Degradation of arylglycerol-␤-aryl ether is the most important process in bacterial lignin catabolism. Sphingobium sp. strain SYK-6 degrades guaiacylglycerol-␤-guaiacyl ether (GGE) to ␣-(2-methoxyphenoxy)-␤-hydroxypropiovanillone (MPHPV), and then the ether linkage of MPHPV is cleaved to generate ␣-glutathionyl-␤-hydroxypropiovanillone (GS-HPV) and guaiacol. We have characterized three enantioselective glutathione S-transferase genes, including two genes that are involved in the ether cleavage of two enantiomers of MPHPV and one gene that is involved in the elimination of glutathione from a GS-HPV enantiomer. However, the first step in the degradation of four different GGE stereoisomers has not been characterized. In this study, three alcohol dehydrogenase genes, ligL, ligN, and ligO, which conferred GGE transformation activity in Escherichia coli, were isolated from SYK-6 and characterized, in addition to the previously cloned ligD gene. The levels of amino acid sequence identity of the four GGE dehydrogenases, which belong to the short-chain dehydrogenase/ reductase family, ranged from 32% to 39%. Each gene was expressed in E. coli, and the stereospecificities of the gene products with the four GGE stereoisomers were determined by using chiral high-performance liquid chromatography with recently synthesized authentic enantiopure GGE stereoisomers. LigD and LigO converted (␣R,␤S)-GGE and (␣R,␤R)-GGE into (␤S)-MPHPV and (␤R)-MPHPV, respectively, while LigL and LigN transformed (␣S,␤R)-GGE and (␣S,␤S)-GGE to (␤R)-MPHPV and (␤S)-MPHPV, respectively. Disruption of the genes indicated that ligD is essential for the degradation of (␣R,␤S)-GGE and (␣R,␤R)-GGE and that both ligL and ligN contribute to the degradation of the two other GGE stereoisomers.

It has been suggested that a novel type of aromatic acid transporter, which is similar to the tripartite tricarboxylate transporter (TTT), is involved in terephthalate (TPA) uptake by Comamonas sp. strain E6. This suggestion was based on the presence of the putative TPA-binding protein gene, tphC, in the TPA catabolic operon. The tphC gene is essential for growth on TPA and is similar to the genes encoding TTT-like substrate-binding proteins. Here we identified two sets of E6 genes, tctBA and tpiBA, which encode TTT-like cytoplasmic transmembrane proteins. Disruption of tctA showed no influence on TPA uptake but resulted in a complete loss of the uptake of citrate. This loss suggests that tctA is involved in citrate uptake. On the other hand, disruption of tpiA or tpiB demonstrated that both genes are essential for TPA uptake. Only when both tphC and tpiBA were introduced with the TPA catabolic genes into cells of a non-TPA-degrading Pseudomonas strain did the resting cells of the transformant acquire the ability to convert TPA. From all these results, it was concluded that the TPA uptake system consists of the TpiA-TpiB membrane components and TPA-binding TphC. Interestingly, not only was the tpiA mutant of E6 unable to grow on TPA or isophthalate, it also showed significant growth delays on o-phthalate and protocatechuate. These results suggested that the TpiA-TpiB membrane components are able to interact with multiple substrate-binding proteins. The tpiBA genes were constitutively transcribed as a single operon in E6 cells, whereas the transcription of tphC was positively regulated by TphR. TPA uptake by E6 cells was completely inhibited by a protonophore, carbonyl cyanide m-chlorophenyl hydrazone, indicating that the TPA uptake system requires a proton motive force.

Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-Omethylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase ␤ and ␣ subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The K m for gallate and the V max were determined to be 66.9 ؎ 9.3 M and 42.7 ؎ 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

Paenibacillus sp. (formerly Bacillus macerans) strain JJ-1b is able to grow on 4-hydroxybenzoate (4HB) as a sole source of carbon and energy and is known to degrade 4HB via the protocatechuate (PCA) 2,3-cleavage pathway. However, none of the genes involved in this pathway have been identified. In this study, we identified and characterized the JJ-1b genes for the 4HB catabolic pathway via the PCA 2,3-cleavage pathway, which consisted of praR and praABEGFDCHI. Based on the enzyme activities of cell extracts of Escherichia coli carrying praI, praA, praH, praB, praC, and praD, these genes were found to code for 4HB 3-hydroxylase, PCA 2,3-dioxygenase, 5-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase, 2-hydroxymuconate-6-semialdehyde dehydrogenase, 4-oxalocrotonate (OCA) tautomerase, and OCA decarboxylase, respectively, which are involved in the conversion of 4HB into 2-hydroxypenta-2,4-dienoate (HPD). The praE, praF, and praG gene products exhibited 45 to 61% amino acid sequence identity to the corresponding enzymes responsible for the catabolism of HPD to pyruvate and acetyl coenzyme A. The deduced amino acid sequence of praR showed similarity with those of IclR-type transcriptional regulators. Reverse transcription-PCR analysis revealed that praABEGFDCHI constitute an operon, and these genes were expressed during the growth of JJ-1b on 4HB and PCA. praR-praABEGFDCHI conferred the ability to grow on 4HB to E. coli, suggesting that praEGF were functional for the conversion of HPD to pyruvate and acetyl coenzyme A. A promoter analysis suggested that praR encodes a repressor of the pra operon.Protocatechuate (PCA) is one of the key intermediate metabolites in the microbial catabolic pathways for various aromatic compounds, including phthalates, hydroxybenzoates, and lignin-derived aromatic compounds such as vanillate and ferulate. It is known that the aromatic ring fission of PCA is catalyzed by one of the three distinct dioxygenases PCA 3,4-dioxygenase (26), PCA 4,5-dioxygenase (36, 41), and PCA 2,3-dioxygenase (7). In the PCA 3,4-cleavage pathway, PCA is converted into 2-carboxy-cis,cis-muconate by the reaction catalyzed by PCA 3,4-dioxygenase, and the catabolic pathway for its product (␤-ketoadipate pathway) has been reported in many bacteria (24,26). In the case of the PCA 4,5-cleavage pathway, PCA is cleaved by PCA 4,5-dioxygenase to yield 4-carboxy-2-hydroxymuconate-6-semialdehyde, and then the product is degraded to 2-pyrone-4,6-dicarboxylate, 4-oxalomesaconate, and 4-carboxy-4-hydroxy-2-oxoadipate before entering the Krebs cycle (36). The genes and enzymes involved in this pathway have been recently characterized for several bacteria, such as Sphingobium (Sphingomonas) (36), Comamonas (47), Pseudomonas (35), and Arthrobacter (13) strains. On the other hand, no genetic information on the PCA 2,3-cleavage pathway has been reported since the finding of this pathway in some bacilli (7,8).In 1979, Crawford et al. reported the PCA 2,3-cleavage pathway of a 4-hydroxybenzoate (4HB) degrader, Paenibacillus sp. ...

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