INTERNATIONALBIODETERIORATION +BIODEGRADATION

International Biodeterioration + Biodegradation 31 "0887# 110Ð117

S9853Ð7294:87:,08[99 Þ 0888 Elsevier Science Ltd[ All rights reserved[PII] S9853Ð7294"87#999424

Pseudomonas C01B\ an SDS degrading strain\ harbours a plasmidcoding for degradation of medium chain length n!alkanesJan Kostala�\ Miloslav Suchaneka\ Hana Klierovaa\ Katerina Demnerovaa\

Blanka Kralovaa\ Dani L[ McBethb

aInstitute of Chemical Technology\ Dept[ of Biochemistry and Microbiology\ Technicka 4\ Praha\ 5 05999\ Czech RepublicbThe City University of New York Medical School\ Dept[ of Microbiology and Immunology\ 027th St[ at Convent Ave[\ New York\

NY 09920\ USA

Received 02 May 0887^ revised 29 June 0887^ accepted 2 July 0887

Abstract

Pseudomonas C01B is able to degrade alkyl sulfates\ alkylbenzene sulfonates\ and linear alkanes and alkenes[ Mitomycin C curingexperiments and conjugation experiments demonstrated that the ability to utilize n!alkanes "C8ÐC01# and n!alkenes "C09 and C01# ofmedium chain length was plasmid!encoded[ The plasmid was designated pDEC[ Its size was estimated at several hundreds kbaccording to mobility in agarose gels[ The plasmid did not confer resistance to the antibiotics tested[ Analysis of alkylsulfatases P0and P1 in original and cured strains con_rmed that both enzymes are encoded by the chromosome[ The ability of PseudomonasC01B to utilize alkylbenzene sulfonates also appears to be encoded by the chromosome[ pDEC could be transferred only to curedderivatives of Pseudomonas C01B\ but not to strains of P[ aeru`inosa\ P[ putida\ or Acinetobacter sp[ Cured derivatives of PseudomonasC01B could not serve as hosts for the broad host range plasmid CAMÐOCT[ The enzyme system encoded by the putative dec genespresent on plasmid pDEC di}ers from the system coded by the alk genes of plasmid OCT in the size range of hydrocarbonspreferentially used[ Þ 0888 Elsevier Science Ltd[ All rights reserved

Keywords] Anionic surfactants\ Biodegradation^ Plasmid^ Pseudomonas

0[ Introduction

The metabolic diversity of Pseudomonas bacteria hasbeen well documented[ Sometimes biodegradativeenzymes are encoded on the chromosome\ but often theyare plasmid encoded[ Chakrabarty "0861# identi_ed aplasmid!encoded pathway for salicylate degradation inP[ putida R0[ Since then\ other degradative plasmids havebeen shown to be involved in the metabolism of camphor"Rheinwald et al[\ 0862#\ naphthalene "Dunn and Gunsa!lus\ 0862#\ toluene\ m! and p!xylenes "Worsey andWilliams\ 0864^ Friello et al[\ 0865#\ octane "Chakrabartyet al[\ 0862# and other substrates "Sayler et al[\ 0889#[Many of these plasmids are transmissible among Pseudo!monas species and compatible with one another"Chakrabarty\ 0865#[ Biodegradative plasmids some!times confer other characteristics\ such as mercury resist!

�Corresponding author[ Tel[] ¦319 1 13242910^ fax] ¦319 12008889^ e!mail] kostaljÝvscht[cz[

Abbreviations] DBS\ Sodium dodecylbenzene sulfonate^ EMS\ Ethyl!methane sulfonate^ SDS\ Sodium n!dodecyl sulfate[

ance "Chakrabarty\ 0865#\ or UV!response enhancement"McBeth\ 0878#[

Several n!alkane degradation pathways have beeninvestigated in bacteria\ but only two plasmids conferringn!alkane dissimilation have been reported[ In early stud!ies of alkane degradation\ the OCT plasmid was foundin P[ putida "van Beilen et al[\ 0883#[ Chakrabarty "0862#constructed a cointegrate of OCT with plasmid CAM[The cointegrate retained properties of both plasmids[Furthermore\ CAMÐOCT was much more readily trans!missible to a wide range of recipients than the poorlytransmitted OCT[ Recently\ another plasmid\ which isvery similar to plasmid OCT\ has been isolated from P[maltophilia N135 "Lee et al[\ 0885#[

We initiated studies of alkane metabolism with severalbacterial strains isolated from oil!contaminated soil inthe Czech Republic "Demnerova et al[\ 0883#[ Originally\Pseudomonas C01B was isolated for its ability to utilizelinear alkyl sulfates and alkylbenzene sulfonates "Payneand Feisal\ 0852^ White and Russell\ 0883#\ however\ itwas recently shown that this strain is also able to utilizen!alkanes "Kostal et al[\ 0884# via the monoterminal oxi!dation pathway "Suchanek et al[\ 0887#[ In this paper\ we

J[ Kostal et al[:International Biodeterioration + Biode`radation 31 "0887# 110Ð117111

describe the identi_cation and initial characterization ofa biodegradative plasmid in Pseudomonas C01B whichspeci_es enzymes required for n!alkane degradation[

1[ Materials and methods

1[0[ Chemicals

n!Alkanes and n!alkenes were obtained from FlukaChemie AG "Buchs\ Switzerland#[ Primary alcohols wereobtained from Fluka Chemie AG or Koch Light Lab!oratories "Colnbrook\ Bucks\ U[K[#[ Sodiumdodecylsulfate "SDS#\ sodium dodecylbenzene sulfonate "DBS#\and decyl aldehyde were supplied by Sigma Chemical Co["St[ Louis\ MO\ U[S[A[#[ n!Decanoic acid was purchasedfrom Fluka Chemie AG[ N!lauroylsarcosine\ ethyl!methane sulfonate "EMS#\ mitomycin C\ rifampin\ car!benicillin\ and lysozyme ðEC 2[1[0[06Ł were obtained fromSigma Chemical Co[ Acrylamide and N\N?!methylene!bis!acrylamide were supplied by Serva FeinbiochemicaGmbH + Co[ and other electrophoretic reagents "elec!trophoresis grade# were purchased from SigmaÐAldrichVertriebs GmbH or Sigma Chemical Co[ Phenol andchloroform were of molecular biology grade from FlukaChemie AG "Buchs\ Switzerland#[

Agar Technical "AGAR No[ 2\ Oxoid\ U[K[# was usedfor media solidi_cation where needed[ All other chemi!cals were the purest available from LachemaÐChemapol"Brno\ Czech Republic#[

1[1[ Bacterial strains

Bacterial strains are listed in Table 0[ Strains weremaintained in 14) "v:v# glycerol at −79>C and were pre!cultivated on nutrient agar plates prior to experiments[

Table 0Bacterial strains

Strain Relevant characteristic"s# Source or reference

Pseudomonas sp[ strainsC01B prototroph\ dec¦\ "pDEC# Payne and Feisal\ 0852aHM0 C01B\ met This studycH0\ cH2\ cH4\ cH6\ cH8\ cV0 C01B cured of pDEC\ Dec! This studymH0\ mH1\ mH2\ mH3\ mH4 exconjugant aHM0×cH0 "pDEC# This study

Pseudomonas putidaPpS034 alcA70\ met!034 "CAMÐOCT# Dunn and Gunsalus\ 0862PpS227 alcA70\ trp!227 Dunn and Gunsalus\ 0862H2r PpS227\ Rifr This studyKT1339 strain mt!1 res− mod¦ Bagdasarian et al[\ 0870KT1339r KT1339\ Rifr This study

Pseudomonas aeru`inosaPB1925 strain PAO0051 leu−27 res− mod¦ Chakrabarty\ 0863

Acinetobacter sp[AcM0 natural isolate\ dec− Demnerova et al[\ 0883

1[2[ Culturing cells in liquid media

Bacteria were grown in 19ml of nutrient broth"HiMedia Laboratories Pvt[ Ltd[\ Bombay\ India# over!night[ 0Ml of this starter was inoculated into variousvolumes of nutrient broth "as required#\ or into 099mlof minimal medium "Payne and Feisal\ 0852# in 499mlerlenmeyer ~asks with ba/es in the wall to achieve betteraeration[ Minimal medium was supplemented withappropriate carbon sources when needed at con!centrations as follows] n!alkane and n!alkene "except n!decene#\ 0) "v:v#^ n!decene\ 9[2) "v:v#^ SDS\ 199mg:l^DBS\ 0 g:l[ Cultures were grown with shaking at 17>Cand growth was monitored by measurement of turbidityat 549 nm[

1[2[ Culturing cells on solid media

Cells were streaked on the surface of solidi_ed minimalmedia "Dodgson et al[\ 0863#[ n!Decanoic acid was addedprior to autoclaving at a _nal concentration of 0 g:l[ SDSand pyruvate were added after autoclaving in the formof _lter sterilized solutions to give concentrations of199mg:l and 09 g:l\ respectively[ Hydrocarbons wereplaced in the lid after inoculation of the plates\ or\ alter!natively\ plates were placed in closed containers withhydrocarbon vapors[ The plates were then incubated at17>C for 0Ð2 days[ To test for antibiotic resistances\ anti!biotic disks supplied by LachemaÐChemapol"Neratovice\ Czech Republic# were placed on freshlyinoculated Mueller Hinton Agar plates "DiagnosticsPasteur\ France#[ Inhibition zones were evaluated after0!day incubation at 17>C[ In conjugation experiments\where appropriate\ medium was supplemented after

J[ Kostal et al[:International Biodeterioration + Biode`radation 31 "0887# 110Ð117 112

autoclaving with methionine\ tryptophan\ or rifampinto give concentrations of 7mg:l\ 19mg:l and 199mg:l\respectively[

1[3[ Isolation of plasmid DNA

Several plasmid isolation procedures and modi!_cations of those procedures were used including] alka!line lysis "Ausubel et al[\ 0876# and the methods of Kadoand Liu "0870#\ Wheatcroft and Williams "0870#\Wheatcroft et al[ "0889#\ Brenner et al[ "0882#\ Hansenand Olsen "0867# and Eckhardt "0867#[

1[4[ Spontaneous loss and plasmid curing

To monitor spontaneous plasmid loss\ cells were grownin nutrient broth for approximately 49 cell divisions withfrequent media replacement[ Cultures were then dilutedand spread on nutrient agar plates[ 419 individual col!onies were picked and tested for growth on n!decane andlauryl sulfate[

For plasmid curing\ the method of Rheinwald et al["0862# was followed[ 092 to 093 cells from an overnightculture were inoculated in several tubes containing 1mlof nutrient broth and mitomycin C at concentrationsranging from 4 to 14mg:ml[ Cultures were shaken at 17>Cuntil visible growth\ as evidenced by turbidity\ occurred[Aliquots from the highest concentration of mitomycin Cthat still permitted bacterial growth were diluted andspread on nutrient agar plates[ Individual colonies werethen tested on solid media for growth on n!decane\ SDS\and pyruvate[ Stability of the cured strains was moni!tored by periodic cycling of cells under non!selective con!ditions and re!testing for the ability to grow on n!decane[

1[5[ Mutant isolation

For isolation of auxotrophic mutants\ cells weremutagenized with EMS "Gerhard et al[\ 0883#[ Mutantswere subsequently enriched by the method of Ornston etal[ "0858# where penicillin was substituted by carbenicillinat a concentration of 649mg:ml[ Rifampin resistantmutants were isolated by the gradient method describedby Gerhard et al[ "0883#[

1[6[ Plasmid transfers

Both the donor and the recipient cells were grown toexponential or late exponential phase in nutrient broth[Then\ 09ml of donor cells were mixed with 09ml of recipi!ent cells on a 34mm membrane _lter placed on the surfaceof a nutrient agar plate and incubated overnight[ Cellswere scraped from the _lter\ suspended in saline waterand several dilutions were plated on minimal mediumagar supplemented with antibiotics or amino acids asrequired[ Rifampin resistance of the recipient\ methionine

auxotrophy of the donor\ or both were used for donorcounterselection[ The ability to utilize n!decane\ camphoror n!octane was used for recipient counterselection[ Con!jugation frequencies were expressed as number of trans!conjugants per one donor cell[

1[7[ Preparation of cell!free extracts

Cells were grown on SDS until mid exponential\ lateexponential\ stationary or late stationary phase[ Har!vested cells were resuspended in 9[0M phosphate bu}er\pH6[9 containing 19) "w:w# sucrose and lysozyme[ Thissuspension was incubated at room temperature for29min with gentle shaking[ Resulting spheroplasts wereallowed to stand in an ice bath for 29min to promotelysis and subsequently disrupted by ultrasonication[ Thelysate was centrifuged "24999 g for 29min at 3>C# andthe supernatant was used as a source of alkylsulfatases[

1[8[ Analysis of alkylsulfatase enzymes

Cell!free extracts were examined for alkylsulfataseactivity by the polyacrylamide gel zymographyprocedure[ Electrophoresis in 5[7) "w:v# non!denaturingpolyacrylamide rod gels and detection of alkylsulfataseswith incubation in 9[0M TrisÐHCl\ pH6[4\ containing09mM SDS\ were conducted as described by White et al["0874#[ Primary alkylsulfatases were revealed as whitebands of insoluble dodecanol released from SDS byenzyme activity[

2[ Results

2[0[ Plasmid isolation

Biodegradative abilities in Pseudomonas are oftenencoded on plasmids[ A variety of plasmid isolation pro!cedures were used in an e}ort to demonstrate the presenceof plasmid"s# in strain C01B[ These included the alkalinelysis method "Ausubel et al[\ 0876# as well as methodsthat have been previously used for isolating large\ lowcopy biodegradative plasmids] Wheatcroft and Williams"0870#\ Brenner et al[ "0882#\ Hansen and Olsen "0867#and Eckhardt "0867#[ The method of Wheatcroft et al["0889# showed a distinct plasmid band of high molecularweight "data not shown#\ whereas the other methodsyielded only a di}use band of DNA of 19Ð29 kb[ Finally\the method of Kado and Liu "0870# provided the bestresults for plasmid visualization "Fig[ 0#\ and was simplerand faster[ Using this method we estimated the size ofthe plasmid found in C01B roughly as several hundredskilobases[ Further puri_cation of the plasmid was notsuccessful\ presumably due to its large size[

J[ Kostal et al[:International Biodeterioration + Biode`radation 31 "0887# 110Ð117113

Fig[ 0[ Visualisation of plasmids in 9[64) agarose gel[A * e� DNAHind III digested molecular size marker "only largest 12 kb fragment isvisible#[B * plasmid DNA of Pseudomonas C01B visualized by themethod of Kado and Liu "0870#[C * DNA of cured strain cH0 vis!ualized by the method of Kado and Liu "0870#[D * plasmid CAMÐOCT visualized by the method of Wheatcroft and Williams "0870#[ Thismethod also produces large di}use bands of sheared chromosomalDNA as can be seen in line D[

2[1[ Plasmid loss

The potential spontaneous loss of plasmid was testedby screening individual colonies from cultures grown forapproximately 49 generations in nutrient media with noselection[ Screening was for the loss of the ability to growon n!decane or SDS[ No colony was found that hadspontaneously lost either of these degradative abilities[

The induced curing of plasmid DNA was attemptedusing mitomycin C treatment of cultures "Rheinwald etal[\ 0862#[ C01B was quite sensitive to this antibiotic\

Table 1Conjugal transfer of plasmid conferring n!decane utilisation in C01B

Medium for selection of Transfer frequencyExperiment Donor strain Recipient strain transconjugants0 "T:D#1

0 aHM0 cH0r Rif\ C09 09−2

1 aHM0 cV0r Rif\ C09 09−2

2 aHM0 cH0 C09 5×09−1

3 mH0\mH3 cH0r Rif\ C09 09−4Ð09−2

4 aHM0 H2r\ KT1339r Rif\ C09 ³09−7

5 aHM0 PB1925 C09 ³09−7

6 aHM0 AcM0 C09 ³09−7

0Transconjugants were selected on minimal medium agar with n!decane "C09# as the sole carbon and energy source[Where appropriate\ the medium contained rifampin "Rif# at 199 mg:ml[1 T:D\ number of transconjugants obtained per one donor cell[

4mg:ml allowed only weak growth that could be observedafter two days of incubation in nutrient broth[ Individualcolonies were again screened for the ability to grow on n!decane\ SDS or pyruvate[ All tested colonies remainedable to grow on SDS and pyruvate[ However\ 7 coloniesout of 0926 lost the ability to grow on n!decane whileretaining the ability to grow on the other carbon sources[The cured strains were designed cV0 and cH0 through 6[

2[2[ Conjugative ability

In order to test the transfer ability of the apparentdecane biodegradative plasmid\ we _rst isolated severalauxotrophic mutants and rifampin resistant mutantsusing EMS mutagenesis and carbenicillin enrichment asdescribed in Materials + Methods[ These strain con!structions were necessary for the development of geneticsystems in which donor and recipient could be coun!terselected[ Strain aHM0\ a methionine auxotroph ofC01B\ was used in subsequent experiments as the donorstrain[ It reverted to prototrophy with a frequency of09−7[ Strains cH0r and cV0r were rifampin resistantmutants of the plasmid!cured strains described above[The ability to utilize n!decane could be transferred athigh frequencies from aHM0 to both cured strains "Table1\ rows 0 and 1#[

Similar experiments were carried out using strain cH0as recipient "Table 1\ row 2#[ Five exconjugants from thismating were analysed for the presence of plasmid DNAalong with donor strain aHM0[ Plasmid DNA from allstrains exhibited identical mobilities on agarose gels tothat seen from the plasmid in aHM0 "data not shown#[Two exconjugants "mH0 and mH3# were tested as donorsof the plasmid in a second round of conjugation[ Again\decane!positive transconjugants were obtained "Table 1\row 3#[

In order to enable proper comparison of properties ofthe plasmid from C01B and plasmid CAMÐOCT\ wetried to transfer them into one bacterial strain[ Attempts

J[ Kostal et al[:International Biodeterioration + Biode`radation 31 "0887# 110Ð117 114

Table 2Conjugal transfer of plasmid CAMÐOCT

Medium for selection of Transfer frequencyExperiment Donor strain Recipient strain transconjugants0 "T:D#1

0 PpS034 H2r Rif\ C7 09−3Ð09−1

1 PpS034 KT1339r Rif\ C7 09−3

2 PpS034 H2r\KT1339r Rif\ CAM 09−3Ð09−1

3 PpS034 cH0r\cV0r Rif\ C7 ³09−8

4 PpS034 cH0r\cV0r Rif\ CAM ³09−7

0Transconjugants were selected on minimal medium agar with n!octane "C7# or camphor "CAM# as the sole carbonand energy source[ Rifampin "Rif# was added at 199 mg:ml[1 T:D\ number of transconjugants obtained per donor cell

to transfer CAMÐOCT into cH0r or cV0r failed "Table2\ rows 3 and 4#[ However\ CAMÐOCT was easily trans!ferred to rifampin resistant derivatives of P[ putida strainsKT1339 and PpS227\ designated as KT1339r and H2r\respectively "Table 2\ rows 0 to 2#[ Attempts to transferthe alkane degradative plasmid from C01B to KT1339rand H2r were not successful "Table 1\ row 4#\ nor couldthe plasmid be transferred to P[ aeru`inosa PB1925 or toAcinetobacter sp[ AcM0 "Table 1\ rows 5 and 6#[

2[3[ Comparison of C01B and its cured derivatives[

When su.cient evidence for the presence of the degrad!ative plasmid in Pseudomonas C01B was gathered andstrains cured of the plasmid were obtained\ we searchedfor di}erences in the properties of the original strain andcured derivatives\ represented by strain cH0[ Growth ona range of substrates in both solid and liquid mediawas tested[ Table 3 summarises di}erences in the growthabilities on normal alkanes and alkenes of varying chainlengths[ C01B was able to grow in liquid media only onn!alkanes with 8 to 01 carbon atoms\ whereas it utilized

Table 3Growth of strains C01B and cH0

Pseudomonas cH0 Pseudomonas C01B Pseudomonas C01BC!source agar plates agar plates liquid medium

n!hexane N[D[ N[D[ !n!heptane − ¦¦ −n!octane * ¦¦ −n!nonane ! ¦¦ ¦n!decane ! ¦¦ ¦¦n!undecane ! ¦¦ ¦¦n!dodecane ! ¦¦ ¦n!tridecane ! ¦¦ −n!tetradecane ¦ ¦¦ −n!pentadecane ¦ ¦¦ −n!hexadecane ¦ ¦¦ !n!decene − ¦¦ ¦¦n!dodecene − ¦¦ ¦¦n!octadecene ¦ ¦ −

a much wider range of tested n!alkanes in the vapourphase "growth on solid media#[ Similarly\ higher n!alkenes did not support growth in liquid media[ Curedderivatives lost the ability to grow on medium chain!length alkanes and alkenes "C²02#[ They maintained theability to grow on longer chain length hydrocarbons\ butthe growth was considerably more feeble than that seenfor C01B[ On the other hand\ both strains grew equallywell with tested anionic surfactants "SDS and DBS# and0!decanal\ 0!decanoic acid and longer primary alcohols"C7ÐC01#^ while shorter primary alcohols "C4ÐC7# were notutilized[

We attempted to _nd out if there is an antibiotic resist!ance gene present on the plasmid[ For this\ we testedstrain C01B and the cured derivative cH0 for growth inpresence of 03 di}erent antibiotics[ Both strains exhibitedan identical pattern[ They were resistant to penicillin\ko!trimoxazol\ chloramphenicol\ ampicillin\ lincomycin\oxacillin\ cephalotin\ and erythromycin\ but sensitive totetracycline\ gentamycin\ neomycin\ kanamycin\ strep!tomycin\ and sulfothioxazole[

Production of alkylsulfatases\ designated as P0 and

J[ Kostal et al[:International Biodeterioration + Biode`radation 31 "0887# 110Ð117115

P1 "Dodgson and White\ 0872#\ was monitored duringgrowth of original "C01B# and cured "cH0# strains onSDS as a sole source of carbon and energy[ Cells ofboth strains yielded extracts containing both enzymes[P1 enzyme occurred transiently during the exponentialand beginning of stationary phases of growth while P0was produced after the cultures reached stationary phase"data not shown#[ These results agreed well with previousstudies "White and Russell\ 0883#[

3[ Discussion

Loss of function at a rate higher than the expected rateof mutation and:or the ability to transfer a function torecipient cells are generally accepted properties that pro!vide genetic evidence for the presence of a plasmidinvolved in conferring a particular function to a cell"Williams\ 0867#[ Physical visualisation of plasmid DNAisolated from cells provides additional evidence\ but canbe quite di.cult when dealing with large plasmids[ Here\evidence of all three types cited above has been presentedfor plasmid involvement in the degradation of mediumchain!length n!alkanes by Pseudomonas C01B\ a strainoriginally isolated for its ability to degrade alkyl sul!phates and alkylbenzene sulfonates "Payne and Feisal\0852^ White and Russell\ 0883#[ The plasmid identi_ed inC01B has been designated pDEC[ Previously\ only twoplasmids encoding genes for alkane utilization have beendescribed\ CAMÐOCT "van Beilen et al[\ 0883# anda plasmid in a strain of P[ maltophilia "Lee et al[\0885#[

The presence of a plasmid in Pseudomonas C01B wasphysically demonstrated by gentle cell lysis followed byagarose gel electrophoresis "Kado and Liu\ 0870#[ A var!iety of procedures were attempted\ as the visualisation ofa large degradative plasmid is often di.cult[ We estimatethe size of pDEC carried by C01B to be several hundredskilobases "Fig[ 0# which is di}erent than the 007 kb ofthe plasmid found in P[ maltophilia N135 "Lee et al[\0885#[ Loss of the ability of cells to utilize medium chainlength n!alkanes and the regaining of that ability viaconjugative plasmid transfer\ as discussed below\ wasassociated with the disappearance and reappearance ofplasmid bands of the same size as those from the originalstrain[ These observations are in agreement with the con!clusion that C01B harbours a plasmid that encodes genesfor the utilization of these alkanes[

Spontaneous loss of n!decane utilization ability amongcells of Pseudomonas C01B was not observed[ However\treatment of cells with mitomycin C "4mg:ml# resulted incuring of this ability[ The frequency of curing "9[7) ofcells after 49 generations of growth# was equivalent tothat reported for curing of a variety of other Pseudomonasplasmids under similar conditions\ including CAM\OCT\ SAL\ NAH and TOL "Williams\ 0867#[

pDEC appeared to be conjugative in that the ability toutilize medium chain length n!alkanes could be trans!ferred to cured cells at high frequencies "Table 1#[However\ the host range for plasmid transfer was rathernarrow in that transfer to strains of P[ putida\ P[ aeru!`inosa and Acinetobacter was not detected "Table 1#[Interestingly\ the CAM− OCT plasmid\ which alsoencodes genes for n!alkane utilization\ could not be trans!ferred to cured derivatives of C01B even though it easilytransfers to other Pseudomonas species "Table 2# andAcinetobacter species "Demnerova et al[\ 0883#[ Thus far\we have been unable to fully classify C01B to a particularPseudomonas species "unpublished results#[ The results ofconjugation experiments suggest that pDEC is a verynarrow host range plasmid\ or limited to groups of soilbacteria not related to the recipient hosts tried here[ Fur!thermore\ these results prevented the construction ofisogenic derivatives carrying either CAMÐOCT or pDECwhich would have facilitated comparative analysis of thee}ects of the two plasmids[

The growth characteristics of C01B and cured deriva!tives were compared for growth on a variety of substrates"Table 3#[ In liquid medium\ pDEC clearly conferred theability to grow on medium chain length alkanes "C8ÐC01#\ whereas cured derivatives no longer supported suchgrowth[ Growth on solid media using alkane sourcesderived from the vapour phase exhibited a much widerrange and more extensive growth in the presence ofpDEC[ Cured derivatives were capable of slow\ feeblegrowth on longer chain alkanes of 01Ð05 carbons[ In ourexperience\ such abilities are not unusual among environ!mental isolates and probably re~ect a more non!speci_cenzyme system "unpublished observations#[ pDEC alsospeci_ed growth on n!alkenes of comparable chain length"Table 3#[ It is not yet clear whether oxidation of bothsubstrates utilizes identical enzymes[ The possible pres!ence of genes conferring antibiotic resistances was alsotested and none was found[ C01B was resistant to 7 of 03antibiotics tested\ but resistance remained after plasmidcuring[ This is consistent with the known high levels ofantibiotic resistance among species of Pseudomonas\ butinconsistent with the common carriage of R plasmidsamong these bacteria "Chakrabarty\ 0865#[

The ability to assimilate n!alkanes and n!alkenes codedfor by plasmid DNA complements an already describedbroad versatility in the degradation of various surfactantspresent in detergent formulations "01\ 26#[ PseudomonasC01B produces two primary alkylsulfatases\ P0 and P1\which act on a similar range of substrates "Dodgsonand White\ 0872#[ It was previously suggested that bothenzymes are chromosomally encoded "White and Russell\0883# and our results with plasmid curing and enzymeanalysis support this[ Cured derivatives of C01B whichno longer grow on n!alkanes are still capable of growth onSDS and related substrates and both P0 and P1 enzymeactivities can be detected[ The enzyme systems involved

J[ Kostal et al[:International Biodeterioration + Biode`radation 31 "0887# 110Ð117 116

in degradation of the di}erent substrates are clearly sep!arate[

Our inability to transfer both pDEC and CAMÐOCTto identical genetic backgrounds unfortunately did notallow a relevant comparison of phenotypes conferred bythe plasmids[ When growth of PpS034 "CAMÐOCT# iscompared with C01B strain "pDEC#\ the most signi_cantdi}erence is a shift of substrate speci_city[ WhereasCAMÐOCT confers dissimilation of n!alkanes with 5 to09 carbon atoms "van Beilen et al[\ 0883#\ pDEC enablesgrowth on n!alkanes with 8 to 01 carbon atoms[ Directin~uence of a host strain on the range of utilized sub!strates speci_ed by a degradative plasmid has not\ to ourknowledge\ been reported[ Thus it is possible that the setof alk genes "OCT# is di}erent from the set of genes foroxidation of n!alkanes residing on pDEC\ designated asdec[

4[ Conclusions

The work describes the role of a large plasmid in initialsteps of utilization of n!alkanes "C8ÐC01# and n!alkenes"C09 and C01# by strain Pseudomonas C01B[ The plasmid\pDEC\ is shown to be conjugative\ although with a verynarrow host range limited to the original host geneticbackground[ Strain C01B is also capable of degradingsome anionic surfactants\ but genes involved in this capa!bility do not reside on the pDEC plasmid[ The plasmiddi}ers from another alkane utilization plasmid\ CAMÐOCT\ in its host range\ size and substrate speci_city[ Tomake a more relevant comparison to other known alkaneutilization genes\ our current work is aimed at furthercharacterisation of dec genes both biochemically and gen!etically[

Acknowledgements

This work was supported by European Union grantPECO Proposal No[ CIPA 2409 PL 818591[We wish tothank V[ Brenner "Czech Academy of Science\ Prague\Czech Republic# for valuable discussions on plasmid iso!lation[

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