Sequencing and Analysis of Plasmids Pav1 and Pav2 of <Emphasis Type

Annals of Microbiology, 57 (4) 521-526 (2007) Sequencing and analysis of plasmids pAV1 and pAV2 of Acinetobacter venetianus VE-C3 involved in diesel fuel degradation Alessio MENGONI1, Sandra RICCI1, Matteo BRILLI1, Franco BALDI2, Renato FANI1* 1Department of Animal Biology and Genetics, University of Florence, Via Romana 17-19, 50125 Firenze; 2Department of Environmental Sciences, Cà Foscari University, Calle Larga Santa Marta, Dorsoduro 21-37, 30123 Venezia, Italy Received 8 August 2007 / Accepted 8 October 2007 Abstract - Acinetobacter venetianus strain VE-C3 was isolated in the Venice lagoon (Italy) as a strain able to degrade diesel fuel oil. This strain possesses genes of the alkane monoxygenase complex responsible for n-alkane degradation and carries two plasmids, pAV1 (10820 bp) and pAV2 (15135 bp), which were supposed from the analysis of Alk- mutant strains to harbour genetic determinants for hydrocarbon degradation. In this work we determined the nucleotide sequence of both plasmids and showed the presence of a putative aldehyde dehydrogenase gene, essential for hydrocarbon degradation, on plasmid pAV2, and of an ORF similar to alkL gene present on pAV1 plasmid. These data, combined with genetic reports indicating that strains lacking one of the two plasmids or carrying transposon insertion on pAV1, are defective in n-alkane degradation, suggest a complex genomic organisation of genes involved in alkane degradation in A. venetianus VE-C3. In this bacterium these genes are carried by both the chromosome and the plasmids, while in Acinetobacter sp. strain ADP1 and M1 all the genes for alkane monoxygenase complex are located only on the chromosome. Key words: Acinetobacter venetianus, biodegradation, alkanes, pAV1, pAV2. INTRODUCTION (Ratajczak et al., 1998). In Acinetobacter sp. M1, an n- alkane degrading strain, several genes encoding the alka- The process of bacterial hydrocarbon degradation consists ne hydroxylase complex were also found (Tani et al., of two main steps, the attachment of hydrocarbons at the 2001); in particular the rubAB operon and two different cell envelope, and the enzymatic degradation of hydrocar- alkane hydroxylase genes, alkMa and alkMb, that are dif- bons to fatty acids (Head et al., 2006). Enzymatic degra- ferentially induced in response to the chain length of the n- dation can be performed both in anaerobic (Young and alkane were disclosed. In the alkMa and alkMb upstream Phelps, 2005) and in aerobic conditions. In this latter case regions, two putative transcriptional regulator genes (alkRa only the terminal carbon oxidation pathway has been iden- and alkRb, respectively) were identified. Acinetobacter sp. tified so far and the process is usually catalysed by the ADP1 and A. sp. M1 share a very similar overall organisa- alkane monoxygenase complex formed by three different tion of alk genes, which is completely different from the subunits (alkane hydroxylase, rubredoxin and rubredoxin arrangement found in P. oleovorans. In fact, these genes reductase). Genetic and biochemical studies conducted on are neither embedded in a large operon nor clustered or alkane utilisation in Pseudomonas oleovorans (van Beilen localised on a plasmid but they are scattered throughout et al., 1994), revealed that the alk genes, encoding pro- the bacterial chromosome. teins for the conversion of alkanes to acyl-coenzyme A The bacterial strain VE-C3, belonging to the species (acyl-CoA), were located in two different regions of the OCT Acinetobacter venetianus (Di Cello et al., 1997; plasmid. The alkBFGHJKL genes are cotranscribed from the Vaneechoutte et al., 1999), was firstly isolated from surface alk promoter and code for the alkane hydroxylase (alkB), water of Venice Lagoon. As shown previously (Di Cello et al., the rubredoxin (alkF and alkG), an aldehyde dehydroge- 1997), this strain is able to grow efficiently in minimal medi- nase (alkH), an alcohol dehydrogenase (alkJ), an acyl-CoA um with diesel-fuel as the sole energy and carbon source. synthetase (alkK) and an outer membrane protein (alkL). In the last years the n-alkane degradation process in this The other region contains alkS and alkR, which encode a bacterium has been studied at both physiological and LuxR-UhpA-like regulation system of the expression of the molecular genetic level (Baldi et al., 1999). The first step of alk and rubredoxin reductase. AlkS is necessary for the hydrocarbon degradation is represented by the interaction activation of the expression of alkBFGHJKL operon. In between the diesel-fuel droplets and the VE-C3 cell enve- Acinetobacter sp. strain ADP1 five genes (alkM, rubA, rubB, lope, a complex process that requires a preliminary cell-to- alkR, xcpR) are essential for n-alkane degradation cell aggregation, which is parallel to an increase of cell envelope hydrophobicity, and followed by the internalisation of * Corresponding author. Phone: +390552288244; diesel-fuel droplets (Baldi et al., 1999). The further enzy- Fax: +390552288250; E-mail: [email protected]. matic degradation of n-alkanes to fatty acids is performed 522 A. Mengoni et al. under aerobic conditions via a terminal carbon oxidation Materials and Methods); in this way, 11 and 18 ORFs were pathway. The structure and organisation of alk genes in A. identified in pAV1 and pAV2, respectively. The aminoacid venetianus VE-C3 has been recently investigated (Decorosi sequence of each of the putative proteins coded for by the et al., 2006). In particular it has been demonstrated that in 29 ORFs was used as a query in a BLASTP search (Altschul this A. venetianus VE-C3 cells the alkM and rubA genes et al., 1997) in order to retrieve the most similar sequences (coding for a alkane hydroxylase and a rubredoxin) are available in databases. Data obtained are reported in scattered in different loci of the bacterial chromosome Tables 1 and 2 and the linear genetic maps of the two plas- (Decorosi et al., 2006). Moreover, a possible involvement of mids are shown in Fig. 1. The analysis of these data the two plasmids harboured by VE-C3 cells, i.e. pAV1 (11 revealed that: Kb) and pAV2 (15 Kb) in the degradation process, was ini- 1) The two plasmids harbour an ORF (ORF1) coding for tially suggested by the presence of hybridization signal with a putative protein of very similar length that shares a high alkBFGH probe of P. oleovorans on the plasmid pAV2 (Di degree of sequence similarity with replication proteins from Cello et al. 1997) and by the analysis of mutants lacking one other plasmids (Tables 1 and 2). Upstream of each ORF1, or both plasmids (Decorosi et al., 2006). an iteron consisting of a 22-nt fourfold-repetition was In the present work, we report the nucleotide sequence found; this sequence very likely represents the origin of and the analysis of pAV1 and pAV2 plasmids. replication of each plasmid. The nucleotide sequence of the two iterons are highly divergent (13 out of 22 nt are different), a finding which is parallel to a low degree of sequence MATERIALS AND METHODS similarity between the putative Rep proteins, which exhibit- ed only 40% of sequence identity between them. This sug- DNA sequencing. Plasmid DNA of pAV1 and pAV2 was gests that the two plasmids have been very likely originat- extracted from A. venetianus VE-C3 actively growing cells ed from different ancestral plasmids, rather than by dupli- using the QiaSpin plasmid purification kit (Qiagen), digest- cation and further divergence of a common ancestor DNA ed with restriction enzymes EcoRI and HindIII (Biolabs) molecule. and cloned in the plasmid vector pUC18. Selected clones 2) The second ORF of both plasmids (ORF2) is locat- were sequenced and nucleotide sequences obtained were ed just downstream from ORF1 and codes for a putative used for designing divergent primer pairs for whole plasmid protein showing a high degree of sequence identity with amplification. Whole plasmid PCR amplification was carried homologous proteins from other Acinetobacter plasmids. out with Long PCR system kit (Expand Long Template PCR The two proteins share a degree of sequence System, Roche), following manufacturer’s instruction, for identity/similarity of 42/57%. It is worth of noticing that templates of about 10 and 15 Kbp for pAV1 and pAV2, the product of ORF2 from pAV2 is almost identical to the respectively on a MJ Research PTC-100 Thermal Cycler. orthologous protein carried by plasmid ptet5605 from Amplification products were sequenced by using a primer Acinetobacter sp. LUH5605, whereas the same protein walking approach on an ABI310 Genetic Analyzer (Applied from pAV1 shares the highest degree of sequence identi- Biosystems) using BigDye v1.1 chemistry (Applied ty with a protein coded for by a gene located on the Biosystems). Sequences were visually inspected and Acinetobacter baumannii pMAC. This finding strongly assembled to obtain the complete plasmids sequence. suggestes that pAV1 and pMAC might have a common ancestry, in that they might have been evolved through Sequence analysis and annotation. The nucleotide different genetic rearrangements from a common ances- sequences of the two plasmids were analysed for the pres- tral plasmid; this appears to be true also for pAV2 and ence of ORF using the program ORF finder ptet5505. (www.ncbi.nlm.nih.gov/gorf). Each ORF and the correspon- 3) The two plasmids did not share any other ORF. ding product were then analysed using the BLASTn and 4) It was not possible to assign a function to most of BLASTp options of BLAST program (Altschul et al., 1997), putative proteins encoded by pAV1 and pAV2 ORFs (Tables respectively. The Clustal W program (Thompson et al., 1 and 2). 1994) with standard parameters was used for multiple 5) None of the pAV1 and pAV2 ORFs encodes a product amino acid sequences alignment followed by careful visual showing a significant degree of sequence similarity with inspection. known enzymes involved in hydrocarbon oxidation. The only exception is represented by pAV2 ORF13, whose prod- Nucleotide sequence accession numbers.

Load more