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Bacterial Cellulose Hydrolysis in Anaerobic

Environmental SubsystemsClostridium

thermocellum andClostridium stercorarium,

Thermophilic Plant-fiber DegradersVLADIMIR V. ZVERLOV AND WOLFGANG H. SCHWARZ

Department of Microbiology, Technische Universit at Munchen, Freising, Germany

Cellulose degradation is a rare trait in bacteria. However, the truly cellulolytic bacteria are ex-

tremely efficient hydrolyzers of plant cell wall polysaccharides, especially those in thermophilic

anaerobic ecosystems. Clostridium stercorarium, a thermophilic ubiquitous soil dweller, has a

simple cellulose hydrolyzing enzyme system of only two cellulases. However, it seems to be

better suited for the hydrolysis of a wide range of hemicelluloses. Clostridium thermocellum,

an ubiquitous thermophilic gram-type positive bacterium, is one of the most successful cellu-

lose degraders known. Its extracellular enzyme complex, the cellulosome, was prepared from

C. thermocellum cultures grown on cellulose, cellobiose, barley -1,3-1,4-glucan, or a mixture of

xylan and cellulose. The single proteins were identified by peptide chromatography and MALDI-

TOF-TOF. Eight cellulosomal proteins could be found in all eight preparations, 32 proteins occur

in at least one preparation. A number of enzymatic components had not been identified previ-

ously. The proportion of components changes if C. thermocellum is grown on different substrates.

Mutants ofC. thermocellum, devoid of scaffoldin CipA, that now allow new types of experiments

with in vitro cellulosome reassembly and a role in cellulose hydrolysis are described. The charac-

teristics of these mutants provide strong evidence of the positive effect of complex (cellulosome)

formation on hydrolysis of crystalline cellulose.

Key words: bacterial; cellulose; hydrolysis; anaerobic; thermophilic; cellulosome; mutant; scaf-foldin; enzyme; complex

Introduction

Climate is changing. One indication for this change

is the large number of strong hurricanes in the United

States in 2005, including Katrina and Rita, the hot

summer in 2006, and the early and extremely dry

spring in Europe in 2007. A long time indicator for

global warming is the melting of the alpine glaciers,

which can be observed by a larger number of people,in contrast to the less familiar melting of the Arctic

and Antarctic ice shields and other indicators.1 The

Furtwangler glacier on mount Kilimandjaro (Tansa-

nia, Africa) has supposedly existed for 11,700 years but

will disappear completely within 20 years.1 The pub-

lication of the Intergovernmental Panel on Climate

Change (IPCC) report,2 the first parts of which present

the scientific evidence for global warming and the hu-

Address for correspondence: Wolfgang H. Schwarz, Department ofMicrobiology, TUM,Am Hochanger4, D-85350 Freising,Germany.+49-

8161-715445.

[email protected]

man impact. The Stern report3 made clear that im-

mediate measures for the reduction of CO2 emissions

would be economically more efficient than subsequent

repair measures and damage.

With the growing prosperity of an increasing num-

ber of people, associated with an advancing depen-

dence on technology, the demand for energy is grow-

ing steadily. More than 90% of our energy is currently

made from stored natural resources that are rapidly de-

pleted. The most important of those sources is crude

(fossil) oil, which is burned with the release of CO 2, a

climate-active greenhouse gas. This leads to a global

increase in CO2 content of the atmosphere. The re-

duction of CO2 release is a prime issue and calls the

scientific world to present solutions for the produc-

tion of sustainable energy.4 New sustainably produced

liquid and gaseous fuels for the energy sector could

contribute considerably to a CO2-neutral energy us-

age. Even more effective measures would be a more

rational use of energy combined with energy saving.

At present, only about1% of the worlds energy need

is met by biofuels. New biofuels are to be produced in

Ann. N.Y. Acad. Sci. 1125: 298307 (2008). C 2008 New York Academy of Sciences.doi: 10.1196/annals.1419.008 298


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Zverlov & Schwarz: Bacterial Cellulose Hydrolysis 299

FIGURE 1. The double role of microbes in biofuelproduction.

greater amounts. This has to include the production of

new types of fuels that are optimized for conversion to

energy with the existing and even more with advancedand specially adapted technology. Both achievements

have to go hand in hand with utilization of new and

more abundant substrates, such as cellulose and hemi-

cellulose, from plant biomass.

Microbiology can contribute to the CO2-neutral

production of biofuels from renewable biomass in two

major fields: (1) the supply of efficient polysaccha-

ride hydrolyzing enzymes, especially for the cheap and

abundant polysaccharides cellulose and hemicellulose,

and (2) the fermentation of the resulting sugars to sol-

vents and fuels (FIG. 1). However, the hydrolysis tech-nology for the production of sugary fermentation sub-

strates is lagging behind and commercial conversion

processes for biomass-to-sugar plants are still in their

infancy. New and more efficient cellulase and hemicel-

lulase preparations have to be developed.

Clostridium stercorarium produces a variety of plant cell

wall hydrolyzing enzymes, especially for the hydrolysis

of hemicellulose. However, one of the most efficient

cellulose-degrading microorganisms known so far is

Clostridium thermocellum.5,6 The cellulosome, an extra-

cellular, remarkably efficient cellulase complex of thisanaerobic, thermophilic bacterium, is one of the tar-

gets of this overview.7 The understanding of the de-

tailed cellulosome function could provide insight into

the mechanism of degradation enzymes for crystalline

(= native) cellulose. This chapter gives a short intro-

duction and summarizes new findings in the enzymatic

polysaccharide hydrolysis and the possible role of the

thermophilic bacteria C. stercorarium and C. thermocellum

therein.

The Role of Biomass HydrolysisBacteria seem to be the major degraders of organic

biomass in various ecosystems, either in the environ-

ment, for example, in biofilms, or in symbiosis with uni-

or multicellular organisms, for example, the intestine

of termites or ruminants. For this task they produce

a multiplicity of enzymes. Especially the hydrolysis of

refractory material, such as crystalline cellulose or the

heterogeneous hemicellulose, is dependent on the si-

multaneous presence of a large number of interacting

(synergistic) enzymes in high local concentration on

the target site. The heterogeneity of hemicellulose ob-

viously requires a variety of enzymes to cleave the dif-

ferent chemical bonds.8 In contrast, the chemically ho-

mogeneous cellulose has to be hydrolyzed by enzymes

that cleave all the same type of chemical bond (-1,4-

glycosidic). However, they attack in a distinct mode of

action, such as progressively from one end (from the

reducing or the nonreducing end) or nonprogressively

in an endomode, by splitting off cellobiose or cellote-traose units, in amorphic or crystalline regions of the

cellulose molecule bundle, in I- or I-type cellulose,

and so on.911

Cellulases are distinguished by two cleavage mech-

anisms, retaining or inverting cleavage. Besides these

mechanisms there are only the above mentioned two

ways to cut an otherwise uniform chemical bond, that

is, the cellulases belong to a number of different struc-

tural enzyme classes, called glycosyl hydrolase (GH)

families.12 However, beyond the retaininginverting

modus, the mode of action is not defined by the GHfamily. Most GH families contain nonprogressive en-

doglucanases as well as progressive cellobiohydrolases

or progressive endoglucanases (CAZY database). The

difference between those two action modes is generally

dependent on the absence or presence of a lid of pro-

truding amino acid residues over the active site pocket

that forms a tunnel for the substrate.13,14

Another way of influencing the mode of cellulase ac-

tivity is the attachment of noncatalytic modules, many

of which are carbohydrate binding modules (CBMs).

These modules bind to single, long or short, cellulosemolecules, or to crystalline structures of a different type

in cellulose.15 Some thread a single molecule into the

active site pocket, as was shown for the Thermonospora

fuscacellulase.16 The direction of activity in progressive

enzymes (from the reducing or nonreducing end) may

also be influenced by those modules. At present such

enzyme mechanisms cannot be predicted by bioinfor-

matics (yet?); they have to be determined experimen-

tally in the laboratory.

Anaerobic hydrolytic bacteria play a major role in

the first step of biomass degradation cascades, suchas in compost heaps, plant-eating animals (especially

in ruminants), or biogas-producing plants. The sugars

produced by enzymatic degradation are metabolized


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300 Annals of the New York Academy of Sciences

FIGURE 2. A compost plant for household biowastewith forced aeration in Germany: temperatures of up to75C are reached. (Photograph provided by W. Hiegl.)

by the cellulolytic microorganisms either aerobically

(by fungi and some bacteria) or anaerobically (mostly

by bacteria). The anaerobic conversion of biomass re-

sults in energy-rich fatty acids and alcohols (plus CO2and H2) that accumulate in some cases to considerable

amounts.

The formation of ethyl- or butyl-alcohol by anaero-

bic fermentation has been exploited for the produc-

tion of biofuels.4 Although no commercially viable

process is yet available to directly ferment cellulosicbiomass into ethanol, a pilot plant with a combina-

tion of weak acid treatment and enzymatic hydroly-

sis is under construction in Japan.17 The process uses

separate conversion of the pentose and hexose sug-

ars formed and involves metabolically engineered bac-

teria for the fermentation step. In contrast, C. ther-

mocellum would be able to directly ferment cellulose

and hemicellulose to ethanol in coculture with Clostrid-

ium thermohydrosulfuricum as has been suggested by Ng

et al.18 Various C. stercorarium strains were applied dur-

ing the early 1990s at a pilot plant of Takara ShuzoInc., Japan, for the direct fermentation of biomass to

ethanol.19

For drawing out single cellulose molecules from the

crystal surface, a rise of temperature should be ener-

getically advantageous for cellulolysis. In fact, at least

some thermophilic cellulolytic bacteria seem to be

very efficient cellulose degraders.5 This is in agree-

ment with the increasing number of thermophilic pro-

cesses for fiber hydrolysis. Thermophilic processes are

developed for composting, that is, in vitro enzymatic di-

gestion of plant material or biogas formation. Ther-mophilic treatment plants for biological household

and agricultural waste are meanwhile state of the art

(FIG.2).

TABLE 1. Comparison of hydrolytic activities inculture supernatants of type strains of Clostrid-ium thermocellum and Clostridium stercorariumgrown in cellobiose medium

C. thermocellum C. stercorarium

Substrate (mu/mL) (mu/mL)

Microcrist. cellulose 2 2

Phosphoric acid swollen cellulose 13 10

Carboxymethylcellulose (CMC) 140 120

1,3-1,4--glucan (lichenan) 6,500 12,000

Arabino-xylan 3,000 20,000

pNP--glucopyranoside 1.3 7

pNP--cellobioside 12 1.7

pNP--xylopyranoside 0.3 2

pNP--arabinofuranoside 1.3 21

Cell-free culture supernatants were incubated with the

substrates indicated and activity was determined by estimating

reducing sugars (upper part of the list) with dinitrosalicylic acidand p-nitrophenol, respectively (lower part) released.

Activity in cell-free culture fluid (grown on cellobiose).

Polysaccharide Degradation byClostridium stercorarium

For a process of direct conversion of lignocellulosic

plant biomass to ethanol, new thermophilic bacterial

strains had been isolated by Takara Shuzo Inc.

19

Threeof those strains have been investigated and found to

be very similar to C. stercorarium in the pattern of ge-

nomic restriction fragments and the hybridization of

cellulase genes to macrorestriction fragments of the

same size.19 The degradation of crystalline cellulose

in these strains was relatively modest as was the case

with the type strain C. stercorarium. However, the new

strains as well as the C. stercorarium type strain were very

good degraders of a wide range of hemicellulosic model

substrates.20

When culture supernatants of cellobiose-grown typestrains ofC. stercorarium and C. thermocellum were com-

pared with different substrates, it became clear that

both strains can degrade the crystalline or amorphic

cellulose, the mixed-linkage glucan, the xylan, and the

arylglycosides (TABLE 1). But apparently the activity of

C. stercorarium was slower with the hydrolysis of cellu-

losic substrates, whereas it was faster, and in many cases

very much faster, with the -glucan, the xylan, and

the arylglycosides. An exception is pNP--cellobioside,

where C. thermocellum has an enzyme system that is

better suited to degrade the aryl bond between thep-nitrophenyl residue and the cellobiose (cellobiohy-

drolase). This agrees well with the better cellulose de-

grading ability, which is also obvious with the shorter


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TABLE 2. Screening of a genomic library of Clostridium stercorarium DNA in E. coli

Substrate Isolates Genes isolated Alternative substrates Ref.

Cellulose (CMC, Avicel) 2 celY, celZ 38, 39

Xylan 17 xynA 8, 40

Lichenan 18 xynB, xynC xylan 8, 40

PNP--xyloside (PNPX) 18 bxlA 8, 40PNP--arabinoside (PNPAf) 4 arfB 8, 40, 41, 42

PNPX + PNPAf 4 arfA, bxlB 8, 40

PNP--glucoside 4 bglZ 8

PNP--rhamnoside 7 ramA 6 on PNPX 43

PNP--fucoside 2

PNP--galactoside 2

PNP--galactoside 3

PNP-maltoside 3

PNP--glucuronoside 2 PNPX

PNP--galacturonoside 4 PNP--glucuronoside

Source:Schwarz et al.40

In all, cell-free extracts from 1139 clones were prepared and assayed on the substrates indicated. Not all clones were tested onall substrates and not all identified active clones were characterized. Designation of identified genes is indicated, together with the

references for the publication of sequence analysis, methods, and biochemical characterization. Some activities found with a clone

were also active on other substrates (alternative substrates).

time in which Whatman filter paper is completely hy-

drolyzed during growth (unpublished observation). C.

stercorarium can thus be regarded as a specialist for hy-

drolysis of hemicellulose, whereas C. thermocellum is a

specialist for cellulose.

This fact persuaded us to screen hydrolytic genes

from a genomic library of C. stercorarium DNA.21 A

library of 1139 clones were obtained by the partial

digest of genomic DNA with different restriction en-

zymes. Cell-free extracts were prepared and screened

with 14 different substrates indicative for enzyme activ-

ities involved in cellulose and hemicellulose hydrolysis

(TABLE 2). Substrates containing, for example, - and

-linkages, hexoses and pentoses, or uronic acids, were

applied. As soon as a handful of active clones on a sub-

strate were identified, the screening with that substrate

was stopped. TABLE 2 thus cannot be used for a sta-

tistical calculation of genes present. On any substrate

tested, a number of active clones could be found. For

those enzyme activities with the greatest importance

for cellulose or hemicellulose hydrolysis, the genes ex-

pressing activity were subcloned, the DNA restriction

maps were compared, and the genes responsible for the

activity were sequenced (TABLE 2). The biochemical

characteristics of the single enzymes were investigated.

Only two genes ofC. stercorarium could be identified

with activity on cellulose and soluble -1,4-glucans:

Cel9Z and Cel48Y. The two enzymes produced were

also the only enzymes isolated from the culture super-

natant. They were found to be responsible for cellu-

lose degradation in a synergistic manner.22 They were

the only cellulase genes of that bacterium identified

so far in a number of screenings of different genomic

libraries and in protein isolations: it can be assumed

that they are indeed the only cellulases of that organ-

ism, although this cannot be said with certainty until

the genome is sequenced.

A number of genes expressing enzyme activities

related to hemicellulose hydrolysis were sequenced

(TABLE 2). A combination of recombinant xylanase

XynA, arabinofuranosidase ArfB, and -xylosidase

BxlB was sufficient to degrade arabinoxylan com-

pletely to the monosaccharides xylose and arabinose

with a mass ratio of about 10:1.8 Other genes express-

ing similar activities (such as ArfA and BxlA) were

not able to substitute these enzymes on arabinoxylan

because they were arylhydrolases and not active on

oligosaccharides (alkyl compounds). Their function is

probably the detoxification of arylglycosides.

In contrast to C. thermocellum, C. stercorarium thus

degrades a wide range of polysaccharides (especially

hemicellulose) and ferments pentoses more readily.

Many strains ofC. thermocellum do not metabolize pen-

toses and even have a long lag phase when glucose is

provided as the only carbon source. However, there are

differences between the isolates (see, e.g., Ref. 23).

The Clostridium thermocellum

CellulosomeC. thermocellum, an anaerobic thermophilic ubiq-

uitous bacterium frequently isolated from decaying


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FIGURE 3. Map of the CipA protein of C. thermocel-lum, the scaffoldin protein. c1 to c9, cohesin modules; d,dockerin module of type 2; carbohydrate binding module(CBM) family III. The leader peptide is indicated by a blackbox. The insertion points, translation direction, and approx-imate size of IS 1447 in four different mutants are indicatedby black arrow boxes.

biomass and soil,5 was reported to be one of the most

efficient bacteria in degradation of native cellulose. Itcan also be found in high numbers in habitats that

are not hot (thermophilic). Recently, C. thermocellum

was shown to be present in a thermophilic biomass

digester that was run at an elevated temperature, as

was the more moderately thermophilic bacterium, C.

stercorarium24 which is closely related to C. thermocellum,

but has a much simpler cellulase system.25 These data

were confirmed in our laboratory when various enrich-

ments of plant fiber degrading bacteria at 55 or 60C

were investigated (Hiegl, personal communication).

The natural substrate of C. thermocellum catabolismare the hydrolysis products of cellulose hydrolysis, the

cellodextrins, which are degraded by the consecu-

tive action of intracellular cellodextrin- and cellobiose-

phosphorylases, as was shown for the type strain ATCC

27405.6,26 This results in a much more energy-efficient

metabolism, which makes up for the costly production

of the large amount of extracellular cellulase complex

needed for the degradation of the refractory substrate.6

For the degradation of crystalline cellulose, C. thermo-

cellum synthesizes a large extracellular protein particle.

It contains a core protein called scaffoldin (the CipAprotein).15,27,28 CipA is a noncatalytic structural pro-

tein of intricate structure, and, among other modules, it

consists of nine binding modules (FIG. 3) that are called

cohesins. One enzyme component, probably randomly

selected, is firmly attached to each cohesin by virtue of

its dockerin module. The dockerins consist of highly

conserved dual 24 aa repeats of amino acids. CipA is

connected to the bacterial cell wall by an S-layer ho-

mologous module; it is also connected to the cellulosic

substrate via a CBM.27

The assembly of the cellulosome is a highly demand-ing task to discover. The sequence of events and the

assembly mechanism have not been uncovered to date.

The cellulosome formation may begin with the secre-

tion of all components to the cell surface, and is suc-

ceeded by the assembly into the enzyme complex on

the cell surface. The steps in between are a black box.

The present working hypothesis is that the exact com-

position of a single particle cannot be predicted. The

particles are apparently composed at random, accord-

ing to the concentration of single components secreted

through the cell membrane. It can be speculated that

the most frequent (and most important) enzyme com-

ponents, such as Cel48S, may have a defined cohesin

where they are preferentially bound. To find proof of

this would need much more sophisticated experiments

than have been performed to date.

The number of cellulosome components is surpris-

ingly large. An analysis of the closed genome sequence

ofC. thermocellum (GenBank No. CP000568, DOE JGI

as of February 16, 2007) detected 74 reading framescontaining a dockerin duplet (unpublished data and

Ref. 29). This unequivocally indicates cellulosomal

components. The high number of components makes

working with an artificial cellulosome difficult. How-

ever, not all components are present in the same mo-

lar amount (TABLE 3 and Ref. 29). It can be assumed

that components less frequently represented in the cel-

lulosome are less important for cellulose hydrolysis.

Moreover, a part of the most frequent components are

xylanases. This reduces the number of components for

in vitro reconstruction of the cellulosome.Besides the cell-bound cellulosome complex, the C.

thermocellum genomic sequence also revealed the pres-

ence of a putative second cellulase system. This system

consists of the cellulases Cel9I and Cel48Y (GHF9 and

GHF48, respectively). Both enzymes cooperate syner-

gistically.30 This soluble cellulase system resembles the

one in C. stercorarium that consists of the two cellu-

lases, Cel9Z and Cel48Y,30 the only cellulases found in

C. stercorarium. Cs-Cel9Z resembles Ct-Cel9I, and Cs-

Cel48Y resembles Ct-Cel48Y in structure and mode of

activity. These soluble cellulases ofC. thermocellum possi-bly assist the cellulosomal cellulose hydrolysis, whereas

the cellulosomes are cell-bound, the soluble enzymes

putatively act on different subsites of the cellulose crys-

tals, and at a greater distance to the producer cell.

The cellulosomes contain not only cellulases but

also a large number of xylanases and glycosidases (in

addition to a chitinase and to pectinases). These en-

zymes are obviously removing covering hemicellulosic

material from the surface of the cellulose crystals that

are the substrate for the cellulases and for the host

bacterium. To test the ability of different C. thermo-cellum strains, such as the various DSMZ strains and

strain VKPM2203, to degrade plant cell wall, the bac-

teria were inoculated to ground plant fibers (2 mm) and


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TABLE 3. Proteins identified in cellulosomes fromClostridium thermocellum produced on differentsubstrates

Protein (GHF) Locus_tag C Cb X+ C G C2D

CipA Cthe 3077

CelS (48) Cthe 2089 CelG (5) Cthe 2872

CelK (9) Cthe 0412

CbhA (9) Cthe 0413

CelR (9) Cthe 0578

CelA (8) Cthe 0269

XynC (10) Cthe 1838

- (9) Cthe 0433

CelJ (9,44) Cthe 0624

CelQ (9) Cthe 0625

CelF (9) Cthe 0543

XynY (10) Cthe 0912

CelV (9) Cthe 2760

CprA Cthe 3136

XynZ (10) Cthe 1963

XghA (74) Cthe 1398

XynD (10) Cthe 2590

ChiA (18) Cthe 0270

CelT (9) Cthe 2812

CelB (5) Cthe 0536

CelU (9) Cthe 2360

- (PL11) Cthe 0246

- (28) Cthe 2038

- (9) Cthe 2761

CelN (9) Cthe 0043

- (5) Cthe 2193

CelL (5) Cthe 0405

CelW (9) Cthe 0745

- (53) Cthe 1400

- (UN) Cthe 3132

CseP (UN) Cthe 0044

The glycosyl hydrolase family is indicated in the first column

in brackets; in one case this is a pectate lyase (PL); unknown

module (UN); no number in brackets is indicating a protein

without depolymerizing function (or a putative functional

protein). The presence of a protein in the protein identification

of culture supernatants is indicated by a black box. The

proteins are sorted according to their frequency of detection.ABBREVIATIONS: C, cellulose; Cb, cellobiose; X + C, xylane +

cellulose; G, barley -glucan; C2D, cellulose experiment 2

(proteins identified with 2D-gel electrophoresis).

incubated in GS2 medium.31After 5 days incubation at

60C, the residual insoluble fibers were washed, dried,

and weighed. Although no drastic volume degradation

was apparent in the medium (the sedimented material

at the bottom of the example bottles in F IG. 4), the

reduction in dry weight was up to 80% compared tothe control with no bacteria in it. A mixture of the

gases CO2 and H2 was formed and cell growth was

observed (turbidity of the medium in FIG. 4); further-

more, short-chain fatty acids (acetic acid), lactate, and

ethanol were formed (data not shown; Hiegl, personal

communication). This result shows that many C. ther-

mocellum strains are able to degrade native plant fibers

well and to a considerable degree.

Differential Expression of CellulosomeComponents

C. thermocellum is usually cultivated on cellobiose as a

carbon source. Cellulosomes are produced well under

these conditions, although they are not necessary for

substrate degradation; cellobiose is a good substrate

for cellulosome expression.44 The protein complexes

can be isolated from the culture supernatant relatively

easily.32 Although it is difficult to detach cellulosomes

in natural form from insoluble cellulose particles, cel-

lulosomes can be isolated in a sufficient amount and

purity if such substrates are digested to completion.

Otherwise, the cellulosomes would be found in the pel-

let fraction, together with the substrate particles and

the cells.

Cellulosome preparations were obtained from

C. thermocellumATCC27405 (type strain) grown on four

different substrates, crystalline cellulose MN300, cel-

lobiose, xylan plus cellulose, and barley -1,3-1,4-D-

glucane.32 Growth on xylan alone was too poor to ob-

tain a sufficient amount of cellulosomes. Soluble bar-

ley -glucan was a good but expensive substrate for

C. thermocellum. The isolated cellulosomes were ana-

lyzed by ICPL peptide mapping and N terminal se-

quencing (J. Kellermann, personal communication).

The C2D fraction of cellulose-grown cellulosomes was

analyzed by MALDI-TOF-TOF from 2D-gel elec-

trophoresis.29

Combining the five preparations investigated with

two different methods, 32 different proteins were iden-

tified in the cellulosomes (TABLE 1):

4 potentially structured, noncatalytic proteins, in-cluding CipA,

19 -glucanases, including 1 each of GHF8 and

GHF48, 4 of GHF5, and 13 of GHF9, 6 xylanases, 7 proteins from not yet cloned genes and not yet

studied enzymes.

All proteins (except CipA) contain a module coding

for a dockerin type I, indicating unambiguously their

localization on the cohesin modules of CipA. This is

corroborated by the fact that they were identified inthe cellulosome complex. Contrarily, all proteins iden-

tified in the cellulosome are containing dockerin mod-

ules, which emphasizes that the major and maybe the


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FIGURE 4. Ground maize silage incubated with C. thermocellum. Anaerobic bottles are filled with10% (w/v dry mass) in GS2 medium and stoppered under anaerobic conditions. The left bottle withoutinoculation (negative control), the right bottle five days after inoculation with C. thermocellum ATCC27405. Incubation was at 65C. (Photograph provided by W. Hiegl.)

only principle for integration in the cellulosome is the

dockerin-cohesin interaction. Even putative structural

proteins (besides CipA itself) are bound by dockerin-

cohesin (type I) interactions. The dockerin module of

CipA is of type II and responsible for binding the com-

plex to the cell surface or to multicomplex integrating

proteins, such as OlpB.

Interestingly, almost 50% of the genes (32/72) con-

taining a dockerin module (Ref. 29 and TABLE 3) are

actually expressed, secreted, and assembled as com-

ponents of the cellulosome. Some components may

be minor proteins in the cellulosome and have not

been detected yet. The peptides, which were most fre-

quently picked up, belonged to cellulases of GHF9, a

hydrolase family containing nonprogressive as well as

progressive -glucanases. GHF5, GHF8, and GHF48

cellulases appear to be less represented. However, given

the uncertainty of quantification by the method, this

cannot be stated with certainty. Nevertheless, the num-

ber of peptide counts coincides with the density of the

corresponding spots in 2D-gel electrophoresis.

The lower part of the list in TABLE 3 represents the

proteins that presumably occur less frequently in cel-

lulosomes; they were not identified in each prepara-

tion. No conclusive picture concerning the substrate-

depending regulation can be drawn, such as the up-

regulation of xylanase genes when xylan is used as a

substrate. This ambiguous result may be due to the

presence of cellulose in the xylan or the inoculum

added to the culture medium. The question of differ-

ential expression of cellulosomal components cannot

be answered conclusively with the data currently avail-

able. Influence on the expression of cellulase genes by

the conditions of the culture were shown earlier, but

with mRNA and not with protein.6,33,34

Knockout Mutants of the Clostridiumthermocellum cipA Gene

For identification of the components necessary for

crystalline cellulose hydrolysis, mutants with reduced

ability to hydrolyze crystalline cellulose were isolated.

They were created by mutagenesis with ethyl-methane-

sulfonate (EMS) treatment of growing cultures of

C. thermocellum. Mutagenized cells were plated in tur-

bid cellulose plates and screened for colonies without a

clear halo around them due to reduced cellulose degra-

dation. Analysis of the cellulosomal proteins should

eventually reveal the components missing in mutants

reduced in cellulase activity.

Six almost completely cellulose-defective mutants

were purified by single-colony isolation and subjected

to SDS-PAGE of their culture supernatant proteins.

The protein pattern was almost identical to that of

cellulosomal proteins, but the most prominent band

of cellulosomes, the scaffoldin band (CipA), was miss-

ing.35 Size exclusion chromatography indicated that


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the mutants were defective in the formation of the

cellulosome complexes that were absent in cleared cul-

ture supernatants. Sequencing of the amplified CipA

region revealed the insertion of a new insertion el-

ement, called IS1447 (gene bank account numbers

AM491039 to AM491042), into the N terminal region

of the cipA gene, with opposite transcription direction.

This should lead to reading-frame interruption, and

thus to seriously shortened CipA fragments missing,

for example, all cohesin modules (as in mutant SM1;

FIG. 3). The location of the insertions of four mutants

is indicated in FIGURE 3.

The lack of a functional scaffoldin has the conse-

quence that the C. thermocellum culture supernatant

contains all cellulosomal proteins except CipA. The

components of the cellulosome are not integrated in a

large protein complex. They are not bound to the cellwall because the cell wall binding of the complex is also

mediated by the now missing CipA. This particle-free

mixture of cellulosomal components will be used for

follow-up experiments that will reveal the mechanism

of the assembly of the cellulosome and the nature of

the cellulase synergism in the complex.

Preliminary experiments have shown that CipA mu-

tants show similar activity compared to the wild type

on soluble -glucans, such as carboxymethylcellulose

(CMC) or barley -glucan. However they have lost

most of their activity (up to 90%) on crystalline cel-lulose.35 It has always been a paradigm of cellulase

research that the complex formation, the combination

of several cellulases in the cellulosome, is responsible

for the outstanding effectiveness of the true cellulase

activity in C. thermocellum and other anaerobic bacteria.

The generally lower specific activity of cellulase sys-

tems produced by other bacteria that do not form a

cellulosome complex, such as C. stercorarium25 or most

aerobic cellulolytic bacteria can perhaps be explained

by the findings on the key role of CipA in maintaining

the cellulosome.These resultstogether with the genomic DNA

sequence completed recently, the possibility for

metabolic engineering through gene transfer,36 the

remarkable cellulase system, and the earlier reports

on cocultures of C. thermocellum with other ther-

mophilic clostridia for ethanol production directly

from biomass37 open chances to make use of im-

proved C. thermocellum strains for the production of

chemicals and biofuels in future white biotechnology

and biorefinery applications. C. thermocellum with its two

independent cellulase systems, the soluble and the in-soluble (cellulosomal), is a good candidate for further

developments in novel cellulases for industrial applica-

tions. C. stercorarium may have an important role for the

production of new hemicellulases for the saccharifica-

tion of plant biomass.

Acknowledgment

This work was supported by a grant from the

Deutsche Forschungsgemeinschaft DFG. Thanks to J.Kellermann for providing the MALDI-TOF results,

and M. Klupp for preparing the Clostridium thermocel-

lum cultures on different substrates. Experimental re-

sults from E. Berger, S. Freiding, W. Hiegl, J. Krauss,

and M. Hosl (all Inst. Microbiology, TUM) were used

for preparation of this manuscript.

Conflict of Interest

The authors declare no conflicts of interest.

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