Annals of Microbiology, 57 (4) 521-526 (2007)

Sequencing and analysis of pAV1 and pAV2 of 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 puta- tive aldehyde dehydrogenase gene, essential for hydrocarbon degradation, on pAV2, and of an ORF similar to alkL gene pres- ent 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 (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 enve- lope 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 differ- ent), 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. The uct showed a limited similarity with the central region of P. nucleotide sequences of plasmids pAV1 (DQ278485) and oleovorans AlkH (see below). pAV2 (DQ278486) were submitted to GenBank. 6) The two plasmids are highly divergent concerning the presence of mobile elements; pAV1 apparently does not harbour any (known) gene involved in transposition. RESULTS AND DISCUSSION On the contrary, plasmid pAV2 contains two identical copies (ORF3 and ORF8) of a gene coding for a putative Sequence analysis and ORF attribution integrase, and two genes coding for putative transposases The nucleotide sequences of plasmids pAV1 and pAV2 (ORFs 15 and 16), suggesting that this plasmid might revealed that they are circular molecules of 10,820 and undergo genetic rearangements more easily than pAV1. 15,135 bp in length, respectively, with a GC content of 35.6 The mobility of one of these genetic elements (ORF15) has and 37.5%, very similar to the GC content of A. venetianus been demonstrated (see below). Lastly, the presence in VE-C3 chromosomal fragments (data not shown). The pAV2 of a gene coding for a putative plasmid mobilisation nucleotide sequence of the two plasmids was analysed for protein also suggests that this plasmid might be horizon- the presence of ORF using the program ORF finder (see tally transferred by conjugation. Ann. Microbiol., 57 (4), 521-526 (2007) 523 protein yltransferase Unknown Unknown Unknwon Unknwon Unknwon Unknwon Hypothetical ypothetical protein meth functrion/structure DNA replication protein DNA Putative cytoplasmic protein cytoplasmic Putative Virulence associated protein B Virulence associated protein C Virulence Conserved hypothetical protein Conserved hypothetical % id** 52.43 35.87 58.67 70.32 87.50 61.04 42.04 42.04 H 96.94 inner membrane Putative DNA 36.62 Cytosine-specific -31 -28 -18 -43 -44 -50 -47 -52 -104 E -- 3.0E E-value E-value - - - - Localization Chromosome 7.0E Chromosome on 1 corresponds to the first nucleotide in iteron. - - 732729 Chromosome 2.0E 732729 Chromosome 34102520 ORF putative product ORF putative - - - Name gi RepM 61199611 PlasmidE 2.0 37699623 Chromosome 37699622 Chromosome 3.0E Chromosome 37699622 VapB 1304404 Chromosome 4.0E VapC 477904 Chromosome 9.0E

- 61199612 Plasmid 7.0 - 68232649 Chromosome 7.0E Chromosome 68232649 - EAN1pec sp. Chromobacterium violaceum Frankia Frankia Acinetobacter baumannii Acinetobacter baumannii Acinetobacter baumannii Acinetobacter baumannii Haemophilus parahaemolyticus Dichelobacter nodosus Dichelobacter Dichelobacter nodosus Dichelobacter 12472 ATCC Similar to organism

76 98 104 181 737 175 311 131 360 128 - 418 - (aa) Length GC pAV1 0.356 * |GCORF- GCpAV1 | ORF Fra me GC ORF (bp) 148 1083 936 1 0.348 0.008 5658 6914 1257 -1 0.302 0.054 3004 3234 231 1 0.471 0.115 4992 5288 297 3 0.395 0.039 1152 546 1697 3 0.398 0.042 6923 9136 2214 -1 0.328 0.028 9422 9808 387 -1 0.401 0.045 1915 528 2442 1 0.322 0.034 5275 5589 3150.009 1 0.365 3231 36263750 396 4832 1083 -3 3 0.331 0.025 0.397 0.041 ORF9 ORF4 ORF7 ORF2 ORF10 Name Start End Length ORF11 ORF3 ORF8 ORF1 ORF5 ORF6 TABLE 1 - Identification of the 11 putative ORFs found in pAV1 plasmid sequence * Absolute value of the difference between ORF GC content and total plasmid pAV1; ** %id = % identity. Positi 524 A. Mengoni et al.

protein Cro-like Cro-like Integrase Integrase Integrase subunit 6 subunit Unknown Unknown Unknwon Unknwon Unknown Unknown Unknown Unknown Unknown Unknown Unknown Hypotetical Transposase Transposase Transposase function /structure Hypothetical protein Hypothetical protein Hypothetical protein Helix-turn-helix motif DNA replication protein DNA id** 96.41 61.28 61.28 83.51 93.22 98.00 48.51 48.00 57.09 59.41 mobilisation 34.04 Plasmid 37.50 -162 -83 -109 -57 -47 -109 -16 -17 -91 -26 -34 -31 - - Plasmid 4.0E Plasmid on 1 corresponds to the first nucleotide in iteron. ------gi Localisation E-value % E-value gi Localisation 61199620 ORF putative product ORF putative

- 15076126 Chromsome 5.0E -0.17 51339712 Chromosome dehydrogenase 36.00 NADH - 27383371 Chromosome 3.0E - 68005149 Chromosome 1,0E Chromosome - 68005149 ------Sea24 49206959 Plasmid Plasmid 1,0E Sea24 49206959 Name MobA 61199621 Plasmid Plasmid 3.0E MobA 61199621 -7.0E Chromosome 67154790 - 61199614 Plasmid 1,0E - Plasmid 61199614 - 61199615 Plasmid 2,0E - Plasmid 61199615 - Sea24 492$06959 Plasmid 1,0E Plasmid 492$06959 Sea24 sp. LUH5605 Rep 58200481 Plasmid 2,0E sp. LUH5605 - 58200480 Plasmid 1,0E Similar to organism Acinetobacter baumanni Sinorhizobium meliloti Azotobacter vinelandii AvOP Azotobacter vinelandii Serratia entomophila Serratia Polysphondylium pallidum Polysphondylium Acinetobacter baumannii Acinetobacter Bradyrhizobium japonicum Bradyrhizobium 110 USDA Acinetobacter baumannii Acinetobacter baumannii Serratia entomophila Serratia Geobacter metallireducens GS- Geobacter metallireducens 15

Acinetobacter (aa) 107 101 318 104 275 107 445 118 307 188 100 445 259 89 - 101 - 101 217 - 217 223 - 223 389 - 389 Length 0.375 GC pAV2

|GCORF- GCpAV2|* ORF Fra GC me ORF (bp) 176 1099 924 2 0.440 0.065 4730 54010.393 672 2 0.018 91659614 9470 99370.410 306 -1 0.412 324 2 0.035 0.037 1929 32660.430 1338 -1 0.055 6019 63750.347 357 1 0.028 3547 47160.295 1170 1 0.080 8385 90380.292 654 -1 0.083 1163 17290.440 567 2 0.065 6368 66700.302 303 2 0.073 9918 10232 315 3 0.315 0.06 6930 82670.312 1338 -1 0.063 Start End Length Start End 10752 110210.388 270 3 0.013 11368 121950.399 828 -3 0.024 10286 10609 324 2 0.364 0.011 12192 12497 306 -1 12686 13642 957 0.330 -2 0.045 0.387 0.012 13972 147510.367 780 1 0.008

ORF14 ORF15 ORF5 ORF10 ORF11 ORF3 ORF13 ORF6 ORF4 ORF9 ORF1 ORF2 ORF16 ORF17 ORF7 ORF12 ORF8 ORF18 Name TABLE 2 - Identification of the 18 ORFs found in pAV2 plasmid sequence * Absolute value of the difference between ORF GC content and total plasmid pAV2; ** %id = % identity. Positi Ann. Microbiol., 57 (4), 521-526 (2007) 525

Inner DNA replication membrane protein protein DNA methyl-transferase Cytoplasmic VapB VapC protein Iteron

123 4 5 6 7 8 9 10 11 pAV1

Acinetobacter venetianus 12 3 4 5 678 9101112131415161718 pAV2

Iteron Transposase Plasmid mobilisation DNA replication Integrase Cro-like Integrase NADH- Transposase protein dehydrogenase

FIG. 1 - Linear genetic maps of pAV1 and pAV2 showing the identified ORFs with the corresponding putative protein. The same shad- ing indicates homologous (orthologous or paralogous) genes.

7) Concerning the organisation of genes along the plas- data, plasmid pAV2 might harbour a gene coding for an mid molecule, most of pAV1 genes are organised in such a aldehyde dehydrogenase activity (Decorosi et al., 2006). way that the direction of transcription and DNA replication This relies on the analysis of mutants lacking pAV2, which is the same. A different organisation was found in pAV2 failed to grow in mineral medium containing different n- where genes are not oriented towards the replication ter- alkanes (diesel fuel, decane, tetradecane, 1-tetradecanhol, mination. tetradecanale, eicosane), aliphatic alcohol (1-eicosanol) or fatty acids (arachidonic acid) as sole carbon sources. Role of plasmid pAV1 and pAV2 in hydrocarbon- However, growth was observed when mutants were grown degradation in presence of myristic acid, suggesting the possible pres- The possible involvement of plasmids pAV1 and pAV2 in the ence on pAV2 of a gene encoding an aldehyde dehydroge- biodegradation of n-alkanes was previously suggested by nase activity. Moreover, Southern blotting experiments (Di the analysis of A. venetianus Alk–mutants lacking pAV1 Cello et al., 1997) showed that plasmid pAV2 harbours DNA and/or pAV2 (Decorosi et al., 2006). According to these sequences complementary to alkBFGH operon of P. oleovo-

A

pAV2 ORF13 ------gi5531408 MTMHTRLAESNSSTESHSVIDIFNAQKKASSARRGKFSLAERIAALNILKEIIQRRESEIIAALAADFRKPASEVKLTEIFPVLQEISHAKRNLKTWMMP

pAV2 ORF13 ------MKHL gi5531408 RRVKAALSVLGTKARLSFEPKGVCLIIAPWNYPFNLSFGPLVSALAAGNSVIIKPSELTPHTASLIGSIIREAFSVDLVTVVEGDAAVSQELLTLPFDHI

pAV2 ORF13 GFVG------KSTEYIG------YTHVEIINSSLRLSFNVPLTNNVEETFKKIVDNN----- gi5531408 FFTGSPRVGKLVMEAAAKTLASVTLELGGKSPTIIGPTANLKKAARNIVWGKFSNNGQTCIAPDHVFVHRSIAHDFHKIIMEEVTRVYGKDFEAQKKSPD

pAV2 ORF13 ------YDLLMSTSAEMSFELENETLEILEKVIR------EQGIETEEFIQNTLIKFINQNGLDTTKSNL------gi5531408 YCRIVNDQHLNRLYELIGDAKSKGAKILQGGEIDALDRFVAPTIISKVTPEMDISREEIFG-PLLPIIEYDDIDHVIKQINDNAKPLALYIFSEDKEFAQ

pAV2 ORF13 ------gi5531408 DIVGRTSSGSVGINLSVVQFLHPNLPFGGVNNSGIGSAHGFYGFQAFSHEKPILLDRFSITHLLFPPYTGKVKRLISMTVRYLS

B

gi416606 MSFSNYKVIAMPVLVANFVLGAATAWANENYPAKSAGYNQGDWVASFNFSKVYVGEELGDLNVGGGALPNADVSIGNDTTLTFDIAYFVSSNIAV pAV1 ORF7 ------MFFM-EQYF--

gi416606 DFFVGVPAR-AKFQGEKSISSLGRVSEVDYGPAILSLQ-YHYDSFERLYPYVGVGVGRVLFF-DKTDGALSSFDIKDKWAPAFQV----GLRY-- pAV1 ORF7 EWDE---AKNRKNQ-KKHDISFETASLVFEDPLRISIQDRHTDGEER-WQTIGKVKGVLMLLVAHTIFDEDDCEIIRIIS-ARQVTKAERNQYEH

FIG. 2 - Clustal W alignment of the aminoacid sequences of the Acinetobacter venetianus VE-C3 putative proteins encoded by ORF7 and ORF13 of pAV2 and pAV1. A: alignment of ORF7 located on plasmid pAV1 and the outer membrane protein encoded by Pseudomonas oleovorans alkL gene; B: alignment of ORF13 located on plasmid pAV1 and the aldehyde dehydrogenase encod- ed by P. oleovorans alkH gene. 526 A. Mengoni et al.

rans, which actually contains one gene (alkH) encoding an degree of sequence similarity between ORF7 and AlnA. aldehyde dehydrogenase. Lastly, several mutant strains in which ORF9 of pAV1 was Data reported in Table 2 showed that many of the 18 interrupted by the insertion of a transposon coded for pAV2 ORFs coded for proteins of unknown function. either by pAV2 ORF15 or by a chromosomal gene showed However, a deeper analysis of those unknown proteins a reduced growth on medium containing diesel fuel as sole revealed that the putative protein of 107 aa encoded by carbon source (data not shown). Therefore plasmid pAV1 ORF13 shared a 20-44% of aminoacid sequence identity- might carry some function related to adhesion or internali- similarity with the central region of the aldehyde dehydro- sation of n-alkane molecules but possibly not related to genase (483 aa) coded for by the P. oleovorans alkH gene emulsification. (Fig. 2A). The whole body of data suggested the involve- In conclusion, A. venetianus VE-C3 shows a new ment of such a plasmid in key steps of aliphatic molecule genomic organisation of genes coding for alkane degrada- degradation (Decorosi et al., 2006). tion (presence of genetic determinants on both chromo- The finding that none of plasmid pAV1 encoded proteins some and plasmids pAV1 and pAV2) compared with that of shared a significant degree of sequence similarity with any Acinetobacter sp. strains ADP1 and M1 where all the genes known Alk protein is in agreement with PCR and Southern for alkane monoxygenase complex are located on the data previously reported (Di Cello et al., 1997; Decorosi et chromosome. al., 2006). On the other hand, plasmid pAV1 should be somehow involved in hydrocarbon degradation because Acknowledgements mutants lacking pAV1 did not grow in mineral minimal We are very grateful to Dr. Emanuele G. Biondi for helpul medium supplemented with different n-alkanes (diesel fuel, discussion and revision of the manuscript. decane, tetradecane, 1-tetradecanhol, tetradecanale, eicosane), aliphatic alcohol (1-eicosanol) or fatty acids REFERENCES (myristic acid, arachidonic acid) (Decorosi et al., 2006). This raises the question of the possible role played by plas- Altschul S.F., Madden T.L., Schaffer A.A., Zhalg J., Zhalg Z., mid pAV1 in n-alkanes degradation. Some clues concerning Miller W., Lipman D.J. (1997). Gapped BLAST and PSI- this issue can be obtained by the cross-analysis of genetic, BLAST: a new generation of protein database search pro- molecular and sequence data; since we did not find in plas- grams. Nucl. Acids Res., 25: 3389-3402. mid pAV1 any gene coding for protein apparently involved Baldi F., Ivosevic N., Minacci A., Pepi M., Fani R., Svetlicic V., in hydrocarbon oxidation, it is plausible that genes localised Zutic V. (1999). Adhesion of Acinetobacter venetianus to on plasmid pAV1 might be involved in the early steps of diesel fuel droplets studid with in situ electrochemical and molecular probes. Appl. Environ. Microbiol., 65: 2041-2048. hydrocarbon process, i.e. the n-alkanes recognition and/or Decorosi F., Mengoni A., Baldi F., Fani R. (2006). Identification uptake, or in the regulation of this process or even emulsi- of alkane monoxygenase genes in Acinetobacter venetianus fication. If this is true, the lack of pAV1 might interfere with VE-C3 and analysis of mutants impaired in diesel fuel degra- cell envelope formation and/or correct assembly; accord- dation. Ann. Microbiol., 56: 207-214. ingly, A. venetianus VEC3 mutants obtained with Di Cello F., Pepi M., Baldi F., Fani R. (1997). Molecular charac- nitrosoguanidine and lacking pAV1 form long cell chains terization of an n-alkane-degrading bacterial community and during growth in complex medium (Decorosi et al., 2006) identification of a new species, Acinetobacter venetianus. suggesting that some cell wall-related function has been Res. Microbiol., 148: 237-249. affected. It is not still clear which of the proteins coded for Head I.M., Jones D.M., Roling W.F. (2006). Marine microorgan- isms make a meal of oil. Nat. Rev. Microbiol., 4: 173-182. by pAV1 might be involved in hydrocarbon uptake; howev- er, some hypotheses can be formulated by the analysis of Ratajczak A., Geißdörfer W., Hillen W. (1998). Expression of alkane hydroxylase from Acinetobacter sp. strain ADP1 is the organisation of genes localised on this plasmid. Two induced by a broad range of n-alkanes and require the tran- genes, ORF 7-8, that encode a putative membrane inner scriptional activator AlkR. J. Bacteriol., 180: 5822-5827. protein and a cytoplasmic protein, respectively, have par- Tani A., Ishige T., Sakai Y., Kato N. (2001). Gene structures and tially overlapping coding sequences, which implies an oper- regulation of the alkane hydroxylase complex in on organisation. These two genes also shared an extreme- Acinetobacter sp. strain M-1. J. Bacteriol., 183: 1819-1823. ly high level of sequence similarity with orthologous genes Thompson J.D., Higgins D.G., Gibson T.J. (1994). CLUSTAL W: from a number of , both gram-negative and gram- improving the sensitivity of progressive multiple sequence positive, coding for an outer membrane and a cytoplasmic alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res., 22: protein, respectively (Table 1). In addition to this, we per- 4673-4680. formed a Clustal W alignment of the aminoacid sequence of Toren A., Orr E., Paitan Y., Ron E.Z., Rosenberg E. (2002). The the putative membrane protein coded for by ORF7 and that active component of the bioemulsifier alasan from of the outer membrane protein involved in alkane degra- Acinetobacter radioresistens KA53 is an OmpA-like protein. J. dation in P. oleovorans and encoded by alkL, which is locat- Bacteriol., 184: 165-170. ed on the OCT-plasmid. As shown in Fig. 2B, the A. vene- van Beilen J.B., Wubbolts M.G., Witholt B. (1994). Genetics of tianus protein shared a significant degree of sequence sim- alkane oxidation in Pseudomonas oleovorans. ilarity (22% identity, 43% similarity) with the second half Biodegradation, 5: 161-174. of the P. olevorans AlkL protein, a finding that might sug- Vaneechoutte M., Tjernberg I., Baldi F., Pepi M., Fani R., Sullivan gest a possible involvement of the A. venetianus VE-C3 E.R., van der Toorn J., Dijkshoorn L. (1999). Oil-degrading Acinetobacter strain RAG-1 and strains described as ORF7 protein in n-alkane degradation. We also compared ‘Acinetobacter venetianus sp. nov.’ belong to the same the A. venetianus ORF7 encoded protein with the genomic species. Res. Microbiol., 150: 69-73. Acinetobacter radioresistens alnA gene product (Toren et Young I.Y., Phelps C.D. (2005). Metabolic biomarkers for moni- al., 2002), a bioemulsifier molecule, referred to as alasan; toring in situ anaerobic hydrocarbon degradation. Environ. however, the comparison did not reveal any significant Health Perspective, 113: 62-67.