ELIZABETH E. M. BATES,1 HARRY J. GILBERT,l* GEOFFREY P. HAZLEWOOD,2 JAMES HUCKLE,1. JUDITH I. LAURIE,) AND STEPHEN P. MANN2.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1989. p. 2095-2097 0099-2240/89/082095-03$02.00/0 Copyright (C 1989, American Society for Microbiology
Vol. 55. No. 8
Expression of a Clostridium thermocellum Endoglucanase Gene in Lactobacillus plantarum ELIZABETH E. M. BATES,1 HARRY J. GILBERT,l* GEOFFREY P. HAZLEWOOD,2 JAMES HUCKLE,1 JUDITH I. LAURIE,) AND STEPHEN P. MANN2 Department of Agriclultuiral Biochemistry a(id Nutr^ition, Univ,ersity of New castle lupon Tyne, Neiv'castle ulpon Tyvne, NE] 7RU,1 and Department of Biochemistry, AFRC Institiute of Animal Physiology and Genetics Research,
Babralham, Cambridge, CB2 4AT,- Englanid Received 7 February 1989/Accepted 8 May 1989
Recombinant plasmid pM25 containing the celE gene of Clostridium thermocellum, which codes for an enzymatically active endoglucanase, was transformed into Lactobacillus plantarum by electroporation. Strains harboring pM25 expressed thermostable endoglucanase, which was found predominantly in the culture medium. Two other plasmids, pGK12 and pSA3, were transformed into L. plantarum, and the stability of each plasmid was evaluated.
Lactobacilluts plantaruim is the primary bacterium used in silage fermentation, whereby summer-grown forage crops are preserved for the winter feeding of ruminants. Variations in the sugar and dry-matter content of herbage are two important factors which affect the quality of silage by influencing the rate of fermentation. It has been customary to add soluble carbohydrate (molasses) to silage to facilitate rapid fermentation. However, the advent of recombinant DNA methodologies has presented an alternative means of increasing the availability of fermentable sugars in ensiled herbage. By using such techniques, it is envisaged that heterologous genes encoding polysaccharide-degrading enzymes could be inserted into, and expressed by, L. plantaruim to produce strains which could degrade plant structural polysaccharides and would therefore be more effective under conditions in which endogenous mono- and disaccharide concentrations are low. Electroporation has been successfully used to transform lactobacilli with plasmid vectors which replicate in several gram-positive bacteria, but there have been no reports of the expression of heterologous genes, apart from antibiotic resistance genes, in Lactobacilllas spp. (1, 10). In this paper, we describe the transformation of L. plantaruim with three plasmids, one of which contains the celE gene from Clostridium thermocellutm, and demonstrate the expression of the encoded endoglucanase. This enzyme is particularly well suited to L. plantarwen since it remains active over a broad pH range (4.5 to 8). Escherichia coli strains used in this study were grown in Luria broth (9); L. plantarlum was cultured, unless otherwise stated, in MRS medium (5). Anaerobic conditions were maintained by using the GasPak system (BBL Microbiology Systems). Media were solidified by the addition of agar to a final concentration of 2% (wt/vol). For measuring endoglucanase activity in liquid cultures, L. plantaruim was grown in a medium containing yeast extract, tryptone, and Casamino Acids (all at 1%, wt/vol) and sodium chloride (1.4%, wt/vol); the pH was adjusted to 5.5 with lactic acid, and glucose was added to a final concentration of 0.2%. Antibiotics, when required, were added to E. coli cultures at the following concentrations: chloramphenicol, 10 ,ug/ml; erythromycin, 100 p.g/ml; and tetracycline, 12.5 [ig/ml. L. planitarium was grown in the presence of S pg of chloramphenicol and 10 pLg *
of erythromycin per ml. Plasmids were isolated from E. coli by the Brij lysis method and purified by cesium chloride density gradient centrifugation (2). For rapid screening, plasmids were isolated from E. coli by the method of Holmes and Quigley (7) and from L. plantaruim by the method of West and Warner (14). The recombinant plasmid pRV1.3 consists of a 3.5-kilobase Sau3A restriction fragment of C. thermnocellutm genomic DNA cloned into the BamHI site of pBR322 (6, 12). The clostridial insert contains the celE gene which directs the synthesis of a thermostable endoglucanase designated EGE (6). An EcoRI fragment from pRV1.3 coding for an enzymatically active endoglucanase has been ligated into pSA3, a shuttle vector which replicates in E. coli and Streptococcuis sp. (4), to produce the recombinant plasmid pM25 (11). The third plasmid used in this study, pGK12, is a cloning vector for lactic streptococci which replicates and is stably maintained in Bacillus suibtilis and E. coli (8). Plasmids pGK12, pSA3, and pM25 were transformed into L. plantaruiim NCDO1193 by using a modification of the protocol described by Luchansky et al. (10). Cells of L. planitaruiim were grown in MRS broth to a density of 2 x 108 to 5 x 108 bacteria per ml (A660, 0.66). Cells from 50 ml of culture were recovered by centrifugation, washed twice in lx PEB buffer (7 mM potassium phosphate [pH 7.4] containing 1 mM MgCl2 and 272 mM sucrose), and suspended in 2.5 ml of 2.5x PEB. Plasmid DNA (1 p.g) was added to 0.8 ml of cells on ice, and electroporation was carried out at 6.25 kV/cm with a capacitance of 25 V1F and a time constant of 3.5 to 4.0 ms. The gene pulser (Bio-Rad Laboratories) was used to electroporate the bacteria. Treated cells were diluted 10-fold in MRS broth and incubated for 4 h at 32°C before being plated onto MRS agar containing erythromycin. Colonies were observed after incubation for 48 h at 37°C. The transformation frequency was higher for pGK12 than for other plasmids (Table 1). To confirm that the L. planitatrutm colonies with the Emr phenotype were true transformants, DNA was extracted from 10 such colonies by the small-scale method of West and Warner (14). Total nucleic acid was then transformed into E. coli LE392 (3), and recombinants were selected on media containing either chloramphenicol (pGK12) or tetracycline (pSA3 and pM25). Plasmids were isolated from the resultant Tc'- and Cmr E. coli colonies, and their structures were
Corresponding author. 2095
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APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 1. Frequency of transformation into L. plantrianr Plasmid"
DNA (CFU/,ug)
pGK12 ... pSA3 ... pM25 .
102 to 5 x 10' 4 x 10i to 7.4 X 102 3 x 101 to6 x 102 2 X
' Erythromycin was used to select all transformants.
determined by restriction mapping. Without exception, restriction analysis showed that the plasmid extracted from L. plantarum transformants was the same as the parent plasmid used for the particular experiment and had not undergone any detectable structural modification. To determine whether recombinant strains of L. plantarium harboring pSA3 or pM25 expressed endoglucanase activity, the bacteria were cultured on plates containing fastidious anaerobe medium supplemented with medium viscosity carboxymethyl cellulose (CMC) 0.2%, wt/vol). After 72 h of incubation at 30°C, cells were washed from each plate under running water, and the plates were stained for carboxymethyl cellulase (CMCase) activity by using Congo red (13). Wild-type strain 1193 and L. plantaarimin transformed with pSA3 were devoid of CMCase activity (Fig. 1), but L. plantarum transformed with pM25 (recombinant pSA3 containing functional celE) hydrolyzed CMC in the area below and immediately surrounding bacterial colonies (Fig. 1). Whole-cell homogenate, prepared from L. plantarum harboring pM25 by treating log-phase cells (4.5 h) in a Mickle shaker, was assayed for CMCase by using the reducing sugar assay described previously (12). Endoglucanase activity was detected (specific activity, 1.4 U/mg of protein, equivalent to 0.125 CMCase units per ml). CMCase activity remaining in the culture medium after the removal of cells was an order of magnitude higher (1.996 CMCase units per ml), indicating that 94% of the endoglucanase activity encoded by the celE gene had been secreted by L. planitarum. Similar fractions obtained from a liquid culture of untransformed L. plantarum 1193 contained no detectable CMCase activity. To investigate plasmid stability, L. plantarlum strains harboring pGK12, pSA3, and pM25 were grown in MRS medium without antibiotics and maintained in mid-log phase throughout 50 generations by serial dilution. At appropriate times, bacteria were plated onto MRS medium and MRS medium containing erythromycin to determine the rate of
a
b
FIG. 1. L. plantarum 1193 (a) and L. plantaruin 1193 transformed with pM25 (b) were streaked out on fastidious anaerobe medium supplemented with CMC (0.2%, wt/vol). Erythromycin (5 ,ug/ml) was added to plate b. After 72 h at 30°C, bacterial growth was removed by washing, and CMC hydrolysis was detected by Congo red staining (11).
a
O.-0 a 1.4
E
ui 1-
0
(9 0 -i
1.1
0
10
30 20 Bacterial generations
40
50
FIG. 2. Stability of plasmids harbored by L. plantaruirm under nonselective growth conditions. L. plantarurn containing pGK12 (A), pSA3 (U), or pM25 (@) was grown in MRS in the absence of antibiotics. The segregation of the plasmids was determined by the loss of the Emr phenotype.
plasmid loss. The results show that pGK12 was very stable, with 0.2% loss of plasmid per generation (Fig. 2); all Em' colonies had retained the Cmr phenotype, and Ems strains were also Cms. Both pM25 and pSA3 were much less stable and segregated at approximately the same rate (pSA3 and pM25 were lost at rates of 5.0 and 5.2% per generation, respectively). Our results confirm that plasmids pGK12 and pSA3 can be transformed into L. plantarum by electroporation, albeit at low frequency, and that they express antibiotic resistance and are stably maintained in the presence of selective pressure. Furthermore, we have shown that pSA3 is a suitable vehicle for the introduction of heterologous genes encoding polysaccharide-degrading enzymes. C. thermocellum endoglucanase was produced by L. plantarum harboring celE at about the same level it was produced by E. coli (6, 12), and 94% of the enzyme was extracellular, suggesting that the endoglucanase signal peptide is efficiently recognized by the protein transport machinery of L. plantar-iim. This provides further evidence for the universal nature of signal peptide recognition by gram-positive bacteria. Previous work suggests that promoters cloned with C. thermocellum eel genes function in a number of hosts (11). Furthermore, the Shine-Dalgarno sequence of celE was shown to be similar to consensus ribosome-binding sequences of other gram-positive genes (6), so there appears to be no obvious barrier to transcription or translation of celE-derived mRNA by L. plantariun. Although there is currently little information on the structure of Lactobaccilllis promoters, it is likely that improved expression of the clostridial endoglucanase by L. platntaruiim would result from the use of an endogenous promoter in preference to the C. therinocellum promoter cloned with celE. Commercial utilization of genetically engineered L. plantari-in7 during ensiling will undoubtedly preclude the use of
VOL. 55, 1989
antibiotics to maintain selective pressure, so plasmid stability in the absence of positive selection is likely to prove an important issue. Given that L. plantaii in used in silage inoculants would be expected to increase in mass 1,000-fold (10 generations) during ensilage, our stability data suggest that the replicons in either pGK12 or pSA3 could be utilized in constructing vectors for introducing heterologous genes into bacterial silage inoculants. Such plasmids should not encode antibiotic resistance genes but should contain alternative selectable markers. This work was supported in part by AFRC grant AG/13/135 awarded to H.J.G. and E.E.M.B. LITERATURE CITED 1. Chassy, B. M., and J. L. Flinckinger. 1987. Transformation of Lactobacill//s casei by electroporation. FEMS Microbiol. Lett. 44:173-177. 2. Clewell, D. B., and D. R. Helinski. 1968. Supercoiled circular DNA-protein complex in E. coli. Purification and induced conversion to an open circular form. Proc. Natl. Acad. Sci. USA 62:1159-1166. 3. Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of E. coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69:
2110-2114. 4. Dao, M. L., and J. J. Ferretti. 1985. St/-eptoc oc c is-Esc heri-(chiia (o/i shuttle vector pSA3 and its use in the cloning of streptococcal genes. Appl. Environ. Microbiol. 49:115-119. 5. De Man, J. C., M. Rogosa, and M. E. Sharpe. 1960. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23:130-135.
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6. Hall, J., G. P. Hazlewood, P. J. Barker, and H. J. Gilbert. 1988. Conserved reiterated domains in Clostr-idiuilm thcrmocellitin endoglucanases are not essential for catalytic activity. Gene
69:29-38. 7. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114: 193-197. 8. Kok, J., J. M. B. M. Van der Vossen, and G. Venema. 1984. Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus siubtilis and Eschierichia (/oli. Appl. Environ. Microbiol. 48:726-731. 9. Lennox, E. S. 1955. Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190-206. 10. Luchansky, J. B., P. M. Muriana, and T. R. Klaenhammer. 1988. Application of electroporation for transfer of plasmid DNA to Lactobac ill/s, Lactococcus, Lei(c(onostoc, Listevriai. Pediococcus. Bacillus, Staphylococcus. Enterococcuis, and Propionibacteriumn. Mol. Microbiol. 2:637-646. 11. Mann, S. P. 1988. Subcloning of ,B-glucanase genes from Rum7lino(occuS a/bius, Clostridiiin thuernocell/lmn and Bitvriviitrin fibrisolv'ens using the shuttle vector pSA3. Lett. AppI. Microbiol. 7:119-122. 12. Romaniec, M. P. M., N. G. Clarke, and G. P. Hazlewood. 1987. Molecular cloning of Clostridiu,n thermocellum DNA and the expression of further novel endo-P-1, 4-glucanase genes in Escherichia coli. J. Gen. Microbiol. 133:1297-1307. 13. Teather, R. M., and P. J. Wood. 1982. Use of Congo redpolysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43:777-780. 14. West, C. A., and P. J. Warner. 1985. Plasmid profiles and transfer of plasmid-encoded antibiotic resistance in Lactobacil/lus p/uintaruu. AppI. Environ. Microbiol. 50:1319-1321.
