Acta Microbiologica et Immunologica Hungarica, 55 (4), pp. 437–446 (2008) DOI: 10.1556/AMicr.55.2008.4.8
EXPRESSION PATTERNS OF CEL5A – CEL5B, TWO ENDOGLUCANASE ENCODING GENES OF THERMOBIFIDA FUSCA* ZITA SASVÁRI, KATALIN POSTA, L. HORNOK** Agricultural Biotechnology Center, Mycology Group of the Hungarian Academy of Sciences, Institute of Plant Protection, Szent István University, Páter K. u.1, H-2103 Gödöllõ, Hungary
(Received: 23 July 2008; accepted: 30 September 2008)
Expression patterns of cel5A and cel5B, two endoglucanase encoding genes of Thermobifida fusca were compared by quantitative real-time PCR. With Avicel as carbon source the transcript level of cel5A continuously increased until the 10th hour of incubation and then a sharp decrease was observed, whereas cel5B presented a slow constitutive expression on this substrate. When the microcrystalline cellulose powder MN300 was used as the inducing carbon source, the expression patterns of the two genes were similar. A low initial level of expression was followed by a rapid increase at the 5th hour of incubation; a transient repression was then observed at the 10th hour but after this sampling time, the expression levels started to increase again. The relative expression levels of cel5A were always higher than those of cel5B. Differences in transcription patterns of these two genes can be explained with the imperfect structure of the CelR binding regulatory region of cel5B. Keywords: Thermobifida fusca, endocellulase, gene expression, qRT-PCR
Introduction Thermobifida fusca is a thermophilic, filamentous soil inhabiting actinomycete, regarded as the major degrader of lignocellulose containing plant residues under aerobic conditions [1]. Biodegradation of plant cell walls by this organism * This paper is written to commemorate the 60th anniversary of foundation of the Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary ** Corresponding author; E-mail:
[email protected]
1217-8950/$20.00 © 2008 Akadémiai Kiadó, Budapest
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is carried out by a complex set of cellulases, including four endoglucanases (Cel5A, Cel5B, Cel6A, Cel9B), two exoglucanases (Cel6B, Cel48A), and an endo/exoglucanase (Cel9A) [2]. These enzymes and the genes coding for them have been characterized in detail [3–8]. T. fusca is also equipped with a number a hemicellulase genes, including two xylanases (xyl11A and xyl10B), a xyloglucanase (xg74A) and a b-mannosidase (manB) [1, 9]. In addition to these glycoside hydrolases, 28 other putative proteins with predicted role in plant cell wall degradation have been annotated in the genome of T. fusca [10]. The cellulase sytem of T. fusca consists of pairs of enzymes from the same glycoside hydrolase (GH) family: Cel5A – Cel5B, Cel6A – Cel6B, Cel9A – Cel9B plus a single family GH48 enzyme. The two members of an enzyme pair may have similar cellulose binding domains (CBD), like Cel6A and Cel6B both having a CBD_2 domain, but other associations may also occur: Cel5A contains a CBD_2 type domain, whereas Cel5B has a CBD_3 domain [8]. Phylogenetic comparisons of the catalytic domain sequences of the T. fusca cellulases to cellulases of other bacteria showed, that the enzyme pairs in the same GH family are not closely related to each other indicating that the complex cellulose system of this organism has evolved by horizontal gene transfer [8]. Synergistic interactions among members of this complex enzyme system result in a highly efficient cellulose degrading capability of this organism [11–13]. The T. fusca cellulases are coordinately regulated by both inducing and repressing factors. Cellulase synthesis is induced by microcrystalline cellulose and cellobiose, at physiological concentrations and repressed by soluble sugars and cellobiose, if the latter is present in high concentrations [14]. All cellulase genes of T. fusca have a 14 bp inverted repeat sequence, TGGGAGCGCTCCCA in their 5’ regulatory regions [15], which serves as the binding site for a regulatory protein, CelR [16]. Cellobiose, when present at physiological concentrations induces transcription of the cellulase genes by causing dissociation of the CelR – DNA complex [17]. Some cellulase genes of T. fusca, like cel6A, cel6B, and cel48A have one or two additional imperfect copies of this inverted repeat sequence. The molar levels of enzymes encoded by these genes have been found several times higher than that of the other cellulases when the actinomycete was grown on inducing carbon sources: this higher level of enzyme synthesis was explained by the presence of additional copies of the regulatory sequence that allowed a cooperative binding/dissociation of the CelR protein resulting in a stronger transcription of these three genes [15]. Cel5B is, however, a special member of the cellulase genes characterized thus far in T. fusca, as it has only a single imperfect copy of the above mentioned
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14 bp inverted repeat sequence [8], suggesting that its transcription may be different from that of its counterpart, cel5A equipped with a perfect copy of this site. In the present work we compared expression patterns of these two genes by using the real-time quantitative reverse transcription – polymerase chain reaction (qRTPCR) approach. The comparison was made under semi-natural conditions, where T. fusca was grown on two different cellulose sources and all cellulose degrading enzymes of this actinomycete were allowed to exert their activity without disturbing the interactions existing among these proteins.
Materials and Methods Growth conditions Luria-Bertani (LB) broth containing 0.2% glucose was used to grow Thermobifida fusca DSM 43792. Cultures were incubated at 45 °C for 72 h on a rotary shaker at 200 rpm. Mycelia collected from a late exponential-phase culture by centrifugation at 5,000g for 10 min were washed with minimal salt medium and used to inoculate Hagerdahl medium [18] complemented with 1.0% MN300 (Macherey-Nagel, Düren, Germany) or 0.5% Avicel (Merck, Darmstadt, Germany) as inducing carbon sources. Cultures were grown in triplicate on the rotary shaker at 45 °C for 1, 5, 10, 20 h on Avicel and for 1, 5, 10, 20, and 30 h on MN300, a microcrystalline cellulose substrate. We wanted to compare gene expression values between the lag phase and the early stationary phase, and therefore extended the sampling period when T. fusca was grown on MN300 cultures; the actinomycete initially grew more slowly on this carbon source and entered into the early stationary growth phase at 30 h.
Extracellular protein assay Samples taken from cultures at different points of time were centrifuged at 3,000g for 5 min and the supernatants were assayed for extracellular protein by using the Total Protein Reagent (Sigma, St. Louis, MO, USA) according to the manufacturer’s protocol. Absorbance was measured at 540 nm and bovine serum albumin fraction V was used as standard.
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Reducing sugar content Culture supernatants were assayed for soluble reducing sugar content by using the Glucose GOD – PAP enzymatic colorimetric method according to the manufacturer’s protocol (Chronolab, Zug, Schwitzerland). The principle of this method is that the glucose component of the reducing substrate is oxidized in the presence of glucose oxidase (GOD). The generated H2O2 reacts with phenol and 4-amino-antipyrine under catalysis of peroxidase (POD). As a result of this reaction quinoneimine is formed: the intensity of the color of this compound measured at 505 nm is proportional to the glucose concentration in the samples.
Quantitative real-time (qrt) PCR Total RNA was extracted by TRI Reagent (Sigma) from the actinomycete samples collected at different time intervals from liquid cultures grown on the two cellulose sources. cDNA was synthesized from 2.5 µg total RNA with the RevertAid cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) following the manufacturer’s instructions. One µl of the 10 fold diluted first-strand cDNA reaction mixture (equivalent to 10 ng total RNA) was used as a template for qrt-PCR in the 25 µl standard PCR mixtures. Primer pairs of cel5AF – cel5AR (5’-CTGGACCTGAAACGGCAT-3’; 5’-GTAGTTCTCGATGCGGCT-3’) and cel5BF – cel5BR (5’-GATGTAAGGTGCTTCTGCT-3’; 5’-CAACAAGAACAT GGCGAG-3’) producing 437 and 449 bp amplicons, respectively were used to quantify the expression of the two endoglucanase genes. A 232 bp fragment of the 16S rDNA gene, generated by primers, 16SRT2 (’5-GTTCCACGGGTTCCG TG-3’) and 16SRT3 (’5-GAGCTGACGACGACCATG-3’) [19] were used as references. Reference gene expression was validated for each experiment to demonstrate that rDNA gene expression was unaffected by the experimental conditions. The comparative CT (DDCT) method [20] was used to calculate the relative changes in cel5A and cel5B expressions at different sampling times and on different carbon sources. To determine the relative expression values of cel5A and cel5B, DDCT values were calculated for each sample according to the following equations: DDCTcel5A = (CTcel5A – CT16SRT)time x – (CTcel5A – CT16SRT)time 0 DDCTcel5B = (CTcel5B – CT16SRT)time x – (CTcel5B – CT16SRT)time 0, where CTcel5A, CTcel5B and CT16SRT are the threshold cycle numbers for targets and reference amplifications, respectively.
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To check the validity of the DDCT calculations the amplification efficiencies (E) of the target cel5 and cel5B fragments, as well as the reference rDNA fragments were compared. Serial dilutions of cDNA samples were amplified by qRT-PCR, using the above-mentioned gene specific primers and CT value were calculated for each cDNA dilution. A plot of cDNA dilutions versus DCT values was constructed. The absolute value of the slope(s) of the regression line was close to zero in (s = 0.031–0.006) in different runs, indicating that DDCT calculations were suitable for measuring the target gene expression. qRT-PCR was carried out using the ABI PRISM SDS 7000 system (Applied Biosystem, Foster City, CA, USA) with SYBR Green (Bio-Rad, Hercules, CA, USA) detection. PCR amplification mixtures (25 µl) contained 20 ng template cDNA, 0.5 µM of each primer, 12.5 µl of 2 × IQ SYBR Green Supermix [100 mM KCl, 40 mM Tris-HCl (pH 8.4), 0.4 mM of each dNTP, 50 units ml–1, Taq DNA polymerase, 60 mM MgCl2, and 20 nM SYBR Green I] purchased by Bio-Rad, Hercules, CA, USA. Amplification conditions were: (i) 95 °C for 10 min and (ii) 40 cycles for 15 s at 95 °C and 60 °C for 60 s.
Results and Discussion Expression patterns of two endoglucanase genes, cel5A and cel5B of T. fusca were monitored with qRT-PCR. Primers cel5AF and cel5AR, as well as cel5BF and Cel5BR based on previously cloned cel5A [6] and cel5B genes [8] were used to amplify an 437 and an 449 bp cDNA product, respectively from the actinomycete, whereas the 16S rDNA gene-specific primers, 16SRT2 and 16SRT3 [19] were used to amplify a 232 bp fragment serving as a control in the qRT-PCR experiments. Mycelia from a late exponentially growing culture of T. fusca DSM 43792 grown in Luria-Bertani (LB) broth was transferred to minimal medium supplemented with inducing carbon sources, Avicel and MN300 cellulose, respectively. Avicel contains both amorphous and crystalline fractions, while MN300 is a native microcrystalline cellulose powder. With Avicel as the carbon source, negligible amounts of cel5A transcripts were detected at 1 h after the actinomycete mycelium was transferred from LB ? at the medium to Avicel containing minimal medium. Transcript levels significantly in10th hour of creased by 5 h of incubation and further continuous increases were observed during the next few hours. The highest amounts of cel5A mRNA were measured at 10 ?? - at the 1st (next h of incubation, when transcript levels increased to 10 times of the levels mea- hour page) Acta Microbiologica et Immunologica Hungarica 55, 2008
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sured at 1 h. From this point, transcript levels started to decrease and by 20 h of incubation, the amount of cel5A mRNA dropped to the original level measured at the start of the experiment. Contrary to this pattern of induction, cel5B transcripts were slightly more abundant that these of cel5A transcripts at 1 h of incubation, but levels of cel5B transcripts remained almost constant in the two subsequent joints at sampling times, at 5 and 10 h of incubation, respectively. This seemingly constitutive expression was then followed by a significant decrease of transcript levels: the difference between the cel5B transcript levels measured at the beginning and at the end of the 20 h incubation period was nearly 40%. Worthy to note, that tran? at the script levels of cel5A were four times higher than those of cel5B at 10 h of incuba- 10th hour of tion when both genes had their expression maxima (Figure 1). ?? A) A. cel5A 16S rDNA cel5B 16S rDNA Time, hours
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Time, hours Figure 1. Expression patterns of cel5A (empty columns) and cel5B (black columns) on Avicel cellulose source. Quantitative real time (qrt) PCR was used to quantify mRNA transcripts from the two genes at different time intervals (A). Data on (B) was expressed in relative units. Representative results of three independent experiments are presented on (A), standard deviations are indicated with error bars on (B)
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The strong expression of cel5A on Avicel could be foreseen, as this gene retained the perfect 14 bp inverted repeat sequence in its 5’ regulatory region allowing the proper binding of the CelR regulatory protein at this site. On the other hand, cel5B has only an imperfect copy of this sequence (5’-CGGGAGCGCA CCCT-3’) 67 nt before the translational start codon [8], a probably unsuitable site for appropriate CelR binding; this deficiency is the most likely cause of the poor induction (quasi constitutive expression) of cel5B. The sudden decrease of transcript levels of cel5A by 20 h of incubation can be explained by depletion of the easily hydrolysable crystalline fraction of Avicel. Previous studies showed that the catalytic domain (CD) of Cel5A binds more intensely to the easily hydrolysable fraction of crystalline cellulose substrates than the CDs of exocellulases (Cel6B, Cel48A) of T. fusca do [21]. When a substrate with mixed fractions like Avicel is used, the crystalline fractions are rapidly saturated by Cel5A. After degradation and depletion of this fraction, expression of Cel5A becomes strongly reduced. The recalcitrant and amorphous fractions of the substrate are then degraded by other members of the complex cellulase system. ? at the When MN300 was used as inducing carbon source, the two genes showed 10th hour of similar expression patterns (Figure 2). A low level of initial expression was fol?? lowed by a rapid increase of expression at the 5th hour of incubation. A transient repression was then observed at 10 h of incubation, but after this sampling time, expression levels started again to increase. This transient repression effect was also observed by Lin and Wilson [14], when measuring endocellulase synthesis of T. fusca. They explained this phenomenon by a secondary regulatory mechanism reducing the rate of endocellulase synthesis when the growth rate of the producer organism increases. In the present experiment we measured the growth rate by protein assay and found that a rapid increase of growth occurred around the 10th hour of incubation (Figure 3) and this coincided with the marked repression of Cel5A expression (Figure 2). With MN300 substrate the relative expression levels of cel5A were also higher than those of cel5B, but the differences measured during the expression maxima of these two genes were lower than those observed when Avicel was used as carbon source. MN300, a crystalline cellulose powder seems to be better inducer of cel5B expression than Avicel. Furthermore, this substrate allowed a more durable operation of endocellulases than Avicel did. A slight decrease of cel5A expression was observed at 30 h ///? at the 30th hour?///, which can be explained by the high levels of reducing sugar content (Figure 3) accumulated by this time. In conclusion, cel5A is a highly efficient member of the cellulase enzyme system of T. fusca and it participates mainly in degradation of the crystalline frac-
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A)
cel5A 16S rDNA cel5B 16S rDNA Time, hours
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Figure 2. Expression patterns of cel5A (empty columns) and cel5B (black columns) on MN300 cellulose source. Quantitative real time (qrt) PCR was used to quantify mRNA transcripts from the two genes at different time intervals (A). Data on (B) was expressed in relative units. Representative results of three independent experiments are presented on (A), standard deviations are indicated with error bars on (B)
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Figure 3. Reducing sugar content of T. fusca cultures grown on Avicel (black column) and MN300 (empty column). Extracellular protein content of cultures grown on Avicel and MN300 are shown by curve (1) and curve (2), respectively
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tions of the cellulose substrate. The role of cel5B seems to be less important in cellulose degradation: this gene was poorly induced and its relative expression levels were always significantly lower than those of its counterpart, cel5A.
Acknowledgements This research was supported by the National Office for Research and Technology (Péter Pázmány Programme). L.H. thanks support from the Office for Subsidized Research Units of the Hungarian Academy of Sciences.
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12. Kim, D. W., Jang, Y. H., Jeong, Y. K.: Adsorption kinetics and behaviour of two cellobiohydrolases from Trichoderma reesei on microcrystalline cellulose. Biotechnol Appl Biochem 27, 97–102 (1998). 13. Watson, D. L., Wilson, D. B., Walker, L. P.: Synergism in binary mixtures of Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A on BMCC and Avicel. Appl Biochem Biotechnol 101, 97–111 (2002). 14. Lin, E., Wilson, D. B.: Regulation of b-1,4-endoglucanase synthesis in Thermomonospora fusca. J Bacteriol 53, 1352–1357 (1987). 15. Spiridonov, N. A., Wilson, D. B.: Regulation of biosynthesis of individual cellulases in Thermomonospora fusca. J Bacteriol 180, 3529–3532 (1998). 16. Spiridonov, N. A., Wilson, D. B.: Characterization and cloning of CelR, a transcriptional regulator of cellulase genes from Thermomonospora fusca. J Biol Chem 274, 13127–13132 (1999). 17. Spiridonov, N. A., Wilson, D. B.: A celR mutation affecting transcription of cellulase genes in Thermobifida fusca. J Bacteriol 182, 252–255 (2000). 18. Hägerdahl, B. G. R., Ferchak, J. D., Pye, E. K.: Cellulolytic enzyme system of Thermomonospora sp. grown on microcrystalline cellulose. Appl Environ Microbiol 36, 606–612 (1978). 19. Ventura, M., Zink, R.: Comparative sequence analysis of the tuf and recA genes and restriction fragment length polymorphism of the internal transcribed spacer region sequences supply additional tools for discriminating Bifidobacterium lactis from Bifidobacterium animalis. Appl Environ Microbiol 69, 7517–7522 (2003). 20. Livak, K. J., Schmittgen, T. D.: Analysis of relative gene expression data using real-time quantitative PCR and the 2-Delta C(T) method. Methods 25, 402–408 (2001). 21. Jung, H., Wilson, D. B., Walker, L. P.: Binding of Thermobifida fusca CDCel5A, CDCel6B and CDCel48A to easily hydrolysable and recalcitrant cellulose fraction on BMCC. Enz Microbial Technol 31, 941–948 (2002).
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