfate (right side) represents the possibility that SoxB catalyzes a sulfate thiohydrolase reaction (28) in T. denitrificans. APAT, APS:phosphate adenylyltransferase.
- FIG. 3.
Phylogenetic relationships among the eight putative DsrC proteins encoded in T. denitrificans ATCC 25259 and the top BLASTP matches from the GenBank nr database for Tbd2480. Of the proteins represented in this figure that are not from T. denitrificans, more than 70% are known or predicted to be DsrC or more broadly related to sulfite reductases (indicated in boldface type). For limbs that show species names rather than GenBank accession numbers, the corresponding accession numbers are as follows: A. vinosum (AAC35399.1), M. magnetotacticum (ZP 00052645.1), Magnetococcus sp. (ZP 00287929.1), C. tepidum (NP 663123.1), T. norvegica (CAC36215.1), D. desulfuricans (ZP 00130056.2), and D. vulgaris (YP 011988).
- FIG.4.
Phylogenetic relationships among predicted amino acid sequences for HynS (A), Isp1 (B), Isp2 (C), and HynL (D) in T. denitrificans and the best BLASTP matches from the GenBank nr database. For limbs that show species names rather than GenBank accession numbers, the corresponding accession numbers are as follows: (HynS) A. vinosum (AAU93828.1), T. roseopersicina (AAC38281.1), A. aeolicus (NP 213658.1); (Isp1) A. vinosum (AAU93829.2), T. roseopersicina (AAC38283.1), A. aeolicus (NP 213657.1), D. vulgaris Hmc5 (YP 009755), A. ambivalens (CAC86885.1), D. desulfuricans NarI (ZP 00128546.1); (Isp2) A. vinosum (AAY89333.1), T. roseopersicina (AAC38284.1), A. aeolicus (NP213656.1), A. ambivalens (CAC86886.1), Polaromonas sp. (ZP 00503323.1), C. aurantiacus (ZP 00356812), A. vinosum DsrK (AAC35401.2), C. tepidum DsrK (NP 663117.1), D. vulgaris Hmc6 (YP 009754.1); (HynL) A. vinosum (AAY89334.1), T. roseopersicina (AAC38282.1), A. aeolicus (NP213655.1).
Tables
- TABLE 1.
General features of the T. denitrificans strain ATCC 25259 genome
Characteristic Value Chromosome size (bp) 2,909,809 G+C ratio (%) 66.07 Coding density (%) 92.5 No. of predicted protein coding genes 2,827 Average CDS length (bp) 952 No. of predicted proteins unique to T. denitrificans (%) 89 (3.1) No. of rRNA operons 2 No. of tRNA genes 43 No. of other small RNAs 3 No. of predicted proteins with putative function (%) 2,183 (77.2) No. of predicted proteins with unknown function (%) 644 (22.8) Protein categories (%) Energy production 6.6 Inorganic ion transport 5.7 Cell envelope biogenesis 6.0 BLASTP comparison against the KEGG completed microbial genomes database (no. of top KEGG hits) β-Proteobacteria 1,624 γ-Proteobacteria 455 α-Proteobacteria 119 δ-Proteobacteria 92 Archaea 22 - TABLE 2.
Regions with uncharacteristic G+C content and Karlin signaturesa
Location in genome (nt)b % G+C CDS BLAST hits 394058-402433 58.87 Tbd0363-Tbd0373 Phage integrase, regulatory protein, hypothetical proteins 979890-1004773 57.39 Tbd0925-Tbd0943 Methylase, transposase, regulatory protein, helicase (Snf2/Rad54 family), type III restriction-modification system (methylase, restriction enzyme), phage integrases, and several hypothetical proteins 1264595-1271704 56.99 Tbd1207-Tbd1215 Phage integrase, conserved imported protein, and hypothetical proteins 1395068-1400129 52.43 Tbd1314-Tbd1318 Phage integrase and hypothetical and conserved hypothetical proteins 1424272-1431083 60.88 Tbd1342-Tbd1354 Mostly hypothetical proteins and a conserved imported protein 1575347-1583813 57.71 Tbd1487-Tbd1492 Type II/III restriction-modification system (helicase, methylase), conserved hypothetical and hypothetical proteins 1767440-1787406c 60.46 Tbd1679-Tbd1695 Phage integrase, conjugal transfer region (TraWBAY), a number of hypothetical proteins, and imported AAA superfamily ATPase and conserved hypothetical protein 1955614-1961331 61.96 Tbd1861-Tbd1865 Type IV pilus proteins (PilEWXV and FimT) 2052544-2061073 59.64 Tbd1958-Tbd1969 Transposases, conserved imported hypothetical proteins, and hypothetical proteins 2089032-2093604c 57.82 Tbd2000-Tbd2005 Phage integrase, plasmid recombination protein, and several hypothetical proteins 2160137-2166983c 62.36 Tbd2066-Tbd2073 Phage integrase, phage replication protein, DNA helicase, and several hypothetical proteins 2750091-2764591 57.46 Tbd2675-Tbd2685 Phage integrase, prophage regulatory protein, type I restriction-modification system (HsdMS, HsdR) 2900270-2905890 61.02 Tbd2817-Tbd2823 Phage integrase, phage primase and phage regulatory protein - TABLE 3.
Differential transcription of selected genes in T. denitrificans that occur in multiple copies
Genes compared Thiosulfate-induced transcription (fold difference)a FeCO3-induced transcription (fold difference)b dsrC (putative) Tbd2480/Tbd2480c 1d 0.38 Tbd2488/Tbd2480c 1.4 0.025 Tbd2658/Tbd2480c 0.069 0.006 Tbd2326/Tbd2480c 0.015 0.002 Tbd2327/Tbd2480c 0.009 0.002 Tbd1365/Tbd2480c 0.039 0.009 Tbd1408/Tbd2480c 0.82 0.031 Tbd1926/Tbd2480c 0.001 0.002 APS reductase α subunit Tbd0872/Tbd2282 80 NAe β subunit Tbd0873/Tbd2283 740 NA ATP sulfurylase Tbd0874/Tbd0210 5 NA Nitric oxide reductase norC Tbd0562/Tbd0822 63 NA norB Tbd0561/Tbd0823 61 NA ↵ a Cells harvested for RNA while carrying out thiosulfate oxidation and denitrification (see Materials and Methods).
↵ b Cells harvested for RNA while carrying out oxidation of Fe(II) (in FeCO3) and denitrification (see Materials and Methods).
↵ c Comparison made to the number of transcripts for Tbd2480 under thiosulfate-induced conditions. The transcript copy number for Tbd2480 under these conditions was relatively high (in the copy number range of Tbd0562 and Tbd0561, which encode subunits of nitric oxide reductase, a key enzyme involved in denitrification).
↵ d By definition.
↵ e NA, not analyzed.
- TABLE 4.
CDS potentially encoding c-type cytochromes in the T. denitrificans genomea
CDS Annotationb Molecular mass (Da)c No. of hemes Tbd0055 Cytochrome c family protein 20,116 1 Tbd0064 Cytochrome c-553 23,727 2 Tbd0070 Probable nirN 63,589 1 Tbd0076 Probable nirC 10,547 1 Tbd0077 nirS (cytochrome cd1) 62,992 1 Tbd0094 Hypothetical protein 17,109 1 Tbd0128 Cytochrome c 38,000 2 Tbd0129 Cytochrome c 21,784 2 Tbd0137 Diheme cytochrome c 19,427 2 Tbd0138 Cytochrome c-type protein 14,395 1 Tbd0146 Probable cytochrome c5 26,734 2 Tbd0187 Cytochrome c 21,387 2 Tbd0219 Flavin adenine dinucleotide-flavin mononucleotide-containing dehydrogenase 138,251 1 Tbd0325 aa 3-type cytochrome c oxidase, subunit II 41,279 1 Tbd0339 cbb 3-type cytochrome c oxidase, subunit II 28,037 1 Tbd0341 cbb 3-type cytochrome c oxidase, subunit III 33,124 2 Tbd0436 Excinuclease ATPase subunit 103,145 1 Tbd0562 norC 15,845 1 Tbd0564 soxA 30,960 1 Tbd0567 soxX 12,712 1 Tbd0571 DMSO reductase chain B 25,909 1 Tbd0640 cbb 3-type cytochrome c oxidase, subunit III 33,873 2 Tbd0642 cbb 3-type cytochrome c oxidase, subunit II 22,314 1 Tbd0723 Possible high-affinity Fe2+/Pb2+ permease 69,511 1 Tbd0752 Mannose-sensitive hemagglutinin pilin biogenesis ATPase protein MshE 62,481 1 Tbd0820 Cytochrome c (in or near nonfunctional nor cluster) 57,261 2 Tbd0822 norC-related (potentially not functional) 30,200 2 Tbd0840 Probable cytochrome c5 16,718 1 Tbd0917 soxX 13,246 1 Tbd0918 soxA 30,875 1 Tbd1169 Ferredoxin, 2Fe-2S 12,259 1 Tbd1357 Unknown 16,635 1 Tbd1398 Putative cytochrome c-type protein 15,842 1 Tbd1404 narH 59,191 1 Tbd1484 Cytochrome c 9,544 1 Tbd1520 Putative Fe-S protein 48,430 1 Tbd1542 ATPase involved in DNA replication 61,297 1 Tbd1564 Probable ribonuclease E 95,354 1 Tbd1585 Putative pyruvate formate-lyase-activating enzyme 40,800 1 Tbd1831 Putative cytochrome c1 27,211 1 Tbd1840 Unknown 11,061 1 Tbd2026 Possible cytochrome c4 or c-553 11,253 1 Tbd2027 Cytochrome c, class IC 11,521 1 Tbd2034 Possible cytochrome subunit of sulfide dehydrogenase 10,395 1 Tbd2060 Possible alpha-mannosidase 64,835 1 Tbd2157 Cytochrome c 18,742 1 Tbd2170 Activase of anaerobic class III ribonucleotide reductase 24,585 1 Tbd2181 Unknown 20,904 1 Tbd2476 dsrJ 17,948 3 Tbd2477 dsrL 71,328 1 Tbd2545 Diheme cytochrome c 37,973 2 Tbd2726 Cytochrome c 11,096 1 Tbd2727 Conserved protein of unknown function 74,616 1 Tbd2738 Zinc-dependent hydrolase 26,287 1 ↵ a As defined by the presence of at least one CXXCH heme-binding motif. Tbd0039 (which encodes ribosomal protein L31) and Tbd1539 (which encodes DnaJ) include the CXXCH motif but were excluded from this table.
↵ b Best attempt at annotation based on examination of best BLASTP hits, top 10 PSI-BLAST hits, and genomic context.
↵ c Molecular mass predicted for the unprocessed gene product without cofactors.
- TABLE 5.
Comparison of encoded efflux-mediated heavy metal resistance systems among the genomes of T. denitrificans and other selected bacteria
Bacterial species Genome size (Mbp) No. of HME RNDs No. of CDF No. of CPx-type ATPases Thiobacillus denitrificans a 2.9 5 3 3 Geobacter sulfurreducens PCAa 3.8 3 2 2 Ralstonia metallidurans b 6.9 12 3 5 Pseudomonas aeruginosa b 6.3 1 3 4 Escherichia coli b 4.6 1 2 2 ↵ a Putative heavy metal-exporting protein families for T. denitrificans and G. sulfurreducens PCA (GenBank AE01780 ) were identified as described by Nies (55). Confirmatory data on metal transport in G. sulfurreducens was obtained from an unpublished source (H. A. Vrionis and D. R. Lovley, 105th Gen. Meet. Am. Soc. Microbiol., poster I-054, 2005).
↵ b From Nies (55).
