Supplementary Figure 3. Degradation of aromatic compounds

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1 Supplementary Figure 3. Degradation of aromatic compounds Degradation of benzoate and hydroxybenzoate proceeds via the β-ketoadipate pathway. The genes of the catechol branch of this pathway (H16_A ), which allows the degradation of benzoate to 3-oxoadipate via the intermediate catechol, are located on chromosome 1, while those of the protocatechuate branch, involved in the degradation of 4-hydroxybenzoate to 3- oxoadipate via protocatechuate, are on chromosome 2 (Supplementary Fig. 4a). The functions for transport and degradation of 4-hydroxybenzoate to 3-oxoadipate probably belong to a single operon. An interesting feature of this operon is the fused gene H16_B2288 predicting a chimeric enzyme. Usually, the conversion of 4-carboxymuconolactone to 3-oxoadipate is catalyzed by separate 4-carboxymuconolactone decarboxylase and 3-oxoadipate enol-lactone hydrolase enzymes. In R. eutropha H16 the coding sequences corresponding to the two enzyme families are coupled in a single CDS, suggesting that a bifunctional enzyme catalyzes both reactions. A similar case has been described for Rhodococcus opacus 1. Genes for the subsequent degradation of 3-oxoadipate (3-oxoadipate CoA-transferase) are located on chromosome 2 and in one additional copy on phg1. The last enzyme of this pathway, β-ketoadipyl-coa thiolase, is also encoded on chromosome 2 (pcaf, H16_B0200). Since genes for the degradation of 3-oxoadipate are not present on chromosome 1, chromosome 2 is essential for complete degradation of aromatic compounds via 3-oxoadipate. In addition, chromosome 2 is required for the degradation of phenol and 4-cresol via meta-cleavage (H16_B ) (Supplementary Fig. 4b) and the degradation of biphenyl (biphenyl-2,3-diol) (H16_ , H16_B0600, H16_B0654) (Supplementary Fig. 4c). Chromosome 2 also encodes both a gentisate pathway, which is necessary for metabolizing 3-hydroxybenzoate (H16_B ), and a pathway for the degradation of 6-chlorohydroxyquinol to 3-oxoadipate. It has already been shown, that R. eutropha H16 is able to degrade chlorinated aromatic pollutants. like 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid (3-CB), when plasmid pjp4 is transferred to the strain 2. Enzymes for the aerobic degradation of benzoate by a novel degradation pathway via CoAligation 3 are present in two clusters on chromosome 1 and chromosome 2 (Supplementary Fig. 4f). 1. Eulberg, D., Lakner, S., Golovleva, L.A. & Schlömann, M. Characterization of a protocatechuate catabolic gene cluster from Rhodococcus opacus 1CP: evidence for a merged enzyme with 4-carboxymuconolactone-decarboxylating and 3-oxoadipate enol-lactone-hydrolyzing activity. J. Bacteriol. 180, (1998). 2. Friedrich, B., Meyer, M. & Schlegel, H.G. Transfer and expression of the herbicide-degrading plasmid pjp4 in aerobic autotrophic bacteria. Arch. Microbiol. 134, (1983). 3. Gescher, J., Zaar, A., Mohamed, M., Schägger, H. & Fuchs, G. Genes coding for a new pathway of aerobic benzoate metabolism in Azoarcus evansii. J Bacteriol 184, (2002).

2 Supplementary Figure 4a. Benzoate degradation via ortho-cleavage - genes for the 3-oxoadipate pathway are located on three replicons H16_A1965 benm Regulator, benm H16_B2292 pcaq PHG386 mmlh PHG385 mmli PHG384 mmlj H16_A1963 bena H16_A1962 benb H16_A1961 benc H16_A1960 bend H16_A1964 cata PHG405 catb1 PHG394 catb2 H16_A1966 catb3 PHG404 catc H16_A1967 catc3 Benzoate 1,2-dioxygenase EC Dihydrodihydroxybenzoate dehydrogenase EC Catechol 1,2-dioxygenase EC Muconate cycloisomerase EC Muconolactone deltaisomerase EC Benzoate 4-hydroxybenzoate 1,6-Dihydroxy-cis- 2,4-cyclohexadiene -1-carboxylic acid Catechol cis,cis Muconate Muconolactone H16_A2422 pcab2 H16_B1584 catc2 Protocatechuate 3-Carboxy-cis,cismuconate 4-Carboxymuconolactone Regulator p-hydroxybenzoate hydroxylase EC Protocatechuate 3,4- dioxygenase EC Carboxy-cis,cis-muconate cycloisomerase EC H16_B2287 pobr H16_B2293 pcak H16_B2286 poba H16_B2290 pcag H16_B2291 pcah H16_B2289 pcab H16_A1968 catd 3-Oxoadipate enol-lactone hydrolase EC Oxoadipate enol-lactone H16_B1583 catd2 (4-Oxoadipate enol-lactone ) 4-Carboxymuconolactone decarboxylase/3-oxoadipate enol-lactone hydrolase/ EC /EC H16_B2288 pcacd Bifunctional gene in R.eutropha H16 PHG388 cati PHG387 catj 3-Oxoadipate 3-Oxoadipate 3-Oxoadipate CoAtransferase EC H16_B0198 pcai H16_B0199 pcaj EC Oxoadipyl-CoA Acetyl-CoA + Succinyl-CoA Beta-ketoadipyl CoA thiolase EC H16_B0200 pcaf

3 Supplementary Figure 4b. Phenol degradation via meta-cleavage forms one cluster on chromosome 2 Phenol Catechol 2-Hydroxymuconate semialdehyde 2-Hydroxymuconate γ-oxalocrotonate 2-Oxopent-4-enoate 4-Hydroxy-2- oxopentenoate Regulator Phenolhydroxylase EC Catechol 2,3-dioxygenase EC Hydroxymuconic semialdehyde dehydrogenase EC Oxalocrotonate tautomerase EC Oxalocrotonate decarboxylase EC Oxopent-4-enoate hydratase EC Hydroxy-2-ketovalerate aldolase EC H16_B0538 poxr H16_0539- H16_B0545 poxabcdef H16_B0546 H16_B0547 H16_B0550 H16_B0549 H16_B0548 mhpd H16_B0552 mhpe Pyruvate + Acetaldehyde Acetyl-CoA Acetaldehyde dehydrogenase EC H16_B0551 mhpf

4 Supplementary Figure 4c. Biphenyl degradation gene cluster on chromosome 2 Biphenyl-2,3-diol H16_B0654 bphc Biphenyl-2,3-diol:oxygen 1,2- oxidoreductase (decyclizing) EC Hydroxy-6-oxo-6-phenylhexa-3-enoate H16_B0600 bphd 2,6-Dioxo-6-phenylhexa-3-enoate hydrolase EC Benzoate + 2-Oxopent-4-enoate H16_B0597 bphh H16_B0595 bphi 4-Hydroxy-2- oxopentenoate 2-Oxopent-4-enoate hydratase EC Hydroxy-2-ketovalerate aldolase EC H16_B0596 Pyruvate + Acetaldehyde Acetyl-CoA Acetaldehyde dehydrogenase EC

5 Supplementary Figure 4d. Gentisate pathway H16_B0877 pcak2 3-Hydroxybenzoate 3-hydroxybenzoate 6- hydroxylase EC H16_B0876 2,5-Dihydroxyphenylacetate (Homogentisate) 3-Maleylacetoacetate 3-Fumarylacetoacetate Fumarate + Acetoacetate Homogentisate 1,2- dioxygenase EC Maleylacetoacetate isomerase EC Fumarylacetoacetate hydrolase EC H16_B1671 hmga H16_A0362 H16_B1670 faha H16_A0361 2,5-Dihydroxybenzoate (Gentisate) 3-Maleylpyruvate 3-Fumarylpyruvate Fumarate + Pyruvate Gentisate 1,2-dioxygenase EC Maleylpyruvate isomerase EC Fumarylpyruvate hydrolase EC H16_B0873 H16_B0875 H16_B0875

6 Supplementary Figure 4e. Degradation of chlorinated aromatic compounds H16_B0968 pcpa 6-Chlorohydroxyquinol 2-Chloromaleylacetate 6-Chlorohydroxyquinol-1,2- dioxygenase/hydroxyquinol-1,2- dioxygenase EC H16_B0970 pcpe Maleylacetate H16_A Chloromaleylacetate/maleylacetate reductase EC Oxoadipate

7 Supplementary Figure 4f. Predicted benzoate degradation via CoA-ligation Regulator H16_A1411 H16_B1919 H16_B1918 Benzoate Benzoyl-CoA Benzoate-CoA ligase H16_A1418 H16_A1417 H16_A1416 H16_A1415 H16_A1414 H16_A1412 H16_B1912 boxa H16_B1913 boxb Benzoyl-CoA oxygenase/reductase H16_A1408 bdxa H16_A1409 bdxb H16_B1914 Benzoyl-CoA dihydrodiol lyase H16_A1410 (6-hydroxy-3-hexenoyl- CoA) cis-3,4-lactone Lactonase H16_A Hydroxyadipyl-CoA 3-Oxoadipyl-CoA Acetyl-CoA + Succinyl-CoA

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