Genomic Analysis of the Aromatic Catabolic Pathways from Silicibacter Pomeroyi DSS-3

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Genomic Analysis of the Aromatic Catabolic Pathways from Silicibacter Pomeroyi DSS-3 Annals of Microbiology, 59 (4) 789-800 (2009) Genomic analysis of the aromatic catabolic pathways from Silicibacter pomeroyi DSS-3 Dazhong YAN1, Jianxiong KANG2, Dong-Qi LIU2* 1School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, 430023; 2Center for Microbiology Engineering, School of Environmental Science and Engineering, Huazhong University of Science and Technology, No. 1037 Luoyue Road, Wuhan 430074, P.R. China Received 16 June 2009 / Accepted 7 October 2009 Abstract - Genomic analysis of the catabolic potentialities of Silicibacter pomeroyi DSS-3 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least seven main pathways for the conversion of the aromatic compounds to the intermediates which enter into TCA cycle, that is, the catechol (cat I and cat II genes) and protocatechuate (pca genes) branches of the β-ketoadipate pathway, the phenylacetate pathway (paa genes), the gentisate pathway (gtd genes), the homogentisate pathway (hmg/hppD genes), as well as the homoprotocatechuate pathway (hpc genes). Furthermore, the genes encoding those enzymes involved in the peripheral pathways leading to the β-ketoadipate central pathway were also mapped, i.e., 4-hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and act), tyrosine (hpp) and n-phenylalkanoic acids (fad). Evidences showed that S. pomeroyi DSS-3 have versatile abili- ties to the catabolism of aromatic compounds either in anaerobic or in aerobic pathway, suggesting such a strain might be a model of heuristic value for the study of the genomic organization, the evolution of genes, as well as the catalytic or transcriptional mechanisms of enzymes for aromatic degradation in marine bacteria. Further, it would provide new insights into the biodegradation of aromatic compounds in marine bacteria and marine environments. Key words: genomic analysis; aromatic catabolic pathways; Silicibacter pomeroyi DSS-3. INTRODUCTION catabolism of central aromatic intermediates in this strain, they are the homogentisate pathway (hmg/fah/mai genes), the phe- Aromatic compounds, originating from biosynthesis, natural nylacetate pathway (pha genes), as well as catechol (cat genes) diagenesis, and human activity (Gibson, 1980), are the most and protocatechuate (pca genes) branches of the β-ketoadipate widespread class of organic compounds after carbohydrates pathway (Jimenez et al., 2002). It presents the possibility to and hence a common carbon source for many microorganisms explore the interconnection relationship of the aromatic catabolic (Harwood and Parales, 1996). It also has been aroused great network in this strain. interests in the mechanisms about microbial degradation of aro- As the genome of Silicibacter pomeroyi DSS-3, a Gram- matic compounds over the last several decades. Numerous bac- negative, and marine water isolated bacterium belonging to teria that are able to use aromatic compounds as sole sources of A-Proteobacteria group which was capable of mineralizing carbon and energy have been isolated from enrichment cultures, dimethylsulfoniopropionate (DMSP) and its related sulfur com- and furthermore, the genes involved in some of the degradative pounds (Gonzalez et al., 2003), has been sequenced (Moran et pathways were cloned and characterized in detail, such as sum- al., 2004), we carried out a genomic analysis of the genes or marized in the Biocatalysis/Biodegradation Database (Ellis et al., gene clusters which might be involved in the catabolism of aro- 2006). However, the genomic view on mineralization of aromatic matic compounds in this strain. The present study would further compounds in the versatile aromatic compounds-degrading bac- insights into aromatic ring cleavage in marine bacteria, and also terium which would facilitate the rational genetic manipulation of provide the possibility to develop a new useful model system for the strain for improving the biodegradation or biotransformation studying the catabolism of aromatic compounds. ability is very few for the lack of the genomic data. Previously, genomic analysis of the aromatic catabolism was performed on Pseudomonas putida KT2440, a soil bacterium, in which it MATERIALS AND METHODS revealed that there existed at least four main pathways for the Genome analysis. The nucleotide sequence of the entire * Corresponding Author. Phone: 86-27-87792512; Silicibacter pomeroyi DSS-3 genome was obtained from Genbank Fax: 86-27-87792172; E-mail: [email protected] (4, 109, 442 bp for the chromosome, accession number 790 D. YAN et al. CP000031; 491, 611 bp for the megaplasmid, accession number the other exists in the magaplasmid (named pcaCII, 125 aa). The NC_006569). amino acid sequence of pcaCI and pcaCII shows 82% and 81% Protein sequence similarities analysis and the protein names sequence identity to the orthologue in Silicibacter sp. TM1040, identification were carried out using the BLAST programs respectively. Previous evidences showed that pcaD and pcaC (Altschul et al., 1997; Krauthammer et al., 2000). locate at different regions of the chromosome in different strains DNA statistics were analyzed by using DNA star (Clewley, (Jimenez et al., 2002), and further, pcaD is either contiguous to 1995). the pcaC gene, or fused to the latter. However, the arrangement Genomic island was predicted by Z curve (Zhang and Zhang, of pcaD and pcaC (I or II) is differing from the known orthologues 2005). described above. As detected in S. pomeroyi DSS-3, the gene encoding β-ketoadipate enolactone hydrolase (encoded by pcaD) is located at the magaplasmid, thus, in S. pomeroyi DSS-3, pcaD RESULTS AND DISCUSSION and pcaCII are presented in different regions of the megaplas- mid. However, pcaD and pcaCI are located at the megaplasmid By sequences analysis it can be predicted that there were at and chromosome, respectively. least seven different pathways for the catabolism of aromatic It’s worth noting that analysis showed some ORFs are simi- compounds (Fig. 1, Table 1-6). Growth experiments showed that lar to the known genes which involved in the two downstream S. pomeroyi DSS-3 is able to grow in minimal medium containing pathways for the catabolism of catechol, named as cat I and cat tyrosine, phenylethylamine, coniferyl alcohol, p-coumarate, feru- II (Fig. 1; Table1 ). However, no ORFs share similarities to the late, benzoate, 3-hydroxybenzoate, p-hydroxybenzoate, phe- known muconolactone isomerase which catalyzes muconolactone nylacetate, phenylalanine, phenylethylamine, phenylalkanoate, to form β-ketoadipate enol-lactone, although ORFSPOA0044, salicylate, catechol, caffeate, vanillate and quinate as sole carbon a putative protocatechuate 3,4-dioxygenase β subunit (242 and energy source. Thereby the seven different pathways for the aa), shows 31% amino acid sequence identity to a catechol catabolism of aromatic compounds were functional substanti- 1,2-dioxygenase (278 aa) from Ralstonia solanacearum UW551 ated. (ZP_00946584). Therefore, whether cat I genes which involved in the catechol branch plays a role in the aromatic metabo- The β-ketoadipate central pathway lism of S. pomeroyi DSS-3, and whether the two branches Analysis of the genome of S. pomeroyi DSS-3 revealed that the (protocatechuate branch and catechol branch) will converge at ORFs (open reading frames) encoding the putative enzymes for β-ketoadipate enol-lactone as those in P. putida (Jimenez et al., the two branches of the β-ketoadipate central pathway (ortho- 2002), remain to be demonstrated. cleavage genes), i.e. the protocatechuate branch (pca genes) and the catechol branch (cat genes), showed significant similarity to Peripheral pathways leading to the β-ketoadipate central proteins from Roseobacter sp. MED193, Silicibacter sp. TM1040, pathway two of A-Proteobacteria isolated from seawaters (Table 1). The pathways of aerobic metabolism of benzoate generally fol- Former reports revealed that most of the cat genes of the low a well-established strategy for aerobic aromatic metabolisms catechol branch and the pca genes of the protocatechuate were described in detail (Dagley, 1978; Harwood and Parales, branch from the finished genomic sequence are organized in one 1996; Gescher et al., 2006). The ben genes which involved in or several clusters on the chromosome (Jimenez et al., 2002). the conversion of benzoate into catechol were also identified in However, in S. pomeroyi DSS-3, neither of the genes involved in P. putida and Acinetobacter sp. ADP1 (Collier et al., 1998; Cowles these two branches forms a gene cluster. They distribute either et al., 2000). As observed on the chromosome of S. pomeroyi in the chromosome and /or in the megaplasmid (Table 1). This DSS-3, the ben orthologues locate at positions of 1518-1523 kb, suggested that chromosome-plasmid cooperation is required these genes are not linked to the genes involved in the catechol for the β-ketoadipate central pathway in S. pomeroyi DSS-3. branch, similar to the arrangement of the ben genes in P. putida Similarly, pcaGH and dxnHI (named pcaIJ in other bacteria), KT2440 (Jimenez et al., 2002). Recently, a new pathway (box which encode separate subunits of ptotocatechuate 3, 4-dioxy- cluster) initiated by activation of benzoate to benzoyl-CoA has genase and 3-oxoadipate CoA-succinyl transferase, respectively, been described (Gescher et al., 2005). ORFs showing similarity are co-transcribed as the other bacteria (Harwood
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