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Keywords:

  • secreted protein;
  • fatty alcohol;
  • protein-like activator

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Extracellular lipase activity from Ralstonia sp. NT80 is induced significantly by fatty alcohols such as stearyl alcohol. We found that when lipase expression was induced by stearyl alcohol, a 14-kDa protein (designated EliA) was produced concomitantly and abundantly in the culture supernatant. Cloning and sequence analysis revealed that EliA shared 30% identity with the protein-like activator protein of Pseudomonas aeruginosa, which facilitates oxidation and assimilation of n-hexadecane. Inactivation of the eliA gene caused a significant reduction in the level of induction of lipase expression by stearyl alcohol. Furthermore, turbidity that was caused by the presence of emulsified stearyl alcohol, an insoluble material, remained in the culture supernatant of the ΔeliA mutant during the late stationary phase, whereas the culture supernatant of the wild type at 72 h was comparatively clear. In contrast, when lipase expression was induced by polyoxyethylene (20) oleyl ether, a soluble material, inactivation of eliA did not affect the extracellular lipase activity greatly. These results strongly indicate that EliA facilitates the induction of lipase expression, presumably by promoting the recognition and/or incorporation of the induction signal that is attributed to stearyl alcohol.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Lipases (EC 3.1.1.3) catalyze both the hydrolysis and the synthesis of glycerides formed from glycerol and long-chain fatty acids, and of various esters. Owing to their substrate specificity, regiospecificity, chiral selectivity, thermostability, and alkaline stability, microbial lipases have been utilized industrially for many purposes, such as the modification of detergents, processing of oil, diagnostics, biotransformations, and separation or synthesis of chiral compounds (Jaeger & Reetz, 1998; Jaeger & Eggert, 2002; Gupta et al., 2004). In many microorganisms, such as Aspergillus japonicus (Vora et al., 1988), Candida rugosa (de María et al., 2005), Geotrichum candidum (Shimada et al., 1992), Oospora fragrans (Ruban et al., 1978), Penicillium simplicissimum (Sztajer et al., 1992), Thermus thermophilus (Deive et al., 2009), and Yarrowia lipolytica (Destain et al., 2005), lipase expression can be stimulated by oils and fatty acids. In addition, various types of detergent, such as Tween (polyoxyethylene sorbitan), are also known to induce lipase in several species, such as Burkholderia glumae (Boekema et al., 2007), Malassezia furfur (Plotkin et al., 1996), Serratia marcescens (Long et al., 2007), and Sulfolobus shibatae (Huddleston et al., 1995).

As mentioned above, oils, fatty acids, and detergents are useful inducers of lipase expression in many microorganisms. In contrast, we have found that stearyl alcohol and several fatty alcohols can significantly induce extracellular lipase activity in Ralstonia sp. NT80 (previously named Pseudomonas sp. NT80), a beta proteobacterium, and related species (Ushio et al., 1996). When stearyl alcohol is added to the culture medium, extracellular lipase activity is induced by up to c. 350-fold (c. 140 U mL−1 by the p-nitrophenyl laurate method) as compared with the case without oily inducers (≤ 0.4 U mL−1; Ushio et al., 1996). The efficiency of induction of extracellular lipase activity by stearyl alcohol was approximately sevenfold higher than that obtained with olive oil (Ushio et al., 1996). However, the pathway by which stearyl alcohol induces lipase expression in Ralstonia sp. NT80 remained unclear.

In many species of Pseudomonas and Burkholderia, which are classified as Proteobacteria, genes that encode lipases are co-transcribed with a gene that encodes a helper protein (Rosenau & Jaeger, 2000; Rosenau et al., 2004). In the past, this lipase helper protein was thought to regulate lipase expression (Frenken et al., 1993). However, at present, it is generally accepted that the lipase helper protein plays a role in the folding of lipase within the periplasm and not in transcriptional regulation (Rosenau & Jaeger, 2000; Rosenau et al., 2004). The transcriptional regulation of the operon that includes the gene for lipase (the lipAB operon) has been studied extensively in Pseudomonas alcaligenes, in which expression is induced by soybean oil (Cox et al., 2001; Krzeslak et al., 2008, 2012). Initially, a σ54 (RpoN-dependent) promoter sequence and upstream activator sequence (UAS) were identified in the upstream region of the lipAB operon and characterized (Cox et al., 2001). Recently, a two-component regulatory system for this operon, LipQR, has been identified (Krzeslak et al., 2008). Moreover, it has been demonstrated that, after phosphorylation at aspartate 52, LipR associates with the UAS and activates the transcription of lipAB, and RpoN is certainly involved in the expression of lipase (Krzeslak et al., 2012). Although transcriptional regulation of lipase has been well documented, as described above, the mechanisms recognizing inducers of lipase expression, which are often insoluble materials, have not been fully investigated. In the present study, we explored an extracellular protein that is involved in lipase expression to elucidate the mechanisms underlying recognition of inducers in Ralstonia sp. NT80 and identified a secreted protein, EliA (effector protein of lipase induction), that facilitates the induction of lipase expression by stearyl alcohol. On the basis of our findings, the possible role of EliA is discussed.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacterial strains and growth conditions

Ralstonia sp. NT80 (previously named Pseudomonas sp. NT80) was isolated in our laboratory from soil as a producer of thermostable lipase (Ushio et al., 1996). Strains were precultured at 30 °C for 16 h in 2 mL of YP medium (in a test tube) with reciprocal shaking (160 rpm). YP is a nutrient-rich medium (pH 7.1–7.2) that contains 0.5% yeast extract, 0.5% polypeptone, 0.1% K2HPO4, and 0.02% MgSO4.7H2O. Subsequently, 500 μL of the culture was inoculated into 50 mL of YP medium. The culture was incubated at 30 °C with reciprocal shaking (160 rpm) in a 500-mL baffled flask. Inducers of lipase expression, namely stearyl alcohol, polyoxyethylene (20) oleyl ether, and soybean oil, were added to the YP medium at a final concentration of 1% (v/v) as required. The following antibiotics were added as necessary: gentamycin (200 μg mL−1) and kanamycin (40 μg mL−1).

Preparation of extracellular proteins

After the cells had been removed by centrifugation at 13 000 g for 2 min, 750 μL of the culture supernatant was recovered. An equal volume of 20% trichloroacetic acid in acetone was added to the culture supernatant, and the mixture was chilled at −20 °C for 20 min. Subsequently, precipitated proteins were harvested by centrifugation at 13 000 g for 10 min at 4 °C. The protein pellet was washed twice with 1 mL of acetone and collected in a similar manner by centrifugation. The resulting pellets were resuspended and analyzed by sodium dodecyl sulfate polyacrylamide gel (15%) electrophoresis (SDS-PAGE).

N-terminal amino acid sequencing

After SDS-PAGE, the proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore) using a semi-dry blotting system (ATTO) and analyzed by Edman degradation on an Applied Biosystems Model 494 HT Procise protein sequencer. Experiments were performed in triplicate, and the same sequences were detected each time.

DNA manipulation

DNA was manipulated in Escherichia coli (Ausubel et al., 1987; Sambrook & Russell, 2001) as previously described. The primers used in the study are listed in Supporting Information, Table S1.

Cloning of eliA

Amino terminal sequencing of EliA by Edman degradation gave the sequence VTVSRVDGQPMNPSGEPFSVTG. On the basis of this sequence, a primer, eliACF (see Table S1 for all primers used in the study), was synthesized. We used this primer and chromosomal fragments of wild-type NT80 that had been partially digested with Sau3AI to amplify the eliA gene and its downstream region with a LA PCR in vitro Cloning Kit (TaKaRa), in accordance with the manufacturer's instructions. Briefly, chromosomal fragments that had been partially digested with Sau3A and ligated to a Sau3AI cassette (TaKaRa) were used as the template in a PCR amplification with the primers eliACF and cassette primer C1 (TaKaRa), and PrimeSTAR polymerase (TaKaRa). The resultant PCR product, which was blunt-ended, was inserted into the SmaI site of pUC119 (Vieira & Messing, 1987), and the constructs sequenced using one of the following primers: M13F, M13R, eliASF3, or eliASR3. To amplify the upstream region of the eliA gene, the primer eliAUF was synthesized on the basis of a sequence from the Ralstonia pickettii 12D genome that contains a gene homologous to eliA and was used for PCR amplification with the primer eliASR1. The resultant amplified DNA fragment was inserted into the SmaI site of pUC119, and the constructs sequenced using one of the following primers: M13R, M13F, eliASF1, eliASF2, or eliASR2.

Construction of the ΔeliA::gen mutant

To inactivate the eliA gene in NT80, it was replaced with a gentamycin resistance gene from pUCP22 (Olsen et al., 1982). For the disruption, oligonucleotide primers were used to amplify the upstream (eliAUF and eliAUR) and downstream (eliADF and eliADR) regions of the eliA gene. Next, the gentamycin resistance gene of pUCP22 was amplified by PCR using the primers eliAGF and eliAGR. The three fragments obtained were used simultaneously as the template for PCR amplification with the primers eliAUF and eliADR. To transform the strain NT80, the resultant DNA fragment, which contained regions homologous to the genomic regions that flanked the eliA gene, was introduced into NT80 by electroporation as follows. To prepare electrocompetent cells, NT80 cells that had been cultivated in SOB (2% Bacto tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 10 mM MgCl2) at 30 °C (OD600 ≈ 0.4) were immediately washed twice with ice-cold 10% glycerol and resuspended in the drop of 10% glycerol that remained after centrifugation, which resulted in a 100-fold concentration of the cells. Electrocompetent cells (100 μL) were transformed with the purified PCR product described above in a 0.2-cm ice-cold electroporation chamber using a MicroPulser electroporator (Bio-Rad) set to 2.4 kV. Shocked cells were incubated in 1 mL of YP medium at 30 °C for 3 h and plated on YP plates that contained gentamycin. A transformant was selected from among the gentamycin-resistant colonies. Correct disruption was verified by PCR (see Fig. S1) and sequence analysis.

Construction of the strain ΔeliA::gen tlpA::eliA kan for the complementation test

The eliA gene, together with a kanamycin resistance gene (kan), was inserted into the genome of the ΔeliA mutant within a gene that encoded a trypsin-like protein (named tlpA) as follows. The DNA sequence of the tlpA region was determined by whole-genome sequencing (DDBJ accession no. AB742038; the detailed procedures will be published elsewhere). The upstream region of tlpA and also the eliA gene together with the probable promoter region was amplified by PCR using the primers tlpAUF (which contains a HindIII site) and tlpAUR, and eliACompF and eliACompR (which contains a BamHI site), respectively. The fragments obtained were used simultaneously as the template for PCR amplification with the primers tlpAUF and eliACompR. The resultant fragment was digested with HindIII and BamHI and cloned into pUC119 (Vieira & Messing, 1987). Separately, the kanamycin resistance gene of pJB861 (Blatny et al., 1997) and the downstream region of the tlpA gene were amplified by PCR using the primers tlpAKF (which contains a BamHI site) and tlpAKR, and tlpADF and tlpADR (which contains an EcoRI site), respectively. The fragments obtained were used simultaneously as the template for PCR amplification with the primers tlpAKF and tlpADR. The resultant fragment was digested with BamHI and EcoRI and then cloned into the pUC119 construct that contained the upstream region of tlpA and the eliA gene, described above. The resultant plasmid was linearized by DraI digestion and transformed into NT80 for integration at the tlpA site of the chromosome by double crossing over and selection for the Kmr phenotype. Correct integration was verified by PCR (see Fig. S1) and DNA sequencing.

Assay for lipase activity

Lipase activity was assayed by the PNPL (p-nitrophenyl laurate) method as described previously (Shabtai & Daya-Mishne, 1992; Ushio et al., 1996) with slight modification. The reagents used were solution A (10 mM p-nitrophenyl laurate in 2-propanol) and solution B, which comprised 50 mM potassium phosphate buffer (KPB, pH 7.0) with 0.4% Triton X-100 and 0.1% gum arabic. A 960-μL aliquot of substrate solution, which was freshly prepared by adding solution A to solution B in a ratio of 1 : 9 with mild mixing, was preincubated at 37 °C for 10 min. To this mixture, 40 μL of culture supernatant that had been diluted to an appropriate concentration was added and further incubated at 37 °C for 10 min. After the incubation, the absorbance at 405 nm was measured (the blank was substrate solution alone). One unit of lipase was defined as 1 μmol p-nitrophenol released per minute. The ε405 (molar extinction coefficient) of p-nitrophenol under the experimental conditions was c. 8350 M−1 cm−1.

Western blot analysis

Extracellular proteins prepared from 150 μL of culture supernatant were loaded onto a sodium dodecyl sulfate polyacrylamide gel (15%) and transferred to PVDF membrane (Millipore). Immunodetection procedures were carried out as described previously (Nanamiya et al., 2003). Anti-LipA antibody, prepared from a rabbit immunized with the synthetic oligopeptides (SAFNDETVRGEQLL and IRTQANRLKTAGL), which correspond to amino acid sequences in the lipase protein, was used at a dilution of 1 : 1000.

RNA extraction and quantitative RT-PCR

For the RNA extraction, cells harvested from 1 mL of culture were lysed in 180 μL of TNES [100 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM EDTA, 0.2% SDS] and incubated for 20 min at 50 °C with 2 mg mL−1 proteinase K, followed by two acid phenol/chloroform extractions. The transcript from the lipase gene (named lipA) was quantified using reverse transcription (RT)-PCR. As an internal standard, we used 16S rRNA gene, whose sequence was determined by whole-genome sequencing (DDBJ accession no. AB740040; the detailed procedures will be published elsewhere). The cDNA was synthesized from 1 μg of DNase-treated RNA using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa) in accordance with the manufacturer's instructions. We confirmed that no PCR amplification of the 16S rRNA gene region was observed when RNA, treated with DNase, was used as the template. All reactions were performed in a SYBR Premix Ex Taq GC (Takara) reaction mixture using a MyiQ2 Real-Time PCR system (Bio-Rad) under the following conditions: 10 s at 95 °C, followed by 40 cycles of 5 s at 95 °C for denaturation and 30 s at 62 °C for annealing and extension. To amplify the internal regions of the 16S rRNA gene and lipA, we used the primers 16SqF and 16SqR, and lipAqF and lipAqR, respectively. All reactions were performed in triplicate, and the data were normalized against the internal standard.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Cloning of eliA

When extracellular proteins were prepared from culture supernatant from Ralstonia sp. NT80 that contained high lipase activity (c. 120 U mL−1, Fig. 3a), as induced by stearyl alcohol, and analyzed by SDS-PAGE, several specific protein bands were observed (Fig. 1). Among these protein bands, which were not detected in the supernatant of cultures that were incubated without stearyl alcohol or with soybean oil, a protein detected near the 14.4-kDa molecular weight marker, designated EliA, was abundant. Thus, the N-terminal amino acid sequence of the protein EliA was analyzed, and a sequence of 22 amino acids (VTVSRVDGQPMNPSGEPFSVTG) was determined successfully. Using a primer that was synthesized on the basis of the determined amino acid sequence (eliACF; see Table S1) and an LA PCR in vitro Cloning Kit (TaKaRa), the DNA region that contained the eliA gene was amplified and sequenced. The resultant data revealed that the region that contained the eliA gene showed a high degree of homology to the corresponding region of the Rpickettii 12D genome (DNA sequence identity is 82% in eliA), for which the sequence has already been determined (NCBI accession no. NC_012856, for genome sequence; YP_002981973, for EliA homologue). We used several primers that were based on the genome sequence of Rpickettii 12D to clone the unidentified region of the NT80 genome and obtained a 2.7-kb DNA sequence that contained the open reading frame of eliA (DDBJ accession no. AB740042). These results show that EliA is a secreted protein with a signal peptide of 25 amino acids and a predicted molecular weight of 16 436 Da (without signal peptide; 14 024 Da). Moreover, it was found that orthologs of EliA are distributed among Proteobacteria (Fig. 2a) and that EliA shows homology with protein-like activator (PA) protein of Pseudomonas aeruginosa PG201 (identity 30%), which is also a secreted protein (Fig. 2b). It has been reported that PA protein facilitates oxidation and is involved in the assimilation of n-hexadecane (Hisatsuka et al., 1972, 1977; Hardegger et al., 1994). Given that EliA was produced when high levels of lipase activity were induced by stearyl alcohol, which contains a long-chain hydrocarbon like n-hexadecane, we assumed that EliA is involved in the expression of lipase, perhaps by helping cells to recognize and/or incorporate stearyl alcohol.

image

Figure 1. SDS-PAGE of extracellular proteins from Ralstonia sp. NT80. Extracellular proteins were prepared from the culture supernatant of NT80, which incubated at 30 °C for 72 h: 1, without inducer; 2, with soybean oil; 3, with polyoxyethylene (10) oleyl ether; 4, with polyoxyethylene (20) oleyl ether; 5, with cetyl alcohol; 6, with stearyl alcohol. M, molecular weight marker, each molecular size is shown on the left side. An arrow indicates the protein band of EliA.

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image

Figure 2. Phylogenetic tree of EliA and alignment between EliA and PA protein. (a) Protein sequences, available at the KEGG database through GenomeNet (http://www.genome.ad.jp/), were aligned and a phylogenetic tree was constructed by the maximum likelihood method, with the use of mega5 software (Tamura et al., 2011). Bootstrapping was performed with 1000 replicates. The scale bar (0.2) indicates the number of changes per site. (b) Alignment of the amino acid sequences of EliA from NT80 and PA protein from Pseudomonas aeruginosa PG201.

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EliA is required for sufficient induction of lipase by stearyl alcohol

To investigate whether EliA is involved in the induction of lipase expression, we constructed a mutant in which the eliA gene was inactivated and characterized this mutant. As expected, extracellular lipase activity was decreased markedly in the ΔeliA mutant as compared with the wild type after induction with stearyl alcohol (Fig. 3a). The lipase activity in the culture supernatant of the wild type was c. 120 U mL−1 at 72 h after inoculation, whereas that of the ΔeliA mutant was c. 10 U mL−1 (Fig. 3a). To determine the amount of extracellular lipase protein, the gene that encoded lipase in NT80, designated lipA, was cloned and sequenced (DDBJ accession no. AB740041; the detailed procedures will be published elsewhere), and a peptide antibody against lipase was prepared. When the ΔeliA mutant was cultivated with stearyl alcohol, the amount of LipA protein in the culture supernatant was significantly decreased as compared with the wild type, in accord with the extracellular lipase activity, whereas no signal was detected in the absence of stearyl alcohol (Fig. 3b). Furthermore, the influence of inactivation of the eliA gene on the transcription of lipA was evaluated by quantitative RT-PCR. In the wild type, stearyl alcohol increased the transcription of lipA by c. 240-fold at 48 h after inoculation, as compared with the absence of stearyl alcohol (Fig. 3c). In contrast, in the mutant ΔeliA, the transcription of lipA increased only c. 15-fold in the presence of stearyl alcohol (Fig. 3c). Despite its effects on transcription, stearyl alcohol did not promote the efficiency of secretion of lipase in NT80 up to at least 7 h after induction (data not shown), even though hexadecane stimulates the secretion of lipase in Bglumae PG1 (Boekema et al., 2007). These results suggest that stearyl alcohol can induce lipA expression at the transcriptional level. To confirm that the lack of induction of lipase in the ΔeliA mutant was caused by the absence of EliA protein, the eliA gene together with the probable promoter region was inserted into the tlpA locus of the ΔeliA mutant. As expected, induction of lipA expression by stearyl alcohol was restored fully by the gene complementation (Fig. 3a–c). It is noteworthy that disruption only of the tlpA gene, which encodes a trypsin-like protein, in the ΔeliA mutant did not affect lipase expression at all (data not shown). These results clearly indicate that EliA is essential for the strong induction of lipA expression by stearyl alcohol.

image

Figure 3. Decrease in the induction of lipase by stearyl alcohol in the ΔeliA mutant. (a) Lipase activity in the culture supernatant of Ralstonia sp. NT80 wild type (circles) or ΔeliA::gen (triangles) or ΔeliA::gen tlpA::eliA kan (squares) in the absence (open symbols) or presence (closed symbols) of stearyl alcohol at the indicated times. The averages of three independent experiments are shown. Error bars indicate standard deviations. (b) Western blot analysis of culture supernatant inoculated after 60 h in the absence or presence of stearyl alcohol with antilipase antibody. W, wild-type; M, ΔeliA::gen; C, ΔeliA::gen tlpA::eliA kan. (c) The expression ratio of the lipase gene (induced by stearyl alcohol/without induction) at the indicated times was determined using quantitative RT-PCR. The averages from three experiments are shown, with error bars indicating the standard deviations.

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A possible role of EliA in lipase induction by stearyl alcohol

The homology between EliA and PA protein, which is involved in the assimilation of n-hexadecane in Paeruginosa (Hisatsuka et al., 1972, 1977; Hardegger et al., 1994), raised the possibility that EliA is involved in the assimilation of stearyl alcohol, which contains a long hydrocarbon chain like n-hexadecane. When the NT80 strain was cultured in the presence of stearyl alcohol, which exists mostly in a solid state at ambient temperature, the culture supernatant had a white cloudy appearance owing to emulsification of the stearyl alcohol. This turbidity was not observed in the supernatant of YP medium that contained stearyl alcohol but no cells (Fig. 4a and Table 1), because no emulsifiers such as biosurfactants and/or proteins were being supplied by cells. In the case of the wild type, the turbid culture supernatant gradually became transparent during late stationary phase. However, in the case of the ΔeliA mutant, the turbidity caused by the stearyl alcohol did not appear to change (Fig. 4a and Table 1). This phenotype of the ΔeliA mutant could be rescued by gene complementation (Fig. 4a and Table 1). These results suggest that NT80 cells can emulsify a stearyl alcohol even in the absence of EliA, but cells require EliA to assimilate a stearyl alcohol.

Table 1. Turbidity in the culture supernatant as indicated by optical density
 MediumWild typeΔeliAΔeliA tlpA::eliA
  1. YP medium containing 1% stearyl alcohol with or without cells was incubated at 30 °C with reciprocal shaking (160 rpm) in a 500-mL baffled flask for the indicated times. Culture supernatants were obtained by centrifugation (8000 g for 5 min), and the OD600 was measured. Means of three independent experiments with the standard deviation are shown.

48 h0.04 ± 0.016.5 ± 0.58.4 ± 0.57.4 ± 0.9
72 h0.08 ± 0.011.5 ± 0.48.6 ± 0.42.3 ± 0.2
image

Figure 4. Effects of inactivation of the eliA gene on the degradation or incorporation of stearyl alcohol and on lipase induction by an insoluble inducer. (a) Culture supernatants of cells grown in YP medium containing 1% stearyl alcohol were obtained at 72 h after inoculation by centrifugation at 8000 g for 5 min: 1, without cells; 2, wild type; 3, ΔeliA::gen; 4, ΔeliA::gen tlpA::eliA kan. (b) Cells were cultivated at 30 °C in YP medium containing 1% of the indicated inducers for 72 h, and lipase activity in the culture supernatant was measured. The averages of three independent experiments are shown. Error bars indicate standard deviations. POE, polyoxyethylene.

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We have previously reported that several compounds containing a long hydrocarbon chain are effective in induction of the lipase in NT80 and related strains (Ushio et al., 1996). Cetyl alcohol, which exists mostly in a solid state at ambient temperature like stearyl alcohol but to a lesser extent, and polyoxyethylene (10) oleyl ether and polyoxyethylene (20) oleyl ether, both of which are completely water-soluble materials, also can induce the extracellular activity of the lipase efficiently in NT80. To investigate whether EliA is important for recognition and/or incorporation of insoluble materials other than stearyl alcohol, we also observed the effect of inactivation of eliA on the induction of extracellular lipase activity by poorly soluble cetyl alcohol and soluble polyoxyethylene derivatives. As shown in Fig. 4b, inactivation of eliA significantly reduced extracellular lipase activity after induction with the insoluble materials stearyl alcohol and cetyl alcohol, but the absence of EliA did not greatly affect the amount of extracellular lipase activity after induction with the soluble materials polyoxyethylene (10) oleyl ether and polyoxyethylene (20) oleyl ether. This result indicates that EliA is required for recognition and/or incorporation of insoluble inducers for lipase expression, but is not necessary for lipase induction by soluble materials. However, EliA was slightly observed in the extracellular fraction even when soluble materials were used as inducers of lipase (Fig. 1). This observation prompted us to investigate whether EliA has another function, such as proper folding of the lipase. We purified a recombinant EliA protein, which was expressed in Ecoli and did not have any tags such as histidine tag, and observed its effect on lipase activity in vitro. However, addition of purified EliA did not enhance the lipase activity contained in the extracellular fraction of the ΔeliA mutant (data not shown). Therefore, EliA is involved in the expression of lipase induced by insoluble materials such as stearyl alcohol, but does not affect the enzyme activity of lipase.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In the present study, we identified the secreted protein, EliA, as a facilitator of the induction of lipase expression by stearyl alcohol in Ralstonia sp. NT80. Given that orthologs of EliA are distributed throughout Proteobacteria, especially in Beta- and Gammaproteobacteria (Fig. 2a), it is likely that EliA orthologs such as PA protein are involved in the induction of lipase by insoluble materials. In fact, the absence of EliA clearly affected even the induction of extracellular lipase activity by soybean oil (slightly soluble oil), which is a well-known inducer of lipase expression in several microorganisms (Shabtai & Daya-Mishne, 1992; Gerritse et al., 1998; Shu et al., 2012; Fig. 4b). Several species belonging to Pseudomonas or Burkholderia genera, such as Palcaligenes and Bglumae, several of which are used to produce lipase industrially (Jaeger & Reetz, 1998; Jaeger & Eggert, 2002; Gupta et al., 2004), also harbored EliA orthologs. Therefore, the findings in this study may be useful for industrial applications.

EliA shares homology with PA protein, which is a secreted protein of Paeruginosa (Fig. 2b). PA protein facilitates oxidation and is involved in the assimilation of n-hexadecane; indeed, its production is induced by long-chain n-alkanes such as n-hexadecane or cetyl alcohol (Hisatsuka et al., 1972, 1977; Hardegger et al., 1994; Holden et al., 2002). Therefore, it is predicted that EliA has the potential to participate in the assimilation of stearyl alcohol, which contains a long-chain (octadecyl) hydrocarbon group. In microorganisms, the assimilation of long-chain n-alkanes that are virtually insoluble in water, including materials that are solid at ambient temperature, is initiated by the emulsification of n-alkanes, which is supported by biosurfactants, and interaction between the substrates and cells (Ochsner et al., 1996; Bouchez-Naitali et al., 1999), followed by terminal hydroxylation of n-alkanes (Kok et al., 1989; Tani et al., 2001; Throne-Holst et al., 2006). The resulting alcohol is further converted to a fatty acid via a pathway that involves an alcohol dehydrogenase, an aldehyde dehydrogenase, and an acyl-CoA synthetase, after which it enters the β-oxidation pathway (van Beilen & Funhoff, 2007). Given that it has been reported that PA protein together with rhamnolipid, a biosurfactant, can emulsify n-hexadecane (Hisatsuka et al., 1977), the emulsification of stearyl alcohol might be one of the functions of EliA. However, the emulsification of stearyl alcohol could be observed even in cultures of the ΔeliA mutant (Fig. 4a and Table 1). Therefore, EliA presumably also plays an important role in the assimilation of stearyl alcohol other than emulsification.

It is expected that stearyl alcohol is also oxidized in a stepwise manner similar to the degradation pathway of n-alkanes described above. On the other hand, stearic acid results in only one-tenth of the induction of extracellular lipase activity in NT80 as compared with stearyl alcohol (data not shown). Therefore, cells must recognize stearyl alcohol itself or derivatives other than the fatty acid as the signal for the significant induction of lipase expression. We have already analyzed the entire genome sequence of this strain. Further investigations that include comprehensive analyses such as proteomics and transcriptomics will reveal the details of the mechanisms by which stearyl alcohol and other compounds induce lipase expression.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to Hirofumi Hara and Saori Kosono for the provision of plasmids and helpful suggestions. This work was supported in part by a Grant-in-Aid for Young Scientists (B) (G.A.), Grant-in-Aid for Exploratory Research (M.I.), Grants-in-Aid for Scientific Research (C) (M.I.), and Grants-in-Aid for Scientific Research (S0801025) (H.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Ausubel FM, Brent R, Kingstone RE, Moore DO, Seidman JS, Smith JA & Struhl K (1987) Current Protocols in Molecular Biology. John Wiley & Sons, New York.
  • van Beilen JB & Funhoff EG (2007) Alkane hydroxylases involved in microbial alkane degradation. Appl Microbiol Biotechnol 74: 1321.
  • Blatny JM, Brautaset T, Winther-Larsen HC, Karunakaran P & Valla S (1997) Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria. Plasmid 38: 3551.
  • Boekema BK, Beselin A, Breuer M, Hauer B, Koster M, Rosenau F, Jaeger KE & Tommassen J (2007) Hexadecane and Tween 80 stimulate lipase production in Burkholderia glumae by different mechanisms. Appl Environ Microbiol 73: 38383844.
  • Bouchez-Naitali M, Rakatozafy H, Marchal R, Leveau JY & Vandecasteele JP (1999) Diversity of bacterial strains degrading hexadecane in relation to the mode of substrate uptake. J Appl Microbiol 86: 421428.
  • Cox M, Gerritse G, Dankmeyer L & Quax WJ (2001) Characterization of the promoter and upstream activating sequence from the Pseudomonas alcaligenes lipase gene. J Biotechnol 86: 917.
  • Deive FJ, Carvalho E, Pastrana L, Rúa ML, Longo MA & Sanroman MA (2009) Strategies for improving extracellular lipolytic enzyme production by Thermus thermophilus HB27. Bioresour Technol 100: 36303637.
  • de María PD, Sánchez-Montero JM, Alcántara AR, Valero F & Sinisterra JV (2005) Rational strategy for the production of new crude lipases from Candida rugosa. Biotechnol Lett 27: 499503.
  • Destain J, Fickers P, Weekers F, Moreau B & Thonart P (2005) Utilization of methyl oleate in production of microbial lipase. Appl Biochem Biotechnol 121: 269277.
  • Frenken LG, Bos JW, Visser C, Muller W, Tommassen J & Verrips CT (1993) An accessory gene, lipB, required for the production of active Pseudomonas glumae lipase. Mol Microbiol 9: 579589.
  • Gerritse GR, Hommes WJ & Quax WJ (1998) Development of a lipase fermentation process that uses a recombinant Pseudomonas alcaligenes strain. Appl Environ Microbiol 64: 26442651.
  • Gupta R, Gupta N & Rathi P (2004) Bacterial lipases: an overview of production, purification and biochemical properties. Appl Microbiol Biotechnol 64: 763781.
  • Hardegger M, Koch AK, Ochsner UA, Fiechter A & Reiser J (1994) Cloning and heterologous expression of a gene encoding an alkane-induced extracellular protein involved in alkane assimilation from Pseudomonas aeruginosa. Appl Environ Microbiol 60: 36793687.
  • Hisatsuka K, Nakahara T & Yamada K (1972) Protein-like activator for n-alkane oxidation by Pseudomonas aeruginosa S7B1. Agric Biol Chem 36: 13611369.
  • Hisatsuka K, Nakahara T, Minoda Y & Yamada K (1977) Formation of protein-like activator for n-alkane oxidation and its properties. Agric Biol Chem 41: 445450.
  • Holden PA, LaMontagne MG, Bruce AK, Miller WG & Lindow SE (2002) Assessing the role of Pseudomonas aeruginosa surface-active gene expression in hexadecane biodegradation in sand. Appl Environ Microbiol 68: 25092518.
  • Huddleston S, Yallop CA & Charalambous BM (1995) The identification and partial characterization of a novel inducible extracellular thermostable esterase from the archaeon Sulfolobus shibatae. Biochem Biophys Res Commun 216: 495500.
  • Jaeger KE & Eggert T (2002) Lipases for biotechnology. Curr Opin Biotechnol 13: 390397.
  • Jaeger KE & Reetz MT (1998) Microbial lipases form versatile tools for biotechnology. Trends Biotechnol 16: 396403.
  • Kok M, Oldenhuis R, van der Linden MP, Raatjes P, Kingma J, van Lelyveld PH & Witholt B (1989) The Pseudomonas oleovorans alkane hydroxylase gene. Sequence and expression. J Biol Chem 264: 54355441.
  • Krzeslak J, Gerritse G, van Merkerk R, Cool RH & Quax WJ (2008) Lipase expression in Pseudomonas alcaligenes is under the control of a two-component regulatory system. Appl Environ Microbiol 74: 14021411.
  • Krzeslak J, Papaioannou E, van Merkerk R, Paal KA, Bischoff R, Cool RH & Quax WJ (2012) Lipase A gene transcription in Pseudomonas alcaligenes is under control of RNA polymerase σ54 and response regulator LipR. FEMS Microbiol Lett 329: 146153.
  • Long ZD, Xu JH & Pan J (2007) Significant improvement of Serratia marcescens lipase fermentation, by optimizing medium, induction, and oxygen supply. Appl Biochem Biotechnol 142: 148157.
  • Nanamiya H, Shiomi E, Ogura M, Tanaka T, Asai K & Kawamura F (2003) Involvement of ClpX protein in the post-transcriptional regulation of a competence specific transcription factor, ComK protein, of Bacillus subtilis. J Biochem (Tokyo) 133: 295302.
  • Ochsner UA, Hembach T & Fiechter A (1996) Production of rhamnolipid biosurfactants. Adv Biochem Eng Biotechnol 53: 89118.
  • Olsen RH, DeBusscher G & McCombie WR (1982) Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome. J Bacteriol 150: 6069.
  • Plotkin LI, Squiquera L, Mathov I, Galimberti R & Leoni J (1996) Characterization of the lipase activity of Malassezia furfur. J Med Vet Mycol 34: 4348.
  • Rosenau F & Jaeger KE (2000) Bacterial lipases from Pseudomonas: regulation of gene expression and mechanisms of secretion. Biochimie 82: 10231032.
  • Rosenau F, Tommassen J & Jaeger KE (2004) Lipase-specific foldases. ChemBioChem 5: 152161.
  • Ruban EL, Ksandopulo GB & Murzina LP (1978) Conditions of exolipase biosynthesis by the fungus Oospora fragrans. Prikl Biokhim Mikrobiol 14: 849857.
  • Sambrook J & Russell D (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  • Shabtai Y & Daya-Mishne N (1992) Production, purification, and properties of a lipase from a bacterium (Pseudomonas aeruginosa YS-7) capable of growing in water-restricted environments. Appl Environ Microbiol 58: 174180.
  • Shimada Y, Sugihara A, Nagao T & Tominaga Y (1992) Induction of Geotrichum candidum lipase by long chain fatty acids. J Ferment Bioeng 74: 7780.
  • Shu ZY, Wu JG, Cheng LX, Chen D, Jiang YM, Li X & Huang JZ (2012) Production and characteristics of the whole-cell lipase from organic solvent tolerant Burkholderia sp. ZYB002. Appl Biochem Biotechnol 166: 536548.
  • Sztajer H, Lundsorf H, Erdmann H, Menge U & Schmid R (1992) Purification and properties of lipase from Penicillium simplicissimum. Biochim Biophys Acta 3: 253261.
  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M & Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 27312739.
  • Tani A, Ishige T, Sakai Y & Kato N (2001) Gene structures and regulation of the alkane hydroxylase complex in Acinetobacter sp. strain M-1. J Bacteriol 183: 18191823.
  • Throne-Holst M, Markussen S, Winnberg A, Ellingsen TE, Kotlar HK & Zotchev SB (2006) Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: the role of two AlkB-type alkane hydroxylases. Appl Microbiol Biotechnol 72: 353360.
  • Ushio K, Hirata T, Yoshida K, Sakaue M, Hirose C, Suzuki T & Ishizuka M (1996) Superinducers for induction of thermostable lipase production by Pseudomonas species NT-163 and other Pseudomonas like bacteria. Biotechnol Tech 10: 267272.
  • Vieira J & Messing J (1987) Production of single-stranded plasmid DNA. Methods Enzymol 153: 311.
  • Vora KA, Bhandare SS, Pradhan RS, Amin AR & Modi VV (1988) Characterization of extracellular lipase produced by Aspergillus japonicas in response to Calotropis gigantea latex. Biotechnol Appl Biochem 10: 465472.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
fml12055-sup-0001-FigS1.epsimage/eps2750KFig. S1. Verification of the inactivation or complementation of eliA gene by PCR.
fml12055-sup-0002-TableS1.docxWord document24KTable S1. Primers used in this study.
fml12055-sup-0003-legend.pdfapplication/PDF68K 

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