Experimental and Genomic Evaluation of the Oestrogen Degrading Bacterium Rhodococcus equi ATCC13557

Rhodococcus equi ATCC13557 was selected as a model organism to study oestrogen degradation based on its previous ability to degrade 17α-ethinylestradiol (EE2). Biodegradation experiments revealed that R. equi ATCC13557 was unable to metabolise EE2. However, it was able to metabolise E2 with the major metabolite being E1 with no further degradation of E1. However, the conversion of E2 into E1 was incomplete, with 11.2 and 50.6% of E2 degraded in mixed (E1-E2-EE2) and E2-only conditions, respectively. Therefore, the metabolic pathway of E2 degradation by R. equi ATCC13557 may have two possible pathways. The genome of R. equi ATCC13557 was sequenced, assembled, and mapped for the first time. The genome analysis allowed the identification of genes possibly responsible for the observed biodegradation characteristics of R. equi ATCC13557. Several genes within R. equi ATCC13557 are similar, but not identical in sequence, to those identified within the genomes of other oestrogen degrading bacteria, including Pseudomonas putida strain SJTE-1 and Sphingomonas strain KC8. Homologous gene sequences coding for enzymes potentially involved in oestrogen degradation, most commonly a cytochrome P450 monooxygenase (oecB), extradiol dioxygenase (oecC), and 17β-hydroxysteroid dehydrogenase (oecA), were identified within the genome of R. equi ATCC13557. These searches also revealed a gene cluster potentially coding for enzymes involved in steroid/oestrogen degradation; 3-carboxyethylcatechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde hydrolase, 3-alpha-(or 20-beta)-hydroxysteroid dehydrogenase, 3-(3-hydroxy-phenyl)propionate hydroxylase, cytochrome P450 monooxygenase, and 3-oxosteroid 1-dehydrogenase. Further, the searches revealed steroid hormone metabolism gene clusters from the 9, 10-seco pathway, therefore R. equi ATCC13557 also has the potential to metabolise other steroid hormones such as cholesterol.

Genome analysis provides insights into microaerobic toluene-degradation pathway of Zoogloea oleivorans BucT

Zoogloea oleivorans, capable of using toluene as a sole source of carbon and energy, was earlier found to be an active degrader under microaerobic conditions in aquifer samples. To uncover the genetic background of the ability of microaerobic toluene degradation in Z. oleivorans, the whole-genome sequence of the type strain BucT was revealed. Metatranscriptomic sequence reads, originated from a previous SIP study on microaerobic toluene degradation, were mapped on the genome. The genome (5.68 Mb) had a mean G + C content of 62.5%, 5005 protein coding gene sequences and 80 RNA genes. Annotation predicted that 66 genes were involved in the metabolism of aromatic compounds. Genome analysis revealed the presence of a cluster with genes coding for a multicomponent phenol-hydroxylase system and a complete catechol meta-cleavage pathway. Another cluster flanked by mobile-element protein coding genes coded a partial catechol meta-cleavage pathway including a subfamily I.2.C-type extradiol dioxygenase. Analysis of metatranscriptomic data of a microaerobic toluene-degrading enrichment, containing Z . oleivorans as an active-toluene degrader revealed that a toluene dioxygenase-like enzyme was responsible for the ring-hydroxylation, while enzymes of the partial catechol meta-cleavage pathway coding cluster were responsible for further degradation of the aromatic ring under microaerobic conditions. This further advances our understanding of aromatic hydrocarbon degradation between fully oxic and strictly anoxic conditions.

Catabolism of alkylphenols inRhodococcusvia ameta-cleavage pathway associated with genomic islands

The bacterial catabolism of aromatic compounds has considerable promise to convert lignin depolymerization products to commercial chemicals. Alkylphenols are a key class of depolymerization products whose catabolism is not well elucidated. We isolatedRhodococcus rhodochrousEP4 on 4-ethylphenol and applied genomic and transcriptomic approaches to elucidate alkylphenol catabolism in EP4 andRhodococcus jostiiRHA1. RNA-Seq and RT-qPCR revealed a pathway encoded by theaphABCDEFGHIQRSgenes that degrades 4-ethylphenol via themeta-cleavage of 4-ethylcatechol. This process was initiated by a two-component alkylphenol hydroxylase, encoded by theaphABgenes, which were up-regulated ~3,000-fold. Purified AphAB from EP4 had highest specific activity for 4-ethylphenol and 4-propylphenol (~2000 U/mg) but did not detectably transform phenol. Nevertheless, a ΔaphAmutant in RHA1 grew on 4-ethylphenol by compensatory up-regulation of phenol hydroxylase genes (pheA1-3). Deletion ofaphC, encoding an extradiol dioxygenase, prevented growth on 4-alkylphenols but not phenol. Disruption ofpcaLin the β-ketoadipate pathway prevented growth on phenol but not 4-alkylphenols. Thus, 4-ethylphenol and 4-propylphenol are catabolized exclusively viameta-cleavage in rhodococci while phenol is subject toortho-cleavage. Putative genomic islands encodingaphgeneswere identified in EP4 and several other rhodococci. Overall, this study identifies a 4-alkylphenol pathway in rhodococci, demonstrates key enzymes involved, and presents evidence that the pathway is encoded in a genomic island. These advances are of particular importance for wide-ranging industrial applications of rhodococci, including upgrading of lignocellulose biomass.ImportanceElucidation of bacterial alkylphenol catabolism is important for the development of biotechnologies to upgrade the lignin component of plant biomass. We isolated a new strain,Rhodococcus rhodochrousEP4, on 4-ethylphenol, an alkylphenol that occurs in lignin-derived streams, including reductive catalytic fractionation products of corn stover. We further demonstrated its degradation via ameta-cleavage pathway (Aph) with transcriptomics. A new class of Actinobacterial hydroxylase, AphAB, acts specifically on alkylphenols. Phylogenomic analysis indicated that theaphgenes occur on putative genomic islands in several rhodococcal strains. These genes were identified in the genetically-tractable strainRhodococcus jostiiRHA1. Strains missing this element cannot metabolise 4-ethylphenol and 4-propylphenol. Overall, we advanced the understanding of how aromatic compounds are degraded by environmental bacteria and identified enzymes that can be employed in the transition away from petro-chemicals towards renewable alternatives.

Biodegradation of 7-Hydroxycoumarin in Pseudomonas mandelii 7HK4 via ipso-Hydroxylation of 3-(2,4-Dihydroxyphenyl)-propionic Acid

A gene cluster, denoted as hcdABC, required for the degradation of 3-(2,4-dihydroxyphenyl)-propionic acid has been cloned from 7-hydroxycoumarin-degrading Pseudomonas mandelii 7HK4 (DSM 107615), and sequenced. Bioinformatic analysis shows that the operon hcdABC encodes a flavin-binding hydroxylase (HcdA), an extradiol dioxygenase (HcdB), and a putative hydroxymuconic semialdehyde hydrolase (HcdC). The analysis of the recombinant HcdA activity in vitro confirms that this enzyme belongs to the group of ipso-hydroxylases. The activity of the proteins HcdB and HcdC has been analyzed by using recombinant Escherichia coli cells. Identification of intermediate metabolites allowed us to confirm the predicted enzyme functions and to reconstruct the catabolic pathway of 3-(2,4-dihydroxyphenyl)-propionic acid. HcdA catalyzes the conversion of 3-(2,4-dihydroxyphenyl)-propionic acid to 3-(2,3,5-trihydroxyphenyl)-propionic acid through an ipso-hydroxylation followed by an internal (1,2-C,C)-shift of the alkyl moiety. Then, in the presence of HcdB, a subsequent oxidative meta-cleavage of the aromatic ring occurs, resulting in the corresponding linear product (2E,4E)-2,4-dihydroxy-6-oxonona-2,4-dienedioic acid. Here, we describe a Pseudomonas mandelii strain 7HK4 capable of degrading 7-hydroxycoumarin via 3-(2,4-dihydroxyphenyl)-propionic acid pathway.

Crystal structures of human 3-hydroxyanthranilate 3,4-dioxygenase with native and non-native metals bound in the active site

3-Hydroxyanthranilate 3,4-dioxygenase (3HAO) is an enzyme in the microglial branch of the kynurenine pathway of tryptophan degradation. 3HAO is a non-heme iron-containing, ring-cleaving extradiol dioxygenase that catalyzes the addition of both atoms of O2to the kynurenine pathway metabolite 3-hydroxyanthranilic acid (3-HANA) to form quinolinic acid (QUIN). QUIN is a highly potent excitotoxin that has been implicated in a number of neurodegenerative conditions, making 3HAO a target for pharmacological downregulation. Here, the first crystal structure of human 3HAO with the native iron bound in its active site is presented, together with an additional structure with zinc (a known inhibitor of human 3HAO) bound in the active site. The metal-binding environment is examined both structurally andviainductively coupled plasma mass spectrometry (ICP-MS), X-ray fluorescence spectroscopy (XRF) and electron paramagnetic resonance spectroscopy (EPR). The studies identified Met35 as the source of potential new interactions with substrates and inhibitors, which may prove useful in future therapeutic efforts.