Expression patterns of \u003ci\u003ecel5A–cel5B\u003c/i\u003e , two endoglucanase encoding genes of \u003ci\u003eThermobifida fusca\u003c/i\u003e

$Page 1: Expression patterns of \u003ci\u003ecel5A–cel5B\u003c/i\u003e , two endoglucanase encoding genes of \u003ci\u003eThermobifida fusca\u003c/i\u003e$
EXPRESSION PATTERNS OF CEL5A – CEL5B,

TWO ENDOGLUCANASE ENCODING GENES OFTHERMOBIFIDA 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 ofThermobifida fusca were compared by quantitative real-time PCR. With Avicel as car-bon source the transcript level of cel5A continuously increased until the 10th hour of in-cubation and then a sharp decrease was observed, whereas cel5B presented a slow con-stitutive expression on this substrate. When the microcrystalline cellulose powderMN300 was used as the inducing carbon source, the expression patterns of the twogenes were similar. A low initial level of expression was followed by a rapid increaseat the 5th hour of incubation; a transient repression was then observed at the 10th hourbut after this sampling time, the expression levels started to increase again. The rela-tive expression levels of cel5A were always higher than those of cel5B. Differences intranscription patterns of these two genes can be explained with the imperfect structureof the CelR binding regulatory region of cel5B.

Keywords: Thermobifida fusca, endocellulase, gene expression, qRT-PCR

Introduction

Thermobifida fusca is a thermophilic, filamentous soil inhabiting actino-mycete, regarded as the major degrader of lignocellulose containing plant residuesunder aerobic conditions [1]. Biodegradation of plant cell walls by this organism

1217-8950/$20.00 © 2008 Akadémiai Kiadó, Budapest

Acta Microbiologica et Immunologica Hungarica, 55 (4), pp. 437–446 (2008)DOI: 10.1556/AMicr.55.2008.4.8

* This paper is written to commemorate the 60th anniversary of foundation of the Institute ofMedical Microbiology, Semmelweis University, Budapest, Hungary** Corresponding author; E-mail: [email protected]

is carried out by a complex set of cellulases, including four endoglucanases(Cel5A, Cel5B, Cel6A, Cel9B), two exoglucanases (Cel6B, Cel48A), and anendo/exoglucanase (Cel9A) [2]. These enzymes and the genes coding for themhave been characterized in detail [3–8]. T. fusca is also equipped with a number ahemicellulase genes, including two xylanases (xyl11A and xyl10B), a xylo-glucanase (xg74A) and a b-mannosidase (manB) [1, 9]. In addition to theseglycoside hydrolases, 28 other putative proteins with predicted role in plant cellwall degradation have been annotated in the genome of T. fusca [10].

The cellulase sytem of T. fusca consists of pairs of enzymes from the sameglycoside hydrolase (GH) family: Cel5A – Cel5B, Cel6A – Cel6B, Cel9A –Cel9B plus a single family GH48 enzyme. The two members of an enzyme pairmay have similar cellulose binding domains (CBD), like Cel6A and Cel6B bothhaving a CBD_2 domain, but other associations may also occur: Cel5A contains aCBD_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 sameGH family are not closely related to each other indicating that the complex cellu-lose system of this organism has evolved by horizontal gene transfer [8]. Synergis-tic interactions among members of this complex enzyme system result in a highlyefficient cellulose degrading capability of this organism [11–13].

The T. fusca cellulases are coordinately regulated by both inducing and re-pressing factors. Cellulase synthesis is induced by microcrystalline cellulose andcellobiose, at physiological concentrations and repressed by soluble sugars andcellobiose, if the latter is present in high concentrations [14]. All cellulase genes ofT. 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 tran-scription of the cellulase genes by causing dissociation of the CelR – DNA com-plex [17]. Some cellulase genes of T. fusca, like cel6A, cel6B, and cel48A have oneor two additional imperfect copies of this inverted repeat sequence. The molar lev-els of enzymes encoded by these genes have been found several times higher thanthat of the other cellulases when the actinomycete was grown on inducing carbonsources: this higher level of enzyme synthesis was explained by the presence ofadditional copies of the regulatory sequence that allowed a cooperative bind-ing/dissociation of the CelR protein resulting in a stronger transcription of thesethree genes [15].

Cel5B is, however, a special member of the cellulase genes characterizedthus far in T. fusca, as it has only a single imperfect copy of the above mentioned

Acta Microbiologica et Immunologica Hungarica 55, 2008

438 SASVÁRI et al.

14 bp inverted repeat sequence [8], suggesting that its transcription may be differ-ent from that of its counterpart, cel5A equipped with a perfect copy of this site. Inthe present work we compared expression patterns of these two genes by using thereal-time quantitative reverse transcription – polymerase chain reaction (qRT-PCR) approach. The comparison was made under semi-natural conditions, whereT. fusca was grown on two different cellulose sources and all cellulose degradingenzymes of this actinomycete were allowed to exert their activity without disturb-ing the interactions existing among these proteins.

Materials and Methods

Growth conditions

Luria-Bertani (LB) broth containing 0.2% glucose was used to growThermobifida fusca DSM 43792. Cultures were incubated at 45 °C for 72 h on arotary shaker at 200 rpm. Mycelia collected from a late exponential-phase cultureby centrifugation at 5,000g for 10 min were washed with minimal salt medium andused to inoculate Hagerdahl medium [18] complemented with 1.0% MN300(Macherey-Nagel, Düren, Germany) or 0.5% Avicel (Merck, Darmstadt, Ger-many) as inducing carbon sources. Cultures were grown in triplicate on the rotaryshaker 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 val-ues between the lag phase and the early stationary phase, and therefore extendedthe sampling period when T. fusca was grown on MN300 cultures; the actino-mycete initially grew more slowly on this carbon source and entered into the earlystationary growth phase at 30 h.

Extracellular protein assay

Samples taken from cultures at different points of time were centrifuged at3,000g for 5 min and the supernatants were assayed for extracellular protein by us-ing the Total Protein Reagent (Sigma, St. Louis, MO, USA) according to the man-ufacturer’s protocol. Absorbance was measured at 540 nm and bovine serum albu-min fraction V was used as standard.


GENES OF THERMOBIFIDA FUSCA 439

Reducing sugar content

Culture supernatants were assayed for soluble reducing sugar content byusing the Glucose GOD – PAP enzymatic colorimetric method according to themanufacturer’s protocol (Chronolab, Zug, Schwitzerland). The principle of thismethod is that the glucose component of the reducing substrate is oxidized in thepresence of glucose oxidase (GOD). The generated H2O2 reacts with phenol and4-amino-antipyrine under catalysis of peroxidase (POD). As a result of this reac-tion quinoneimine is formed: the intensity of the color of this compound measuredat 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 actinomycetesamples collected at different time intervals from liquid cultures grown on the twocellulose sources. cDNA was synthesized from 2.5 µg total RNA with theRevertAid cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) following themanufacturer’s instructions. One µl of the 10 fold diluted first-strand cDNA reac-tion mixture (equivalent to 10 ng total RNA) was used as a template for qrt-PCR inthe 25 µl standard PCR mixtures. Primer pairs of cel5AF – cel5AR(5’-CTGGACCTGAAACGGCAT-3’; 5’-GTAGTTCTCGATGCGGCT-3’) andcel5BF – cel5BR (5’-GATGTAAGGTGCTTCTGCT-3’; 5’-CAACAAGAACATGGCGAG-3’) producing 437 and 449 bp amplicons, respectively were used toquantify the expression of the two endoglucanase genes. A 232 bp fragment of the16S rDNA gene, generated by primers, 16SRT2 (’5-GTTCCACGGGTTCCGTG-3’) and 16SRT3 (’5-GAGCTGACGACGACCATG-3’) [19] were used as ref-erences. Reference gene expression was validated for each experiment to demon-strate that rDNA gene expression was unaffected by the experimental conditions.

The comparative CT (DDCT) method [20] was used to calculate the relativechanges in cel5A and cel5B expressions at different sampling times and on differ-ent carbon sources. To determine the relative expression values of cel5A andcel5B, DDCT values were calculated for each sample according to the followingequations:

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 andreference amplifications, respectively.


440 SASVÁRI et al.

To check the validity of the DDCT calculations the amplification efficien-cies (E) of the target cel5 and cel5B fragments, as well as the reference rDNA frag-ments were compared. Serial dilutions of cDNA samples were amplified byqRT-PCR, using the above-mentioned gene specific primers and CT value werecalculated for each cDNA dilution. A plot of cDNA dilutions versus DCT valueswas constructed. The absolute value of the slope(s) of the regression line was closeto zero in (s = 0.031–0.006) in different runs, indicating that DDCT calculationswere suitable for measuring the target gene expression.

qRT-PCR was carried out using the ABI PRISM SDS 7000 system (Ap-plied Biosystem, Foster City, CA, USA) with SYBR Green (Bio-Rad, Hercules,CA, USA) detection. PCR amplification mixtures (25 µl) contained 20 ng tem-plate cDNA, 0.5 µM of each primer, 12.5 µl of 2 × IQ SYBR Green Supermix [100mM KCl, 40 mM Tris-HCl (pH 8.4), 0.4 mM of each dNTP, 50 units ml–1, TaqDNA polymerase, 60 mM MgCl2, and 20 nM SYBR Green I] purchased byBio-Rad, Hercules, CA, USA. Amplification conditions were: (i) 95 °C for 10 minand (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 ascel5BF 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 theactinomycete, whereas the 16S rDNA gene-specific primers, 16SRT2 and16SRT3 [19] were used to amplify a 232 bp fragment serving as a control in theqRT-PCR experiments.

Mycelia from a late exponentially growing culture of T. fusca DSM 43792grown in Luria-Bertani (LB) broth was transferred to minimal medium supple-mented with inducing carbon sources, Avicel and MN300 cellulose, respectively.Avicel contains both amorphous and crystalline fractions, while MN300 is a na-tive microcrystalline cellulose powder.

With Avicel as the carbon source, negligible amounts of cel5A transcriptswere detected at 1 h after the actinomycete mycelium was transferred from LBmedium to Avicel containing minimal medium. Transcript levels significantly in-creased by 5 h of incubation and further continuous increases were observed dur-ing the next few hours. The highest amounts of cel5A mRNA were measured at 10h of incubation, when transcript levels increased to 10 times of the levels mea-



? at the10th hour of?? - at the 1sthour (nextpage)

sured at 1 h. From this point, transcript levels started to decrease and by 20 h of in-cubation, the amount of cel5A mRNA dropped to the original level measured atthe start of the experiment. Contrary to this pattern of induction, cel5B transcriptswere slightly more abundant that these of cel5A transcripts at 1 h of incubation, butlevels of cel5B transcripts remained almost constant in the two subsequent jointsat sampling times, at 5 and 10 h of incubation, respectively. This seemingly consti-tutive expression was then followed by a significant decrease of transcript levels:the difference between the cel5B transcript levels measured at the beginning and atthe end of the 20 h incubation period was nearly 40%. Worthy to note, that tran-script levels of cel5A were four times higher than those of cel5B at 10 h of incuba-tion when both genes had their expression maxima (Figure 1).


442 SASVÁRI et al.

? at the10th hour of??

A.

cel5A

16S rDNA

cel5B

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Time, hours 1 5 10 20

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0,5

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1,5

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Figure 1. Expression patterns of cel5A (empty columns) and cel5B (black columns) on Avicel cellulosesource. Quantitative real time (qrt) PCR was used to quantify mRNA transcripts from the two genes at dif-ferent time intervals (A). Data on (B) was expressed in relative units. Representative results of three inde-

pendent experiments are presented on (A), standard deviations are indicated with error bars on (B)

A)

B)

The strong expression of cel5A on Avicel could be foreseen, as this gene re-tained the perfect 14 bp inverted repeat sequence in its 5’ regulatory region allow-ing the proper binding of the CelR regulatory protein at this site. On the otherhand, cel5B has only an imperfect copy of this sequence (5’-CGGGAGCGCACCCT-3’) 67 nt before the translational start codon [8], a probably unsuitable sitefor appropriate CelR binding; this deficiency is the most likely cause of the poorinduction (quasi constitutive expression) of cel5B. The sudden decrease of tran-script levels of cel5A by 20 h of incubation can be explained by depletion of theeasily hydrolysable crystalline fraction of Avicel. Previous studies showed thatthe catalytic domain (CD) of Cel5A binds more intensely to the easily hydro-lysable fraction of crystalline cellulose substrates than the CDs of exocellulases(Cel6B, Cel48A) of T. fusca do [21]. When a substrate with mixed fractions likeAvicel is used, the crystalline fractions are rapidly saturated by Cel5A. After deg-radation and depletion of this fraction, expression of Cel5A becomes strongly re-duced. The recalcitrant and amorphous fractions of the substrate are then degradedby other members of the complex cellulase system.

When MN300 was used as inducing carbon source, the two genes showedsimilar 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 transientrepression was then observed at 10 h of incubation, but after this sampling time,expression levels started again to increase. This transient repression effect wasalso observed by Lin and Wilson [14], when measuring endocellulase synthesis ofT. fusca. They explained this phenomenon by a secondary regulatory mechanismreducing the rate of endocellulase synthesis when the growth rate of the producerorganism increases. In the present experiment we measured the growth rate byprotein 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 ofCel5A expression (Figure 2).

With MN300 substrate the relative expression levels of cel5A were alsohigher than those of cel5B, but the differences measured during the expressionmaxima of these two genes were lower than those observed when Avicel was usedas carbon source. MN300, a crystalline cellulose powder seems to be better in-ducer of cel5B expression than Avicel. Furthermore, this substrate allowed a moredurable operation of endocellulases than Avicel did. A slight decrease of cel5A ex-pression was observed at 30 h ///? at the 30th hour?///, which can be explained bythe high levels of reducing sugar content (Figure 3) accumulated by this time.

In conclusion, cel5A is a highly efficient member of the cellulase enzymesystem of T. fusca and it participates mainly in degradation of the crystalline frac-



? at the10th hour of??


444 SASVÁRI et al.

cel5A

16S rDNA

cel5B

16S rDNA

Time, hours 1 5 10 20 30

0

0,5

1

1,5

2

2,5

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Time, hours

Re

lative

exp

ressio

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Figure 2. Expression patterns of cel5A (empty columns) and cel5B (black columns) on MN300 cellulosesource. Quantitative real time (qrt) PCR was used to quantify mRNA transcripts from the two genes at dif-ferent time intervals (A). Data on (B) was expressed in relative units. Representative results of three inde-

pendent experiments are presented on (A), standard deviations are indicated with error bars on (B)

0

5

10

15

20

25

1 5 10 20 30

Time, hours

Reducin

gsugar,

µg

ml-1

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50

100

150

200

250

300

350

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500

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,µg

ml-1

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Figure 3. Reducing sugar content of T. fusca cultures grown on Avicel (black column) and MN300 (emptycolumn). Extracellular protein content of cultures grown on Avicel and MN300 are shown by curve (1) and

curve (2), respectively

A)

B)

tions of the cellulose substrate. The role of cel5B seems to be less important in cel-lulose degradation: this gene was poorly induced and its relative expression levelswere always significantly lower than those of its counterpart, cel5A.

Acknowledgements

This research was supported by the National Office for Research and Tech-nology (Péter Pázmány Programme). L.H. thanks support from the Office for Sub-sidized Research Units of the Hungarian Academy of Sciences.

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Expression patterns of \u003ci\u003ecel5A–cel5B\u003c/i\u003e , two endoglucanase encoding genes of \u003ci\u003eThermobifida fusca\u003c/i\u003e

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