1. central carbohydrate metabolism - media.nature.com · tes that no ortholog is present in the...

Carbohydrate metabolism

Supplementary Figure 2. Pathway reconstruction, context analysis and colocalization analysis of key metabolic pathways of Ralstonia eutropha H16.

1. Central carbohydrate metabolism

Note: • R. eutropha H16 is limited in sugar degradation, only fructose, 2-ketogluconate and N-acetyl-glucosamine can be

metabolized. • Fructose is catabolized via the Entner-Doudoroff (2-keto-3-deoxy-6-phosphogluconate, KDPG) pathway. • No phosphofructokinase (key enzymes of Embden-Meyerhoff pathway) or 6-phosphogluconate dehydrogenase (key

enzyme of oxidative pentose phosphat cycle) are present in the genome. • The genes for the degradation of sugars via KDPG are located in three clusters on chromosome 2. These clusters contain

the genetic determinants for the degradation of fructose, glucose and glusoaminate. • Glucose is not supporting growth, probably by limited glucose transport into the cell. • Genes for the utilization of the aminosugar N-acetyl-glucosamine are encoded on chromosome 1. The uptake of N-acetyl-

glucosamine is probably mediated by a PTS-type transport system. • Autotrophic fixation of CO2 in R. eutropha H16 proceeds via the Calvin-Benson-Bassham (CBB) cycle. Duplicated cbb

operons are present in R. eutropha H16. Genes for the key enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase are lacking in R. eutropha JMP134.

1.1. Pathway reconstruction of central carbohydrate metabolism in Ralstonia eutropha H16.

Glyceraldehyde-3-phosphate

Fructose-1,6-bisphosphate

Fructose-6-phosphate

PHG422H16_B1390

Phosphofructokinase EC2.7.1.11 not present in H16

H16_B01384PHG416

pHG1

Chromosome 2Chromosome 1

Dihydroxy-acetone

phosphate

1,3-bisphsophoglycerate

H16_B1386PHG418

H16_B1385PHG417

3-phosphoglycerate

2-phosphoglycerate

Phosphoenol-pyruvate

Pyruvate

H16_A0332H16_A0493

H16_A1188

H16_A3602H16_B0961

Gluconate

H16_A1179

6-Phosphogluconate

H16_A1178

2-Keto-3-desoxy-6-phospho-gluconate

H16_A1502

H16_A0568

H16_A3146

H16_A0566

H16_A1047

Erythrose-4-phosphate

Xylulose-5-phosphate

PHG420H16_B1388

H16_A3147

Sedoheptulose-1,7-bisphosphate


Sedoheptulose-7-phosph

PHG420H16_B1388

Xylulose-5-phosphat

Ribose-5-sphate

Ribulose-5-phosphate

PHG423H16_B1391

CO2

PHG426/427H16_B1394/1395

+

PHG422H16_B1390

H16_B01384PHG416

RuBisCo, Ribulose-1,5-bisphosphate carboxylase/oxygenasePGK, Phosphoglycerate kinaseGAP, Glyceraldehyde-3-phosphate dehydrogenaseFSBA, Fructose-1,6-/sedoheptulose-1,7-bisphosphate aldolaseFSBP, Fructose-1,6-/sedoheptulose-1,7-bisphosphataseTKT, TransketolasePPE, Pentose-5-phosphate 3-epimerasePPI, Pentose-5-phosphate isomerase

PGK

GAP

FSBA

FSBP

TKT

FSBP

TKT

FSBA

PPE PPI

GAP, Glyceraldehyde-3-phosphate dehydrogenasePGK, Phosphoglycerate kinaseFBA, Fructose-1,6-bisphosphate aldolaseFSBP, Fructose-1,6-/sedoheptulose-1,7-bisphosphataseTPI, Triose phosphate isomerasePGM, Phosphoglycerate mutaseENO, EnolasePK, Pyruvate kinase

PK

ENO

PGM

Glucose-6-phosphate

FRK, FructokinaseGPI, Glucose-6-phosphate isomeraseGPD, Glucose-6-phosphate 1-dehydrogenasePGL, 6-PhosphogluconolactonasePGD, Phosphogluconate dehydrataseKDPG, KDPG aldolaseKDG, KDG kinase

Fructose

2-Keto-3-deoxy-6-spho-gluconate


Pyruvate +

H16_B1213

H16_B2567

phogluconate

ose-6-phosphate

ose-6-phosphate

H16_B1502

Glucono-1,5-lactone 6-phosphate

H16_B1501H16_B2566

H16_B2565

H16_B1503

H16_A0316

GPI

FRK

GPD

PGL

PGD

KDPG

GK, Gluconate kinasePGD, Phosphogluconate dehydratase

PGD

GK

N-Acetyl-glucosamine (extracellular)

H16_A0311H16_A0312

N-Acetyl-glucosamine 6-phosphate

H16_A0314

Glucosamine 6-phosphate

GPA

PTS-Transport

PTS, Glucosamine specific phosphotransferase systemGPA, N-acetylglucosamine-6-phosphate deacetylaseGPD, Glucosamine-6-phosphate deaminase

H16

GPD

Ribulose-1,5-bisphosphate

PHG421H16_B1389PRK

H16_A1502

+

H16_A2345

Ribulose-5-sphate

Fructose ABC-Transporter

H16_B1498-1500

H16_B1497 Regulator

Fructose (

GlucoseG

H16_B2564

2-Keto-3-deoxy-6-glucona

H16_B1212

K

Glucosaminate

H16_B1211

H16_A0999

H16_A0567

ate

e pho

pho

6-Phos

Fruct

Gluc

_A0315

pho

extracellular)

K

teDG


1.2. Relevant gene clusters 1.2.1. Calvin-Benson-Bassham cycle

CDS2 Organism1

RuBisCo PGK GAP FSBA FSBP TKT PPE PPI TA PRK Regulator cbbR

H16 H16_B1394/1395 PHG426/427

H16_B1385 PHG417

H16_B1386 PHG418

H16_B1384 PHG416

H16_B1390 PHG422

H16_B1388 PHG420

H16_B1389 PHG421

H16_A2345

H16_A2346

H16_B1391 PHG423

H16_B1396 PHG428

JMP134 - + + + + + + + + - - Parden +

+ +

+

+ + +

+ + + +

1H16, Ralstonia eutropha H16; JMP134, Ralstonia eutropha JMP134; Parden, Paracoccus denitrificans 2RuBisCo, Ribulose-1,5-bisphosphate carboxylase/ oxygenase; PGK, Phosphoglycerate kinase; GAP, Glyceraldehyde-3-phosphate dehydrogenase; FSBA, Fructose-1,6-/sedoheptulose-1,7-bisphosphate aldolase; FSBP, Fructose-1,6-/seduheptulose-1,7-bisphosphatase; TKT, Transketolase; PPE, Pentose-5-phosphate 3-epimerase; PPI, Pentose-5-phosphate isomerase; TA, Transaldolase; (+), indicates that an ortholog is present in the organism; (-), indicates that no ortholog is present in the organism Cluster cbb CO2 fixation 1

1The red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues 1.2.2. Embden-Meyerhof-Parnas pathway

CDS2 Organism1

GPI PFK FSBA GAP TRI PGK PGM ENO PK

H16 H16_A1502 - H16_A0568

H16_A3146

H16_A1047

H16_A0566

H16_A0332 H16_A0493

H16_A1188

H16_A0567 H16_A3602 H16_B0961

JMP134 + - + + + + + + + Psae + - +

+ + + + + +

Eco + + + + + + + + + 1H16, Ralstonia eutropha H16; JMP134, Ralstonia eutropha JMP134; Psae, Pseudomonas aeruginosa; Eco, Escherichia coli 2GPI, Glucose phosphate isomerase; PFK, 6-Phosphofructokinase; FBA, Fructose-1,6-bisphosphate aldolase; GAP, Glyceraldehyde-3-phosphate dehydrogenase; PGK, Phosphoglycerate kinase; TPI, Triose phosphate isomerase; PGM, Phosphoglycerate mutase;ENO, Enolase; (+), indicates that an ortholog is present in the organism; (-), indicates that no ortholog ist present in the organism


1.2.3. N-Acetylglucosamine/Gluconate degradation

CDS2 Organism1

putative Regulator

PTS Porin GPA GPD GK PGD putative Regulator

H16 H16_A0310

H16_A0311 H16_A0312

H16_A0313

H16_A0314

H16_A0315

H16_A1179

H16_A1178

H16_A1177

JMP134 - - - - - + + + Psae + + - + + - + - Eco - + - + + + + - 1H16, Ralstonia eutropha H16; JMP134, Ralstonia eutropha JMP134; Psae, Pseudomonas aeruginosa; Eco, Escherichia coli 2PTS, Glucosamine specific phosphotransferase system; GPA, N-acetylglucosamine-6-phosphate deacetylase; GPD, Glucosamine-6-phosphate deaminase; GK, Gluconate kinase; PGD, Phosphogluconate dehydratase; (+), indicates that an ortholog is present in the organism; (-), indicates that no ortholog is present in the organism Cluster: H16_A0310-03155: Degradation of N-acetyl-Glucosamine1

nagC ,outer membrane porin, H16_A0310, Transcriptional regulator, GntR-family

1The red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues Cluster H16_A1177-1179: Degradation of Gluconate to KDPG1

1The red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues 1.2.4. Entner-Doudoroff pathway


CDS2

rganism

AGH KDG, KDPG putative GK PGD PGL GPD O 1 Regulator

H16 H16_B1211 H16_B1212 H16_B1213 _B2562

16_B2564

_B2567

16_B2565

16_B2566 H16

-B2563

H

H16

H

HH16_B1501

JMP134 + + + + + + + +Psae - + + - + + + + Eco - + + - - + + + 1H16, Ralstonia H16; 134, Rals eutropha 134; Psae eudomonas aeruginosa; Escherich li

PG, KDPG-Aldolase; GK, Glucokinase; KDG,

CDS

rganism GPI FRK Transporter Regulator

eutropha JMP tonia JMP , Ps Eco, ia co2GPD, Glucose-6-phosphate 1-dehydrogenase; PGL, 6.-Phosphogluconolactonase; PGD, Phosphogluconate dehydratase; KDKDG Kinase; AGH, 2-Amino-2-keto-gluconate hydrolase; (+), indicates that an ortholog is present in the organism; (-), indicates that no ortholog is present in the organism

2

O 1frcACB frcR

H16

16_B1502

16_B1503

_B1498-

16_B1497 H

H

H16B1500

H

JMP134 + + + +

Psae + + rbsBAC -

Eco + + rbsACB - 1H16, R2

alstonia opha H16; 134, Rals opha JM sae, Pseudomonas aeruginosa; Eco, Escherichia coli tes that no ortholog is present in the organism

eutr JMP tonia eutr P134; PFRK, Fructokinase; GPI, Glucose-6-phosphate isomerase; (+), indicates that an ortholog is present in the organism; (-), indica


Cluster 1 H16_B1497-1501 Fructose transport and degradation to Glucono-1, 5-lactone 6-phosphate1

1The red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues frcR, putative Regulator, NagC family frcACB, orthologs in E. coli and R. solanacearum putative ribose transporters


red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues

16_B1211, putative 2-amino-2-deoxy-D-gluconate hydrolyase; H16_B1210, putative transcriptional regulator, GlvR-family

Cluster 2 H16_B2562-2566 Degradation of Glucose via Glucono-1, 5-lactone 6-phosphate to 2-Keto-3-deoxy-6-phospho-gluconate1

1The red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues H16_B2562, Signal transduction histidine kinase, H16_B2563, Response regulator, OmpR family; McsC, Small-conductance mechano-sensitive channel Cluster 3 H16_B1211-1213 Degradation of Glucosaminate via 2-Keto-3-deoxy-6-phospho-gluconate to Pyruvate and Glyceraldehyde-3-phosphate 1

1The HH16_1209, putative N-acyl-D-amino acid deacylase


1.3. Overview of genes involved in sugar degradation via KDPG.

Fructose

2-Keto-3-deoxy-6-phospho-gluconate

Glyceraldehyde-3-phosphatePyruvate +

6-Phosphogluconate

Fructose-6-phosphate

Glucose-6-phosphate


GPI

FRK

GPD

PGL

PGD

KDPG

Fructose ABC-Transporter

Fructose (extracellular)

GK

2-Keto-3-deoxy-6-gluconate

KDG

Glucosaminate

Glucose-6-phosphate

Glucose

Cluster 1 Cluster 2


2-Keto-3-deoxy-6-phospho-gluconate

GPD

Cluster 3

2-Ketogluconate 6-phosphate

KGR

2-Ketogluconate

6-Phosphogluconate

Cluster 4

KDK

FRK, FructokinaseGPI, Glucose-6-phosphate isomeraseGPD, Glucose-6-phosphate 1-dehydrogenasePGL, 6-PhosphogluconolactonasePGD, Phosphogluconate dehydrataseKDPG, KDPG aldolaseKDG, KDG kinaseKGK, 2-Ketogluconate kinaseKGR, 2-Ketogluconate 6-phosphate reductase

H16_B1213

H16_B2567

H16_B1502

H16_B1501

H16_B2565

H16_B1503

H16_B1498-1500

H16_B1497 Transcriptional regulator, NagC-family

H16_B2564

H16_B1212

H16_B1211

H16_B2566H16_B1210 Transcriptional regulator, GlvR-family

H16_B2562 Signal transduction histidine kinase, H16_B2563 Response regulator, OmpR family

H16_B1813

H16_B1812

Biosynthesis of cofactors

2. Biosynthesis of cofactors

Note: • R. eutropha H16 contains genes for the de novo biosynthesis of all cofactors/vitamines, except for

Coenzyme B12. • Coenzyme B12: R. eutropha H16 is unable to synthesize coenzyme B12 de novo. Nevertheless, the

cells are able to assimilate cobalamin and its precursors. • Biotin: R. eutropha H16 contains no BioY-type Biotin transporter. • Riboflavin: R. eutropha H16 contains a bifunctional RibBA riboflavin biosynthesis gene • NAD+: Two pathways for the biosynthesis of NAD+ are present in R. eutropha H16, starting from

aspartate or tryptophan, respectively. Genes for four transhydrogenases for the synthesis of NAD(P)H could be identified

• Fe-S Cluster assembly: R. eutropha H16 contains two pathways for Fe-S Cluster Assembly. One region is located on chromsome 1 (isc) and one cluster is encoded by chromsome 2 (suf). R. eutropha JMP 134 contains no suf gene region.

2.1. Thiamine biosynthesis

Purine metabolismAminoimidazoleribotide (AIR)

5-(2-Hydroxyethyl)-4-methylthiazole

4-Amino-5-hydroxymethyl-2-methylpyrimidine

H16_A0235 Thiamine biosynthesis protein, ThiC


Pyruvate


Unidentified kinase (ThiM not present in Burkholderiaceae except B. pseuodomallei)

4-Methyl-5-(2-phosphoethyl)-thiazole

H16_A0243

4-Amino-2-methyl-5-diphosphomethylpyrimidine

Hydroxymethylpyrimidine/phosphomethylpyrimidine kinase, ThiD

Thiaminephosphate

Thiamine monophosphate synthase, ThiEH16_A0239

ThiOSGH16_A0236H16_A0237H16_A0238

Thiaminepyrophosphate

Thiamine-phosphate kinase, ThiLH16_A3154

LocalizationChromosome 1

2.1.1. Key enzymes of thiamine biosynthesis in R. eutropha H16 2.1.2. Relevant gene cluster analysis

n IS element separates thiS and thiG In R. solanacearum.

g replicon; numbers indicate percentage of identical residues

ADifferent clustering of thi genes in Burkholderia strains

ondinThe red dots represent the location of the cluster on the corresp


2.2. Riboflavin biosynthesis 2.2.1. Key enzymes of riboflavin biosynthesis in R. eutropha H16

Purine metabolism

GTP

2,5-diamino-6-hydroxy-4-(5-phosphoribosylamino)

pyrimidine

H16_A2854

H16_A2848

5-Amino-6-(5-phosphoribosylamino) uracil

Pentose-phosphate Pathway

GTP cyclohydrolase, RibA

Pyrimidine deaminase/reductase, RibD

In R. eutropha H16 bifunctional riboflavin biosynthesis protein, RibBA

3,4-DHBP synthase, RibB

3,4-Dihydroxy-2-butanone-4-phosphate

Ribulose-5-phosphate

5-Amino-6-(5-phosphoribitylamin) uracil

Pyrimidine deaminase/reductase, RibDH16_A2848

H16_A2854

6,7-Dimethyl-8-(1-D-ribityl) lumazine

Riboflavin synthase beta chain, RibH

Riboflavin synthase alpha chain, RibC

H16_A2855

H16_A2849

Riboflavin


2.2.2. Relevant gene cluster analysis

The red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues


2.3. Biotin biosynthesis 2.3.1. Key enzymes of biotin biosynthesis in R. eutropha H16

Pimelate

6-carboxyhexanoate-CoA ligase

Biotin-CoA ligaseEC6.2.1.11

Pimeloyl-CoA

8-Amino-7-oxononanoate

7,8-Diaminononanoate

Dethiobiotin

Biotin Biotinyl-CoA

H16_A2946H16_0135

Biotin-[acetyl-CoA-carboxylase] ligase, EC6.3.4.15

Biotinsynthase, BioBH16_A0183

Desthiobiotinsynthetase, BioD

H16_A0182

Adenosylmethionine-8-amino-7-oxononanoate transaminase, BioA

H16_A0180

8-amino-7-oxononanoate synthase, BioF

H16_A0181

Biotinyl-CoA

Chromosome 1

H16_B1756

H16_B0928H16_B1438

Chromosome 2


1The red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues


2.4. Uptake and assimilation of coenzyme B12 precursors 2.4.1. Key enzymes of assimilation of coenzyme B12 precursors in R. eutropha H16

Adenosyl-GDP-cobinamide

Adensosylcobinamide

Adenosylcobyrinic acid

Adenosylcobyrinic acida,c diamide

Adenosylcobalamin

Corrinoidtransport

CobalaminbtuFCDEbtuR

Cobinamide

Cobyrinic acid

btuFCDEbtuR

btuR

cbiP

cbiB

cobU

cobS

Aminopropanol-2-P

L-threonine-3P

cobDcobC

α-Ribazole

α-Ribazole-5-P

cobC

Dimethylbenzimidazole

cobTcobU

Localization:

Chromosome 1




2.5. NAD+/NAD(P)H+ biosynthesis 2.5.1. Key enzymes of NAD+/NAD(P)H+ biosynthesis in R. eutropha H16

Nicotinate D-ribonukleotide

Aspartate oxidase, NadBH16_A3036

Quinolinate

Imonoaspartate

L- Aspartate

Quinolinate synthetase, NadAH16_A3038

Quinolinate phosphoribosyl transferase, NadC

H16_A3037

Kynurenine formamidaseH16_A2814

3-Hydroxyanthranilate

3-Hydroxy-L-kynurenine

L- Tryptophan

Kynureninase, KynH16_A2815

Tryptophane 2,3-dioxygenase, Tdo

Formylkynurenine

H16_A2816

Kynurenine

Kynurenine monooxygenase (not present in Proteobacteria)

Quinolinate

Deamido-NAD+

Nicotinate-nucleotide adenylyltransferase, NadD

NAD+ diphosphatase

Nicotinamid D-ribonukleotide

NAD+

H16_A0913

H16_A0749

NAD+ synthase, NadE

H16_A2761 H16_A2761

Nicotinate

Nicotinate phosphoribosyl transferase

H16_A2589Nicotinamide

Nicotinamidase

H16_A1527

NADH+

NAD(P)+ transhydrogenase

H16_A0850-0852H16_A1264-1266H16_A3128-3131


H16_B0560

H16_B1701-1702

Chromosome 2

2.5.2. Relevant gene cluster analysis kyn gene cluster 1



nad gene cluster 1



2.6. Fe-S cluster assembly 2.6.1. Key enzymes of Fe-S cluster assembly in R. eutropha H16

Isc-system

H16_A1158

Cysteine desulfurase, IscS

Proteins

SIscU, IscA, HscA, HscB, Fdx

H16_A1159-A1165

H16_A1157

Transcriptional regulator, IscR

Fe

Fe-S Proteins

Suf-system

Cysteine desulfurase, SufS

Proteins

SSufBCD SufUTR

Transcriptional regulator, SufA

Fe

Fe-S Proteins


H16_B1515 H16_B1512-1514,H16_B1516-1518

H16_A1511

Chromosome 2

2.6.2. Relevant gene cluster analysis isc gene cluster 1

1The red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues suf gene cluster 1

e location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues 1The red dots represent th


2.6.3. Involved CDS Gene Locustag Product

iscR H16_A1157 tional regulator of iron sulfur clusterassembly (IscR) Transcrip

iscS H16_A1158 Cysteine desulfurase (IscS)

iscU H16_A1159 Iron sulfur cluster assembly protein (IscU)

iscA H16_A1160 Iron sulfur cluster assembly protein (IscA) H16_A1161 H16_A1161 Conserved hypothetical protein

hscB H16_A1162 Chaperone protein (HscB)

hscA H16_A1163 Molecular chaperone (HscA)

fdx H16_A1164 Ferredoxin, 2Fe-2S

sufA embly transcriptional regulator (SufA) H16_B1511 Iron sulfur cluster ass

sufB H16_B1512 Iron sulfur cluster assembly protein (SufB)

sufC H16_B1513 Iron sulfur cluster assembly protein (SufC), ABC-type transporter ATPase component: Fe-S family

sufD H16_B1514 Iron sulfur cluster assembly protein (SufD), ABC-type transporter involved in Fe-S cluster assembly, Fe-S family

sufS H16_B1515 Cysteine desulfurase (SufS)

sufU H16_B1516 Iron sulfur cluster assembly protein (SufU)

sufT H16_B1517 Iron sulfur cluster assembly protein (SufT)

sufR H16_B1518 Ferredoxin containing [2Fe-2S] Rieske domain (SufR)

Biosynthesis of aromatic amino acids

3. Biosynthesis of aromatic amino acids

Note: • R. eutropha H16 contains a bifunctional chorismate mutase/prephenate dehydratase. • R. eutropha H16 is able to synthesize tyrosine from phenylalanine by a phenylalanine hydroxylase.

3.1. Key enzymes of aromatic amino acid biosynthesis in R. eutropha H16

Phosphoenolpyruvate + Erythrose-4-phosphate

3-Dehydroquinate

3-Dehydroshikimate

Shikimate 5-dehydrogenase, AroE

Shikimate

DHAP synthase, AroG

3-Dehydroquinate dehydratase, AroQ

Shikimate kinase, AroK/AroL

3-Phosphoshikimate 1-carboxyvinyltransferase, AroA

3-Dehydroquinate synthase, AroB

Shikimate 3-phosphate

3-deoxy-D-arabino-hept-2-ulosonate 7-phosphate

DAHP

PEP

5-O-(1-carboxyvinyl)-3-phosphoshikimate

Chorismate

Chorismate synthase, AroC

H16_A1122

H16_A3434

H16_A3170

H16_A3161

H16_A1411H16_A3435

H16_A0795

H16_A1317

Chorismate

Anthranilate synthase, TrpEG

Anthranilate phosphoribosyltransferase, TrpD

Phosphoribosylanthranilate isomerase, TrpF

Indole-3-glycerol-phosphate synthase, TrpC

Tryptophan synthase, TrpAB

H16_A3319H16_A3320

Antranilate

H16_A3321H16_A0356

Phosphribosylantranilate

Carboxyphenylamino) deoxyribulose 5'-phosphate

H16_A2615

Indole-3-glycerol-phosphate

H16_A3322

H16_A2612H16_A2614

Tryptophan

Aminotransferases, TyrB, AspC

Prephenate dehydratase/Chorismate mutase, PheABifunctional enzyme

Chorismate

H16_A0792

Prephenate

Phenylpyruvate

Prephenate dehydrogenase. TyrA

4-Hydroxyhenylpyruvate

H16_A0794

H16_A1494H16_A1151

Tyrosine

H16_A1104H16_A2267H16_A2557

Phenylalanine

Phenylalanine hydroxylase, PhhA

H16_A3678 H16_A3679Hydroxytetrahydrobiopterin dehydratase, PhhB

H16_A3680-81

Response regulator + Histidine kinase, PhhRS


H16_B1076

H16_B0465

H16_B1081

Chromosome 2

Energy metabolism

4. Energy metabolism

Note: • R. eutropha H16 contains genes for eight distinct terminal oxidases • R. eutropha H16 genome encodes at least four different formate dehydrogenases for the use of formate as

energy source for organoautotrophic growth

4.1. Terminal oxidases 4.1.1. Terminal oxidases encoded in R. eutropha H16 Product Genes in R. eutropha H161

aa3-type cytochrome oxidase ctaA-D (chr1)

bo3-type quinol oxidase cyoA1-D1 (chr1)



cbb3-type cytochrome oxidase ccoNPQO (chr1)

bb3-type cytochrome oxidase coxM-Q (chr2)

bd-type quinol oxidase subunit cydB1A1 (chr2)

bd-type quinol oxidase subunit cydA2B2 (chr2) 1chr1, chromosome 1, chr2, chromosome 2 4.1.2. Distribution of terminal oxidases Organism1

Terminal oxidase H16 JMP134 Rasol Bumal Psae Eco

Heme-copper cytochrome oxidases 2 3 2 1 1 0

Heme-copper quinol oxidases 3 3 2 2 1 1

cbb3-type cytochrome oxidases 1 1 1 1 1 0

bd quinol oxidases 2 2 2 1 1 1 1H16, Ralstonia eutropha H16; JMP134, Ralstonia eutropha JMP134; Rasol, Ralstonia solanacearum; Bumal, Burkholderia mallei; Psae, Pseudomonas aeruginosa; Eco, Escherichia coli

Energy metabolism

4.1.3. Relevant gene cluster analysis Chromosome 1 aa3-type cytochrome oxidase 1

bb -type cytochrome oxidase1


c 3

1The

Energy metabolism

bo3-type quinol oxidase1

Energy metabolism

bd type oxidases 1

red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues 1The

Energy metabolism

4.2. Formate dehydrogenase 4.2.1 Involved CDSs Gene Locustag Product fdsR H16_A0639 Transcriptional regulator of formate dehydrogenase operon

fdsG H16_A0640 NAD-dependent formate dehydrogenase gamma subunit

fdsB H16_A0641 NAD-dependent formate dehydrogenase beta subunit

fdsA H16_A0642 NAD-dependent formate dehydrogenase alpha subunit

fdsC H16_A0643 Uncharacterized protein required for formate dehydrogenase activity

fdsD H16_A0644 NAD-dependent formate dehydrogenase delta subunit fdhD1 H16_A2932 Protein required for formate dehydrogenase activity

fdhC H16_A2934 Cytochrome b subunit of formate dehydrogenase

fdhB1 H16_A2936 Formate dehydrogenase iron-sulfur subunit

fdhA1 H16_A2937 Formate dehydrogenase alpha subunit (Fe4S4 domain)

fdoG H16_B1452 Formate dehydrogenase alpha subunit

fdoH H16_B1453 Formate dehydrogenase beta subunit fdoI H16_B1454 Formate dehydrogenase gamma subunit

fdoE H16_B1455 Uncharacterized protein involved in formate dehydrogenase formation

fdhD2 H16_B1470 Protein required for formate dehydrogenase activity

fdhA2 H16_B1471 Formate dehydrogenase alpha chain

fdwA H16_B1700 Tungsten-containing formate dehydrogenase alpha subunit

fdwB H16_B1701 Tungsten-containing formate dehydrogenase beta subunit

4.2.2. Relevant gene cluster analysis fds gene region (chromosome 1) 1

1The red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues Soluble NAD+-reducing enzyme; fdsR; Transcriptional regulator of formate dehydrogenase operon fdh gene region (chromosome 1) 1

1The red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues Membrane-bound, TAT-dependent enzyme torD, chaperone; ldcD, Arginine/lysine/ornithine decarboxylase

Energy metabolism

fdo gene region (chromosome 2) 1

1The red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues Membrane-bound, TAT-dependent enzyme fdw gene region (chromosome 2) 1


embrane-bound, TAT-dependent enzyme, putative tungsten-dependent

xiT, Oxalate / formate antiporter, MFS-type, OFA family

1The M oH16_B1704+1705 Transcriptional regulators, LysR-family

Denitrification – Anaerobic metabolism

5. Denitrification – Anaerobic metabolism

Note: • R. eutropha H16 forms a complete denitrification pathway under anoxic conditions. • Nearly identical copies of nitrate reductase (nar) and nitric oxide reductase (nor) genes are present on

chromosome 2 and pHG1. • A single gene region for cytochrome cd1 nitrite reductase (nir) is located on chromosome 2. • Genes encoding nitrous oxide reductase (nos) and periplasmic nitrate reductase (nap) are present

only on pHG1 • Adjacent to the nitrous oxide gene cluster on pHG1, genes for an anaerobic ribonucleotide reductase

are present. • Remnants of a gene coding for a copper-containing nitrite reductase (pseudogene PHG241) are

present adjacent to the nitric oxide reductase gene cluster on pHG1, suggesting that genes for a complete denitrification pathway were encoded on pHG1, of which the nitrite reductase was inactivated.

• The anaerobic conversion of coproporphyrinogen III is conducted by an oxygen-independent coproporphyrinogen III oxidase encoded on chromosome 1.

• Two flavohemoglobins (fhp, hmp), possibly involved in the detoxification of NO, are encoded in the R. eutropha H16 genome, the latter is probably regulated by a colocalized nsrR-like transcriptional regulator

• DnrD and NarXL were identified as putative regulators of the nitrite and nitrate reductase regulon, respectively. NorR was previously described as transcriptional regulator of nitric oxide reductase.

5.1. Localization of key enzymes of the dentrification pathway in R. eutropha H16

N2O

NO

NO2-

NO3-

N2

narnap

nor

nos

Nitrate reductase

Nitrite reductase

Nitric oxide reductase

Nitrous oxide reductase

narX1L1dnrDfnr

norR1

nosR

Regulators

NONO3- N2O

fhp fhp

hmp hmp nsrRnnr ?

Regulators

Coproporphyrinogen III hemN

NTPnrdD

dNTP

Protoporphyrinogen II

Denitrification

Anaerobic class III ribonucleotide reductase

NO deoxygenase/reductase

NO-Detoxification

Heme and desoxynucleotide synthesis under anaerobic conditions

Oxygen-independent coproporphyrinogen III oxidase


pHG1

N2O

NO

NO2-

NO3-

N2

narnap

nor

nos

Nitrate reductase

Nitrite reductase

Nitric oxide reductase

Nitrous oxide reductase

narX1L1dnrDfnr

norR1

nosR

Regulators

NONO3- N2O

fhp fhp

hmp hmp nsrRnnr ?

Regulators

Coproporphyrinogen III hemN

NTPnrdD

dNTP

Protoporphyrinogen II

Denitrification

Anaerobic class III ribonucleotide reductase

NO deoxygenase/reductase

NO-Detoxification

Heme and desoxynucleotide synthesis under anaerobic conditions

Oxygen-independent coproporphyrinogen III oxidase


pHG1

nir

nar

nor

? narX2L2

norR2

Regulators

Chromosome 2

nir

nar

nor

? narX2L2

norR2

Regulators

Chromosome 2


5. 2. Cluster analysis narG gene region 1

napA gene region1

irS gene region1

n


orB gene region1

orR gene region1

n

n

nosZ gene region1



5.3. Involved CDSs Gene Replicon Locustag Product nnr Chr1 H16_A1838 Transcriptional regulator, NNR like H16_A1839 Chr1 H16_A1839 Truncated hemoglobin hmp2 Chr1 H16_A3533 Flavohemoprotein (hemoglobin-like) nsrR Chr1 H16_A3534 Putative transcriptional regulator hemN Chr1 H16_A3615 Oxygen-independent coproporphyrinogen III oxidase nnrS Chr2 H16_B2262 NnrS protein narK3 Chr2 H16_B2263 Nitrate / nitrite transporter, MFS-type, NNP family narK4 Chr2 H16_B2264 Nitrate / nitrite transporter, MFS-type, NNP family narG2 Chr2 H16_B2265 Respiratory nitrate reductase alpha chain (NarG) narH2 Chr2 H16_B2266 Respiratory nitrate reductase beta chain (NarH) narJ2 Chr2 H16_B2267 Respiratory nitrate reductase delta chain (NarJ) narI2 Chr2 H16_B2268 Respiratory nitrate reductase gamma chain (NarI) ppiC2 Chr2 H16_B2269 Peptidyl-prolyl cis-trans isomerase can2 Chr2 H16_B2270 Beta-carbonic anhydrase mobB3 Chr2 H16_B2271 Molybdopterin-guanine dinucleotide biosynthesis protein B nirS Chr2 H16_B2277 Cytochrome cd1 nitrite reductase (NirS) nirC Chr2 H16_B2278 Cytochrome c, nitrite reductase associated (NirC) nirF Chr2 H16_B2279 Heme d1 biosynthesis protein (NirF) nirD Chr2 H16_B2280 Heme d1 biosynthesis protein (NirD) nirG Chr2 H16_B2281 Heme d1 biosynthesis protein (NirG) nirH Chr2 H16_B2282 Heme d1 biosynthesis protein (NirH) nirJ Chr2 H16_B2283 Heme d1 biosynthesis protein (NirJ) nirN Chr2 H16_B2284 C-type cytochrome (NirN) nirE Chr2 H16_B2285 Uroporphyrin-III C-methyltransferase norB2 Chr2 H16_B2323 Nitric oxide reductase qNor type (NorB2) norA2 Chr2 H16_B2324 NorA2 protein (NorA2) norR2 Chr2 H16_B2325 Nitric oxide reductase regulator (NorR2) narX2 Chr2 H16_B2333 Signal transduction histidine kinase, nitrate/nitrite-specific narL2 Chr2 H16_B2334 Response regulator, NarL-family fhp phg1 PHG200 flavohemoprotein napE phg1 PHG209 periplasmic nitrate reductase accessory protein napD phg1 PHG210 periplasmic nitrate reductase accessory protein napA phg1 PHG211 periplasmic nitrate reductase large subunit napB phg1 PHG212 periplasmic nitrate reductase small subunit napC phg1 PHG213 periplasmic nitrate reductase transmembrane protein nrdG phg1 PHG238 activase of anaerobic class III ribonucleotide reductase nrdD phg1 PHG240 anaerobic class III ribonucleotide reductase norB1 phg1 PHG244 nitric oxide reductase qNor type norA1 phg1 PHG245 NorA norR1 phg1 PHG246 sigma 54 dependent transcriptional activator nosL phg1 PHG247 putative lipoprotein nosY phg1 PHG248 putative transmembrane protein nosF phg1 PHG249 putative copper ATP-binding ABC transporter nosD phg1 PHG250 putative copper binding periplasmic protein nosR phg1 PHG251 putative transcription regulator nosZ phg1 PHG252 nitrous oxide reductase nosC phg1 PHG253 putative cytochrome c-type protein nosX phg1 PHG254 NosX


dnrD phg1 PHG262 transcriptional regulator PHG263 phg1 PHG263 conserved hypothetical protein PHG264 phg1 PHG264 conserved hypothetical protein narX1 phg1 PHG265 transmembrane nitrate/nitrite sensor kinase narL1 phg1 PHG266 nitrate/nitrite response regulator PHG267 phg1 PHG267 conserved hypothetical protein narK1 phg1 PHG268 putative nitrite/nitrate transporter narK2 phg1 PHG269 putative nitrite/nitrate transporter narG1 phg1 PHG270 respiratory nitrate reductase catalytic subunit narH1 phg1 PHG271 respiratory nitrate reductase subunit narJ1 phg1 PHG272 respiratory nitrate reductase subunit narI1 phg1 PHG273 respiratory nitrate reductase transmembrane subunit PHG274 phg1 PHG274 probable peptidyl-prolyl cis-trans isomerase mobB1 phg1 PHG275 putative molybdopterin cofactor biosysnthesis protein fnr1 phg1 PHG276 transcriptional regulator

Cell motility

6. Cell motility

5.1. Cluster analysis flg gene region 1

Cluster 1 fli gene region 1

red dots represent the location of the cluster on the corresponding replicon; numbers indicate percentage of identical residues 1The

Note: • Genes for the synthesis of flagella and for chemotaxis proteins are localized on chromosome 2 in R.

eutropha H16. In R. eutropha JMP 134 and R. solanacearum the determinants for cell motility are encoded on the corresponding small replicons. The genes for cell motility are located on chromosome 1 in Burkholderia mallei.

Cell motility

Cluster 2 fli gene region 1


Cell motility

Cluster 1 che gene region1

Cluster 2 che gene region1


1. central carbohydrate metabolism - media.nature.com · tes that no ortholog is present in the...

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