Annals of Microbiology, 56 (3) 207-214 (2006) Identification of alkane monoxygenase genes in Acinetobacter venetianus VE-C3 and analysis of mutants impaired in diesel fuel degradation Francesca DECOROSI2, Alessio MENGONI1, Franco BALDI3, Renato FANI1* 1Department of Animal Biology and Genetics, University of Florence, Via Romana 17-19, 50125 Firenze; 2Department of Agricultural Biotechnology, University of Florence, Piazzale delle Cascine 24, 50144 Firenze; 3Department of Environmental Sciences, Cà Foscari University, Calle Larga Santa Marta, Dorso-Duro 21-37, 30123 Venezia, Italy Received 27 February 2006 / Accepted 18 May 2006 Abstract - Cells of Acinetobacter venetianus strain VE-C3 are able to degrade diesel fuel oil by a complex mechanism requiring the formation of cell aggregates and their further adhesion to fuel oil drops. In this work the biodegradation process in A. venetianus was studied by a combination of genetic, molecular and physiological methods. PCR amplification, sequencing and Southern blot analysis of alkM and rubA genes coding for the alkane hydroxylase and rubredoxin were carried out. Then, 22 Alk- mutants impaired in diesel fuel degradation were obtained by nitrosoguanidine mutagenesis and characterised by i) growth on alkanes as sole carbon and ener- gy sources, ii) modification of cell electrophoretic properties, and iii) analysis of plasmid content. Data obtained revealed that the genet- ic determinants for alkane degradation are located on both the chromosome and the two plasmids harboured by VE-C3 strain (pAV1 and pAV2, 11 Kbp and 15 kbp, respectively). This organization of genes coding for alkane monoxygenase complex seems to be simi- lar to the arrangement found in Acinetobacter sp. strains ADP1 and M1, where genes are scattered through the chromosome but, as a novelty, that some genes involved in hydrocarbon degradation are plasmid borne also. Key words: Acinetobacter, biodegradation, alkanes, alkane hydroxylase, rubredoxin. INTRODUCTION three different subunits: i) alkane hydroxylase, ii) rubredoxin, and iii) rubredoxin reductase. Extensive genetic and bio- The process of hydrocarbon degradation consists of two chemical studies conducted on alkane utilization in main steps: the former is handling hydrocarbons at the Pseudomonas oleovorans (van Beilen et al., 1994), revealed envelope, and the latter is the enzymatic degradation of that the alk genes, encoding proteins for the conversion of hydrocarbons to fatty acids. Hydrocarbon uptake is gener- alkanes to acyl coenzyme A (acyl-CoA), are located in two ally mediated by the synthesis of biosurfactants that avoid different regions of the OCT (octane utilization) plasmid. the direct contact between oil and membrane phospholipids. The alkBFGHJKL genes are cotranscribed from the alk pro- Biosurfactants can be secreted in the growth medium (Desai moter and code for the alkane hydroxylase, the rubredox- and Banat, 1997; Bach et al., 2003) as amphipathic mole- in, an aldehyde dehydrogenase, an alcohol dehydrogenase, cules able to decrease the interfacial tension between oil and an acyl-CoA synthetase and an outer membrane protein. The water, allowing hydrocarbon solubilisation. Alternatively, in other region contains alkS and alkR, which encode a LuxR- some Acinetobacter species (Navon-Venezia et al., 1995; UhpA-like regulator of alk operon expression and rubredox- Baldi et al., 2003), biosurfactant molecules can be embed- in reductase. AlkS is necessary for the activation of the ded in the cell outer membrane to control hydrocarbon expression of alkBFGHJKL operon. uptake without damaging phospholipid membrane. Once In Acinetobacter sp. strain ADP1 five genes essential for hydrocarbon molecules have bound the cell envelope, they n-alkane degradation have been found (Ratajczak et al., (may) undergo the degradation process. In aerobic condi- 1998): i) alkM, encoding the alkane hydroxylase; ii-iii) rubA tions only the terminal carbon oxidation pathway has been and rubB arranged in a bicistronic operon and coding for the identified so far, whereas other pathways have been eluci- rubredoxin and the rubredoxin reductase; iv) alkR, encod- dated in bacteria growing under anaerobic conditions (Yung ing a protein sharing a high degree of sequence similarity and Phelps, 2005). The aerobic process is usually catalysed with AraC-XylS-like transcriptional regulators, and v) xcpR, by the alkane monoxygenase complex that is formed by which is part of the general secretory pathway. In the n-alka- ne degrading Acinetobacter sp. M-1 genes coding the alka- ne hydroxylase complex were also found (Tani et al., 2001): * Corresponding author. Phone: +390552288244; the rubAB operon and two different alkane hydroxylase Fax: +390552288250; E-mail: [email protected], genes, alkMa and alkMb differentially induced in response to [email protected] the chain length of the n-alkane. In the regions upstream of 208 F. Decorosi et al. alkMa and alkMb, two putative transcriptional regulator MATERIALS AND METHODS genes (alkRa and alkRb, respectively) were found. Thus, Acinetobacter sp. ADP1 and Acinetobacter sp. M-1 share a Bacterial strains and growth conditions. The Acineto- very similar overall organization of alk genes, which, in turn, bacter venetianus strains used in this work were the wild type is completely different from the arrangement found in P. VE-C3 (Di Cello et al., 1997) and twenty-two Alk- mutant oleovorans. In fact these genes are neither embedded in a strains, referred to as C3NG (this work), which were obtained large operon nor clustered or localized on a plasmid but they from strain VE-C3 by nitrosoguanidine mutagenesis as are scattered throughout the bacterial chromosome. described below. The bacterial strain VE-C3, belonging to the species Bacterial strains were grown either on Luria-Bertani (LB) Acinetobacter venetianus (Di Cello et al., 1997; Vanee- or minimal medium MMV (Mills et al., 1978) (1.0 g of choutte et al., 1999), was firstly isolated from surface water MgSO4◊7H2O, 0.7 g of KCl, 2.0 g of KH2PO4, 3.0 g of of Venice Lagoon. As shown previously (Baldi et al., 1997; Na2HPO4, 1.0 g of NH4NO3, and 24.0 g of NaCl per litre of Di Cello et al., 1997), this strain is able to grow efficiently in deionised water) containing 0.4% diesel fuel as the sole minimal medium with diesel-fuel as the sole energy and carbon and energy source (MMG). Diesel fuel (Esso Italiana) carbon source. The structure and organization of alk genes was previously filtered through a 0.2 mm-pore-size filter in A. venetianus VE-C3 is not known. However, hybridisation (Sartorius) for sterilization and particle removal. The agarised experiments using the P. oleovorans alkBFGH as a probe mineral medium (agar 16 g per litre) was supplemented with revealed that some of the genes involved in n-alkanes degra- one of the following different carbon and energy sources: dation were very likely located on both the bacterial chro- 0.5% (w/v) succinic acid (Sigma), 2% (v/v) diesel fuel, 2% mosome and on the largest of the two plasmids harboured (v/v) n-decane, 2% (v/v) n-decanol, 2% (v/v) n-decanal, by VE-C3 cells, i.e. pAV1 (11 Kb) and pAV2 (15 Kb) (Di Cello 0.5% (w/v) caprinic acid, 2% (v/v) n-tetradecane, 0.5% et al., 1997). It is also known (Baldi et al., 1999) that A. vene- (w/v) n-tetradecanol, 0.5% (w/v) n-tetradecanal, 0.5% tianus VE-C3 envelope is hydrophilic when grown in complex (w/v) miristic acid, 2% (v/v) n-eicosane, 0.5% (w/v) n- medium. Conversely, in mineral medium containing diesel- eicosanol, 0.5% (w/v) arachidic acid (Fluka). n-decan, n- fuel as the sole carbon and energy source, the envelope decanol,-n-decanal, and n-tetradecane are liquid at room becomes hydrophobic, changing the electrophoretic mobili- temperature and they were filtered through a 0.2 mm-pore- ty of cells due to the direct contact between cells and oil drops size filter (Sartorius) and spread onto MMV plates. Caprinic (Baldi et al., 1999). The n-alkanes induce glycolisation of acid, n-tetradecanol, n-tetradecanal, miristic acid, n- membrane proteins involved in oil uptake (Baldi et al., 2003) eicosane, n-eicosanol and arachidic acid are solid at room and in biofilm formation due to a cell-to-cell contact and to temperature. In order to spread these substrates on agarised the synthesis of a composite material constituted by medium they were solubilised in ether (Sigma) and the solu- exopolyshaccarydes (EPS) and n-alkanes (Baldi et al., 1999). tions were then spread on the medium. Ether was removed In the present work the investigation was focused on the from the medium by putting the plates under vacuum for two two major steps of alkane degradation process in A. vene- hours. All the cultures were incubated at 28 °C for 48 h. tianus VE-C3 cells: hydrocarbon uptake and oxidation. In par- ticular the presence and localization of alkM and rubA genes Amplification and sequencing of alkM and rubA genes. were investigated and Alk- mutants impaired in diesel fuel PCR primers employed to amplify alkane hydroxylase and degradation were obtained by random chemical mutagene- rubredoxin partial coding sequence from the VE-C3 genome sis and characterised by checking their ability to grow in min- are listed in Table 1. imal medium with different alkanes and their products as the PCR was carried out using a Perkin ElmerGeneAmp PCR sole carbon and energy source, by testing their adhesion to System 9600 in a 1X PCR buffer containing 2 mM MgCl2, 200 hydrocarbons (MATH test), and by the analysis of plasmid µM of each dNTPs, 1 µM of each primer, 0.025 U/µl of Taq content. DNA polymerase (Finnzyme), and 10 ng of genomic VE-C3 DNA in a final volume of 20 µl. The presence of the alkane hydroxylase gene in the A. venetianus VE-C3 genome was checked using the degener- ate primers and the PCR program described by Smits et al.