Short Communication Identification of aldolase and ferredoxin reductase within the dbt operon of Burkholderia fungorum DBT1 Stefano Piccoli*, Marco Andreolli*, Alejandro Giorgetti, Fabio Zordan, Silvia Lampis and Giovanni Vallini Department of Biotechnology, University of Verona, Strada Le Grazie 1537134, Verona, Italy Burkholderia fungorum DBT1, first isolated from settling particulate matter of an oil refinery wastewater, is a bacterial strain which has been shown capable of utilizing several polycyclic aromatic hydrocarbons (PAHs) including dibenzothiophene (DBT). In particular, this microbe is able to efficiently degrade DBT through the Kodama pathway. Previous investigations have lead to the identification of six genes, on a total of eight, required for DBT degradation. In the present study, a combined experimental/computational approach was adopted to identify and in silico characterize the two missing genes, namely a ferredoxin reductase and a hydratase-aldolase. Thus, the finding of all enzymatic components of the Kodama pathway in B. fungorum DBT1 makes this bacterial strain amenable for possible exploitation in soil bioremediation protocols. Keywords: Burkholderia fungorum DBT1 / Dibenzothiophene / Polycyclic aromatic hydrocarbons / Protein structure bioinformatics / Targeting gene walking Received: July 17, 2012; accepted: February 7, 2013 DOI 10.1002/jobm.201200408 Introduction Sulfur heterocycles are toxic compounds commonly found in crude oil, where they can represent up to 5% of the total polycyclic aromatic hydrocarbons (PAHs) content. Among these sulfur-containing hydrocarbons, dibenzothiophene (DBT) has long been taken as model compound in biotransformation studies [1]. Recently, a novel and unusual organization of the catabolic genes (defined as dbt) responsible for the Kodama oxidative degradation of DBT [2, 3] has been described in the bacterial strain Burkholderia sp. DBT1 [4], then attribut- ed to the fungorum species [5]. Actually, B. fungorum DBT1 harbors dbt catabolic genes in two separate operons (pH1A and p51), contrary to what occurs in Pseudomonas sp. strain C18 where the same are located in a single operon [6]. These genes show low similarity to the corresponding conserved isofunctional oxidative nah-like and phn-like genes. Moreover, while both promoter and terminator were identified in the pH1A genomic fragment (containing dbtD, Ac, Ad, B, and ORF5), the transcription terminator of the p51 genetic fragment (containing dbtC, Ab, ORF6, and ORF7) had not been identified so far [4]. Therefore, the two still missing gene sequences (namely the ferredox- in reductase component of the Rieske oxygenase complex and the hydratase-aldolase) were hypothe- sized to be located on the p51 operon. Functionally, the ferredoxin reductase is involved in single electron transfer from NAD(P)H to FAD within the Rieske oxygenase complex [7], while the hydratase-aldolase catalyses the last step of the pathway by transforming trans-4[2-(3-hydroxy)-thionaphthenyl]-2-oxo-3-bute- noate (HTOB) in 3-hydroxy-2-formyl benzothiophene (HFBT) [2, 3]. Materials and methods Bacterial strains and culture conditions B. fungorum DBT1 and B. fungorum LMG 16225 T , this latter provided by DSMZ and used as negative control, were grown through incubation at 27 °C in YMB (Yeast Mannitol Broth) medium [5].  Both authors contributed equally to the work. Correspondence: Silvia Lampis, Department of Biotechnology, Strada Le Grazie 15, 37134 Verona, Italy E-mail: silvia.lampis@univr.it Phone: 39 045 8027095 Fax: 39 045 8027051 Environment  Health  Techniques An in silico study of Burkholderia fungorum DBT1 1 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim www.jbm-journal.com J. Basic Microbiol. 2013, 00,1–6