Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation
- Takamitsu Arai*,†,
- Satoshi Matsuoka*,
- Hee-Yeon Cho*,‡,
- Hideaki Yukawa†,
- Masayuki Inui†,
- Sui-Lam Wong§, and
- Roy H. Doi*,¶
- *Section of Molecular and Cellular Biology, University of California, Davis, CA 95616-8535;
- †Research Institute of Innovative Technology for the Earth, Kyoto 619-0292, Japan; and
- §Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4
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Contributed by Roy H. Doi, December 7, 2006 (received for review September 25, 2006)
Abstract
The ability of two strains of bacteria to cooperate in the synthesis of an enzyme complex (a minicellulosome) was examined. Three strains of Bacillus subtilis were constructed to express Clostridium cellulovorans genes engB, xynB, and minicbpA. MiniCbpA, EngB, and XynB were synthesized and secreted into the medium by B. subtilis. When the strains with the minicbpA and engB genes or with xynB were cocultured, minicellulosomes were synthesized, consisting in one case of miniCbpA and EngB and in the second case of miniCbpA and XynB. Both minicellulosomes showed their respective enzymatic activities. We call this phenomenon “intercellular complementation.” Interesting implications concerning bacterial cooperation are suggested from these results.
Bacteria have been shown to communicate within a bacterial population by sophisticated means called “quorum sensing.” This intercellular communication is carried out by the production of small compounds that, upon reaching a certain level of concentration, signal the cells to act at times in synchrony or individually. The mechanisms for various types of behavior under quorum sensing have been reviewed recently (1).
We have been investigating the cooperativity between bacteria in a similar but different mechanistic mode. In preliminary experiments, we determined that Clostridium cellulovorans genes could be expressed by Bacillus subtilis and their protein products secreted into the medium (2). For the present study, we asked whether two B. subtilis strains could produce individual components of an enzyme complex that could then react to form an active complex in the growth medium. For this purpose, we have constructed strains of B. subtilis that could each produce one part of an enzyme complex and secrete the components into the growth medium. Could the individual components then associate in the medium to form an active enzyme complex?
As a model system for studying this phenomenon, we used the subunits of the C. cellulovorans cellulosome (3) that could bind to each other to form an active enzyme complex. The cellulosome is formed by the interaction of a scaffolding protein, CbpA, which has enzyme-binding sites called cohesins, with cellulosomal enzymes that contain cohesin-binding domains called dockerins (4). The dockerin of the C. cellulovorans cellulosomal enzymes is a 22-aa repeat usually located at the C terminus of the enzymes. The cohesin–dockerin interaction is quite stable once formed.
By coculturing two strains of B. subtilis, one expressing miniCbpA and another expressing either an endoglucanase or a xylanase, we wished to determine whether a minicellulosome was formed that had endoglucanase or xylanase activity. Indeed, active minicellulosomes were obtained, and these results are examples of intercellular complementation.
Results and Discussion
To determine whether two strains of B. subtilis could cooperate to synthesize an active enzyme complex (minicellulosome), strains of B. subtilis were constructed that contained either a C. cellulovorans gene for miniCbpA or genes for cellulosomal enzymes EngB (5), XynB (6), or derivatives of EngB and XynB, which were missing their dockerin domains (Fig. 1). The miniCbpA was a small derivative of CbpA with only two cohesins (7) instead of the nine cohesins found in the normal CbpA (8). Only cellulosomal enzymes containing dockerin domains would bind to the miniCbpA. The strategy was to test initially for expression and secretion of these proteins by B. subtilis and then to determine whether cocultivation of strains expressing miniCbpA and the cellulosomal enzymes would result in a minicellulosome containing both miniCbpA and the cellulosomal enzymes.
Molecular architecture of EngB, XynB, miniCbpA, and their derivatives. CBM, carbohydrate-binding module; Cat., catalytic domain.
Construction of Plasmids for Expression of C. cellulovorans Genes in B. subtilis WB800.
An expression vector for each gene of interest was constructed (see Materials and Methods). This vector pWB980 contained a P43 promoter (9), a sacB signal sequence (10), a multiple cloning site, and a kanamycin resistance marker from B. subtilis. The P43 promoter is a strong and constitutively expressed promoter used to direct the transcription of the C. cellulovorans genes in B. subtilis during growth. The sacB signal sequence allowed the recombinant protein to be secreted into the culture broth. The minicbpA gene that was constructed consisted of a carbohydrate-binding module, a hydrophilic domain, and two cohesin domains, cohesins 1 and 2 (8).
The C. cellulovorans genes were expressed in B. subtilis (Fig. 2), but some of their products were secreted poorly into the medium. For those genes whose products were not secreted efficiently, derivatives were constructed containing a Strep-Tactin (ST) sequence. ST is a short peptide (8 aa, WSHPQFEK), which binds with high selectivity to ST, an engineered streptavidin (11). The proteins modified by the addition of the ST sequence were expressed and secreted well (Fig. 2 B, lanes 3–5). The reason for ST increasing the secretion of proteins is not known.
Production of EngB, Xyn B, miniCbpA, and their derivatives by B. subtilis. (A) Purified proteins analyzed by SDS/PAGE and stained with Coomassie blue. Each lane contained 10 μg of protein. The numbers on the left are in kilodaltons. Lane 1, molecular weight markers; lane 2, EngB-His; lane 3, XynB-His; lane 4, ST-miniCbpA-His. (B) Secreted proteins in growth medium of B. subtilis analyzed by Western blots. Lane 1, EngB; lane 2, EngBΔdoc; lane 3, ST-XynB; lane 4, ST- XynBΔdoc; and lane 5, ST-miniCbpA-His.
The minicbpA was modified by adding an ST sequence at its N terminus for efficient secretion and a His-tag at its C terminus for binding of miniCbpA-His to a Ni-NTA agarose column for ready isolation of cellulosomes. This miniCbpA, which was expressed and secreted efficiently, was designated as ST-miniCbpA-His (Fig. 1).
Strains containing the cellulosomal enzyme genes expressed either EngB or ST-XynB (Figs. 1 and 2). If intercellular complementation occurred, EngB and ST-XynB would bind through their dockerin domains to the cohesins present in ST-miniCbpA-His to form minicellulosomes, which would bind to the Ni-NTA agarose column. Because EngB and ST-XynB did not contain a His-tag, they would not bind to the Ni-NTA agarose column. Strains expressing EngBΔdoc and ST-XynBΔdoc were used as control cultures. Because they did not have dockerins, they were not expected to bind to ST-miniCbpA-His to form cellulosomes.
When we tried to express XynB and XynBΔdoc in B. subtilis WB800, these proteins were secreted, but poorly, in the culture broth at 30°C (Fig. 2 B, lanes 1 and 2). When we placed an ST sequence at the N termini of XynB and XynBΔdoc to form ST-XynB and ST-XynBΔdoc, the cells grew well, and ST-XynB and ST-XynBΔdoc were secreted into the growth medium (Fig. 2 B, lanes 3 and 4). MiniCbpA-His also was not secreted well. After adding ST at its N terminus, ST-miniCbpA-His was secreted much better than miniCbpA-His (Fig. 2 B, lane 5).
Heterologous Expression of EngB, EngB-His, XynB-His, and ST-miniCbpA-His by Protease-Deficient B. subtilis WB800.
When recombinant B. subtilis WB800 harboring pWB980-ST-miniCbpA-His, pWB980-EngB-His, or pW980-XynB-His was grown at 37°C, most of the EngB-His and XynB-His was expressed as inclusion bodies or were degraded (data not shown). In contrast, when the cells were grown at 30°C (Fig. 3 A–C), ST-miniCbpA-His, EngB-His, and XynB-His were secreted into the culture broth. The culture supernatants of B. subtilis WB800 (pWB980-EngB-His, pWB980-ST-miniCbpA-His, and pWB980-XynB-His) collected at different time points of growth were analyzed by Western blot with anti-EngB, -XynB, and -CbpA (Fig. 3 D–F).
Growth curves and Western immunoblots of culture supernatants of B. subtilis grown at 30°C. Growth curves of cultures expressing EngB-His (A), XynB-His (B), and ST-miniCbpA-His (C). Western blot analyses of proteins from culture supernatants containing EngB-His with anti-EngB (D), XynB-His with anti-XynB (E), and ST-miniCbpA-His with anti-CbpA (E) at various times during growth.
As shown in Fig. 3 D, after 6 h, one band of EngB-His could be detected in the supernatant of the growth medium. This band appeared at the position of 48 kDa. The molecular mass of this band was in good agreement with the calculated molecular weight of the EngB-His (48,500). These results indicated that the EngB-His secreted into the culture broth was stable during the culture period until 15 h. EngB was also secreted in similar fashion as EngB-His (data not shown). With the strain expressing XynB-His, one band appeared at the position of 64 kDa (Fig. 3 E). The molecular mass of this band was in good agreement with the calculated molecular weight (64,000) of the XynB-His.
With the culture expressing ST-miniCbpA-His (Fig. 3 F), two bands appeared around the position of 64 kDa. The molecular mass of the major band was in good agreement with the calculated molecular weight (64,000) of the miniCbpA-His. A favorable time to extract proteins from the supernatant of the growth medium was at the 12th hour of growth. We were able to obtain purified EngB-His (1.3 mg/liter), XynB-His (2.2 mg/liter), and ST-miniCbpA-His (1. 1 mg/liter). We verified the production of the secreted proteins by SDS/PAGE (Fig. 2 A) and Western blots (Fig. 2 B).
Coculture of B. subtilis for the Cooperative Synthesis of Minicellulosomes.
With the demonstration that individual cellulosomal proteins could be synthesized and secreted into the growth medium by B. subtilis, we asked whether minicellulosomes could be assembled by the interaction of miniCbpA and cellulosomal enzymes by intercellular complementation. We tested this idea with two sets of experiments in which two strains of B. subtilis were cocultured, one expressing miniCbpA and the other a cellulosomal enzyme. In one set, a culture expressing ST-miniCbpA-His and another expressing EngB were cocultured for 10 h. In a second set, a culture expressing ST-miniCbpA-His and another culture expressing ST-XynB were cocultured for 10 h. The expression and secretion of EngB and ST-XynB were similar to that of EngB-His and XynB-His (Fig. 2 A). We then tested for the presence of minicellulosomes in the culture supernatants.
For both sets of experiments, the cells were separated from the supernatant fraction of the growth medium by centrifugation, and the supernatant fraction was loaded onto the Ni-NTA column. The column was washed with wash buffer, and then attached proteins were eluted with elution buffer (see Materials and Methods for details). The proteins eluted from the column were analyzed for the presence of ST-miniCbpA-His and EngB in the first set and for the presence of ST-miniCbpA and ST-XynB in the second set, because any minicellulosomes formed in these respective experimental sets would bind to the Ni-NTA column and contain these two subunits.
For the purpose of determining protein sizes and complex subunit composition, the samples were subjected to SDS/PAGE analysis and verified with Western blot analysis by using anti-CbpA, -EngB, and -XynB antisera. Also zymograms were performed to demonstrate the enzyme activities present in the minicellulosomes.
When the supernatant of the coculture containing ST-minicbpA-His and EngB was eluted from the Ni-NTA column and analyzed by SDS/PAGE, two bands (Fig. 4, lane 1) were observed with molecular masses of 65 (Fig. 4, lane 2) and 45 kDa (Fig. 4, lane 3), which are similar to the calculated molecular masses of ST-miniCbpA-His and EngB, respectively. A zymogram analysis indicated that the 45-kDa band had endoglucanase activity (Fig. 4, lane 4). Control experiments with cocultures containing ST-miniCbpA and EngBΔdoc showed they did not form a minicellulosome (data not shown).
Analysis of minicellulosomes from cocultures of B. subtilis expressing ST-mini-CbpA and EngB. Lane M, molecular mass marker. Lane 1, SDS/PAGE of Ni-NTA-bound minicellulosomes containing EngB and ST-miniCbpA-His from a coculture of B. subtilis strains containing pWB980-ST-minicbpA-his and pWB980-engB. Lane 2, Western blot analysis of minicellulosome with anti-CbpA; the strains are described in the legend of lane 1. Lane 3, Western blot analysis of minicellulosome with anti-EngB; the strains are described in the legend of lane 1. Lane 4, Zymogram analysis for endoglucanase activity of lane 3; the strains are described in the legend of lane 1.
In similar experiments, minicellulosomes were also formed between ST-miniCbpA-His and ST-XynB. A Western blot analysis of the supernatant of the coculture indicated the presence of both ST-miniCbpA-His and XynB (Fig. 5 A, lane 1). The analysis of the eluate from the Ni-NTA column by SDS/PAGE showed the presence of one band with a mass of 64 kDa (Fig. 5 B, lane 1). Because ST-miniCbpA-His and ST-XynB have approximately the same molecular mass, they cannot be distinguished by SDS/PAGE. However, by Western blot analysis with the antiserum directed against CbpA and XynB, each immunoreactive band with an apparent mass of 64 kDa was detected in the Ni-NTA-binding fraction (Fig. 5 A, lane 2). The sizes of the immunoreactive proteins were in good agreement with that of miniCbpA and XynB. The zymogram indicated that the 64-kDa protein had xylanase activity (Fig. 5 B, lane 1). Furthermore, ST-XynB alone did not bind to the Ni-NTA column (data not shown).
Analysis of cocultures ST-miniCbpA-His and ST-XynB and of ST-miniCbpA-His and ST-XynBΔDoc. (A) Western blot of minicellulosomes probed with antibodies raised against CbpA (Upper) and ST-XynB (Lower), respectively. Lane 1, supernatant of ST-XynB and ST-miniCbpA-His cultures. Lane 2, purified minicellulosomes with ST-XynB and ST-miniCbpA-His. Lane 3, supernatant of ST-XynBΔDoc and ST-miniCbpA-His cultures. Lane 4, ST-miniCbpA-His bound to Ni-NTA column; note no ST-XynBΔDoc was bound to ST-miniCbpA-His in lower frame. (B) (Upper) M, molecular mass marker. Lane 1 (Upper), SDS/PAGE of purified minicellulosome from cocultures containing ST-miniCbpA-His and ST-XynB (both of these proteins have the same molecular weight, so there is one band); zymogram (Lower) shows the presence of xylanase activity in this band. Lane 2, SDS/PAGE of ST-miniCbpA-His bound to Ni-NTA column (Upper). No ST-XynBΔDoc was bound to ST-miniCbpA-His, and therefore no xylanase zymogram was obtained (Lower).
As controls, when strains containing ST-miniCbpA-His and EngBΔdoc (data not shown) or ST-miniCbpA-His and ST-XynBΔdoc were cocultured, no cellulosomes were observed (Fig. 5 A, lane 4). However, ST-miniCbpA-His and ST-XynBΔdoc were observed in the supernatant (Fig. 5 A, lane 3).
The results presented in Figs. 4 and 5 indicate that minicellulosomes were synthesized when two strains of B. subtilis were cocultured. This interaction demonstrates that two strains can cooperate to form an active enzyme complex by intercellular complementation.
Although this demonstration depended on the two strains that were constructed to illustrate this phenomenon, it is possible such situations can exist in nature. The many unculturable strains of bacteria not only may provide small metabolites that allow cross-feeding, but they may also produce complementary proteins that form active enzymes that facilitate mutual growth on recalcitrant substrates. A further investigation of this phenomenon should reveal whether this type of complementation occurs in the microenvironment of bacteria in nature.
The current interest in developing biofuels from agricultural biomass and other cellulosic materials also suggests that a consortium of bacteria may be suitable for this purpose (12). The sequential synthesis of enzymes for degradation of biomass may also be accompanied by microorganisms that cooperatively synthesize active enzyme complexes. The construction of suitable complementary bacterial strains could also be considered in this context, and designer enzyme complexes with useful functions may be obtained by manipulating bacterial strains.
Materials and Methods
Bacterial Strains and Media.
B. subtilis WB800 (13), a strain that contains deletions of all eight extracellular protease genes, was used as host for the expression of C. cellulovorans EngB, XynB, miniCbpA, and their derivatives. Cells were cultivated in superrich kanamycin medium (25 g/liter yeast extract/15 g/liter tryptone/3 g/liter K2HPO4/10 g/liter glucose supplemented with 50 μg/ml kanamycin) (7).
Plasmid Construction for Expression of EngB, XynB, miniCbpA, and Their Derivatives by B. subtilis WB800.
The plasmid vector pWB980, which contained the P43 promoter, the engineered levansucrase signal sequence, sacB SP, and a multiple cloning site that allowed efficient expression of the genes and secretion of the gene product (10), was used to produce rEngB, rXynB, and miniCbpA, and their derivatives were constructed as follows: DNA fragments encoding each gene were amplified by PCR from the C. cellulovorans genomic DNA with LA TaqDNA polymerase (Takara Bio, Tokyo, Japan), and appropriate combinations of primers (Table 1) containing multiple cloning sites for inserting the PCR fragments into the plasmid vector were constructed.
Oligonucleotides used for cloning
After sequencing the DNA fragments for confirmation of the absence of mutations, the expression vectors pWB980-EngB, pWB980-ST-XynB, and pWB980-ST-miniCbpA and their derivatives were constructed (Table 1 and Fig. 1), and the plasmids were transferred to B. subtilis WB800. The miniCbpA construct included the cellulose-binding module, a hydrophilic domain, and two cohesin domains (cohesins 1 and 2) (7).
Purification of EngB-His, XynB-His, and miniCbpA-His Expressed by B. subtilis WB800.
B. subtilis WB800 containing pWB980 plasmids (10) was cultivated in 500 ml of superrich medium for 12 h at 30°C. The cultures expressing proteins with His-tags were centrifuged at 9,820 × g for 20 min at 4°C, and the supernatant from each culture was collected and purified with a Ni-NTA column (Qiagen, Valencia, CA). The columns were washed with 200 ml of wash buffer containing 50 mM NaH2PO4, 300 mM NaCl, and 10 mM Imidazole (pH 7.4), and the bound fraction was eluted with 15 ml of elution buffer [50 mM NaH2PO4/300 mM NaCl/250 mM Imidazole (pH 8.0)]. The eluates were concentrated to ≈200 μl with a 30,000 M r polyethersulfone centrifugation concentrator (Vivascience, Goettingen, Germany).
Expression and Purification of Cellulosomes from B. subtilis WB800.
The clones expressing EngB, XynB, and miniCbpA and their derivatives were streaked on LB kanamycin plates (10 g/liter Bacto-tryptone/5 g/liter yeast extract/10 g/liter NaCl/2% Bacto-agar, supplemented with 50 μg/ml kanamycin) and incubated overnight at 37°C.
One colony each of B. subtilis strains expressing EngB, EngBΔdoc, ST-XynB, ST-XynBΔdoc, or ST-miniCbpA-His was harvested and inoculated in 2 ml of superrich kanamycin medium (2) and grown overnight at 37°C with shaking at 250 rpm. A 1-ml sample from each overnight culture was inoculated into 250-ml flasks containing 25 ml of superrich kanamycin medium and incubated with shaking at 250 rpm at 30°C for 6 h. The ST-MiniCbpA-His culture (2.5 ml) was inoculated into four 2-liter flasks containing 500 ml of superrich kanamycin medium, and each of the ST-miniCbpA-His inoculated flasks was coinoculated with a 10-ml sample from the EngB, EngBΔdoc, ST-XynB, and ST-XynBΔdoc cultures. The 500-ml cultures were grown for 10 h in 30°C with shaking at 250 rpm.
The cultures were centrifuged at 9,820 × g for 20 min at 4°C, and the supernatant from each culture was collected and the His-tagged cellulosomes present in the supernatant were purified with a Ni-NTA column (Qiagen). The columns were washed with 200 ml of wash buffer containing 50 mM NaH2PO4, 300 mM NaCl, and 10 mM Imidazole (pH 7.4), and the bound fraction was eluted with 15 ml of elution buffer [50 mM NaH2PO4/300 mM NaCl/250 mM Imidazole (pH 8.0)]. The eluates were concentrated to ≈200 μl with a 30,000 M r polyethersulfone centrifugation concentrator (Vivascience).
For the purpose of determining protein size and subunit composition of the protein complex, the samples were subjected to SDS/PAGE analysis and verified by Western blot analysis. The antibodies used were anti-EngB, -CbpA, and -XynB produced by rabbits. The blots were probed with goat anti-rabbit IgG (H+L) AP conjugate (Bio-Rad, Hercules, CA) or with donkey anti-rabbit IgG horseradish peroxidase conjugate (Amersham Biosciences, Piscataway, NJ) and detected with the alkaline phosphatase conjugate substrate kit (Bio-Rad) or with ECL-Plus Western blotting detection system (Amersham Biosciences).
Acknowledgments
We thank Helen Chan for technical assistance and John Roth for helpful discussions and suggesting the term “intercellular complementation.” This research was supported in part by U.S. Department of Energy Grant DDF03-92ER20069 and by a RITE Institute (Kyoto, Japan) grant.
Footnotes
- ¶To whom correspondence should be addressed. E-mail: rhdoi{at}ucdavis.edu
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This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 25, 2006.
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Author contributions: R.H.D. designed research; T.A., S.M., and H.-Y.C. performed research; S.-L.W. contributed new reagents/analytic tools; T.A., H.Y., M.I., S.-L.W., and R.H.D. analyzed data; and R.H.D. wrote the paper.
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↵ ‡Present address: Dental Research Institute, University of California School of Dentistry, Los Angeles, CA 90095.
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The authors declare no conflict of interest.
- Abbreviation:
- ST,
- Strep-Tactin.
- © 2007 by The National Academy of Sciences of the USA
