Characterization of p-hydroxycinnamate catabolism in a soil Actinobacterium.

Characterization of p-Hydroxycinnamate Catabolism in a Soil Actinobacterium Hiroshi Otani, Young-Eun Lee, Israël Casabon, Lindsay D. Eltis

p-Hydroxycinnamates, such as ferulate and p-coumarate, are components of plant cell walls and have a number of commercial applications. Rhodococcus jostii RHA1 (RHA1) catabolizes ferulate via vanillate and the ␤-ketoadipate pathway. Here, we used transcriptomics to identify genes in RHA1 that are upregulated during growth on ferulate versus benzoate. The upregulated genes included three transcriptional units predicted to encode the uptake and ␤-oxidative deacetylation of p-hydroxycinnamates: couHTL, couNOM, and couR. Neither ⌬couL mutants nor ⌬couO mutants grew on p-hydroxycinnamates, but they did grow on vanillate. Among several p-hydroxycinnamates, CouL catalyzed the thioesterification of p-coumarate and caffeate most efficiently (kcat/Km ⴝ ⬃400 mMⴚ1 sⴚ1). p-Coumarate was also RHA1’s preferred growth substrate, suggesting that CouL is a determinant of the pathway’s specificity. CouL did not catalyze the activation of sinapate, in similarity to two p-coumaric acid:coenzyme A (CoA) ligases from plants, and contains the same bulged loop that helps determine substrate specificity in the plant homologues. The couO mutant accumulated 4-hydroxy-3-methoxyphenyl-␤-ketopropionate in the culture supernatant when incubated with ferulate, supporting ␤-oxidative deacetylation. This phenotype was not complemented with a D257N variant of CouO, consistent with the predicted role of Asp257 as a metal ligand in this amidohydrolase superfamily member. These data suggest that CouO functionally replaces the ␤-ketothiolase and acyl-CoA thioesterase that occur in canonical ␤-oxidative pathways. Finally, the transcriptomics data suggest the involvement of two distinct formaldehyde detoxification pathways in vanillate catabolism and identify a eugenol catabolic pathway. The results of this study augment our understanding of the bacterial catabolism of aromatics from renewable feedstocks.

T

here is burgeoning interest in the bacterial catabolism of lignocellulose-derived aromatic compounds. One class of the aromatic compounds that occur abundantly in biomass consists of p-hydroxycinnamates, such as ferulate and p-coumarate (Fig. 1). These compounds occur either in free form or esterified to lignin and other polymers in the plant cell walls (1). p-Hydroxycinnamates are precursors of various antimicrobial compounds, including several that play important roles in plant defense (2). Commercial applications include their use as antioxidants (3). More recently, dihydroferulate has been touted as a precursor for biorenewable thermoplastics (4). Therefore, the characterization of the bacterial catabolism of p-hydroxycinnamates should facilitate the development of biocatalysts to transform biomass for various applications. Rhodococcus is a genus of mycolic-acid-producing soil actinobacteria that have been studied in part for their considerable biocatalytic potential (5). Rhodococcus jostii RHA1 (RHA1), isolated from lindane-contaminated soil, degrades a wide variety of aromatic compounds, including biphenyls and lignin-derived monoaryls (5, 6). Genomic analyses of RHA1 (7) and other rhodococci have revealed that their aromatic-compound catabolic pathways are modular: peripheral pathways degrade compounds such as biphenyl, benzoate, and vanillate to common intermediates such as protocatechuate, while central pathways transform common intermediates to central metabolites (5). For example, RHA1 degrades vanillate to protocatechuate via a peripheral pathway and then to central metabolites by the ␤-ketoadipate pathway (6). RHA1 also contains a dye-decolorizing peroxidase (DyP) that catalyzes the H2O2-dependent oxidation of lignin (8, 9). More recently, we have shown that ferulate and p-coumarate were produced when RHA1 was cultivated in the presence of wheat

December 2014 Volume 196 Number 24

straw lignocellulose and that RHA1 metabolizes p-hydroxycinnamates (10). Two p-hydroxycinnamate catabolic pathways have been identified in bacteria. Both pathways involve the initial activation of the acid by coenzyme A (CoASH) in a reaction catalyzed by an aryl-CoA synthetase, and both transform ferulate to vanillate and acetyl-CoA (11, 12, 13, 14, 15). The more commonly described of the two pathways has been experimentally verified. In this pathway, feruloyl-CoA is transformed via a dual enoyl-CoA hydratase/ aldolase to vanillin and acetyl-CoA via 4-hydroxy-3-methoxyphenyl-␤-hydroxypropionyl-CoA. The vanillin is then oxidized to vanillate by a dehydrogenase (see Fig. S1 in the supplemental material) (1, 15). A second pathway involving ␤-oxidation has been proposed to occur in Rhodotorula rubra, Rhodococcus sp. strain I24, white-rot fungus Pycnoporus cinnabarinus, and Agrobacterium fabrum (11, 12, 13, 14). In this pathway, feruloyl-CoA is transformed by a hydratase and a dehydrogenase to yield a ␤-ketoacyl-CoA. It has been proposed that this is cleaved by a ␤-ketothiolase to acetyl-CoA and vanillyl-CoA and that the latter is hydrolyzed by an acyl-CoA thioesterase (15). The last two steps have not been experimentally verified. However, it was recently proposed that they are catalyzed by a novel ␤-ketothiolase in A. fab-

Received 27 August 2014 Accepted 20 September 2014 Published ahead of print 29 September 2014 Address correspondence to Lindsay D. Eltis, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JB.02247-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JB.02247-14

Journal of Bacteriology

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Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada

Otani et al.

O

OH

O

OH

O

O

OH

OH

OH

OCH3

O

OCH3

OH

OH

OH

OH

1

2

3

4

5

OH

O

OH

O

O

OH

O

H 3CO

OCH3

OCH3

6

OCH3

OH

OH

7

8

OH

9

10

FIG 1 Structures of the aromatic compounds used in this study. 1, p-coumarate; 2, ferulate; 3, caffeate; 4, dihydroferulate; 5, dihydro-p-coumarate; 6, sinapate; 7, dihydrocoumarin; 8, eugenol; 9, benzoate; 10, vanillate. Compounds 1 to 6 are p-hydroxycinnamates.

rum (11). Studies performed with the deletion strains of vanA and vdh, essential for vanillate and vanillin catabolism, respectively, established that RHA1 catabolizes ferulate via vanillate but not via vanillin, suggesting that this strain catabolizes ferulate via ␤-oxidation (10). In contrast, Rhodococcus opacus PD630, a closely related strain, is proposed to catabolize ferulate via the non-␤-oxidation pathway (14). Here, we investigated the p-hydroxycinnamate catabolic pathway of RHA1. We conducted transcriptomic and bioinformatics analyses to identify genes potentially involved in p-hydroxycinnamate catabolism or related processes, such as formaldehyde detoxification. Candidate genes predicted to encode p-hydroxycinnamoyl-CoA synthetase and a metal-dependent amidohydrolase, respectively, were deleted, and their metabolites were analyzed to validate the pathway. The purified aryl-CoA synthetase was characterized with respect to its specificity for a range of p-hydroxycinnamates. The results were used to inform further growth studies of RHA1. The elucidated p-hydroxycinnamate catabolic pathway is discussed with respect to the organization of catabolic pathways in Rhodococcus and what has been proposed in other bacteria. MATERIALS AND METHODS Chemicals. Dihydroferulate was purchased from Alfa Aesar (Ward Hill, MA). Other aromatic compounds, ATP, and CoA were purchased from Sigma-Aldrich. Growth conditions. Rhodococcus strains were routinely cultivated at 30°C in 50 ml M9 mineral medium supplemented with a salt stock solution (16) and 2.5 mM aromatic compound unless otherwise mentioned. Cultures were inoculated with 50 ␮l of a cell suspension prepared by growing cells on 5 mM pyruvate M9 for 4 days, harvesting the cells by centrifugation, and suspending them in the same volume of M9. Escherichia coli was grown as described by Maniatis et al. (17). Media were supplemented with ampicillin (50 mg liter⫺1), kanamycin (50 mg liter⫺1), neomycin (10 mg liter⫺1), and apramycin (50 mg liter⫺1) as appropriate. DNA manipulation and generation of mutants. DNA was propagated, amplified, purified, digested, and ligated using standard protocols (17). Oligonucleotides used in this study are listed in Table S1 in the supplemental material. Strains and plasmids used and generated in this

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study are listed in Table S2. To clone couL, the gene was amplified using primers couL-F and couL-R. The amplicon was digested using NdeI and BamHI and cloned into pET41b to yield pET41couL. To construct the ⌬couL mutant strain, upstream and downstream regions of couL were amplified using primer pair couL-FF and couL-FR and primer pair couL-RF and couL-RR, respectively. The two amplicons were combined using splicing by overlap extension-PCR (SOE-PCR). This fragment was digested using XbaI and EcoRI and cloned into pK18mobsacB to yield pK18⌬couL. The ⌬couO strain was constructed using the same strategy. Upstream and downstream regions were amplified using primer pair couO-FF and couO-FR and primer pair couO-RF and couO-RR, respectively, and results were combined using SOE-PCR. This fragment was digested using XbaI and HindIII and cloned into pK18mobsacB to yield pK18⌬couO. RHA1 cells were transformed with either pK18⌬couL or pK18⌬couO by electroporation (18). Neomycin resistance colonies resulting from a single crossover were isolated and replica plated on LB supplemented with 10% (wt/vol) sucrose (19). Sucrose resistance colonies resulting from a second crossover were isolated, and the (couL or couO) deletion was confirmed by Southern hybridization (data not shown). Complementation was performed using pSET152, an integration vector (20). For couL, DNA fragments for complementation were amplified using couH-Fc plus couL-Rc for pSETcouHTL, couH-Fc plus couT-Rc for pSETcouHT, couT-Fc plus couL-Rc for pSETcouTL, and couL-Fc plus couL-Rc for pSETcouL. Amplicons were digested with EcoRI and XbaI and cloned into pSET152. For couO, DNA fragments for complementation were amplified using couN-Fc plus couN-Rc for pSETcouN and couN-Fc plus couO-Rc for pSETcouNO. Amplicons were digested with HindIII and cloned into pSET152 digested with HindIII and EcoRV. The pSETcouNO_D257N construct was generated by PCR-based directed mutagenesis using pSETcouNO as the template together with the oligonucleotides couO_D257N_S and couO_D257N_A. The amplified DNA was treated with DpnI (New England BioLabs) and introduced into E. coli DH5␣. The resulting plasmids were integrated into the genomes of the ⌬couL strain or the ⌬couO strain as appropriate. RNA isolation. Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. The RNA was treated with Turbo DNase (Invitrogen) and extracted with phenol-chloroform. Quantitative RT-PCR (RT-qPCR). cDNAs were synthesized using SuperScript III reverse transcriptase (RT; Invitrogen) according to the manufacturer’s instructions. Reactions were performed using SYBR Select master mix (Invitrogen) under the following conditions: 2 min at 50°C and 2 min at 95°C followed by 40 cycles of 15 s at 95°C, 15 s at 60°C, and 1 min at 72°C. The internal control was sigA. All assays were performed in triplicate, and the data were normalized using the average of the values of the internal standard. RNA-seq analysis. A total of 10 ␮g of total RNA was enriched in mRNA using a Ribominus transcriptome isolation kit (Invitrogen). Libraries for transcriptome sequencing (RNA-seq) samples were prepared from 500 ng of rRNA-depleted RNA samples using Ion Total RNA-Seq kit v2 (Life Technologies) according to the manufacturer’s instructions. The samples were analyzed using an Ion PGM system and an Ion 318 chip (Life Technologies). Data were processed using Torrent Suite software. Single-ended sequence reads were scanned from 3= to 5=. Those with a Phred quality value below 20 were trimmed using the FASTQ Quality Trimmer tool of the FASTX-toolkit (http://hannonlab.cshl.edu/fastx _toolkit/index.html). Reads consisting of fewer than 35 bases after trimming were discarded from further analysis. Trimmed reads were aligned to the RHA1 genome (7) using Bowtie 2 in end-to-end mode with the “very sensitive” option (21). Mapped read counts per gene were obtained using the HTSeq package (22). Differential expression was analyzed using the DESeq package (23). The RNA-seq data set was deposited in the Gene Expression Omnibus database (24) (see below).



OH

O

OH

Novel ␤-Oxidative Catabolism of p-Hydroxycinnamate


Metabolites were eluted using a gradient of 0% to 90% methanol over 60 min and were detected at 280 nm. To confirm the masses of the different metabolites, samples were diluted in 3% acetonitrile and supplemented with 0.1% formic acid and then injected into a large-capacity-chip (II) Zorbax SB300-C18 column (Agilent Technologies) (150 by 0.075 mm) operated at room temperature and equilibrated with 3% acetonitrile and 0.1% formic acid. The metabolites were eluted at 0.3 ␮l/min with a linear gradient of 3% to 90% acetonitrile– 0.1% formic acid over 10 min and then injected into an Agilent 6550 iFunnel quadrupole time-of-flight (Q-TOF) mass spectrometer (MS) operated in the positive-ion mode, with the gas temperature set at 290°C, the drying gas flow rate at 11 liters/min, and the fragmentor voltage at 175 V. The data were collected at 50 to 1,500 m/z with the capillary voltage set at 1,925 V. Data were analyzed with Agilent MassHunter Qualitative Analysis B.06.00 software. Accession number. The RNA-seq data set was deposited in the Gene Expression Omnibus database (24) under accession number GSE56355.

RESULTS

Growth of RHA1 on p-hydroxycinnamates. Having previously established that RHA1 grows on ferulate (10), we investigated the growth of the bacterium on other p-hydroxycinnamates (Fig. 1). In addition to ferulate, RHA1 used each of p-coumarate, dihydroferulate, and dihydro-p-coumarate as the sole growth substrate. Under similar conditions, RHA1 did not grow on sinapate. Comparison of the levels of growth on p-coumarate and ferulate revealed that although the growth yields were comparable, the lag time was slightly shorter with the former (24 h versus 36 h). In addition, the doubling time was significantly shorter on p-coumarate than on ferulate (2.8 ⫾ 0.6 h versus 4.3 ⫾ 0.6 h [n ⫽ 4]) (see Fig. S2 in the supplemental material) as confirmed by a twotailed two-sample Student t test (P ⫽ 0.010). These data indicate that p-coumarate is a better growth substrate than ferulate for RHA1. Identification of p-hydroxycinnamate catabolic genes by RNA-seq. Candidate genes responsible for p-hydroxycinnamate catabolism were identified by comparing the transcriptomes of RHA1 cells growing on 2.5 mM ferulate and benzoate, respectively. Benzoate was chosen as a control since, like ferulate, it is catabolized via the convergent ␤-ketoadipate pathway of RHA1 (26). RNA was isolated from cells at 60 and 36 h, respectively, for ferulate and benzoate. At these time points, the growth substrate was ⬃50% depleted from the culture media (see Fig. S3 in the supplemental material). cDNA was prepared and validated using RT-qPCR to examine the transcriptional levels of vanA and benA. These genes encode the oxygenase components of vanillate O-demethylase and benzoate 1,2-dioxygenase, respectively, and are highly transcribed in the presence of vanillate and benzoate, respectively (6, 27). As expected, vanA and benA were upregulated over 105-fold during growth on ferulate and benzoate, respectively (Table 1). Analysis of the RNA-seq data from these same samples using a threshold of a false-discovery-rate-adjusted P value of ⬍0.1 and a fold change value of ⬎10 resulted in the identification of 38 upregulated genes and 12 downregulated genes during growth on ferulate versus benzoate (Table 2; see also Tables S3 and S4). Data for vanA and benA were consistent with the RT-qPCR data. The upregulated genes were arranged in 7 clusters (see Table S3), including the vanACBR genes, known to be involved in ferulate catabolism (10). The largest cluster of upregulated genes comprises seven genes that span ro05122 to ro05132 (Fig. 2A). Bioinformatic analyses

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Protein purification and analysis. E. coli BL21(DE3) cells freshly transformed with pET41couL were cultivated in 25 ml LB at 30°C overnight. One liter of fresh LB was inoculated with 17 ml of the overnight culture and incubated at 30°C. After 2 h, IPTG (isopropyl-␤-D-1-thiogalactopyranoside) was added to reach a final concentration of 0.1 mM and incubation was continued for 24 h. Cells were harvested by centrifugation, suspended in 0.1 M potassium phosphate (pH 7.0), and then lysed at 4°C using an Emulsi Flex-C5 homogenizer (Avestin Inc.). Cell debris was removed by centrifugation (10,000 ⫻ g for 30 min), and ammonium sulfate was added to the cellular extract to reach a final concentration of 2.5 M. The solution was allowed to equilibrate for 30 min and was then centrifuged (10,000 ⫻ g for 30 min at 4°C). The pellet was recovered, suspended in 0.1 M potassium phosphate (pH 7.0) containing 1 M ammonium sulfate, and filtered. This solution was loaded onto a Source 15 PHE column (GE Healthcare) (1 by 10 cm) equilibrated with 0.1 M potassium phosphate (pH 7.0) containing 1 M ammonium sulfate. CouL was eluted using a linear gradient of 1 to 0 M ammonium sulfate over 100 ml. Fractions containing CouL and approximately 0.1 M ammonium sulfate were pooled, dialyzed against 50 mM Tris (pH 7.0), and loaded onto a Mono Q GL anion exchanger (GE Healthcare) equilibrated with 50 mM Tris (pH 7.0). CouL was eluted using a linear gradient of 0 to 1 M sodium chloride over 100 ml. Fractions containing CouL and approximately 0.3 M sodium chloride were pooled, dialyzed against 50 mM Tris (pH 7.0), concentrated to 11 mg ml⫺1, and stored at ⫺80°C until use. CouL-containing fractions were evaluated using SDS-PAGE. The concentration of CouL in purified preparations was determined using a molar absorptivity (ε280) value of 59.2 mM⫺1 cm⫺1. Enzymatic assay. Transformation assays were performed using 100 mM potassium phosphate (pH 7.0) containing 0.2 mM p-hydroxycinnamate, 0.2 mM CoASH, 5 mM ATP, and 10 mM magnesium chloride. Assay mixtures were incubated with or without 0.1 ␮M CouL overnight at 30°C and then quenched with methanol (50% [vol/vol]) followed by centrifugation. Products were separated by reverse-phase high-performance liquid chromatography (HPLC) using a Luna PFP(2) column (Phenomenex, Inc.) (50 by 4.6 mm) equilibrated with 100 mM ammonium acetate (pH 4.5) and operated at a flow rate of 0.7 ml min⫺1. CoA thioesters were eluted using a gradient of 0% to 90% methanol over 40 min and were detected at 320 nm (ferulate), 310 nm (p-coumarate), 325 nm (caffeate), and 280 nm (dihydroferulate). For steady-state kinetic analyses, reactions were performed in a total volume of 100 ␮l of 100 mM potassium phosphate (pH 7.0) containing 10 mM magnesium chloride, 1 mM ATP, 2 mM CoASH, and 7.8 to 250 ␮M p-hydroxycinnamate unless otherwise noted. Reactions were initiated by adding CouL to reach a final concentration of 50 nM, reaction mixtures were incubated for 1 min at 30°C, and reactions were quenched with trichloroacetate (10% [wt/vol]). The reaction mixtures were analyzed by reverse-phase HPLC to quantify the concentration of p-hydroxycinnamate. Steady-state kinetic equations were fitted to the data using LEONORA (25). CoA thioester analyses. HPLC-purified CoA thioesters were analyzed using a 6460 triple-quadrupole liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) system (Agilent Technologies) in positive-ion electrospray mode. The instrument was tuned for a range of 200 to 1,300 m/z. CoA thioesters were separated using a Zorbax 300SB-C18 column (Agilent Technologies) operated at a flow rate of 0.3 ␮l min⫺1 and a linear gradient of 3% to 90% acetonitrile– 0.1% formic acid. Metabolite analyses. Strains were cultivated at 30°C in M9 containing 20 mM glucose to the late exponential phase, supplemented with 2 mM p-coumarate or ferulate, and incubated for a further 1 or 2 days, respectively. Culture supernatants were collected by centrifugation and acidified. Samples were analyzed by reverse-phase HPLC using a Luna PFP(2) column (Phenomenex, Inc.) (50 by 4.6 mm) equilibrated with 0.1 mM ammonium acetate (pH 4.5) and operated at a flow rate of 0.3 ml min⫺1.

Otani et al.

TABLE 1 RT-qPCR analysis of selected genes during growth on different substratesa Fold change for: benA

vanA

couL

couT

couN

couR

couH

Ferulate p-Coumarate Dihydroferulate Vanillate

⬍10⫺4b ⬍10⫺4b ⬍10⫺4b ⬍10⫺4b

2.0 ⫻ 105 24 9.9 ⫻ 105 9.3 ⫻ 105

170 300 380 0.8c

1,900 1,100 1,100 2.6

1.6 ⫻ 104 4.3 ⫻ 104 2.0 ⫻ 105 1.5c

48 82 140 0.8c

290 390 790 9.8

a Values for mRNA levels relative to sigA mRNA levels were divided by those from the benzoate culture. P values of a two-tailed Student t test were ⬍0.05 except where otherwise noted. b P value, 0.06. c P value, 0.4 to 0.5.

indicated that these genes, annotated as cou, are the best candidates for p-hydroxycinnamate catabolism (Fig. 2B and Table 2). These analyses, combined with the genomic organization and the RNA-seq data, further indicate that the cou genes are arranged in three probable operons: couHTL, encoding uptake and initial transformation; couNOM, encoding transformation to vanillate; and couR, encoding a regulator. Remarkably, CouL shares ⬎90% amino acid sequence identity with feruloyl-CoA synthetase from Rhodococcus opacus PD630, predicted to catabolize ferulate via the non-␤-oxidation pathway (14). CouT is a predicted metaboliteproton symporter belonging to the major facilitator superfamily and is presumably a p-hydroxycinnamate permease. CouH’s closest characterized homologue, AtuD, catalyzes the dehydrogenation of citronelloyl-CoA (28) and is therefore predicted to catalyze the dehydrogenation of dihydro-p-hydroxycinnamoyl-CoAs. Similarly, CouM and CouN are predicted to be an (R)-specific enoyl-CoA hydratase (29) and a dehydrogenase (30, 31) that act successively to transform p-hydroxycinnamoyl-CoA to its ␤-ketoacyl-CoA derivative. The cluster does not include genes predicted to encode a ␤-ketothiolase or an acyl-CoA thioesterase, respectively. However, CouO is a predicted zinc-dependent metalloenzyme belonging to amidohydrolase superfamily 2. Its closest characterized homologue, LigJ, transforms 4-oxalomesaconate to 4-carboxy-4-hydroxy-2-oxoadipate in the protocatechuate-4,5 cleavage pathway (32). Finally, CouR is similar to CouR of Rhodopseudomonas palustris and FerC of Sphingobium sp. strain SYK-6, two MarR family transcriptional regulators whose ligands are p-hydroxycinnamoyl-CoAs (33, 34). Therefore, it is likely that CouR represses the transcription of the cou genes and that p-hydroxycinnamoyl-CoA acts as its effector. Three other clusters of upregulated genes are predicted to encode formaldehyde metabolic pathways, consistent with the production of this toxic metabolite in the transformation of vanillate to protocatechuate (6). Two of the clusters, spanning ro00487 to ro00489 and ro11327 to ro11332 (i.e., 9 genes in total), are predicted to encode partially overlapping ribulose monophosphate pathways (35). In this pathway, formaldehyde is captured by ribulose 5-phosphate in a reaction catalyzed by 3-hexulose-6-phosphate synthase (HPS; Ro00489 and Ro11329). The product, D-arabino-3-hexulose-6-phosphate, is isomerized by 6-phospho3-hexuloisomerase (PHI; Ro00488 and Ro11330) to fructose 6-phosphate. Ribulose 5-phosphate is regenerated from fructose 6-phosphate via pentose phosphate isomerization. The enzymes of the pentose phosphate isomerization pathway are predicted to be encoded by genes ro11327 to ro11332. The third cluster of formaldehyde detoxification genes, spanning ro02584 to ro02587, is highly similar to those corresponding

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to the mycothiol-dependent pathway that has been described in actinobacteria (Fig. 2C and Table 2) (36, 37) and has been annotated accordingly. In this pathway, formaldehyde reacts nonenzymatically with mycothiol to form S-hydroxymethyl-mycothiol, which is converted by a mycothiol-dependent formaldehyde dehydrogenase to S-formyl-mycothiol. This mycothiol formate ester is hydrolyzed to formate and mycothiol in a reaction that may be catalyzed by S-formyl-mycothiol hydrolase. The formate is oxidized to carbon dioxide by a formate dehydrogenase, FdhF. The latter requires an accessory protein, FdhD, which is thought to transfer sulfur from a desulfurase to FdhF during assembly of the molybdenum cofactor (38). Three genes spanning ro03282 to ro03285 whose expression was upregulated during growth on ferulate versus benzoate were predicted to be involved in eugenol catabolism (see Table S3 in the supplemental material). The first gene, eugO, encodes eugenol oxidase. This enzyme has been purified from RHA1 and oxidizes eugenol to coniferyl alcohol (39). Coniferyl alcohol is presumably oxidized to coniferyl aldehyde and then to ferulate (40). The products of ro03285 and ro03284 share low but significant amino acid sequence identity with coniferyl alcohol dehydrogenase (CalA; 26%) and coniferyl aldehyde dehydrogenase (CalB; 41%) from Pseudomonas sp. strain HR199 (41, 42). Although eugenol is not produced during ferulate catabolism, ferulate may induce transcription of eugO, calA, and calB in RHA1. Finally, the other genes that were upregulated during growth on ferulate versus benzoate are known or predicted to be involved in vanillate catabolism. In addition to the vanACBR genes noted above, ro02923 expression was upregulated 90-fold. The corresponding gene product has 46% amino acid sequence identity with VanK, the vanillate transporter in Corynebacterium glutamicum ATCC 13032 (43, 44). The upstream regions of the vanA and vanK genes have a conserved inverted repeat sequence in RHA1, TTCCGA-N3-TCGGAA, suggesting that these genes are regulated by the same transcription factor, presumably VanR. Transcription of the cou genes during growth on other substrates. In light of the bioinformatics predictions (Table 2) and the growth data, the upregulation of selected cou genes during growth of RHA1 on a variety of p-hydroxycinnamates was investigated using RT-qPCR. As summarized in Table 1, each of couL, couT, couH, couN, and couR was upregulated during growth on each of ferulate, p-coumarate, and dihydroferulate but not vanillate. Consistent with the predicted Cou pathway, vanA was also highly upregulated during growth on ferulate and dihydroferulate but not p-coumarate. We predict that p-coumarate is instead transformed to 4-hydroxybenzoate and then hydroxylated to protocatechuate (Fig. 2B). Consistent with this hypothesis, a ⌬vanA



Substrate


TABLE 2 Annotated p-hydroxycinnamate catabolic genes and mycothiol-dependent formaldehyde detoxification genes of RHA1a ORF

Gene product

Closest homologueb

IDc

Ratiod

p-Hydroxycinnamate catabolism couH

ro05132

AtuD, Pseudomonas aeruginosa PAO1 (NP_251579) ShiA, Escherichia coli K-12 (AAC75045) Fcs, Rhodococcus opacus PD630 (AAV91588) CouR, Rhodopseudomonas palustris CGA009 (NP_947139) 3␣,20␤-Hydrosteroid dehydrogenase, Streptomyces hydrogenans (1611236A) LigJ, Sphingobium sp. strain SYK-6 (YP_004834384) HtdZ, Mycobacterium tuberculosis H37Rv (NP_214644)

33

24

35

⬎107

92

34

25

30

30

⬎374

23

243

51

176

45

33

48

23

20

46

84

28

48

41 ⬎324

65

246

70

23

23

86

66

⬎360

49

⬎147

54

70 127

couT

ro05131

Dihydro-p-hydroxycinnamoy-CoA dehydrogenase p-Hydroxycinnamate permease

couL

ro05130

p-Hydroxycinnamoyl-CoA synthetase

couR

ro05125

MarR family transcriptional repressor

couN

ro05124

4-Hydroxyphenoxy-␤-hydroxyacyl-CoA dehydrogenase

couO

ro05123

couM

ro05122

4-Hydroxyphenoxy-␤-ketoacyl-CoA hydrolase p-Hydroxycinnamoyl-CoA hydratase

Mycothiol-dependent formaldehyde detoxification pathway fdhD

ro02584

fdhF

ro02585

Formate dehydrogenase accessory protein Formate dehydrogenase

fmhA

ro02586

S-Formyl-mycothiol hydrolase

adhE

ro02587

Mycothiol-dependent formaldehyde dehydrogenase

phiA

ro00487 ro00488

LuxR family transcriptional regulator 6-Phospho-3-hexuloisomerase

hpsA

ro00489

3-Hexulose-6-phosphate synthase

pgi

ro11327

Glucose-6-phosphate isomerase

gnd4

ro11328

Phosphogluconate dehydrogenase

hpsB

ro11329

3-Hexulose-6-phosphate synthase

phiB

ro11330

6-Phospho-3-hexuloisomerase

zwf5

ro11331 ro11332

LuxR family transcriptional regulator Glucose-6-phosphate 1-dehydrogenase

AdhD, Corynebacterium glutamicum ATCC 13032 (NP_599766.1) AdhF, Corynebacterium glutamicum ATCC 13032 (NP_599768.2) GxlII, Escherichia coli K-12 (NP_414748.1) MD-FALDH, Acidomonas methanolica 239 (P80094)

Ribulose monophosphate pathways and pentose phosphate isomerization pathway No characterized close homologue PHI, Mycobacterium gastri MB19 (BAA90545) HPS, Mycobacterium gastri MB19 (BAA90546) PGI, Mycobacterium tuberculosis H37Rv (NP_215461) GND, Corynebacterium glutamicum ATCC 13032 (NP_600669) HPS, Mycobacterium gastri MB19 (BAA90546) PHI, Mycobacterium gastri MB19 (BAA90545) No characterized close homologue ZWF, Corynebacterium glutamicum ATCC 13032 (NP_600790)

False-discovery-rate-adjusted P values ⫽ ⬍0.1 using RNA-seq data. ORF, open reading frame. Closest homologue with an experimentally verified function (gene identifier). ShiA, shikimate transporter; HtdZ, 3-hydroxyl-thioester dehydratase; AdhD, formate dehydrogenase accessory protein; AdhF, formate dehydrogenase; GxlII, hydroxyacylglutathione hydrolase; MD-FALDH, S-(hydroxymethyl)mycothiol dehydrogenase; PGI, glucose-6-phosphate isomerase; GND, phosphogluconate dehydrogenase; ZWF, glucose-6-phosphate 1-dehydrogenase. c Percent amino acid sequence identity calculated over the entire length of the proteins. d Upregulation during growth on ferulate versus benzoate. a b

mutant grew on p-coumarate to the same growth yield as wildtype (wt) RHA1 but the ⌬pcaL mutant did not (data not shown). As established previously, pcaL encodes the ␤-ketoadipate enollactone hydrolase/4-carboxymuconolactone decarboxylase of the ␤-ketoadipate pathway (26). couL and couO are essential for growth on p-hydroxycinnamate. To validate the role of the cou genes, ⌬couL and ⌬couO deletion mutants were constructed and their growth was investi-


gated (Table 3). Neither the ⌬couL strain nor the ⌬couO strain grew on p-coumarate, ferulate, dihydroferulate, or dihydro-pcoumarate. In contrast, both mutants grew on vanillate, dihydrocoumarin, and benzoate. Dihydrocoumarin thus is catabolized by a different pathway even though it is likely to be transformed to o-hydroxy dihydrocinnamate. Since the couHTL genes presumably comprise an operon, we complemented the ⌬couL strain by introducing a DNA fragment carrying these three

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FIG 2 (A) The cou gene cluster. Black arrows indicate genes highly transcribed in ferulate-grown cells. (B) The Cou pathway of RHA1. Ferulate (R ⫽ OCH3) is activated to feruloyl-CoA by CouL. Feruloyl-CoA is hydrated to 4-hydroxy-3-methoxyphenyl-␤-hydroxypropionyl-CoA and then oxidized to 4H3MPKP-CoA in reactions catalyzed by CouM and CouN, respectively. Finally, CouO catalyzes the hydrolysis of 4H3MPKP-CoA to vanillate. Formaldehyde is formed by the demethylation of vanillate to protocatechuate (see Fig. S1 in the supplemental material). p-Coumarate (R ⫽ H) is catabolized in the same manner, forming 4-hydroxybenzoate. 4H3MPKP-CoA and 4HPKP-CoA may be hydrolyzed if they accumulate intracellularly, resulting in 4H3MPKP and 4HPKP, respectively, which are then excreted. (C) Mycothiol-dependent formaldehyde detoxification gene cluster. (D) RT-PCR analysis of the couHTL genes. Total RNA used to synthesize the cDNA was isolated from the RHA1 cells grown on ferulate. Positions of the amplicons are indicated in panel B. G, genomic DNA; ⫹, reverse transcriptase positive; –, reverse transcriptase negative.

genes into the chromosome of the ⌬couL mutant, yielding the ⌬couL::pSETcouHTL strain. This strain grew on p-hydroxycinnamates. In contrast, the ⌬couL::pSETcouHT strain, complemented with couHT alone, did not grow on p-hydroxycinnamates. The growth deficiency of the ⌬couO strain was recovered by integrating a plasmid, pSETcouNO, into the chromosome of the ⌬couO strain (the ⌬couO::pSETcouNO strain). In contrast, the ⌬couO::pSETcouN strain, complemented only with couN, did not grow on p-hydroxy-cinnamates. These results establish that couL and couO are each essential for growth on p-hydroxycinnamate. The couHTL operon. Bioinformatic analyses suggest that the

couNOM genes likely constitute an operon but that this is less clear for couHTL. More specifically, there are no nucleotides between couN and couO and only 32 between couO and couM. Moreover, the RNA-seq data indicate that the intergenic region between couO and couM is transcribed. In contrast, there are 128 nucleotides (nt) between couH and couT and 54 between couT and couL. We therefore performed RT-PCR and further complementation studies to experimentally validate the couHTL operon. The RTPCR experiments performed using cDNA synthesized from total RNA isolated from ferulate-grown cells yielded intergenic amplicons consistent with couL being part of an operon with couH and couT (Fig. 2D). This experiment further established that the up-

TABLE 3 Growth of mutant and complemented strains on p-hydroxycinnamatesa Result Strain

FA

CA

DFA

DCA

VA

DC

BA

R. jostii Wt R. jostii ⌬couL R. jostii ⌬couL::pSETcouHTL R. jostii ⌬couL::pSETcouHT R. jostii ⌬couL::pSETcouTL R. jostii ⌬couL::pSETcouL R. jostii ⌬couL::pSET152 R. jostii ⌬couO R. jostii ⌬couO::pSETcouNO R. jostii ⌬couO::pSETcouN R. jostii ⌬couO::pSETcouNO_D257N

⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺

⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ND

⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ND

⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ND

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ND

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ND

a FA, ferulate; CA, p-coumarate; DFA, dihydroferulate; DCA, dihydro-p-coumarate; VA, vanillate; DC, dihydrocoumarin; BA, benzoate; Wt, wild type; ⫹, growth rate and yield indistinguishable from wild-type growth; ⫺, no growth detected; ND, not determined.

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Retention time (min) FIG 3 HPLC analysis of p-hydroxycinnamate-derived metabolites. (A) Culture supernatants of the wt strain and the ⌬couL and ⌬couO mutants after 0 and 2 days of incubation with ferulate. Peak 1 corresponds to ferulate. Peaks 2 (m/z ⫽ 211.06) and 3 (m/z ⫽ 167.07) were identified as protonated 4-hydroxy3-methoxyphenyl-␤-ketopropionate and acetovanillone, respectively. (B) The corresponding experiment after 0 and 1 day of incubation with p-coumarate. Peak 1 corresponds to p-coumarate. Peaks 2 (m/z ⫽ 181.05) and 3 (m/z ⫽ 137.06) were identified as protonated 4-hydroxyphenyl-␤-ketopropionate and 4=-hydroxyacetophenone, respectively.

stream gene, ro05133, is not part of the operon. Finally, a DNA fragment containing only couL failed to restore the ability of the ⌬couL mutant to grow on ferulate (Table 3). In contrast, a fragment containing couTL restored the growth phenotype of the mutant (Table 3). Both strains grew normally on benzoate (Table 3). These results, taken together with those of the previous section, strongly suggest that there are promoters upstream of each of couH and couT but not upstream of couL. Characterization of pathway metabolites. To further validate the proposed pathway, the wt and mutant strains were analyzed for the accumulation of metabolites upon incubation in the presence of either ferulate or p-coumarate. Analysis of the culture supernatants using reverse-phase HPLC revealed that ferulate (tR ⫽ 42 min) was completely depleted by the wt and ⌬couO strains within 2 days (Fig. 3A). In contrast, ferulate was not detectably depleted by the ⌬couL mutant (Fig. 3A). Two additional compounds were detected in the supernatant of ⌬couO cultures. The first compound (tR ⫽ 16 min) was identified as 4-hydroxy-3-methoxyphenyl-␤-ketopropionate (4H3MPKP) based on mass spectrometric analyses (m/z ⫽ 211.06). The second compound (tR ⫽


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0 2

s)

3

45 min) was identified as acetovanillone based on its mass spectrum (m/z ⫽ 167.07) and coelution with an authentic standard. Similarly, p-coumarate (tR ⫽ 36 min) was completely depleted by the wt and ⌬couO strains but not by the ⌬couL mutant within 1 day (Fig. 3B). Two compounds that were unique to the ⌬couO cultures were identified as 4-hydroxyphenyl-␤-ketopropionate (4HPKP; tR ⫽ 9 min, m/z ⫽ 181.05) and 4=-hydroxyacetophenone (tR ⫽ 39 min, m/z ⫽ 137.06), respectively. 4H3MPKP and 4HPKP transform nonenzymatically to acetovanillone and 4=-hydroxyacetophenone, respectively (45). Therefore, we conclude that 4H3MPKP and 4HPKP are the major metabolites accumulating in the ⌬couO culture supernatant upon incubation with ferulate and p-coumarate, respectively. A predicted metal-coordinating residue of CouO is essential for function. As noted above, sequence analysis indicates that CouO is a member of the amidohydrolase superfamily. These enzymes contain either a mononuclear or a binuclear metallocenter that is catalytically essential (46). Sequence analysis further indicates that CouO is a subtype I amidohydrolase and harbors all of the residues that coordinate the binuclear metallocenter in these enzymes: His19, His21, Lys131, His163, His205, and Asp257. In subtype I enzymes, the carboxylate side chain of the aspartate is hydrogen bonded to one of the metal ions as well as to the hydroxide that bridges the two metal ions and that initiates hydrolysis of the substrate. To investigate the importance of Asp257 in CouO, we mutated pSETcouNO to encode a D257N variant of CouO. As summarized in Table 3, the D257N mutant was unable to grow on ferulate but grew on vanillate. These data support the bioinformatics prediction that Asp257 is an essential residue in CouO. CouL is a p-hydroxycinnamoyl-CoA synthetase. To experimentally validate the physiological role of CouL, the enzyme was heterologously produced in E. coli. CouL was purified to apparent homogeneity using hydrophobic interaction and anion-exchange chromatographies (see Fig. S4 in the supplemental material). Approximately 10 mg of purified CouL was obtained per liter of E. coli culture. The ability of CouL to catalyze the thioesterification of p-hydroxycinnamates was initially tested by incubating ferulate, ATP, and CoASH with the purified protein. Analysis of the incubated mixtures using reverse-phase HPLC (Fig. 4A) revealed that, compared to a control incubation in the absence of CouL, peaks corresponding to ferulate (tR ⫽ 25 min; peak 1), CoASH (tR ⫽ 14 min), and ATP (tR ⫽ 1 min) were depleted and a new peak with tR ⫽ 26 min (peak 2) was detected. The purified product had an m/z of 944.0 as shown by analysis using LC-ESI-MS, which corresponds to protonated feruloyl-CoA. Similar analyses revealed that CouL also catalyzed the thioesterification of p-coumarate, dihydroferulate, and caffeate (Fig. 4B to D). However, CouL did not detectably catalyze the thioesterification of sinapate, vanillate, or benzoate (data not shown). We conclude that CouL is a p-hydroxycinnamoyl-CoA synthetase. Substrate specificity of CouL. Steady-state kinetics analyses were performed to evaluate the substrate specificity of CouL. An HPLC-based assay was developed to facilitate the evaluation of a broader range of substrates. Reactions were initially performed using 0.25 mM p-coumarate and various concentrations of ATP and CoASH. Under these conditions, the Km values of CouL for ATP and CoASH were 110 ⫾ 10 ␮M and 210 ⫾ 60 ␮M, respectively. Based on these values, the specificity of CouL for p-hydroxycinnamates was evaluated in reaction mixtures containing 1

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FIG 4 CouL is a p-hydroxycinnamoyl-CoA synthetase. Data represent the results of reverse-phase HPLC analysis of CouL reaction with ferulate (A), p-coumarate (B), caffeate (C), or dihydroferulate (D). ATP, CoASH, and phydroxycinnamate were incubated with (red line) or without (black line) CouL at 30°C and analyzed by reverse-phase HPLC. Peak 1 corresponds to p-hydroxycinnamate. The insets show the MS spectra of peak 2 analyzed by LC-ESI-MS. (A) The peak of the inset at the m/z of 944.0 corresponds to protonated feruloyl-CoA. The peak at the m/z of 921.9 was also observed in a blank sample. (B) The peak of the inset at the m/z of 914.1 corresponds to protonated p-coumaroyl-CoA. The peak at the m/z of 921.9 was also observed in a blank sample. (C) The peak of the inset at the m/z of 930.0 corresponds to protonated caffeoyl-CoA. The peak at the m/z of 921.9 was also observed in a blank sample. (D) The peak of the inset at the m/z of 946.1 corresponds to protonated dihydroferuloyl-CoA. The peaks at the m/z of 921.9 and 913.9 were also observed in a blank sample.

mM ATP and 2 mM CoASH. The enzyme displayed MichaelisMenten behavior for all substrates tested (Fig. 5). The accuracy of this assay was validated using a spectrophotometric assay (47). This yielded a kcat/Km value of 370 ⫾ 10 mM⫺1 s⫺1 for p-couma-

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The current data establish that RHA1 catabolizes p-hydroxycinnamates via p-hydroxy-benzoates using a CoA-dependent, ␤-oxidative deacetylation pathway encoded by the cou genes (Fig. 1). These genes were highly upregulated during growth on ferulate, dihydroferulate, and p-coumarate but not during growth on vanillate (Table 1). Catabolism of p-hydroxycinnamates is initiated by CouL, which catalyzes the CoA thioesterification of these growth substrates (Fig. 3). Consistent with the substrate specificity of CouL, a ⌬couL mutant was unable to grow on p-hydroxycinnamates. Moreover, a ⌬couO mutant was also unable to grow on p-hydroxycinnamates, transforming them to the corresponding 4HPKP. This indicates that CouO, a predicted metal-dependent hydrolase, catalyzes the last step of a ␤-oxidative pathway whereby 4HPKP-CoAs are hydrolyzed to vanillate and acetylCoA (Fig. 2B). The growth data established that p-coumarate is a better growth substrate for RHA1 than ferulate. This preference is consistent with the substrate specificity of CouL, as is the inability of RHA1 to grow on sinapate, suggesting that CouL is a determinant for the pathway. Nevertheless, the preferred growth substrate may also reflect the substrate specificity of the CouT permease, the differential activation of the cou genes by ferulate and p-coumarate, and/or the toxicity of formaldehyde. The failure of RHA1 to grow on caffeate may be due to the oxidation of this catechol under the experimental conditions. Analysis of sequenced genomes indicates that the Cou pathway occurs in a number of other actinobacteria as summarized in Fig. S5 in the supplemental material. Among six clades of Rhodococcus (48), it appears to be conserved only within the clade of the R. opacus and jostii strains, which includes PD630, B4, and RHA1. That is, none of the other sequenced rhodococcal genomes contain the cou genes. The occurrence of a gene cluster (OPAG_02409 to OPAG_02420) in PD630 (49) sharing ⬎90% nucleotide identity with the cou genes in RHA1 contradicts a previous conclusion that ferulate is catabolized via a non-␤-oxidation pathway in PD630 (14). Curiously, the previously identified gene cluster does not occur in the published R. opacus PD630 genome (49), although Plaggenborg et al. identified the feruloyl-CoA synthetase. The reasons for this discrepancy are unclear. The actinobacterial Cou pathway is similar but not identical to the Rhizobiales ␤-oxidative deacetylation pathway recently substantiated in A. fabrum C58 (11) and predicted to occur in two Bradyrhizobium species (50). The gene cluster in A. fabrum C58 does not encode the permease of the actinobacterial pathway but does encode a complete ABC transporter. Although this transporter was not characterized (11), it seems likely that it takes up p-hydroxycinnamates. The catabolic enzymes in A. fabrum C58 share ⬃20% amino acid sequence identity with the RHA1 enzymes, with the exception of Atu1421, which shares just over 50% amino acid sequence identity with CouO. Similarly to the ⌬couO mutant, a deletion mutant of atu1421 accumulated 4HPKPs when incubated in the presence of p-hydroxycinnamates. In both cases, the 4HPKP is the expected off-pathway



0

(B)

rate, similar to that obtained using the HPLC-based assay. Although the kcat and Km values did not vary tremendously (Table 4), CouL had greatest specificity for p-coumarate, with a kcat/Km value approximately twice those for ferulate and dihydroferulate.


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FIG 5 Steady-state kinetics of CouL-catalyzed thioesterification of p-hydroxy-cinnamates. Reactions were performed using 1 mM ATP, 2 mM CoASH, 10 mM MgCl2, and 0.1 M potassium phosphate (pH 7.0) at 30°C. The solid line represents the best fits of the Michaelis-Menten equation to the data using LEONORA.

hydrolysis product of 4HPKP-CoA, the predicted substrate of CouO/Atu1421. Although Atu1421 was proposed to be a ␤-ketothiolase (11), it clearly belongs to the amidohydrolase superfamily, just like CouO. Importantly, the predicted metal-coordinating residues are conserved in Atu1421, including Asp257, which is essential for CouO function. Interestingly, the superfamily includes enzymes such as ␣-amino-␤-carboxy-muconate-ε-semialdehyde decarboxylase that also cleave a C-C bond (51). Nevertheless, additional studies are required to directly demonstrate that CouO and Atu1421 hydrolyze the bond between the ␣ and ␤ carbons of 4HPKP-CoAs (Fig. 2B). The substrate specificity of CouL is remarkably similar to that of two 4-coumaric acid:CoA ligases (4CL) from plants: 4CL1 from Populus tomentosa and 4CL2 from Arabidopsis thaliana. CouL shares 26% to 29% amino acid sequence identity with these enzymes, including the catalytic Lys475. Like CouL, neither of these enzymes transforms sinapate. Structural analyses of 4CL1 and 4CL2 revealed that their substrate binding pockets harbor a threeresidue loop, GPV, which occludes the binding of the second methoxy group of sinapate (52, 53). This loop is one residue shorter in 4CL4 of Arabidopsis, which is able to transform sinapate. Moreover, deletion of a single residue from this loop in 4CL2 conferred sinapyl-CoA synthetase activity, presumably by enlarging the pocket (53). Alignment of the CouL amino acid sequence with those of the plant enzymes reveals that CouL has three amino acid residues in its predicted loop: Ala-Pro-Ser (see Fig. S6 in the supplemental material). This suggests that this loop helps to determine the substrate specificity of CouL. The design of the transcriptomic experiment facilitated the identification of two processes related to p-hydroxycinnamate catabolism: formaldehyde detoxification and eugenol catabolism. The eugenol catabolic pathway is substantiated by a previous characterization of eugenol oxidase (39). Nevertheless, the induction of these genes by ferulate is unexpected. The apparent coregu-


lation of these pathways may reflect the fact that soil bacteria such as RHA1 typically grow on mixtures of related compounds as suggested by studies of alkylbenzene catabolism (27). It is unclear whether CouT transports eugenol and coniferyl alcohol. An E. coli strain that heterologously produces EugO was able to transform eugenol (42), suggesting that a specialized transporter is not required. Moreover, a number of other genes encoding transporters were upregulated in RHA1 during growth on ferulate, including two members of the major facilitator superfamily (ro01949 and ro05369) and ABC transporters (ro02013 and ro02014). The results of this study strongly suggest that two pathways are involved in detoxifying formaldehyde produced during vanillate catabolism (6) that are dependent on ribulose 5-phosphate and mycothiol, respectively. In the ribulose 5-phosphate-dependent pathway, it is unclear whether the apparently redundancy of hpsB and phiB is essential (Table 2). This redundancy extends to the LuxR family transcriptional regulators encoded by the two gene clusters. The high (⬎93%) amino acid sequence identity of the encoded homologues and the occurrence of one of the gene clusters on plasmid pRHL3 suggest that the redundancy is not essen-

TABLE 4 Kinetic parameters of CouL for hydroxycinnamate derivativesa Hydroxycinnamate derivative

kcat (s⫺1)

Km (␮M)

kcat/Km (s⫺1 mM⫺1)

Ferulate p-Coumarate Dihydroferulate Caffeate Sinapate

12 ⫾ 1 16 ⫾ 1 9⫾1 17 ⫾ 1 NA

76 ⫾ 6 38 ⫾ 6 47 ⫾ 4 50 ⫾ 3 NA

160 ⫾ 5 400 ⫾ 40 200 ⫾ 10 340 ⫾ 10 NA

a

Reactions were performed using 1 mM ATP, 2 mM CoASH, 10 mM MgCl2, and 0.1 M potassium phosphate (pH 7.0) at 30°C. Data are average values of the results from three independent experiments with standard errors of the means. NA, no activity.

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16. 17.

ACKNOWLEDGMENTS This work was supported by a Genome Canada Large-Scale Research Project. I.C. is a recipient of a postdoctoral fellowship from the Fonds de Recherche en Santé du Québec and the Michael Smith Foundation for Health Research. Nicolas Seghezzi and Anders Klaus assisted with the RNA-seq experiment. Jie Liu constructed plasmid pK18⌬couL.

18.

19.

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Saccharothrix carnea sp. nov., an actinobacterium isolated from soil.

Regulation of a nickel-cobalt efflux system and nickel homeostasis in a soil actinobacterium Streptomyces coelicolor.

Nesterenkonia rhizosphaerae sp. nov., an alkaliphilic actinobacterium isolated from rhizosphere soil in a saline-alkaline desert.

Rhodococcus soli sp. nov., an actinobacterium isolated from soil using a resuscitative technique.

Rhodococcus agglutinans sp. nov., an actinobacterium isolated from a soil sample.

A soil actinobacterium scavenges atmospheric H2 using two membrane-associated, oxygen-dependent [NiFe] hydrogenases.

Streptomyces antioxidans sp. nov., a Novel Mangrove Soil Actinobacterium with Antioxidative and Neuroprotective Potentials.

D-Amino Acid Catabolism Is Common Among Soil-Dwelling Bacteria.

Streptomyces gilvigriseus sp. nov., a novel actinobacterium isolated from mangrove forest soil.

Genetic characterization of 4-cresol catabolism in Corynebacterium glutamicum.

Sinomonas humi sp. nov., an amylolytic actinobacterium isolated from mangrove forest soil.

Draft Genome Sequence of Streptacidiphilus oryzae TH49T, an Acidophilic Actinobacterium Isolated from Soil.

Impact of activated carbon on the catabolism of (14)C-phenanthrene in soil.

Bounagaea algeriensis gen. nov., sp. nov., an extremely halophilic actinobacterium isolated from a Saharan soil of Algeria.

Characterization of a Unique Pathway for 4-Cresol Catabolism Initiated by Phosphorylation in Corynebacterium glutamicum.

Characterization of an Unconventional Rhodopsin from the Freshwater Actinobacterium Rhodoluna lacicola.

Negative Regulation of Ectoine Uptake and Catabolism in Sinorhizobium meliloti: Characterization of the EhuR Gene.

Cloning of Azorhizobium caulinodans nicotinate catabolism genes and characterization of their importance in N2 fixation.

Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism.

Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism.

Niacin catabolism in rodents.

Characterization of a carbofuran-degrading bacterium and investigation of the role of plasmids in catabolism of the insecticide carbofuran.

Salternamides A-D from a Halophilic Streptomyces sp. Actinobacterium.

Streptomyces malaysiense sp. nov.: A novel Malaysian mangrove soil actinobacterium with antioxidative activity and cytotoxic potential against human cancer cell lines.