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Characterization of two long-chain fatty acid CoA ligases in the Gram-positive

Microbiological Research 167 (2012) 602–607

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Microbiological

Research

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.d e /m i c r e

Characterization of two long-chain fatty acid CoA ligases in the Gram-positive bacterium Geobacillus thermodenitri?cans NG80-2

Yanpeng Dong a ,b ,c ,d ,e ,Huiqian Du a ,b ,c ,Chunxu Gao a ,e ,Ting Ma e ,f ,Lu Feng a ,b ,c ,d ,e ,f ,?

TEDA School of Biological Sciences and Biotechnology,Nankai University,23Hongda Street,TEDA,Tianjin 300457,PR China b

Tianjin Key Laboratory of Microbial Functional Genomics,TEDA,Tianjin 300457,PR China c

Tianjin Research Center for Functional Genomics and Biochip,TEDA,Tianjin 300457,PR China d

The Engineering and Research Center for Microbial Functional Genomics and Detection Technology,Ministry of Education,PR China e

College of Life Science,Nankai University,Tianjin 300071,PR China f

The Key Laboratory of Molecular Microbiology and Technology,Ministry of Education,PR China

a r t i c l e

i n f o

Article history:

Received 5December 2011

Received in revised form 27March 2012Accepted 6May 2012

Keywords:CoA ligase FACL

Geobacillus thermodenitri?cans NG80-2Long-chain fatty acid Thermophilic

a b s t r a c t

The functions of two long-chain fatty acid CoA ligase genes (facl )in crude oil-degrading Geobacillus ther-modenitri?cans NG80-2were characterized.Facl1and Facl2encoded by GTNG 0892and GTNG 1447were expressed in Escherichia coli and puri?ed as His-tagged fusion proteins.Both enzymes utilized a broad range of fatty acids ranging from acetic acid (C 2)to melissic acid (C 30).The most preferred substrates were capric acid (C 10)for Facl1and palmitic acid (C 16)for Facl2,respectively.Both enzymes had an optimal temperature of 60?C,an optimal pH of 7.5,and required ATP as a cofactor.Thermostability of the enzymes and effects of metal ions,EDTA,SDS and Triton X-100on the enzyme activity were also investigated.When NG80-2was cultured with crude oil rather than sucrose as the sole carbon source,upregulation of facl1and facl2mRNA was observed by real time RT-PCR.This is the ?rst time that the activity of fatty acid CoA ligases toward long-chain fatty acids up to at least C 30has been demonstrated in bacteria.

1.Introduction

Fatty acid CoA ligase (Facl,fatty acid CoA synthetase;AMP form-ing;EC 6.2.1.)catalyzes the formation of acyl-CoA through a process which requires fatty acid,ATP and coenzyme A as substrates.It performs key roles in various metabolic and regulatory processes,including lipid biosynthesis and fatty acid degradation,interme-diary metabolism and gene expression,triglyceride incorporation and signal transduction/gene regulation (Black and DiRusso 2003;Faergeman and Knudsen 1997),by activating free fatty acids to their CoA thioesters.The enzymatic mechanism is a two-step reaction that proceeds via the formation of an acyl-adenylate (acyl-AMP)intermediate:

Step 1:fatty acid +ATP →fatty acyl-AMP +PPi.

Step 2:fatty acyl-AMP +CoA →fatty acyl-CoA +AMP.

Fatty acids may be classi?ed into three types,short (C 1–C 4),medium (C 5–C 16)and long (C 16+)with respect to the lengths of the

?Corresponding author at:TEDA School of Biological Sciences and Biotechnology,Nankai University,23Hongda Street,TEDA,Tianjin,300457,PR China.Tel.:+862266229592;fax:+862266229596.

E-mail address:fenglu63@https://www.doczj.com/doc/6d12976497.html, (L.Feng).

aliphatic chain (Hisanaga et al.2004;Zhu et al.2004;Soupene and Kuypers 2008;Zhu et al.2010).In bacteria,the most common fatty acids are those of C 14to C 18chain lengths,and bacterial Facls that utilize fatty acids ranging from C 2to C 18have been characterized (Fernandez-Valverde et al.1993;Kameda and Nunn 1981;Hisanaga et al.2004;Morgan-Kiss and Cronan 2004).Consistent with the diverse roles of Facls in cellular metabolisms,many organisms contain several isozymes that can use different chain length sub-strates (Faergeman et al.1997;Fujino et al.1997).In Escherichia coli ,two Facls have been characterized,FadD which has a broad sub-strate speci?city for fatty acids of medium and long chain lengths (C 6–C 18)and is involved in aerobic metabolic roles (Zhang et al.2006)and FadK which has a preference for short-chain length fatty acid substrates (below C 10)and is used for anaerobic metabolism of fatty acids (Morgan-Kiss and Cronan 2004).To our knowledge,Facl enzymes that catalyze fatty acids with chain-length longer than C 18have not been reported in bacteria.

Geobacillus thermodenitri?cans NG80-2is a long-chain alkane degrading thermophilic bacillus that was isolated from a deep sub-terranean oil-reservoir in northern China (Wang et al.2006).In our previous studies,using genomics and proteomics approaches,we investigated the metabolic process for the utilization of long-chain alkanes (C 15–C 36)in NG80-2.The result showed that alkanes are ?rst converted to corresponding fatty acids by a long-chain alkane monooxygenase (LadA),alcohol dehydrogenase (ADH)and

Y.Dong et al./Microbiological Research167 (2012) 602–607603

aldehyde dehydrogenase(ALDH),before entering the?-oxidation pathway as the CoA-activated form(fatty acid CoA)to be further degraded(Feng et al.2007).The functions of LadA,two long-chain ADHs,and a long-chain ALDH were also characterized using bio-chemical means(Feng et al.2007;Liu et al.2009;Li et al.2010).

In this study,two Facls encoded by GTNG0892(Facl1)and GTNG1447(Facl2),which were predicted to be involved in the con-version of long-chain fatty acids derived from alkane degradation to acyl-CoAs based on proteomics analysis(Feng et al.2007),were expressed in E.coli,puri?ed as His-tagged fusion proteins and char-acterized biochemically.The regulation of facl1and facl2mRNA level by long-chain fatty acids present in crude oil was examined by real time RT-PCR.This is the?rst report of fatty acid ligases which can utilize very long-chain fatty acid substrates up to at least C30,and also the?rst report of biochemically well-characterized thermophilic bacterial Facl enzymes.

2.Materials and methods

2.1.Materials and reagents

Crude oil was obtained from Dagang oil?eld69-8in northern China.The detergent Plysurf A-210G was kindly provided by Dai-ichi Kogyo Seiyagu Co.in Japan.Primers were synthesized by AuGCT Biotechnology Corporation,Beijing,China.Restriction enzymes and rTaq DNA polymerase were purchased from TaKaRa,T4DNA ligase from Promega,and DNase I from Roche.Phenylmethanesulfonyl ?uoride(PMSF)was purchased from Sigma.A chelating sepharose fast?ow column,low range SDS-PAGE molecular weight stan-dards and high molecular weight standards were purchased from Amersham Biosciences.An EnzChek?pyrophosphate assay kit was purchased from Molecular Probes.Other chemicals and reagents were obtained from Sangon,Shanghai,China.All reagents used were of analytical grade.

2.2.Bacterial strains and growth conditions

G.thermodenitri?cans NG80-2and E.coli BL21(DE3)(Novagen) were grown in Luria–Bertani(LB)medium with shaking at60?C and 37?C,respectively.When necessary,50mg kanamycin/l was added to the medium.NG80-2cells grown in mineral medium(Wang et al. 2006)supplemented with1%(w/v)sucrose or crude oil as a sole carbon source were used in real-time RT-PCR.

2.3.Cloning and plasmid construction

Genomic DNA from G.thermodenitri?cans NG80-2was extracted as described previously(Wang et al. 2006).NG80-2facl1was ampli?ed by PCR using primers5 -GGAATTCCATATGATGAGCGAAAAACGGGCCTTGT-3 and5 -CCGGAATTCTCATTGCGGGCGAGACGCCTCT-3 . NG80-2facl2was ampli?ed by PCR using primers5 -GGAATTCCATATGATGTTAACGGTGACTGTGGGGA-3 and 5 -CCCAAGCTTTTAAGTCGTCAAGCCGAGGCGC-3 .PCR was ini-tiated by denaturation at95?C for5min,followed by25cycles of95?C for30s,50?C for45s and72?C for1.5min,and a?nal extension at72?C for10min.The facl1product(1620bp)was digested with Nde I and Eco RI,and the facl2product(1635bp) was digested with Nde I,and Hin dIII,and the resulting fragments were ligated into pET-28a(Novagen)to generate pLW1217and pLW1220,respectively.The presence of the inserts in pLW1217 and pLW1220were veri?ed by sequencing using an ABI3730 automated DNA sequencer(ABI,Foster City,USA).2.4.Protein expression and puri?cation

E.coli BL21carrying pLW1217and pLW1220were grown in LB containing50mg/l kanamycin to an A600of0.6,and expression of the Facls were both induced by IPTG(0.1mM)at26?C for4h.

All steps in the following procedures were carried out aero-bically at4?C.Cells were harvested by centrifugation at6000×g for5min,washed with lysis buffer(50mM Tris–HCl buffer,pH 8.0,300mM NaCl and10mM imidazole),resuspended in the same buffer containing1mM PMSF and1g lysozyme/l,and broken up by sonication using a Hielscher UP200s ultrasonic processor(0.5cycle, 90%amplitude).After centrifugation at14,000×g for20min,the crude extract obtained was applied to a chelating sepharose fast ?ow column according to the manufacturer’s instruction.Unbound proteins were washed out with wash buffer(50mM Tris–HCl,pH 8.0,300mM NaCl and20mM imidazole).His-tagged fusion pro-teins were eluted with elution buffer(50mM Tris–HCl,pH8.0, 300mM NaCl and250mM imidazole)and dialyzed against dialysis buffer(50mM Tris–HCl buffer,pH8.0,containing20%glycerol).

Protein concentration was determined by the Bradford method (Bradford1976).SDS-PAGE and native-PAGE were performed using the methods of Laemmli(1970)and Tulchin et al.(1976),respec-tively.Low range SDS-PAGE molecular weight standards and high molecular weight standards were used as molecular weight mark-ers.

2.5.Coupled spectrophotometric assay for Facl activity

Facl activity was measured spectrophotometrically at360nm by following the protocol by Morgan-Kiss et al.with some modi?cation(Morgan-Kiss and Cronan2004).Facl activity was assayed by monitoring the inorganic pyrophosphate(PP i)pro-duced in the?rst half-reaction(Upson et al.1996).Pyrophosphate production was assayed as phosphate using a commercial spec-trophotometric assay in the presence of pyrophosphatase.The assay was very sensitive to the presence of contaminating inor-ganic phosphate(P i);therefore,to remove contaminant P i all enzyme preparations were dialyzed in dialysis buffer(see above) prior to use,and the presence of exogenous P i was assayed in all enzyme preparations as well as all other reaction components by performing the assay in the absence of pyrophosphatase.The standard200?l Facl reaction contained:20mM Tris–HCl(pH7.5), 1mM MgCl2,0.4mM ATP,0.4mM CoA,1mM fatty acid,0.2mM 2-amino-6-mercapto-7-methylpurine ribonucleoside,1unit of purine nucleoside phosphorylase,0.3units of pyrophosphatase and variable amounts of enzyme.For fatty acids ranging from C10to C30,0.001%(w/v)of plysurf A-210G was added into the reaction mixture as the dispersant.Because the unreacted2-amino-6-mercapto-7-methylpurine ribonucleoside substrate contributed to a signi?cant background absorbance(A360>0.3),the spectropho-tometer was calibrated in the presence of a solution containing 20mM Tris–HCl and0.2mM2-amino-6-mercapto-7-methylpurine ribonucleoside prior to assay measurements.For end point assays, the reaction containing20mM Tris–HCl(pH7.5),1mM MgCl2, 0.4mM ATP,0.4mM CoA,and1mM fatty acid was started by the addition of variable amounts of enzyme,and the reaction was incubated at optimal(60?C)or other temperatures for10min. Then0.2mM2-amino-6-mercapto-7-methylpurine ribonucleo-side,1unit of purine nucleoside phosphorylase,and0.3units of pyrophosphatase were added into the mixture and the reaction was continued at22?C for30min to1h.The reaction was stopped by adding200?l chloroform.The mixture was centrifuged and the hydrophilic layers were taken out as samples.The change in absorbance at360nm was measured on a UV-2550UV–visible spectrophotometer(Shimadzu,Kyoto,Japan).In all experiments, the amount of PP i produced during the reaction was determined by

604Y.Dong et al./Microbiological Research167 (2012) 602–607

subtracting the background absorbance in a control reaction con-taining dialysis buffer in place of the enzyme preparation.One unit of enzyme activity was de?ned as1?mol of fatty acid catalyzed per min per mg protein.All assays were carried out in triplicates.

2.6.Effects of temperature,pH,metal ions,EDTA,SDS and Triton

X-100on Facl activity

The temperature optimum was determined by measuring Facl activities at pH7.5and temperatures ranging from45?C to85?C. The pH optimum was determined by measuring Facl activities at 60?C in buffers with pH values ranging from5to11.The buffers used were:citrate–sodium citrate(pH5.0to6.6),sodium barbitu-rate/HCl(pH6.8to7.2),Tris/HCl(pH7.1to9.0),and glycine/NaOH buffer(pH9.0to11.0).To determine the effect of metal ions on Facl activity,reactions were carried out in the standard reac-tion mixture supplemented with metal ions,all in the form of chloride salt,at a?nal concentration of1mM(divalent cations) or100mM(monovalent cations).To determine the effects of EDTA, SDS and TritonX-100,1mM EDTA,0.05%SDS or1%TritonX-100 was included in the standard reaction mixture.For all assays, the substrate used was capric acid(1mM)for Facl1and palmitic acid(1mM)for Facl2.A reaction mixture without corresponding metal ions,EDTA,SDS or Triton X-100was used as a control.The extinction coef?cient for the change in absorbance when MESG (2-amino-6-mercapto-7-methylpurine ribonucleoside)and P i are converted to their reaction products by PNP(purine nucleoside phosphorylase)has been determined as11,000/M cm at pH7.6and 360nm(Webb1992).

2.7.Thermostability of Facl1and Facl2

The thermostability of the activity of Facl1and Facl2was exam-ined at60?C.The two enzymes were incubated at this temperature for a period of time(5min to24h)and then activity assays were taken as previously described,using1mM capric acid(Facl1)or palmitic acid(Facl2)as the substrate.

2.8.Bioinformatic methods

Homologous genes were found by NCBI BLAST search (https://www.doczj.com/doc/6d12976497.html,/BLAST/).Putative signatures were deduced by Prosite(https://www.doczj.com/doc/6d12976497.html,/prosite/).The pI and Mw were calculated using the compute pI/Mw tool (http://www.expasy.ch/tools/pi tool.html).The amino acid sequences were aligned with Clustal X.

2.9.Real-time RT-PCR

Real-time RT-PCR expression levels of facl1and facl2in NG80-2cells grown in a mineral medium supplemented with 1%(w/v)sucrose and crude oil as a sole carbon source were compared by real-time RT-PCR.The cells were grown at60?C with shaking for12h and pelleted by centrifugation.Total RNA was extracted with Trizol(Sangon,Shanghai,China)and pre-cipitated with isopropanol.Purity and concentration of RNA were checked by agarose gel electrophoresis and densitometry (OD260/280).RNA(500ng)was denatured at80?C for5min and reverse transcribed at42?C for20min using the RT Reagents (for Real Time RT-PCR Kit)from Takara.Real-time PCR for quan-ti?cation of cDNA was performed using the SYBR Green PCR master mix(Applied Biosystems)and the ABI7300Real-Time PCR System according to the manufacturer’s instructions.The following PCR conditions were used:50?C for2min,94?C for 10min,followed by40cycles of94?C for15s and60?C for1min. 16S rRNA was used as an internal control for normalization.Primers used were:5 -GCAAGGCTGAAACTCAAAGGA-3 and5 -GCGGGACTTAACCCAACATC-3 for 16S rRNA,5 -AGCGTGAACTGGCGTATGTG-3 and 5 -GGCGAGAAACTCATCAAATGTG-3 for facl1, and5 -GCAACCTGCGCCTGACATAC-3 and5 -GCCGCTTGATAGTTCGTATTGAC-3 for facl2.The average cycle number at the threshold(CT)was normalized against16S rRNA. The fold change of facl1and facl2expression level was calculated using the comparative CT method(User’s Manual for ABI7300 Real-Time PCR System)based on the change in the cycle numbers between the crude oil and sucrose samples.The expected prod-ucts of16S rRNA,facl1and facl2are206bp,187bp and195bp, respectively.

3.Results

3.1.Heterologous expression and puri?cation of NG80-2Facls

The NG80-2facl s were cloned into E.coli BL21(DE3).The Facls were expressed as His-tagged fusion proteins by IPTG induction with good yield detected in the soluble fraction,and puri?ed to apparent homogeneity by a Ni-column(Fig.1).The puri?ed Facl1 migrated as a band of59kDa in SDS-PAGE and118kDa in native-PAGE gels(Fig.1),indicating that it was present as a dimer,in good agreement with Facls of other origins(Kameda and Nunn 1981;Soupene and Kuypers2006;Li et al.2007),The puri?ed Facl2 migrated as a band of61kDa in SDS-PAGE and87kDa in native-PAGE gels,suggesting that it is present as either a non-spherical monomer or a dimer(Fig.1).

3.2.Characterization of Facls

Activity of the two puri?ed Facls was detected with fatty acids ranging from C2to C30(Fig.2).Fatty acids longer than C30were not tested due to these compounds being commercially unavail-able.The most preferred substrate was capric acid(C10)for Facl1 and palmitic acid(C16)for Facl2.Under the optimal assay conditions (60?C,pH7.5;see below),the speci?c activity of the Facls measured was2.8±0.13U/mg for capric acid(Facl1)and8.7±0.40U/mg for palmitic acid(Facl2).Unsaturated fatty acids such as14:1,16:1, 18:1,22:1,18:3(n-3),20:3(n-6)fatty acids and arachidonic acid were also tested,but none was found to be a substrate of Facl1 or Facl2.The kinetic parameters of Facl1and Facl2for representa-tive substrates were determined(data not shown).The apparent K m and catalytic ef?ciency(k cat/K m)of Facl1for capric acid were 0.0064±0.0007mM and37.5±0.10mM?1s?1,respectively.The apparent K m and catalytic ef?ciency(k cat/K m)of Facl2for palmitic acid were0.032±0.006mM and9.6±0.3mM?1s?1,respectively.

3.3.Effects of temperature,pH,metal ions,EDTA,SDS and Triton

X-100on Facl activity

The activity of both Facls was detected at temperatures ranging from25to80?C,and the highest activity was obtained at60?C for both enzymes(Fig.3a).At the tested pH levels from5to11,Facl activity increased along with an increase of pH,with the maximum activity obtained at pH7.5(Fig.3b),and about40%of the highest activity still remained at pH10for Facl1,and about40%of the high-est activity still remained at pH9.5for Facl2.The pH preference is consistent with Facls from other sources(Philipp and Parsons1979; Beaumelle and DVial1988;Fujino et al.1996).

The effect of metal ions on the enzyme activity was also exam-ined(Table1).The activity of Facl1was strongly inhibited by Ni+, Mn2+and Zn2+(more than70%inhibition).Co2+,Cu2+,Fe2+,and Ag+ also inhibited the activity of Facl1to different extents(51–36%of inhibition),but K+,NH4+and Na+showed no effects on the enzyme

Y.Dong et al./Microbiological Research 167 (2012) 602–607

605

Fig.1.(a)SDS-PAGE analysis of NG80-2Facl1and Facl2expressed in E.coli https://www.doczj.com/doc/6d12976497.html,ne 1,crude extract (soluble proteins)of E.coli BL21carrying pET-28a after IPTG induction;lane 2,total proteins (soluble and insoluble proteins)of E.coli BL21carrying pLW1217after IPTG induction;lane 3,crude extract of E.coli BL21carrying pLW1217after IPTG induction;lane 4,pellet proteins (insoluble proteins)of E.coli BL21carrying pLW1217after IPTG induction;lane 5,puri?ed His-tagged Facl1;lane 6,total proteins of E.coli BL21carrying pLW1220after IPTG induction;lane 7,crude extract of E.coli BL21carrying pLW1220after IPTG induction;lane 8,pellet proteins of E.coli BL21carrying pLW1220after IPTG induction;lane 9,puri?ed His-tagged Facl2;lane M,low range SDS-PAGE molecular weight standards.(b)Native-PAGE analysis of NG80-2Facl1and https://www.doczj.com/doc/6d12976497.html,ne 1,puri?ed His-tagged Facl1;lane 2,puri?ed His-tagged Facl2;lane M,high molecular weight

standards.

Fig.2.Substrate spectrum of NG80-2Facl1and Facl2.Enzyme activities were assayed at pH 7.5and 60?C.Each substrate was measured in triplicates.The relative activity of 100%corresponds to 2.8±0.13U/mg for Facl1and 8.7±0.40U/mg for Facl2,respectively.

Table 1

Effects of Triton X-100,EDTA and metal ions on Facl activity.The relative activ-ity of 100%corresponds to 2.8±0.13U/mg for Facl1and 8.7±0.40U/mg for Facl2,respectively.

Metal ion/chelator

Relative activity (%)

FACL1

FACL2None 100100Ag +6471Ca 2+156193Co 2+50191Cu 2+6433Fe 2+5736K +95104NH 4+9598Mn 2+2840Na +98100Ni 2+ 1.30Zn 2+

130.36Triton X-100198232EDTA 1226SDS

1.3

2.6

activity.The activity of Facl2was completely blocked by Ni +and Zn 2+,and strongly inhibited by Fe 2+,Cu 2+,and Mn 2+(more than 60%inhibition).Ag +inhibited 29%of the Facl2activity,but K +,NH 4+and Na +showed no effect on the activity.In contrast to the inhibition by most of the metal ions tested,Ca 2+enhanced the activity of Facl1and Facl2by 56%and 93%,respectively.Co 2+enhanced the activity of Facl2by 91%.

Treatment of the two enzymes with EDTA before dialysis inhib-ited the activity of the two enzymes by 88%and 74%,respectively,suggesting that metals are required for the activity of Facl1and Facl2.SDS inhibited Facl1and Facl2activity by 98%and 97%,respec-tively.Triton X-100enhanced the activity of Facl1and Facl2by 98%and 132%,respectively,suggesting that the enzymes may be membrane associated.

3.4.Thermostability of Facl1and Facl2

The thermostability of the activity of Facl1and Facl2was exam-ined at 60?C.About 40%of the highest activity of Facl1still

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607

Fig.3.Effects of temperature and pH on the activity of Facl1and Facl2.Enzyme activ-ities were assayed at pH 7.5in the range of 45–85?C (a),and at 60?C in the range of pH 5–11(b).The concentrations of Facl1and Facl2were both 0.1mg/ml.The rela-tive activity of 100%corresponds to 2.8±0.13U/mg for Facl1and 8.7±0.40U/mg for Facl2,respectively.The solid boxes represent Facl1and the hollow boxes represent Facl2.

remained after incubation at 60?C for 16h,and above 50%of the highest Facl2activity still remained after incubation at 60?C for 8h (Fig.4).

3.5.Real-time RT-PCR analysis of Facl1and Facl2

Real time RT-PCR showed a 6.7-fold increase and a 10.8-fold increase in the mRNA levels of facl1and facl2,respectively,when crude oil was used as a sole carbon source instead of sucrose (Fig.5),thereby further suggesting the physiological role of the Facls in crude oil degradation.

4.Discussion

In this study,the function of Facl1and Facl2was character-ized in vitro ,and the regulation of their expression by long-chain fatty acids in crude oil was evaluated using RT-PCR.We also eval-uated ?ve other putative Facls encoded on the NG80-2genome (GTNG 0575,GTNG 1170,GTNG 1337,GTNG 1356and GTNG 2617).In contrast to Facl1and Facl2,none of the other Facl enzymes showed activity on fatty acids longer than C 14(data not

shown).

20406080100

120Time(h)

R e l a t i v e a c t i v i t y (%)

Fig.4.Temperature stability of the activity of Facl1and Facl2.Enzyme activities were assayed at pH 7.5and at 60?C.The concentrations of Facl1and Facl2were both 0.1mg/ml.The relative activity of 100%corresponds to 2.8±0.13U/mg for Facl1and 8.7±0.40U/mg for Facl2,respectively.The solid boxes represent Facl1and the hollow boxes represent Facl2.

The deduced sequences of NG80-2Facls both show a conserved ligand of the ATP-binding motif.ScanProsite Results Viewer shows that LQYTGGTTGrSK from 191to 202and MQYTSGTTGfPK from 192to 203are putative AMP-binding domain signatures for Facl1and Facl2,respectively.Both are typical characteristics of the Facl family.The most similar protein of Facl1in the GenBank database is FadD isolated from E .coli (Weimar et al.2002).The identity of amino acids between Facl1and FadD is 39%with few gaps.The most similar protein of Facl2in GenBank is FadD isolated from humans (Watkins et al.2007).The identity of amino acids between these two proteins is 46%.As G.thermodenitri?cans is a facultative anaer-obe,capable of oxygen and nitrate respiration (Wang et al.2006),the seven Facls play different parts in aerobic or anaerobic path-ways.Like FadD in E.coli (Morgan-Kiss and Cronan 2004),the two long-chain Facls are involved in aerobic FA degradation in NG80-2,while other NG80-2Facls are expected for anaerobic functions.Noticeably,both Facls showed no activity with unsaturated FA substrates.The same was also found for the Facl from Thermus ther-mophilus HB8(Hisanaga et al.2004).This is in agreement with the

20406080100120140160

Sucr ose Crude Oil

m R N A r e l a t i v e e x p r e s s i o n (*10-7

)

Fig.5.Expression of facl 1and facl 2genes in G.thermodenitri?cans NG80-2grown

with sucrose and crude oil.Relative mRNA levels of facls were determined by real-time RT-PCR.RNA samples containing equal amounts of 16s rRNA were analyzed.The data were normalized with the expression of the control gene (16s rRNA).All reactions were performed in triplicates.

Y.Dong et al./Microbiological Research167 (2012) 602–607607

notion that thermophilic bacteria have predominantly saturated phospholipids in their membranes.The ability to utilize a broad range of FAs with different chain lengths indicates that the two NG80-2Facls may also execute other cellular functions such as fatty acid uptake for the purposes of phospholipid incorporation,as well as FA degradation.

The optimum temperatures for Facl1and Facl2are both60?C, much higher than the isoenzymes reported in Rattus norvegicus, which showed the highest optimum temperature(45?C)(Reddy et al.1984)among other characterized enzymes so far.Optimal temperature values have been reported for the Facl puri?ed from Pseudomonas putida(40?C),rat liver(38?C),Streptomyces coelicolor (37?C),Penicillium chrysogenum(37?C),and Methanothrix soehn-genii(35?C),as well as for the Facl from Pseudomonas fragi(37?C) and E.coli(35?C).However,in other cases,the optimal assay tem-perature was considerably lower(Fernandez-Valverde et al.1993). Although a Facl from T.thermophilic was also reported,the activity assay was only conducted at25?C(Hisanaga et al.2004).Further-more,the two Facls are the most heat stable Facls reported with more than50%activity detected at60?C after14h and8h for Facl1and Facl2,respectively,while the most heat stable isoenzyme reported in the literature is the enzyme acyl-CoA synthetase-4.6 from E.coli with78%activity retained at46?C after5min(Kameda and Imai1985).Apparently,the thermophilic characteristics of Facl1and Facl2are due to the thermophilic nature of NG80-2.

Alkanes ranging from C1to C35are the major components of crude oils.As long-chain alkanes(C16+)are more persistent in the environment than short-(C1–C4)and medium-chain alkanes (C5–C16),microorganisms capable of degrading those compounds, such as NG80-2,are advantageous in regard to their potential biotechnological applications for the treatment of oil spills.Up to this stage,we characterized all the genes involved in the degrada-tion of long-chain alkanes in NG80-2(Feng et al.2007;Liu et al. 2009;Li et al.2010).Those genes may serve as enzyme sources for oil bioremediation and other bioconversion processes.The ther-mophilic feature of NG80-2enzymes has many advantages over their mesophilic counterparts such as heat stability.

To our knowledge,this is the?rst report on Facls that oxidize long-chain fatty acids up to at least C30in bacteria. Acknowledgments

This work was supported by the Chinese National Science Fund for Distinguished Young Scholars(30788001),the National Natural Science Foundation of China(NSFC)Program Grants(30870070, 30800025,and81071392),and the National863Program of China Grant(2007AA02Z106and2009AA063502).

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