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Novel biphenyl-oxidizing bacteria and dioxygenase genes from a korean tidal mudflat.

ABSTRACT: Gene-targeted FLX titanium pyrosequencing integrated with stable isotope probing (SIP) using [(13)C]biphenyl substrate revealed that tidal mudflat sediments harbor novel aromatic ring hydroxylating dioxygenases (ARHD). More than 80% of the detected ARHD genes comprise four clades (0.5 distance) with 49 to 70% amino acid identity to sequences in public databases. The 16S rRNA sequences enriched in the (13)C fraction were from the Betaproteobacteria, bacilli (primarily Paenibacillus-like), and unclassified phyla.

SUBMITTER: Lee TK

PROVIDER: S-EPMC3127585 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Novel biphenyl-oxidizing bacteria and dioxygenase genes from a korean tidal mudflat.

Lee Tae Kwon TK Lee Jaejin J Sul Woo Jun WJ Iwai Shoko S Chai Benli B Tiedje James M JM Park Joonhong J

Applied and environmental microbiology 20110415 11

Gene-targeted FLX titanium pyrosequencing integrated with stable isotope probing (SIP) using [(13)C]biphenyl substrate revealed that tidal mudflat sediments harbor novel aromatic ring hydroxylating dioxygenases (ARHD). More than 80% of the detected ARHD genes comprise four clades (0.5 distance) with 49 to 70% amino acid identity to sequences in public databases. The 16S rRNA sequences enriched in the (13)C fraction were from the Betaproteobacteria, bacilli (primarily Paenibacillus-like), and unc ...[more]

PMID: 21498748

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Project description:Many but not all ammonia-oxidizing bacteria (AOB) produce urease (urea amidohydrolase, EC 3.5.1.5) and are capable of using urea for chemolithotrophic growth. We sequenced the urease operons from two AOB, the beta-proteobacterium Nitrosospira sp. strain NpAV and the gamma-proteobacterium Nitrosococcus oceani. In both organisms, all seven urease genes were contiguous: the three structural urease genes ureABC were preceded and succeeded by the accessory genes ureD and ureEFG, respectively. Green fluorescent protein reporter gene fusions revealed that the ure genes were under control of a single operon promoter upstream of the ureD gene in Nitrosococcus oceani. Southern analyses revealed two copies of ureC in the Nitrosospira sp. strain NpAV genome, while a single copy of the ure operon was detected in the genome of Nitrosococcus oceani. The ureC gene encodes the alpha subunit protein containing the active site and conserved nickel binding ligands; these conserved regions were suitable primer targets for obtaining further ureC sequences from additional AOB. In order to develop molecular tools for detecting the ureolytic ecotype of AOB, ureC genes were sequenced from several beta-proteobacterial AOB. Pairwise identity values ranged from 80 to 90% for the UreC peptides of AOB within a subdivision. UreC sequences deduced from AOB urease genes and available UreC sequences in the public databases were used to construct alignments and make phylogenetic inferences. The UreC proteins from beta-proteobacterial AOB formed a distinct monophyletic group. Unexpectedly, the peptides from AOB did not group most closely with the UreC proteins from other beta-proteobacteria. Instead, it appears that urease in beta-proteobacterial autotrophic ammonia oxidizers is the product of divergent evolution in the common ancestor of gamma- and beta-proteobacteria that was initiated before their divergence during speciation. Sequence motifs conserved for the proteobacteria and variable regions possibly discriminatory for ureC from beta-proteobacterial AOB were identified for future use in environmental analysis of ureolytic AOB. These gene sequences are the first publicly available for ure genes from autotrophic AOB.

Project description:Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs). In three different assays, BPDO(B356) from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than BPDO(LB400) from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3',4-trichloro approximately 2,3,4'-trichloro > 3,3'-dichloro > 2,4,4'-trichloro > 4,4'-dichloro approximately 2,2'-dichloro > 2,6-dichloro > 2,2',3,3'-tetrachloro approximately 2,2',5,5'-tetrachloro. Except for 2,2',5,5'-tetrachlorobiphenyl, BPDO(B356) transformed each congener at a higher rate than BPDO(LB400). The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized BPDO(B356) activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. BPDO(LB400) had a greater apparent specificity for biphenyl than BPDO(B356) (k(cat)/K(m) = 2.4 x 10(6) +/- 0.7 x 10(6) M(-1) s(-1) versus k(cat)/K(m) = 0.21 x 10(6) +/- 0.04 x 10(6) M(-1) s(-1)). However, the latter transformed biphenyl at a higher maximal rate (k(cat) = 4.1 +/- 0.2 s(-1) versus k(cat) = 0.4 +/- 0.1 s(-1)). A variant of BPDO(LB400) containing four active site residues of BPDO(B356) transformed para-substituted congeners better than BPDO(LB400). Interestingly, a substitution remote from the active site, A267S, increased the enzyme's preference for meta-substituted congeners. Moreover, this substitution had a greater effect on the kinetics of biphenyl utilization than substitutions in the substrate-binding pocket. In all variants, the degree of coupling between congener depletion and O(2) consumption was approximately proportional to congener depletion. At 2.4-A resolution, the crystal structure of the BPDO(B356)-2,6-dichlorobiphenyl complex, the first crystal structure of a BPDO-PCB complex, provided additional insight into the reactivity of this isozyme with this congener, as well as into the differences in congener preferences of the BPDOs.

Dataset Information

Novel biphenyl-oxidizing bacteria and dioxygenase genes from a korean tidal mudflat.

Publications

Novel biphenyl-oxidizing bacteria and dioxygenase genes from a korean tidal mudflat.

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