G Model

ARTICLE IN PRESS

BIOTEC-6796; No. of Pages 8

Journal of Biotechnology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Genetic analysis of lipolytic activities in Thermus thermophilus HB27 Benedikt Leis, Angel Angelov, Haijuan Li, Wolfgang Liebl ∗ Department of Microbiology, Technische Universität München, Emil-Ramann-Straße 4, D-85354 Freising-Weihenstephan, Germany

a r t i c l e

i n f o

Article history: Received 30 April 2014 Received in revised form 17 July 2014 Accepted 25 July 2014 Available online xxx Keywords: T. thermophilus HB27 Lipolytic activity Thermostable esterase Genetic analysis Novel clean deletion system

a b s t r a c t The extremely thermophilic bacterium Thermus thermophilus HB27 displays lipolytic activity for the hydrolysis of triglycerides. In this study we performed a mutational in vivo analysis of esterases and lipases that confer growth on tributyrin. We interrupted 10 ORFs suspected to encode lipolytic enzymes. Two chromosomal loci were identified that resulted in reduced hydrolysis capabilities against tributyrin and various para-nitrophenyl acyl esters. By implementation of a convenient new one-step method which abstains from the use of selectable markers, a mutant strain with multiple scar-less deletions was constructed by sequentially deleting ORFs TT C1787, TT C0340, TT C0341 and TT C0904. The quadruple deletion mutant of T. thermophilus exhibited significantly lower lipolytic activity (approximately 25% residual activity compared to wild type strain) over a broad range of fatty acyl esters and had lost the ability to grow on agar plates containing tributyrin as the sole carbon source. Furthermore, we were able to determine the impact of each gene disruption on the lipolytic activity profile in this model organism and show that the esterase activity in T. thermophilus HB27 is due to a concerted action of several hydrolases having different substrate preferences and activities. The esterase-less T. thermophilus multideletion mutant from this study can be used as a screening and expression host for esterase genes from thermophiles or metagenomes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Lipolytic enzymes are secreted by a variety of microorganisms for the degradation of extracellular substrates and may also be involved in bacterial pathogenicity (Bender and Flieger, 2010). Various Thermus species are known to produce lipolytic activities (Berger et al., 1995; Sigurgísladóttir et al., 1993). For example, Thermus thermophilus HB27 (Oshima and Imahori, 1974), a Gram-negative, aerobic, naturally competent and extremely thermophilic organism (Cava et al., 2009), can express extracellular esterase/lipase activities in significant amounts (Domínguez et al., 2004). Most studies conducted so far have focused on the improved production of lipolytic activities for biotechnological purposes ˜ (Deive et al., 2009; Domínguez et al., 2010, 2007; Fucinos et al., 2008, 2005a,b). These studies have shown that esterase and lipase expression levels dramatically depend on the culture conditions, the medium constituents and the presence of lipid compounds (e.g., olive oil). The regulation of gene expression involved in fatty acid degradation (fad-genes) in the T. thermophilus strain HB8 has been studied in detail (Agari et al., 2011). Nevertheless, the picture about

∗ Corresponding author. Tel.: +49 8161 7154 50; fax: +49 8161 7154 75. E-mail address: [email protected] (W. Liebl).

which genes are necessary for hydrolysis of triglycerides in Thermus is scarce. For this reason we performed a mutational in vivo analysis of lipolytic activities and substrate specificities in this bacterium. We interrupted 10 ORFs suspected to encode esterase- or lipase-active proteins. Furthermore we constructed a quadruple markerless knockout mutant strain lacking major extra- and intracellular lipolytic activities. The esterase-diminished strain was no longer able to grow on minimal medium supplemented with tributyrin as defined carbon and energy source and showed a largely reduced capability to hydrolyze a broad range of fatty acid esters.

2. Materials and methods 2.1. Bacterial strains and media The Escherichia coli strain XL1-Blue, XL10 GOLD® ultracompetent cells (Stratagene, La Jolla, USA) and DH10B (Invitrogen, Carlsbad, USA) were used for transformation and propagation of recombinant plasmids. Bacterial cultures were grown in lysogenic broth (LB) or LB agar plates with ampicillin (100 ␮g/ml) or kanamycin (20 ␮g/ml) at 37 ◦ C. Staphylococcus carnosus TM300 with pLipPS (Liebl and Götz, 1986) was cultured with 10 ␮g/ml chloramphenicol. For T. thermophilus HB27 (DSMZ 7039), culturing was performed using TB complex medium (Ramírez-Arcos

http://dx.doi.org/10.1016/j.jbiotec.2014.07.448 0168-1656/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Leis, B., et al., Genetic analysis of lipolytic activities in Thermus thermophilus HB27. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.448

G Model BIOTEC-6796; No. of Pages 8 2

ARTICLE IN PRESS B. Leis et al. / Journal of Biotechnology xxx (2014) xxx–xxx

et al., 1998) and SH minimal medium (Supplementary Table S2) with high-carbonate mineral water Aqua Purania (TSI, Zeven, Germany) adjusted at pH 7.5. Cells were cultured at 70 ◦ C or at 60 ◦ C when using kanamycin (final concentration 20 ␮g/ml), according to Angelov et al. (2009). Optical densities were measured at 600 nm wavelength (OD600 nm ) using an Ultrospec 2100pro UV/Vis Spectrophotometer (Amersham Bioscience, UK). Bacterial suspensions from overnight-grown liquid cultures were washed three times in 50 mM phosphate buffer (pH 7.5), adjusted to equal optical densities, and spotted on complex and minimal medium. Lipid additives like tributyrin, triolein and olive oil (all obtained from Sigma–Aldrich, St. Louis, USA) were added at final concentrations of 1.0% (v/v) and mixed using an Ultra-Turrax emulsifier (IKA, Staufen, Germany) immediately before autoclaving. 2.2. Single gene disruption mutants and generation of a T. thermophilus multiple clean deletion strain (BL03) For the generation of the suicide plasmid pBKat, the thermostable kanamycin nucleotidyl transferase (kat) cassette from vector pMK18 (de Grado et al., 1999) was isolated by PCR amplification (Pfu DNA polymerase, Fermentas, Vilnius, Lithuania) using the primer pair pMK18-Kat-f/r (all primer sequences and cloning sites are listed in Supplementary Table S1) and cloned into the Ecl136 site of pBlueskript SK + (Stratagene, La Jolla, USA). Several esterase/lipase gene candidates (Supplementary Table S3) were targeted for gene disruption experiments in T. thermophilus HB27. Homologous sequences from the target ORFs were amplified by PCR and cloned into the suicide vector pBKat. After transformation of the knockout plasmids in T. thermophilus, resistant colonies were selected after 2 days of growth on TB plates with 20 ␮g/ml kanamycin at 60 ◦ C. Upon integration by homologous recombination of the respective suicide vector constructs, the ORF integrity (e.g., disruption of known catalytic domains and predicted catalytic sites) was determined by analytical PCR. For phenotypic characterization of the single gene mutants, fresh cultures were adjusted to similar cell densities (OD600 nm ) and spotted onto agar plates supplemented with 1.0% tributyrin (v/v). Genomic DNA was isolated using the Masterpure DNA/RNA Extraction Kit (Epicentre, Madison, USA) according to the manufacturer’s instructions. For the generation of the multiple knockout mutants (summarized in Supplementary Table S4), approximately 1 kbp homologous regions up- and downstream from the target gene locus were amplified by PCR and cloned into pCR® 2.1-XL-TOPO® (Invitrogen, Carlsbad, USA) or pUC18. Two BglII sites were included flanking ORFs TT P0042, TT C0340 and TT C0341, using the ChangeIT Multiple Mutation Site Directed Mutagenesis Kit (Affymetrix, Santa Clara, USA). The sequences of the two ORFs could be deleted upon religation of the BglII sites after digestion, resulting in vector pCR 340-1. The knockout vector for allelic gene exchange in T. thermophilus was generated by cloning the kat-cassette from pBKat as BamHI fragment between the using BglII. Fusion constructs with the flanking regions of TT C0904 and TT C1787 were merged via splicing by overlap extension PCR (Heckman and Pease, 2007) to yield suicide vectors pUC18 0904 and pUC18 1787, respectively. In all cases, the resulting knockout vectors contained approximately 1 kbp regions flanking the target ORFs on the upand downstream side. In order to generate multiple markerless clean-deletions, approximately 10 ␮g of suicide vector was used to transform T. thermophilus, before analyzing several hundred colonies (grown on TB medium without an antibiotic) for the presence of the knockout allele by colony PCR. Potential candidates were confirmed by Southern blot analysis. Each probe (Supplementary Table S4) was biotin-labeled (Decalabel, Fermentas), hybridized at 42 ◦ C overnight and visualized according to the manufacturer’s instructions (Chromogenic Detection Kit, Fermentas).

Probes for the detection of ORFs TT C1787 and TT C0340-0341 were generated by PCR and biotin-labeling (see primer sequences, Supplementary Table 1). The probe for TT C0904 was the 825 bp biotin-labeled fragment generated by XhoI/HindIII digestion of the suicide vector pUC 904 that was also used for the deletion of ORF TT C0904. 2.3. Activity measurements Lipolytic activity measurements were conducted in para˜ et al. nitrophenol (pNP-) enzymatic assays based on Fucinos (2005a) with modifications. T. thermophilus cells from 5 to 30 ml overnight grown cultures were harvested by centrifugation and the supernatants were saved for the determination of extracellular hydrolytic activities. The following steps were performed on ice. The cells were washed twice and finally resuspended in 2 ml of 50 mM Tris–HCl (pH 7.5) buffer and disrupted by sonication (Dr. Hielscher Ultrasonics GmbH, Teltow, Germany) until the turbid suspensions became clear. Cell debris was removed by centrifugation (15,000 rpm, 10 min at 4 ◦ C) and the supernatants were analyzed for cell-bound, intracellular lipolytic activities. The para-nitrophenyl (pNP) assays were performed at 60, 65 and 70 ◦ C by mixing 0.1 ml of crude lysates in 0.9 ml pre-warmed 50 mM Tris–HCl (pH 7.5) buffer containing 0.25–2.5 mM pNP-substrates of different acyl chain lengths [(pNP-propionate (C3 ), -butyrate (C4 ), valerate (C5 ), -caproate (C6 ), -caprylate (C8 ), caprate (C10 ), -laurate (C12 ), -myristate (C14 ) and -palmitate (C16 ), all obtained from Sigma–Aldrich)]. After incubating the mixtures in a Thermomixer comfort (Eppendorf, Hamburg, Germany), the reactions were stopped by the addition of 0.25 ml of 2 M sodium carbonate and the absorbance (extinction coefficient ε of 16,700 M−1 cm−1 ) was measured spectrophotometrically at 400 nm. One unit of lipolytic activity was defined as 1 ␮mol product released per minute and mg protein. The protein concentration of the cell lysate and extracellular fractions was determined using Bradford Reagent (Fermentas). If necessary, samples were concentrated using Vivaspin 15 centrifugal ultrafiltration tubes (MWCO 5000, Sartorius Stedim, Göttingen, Germany). For measurements of extracellular activities and plate assays, more stable and sensitive 5-bromo-4-chloro-3-indoxylbutyrate (BCI-C4 ) -caprylate (BCI-C8 ) and -palmitate (BCI-C16 ) were used as the substrates (Biosynth, Staad, Switzerland). After stopping the reactions on ice, precipitates were spun down and the absorbance of the chromophor released by hydrolysis was measured at 615 nm wavelength. 3. Results 3.1. Lipolytic activities of T. thermophilus HB27 wild type on various triglycerides Tributyrin, triolein and olive oil emulsified in liquid and solid medium were used as substrates for monitoring triglyceride degradation and growth behavior of T. thermophilus HB27. In liquid cultures, it was difficult to keep the substrate emulsified even at elevated shaking speeds. Furthermore, the addition of tributyrin had an inhibitory effect on the growth in liquid cultures. Therefore, we focused on solid medium for growth experiments. On triolein and olive oil, no conversion of the substrate could be monitored. On tributyrin plates at 60 and 70 ◦ C, a clear substrate hydrolysis zone was accompanied by growth of the cells after one day of incubation (Fig. 1). Furthermore, we supplemented indigoid indicator substrates based on 5-bromo-4-chloro-3-indoxyl (BCI)butyrate and -palmitate in final concentrations of 50 ␮g/ml to the medium. The uncleaved BCI-substrate is not toxic, but upon its hydrolysis hydroxyindole is released and causes severe growth

Please cite this article in press as: Leis, B., et al., Genetic analysis of lipolytic activities in Thermus thermophilus HB27. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.448

G Model BIOTEC-6796; No. of Pages 8

ARTICLE IN PRESS B. Leis et al. / Journal of Biotechnology xxx (2014) xxx–xxx

Fig. 1. Assessment of lipolytic degradation ability of T. thermophilus HB27 on agar plates containing the triglycerides tributyrin (TB), triolein (TO) and olive oil (OO). As a control, a lipase-secreting S. carnosus TM300 strain bearing a Staphylococcus. hyicus lipase gene on plasmid pLipPS1 (Liebl and Götz, 1986) was grown on the substrate plates. This strain showed a positive reaction on all substrates tested, i.e. a clear hydrolysis zone around the colony on tributyrin, while on triolein and olive oil a reduction of the turbidity around the colony and slight halo formation was observed. Incubation with rhodamine B (0.005% w/v) was used to confirm the detection of free fatty acids resulting in fluorescence signals under UV light exposure (Kouker and Jaeger, 1987). E. coli served as a negative control.

inhibition of T. thermophilus HB27 (Angelov et al., 2013). In our study, cell growth on BCI-palmitate without the formation of blue indigo dye was observed. In contrast, no growth on BCI-butyrate was obtained, probably due to release of toxic hydrolysis products (data not shown). To elucidate which genes are responsible for the extracellular tributyrase activity in T. thermophilus, we decided to perform single and multiple disruptions of ORFs encoding possible lipolytic enzymes. 3.2. In silico analysis and single gene disruptions A bioinformatic analysis of all known protein domains and families (annotated Pfam database entries) encoded by the T. thermophilus HB27 genome revealed a considerable number of putative lipolytic enzymes (Table 1). Ten candidate ORFs could be assigned to code for ␣/␤-hydrolases, carboxylesterases, phospholipases and other esterase-related activities (e.g., hydrolysis of pectinacetylesters and thioesters). All ORFs listed in Table 1

3

could be disrupted via recombinatorial integration of the respective suicide plasmids into the chromosome (Table 2). Only two mutants conferred significantly smaller substrate hydrolysis halos on tributyrin agar plates compared to the wild type. Diminished halo formation was observed for the disruption mutants of ORF TT C0904, annotated as esterase (GenBank protein accession number YP 004875.1, Pfam pectinacetylesterase family), and of TT C1787, an ␣/␤-hydrolase family 3 carboxylesterase (EC 3.1.1.1, accession number YP 005756.1) resulting in 67.8 ± 9.8% and 74.1 ± 2.7% residual halo formation, respectively. The lipolytic activity of these strains was tested with a spectrum of different para-nitrophenyl substrates (Fig. 2). Upon disruption of TT C0904, mainly extracellular activity towards medium chain length pNP substrates was affected (on pNP-C10 , 37.9% residual extracellular activity was measured). The disruption of ORF TT C1787 led to a decreased lipolytic activity of the intracellular or cell-bound fraction: on C12 and C14 substrates only 57.4% and 54.4% residual activity was measured, respectively. The replacement of ORFs TT C0340-1 with a kanamycin cassette did not lead to significant phenotypic changes on tributyrin plates (91.6 ± 3.7% residual halo formation).

3.3. Generation of multiple esterase knockout strains of T. thermophilus HB27 A strain which combined deletions in all three relevant genetic loci which first had been inactivated individually (see above) was constructed. After transformation with a deletion-causing DNA construct, the bacterial suspension was plated in dilutions on selection-free TB medium and incubated until single colonies were visible. Several hundred of grown colonies were screened by PCR in order to identify deletion mutants. Approximately 0.3–1.0% of all colonies analyzed carried the knockout allele. All four targeted genetic loci could be deleted sequentially, beginning from a TT P0042 mutant (bgl) devoid of ␤-glucosidase activity (Angelov et al., 2013). In succession, the knockout mutants were generated and their genotype was confirmed by PCR and Southern blot analysis (Fig. 3). In summary,

Table 1 Proteome-based prediction of esterase and lipase candidates in T. thermophilus HB27 that were knocked out by single gene disruption or allelic exchange with a kanamycin resistance cassette. The reduction of lipolytic capability of each mutant on tributyrin agar plates is shown as + or − symbols. Pfam family targets

Pfam description

ORF annotation, lipase family classification† , additional information and references

Protein accession number

ORF number

Reduced halo on tributyrin plate upon interruption

Abhydrolase 6 Abhydrolase 6

Alpha/beta hydrolase family Alpha/beta hydrolase family

YP 004315.1 YP 004316.1

TT C0340 TT C0341

+/−

Abhydrolase 6

Alpha/beta hydrolase family

YP 005310.1

TT C1341

−

CO esterase

Carboxylesterase family

YP 005756.1

TT C1787

+

Esterase

Putative esterase

YP 004722.1

TT C0749

−

PAE

Pectinacetylesterase

YP 004875.1

TT C0904

++

Patatin Two PLDc 2

Patatin-like phospholipase Phospholipase D-like domain Phospholipase D-like domain Thioesterase superfamily

Hydrolase, lipase family V, esterase precursor EstTs1 in T. scotoductus SA-01 was characterized (homolog to TT C0341) (du Plessis et al., 2010) Carboxylesterase, lipase family LipS Carboxylesterase, lipase family VII, signal peptide predicted‡ Ferric enterobactin esterase-related protein Extracellular esterase, lipase family LipT, signal peptide predicted‡ , characterized by ˜ et al. (2011) Fucinos Hypothetical protein Phospholipase

YP 004770.1 YP 004797.1

TT C0797 TT C0824

− −

YP 005054.1

TT C1085

−

YP 005421.1

TT C1452

−

Two PLDc 2 4HBT † ‡

Hypothetical protein, signal peptide predicted‡ Acyl-CoA hydrolase

Lipase family classification according to Arpigny and Jaeger (1999) and Chow et al. (2012). According to SignalP prediction server from http://www.cbs.dtu.dk/services/SignalP (Petersen et al., 2011).

Please cite this article in press as: Leis, B., et al., Genetic analysis of lipolytic activities in Thermus thermophilus HB27. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.448

G Model BIOTEC-6796; No. of Pages 8

ARTICLE IN PRESS B. Leis et al. / Journal of Biotechnology xxx (2014) xxx–xxx

4

Table 2 T. thermophilus HB27 strains and mutants generated in this study. Abbreviation

Genotype

Remarks

WT 340-1 749 797 824 904 1085 1341 1452 1787 bgl BL01 BL02 BL03

Wild type strain TT C0340-1::kat TT C0749:kat TT C0797:kat TT C0824:kat TT C0904:kat TT C1085:kat TT C1341:kat TT C1452:kat TT C1787:kat TT P0042 TT P0042, TT C1787 TT P0042, TT C1787, TT C0340-1 TT P0042, TT C1787, TT C0340-1, TT C0904

HB27, DSM7039 Kanamycin cassette replacement of locus TT C0340-TT C0341 with suicide vector in HB27 Interruption of locus TT C0749 with suicide vector in HB27 Interruption of locus TT C0797 with suicide vector in HB27 Interruption of locus TT C0824 with suicide vector in HB27 Interruption of locus TT C0904 with suicide vector in HB27 Interruption of locus TT C1085 with suicide vector in HB27 Interruption of locus TT C1341 with suicide vector in HB27 Interruption of locus TT C1452 with suicide vector in HB27 Interruption of ORF TT C1787 with kan cassette in HB27 HB27 wild type strain with markerless clean deletion of beta-glucosidase gene (Ohta et al., 2006) Markerless clean deletion of carboxylesterase (encoded by TT C1787) in bgl strain Markerless clean deletion of both hydrolases (encoded by TT C340-TT C341) in BL01 Markerless clean deletion of characterized extracellular esterase (encoded by TT C0904) in BL02

Fig. 2. Lipolytic footprints of single gene disruption mutants of the two major esterase-encoding genes, TT C0904 and TT C1787 in T. thermophilus HB27. Intracellular/cell bound and extracellular enzymatic activities in the respective single gene mutant are shown as relative lipolytic activities (in %). Wild type activity corresponds to 100%. Cell cultures were grown at 60 ◦ C in TB medium containing 20 ␮g/ml kanamycin as antibiotic. Experiments were conducted in triplicate at 60 ◦ C for enzymatic reactions using para-nitrophenyl substrates (the data represents average values and standard deviations).

Fig. 3. Confirmation of the genotype of the strain BL03 (bgl 1787 0340-0341 0904) by PCR with genomic DNA as a template and primers flanking the deleted region (left) and by Southern blot hybridization (right). For the hybridization with probe TT P0042, genomic DNA was digested with BamHI, while for the TT C1787 and TT C03400341 probes the genomic DNA was digested with SacII. For probe TT P0042, the in silico predicted sizes are 1.88 kbp for the wild type (lane 1) and 1.21 kbp for the bgl allele (lane 2); for probe TT C1787, the predicted sizes are 2.55 kbp for the wild type (lane 3) and 1.80 kbp for 1787 (lane 4); for probe TT C0340-0341, the predicted sizes are 4.80 kbp for the wild type (lane 5) and 3.33 kbp for 0340-0341 (lane 6); and for probe TT C0904, the predicted sizes are 5.47 kbp for the wild type (lane 7) but 4.48 kbp for the 0904 allele (lane 8).

Please cite this article in press as: Leis, B., et al., Genetic analysis of lipolytic activities in Thermus thermophilus HB27. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.448

G Model BIOTEC-6796; No. of Pages 8

ARTICLE IN PRESS B. Leis et al. / Journal of Biotechnology xxx (2014) xxx–xxx

5

Fig. 4. Summary of the esterase activity patterns of each sequential knockout mutant of T. thermophilus HB27 generated in this study. (A) Extracellular volumetric esterase activity (normalized to the cell density), measured with BCI-butyrate (black bars), BCI-caprylate (dark gray bars) and pNP-laurate (light gray bars). The activity of the wild type with each substrate is set to 100%. Experiments were conducted in triplicates (n = 3). Cell culturing and assays were performed at 70 ◦ C. (B) Spots of cell suspensions on agar plates supplemented with tributyrin (1% v/v) were grown for 3 days at 60 ◦ C. Upon hydrolysis of the turbid substrate, the extent of the halos around the colonies corresponds to the amount of lipolytic activity released (or secreted) by the cells. Equal amounts of cells of each strain were used in both (B) and (C). (C) Growth of cell suspensions on SH minimal medium agar plates containing 1% (v/v) tributyrin as the sole carbon source after 3 days of growth at 70 ◦ C.

we obtained a double mutant BL01 (genotype TT P0042 and TT C1787), a triple mutant BL02 (TT P0042, TT C1787 and TT C0340-1) and a quadruple knockout strain BL03 (genotype TT P0042, TT C1787, TT C0340-1 and TT C0904) of T. thermophilus HB27 (Table 2). These mutant strains were characterized in terms of halo formation on tributyrin plates, specific activities on BCI- and pNP-substrates and growth on minimal medium supplemented with tributyrin (Fig. 4). BL01 (genotype bgl, TT C1787) showed a trend of activity reduction (10–20% less activity) with the substrates tested. With the increasing number of additional gene knockouts, the esterase activity gradually decreased for the BCI-C4 substrate, whereas hydrolysis of substrates with longer acyl chain length like caprylate and laurate was only severely affected after finally deleting ORF TT C0904. The quadruple mutant BL03 exhibited approximately 75% lower extracellular specific esterase/lipase activity on the short as well as long acyl chain substrates (Fig. 4A). The intracellular activity of the BL03 strain with pNP-butyrate was 54.9 ± 4.1 mU/mg, which is a fourfold reduction compared to the wild type strain (207.4 ± 17.8 mU/mg crude extract). With longer substrates however, the reduction in intracellular activity in extracts from the multiple knockout mutant was less pronounced, i.e. 99.9 ± 17.7 mU/mg for pNP-laurate (128.4 ± 10.8 mU/mg in the wild type) and

3.3 ± 0.9 mU/mg for pNP-palmitate (4.5 ± 0.8 mU/mg in the wild type). On TB tributyrin agar plates, a considerable reduction of halo formation compared to the wild type HB27 strain was observed (Fig. 4B), in agreement with the results obtained from the colorimetric assays. With an increasing number of esterase gene deletions, the area of the hydrolysis zone around the colonies gradually became smaller. In batch culture experiments, we could show that after 24 and 48 h growth at 70 ◦ C in TB broth T. thermophilus HB27 accumulated twofold higher lipolytic activity in the growth medium (volumetric activity 61.3 mU × L−1 × h−1 ) than BL03 (32.6 mU × L−1 × h−1 ). The multiple gene deletions had no detectable negative impact on the growth behavior of BL03 when grown in nutrient-rich TB medium (Fig. 5). The ability of various deletion mutant strains to utilize tributyrin was assessed on minimal medium plates containing tributyrin as defined carbon source. All of the single-gene disruption mutants showed growth on the substrate (strains 1787:kat, 0904:kat, strain BL01) after 3 days of incubation at 60–70 ◦ C, which was also the case for the triple mutant BL02 (genotype bgl, 1787, 0340-1). In contrast, the quadruple mutant BL03 (genotype bgl, 1787, 0340-1 and 0904) could not grow even after prolonged incubation periods on the substrate (up to 7 days) (Fig. 4C).

Please cite this article in press as: Leis, B., et al., Genetic analysis of lipolytic activities in Thermus thermophilus HB27. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.448

G Model BIOTEC-6796; No. of Pages 8

ARTICLE IN PRESS B. Leis et al. / Journal of Biotechnology xxx (2014) xxx–xxx

6

Fig. 5. Comparison of the growth behavior and extracellular lipolytic activity between T. thermophilus wild type (black) and the multiple knockout mutant BL03 (grey). The cell cultures were grown at 70 ◦ C and samples were withdrawn for cell density measurements and for determination of the extracellular esterase activity (U × L−1 )with pNP-laurate after 3, 24 and 48 h.

4. Discussion 4.1. Contribution of individual putative esterase ORFs to lipolytic activity T. thermophilus HB27 is known to have extracellular and cell˜ et al., associated lipolytic enzymes (Domínguez et al., 2004; Fucinos 2005a,b). Highest activities have been observed on short chain acyl esters, whereas substrates with long fatty acyl chains are degraded less efficiently. The property of lipid substrate hydrolysis has also been reported for other Thermus species (Berger et al., 1995; Domínguez et al., 2004). On the other hand, not much is known which genes and gene products are involved in the breakdown of triglycerides by T. thermophilus. Until now, two extracellular pro˜ teins of 34 and 62 kDa size have been reported (Fucinos et al., 2005a). The esterase YP 004875.1 (encoded by ORF TT C0904; Pfam pectinacetylesterase family) has been purified and characterized in ˜ et al., 2011). The protein YP 005756.1 (gene proddetail (Fucinos uct of TT C1787) has been annotated as a carboxylesterase (BioCyc database, www.biocyc.org, Caspi et al., 2012). Furthermore it shares 97% amino acid sequence identity with a characterized polyhydroxybutyrate (PHB) depolymerase (protein accession YP 143465.1) of T. thermophilus strain HB8 (Papaneophytou et al., 2009). Our mutational in vivo analysis of esterases and lipases allowed the identification of ORFs that are related to triglyceride utilization in T. thermophilus HB27. From 10 single disruption mutants, the inactivation of only two ORFs, TT C0904 and TT C1787, led to significantly lower tributyrase activity. Disruption of ORF TT C0904 resulted in the highest reduction of activity on the mediumchain acyl substrate pNP-C10 , which is in accordance with the ˜ data from heterologous expression and characterization by Fucinos et al. (2011). Also upon inactivation of the carboxylesterase ORF TT C1787, the halo formation on the tributyrin plates was unambiguously reduced. Halo formation can arise from true secretion of the active enzyme to the cell surface or the surrounding medium but also from the release of cytoplasmically located enzyme due to cell lysis during growth of colonies on the agar. The predicted signal peptide/N-terminal transmembrane residues of the carboxylesterase (SignalP, Petersen et al., 2011) suggest a membrane-bound localization or export to the periplasm or into

multicellular bodies (rotund bodies). These peculiar cell assemblages with large extracytoplasmic compartments are regularly observed in Thermus species and certain other thermophilic bacteria (Brock and Freeze, 1969; Hoppert et al., 2012). It has been shown that rotund bodies contain lipolytic enzymes that could only be released upon their disruption, e.g., by a freeze–thaw treatment ˜ et al., 2005b). (Fucinos The knockout of the putative esterase gene couple consisting of TT C0340 and TT C0341 in T. thermophilus HB27 wild type genetic background did not result in significant phenotypic changes. However, it must be considered that phenotypic changes may not be visible in single mutant strains as long as other lipolytic enzymes are still present in the cell/periplasm whose activities may override relatively small activity losses. In our case, tributyrin hydrolysis in the plate assay was clearly affected by the TT C0340-0341 disruption only in the TT C1787 strain BL01, which resulted in the triple mutant strain BL02 (see Fig. 4). The substrate specificity of TT C0340-0341 was confirmed by enzymatic assays. The data is in agreement with du Plessis et al. (2010) who characterized the esterase precursor protein EstTs1 from Thermus scotoductus SA-01 (accession number ACS36170.1), which shares 76% identity with ORF TT C0341 from T. thermophilus HB27 (annotated as 3oxoadipate enol-lactone hydrolase). The T. scotoductus enzyme has been heterologously expressed in E. coli and was found to preferably hydrolyze short-chain fatty acid esters. The adjacent ORF encoding EstTs2 (homolog to TT C0340) showed no activity upon expression in E. coli (du Plessis et al., 2010). Due to the simultaneous deletion of both ORFs at once, it was not possible to distinguish the contribution of each single ORF to the lipolytic activity. Interestingly, both ORFs TT C0340 and TT C0341 are embedded in a gene cluster related to fatty acid metabolism. It comprises a putative 3-oxoacylacyl carrier protein (ACP)- synthase III (encoded by TT C0343), an acyl-CoA ligase (TT C0342) and a (R)-specific enoyl-CoA hydratase (also known as crotonase, encoded by TT C0339). 4.2. Generation and phenotype of multiple mutant strain T. thermophilus BL03 Different counter-selection strategies for the generation of markerless knockout mutants are available for Thermus. The pyrE

Please cite this article in press as: Leis, B., et al., Genetic analysis of lipolytic activities in Thermus thermophilus HB27. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.448

G Model BIOTEC-6796; No. of Pages 8

ARTICLE IN PRESS B. Leis et al. / Journal of Biotechnology xxx (2014) xxx–xxx

7

Fig. 6. Activity profile of main esterases/lipases and their contribution to the overall extracellular lipolytic activity in T. thermophilus HB27 over three different acyl chain substrate lengths (C4 : BCI-butyrate, C8 : BCI-caprylate and C12 : pNP-laurate). Single volumetric activities were calculated from the results of the mutational in vivo analysis (corresponding to data from Fig. 4A). The observed substrate specificities are visualized by the colored background (TT C0904: red, TT C0340-TT C0341: green, TT C1787: blue). The maximum extracellular lipolytic activity of the wild type strain corresponds to 100%. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

system using 5-fluoro-orotic acid (FOA) (Tamakoshi et al., 1999), rpsL1 allele selection in the presence of streptomycin (Blas-Galindo et al., 2007) and 5-bromo-4-chloro-3-indoxyl substrates (Angelov et al., 2013). As an interesting alternative which should also be very useful for other studies in the future, we used a new direct knockout strategy for deletion of the esterase-encoding loci TT C1787, TT C0340-0341 and TT C0904. This strategy, which does not require any additional counter-selection step, was shown here to be a fast and efficient way to introduce multiple genome modifications in T. thermophilus HB27. The observed frequencies of detecting knockout mutants after transformation with a knockout allele in the absence of selection pressure (approximately 1 × 10−2 to 3 × 10−3 of all colonies) make this new approach applicable in cases where the desired mutations do not negatively affect the growth of the strain. An additional advantage of this method is that no prior modifications of the wild type strain are needed. The multiple genetic lesions in the T. thermophilus strain BL03 resulted in significantly reduced extracellular lipolytic activities by pNP-assays as well as reduced halo formation on tributyrin agar plates without affecting the growth behavior in nutrient-rich medium compared to the wild type strain. On the other hand, strain BL03 revealed severely impaired growth on minimal medium containing tributyrin as defined carbon and energy source. For triglyceride utilization, the wild type strain HB27 obviously contains esterases for triacylglycerol hydrolysis and also clusters of genes for fatty acid metabolism and multiple gene copies coding for enzymes of the ␤-oxidation pathway (Cantu et al., 2011; Henne et al., 2004; Kanehisa et al., 2008). Our results demonstrate that in strain BL03 the enzyme activities for triglyceride ester hydrolysis were reduced below the level necessary for growth on tributyrin. Lipolytic enzymes are regularly found in thermophilic and extremely thermophilic microorganisms (Levisson et al., 2009) and in metagenomic communities (Chow et al., 2012; Henne et al., 2000). The presence of multiple esterase and lipase-encoding enzymes has been reported in various microorganisms such as Geobacillus stearothermophilus (Ewis et al., 2004), Thermosyntropha lipolytica (Salameh and Wiegel, 2007) and the archaeon Sulfolobus solfataricus P2 (Chung et al., 2000; Kim and Lee, 2004; Mandrich et al., 2007, 2005). The present study indicates that the ability of T. thermophilus HB27 to degrade triacylglycerol esters is due to

the concerted action of several lipolytic enzymes. The sequential introduction of deletions in selected genes, accompanied by measurements of esterase activities towards different substrates allowed the reconstruction of substrate preferences and the analysis of the contribution of each esterase/lipase encoding ORF for the extracellular in vivo activity (Fig. 6). We could identify three loci in T. thermophilus HB27 which account for approx. 75% of the total activity measured. Short acyl chain pNP-substrates (C4 ) were hydrolyzed by all enzymes at the same time, whereas longer to medium acyl chain substrate esters (C8 and C12 ) were preferably degraded by the esterase YP 004875.1 (TT C0904). In comparison to single gene interruptions (Fig. 2), we observe clearly different activity patterns from the multiple deletion mutants. This can mainly be accounted to the different culturing and assay conditions (e.g., incubation temperature, kind of substrates tested). Furthermore the genetic background (presence/dependence of certain genes) may also contribute to the additive lipolytic activity in T. thermophilus (epistatic effects). From our data we conclude that the presence of multiple enzymes with distinct acyl ester preferences and activity patterns permits the degradation and metabolic utilization of a broad substrate spectrum of triglycerides. This in turn may be beneficial for survival, especially in extreme thermophilic habitats that are characterized by low nutrient availability. The three identified loci encode for highly conserved proteins among the known Thermus species for which genomic sequences are available, and were shown to be essential for growth of T. thermophilus HB27 on tributyrin. The esterase-deficient multiple knockout strain BL03 reported here may also serve as a platform for the molecular functional analysis of further thermostable lipolytic enzymes in vivo, for further mutational knockouts studies in T. thermophilus (Liebl, 2004), or as a host for (meta)genomic screening for new esterase and lipase genes (Leis et al., 2013). Acknowledgments The authors gratefully acknowledge the support by the Faculty Graduate Center Weihenstephan of TUM Graduate School at Technische Universität München, Germany. This work was funded by the German Federal Ministry of Education and Research (BMBF)

Please cite this article in press as: Leis, B., et al., Genetic analysis of lipolytic activities in Thermus thermophilus HB27. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.448

G Model BIOTEC-6796; No. of Pages 8

ARTICLE IN PRESS B. Leis et al. / Journal of Biotechnology xxx (2014) xxx–xxx

8

in the collaborative project ExpresSys (FKZ 0315586A) within the funding measure GenoMik-Transfer. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec. 2014.07.448. References Agari, Y., Agari, K., Sakamoto, K., Kuramitsu, S., Shinkai, A., 2011. TetR-family transcriptional repressor Thermus thermophilus FadR controls fatty acid degradation. Microbiology 157, 1589–1601. Angelov, A., Li, H., Geissler, A., Leis, B., Liebl, W., 2013. Toxicity of indoxyl derivative accumulation in bacteria and its use as a new counterselection principle. Syst. Appl. Microbiol. 36, 585–592. Angelov, A., Mientus, M., Liebl, S., Liebl, W., 2009. A two-host fosmid system for functional screening of (meta)genomic libraries from extreme thermophiles. Syst. Appl. Microbiol. 32, 177–185. Arpigny, J.L., Jaeger, K.E., 1999. Bacterial lipolytic enzymes: classification and properties. Biochem. J. 343 (Pt 1), 177–183. Bender, J., Flieger, A., 2010. Lipases as pathogenicity factors of bacterial pathogens of humans. In: Timmis, K. (Ed.), Handbook of Hydrocarbon and Lipid Microbiology. Springer, Berlin, pp. 3241–3258. Berger, J.L., Lee, B.H., Lacroix, C., 1995. Identification of new enzyme activities of several strains of Thermus species. Appl. Microbiol. Biotechnol. 44, 81–87. Blas-Galindo, E., Cava, F., Lopez-Vinas, E., Mendieta, J., Berenguer, J., 2007. Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus. Appl. Environ. Microbiol. 73, 5138–5145. Brock, T.D., Freeze, H., 1969. Thermus aquaticus gen. n. and sp. n., a nonsporulating extreme thermophile. J. Bacteriol. 98, 289–297. Cantu, D.C., Chen, Y., Lemons, M.L., Reilly, P.J., 2011. ThYme: a database for thioesteractive enzymes. Nucleic Acids Res. 39, D342–D346. Caspi, R., Altman, T., Dreher, K., Fulcher, C.A., Subhraveti, P., Keseler, I.M., Kothari, A., Krummenacker, M., Latendresse, M., Mueller, L.A., Ong, Q., Paley, S., Pujar, A., Shearer, A.G., Travers, M., Weerasinghe, D., Zhang, P., Karp, P.D., 2012. The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Res. 40, D742. Cava, F., Hidalgo, A., Berenguer, J., 2009. Thermus thermophilus as biological model. Extremophiles 13, 213–231. Chow, J., Kovacic, F., Dall Antonia, Y., Krauss, U., Fersini, F., Schmeisser, C., Lauinger, B., Bongen, P., Pietruszka, J., Schmidt, M., Menyes, I., Bornscheuer, U.T., Eckstein, M., Thum, O., Liese, A., Mueller-Dieckmann, J., Jaeger, K.-E., Streit, W.R., 2012. The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases. PloS One 7, e47665. Chung, Y.M., Park, C.B., Lee, S.B., 2000. Partial purification and characterization of thermostable esterase from the hyperthermophilic archaeon Sulfolobus solfataricus. Biotechnol. Bioprocress Eng. 5, 53–56. Deive, F.J., Carvalho, E., Pastrana, L., Rúa, M.L., Longo, M.A., Sanroman, M.A., 2009. Assessment of relevant factors influencing lipolytic enzyme production by Thermus thermophilus HB27 in laboratory-scale bioreactors. Chem. Technol. 32, 606–612. ˜ Domínguez, A., Sanromán, A., Fucinos, P., Rúa, M.L., Pastrana, L., Longo, M.A., 2004. Quantification of intra- and extra-cellular thermophilic lipase/esterase production by Thermus sp. Biotechnol. Lett. 26, 705–708. Domínguez, A., Deive, F.J., Pastrana, L., Rúa, M.L., Longo, M.A., Sanroman, M.A., 2010. Thermostable lipolytic enzymes production in batch and continuous cultures of Thermus thermophilus HB27. Bioproc. Biosys. Eng. 33, 347–354. ˜ Domínguez, A., Fucinos, P., Rúa, M.L., Pastrana, L., Longo, M.A., Sanromán, M.A., 2007. Stimulation of novel thermostable extracellular lipolytic enzyme in cultures of Thermus sp. Enzyme Microb. Technol. 40, 187–194. du Plessis, E.M., Berger, E., Stark, T., Louw, M.E., Visser, D., 2010. Characterization of a novel thermostable esterase from Thermus scotoductus SA-01: evidence of a new family of lipolytic esterases. Curr. Microbiol. 60, 248–253. Ewis, H.E., Abdelal, A.T., Lu, C.-D., 2004. Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus. Gene 329, 187–195.

˜ Fucinos, P., Abadín, C., Sanromán, A., Longo, M., Pastrana, L., Rúa, M., 2005a. Identification of extracellular lipases/esterases produced by Thermus thermophilus HB27: partial purification and preliminary biochemical characterization. J. Biotechnol. 117, 233–241. ˜ Fucinos, P., Atanes, E., López-López, O., Esperanza Cerdán, M., Isabel González-Siso, M., Pastrana, L., Luisa Rúa, M., 2011. Production and characterization of two Nterminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: effect of N-terminus in thermal activity and stability. Prot. Express. Purif. 78, 120–130. ˜ Fucinos, P., Domínguez, A., Angeles Sanromán, M., Longo, M.A., Luisa Rúa, M., Pastrana, L., 2005b. Production of thermostable lipolytic activity by Thermus species. Biotechnol. Prog. 21, 1198–1205. de Grado, M., Castán, P., Berenguer, J., 1999. A high-transformation-efficiency cloning vector for Thermus thermophilus. Plasmid 42, 241–245. Heckman, K.L., Pease, L.R., 2007. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat. Protoc. 2, 924–932. Hoppert, M., Valdez, M., Enseleit, M., Theilmann, W., Valerius, O., Föst, C., Liebl, W., 2012. Structure-function analysis of the Dictyoglomus cell envelope. Syst. Appl. Microbiol. 35, 279–290. Henne, A., Schmitz, R.A., Bomeke, M., Gottschalk, G., Daniel, R., 2000. Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli. Appl. Environ. Microbiol. 66, 3113–3116. Henne, A., Brüggemann, H., Raasch, C., Wiezer, A., Hartsch, T., Liesegang, H., Johann, A., Lienard, T., Gohl, O., Martinez-Arias, R., Jacobi, C., Starkuviene, V., Schlenczeck, S., Dencker, S., Huber, R., Klenk, H.-P., Kramer, W., Merkl, R., Gottschalk, G., Fritz, H.-J., 2004. The genome sequence of the extreme thermophile Thermus thermophilus. Nat. Biotechnol. 22, 547–553. Kanehisa, M., Araki, M., Goto, S., Hattori, M., Hirakawa, M., Itoh, M., Katayama, T., Kawashima, S., Okuda, S., Tokimatsu, T., Yamanishi, Y., 2008. KEGG for linking genomes to life and the environment. Nucleic Acids Res. 36, D480–D484. Kim, S., Lee, S.B., 2004. Thermostable esterase from a thermoacidophilic archaeon: purification and characterization for enzymatic resolution of a chiral compound. Biosci. Biotechnol. Biochem. 68, 2289–2298. Kouker, G., Jaeger, K.E., 1987. Specific and sensitive plate assay for bacterial lipases. Appl. Environ. Microbiol. 53, 211–213. Leis, B., Angelov, A., Liebl, W., 2013. Screening and expression of genes from metagenomes. Adv. Appl. Microbiol. 83, 1–68. Levisson, M., Oost, J., Kengen, S.W.M., 2009. Carboxylic ester hydrolases from hyperthermophiles. Extremophiles 13, 567–581. Liebl, W., Götz, F., 1986. Studies on lipase directed export of Escherichia coli betalactamase in Staphylococcus carnosus. Mol. Gen. Genet. 204, 166–173. Liebl, W., 2004. Genomics taken to the extreme. Nat. Biotechnol. 22, 524–525. Mandrich, L., Merone, L., Pezzullo, M., Cipolla, L., Nicotra, F., Rossi, M., Manco, G., 2005. Role of the N terminus in enzyme activity, stability and specificity in thermophilic esterases belonging to the HSL family. J. Mol. Biol. 345, 501–512. Mandrich, L., Pezzullo, M., Rossi, M., Manco, G., 2007. SSoNDelta and SSoNDeltalong: two thermostable esterases from the same ORF in the archaeon Sulfolobus solfataricus? Archaea 2, 109–115. Ohta, T., Tokishita, S., Imazuka, R., Mori, I., Okamura, J., Yamagata, H., 2006. Beta-Glucosidase as a reporter for the gene expression studies in Thermus thermophilus and constitutive expression of DNA repair genes. Mutagenesis 21, 255–260. Oshima, T., Imahori, K., 1974. Description of Thermus thermophilus (Yoshida and Oshima) comb. nov., a nonsporulating thermophilic bacterium from a Japanese Thermal Spa. Int. J. Syst. Bacteriol. 24, 102–112. Papaneophytou, C.P., Pantazaki, A.A., Kyriakidis, D.A., 2009. An extracellular polyhydroxybutyrate depolymerase in Thermus thermophilus HB8. Appl. Microbiol. Biotechnol. 83, 659–668. Petersen, T.N., Brunak, S., von Heijne, G., Nielsen, H., 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8, 785–786. Ramírez-Arcos, S., Fernández-Herrero, L.A., Marín, I., Berenguer, J., 1998. Anaerobic growth, a property horizontally transferred by an Hfr-like mechanism among extreme thermophiles. J. Bacteriol. 180, 3137–3143. Salameh, M.A., Wiegel, J., 2007. Purification and characterization of two highly thermophilic alkaline lipases from Thermosyntropha lipolytica. Appl. Environ. Microbiol. 73, 7725–7731. Sigurgísladóttir, S., Konráðsdóttir, M., Jónsson, Á., Kristjánsson, J.K., Matthiasson, E., 1993. Lipase activity of thermophilic bacteria from icelandic hot springs. Biotechnol. Lett. 15, 361–366. Tamakoshi, M., Yaoi, T., Oshima, T., Yamagishi, A., 1999. An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus. FEMS Microbiol. Lett. 173, 431–437.

Please cite this article in press as: Leis, B., et al., Genetic analysis of lipolytic activities in Thermus thermophilus HB27. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.07.448