Supporting Information
Yan et al. 10.1073/pnas.0801093105 SI Methods ment was Ϸ200–500 bp in length. The microarray featured 3,988 Genome Assembly. The initial sequences assembly was carried out of the 4,146 CDS identified in P. stutzeri A1501, excluding by using the Phred/Phrap package (www.phrap.org) (1, 2). Gaps multiple copies of transposases, unamplified genes, low- were closed using a combination of primer walking and multiplex concentration products, and unpurified products (158 CDSs in PCR. The Consed program (3) was used to facilitate sequence total). finishing. The overall sequence quality of the genome was PCR products were analyzed on agarose gels to confirm the further improved, inspecting the following criteria for each base: success of each reaction and subsequently purified by using (i) covered by at least two independent valid reads accountable MultiScreen-PCR plates (Millipore). DNA was then resus- from both strands and (ii) the final consensus quality score pended in 12 l of spotting solution containing 50% dimethyl generated by Phrap Ͼ40. sulfoxide. A set of microarrays containing a total of 4,352 spots, including 3,988 PCR products and 364 controls in each block, Genome Annotation. Prediction of coding sequences (CDS) was including blank, negative, and positive controls, were spotted generated wih the program Glimmer (4), and location of start onto CMT-GAPSII-coated slides (Corning), using a 16-pin codons were corrected by RBSfinder (5). Overlapping and configured MicroGrid II array printer (BioRobotics) controlled closely clustered CDS were manually inspected. The functional by the MicroGrid TAS Application Suite, Version 2.2.0.6. Neg- annotation of the predicted CDS was carried out by a BLASTP ative control genes consisted of the type III secretion system (6) search of the translations versus GenBank’s nonredundant genes of Shigella flexneri 2a strain 301 (GenBank accession nos. protein database (NR) and manual curation. Transfer RNA AE005674 and AF386526) and the commercial Arabidopsis genes were identified by the program tRNAscan-SE (7). Ribo- genes (SpotReport cDNA Array Validation System, Stratagene). somal RNAs were identified by a BLASTN (6) search against a A positive control gene was 16S rRNA of P. stutzeri A1501. database of all publicly available rRNA sequences. Spotted DNA was UV cross-linked to slides, using a CL-1000 Repeat sequences (RPs) were primarily detected by Repseek UV cross-linker and subsequently placed in a blocking solution (8). After manual curation a nonredundant set of potential RPs containing 200 mM succinic anhydride and 50 mM N- was given to RepeatMasker (www.repeatmasker.org) to identify methylimidazol prepared in 1-methyl-2-pyrrolidinone for 60 all intact or partial copies within A1501 genome. PhageFinder min, washed for 2 min in 95°C distilled water, and rinsed five (9) program was used to identify intact or nearly intact prophage times in 95% ethanol. Slides were spin dried at 185 ϫ g for 1 min regions in the genome. The IslandPath software (10) was used to and stored for future hybridizations. screen A1501 genome for potential anomalous gene clusters first, and then genomic islands were identified manually based on cDNA Synthesis, Labeling, and Hybridization. Cells from an over- general characteristics of typical genomic islands. night culture were centrifuged and resuspended in a 50-ml flask containing 10 ml of N-free minimal medium K at an OD600 of Comparative Genomics. Genomic comparisons were carried out by 0.1. The suspensions were incubated for6hat30°C under ϩ bidirectional BLASTP comparisons of whole genome protein nitrogen fixation conditions (0.5% O2, 0.1 mM NH4 ) or nitro- ϩ databases. Ortholog groups among P. stutzeri A1501 and five gen excess growth conditions (0. 5% O2 and 20 mM NH4 ), other Pseudomonas species, P. aeruginosa PAO1 (AE004091), P. respectively. Total RNA was extracted using the Promega SV putida KT2440 (AE015451), P. entomophila L48 (CT573326), P. Total RNA isolation system and then 5 g of total RNA was used fluorscens Pf-5 (CP000076) and P. syringae pv. tomato DC3000 for cDNA synthesis. Genomic DNA and cDNA were fluores- (AE016853, AE016854, AE016855), were identified by the Or- cently labeled by using the BioPrime DNA Labeling System (Life thoMCL program (11) with an e value cutoff of 10Ϫ5. Technologies) with random hexamers. Cy5 dye-labeled cDNA and Cy3-labeled genomic DNA samples were mixed and hybrid- Phylogenetic Analysis. Twelve conserved housekeeping proteins ized at 65°C for 16 h. Genomic DNA was used as a universal were used for whole genome based phylogenetic analysis of internal control for the quality of the microarrays allowed for the Pseudomonas (Fig. S2). Total length of alignment was 7,003 aa comparison of results across multiple experiments. Biological after removing ambiguously aligned regions. Protein sequence experiments were carried out three times, which provided three alignment was carried out for each individual protein, using biological repeats. ClustalW. The multialignments were then manually checked and trimmed with BioEdit. The dataset with concatenation of the 12 Microarray Data Analysis. Processed slides were scanned with a proteins was fed to TREE-PUZZLE software to construct the GenePix 4000B scanner (Axon). Fluorescent spots and local maximum likelihood (ML) tree using the JTT model of amino background intensities were quantified with GenePix Pro 6.0 acid substitution. The rate heterogeneity was estimated by software (Axon). Before data analysis, signals were normalized gamma distribution with eight rate categories, and the alpha- using a locally weighted scatterplot smoothing regression (LOW- parameter estimated from the dataset. Reliability of the dataset ESS) algorithm in the MIDAS software package (www.tigr.org/ was assessed by bootstrap. One thousand permutation datasets software/tm4) with smoothing parameter set to 0.33. Local were generated by using the SEQBOOT program from the background value was subtracted from intensity of each spot. PHYLIP package. For each of the 1,000 datasets ML tree was The mean of the signal intensities of the control spots hybridized constructed using the same parameters described above. TREE- with labeled reference genomic DNA in each experiment was PUZZLE was then used with the ‘‘Consensus of user defined calculated. The mean Cy5/Cy3 (sample/reference) ratios of trees’’ option to generate a consensus tree. signal intensity were calculated for analysis. Genes were consid- ered to be differentially expressed if (i) average expression Microarray Fabrication. PCR fragments used for printing microar- changed by at least 2-fold in three independent experiments or ray chips were amplified as described in ref. 12 using ologo- (ii) the change in gene expression was in the same direction nucleotides designed from A1501 genome sequence. Each frag- (‘‘increased’’ or ‘‘decreased’’) in three experiments.
Yan et al. www.pnas.org/cgi/content/short/0801093105 1of18 1. Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of automated sequencer 8. Achaz G, Boyer F, Rocha EP, Viari A, Coissac E (2007) Repseek, a tool to retrieve traces using phred. I. Accuracy assessment. Genome Res 8:175–185. approximate repeats from large DNA sequences. Bioinformatics 23:119–121. 2. Ewing B, Green P (1998) Base-calling of automated sequencer traces using phred. II. 9. Fouts DE (2006) PhageFinder: Automated identification and classification of pro- Error probabilities. Genome Res 8:186–194. phage regions in complete bacterial genome sequences. Nucleic Acids Res 34:5839– 3. Gordon D, Abajian C, Green P (1998) Consed: A graphical tool for sequence finishing. 5851. Genome Res 8:195–202. 10. Hsiao W, Wan I, Jones SJ, Brinkman FS (2003) IslandPath: Aiding detection of genomic 4. Delcher AL, Bratke KA, Powers EC, Salzberg SL (2007) Identifying bacterial genes and islands in prokaryotes. Bioinformatics 19:418–420. endosymbiont DNA with Glimmer. Bioinformatics 23:673–679. 11. Li L, Stoeckert CJ, Jr, Roos DS (2003) OrthoMCL: Identification of ortholog groups for 5. Suzek BE, Ermolaeva MD, Schreiber M, Salzberg SL (2001) A probabilistic method for eukaryotic genomes. Genome Res 13:2178–2189. identifying start codons in bacterial genomes. Bioinformatics 17:1123–1130. 12. Peng J, et al. (2006) The use of comparative genomic hybridization to characterize 6. Altschul SF, et al. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. genome dynamics and diversity among the serotypes of Shigella. BMC Genomics 7:218. 7. Lowe TM, Eddy SR (1997) tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955–964.
Yan et al. www.pnas.org/cgi/content/short/0801093105 2of18 Fig. S1. Pairwise genome comparisons of P. stutzeri A1501 with five other representative Pseudomonas species in dot–matrix style. Orthologs between A1501 (always shown on the x axis) and the five other Pseudomonas species (shown on the y axis) are marked as dots and color-coded for similarity. Red, Ͼ80%; blue, 50–80%; cyan, Ͻ50%. The start of the KT2440 genome was adjusted to make it begin with dnaA in accordance with the others.
Yan et al. www.pnas.org/cgi/content/short/0801093105 3of18 Fig. S2. Maximum likelihood (ML) tree of sequenced Pseudomonas species based on combined dataset of 12 housekeeping proteins. E. coli K-12 MG1655 is used as outgroup. Numbers along the branches show support value of nodes estimated by 1,000 bootstrap replicates. The 12 housekeeping proteins used for phylogenetic analysis are aspartyl-tRNA synthetase (AspS), ATP synthase subunit B (AtpD), carbamoyl-phosphate synthase small subunit (CarA), glycogen phosphorylase (GlgP), DNA gyrase subunit B (GyrB), uroporphyrinogen decarboxylase (HemE), amidophosphoribosyltransferase (PurF), recombinase A (RecA), DNA-directed RNA polymerase beta subunit (RpoB), RNA polymerase sigma factor (RpoD), seryl-tRNA synthetase (SerS), and dihydrolipoamide acetyltransferase (SucB).
Yan et al. www.pnas.org/cgi/content/short/0801093105 4of18 Fig. S3. Physical organization of the highly conserved region containing the cob gene cluster and putative glutathione peroxidase in sequenced Pseudomonas strains. Genes of the same color indicate corresponding orthologous genes with high homology (Ͼ80%) at the nucleic acid level and arrow shows transcriptional directions of genes. Inserted genes are marked in white. Wedge marks indicate that insertion occurs at this spot. The number of insertions is listed above each wedge.
Yan et al. www.pnas.org/cgi/content/short/0801093105 5of18 Fig. S4. Genetic organization of the nitrogen-fixation island in P. stutzeri A1501. Genes are not drawn to scale; number of nucleotides in the non-coding regions or overlap between genes is shown in parentheses. Related genes are color-coded as follows: red, core nif and rnf genes previously characterized; black, additional genes encoding proteins of unknown function; white, molybdenum transport. The relative increase in gene expression (determinded by real-time RT-PCR) under nitrogen fixation conditions vs. ammonia excess conditions is indicated within the arrows. nifH was used as control. The localization of insertion mutations in 16 genes by homologous suicide plasmid integration is shown by wedge above the genes and nitrogenase activity is indicated as percentage of that of wild-type strain A1501.
Yan et al. www.pnas.org/cgi/content/short/0801093105 6of18 Table S1. Functional description of predicted genes inserted between cobalamin 5-phosphate synthase and glutathione peroxidase of sequenced Pseudomonas strains Strain Gene ID Length, bp G ϩ C, % Functional description
P. aeruginosa PAO1 PA1282 1,506 71.3 Major facilitator superfamily (MFS) transporter PA1283 561 68.3 Transcriptional regulator, TetR family PA1284 1,821 67.3 Acyl-CoA dehydrogenase domain protein PA1285 450 68.4 Transcripitional regulator, MarR family PA1286 1,200 71.0 Major facilitator superfamily transporter P. fluorescens Pf-5 PFl4421 405 61.7 Transcriptional regulator, MarR family PFl4420 1,203 63.0 Major facilitator superfamily transporter P. fluorescens PfO-1 Pfl011651 405 63.0 Transcriptional regulator, MarR family Pfl011652 1,209 63.6 Major facilitator superfamily transporter Pfl011653 123 66.7 Conserved hypothetical protein P. syringae pv. tomato DC3000 PSPTO1718 1,209 62.2 Major facilitator superfamily transporter P. syringae pv. phaseolicola 1448A PSPPH3692 1,209 61.2 Major facilitator superfamily transporter PSPPH3691 123 66.7 Hypothetical protein P. entomophila L48 PSEEN1388 1,179 64.8 Glycosyl hydrolase, family 5 PSEEN1389 405 66.4 Transcriptional regulator, MarR family PSEEN1390 1,203 65.2 Major facilitator superfamily transporter PSEEN1391 123 64.2 Hypothetical protein P. putida KT2440 PP1682 1,305 63.4 Glycosyl hydrolase, family 5 PP1683 405 66.2 Transcriptional regulator, MarR family PP1684 1,221 63.7 Major facilitator superfamily transporter PP1685 123 59.4 Hypothetical protein
Yan et al. www.pnas.org/cgi/content/short/0801093105 7of18 Table S2. Functional description of genes upregulated in nitrogen-fixing conditions in comparison to nitrogen-excess conditions Gene Relative Gene ID Functional description name increase PST0039 katE + catalase PST0167 + formate dehydrogenase-O, major subunit PST0179 ectC + L-ectoine synthase PST0200 + 4-hydroxyphenylpyruvate dioxygenase PST0241 + arsenic resistance transcriptional regulator PST0265 osmC + osmotically inducible protein OsmC PST0349 ntrC + nitrogen regulation protein NtrC PST0350 ntrB + nitrogen regulation protein NtrB PST0353 glnA + glutamine synthetase
*PST0502 GlnK ++ nitrogen regulatory protein PII PST0503 amtB1 + ammonium channel PST0504 amtB2 + ammonium channel PST0565 + major facilitator family transporter PST0579 + GMP synthase, PP-ATPase domain/subunit PST0585 + site-specific recombinase, phage integrase family PST0632 + type I restriction-modification system, S subunit PST0692 phaP + phasin PhaP PST0722 prkA + serine protein kinase PrkA PST0752 + membrane protein PST0754 ++ membrane protein PST0757 + peptidase, M23/M37 family PST0811 katA + catalase PST0813 + major facilitator family transporter PST0874 pctA + chemotactic transducer PctA PST0970 pilY1 + type 4 fimbrial biogenesis protein PilY1 PST0972 pilW + type 4 fimbrial biogenesis protein PilW PST0984 + putative lipoprotein PST1000 + putative membrane protein PST1054 + fimbrial protein ecpC precursor phosphomannose isomerase / guanosine 5'-diphospho-D- PST1169 algA + mannose pyrophosphorylase PST1301 cobS +++ cobalamin (5'-phosphate) synthase PST1302 ++++ glutaredoxin-related protein PST1303 ++++ thiosulfate sulfurtransferase GlpE PST1304 nifQ ++++ nitrogen fixation protein NifQ PST1305 ++++ arsenate reductase related protein PST1306 nifB ++++ FeMo cofactor biosynthesis protein NifB PST1308 + transcriptional regulator, LysR family
Yan et al. www.pnas.org/cgi/content/short/0801093105 8of18 Table S2. (Continued) PST1312 tpmA + thiopurine S-methyltransferase PST1313 nifA ++ nitrogen fixation positive regulatory protein PST1314 nifL ++ nitrogen fixation negative regulatory protein electron transport complex, RnfABCDGE type, A PST1315 rnfA + subunit electron transport complex, RnfABCDGE type, B PST1316 rnfB +++ subunit electron transport complex, RnfABCDGE type, C PST1317 rnfC + subunit electron transport complex, RnfABCDGE type, D PST1318 rnfD ++ subunit electron transport complex, RnfABCDGE type, G PST1319 rnfG ++ subunit electron transport complex, RnfABCDGE type, E PST1320 rnfE +++ subunit electron transport complex, RnfABCDGE type, H PST1321 rnfH ++++ subunit PST1322 nifY2 ++++ dinitrogenase iron-molybdenum cofactor biosynthesis PST1323 +++ nitrogen fixation-related protein PST1324 ++++ conserved hypothetical protein PST1325 +++ conserved hypothetical protein PST1326 nifH ++++ Fe protein, nitrogenase reductase NifH PST1327 nifD ++++ MoFe protein, alpha subunit PST1328 nifK ++++ MoFe protein, beta subunit PST1329 nifT ++ nitrogen fixation protein PST1330 nifY +++ nitrogenase iron-molybdenum cofactor biosynthesis PST1331 +++ conserved hypothetical protein PST1332 + leucine-rich repeat domain protein nitrogenase iron-molybdenum cofactor biosynthesis PST1333 nifE ++++ protein nitrogenase iron-molybdenum cofactor biosynthesis PST1334 nifN +++ protein nitrogenase iron-molybdenum cofactor biosynthesis PST1335 nifX ++++ protein PST1336 ++ protein of unknown function DUF269 PST1337 ++++ protein of unknown function DUF683 PST1338 ++++ ferredoxin, 4Fe-4S PST1339 + ferredoxin, 2Fe-2S PST1342 + conserved hypothetical protein PST1343 + conserved hypothetical protein PST1344 ++ conserved hypothetical protein PST1346 modB + molybdate ABC transporter, permease protein
Yan et al. www.pnas.org/cgi/content/short/0801093105 9of18 Table S2. (Continued) molybdenum ABC transporter, periplasmic binding PST1347 modA ++ protein PST1348 + putative molybdenum-binding protein PST1349 hesB ++++ Fe-S cluster assembly protein PST1350 nifU +++ Fe-S cluster assembly protein NifU PST1351 nifS ++++ nitrogenase metalloclusters biosynthesis proteinNifS PST1352 nifV ++++ NifV protein, encodes a homocitrate synthase PST1353 cysE ++++ serine acetyltransferase (CysE-like) PST1354 +++ conserved hypothetical protein PST1355 nifW ++++ nitrogenase stabilizing/protective protein NifW PST1356 nifZ ++++ Fe-S cofactor synthesis protein NifM protein, putative a peptidyl-prolyl cis/trans PST1357 nifM ++++ isomerase PST1358 +++ ATP-dependent Clp protease, ATP-binding subunit ClpX flavodoxin required for electron transfer to the Fe PST1359 nifF +++ protein PST1360 ++ glutathione peroxidase PST1379 algZ + alginate biosynthesis transcriptional activator PST1404 fliC + flagellin type B PST1471 hsdS + type I restriction-modification system, S subunit PST1495 + TonB-dependent siderophore receptor PST1521 oprF + outer membrane protein OprF PST1561 + ribosomal subunit interface protein, putative PST1563 adhC + alcohol dehydrogenase class III PST1610 + flavin monoamine oxidase-related protein PST1633 + DNA topoisomerase I: Restriction endonuclease PST1641 + mechanosensitive ion channel family protein PST1673 catC + muconolactone isomerase PST1675 benE + benzoate transport protein PST1711 exbD1 + TonB system transport protein ExbD1 PST1712 exbB1 + TonB system transport protein ExbB1 PST1713 ++++ TonB protein PST1714 + predicted Zn-dependent protease PST1715 ++ peptidase, TldD/PmbA family PST1716 mvaB + hydroxymethylglutaryl-CoA lyase PST1733 + cell division inhibitor-related protein PST1753 + glycosyl transferase, group 1 family protein PST1760 + glycosyl transferase, group 2 family protein PST1762 + aminotransferase, DegT/DnrJ/EryC1/StrS family PST1889 + methyl-accepting chemotaxis protein
Yan et al. www.pnas.org/cgi/content/short/0801093105 10 of 18 Table S2. (Continued) PST1941 + transcriptional regulator, LysR family PST1954 ++ nitrite reductase [NAD(P)H], small subunit PST1955 ++++ nitrite reductase [NAD(P)H] large subunit PST1992 + ABC transporter, periplasmic substrate-binding protein PST1993 ++ glutamate-ammonia ligase chain F, crystal structure of creatinine amidohydrolase PST1998 + (creatininase) ABC-type nitrate/sulfonate/bicarbonate transport PST2003 ++ systems, periplasmic component PST2011 + ABC transporter, permease protein PST2048 + universal stress family protein PST2137 glgA + glycogen synthase PST2154 + alpha-amylase family protein PST2179 strU + NAD(P)-dependent oxidoreductase PST2192 mtlG + mannitol ABC transporter, permease protein maltose/maltodextrin ABC transporter, ATP-binding PST2193 mtlK + protein putative ABC transporter periplasmic solute-binding PST2199 + protein PST2234 + heat-shock protein, HSP20 family PST2335 + aminotransferase PST2400 nasS + nitrate-binding protein NasS PST2406 nasA ++ nitrate transporter PST2409 +++ assimilatory nitrite reductase large subunit PST2410 ++ assimilatory nitrite reductase small subunit PST2411 + assimilatory nitrate reductase PST2500 + N-acetyl-mannosamine transferase PST2501 + capsular polysaccharide biosynthesis protein PST2508 ++ methyl-accepting chemotaxis transducer PST2575 fliR + flagellar biosynthetic protein FliR PST2748 + OmpA family protein PST2862 ++ nucleoside-binding outer membrane protein PST2885 + similar to circadian oscillation regulator PST2897 + probable oxidoreductase PST2899 ++ hypothetical protein PST2900 + probable ABC transporter, ATP-binding component PST2907 + ABC transporter, ATP-binding protein PST2913 topB ++ DNA topoisomerase III PST2922 + hydrolase, alpha/beta fold family PST2982 braC + branched-chain amino acid transport protein BraC
Yan et al. www.pnas.org/cgi/content/short/0801093105 11 of 18 Table S2. (Continued) PST3079 +++ membrane protein, bmp family PST3080 + oxidoreductase, 2OG-Fe(II) oxygenase family PST3101 + transcriptional regulator, TetR family PST3106 + NAD-dependent aldehyde dehydrogenases PST3127 estA + esterase EstA PST3130 iciA + chromosome initiation inhibitor PST3159 + prolyl oligopeptidase family protein PST3246 + PqiB family protein PST3247 + paraquat-inducible protein A PST3253 + membrane protein, putative PST3305 ++ OsmY-related protein PST3319 ftsH + cell division protein FtsH PST3361 czcA + heavy metal efflux pump CzcA permease of the drug/metabolite transporter (DMT) PST3408 + superfamily PST3416 + Co/Zn/Cd efflux system component PST3417 + predicted transcriptional regulators PST3422 + outer membrane porin PST3428 + cation efflux system protein PST3455 + ATPase of the AAA+ class PST3503 + probable periplasmic protein PST3510 dnrS + transcriptional regulator DnrS PST3521 norB + nitric-oxide reductase subunit B PST3522 norC + nitric-oxide reductase subunit C PST3532 nirS + cytochrome cd1 nitrite reductase precursor PST3533 nirQ + denitrification regulatory protein NirQ PST3536 nirJ + heme d1 biosynthesis protein NirJ PST3566 cynS ++ cyanate lyase PST3569 codB + cytosine transporter PST3570 codA + cytosine deaminase PST3584 + oxidoreductase, 2OG-Fe(II) oxygenase family PST3597 +++ bacterial luciferase family protein PST3598 ++ isochorismatase family protein PST3621 + transcriptional regulator, AraC family PST3680 + ethanolamine transporter PST3681 eutB + ethanolamine ammonia-lyase large subunit branched-chain amino acid ABC transporter, PST3720 +++ periplasmic amino acid-binding protein PST3726 ureD-2 ++ urease accessory protein UreD PST3727 ureA ++ urease, gamma subunit UreA
Yan et al. www.pnas.org/cgi/content/short/0801093105 12 of 18 Table S2. (Continued) PST3736 ureE ++ urease accessory protein UreE PST3737 ureF-2 + urease accessory protein UreF PST3738 ureG + urease accessory protein UreG PST3780 rodA + rod-shape-determining protein RodA PST3781 mrdA-2 + penicillin-binding protein 2 PST3795 + ribosome-associated GTPase PST3929 + ABC transporter, ATP-binding protein PST3971 metW + methionine biosynthesis protein MetW ABC-type amino acid transport/signal transduction PST4084 + systems, periplasmic component PST4091 nasR + nitrate-and nitrite-responsive positive regulator NrtA-type periplasmic nitrate transport binding protein, PST4092 nasF ++++ probable PST4093 nasE ++ nitrate ABC transporter permease protein PST4094 nasD ++++ nitrate ABC transporter, ATP-binding protein, putative PST4095 ++ putative acetyltransferase PST4124 + periplasmic binding protein, putative PST4164 coxA + cytochrome c oxidase, subunit I
Genes/products coded by color as follows (genes coding for hypothetical protein outside the nif island were excluded from this table): red, nitrogen fixation island; green, nitrogen metabolism; deep yellow, chemotaxis or adhesion; pink, related to osmotolerance and oxidative stress; blue, transporters/membrane proteins; black, other enzymes, regulators or functional proteins; Upregulation factor between 2–4, ϩ; between 4–8, ϩϩ; between 8–16, ϩϩϩ; above 16, ϩϩϩϩ. *PST0502: increased expression of glnK. We recently established that a glnK mutant is NifϪ and that GlnK controls nifA expression. [He, et al. (2009) Involvement of GlnK, a PII protein, in control of nitrogen fixation and ammonia assimilation in Pseudomonas stutzeri A1501. Arch Microbiol, 10.1007/s00203-008-0354-x].
Yan et al. www.pnas.org/cgi/content/short/0801093105 13 of 18 Table S3. Putative iron transporters identified in the P. stutzeri A1501 genome Gene ID Gene name Functional description
PST0031 TonB-dependent receptor PST0147 TonB-dependent ferric siderophore receptor PST0819 TonB system biopolymer transport component PST0820 exbB2 TonB system transport protein ExbB2 PST0821 exbD2 TonB system transport protein ExbD2 PST0822 TonB family protein PST0846 TonB-dependent receptor, B12 family PST0996 TonB-dependent siderophore receptor PST1245 TonB-dependent receptor protein PST1289 TonB-dependent receptor PST1495 TonB-dependent siderophore receptor PST1710 TonB-dependent receptor PST1711 exbD1 TonB system transport protein ExbB1 PST1712 exbB1 TonB system transport protein ExbB1 PST1713 TonB protein, C-terminal domain PST1944 TonB-dependent receptor PST1948 TonB protein, C-terminal domain PST2208 TonB-dependent siderophore receptor PST2276 TonB-dependent receptor PST3505 Iron permease, FTR1 family PST3955 TonB protein, C-terminal domain PST4065 Iron ABC transporter, permease protein PST4066 Iron ABC transporter, periplasmic iron-binding protein PST4174 TonB-dependent siderophore receptor
Yan et al. www.pnas.org/cgi/content/short/0801093105 14 of 18 Table S4. Genes involved in denitrification process Gene ID Gene name Functional description
PST0891 narL Two-component response regulator NarL PST0892 narX Two-component sensor NarX PST0893 narK Nitrite extrusion protein PST0895 narM Nitrate transporter PST0896 narG Respiratory nitrate reductase alpha chain PST0897 narH Respiratory nitrate reductase beta chain PST0898 narJ Respiratory nitrate reductase delta chain PST0899 narI Respiratory nitrate reductase gamma chain PST1266 napC Periplasmic nitrate reductase c subunit PST1267 napB Periplasmic nitrate reductase b subunit PST1268 napA Periplasmic nitrate reductase large subunit precursor PST1269 napD Uncharacterized protein involved in formation of periplasmic nitrate reductase PST1270 napE Periplasmic nitrate reductase NapE subunit PST3516 norR Nitric oxide reductase regulator PST3515 dnrN Regulator of cell morphogenesis and NO signaling PST3517 dnrO DnrO protein, function unknown PST3518 dnrD Transcription factor for denitrification gene expression elicited by NO PST3519 dnrP DnrP protein, function unknown PST3520 norD Nitric-oxide reductase activation protein PST3521 norB Nitric-oxide reductase subunit B PST3522 norC Nitric-oxide reductase subunit C PST3523 nirH Nitrite reductase heme biosynthesis H protein PST3524 nirG Putative heme biosynthesis protein PST3525 nirL Heme d1 biosynthesis protein NirL PST3526 nirD Transcriptional regulators PST3527 nirF Heme d1 biosynthesis protein NirF PST3528 nirC c-type cytochrome precursor PST3529 nirM Cytochrome c-551 precursor PST3530 nirB Denitrification system component cytochrome c-552 PST3531 nirT Tetraheme protein NirT precursor PST3532 nirS Cytochrome cd1 nitrite reductase precursor PST3533 nirQ Denitrification regulatory protein NirQ PST3534 nirO Cytochrome c oxidase subunit III family protein PST3535 nirP Membrane protein NirP PST3536 nirJ Heme d1 biosynthesis protein NirJ PST3537 nirE Uroporphyrinogen-III C-methyltransferase PST3538 nirN Probable c-type cytochrome PST3539 nirY Transcriptional Regulator, LysR family PST3546 nosL Lipoprotein involved in nitrous oxide reduction PST3547 nosY ABC-type transport system involved in multi-copper enzyme maturation PST3548 nosF ABC-type multidrug transport system, ATPase component PST3549 nosD Nitrous oxidase accessory protein PST3550 nosZ Nitrous-oxide reductase precursor PST3551 nosR Regulatory protein NosR
Yan et al. www.pnas.org/cgi/content/short/0801093105 15 of 18 Table S5. Genes potentially involved in resistance to the toxic or oxygen stresses Gene ID Gene name Functional description
Putative catalases PST0039 katE Catalase PST0092 Catalase PST0811 katA Catalase PST1629 katG Catalase/peroxidase HPI PST3568 katB Catalase Superoxide dismutases PST2573 sodC Superoxide dismutase, Cu-Zn PST3132 sodB Superoxide dismutase Peroxidases PST1129 Glutathione peroxidase PST1360 Glutathione peroxidase PST2181 tpx Thiol peroxidase PST2535 Glutathione peroxidase Hydroperoxide reductases PST0005 ahpF-1 Alkyl hydroperoxide reductase subunit F PST0169 ohr Organic hydroperoxide resistance protein PST3161 ahpF-2 Alkyl hydroperoxide reductase subunit F PST3162 ahpC Alkyl hydroperoxide reductase subunit C Glutathione S-transferases PST0872 Glutathione S-transferase PST1982 Glutathione S-transferase domain protein PST2334 Glutathione S-transferase family protein PST2471 gstA Glutathione S-transferase PST2533 Glutathione S-transferase PST2735 Glutathione S-transferase PST3073 Glutathione S-transferase PST3164 Glutathione S-transferase PST3481 Glutathione S-transferase family protein PST4136 Glutathione S-transferase family protein
Yan et al. www.pnas.org/cgi/content/short/0801093105 16 of 18 Table S6. Conserved regulatory genes involved in alginate biosynthesis in P. aeruginosa Gene ID Gene name Functional description
PST0522 algZ Alginate biosynthesis regulatory protein AlgZ/FimS PST0523 algR Alginate biosynthesis regulatory protein AlgR PST0529 algQ Alginate regulatory protein AlgQ PST0530 algP Alginate regulatory protein AlgP PST1225 mucB Negative regulator for alginate biosynthesis MucB PST1226 mucC Positive regulator for alginate biosynthesis MucC PST1379 algZ Alginate biosynthesis transcriptional activator PST3956 algH Alginate biosynthesis nucleoside diphosphate kinase regulator AlgH PST4053 algB Alginate biosynthesis transcriptional regulatory protein AlgB
Yan et al. www.pnas.org/cgi/content/short/0801093105 17 of 18 Table S7. Type I restriction-modification systems identified in the P. stutzeri A1501 genome Gene ID Functional description
PST0248 Type I restriction-modification system endonuclease PST0251 Type I restriction-modification system, S subunit PST0252 Type I restriction-modification system, M subunit PST0632 Type I restriction-modification system, S subunit PST0633 Type I restriction-modification system, M subunit PST0635 Type I restriction-modification system, R subunit PST0639 Type I restriction-modification system endonuclease PST0646 Type I restriction-modification system, S subunit, truncation PST0647 Type I restriction-modification system, M subunit, putative PST1467 Type I restriction-modification system, R subunit PST1469 Type I restriction-modification system, M subunit PST1471 Type I restriction-modification system, S subunit
Yan et al. www.pnas.org/cgi/content/short/0801093105 18 of 18