characterization and spreadability of protocatechuate 4,5-cleavage

Appl Microbiol Biotechnol 

DOI 10.1007/s00253-012-4402-8 

ENVIRONMENTAL BIOTECHNOLOGY 

Assimilation of aromatic compounds by Comamonas testosteroni: 

characterization and spreadability of protocatechuate 4,5-cleavage 

pathway in bacteria 

Bin Ni & Yun Zhang & Dong-Wei Chen & Bao-Jun Wang & 

Shuang-Jiang Liu 

Received: 25 June 2012 / Revised: 25 August 2012 /Accepted: 29 August 2012 

# Springer-Verlag 2012 

Abstract Comamonas testosteroni strain CNB-1 was isolated 

from activated sludge and has been investigated for its ability 

to degrade 4-chloronitrobenzene. Results from this study 

showed that strain CNB-1 grew on phenol, gentisate, vanillate, 

3-hydroxybenzoate (3HB), and 4-hydroxybenzoate (4HB) as 

carbon and energy sources. Proteomic data and enzyme activity 

assays suggested that vanillate, 3HB, and 4HB were degraded 

in strain CNB-1 via protocatechuate (PCA) 4,5- 

cleavage pathway. The genetics and biochemistry of the 

PCA 4,5-cleavage pathway were investigated. Results 

showed that the 4-oxalomesaconate (OMA) hydratase 

from C. testosteroni takes only enol-OMA as substrate. 

A previously functionally unknown gene pmdU encodes 

an OMA tautomerase and catalyzes conversion of 

OMA keto into OMA enol . The 4-carboxy-4-hydroxy-2- 

oxoadipate (CHA) aldolase is encoded by pmdF and catalyzes 

the last step of the PCA 4,5-cleavage pathway. We 

explored the 1,183 microbial genomes at GenBank for 

potential PCA 4,5-cleavage pathways, and 33 putative pmd 

Electronic supplementary material The online version of this article 

(doi:10.1007/s00253-012-4402-8) contains supplementary material, 

which is available to authorized users. 

B. Ni : Y. Zhang : D.-W. Chen : B.-J. Wang : S.-J. Liu 

State Key Laboratory of Microbial Resources, 

Institute of Microbiology, Chinese Academy of Sciences, 

Beijing 100101, People’s Republic of China 

B.-J. Wang : S.-J. Liu 

Environmental Microbiology Research Center, 



S.-J. Liu (*) 


Beichen Xilu 1, Chaoyang District, 


e-mail: liusj@im.ac.cn 

clusters were found. Results suggest that PCA 4,5-cleavage 

pathways are mainly distributed in α- andβ-Proteobacteria. 

Keywords Comamonas testosteroni . Protocatechuate 

4,5-cleavage . Vanillate . 3-/4-Hydroxybenzoate . 

4-Oxalomesaconate tautomerase 

Introduction 

Comamonas species are metabolically diverse and dwell in 

a wide range of habitats such as activated sludge (Gumaelius 

et al. 2001), soils (Yu et al. 2011), fresh and marine sediments 

(Shinomiya et al. 1997), or they associate with plant and 

animal tissues (Chou et al. 2007; Gul et al. 2007). The 

majority of Comamonas species does not assimilate sugars 

and thus, does not compete for carbohydrates with other 

environmental microbes such as members of the genus 

Pseudomonas; they rather remove toxic metabolites resulted 

from those microbes (Ornston and Ornston 1972; Parke et al. 

2000). Comamonas species are genetically dynamic and are 

equipped for adaptation to changing environments and xenobiotic 

compounds such as chloronitrobenzenes (Katsivela et 

al. 1999;Wuetal.2005). Genome data mining has suggested 

that Comamonas testosteroni is potentially a robust aromatic 

degrader. For example, 37 and 18 genes, respectively, encoding 

putative dioxygenases and hydroxylases were identified 

from the genome of C. testosteroni (Ma et al. 2009). 

Protocatechuate (PCA) is one of the key metabolic intermediates 

during biodegradation of aromatic compounds 

(Hara et al. 2000; Harwood et al. 1994; Kamimura et al. 

2010a; Providenti et al. 2006). PCA is aerobically degraded 

via ring cleavage at either 2,3 (Crawford 1975; Kasai et al. 

2009), or 3,4 (Harwood and Parales 1996) or 4,5 positions 

(Kamimura et al. 2010a; Masai et al. 2007). The ring


cleavage at the 4,5 position is catalyzed by PCA 4,5-dioxygenase 

(P45D). Various P45Ds have been found in several 

bacterial groups such as Comamonas species (Kamimura et 

al. 2010a; Mampeletal.2005; Providentietal.2001), 

Pseudomonas species (Maruyama et al. 2004), Sphingobium 

species (Masai et al. 1999; Wattiau et al. 2001), and Delftia 

sp. strain TBKNP-05 (Patil et al. 2006). Following PCA 

ring cleavage, the product 4-carboxy-2-hydroxymuconate- 

6-semi-aldehyde (CHMS) is spontaneously converted to its 

intramolecular hemiacetal form and is sequentially oxidized 

into 2-pyrone-4,6-dicarboxylate (PDC) (Hara et al. 2000, 

2003; Kamimura et al. 2010a; Maruyama et al. 2004; Masai 

et al. 1999, 2000; Providenti et al. 2001; Fig. 1a). PDC is 

hydrolyzed into 4-oxalomesaconate (OMA), which then 

undergoes hydration to 4-carboxy-4-hydroxy-2-oxoadipate 

(CHA). CHA is converted into oxaloacetic and pyruvic 

acids via aldolic cleavage (Hara et al. 2003; Maruyama et 

al. 2001; Wang et al. 2010). Although the genes involved in 

PCA 4,5-cleavage have been investigated extensively in 

Sphingobium paucimobilis SYK-6 (Hara et al. 2000, 2003; 

Kamimura et al. 2010b; Masai et al. 1999, 2000) and in 

Pseudomonas (Noda et al. 1990) and Comamonas species 

(Kamimura et al. 2010a; Mampel et al. 2005; Providenti et 

al. 2001), the lower pathway reactions and genes involved in 

conversion of PDC into oxaloacetic and pyruvic acids in 

Comamonas species are still hypothetical and have not been 

experimentally identified. 

In this report, we present the results of assimilation of 

aromatic compounds by C. testosteroni strain CNB-1 grown 

on phenol, gentisate, vanillate, 3-hydroxybenzoate (3HB), 

and 4-hydroxybenzoate (4HB). Vanillate, 3HB, and 4HB 

were degraded via PCA 4,5-cleavage pathway. A previously 

hypothetical gene pmdU with unknown function was characterized 

to be an OMA tautomerase and catalyzes the 

conversion of OMA keto into OMA enol . The spreadability of 

PCA 4,5-cleavage pathway in bacteria was explored via data 

mining of microbial genomes. 

Material and methods 

Bacterial strains, plasmids, and cultural conditions 

Bacterial strains and plasmids used in this study are listed in 

Table 1. C. testosteroni strain CNB-2 is a plasmid-curing 

derivative from C. testosteroni strain CNB-1 (CGMCC 

1.12282;Wu et al. 2005, 2006). Escherichia coli was grown 

aerobically on a rotary shaker (200 rpm) at 37 °C in Luria- 

Bertani (LB) broth or on LB plate with 1.5 % (w/v) agar. C. 

testosteroni strains were cultivated and maintained in LB 

medium or in minimal salt broth (MSB; Wu et al. 2006) 

containing 1 gL −1 ammonium chloride as nitrogen source. 

Aromatic compounds were added at final concentrations of 

0.2 gL −1 when they were used as sole carbon and energy 

sources. Cellular growth was monitored by measuring turbidity 

(optical density) at 600 nm. If necessary, antibiotics 

were used at the following concentrations: kanamycin, 

50 mgL −1 for E. coli and 150 mgL −1 for strain CNB-1; 

tetracycline, 20 mgL −1 for both E. coli and strain CNB-1. 

Comparative proteomic analysis 

Comparative proteomic studies were conducted as previously 

described (Zhang et al. 2009). In brief, cells 

were harvested at the late exponential phase of growth 

and were broken by sonication on ice. Supernatant of 

cellular lysate (ca. 300 μg proteins) were analyzed by 

2-DE. IEF and SDS-PAGE were operated at the same 

conditions as described previously (Zhang et al. 2009). 

Proteins were digested with trypsin and the resulting 

peptides were detected by mass spectrometry. Protein 

identification was carried out according to their peptide 

fingerprints. A two-tailed Student's t test was adopted to 

evaluate the spot differences between the control and 

experimental gels (P


Fig. 1 The degradation of vanillate, 3-, and 4-hydroxybenzoate in 

Comamonas testosteroni CNB-1 (a) and gene clusters of protocatechuate 

4,5-cleavage pathway in strain CNB-1 and other bacterial strains 

(b). In a, proteins shown in bold indicates that proteins were detected 

from proteomes of strain CNB-1 grown on vanillate, 3HB, or 4HB. In 

b, species shown in bold indicate the gene cluster was identified 

experimentally. The numbers in the brackets indicate the strains found 

in the species. Abbreviations: for enzymes: PmdA protocatachuate 4,5- 

dioxygenase α-subunit, PmdB protocatachuate 4,5-dioxygenase β- 

subunit, PmdC 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase, 

PmdD PDC hydrolase, PmdU 4-oxalomesaconate tautomerase, 

PmdE 4-oxalomesaconate hydratase, PmdF 4-carboxy-4- 

hydroxy-2-oxoadipate aldolase/oxaloacetate decarboxylase. For compounds: 

4HB 4-hydroxybenzoate, PCA protocatechuate, CHMS 4- 

carboxy-2-hydroxymuconate-6-semialdehyde, PDC 2-pyrone-4, 6- 

dicarboxylate, OMAketo 4-oxalomesaconate keto form, OMAenol 4- 

oxalomesaconate enol form, CHA 4-carboxy-4-hydroxy-2-oxoadipate 

Various derivative plasmids from these vectors were constructed, 

and their relevant characteristics are listed in 

Table 1. Plasmids were electroporated into CNB-1 and the 

mutants were screened according to Schäfer et al. (1994), 

except that LB medium supplemented with a final sucrose 

concentration of 20 % was used for selection. The deletion


Table 1 Bacterial strains and plasmids used in this study 

Strain/plasmid/ 

oligonucleotide 

Description 

Sources/ 

references 

Strains 

Comamonas testosteroni 

CNB-1 

Wu et al. 

(2005) 

CNB-1ΔpmdF A fragment of DNA coding for amino acids 105 to 215 of pmdF was deleted This study 

CNB-1ΔpmdF/ 

This study 

pBBR1MCS3-pmdF 

CNB-1ΔpmdE A fragment of DNA coding for amino acids 32 to 296 of pmdE was deleted This study 

CNB-1ΔpmdE/ 

This study 

pBBR1MCS2-pmdE 

CNB-1ΔpmdE/ 

This study 

pBBR1MCS2-galB 

CNB-1ΔpmdU A fragment of DNA coding for amino acids 6 to 226 of pmdU was deleted This study 

CNB-1ΔpmdU/ 

This study 

pBBR1MCS2-pmdU 

CNB-1ΔpmdU/ 

This study 

pBBR1MCS2-galD 

E. coli strains This study 

BL21(DE3) 

F − ompT hsdSB(rB − mB − ) gal dcm λDE3 (harboring gene 1 of the RNA polymerase from the 

phage T7 under the PlacUV5 promoter) 

Maniatis et al. 

(1982) 

DH5α (f80lacZΔM15)endA1 recA1 hsdR17 (rK − mK − ) supE44ΔlacU169 Maniatis et al. 

(1982) 

Pseudomonas putida 

KT2440 

Plasmid 

Franklin et al. 

(1981) 

pK18mobSacB Mobilizable vector, allows for selection of double crossover in CNB-1 Schäfer et al. 

(1994) 

pK18mobSacB-ΔpmdE 

pK18mobSacB-ΔpmdU 

pK18mobSacB-ΔpmdF 

pBBR1MCS3 Tc r , lacPOZ′ broad host vector with R type conjugative origin Kovach et al. 

(1995) 

pBBR1MCS2 Km r , lacPOZ′ broad host vector with R type conjugative origin Kovach et al. 

(1995) 

pBBR1MCS3-pmdF Carrying pmdF (to generate complementation for pmdF) This study 

pBBR1MCS2-pmdE Carrying pmdE (to generate complementation for pmdE) This study 

pBBR1MCS2-pmdU Carrying pmdU (to generate complementation for pmdU) This study 

pBBR1MCS2-galD Carrying galD (to generate expression for galD) This study 

pBBR1MCS2-galB Carrying galB (to generate expression for galB) This study 

pET-28a(+) Expression vector Novagen 

pET-28a-pmdAB pET28a derivative for expression of pmdAB This study 

pET-28a-pmdU pET28a derivative for expression of pmdU This study 

pET-28a-pmdE pET28a derivative for expression of pmdE This study 

pET-28a-pmdF pET28a derivative for expression of pmdF This study 

pET-28a-galB pET28a derivative for expression of galB This study 

pET-28a-galD pET28a derivative for expression of galD This study 

of the target genes in pK18mobsacB derivatives and in 

CNB-1 mutants were verified by PCR amplification. The 

complementation of these genes was conducted by introducing 

pBBR1MCS2 or pBBR1MCS3 derivatives into the 

mutants. 

Cloning, expression, and purification of pmd and gal genes 

in E. coli 

Each gene of the pmd gene cluster of strain CNB-1 and gal 

cluster of strain KT2440 was amplified by PCR from the


genomes of strains CNB-1 and KT2440. Purified PCR products 

were treated with restriction enzymes and then ligated into the 

similarly treated pET28a (+). The resulting plasmids were transformed 

in E. coli BL21 (DE3) for expression of the genes. 

Expression of the genes in E. coli was induced with 0.1 mM 

isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 °C for 10– 

12 h, when the optical density at 600 nm reached 0.6. Cellular 

lysates of recombinant E. coli were prepared by sonication of 

cell suspensions in 50 mM phosphate buffer (pH 8.0) or in 

50 μM Fe(NH 4 ) 2 (SO 4 ) 2 solution when purifying PmdAB or 

20 μM ZnSO 4 solution when purifying PmdU and GalD. 

Sonication was conducted (at 300 W, 3 s, interval of 5 s, for 

90 cycles). Cell debris was removed by centrifugation at 

12,000 rpm for 20 min, and the supernatant was used for further 

purification. Proteins were purified with His-affinity resin chromatography 

by following the instruction from the manufacturers 

(Novagen). 

Enzymatic synthesis of OMA keto , OMA enol , and CHA 

OMA keto ,OMA enol , and CHA were prepared as previously 

described (Nogales et al. 2011) with the following modification. 

OMA keto was freshly prepared from gallic acid in a 

system containing (totally 2 mL): 0.2 mM gallic acid, 

50 μM Fe(NH 4 ) 2 (SO 4 ) 2, 24 μg recombinant PmdAB from 

E. coli, 50 mM potassium phosphate buffer, pH 7.0. After 

incubation at room temperature for 5 min, this mixture solution 

was used as the crude OMA keto preparation without any 

further purification. OMA enol was prepared by incubating the 

freshly prepared OMA keto with GalD and 20 μM ZnSO 4 for 

5 min. CHA was prepared by adding PmdE or GalB (Nogales 

et al. 2011) into the freshly prepared OMA enol solution and 

incubated at room temperature for another 5 min. 

Enzyme activity assays 

The OMA hydratase activity of PmdE was assayed spectrophotometrically 

at room temperature by measuring the decrease of 

A 265 due to the disappearance of OMA enol (Nogales et al. 2011). 

The OMA tautomerase activity of PmdU was qualitatively 

determined at room temperature by adding 0.8 μg purified 

PmdU in the 2.0 mL freshly prepared OMA keto .WhenPmdU 

was added in the system, an increase in the absorbance at 

265 nm can be observed. The CHA aldolase activity of PmdF 

was assayed at room temperature by monitoring the decrease in 

A 340 derived from NADH oxidation in a coupled assay by 

adding 1 mM MgSO 4 ,140μM NADH, 30 U lactate dehydrogenase 

(Sigma), 30 U malate dehydrogenase (AMRESCO), and 

2.6 μg recombinant PmdF from E. coli (Hara et al. 2003; 

Nogales et al. 2011). Catechol 2,3-dioxygenase (C23D) activity 

was assayed by monitoring the absorbance changes at 375 nm to 

monitor the formation of catechol ring cleavage product (Cerdan 

et al. 1994), and PCA P45D activity was determined 

spectrophotometrically by measuring the increase in the absorbance 

at 410 nm due to the formation of 4-carboxy-2-hydroxymuconate-6-semialdehyde 

(Kamimura et al. 2010a;Mampelet 

al. 2005) and gentisate 1,2-dioxygenase (G12D) was measured 

spectrophotometrically by measuring the increase of absorption 

at 330 nm derived from maleylpyruvate (Shen et al. 2005). 

Results 

C. testosteroni strain CNB-1 grew on various aromatic 

compounds 

Strain CNB-1 had been isolated for 4CNB degradation, and it 

takes 4CNB as sole carbon and nitrogen sources (Wu et al. 

2005). Previous results showed that CNB-2 grew on benzoate, 

phenol, 3- and 4HB, PCA, and vanillate (Ma et al. 2009). In 

this study, the ability of strain CNB-1 (the parent of strain 

CNB-2) to grow on a range of aromatic compounds was 

tested. Results showed that CNB-1 also grew on the following 

aromatic compounds, including phenol, benzoate, 3HB, 4HB, 

vanillate, and gentisate, but not on 4-cresol, 2,4-dihydroxybenzoate, 

resorcinol, and phenylacetate. Further, aromatic ring 

cleavage dioxygenase activities were determined. It was found 

that C23D activity was associated with cells grown on phenol. 

P45D activity was associated with cells grown on vanillate, 

3HB, and 4HB. G12D activity was associated with cells 

grown on gentisate, benzoate, and 3HB (Table 2). These 

results suggested that strain CNB-1 possessed multiple degradative 

pathways for various aromatic compounds. 

Identification of proteins involving in vanillate, 3HB, 

and 4HB degradation 

In order to identify enzymes/proteins involved in the degradation 

of these compounds, comparative proteomic analyses 

were carried out on CNB-1 cells grown with vanillate, 3HB, 

or 4HB versus cells grown with succinate. With cells grown 

on vanillate, 3HB, and 4HB, 704±5, 705±12, and 692±17 

spots were recognized, respectively (Supplementary Fig. 

S1). Compared with cells grown on succinate, 40 proteins 

in total were differently synthesized, of which 30 were 

induced and ten were upregulated. Thirty-seven out of the 

40 protein spots were successfully identified with MALDI- 

TOF mass spectrometry (Table 3). 

Most of the identified proteins were functionally related 

to degradation of vanillate, 3HB, and 4HB. They are either 

involved in the peripheral routes leading to PCA or involved 

in the PCA 4,5-cleavage pathway (Fig. 1a). The following 

enzymes involved in the peripheral routes were detected: 

vanillate monooxygenase (V2 and V3) and vanillate O- 

demethylase subunit (V5), 4HB-3-hydroxylase (O3 and 

O4), and 3HB hydroxylase (M1). Four enzymes of the


Table 2 Assimilation of various 

aromatic compounds and determination 

of ring cleavage activities 

in strain CNB-1 

+ indicates the enzymatic activity 

was positive; − indicates the 

enzymatic activity was negative 

C23D catechol 2,3-dioxygenase, 

P45D protocatechuate 4,5-dioxygenase, 

G12D genticsate 1,2- 

dioxygenase, n.d. enzymatic activity 

was not detected 

Aromatic compounds Growth (OD 600 ) Dioxygenase activities 

At 0 h At 24 h C23D P45D G12D 

Resorcinol 0.02 0.09 n.d. n.d. n.d. 

2,4-Dihydroxybenzoate 0.03 0.10 n.d. n.d. n.d. 

4-Cresol 0.05 0.11 n.d. n.d. n.d. 

Phenylacetate 0.04 0.09 n.d. n.d. n.d. 

Phenol 0.05 0.47 + − − 

Benzoate 0.03 0.30 − − + 

3-Hydroxybenzoate 0.04 0.46 − + + 

4-Hydroxybenzoate 0.03 0.41 − + − 

Vanillate 0.02 0.36 − + − 

Gentisate 0.05 0.43 − − + 

PCA 4,5-cleavage pathway were detected. They were PmdA 

(V13/O12/M10), PmdD (V7/O10/M5), PmdF (V9/O6/M7), 

and PmdE (V6/O9/M3). A functionally unknown protein 

and putatively involving in PCA degradation, PmdU (V11/ 

O5/M2), was also detected. In addition, a LysR-type transcriptional 

regulator (V8/O8/M4) was induced when vanillate, 

3HB, and 4HB were used as carbon sources for growth. 

Based on the proteomic data, it was deduced that vanillate, 

3HB, and 4HB were degraded via a common PCA 4,5- 

cleavage pathway and this pathway was likely to be regulated 

by the putative LysR-type transcriptional regulator. 

Annotation of genes involved in PCA 4,5-cleavage pathway 

from the genome of C. testosteroni 

In silico analysis of the C. testosteroni CNB-2 genome (Ma 

et al. 2009) revealed a gene cluster (pmd) that is potentially 

involved in PCA 4,5-cleavage pathway (Fig. 1b). This pmd cluster 

carries genes putatively encoding P45D (CtCNB1_2741-2742/ 

PmdAB), CHMS dehydrogenase (CtCNB1_2740/PmdC), PDC 

hydrolase (CtCNB1_2743/PmdD), OMA hydratase 

(CtCNB1_2745/PmdE), CHA aldolase/oxaloacetate decarboxylase 

(CtCNB1_2744/PmdF), transporter (CtCNB1_2746/PmdK), 

and a hypothetical protein (CtCNB1_2747/PmdU) orthologous to 

the functionally unknown PmdU of Comamonas sp. strain E6. 

Results from bioinformatic analysis on these genes are listed in 

Supplementary Table S2. Except PmdB, PmdC, and the putative 

transporter PmdK, all the other translational products from these 

genes were detected from the proteomes of cells grown on vanillate,3HB,and4HB. 

Genetic identification of the genes involved 

in PCA 4,5-cleavage pathway in strain CNB-1 

To further identify if these genes were involved in PCA 4,5- 

cleavage pathway, pmdE, pmdF, and pmdU were knocked 

out. The three resulting mutants lost the ability to grow on 

vanillate, 3HB, and 4HB as carbon sources. This ability to 

grow on vanillate, 3HB, and 4HB was restored by genetic 

complementation (Fig. 2). All these results indicated that 

pmdE, pmdF, and pmdU were involved in PCA 4,5-cleavage 

pathway and are essential to vanillate, 3HB, and 4HB assimilation 

with strain CNB-1. 

PmdE is an OMA hydratase and takes OMA enol 

as its substrate 

PmdE had 63 % identity to a previously identified OMA 

hydratase (LigJ) from S. paucimobilis that was proposed to 

take the keto form of OMA, i.e., OMA keto , as substrate (Hara 

et al. 2000). PmdE also showed 12 % identity to GalB, a 

previously characterized OMA hydratase from P. putida that 

took enol form of OMA, i.e., OMA enol , as substrate (Nogales 

et al. 2011). In order to determine the catalytic stereochemistry 

of PmdE, OMA keto and OMA enol were prepared enzymatically 

in this study. The hydratase activity of PmdE towards 

OMA keto or OMA enol was determined. Results showed that 

the purified PmdE catalyzed the conversion of OMA enol ,but 

not OMA keto , into CHA (Fig. 3a and b). Thus, it was concluded 

that the substrate of PmdE was OMA enol , not OMA keto . 

This conclusion is also supported by the observation that galB 

from P. putida partially restored the phenotype of CNB- 

1ΔpmdE to grow on 4HB (Fig. 2b). 

PmdU is an OMA tautomerase that catalyzes OMA keto 

to OMA enol 

As the PmdE in strain CNB-1 took OMA enol for substrate, 

an enzyme that catalyzes the conversion of OMA keto into 

OMA enol would be needed. A tautomerase that converts 

OMA enol into OMA keto was proposed for Comamonas sp. 

strain E6, but had not been experimentally confirmed 

(Kamimura et al. 2010a). In this study, we observed that 

galD, encoding a tautomerase that converts OMA keto into


Table 3 Proteins differentially expressed in cells grown with vanillate, 3-, or 4-hydroxybenzoate compared to that from succinate 

Number orf Protein names/putative function Score Match Coverage pI/ 

MW (T) 

pI/ 

MW (E) 

P 

value 

Ratio 

V1 CtCNB1_0452 FlgL/flagellin and related hook-associated 

proteins 

147 29 69 % 5.04/ 

41 

V2 CtCNB1_4192 VanA2/vanillate monooxygenase 100 19 48 % 5.05/ 

40 

V3 CtCNB1_4192 VanA2/vanillate monooxygenase 101 20 48 % 5.05/ 

40 

V4 CtCNB1_1380 TtdA/tartrate dehydratase alpha subunit/ 52 9 18 % 6.04/ 

fumarate hydratase class I N-terminal domain 

56 

V5 CtCNB1_4189 VanA1/vanillate O-demethylase oxygenase 55 19 40 % 5.87/ 

subunit 

40 

V6 a CtCNB1_2745 PmdE/putative 4-oxalomesaconate hydratase 

76 20 40 % 5.81/ 

38 

V7 a CtCNB1_2743 PmdD/putative 2-pyrone-4,6-dicarboxylic 78 15 49 % 6.06/ 

acid hydrolase 

34 

V8 a CtCNB1_2864 LysR/transcriptional regulator 53 13 42 % 6.43/ 

35 

V9 a CtCNB1_2744 PmdF/4-hydroxy-4-methyl-2-oxoglutarate 59 15 45 % 5.78/ 

aldolase 

24 

V10 a CtCNB1_2820 SodA/superoxide dismutase 66 9 47 % 6.11/ 

25 

V11 a CtCNB1_2747 PmdU/OMA keto-enol tautomerase 109 11 59 % 4.95/ 

23 

V13 a CtCNB1_2742 PmdA/protocatechuate 4,5-dioxygenase 66 15 68 % 5.27/ 

alpha subunit 

17 

V14 CtCNB1_3292 Ndk/nucleoside diphosphate kinase 64 7 27 % 5.91/ 

15 

V15 a CtCNB1_2844 UspA/universal stress protein UspA and 79 11 62 % 6.28/ 

related nucleotide-binding proteins 

16 

O1 CtCNB1_1128 Eno/enolase 71 10 32 % 4.88/ 

46 

O2 CtCNB1_2183 KatG/catalase 162 36 46 % 5.61/ 

80 

O3 CtCNB1_2156 PobA1/4-hydroxybenzoate 3-hydroxylase 91 20 59 % 5.50/ 

44 

O4 CtCNB1_2156 PobA1/4-hydroxybenzoate 3-hydroxylase 100 14 29 % 5.50/ 

44 

M1 CtCNB1_3410 MobA/m-hydroxybenzoate hydroxylase 193 39 66 % 5.69/ 

70 

M6 CtCNB1_2778 GodA/gentisate 1,2-dioxygenase 52 16 42 % 6.10/ 

42 

M9 CtCNB1_2779 Fah/fumarylacetoacetate (FAA) hydrolase 77 12 58 % 5.31/ 

26 

5.08/ 

45 

5.15/ 

40 

5.21/ 

40 

6.38/ 

64 

6.27/ 

49 

6.23/ 

38 

6.56/ 

36 

5.78/ 

28 

6.06/ 

25 

6.12/ 

21 

5.00/ 

23 

5.30/ 

17 

6.36/ 

16 

6.48/ 

15 

4.92/ 

52 

5.95/ 

78 

5.78/ 

53 

5.79/ 

50 

5.96/ 

72 

6.42/ 

48 

5.47/ 

27 

0.000 −3.22 

+ + 

+ + 

+ + 

+ + 

+ + 

+ + 

+ + 

+ + 

0.000 2.39 

+ + 

+ + 

0.000 8.52 

+ + 

0.010 2.01 

0.000 3.55 

+ + 

+ + 

+ + 

+ + 

+ + 

V1–V15 spots from cells grown on vanillate as sole carbon source, O1–O13 spots from cells grown on 4HB as sole carbon source, M1–M11 spots 

from cells grown on 3HB as sole carbon source, Score the Mascot protein score among the fractions where the protein was identified, Match the 

number of spectra matched to the protein, Coverage the protein sequence coverage for the fraction, pI/MW (T) theoretical values from Mascot 

search, pI/MW (E) experimental values, P value the change of protein is significant (


Fig. 2 Gene disruption and complementation of pmdE, pmdF, and 

PmdU in C. testosteroni wildtype and mutants. Vanillate, 3-, and 4- 

hydroxybenzoate were used as carbon sources, and the phenotypes 

were the same. Only the data with 4-hydroxybenzoate were showed. 

a CNB-1 (filled square), CNB-1ΔpmdU (filled circle), CNB- 

1ΔpmdU/pBBR1MCS2-pmdU (filled triangle), and CNB-1ΔpmdU/ 

pBBR1MCS2-galD (filled inverted triangle). b Growth of CNB-1 

(filled square), CNB-1ΔpmdE (filled circle), CNB-1ΔpmdE/ 

pBBR1MCS2-pmdE (filled triangle), and CNB-1ΔpmdE/ 

pBBR1MCS2-galB (filled invertedtriangle).c Growth of CNB-1 

(filled square), CNB-1ΔpmdF (filled circle), and CNB-1ΔpmdF/ 

pBBR1MCS3-pmdF (filled triangle) 

the above functionally identified PmdE. When OMA keto 

was used as substrate and in the presence of PmdU and 

PmdE, OMA keto was converted to CHA (Fig. 3c). Combining 

the observation that PmdE only took OMA enol as 

substrate, it was deduced that PmdU catalyzed the conversion 

of OMA keto into OMA enol . We further demonstrated 

that PmdU directly converted OMA keto into OMA enol 

(Fig. 3d). Thus, it was concluded that PmdU is an OMA 

Fig. 3 Determination of the 

stereochemistry of PmdE and 

PmdU from C. testosteroni. a 

Time-course of the enzymatic 

activity of PmdE using 

OMA enol as substrate. b Timecourse 

of the enzymatic activity 

of PmdE using OMA keto as 

substrate. c The conversion of 

OMA keto into CHA by coupling 

the catalysis of PmdU and 

PmdE. d The conversion of 

OMA keto into OMA enol by 

PmdU. Filled circle with 

PmdU, filled square without 

PmdU, showing the spontaneously 

conversion of OMA keto 

into OMA enol


tautomerase and catalyzes the conversion of OMA keto into 

OMA enol . 

PmdF shows both CHA aldolase and oxalacetate 

decarboxylase activities 

The deduced amino acid sequence of PmdF showed 98.7 %, 

65.1 %, 56.7 %, and 55.9 % identities to CHA aldolases 

from P. ochraceae (ProA), S. paucimobilis (LigK), P. putida 

(HMG), and P. putida (GalC), respectively (Hara et al. 2003; 

Maruyama et al. 2001; Nogales et al. 2011; Wang et al. 

2010). The pmdF gene was cloned and expressed in E. coli, 

and the recombinant PmdF was purified and used for enzymatic 

activity assays. The PmdF showed obvious aldolase 

activity when CHA was used as substrate and obvious 

decarboxylase activity when oxaloacetate was used as substrate 

(Supplementary Fig. S4). 

Discussion 

Being a key intermediate during biodegradation of aromatic 

compounds (Harwood et al. 1994; Haraetal.2000), PCA is 

aerobically degraded via ring cleavage at either 2,3 (Crawford 

1975; Kasai et al. 2009), or 3,4 (Harwood and Parales 1996) 

or 4,5 positions (Kamimura et al. 2010a; Masai et al. 2007). In 

order to explore the distribution of the PCA 4,5-cleavage 

pathway in various microbial groups, we conducted extensive 

searches for putative pmd clusters from currently sequenced 

organisms. In silico searches from 1,183 completed microbial 

genomes revealed that 33 genomes carry gene clusters orthologous 

to pmdBCDEF. About91%ofthepmd clusters were 

found in Proteobacteria (genera Bradyrhizobium, Comamonas, 

Delftia, Marinomonas, Novosphingobium, Rhodopseudomonas, 

Sphingobium, etc.) and the remaining 9 % were 

found in Actinobacteria (Arthrobacter and Kitasatospora). 

Interestingly, seven of the 33 microorganisms can colonize 

higher plants (Giraud et al. 2007;Krauseetal.2006; Liu et al. 

2007a; Lucas-Elio et al. 2011; Rawlings and Bateman 2009; 

Vandamme et al. 2002), two can colonize animal (Pinel et al. 

2008; Roh et al. 2012), and one was in association with marine 

algae (Akagawamatsushita et al. 1992). 

Two types of genetic organization/cluster were recognized 

for PCA 4,5-cleavage pathway: The Sphingobium-type gene 

cluster constitutes several transcriptional units (Hara et al. 

2003). The Comamonas–Pseudomonas type constitutes only 

one transcriptional unit (Masai et al. 2007). According to our 

alignment on the pmd clusters in Fig. 1b, the gene organizations 

of pmd clusters are more diverse among various bacterial 

groups. The positions of the putative regulator, the PMA tautomerase, 

and a functionally unknown gene are apparently localized 

differently at the pmd gene clusters among the 

Acitinobacteria, α-Proteobacteria, β-Proteobacteria, and γ- 

Proteobacteria. Thus, we deduce that the transcriptions of pmd 

genes are also diverse and they are controlled by specific 

regulators in each bacterial group. In Sphingobium sp. strain 

SYK-6, a LysR-type transcriptional regulator, LigR, is responsible 

for the activation of pmd genes (Kamimura et al. 2010b). 

AsshowninFig.1b andreportedbyKamimuraetal.(2010b), 

there is no putative regulator gene within the pmd cluster of 

Comamonas species. In this study, we found that the expression 

level of a LysR-type regulator (CtCNB1_2864) was induced 

when vanillate, 3HB, or 4HB served as carbon source. It is 

deduced that this CtCNB1_2864 is the putative LysR-type 

regulator and activates the pmd operon in Comamonas species. 

C. testosteroni strain CNB-1 has been extensively investigated 

for its ability to grow on chloronitrobenze and various 

aromatic compounds (Liu et al. 2007a, b;Maetal.2007;Wuet 

al. 2005, 2006; Zhang et al. 2009). This study further disclosed 

that strain CNB-1 adopts a PCA 4,5-cleavage for the degradation 

of vanillate, 3HB, and 4HB. The genes involved in this 

PCA 4,5-cleavage pathway were located at a pmd gene cluster 

and their translational products were detected in proteomes of 

strain CNB-1 cells grown on vanillate, 3HB, and 4HB. Based 

on the results obtained from this study, the degradative pathways 

for vanillate, 3HB, and 4HB were proposed, and the central 

PCA 4,5-cleavage pathway is modified from that proposed for 

Comamonas species strain E6 (Kamimura et al. 2010b). This 

modified PCA 4,5-cleavage pathway in strain CNB-1 includes 

tautomerization and is similar to the gallic acid degradation 

pathway from P. putida (Nogales et al. 2011). Based on our 

observation that the genetic organizations of pmd clusters are 

similar in Comamonas and Sphingomonas species, we further 

proposed that the PCA 4,5-cleavage pathway in C. testosteroni 

strainsCNB-1andE6andinS. paucimobilis SYK-6 should be 

the same. This proposal is also supported by the previous results 

that the GalB was able to replace the function of LigJ in S. 

paucimobilis (Nogales et al. 2011). 

Acknowledgements This work was supported by a grant from the 

National Natural Science Foundation of China (31230003). 

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characterization and spreadability of protocatechuate 4,5-cleavage

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