Biofechnol. hog. 1992, 8, 78-80
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NOTES Production of Catechol by a Bacillus stearothermophilus Transpositional Mutant M. R. Natarajan and P. Oriel* Department of Microbiology, Michigan State University, East Lansing, Michigan 48824
The phenol-utilizing thermophile Bacillus stearothermophilus BR219 was mutagenized with transposon Tn916 introduced on a suicide plasmid using electroporation. The resulting mutant BR321 carried Tn916 integrated in the resident plasmid pGGOl and accumulated catechol throughout the growth phase.
Introduction Since enzymes possessing thermostability tend to be more resistant to chemical denaturation than their thermolabile counterparts (see Tombs, 19851, thermophilic microorganisms provide interesting opportunities for development of specialty chemical bioprocesses utilizing substrates or products which are toxic to the producing organism. We have isolated a phenol-tolerant Bacillus stearothermophilus strain designated BR219 which is capable of growth at 70 "C and phenol degradation at an optimal concentration of 10mM, a level significantlyhigher than that reported for most mesophiles (Gurujeyalakshmi and Oriel, 1989). Addition of tetracycline to stationaryphase BR219 cells inhibits catechol 2,3-dioxygenasein the plasmid-encoded phenol meta pathway of this organism, resulting in accumulation of catechol, a specialty chemical of value in photographic and other applications (Gurujeyalakshmi and Oriel, 1989b). In order to eliminate the requirement for tetracycline addition and permit catechol production during growth, it was desirable to improve the process by disabling the catechol 2,3-dioxygenasegene. This report describes the use of broad host-range conjugating transposon Tn916 for this purpose.
Materials and Methods Organisms and Growth Conditions. The isolation and growth characteristics of Bacillus stearothermophilus BR219 have been described (Gurujeyalakshmi and Oriel, 1989). DP medium is a minimal salts medium supplemented with 0.2 g of yeast extract and 0.1 g of casamino acids per liter (Buswell, 1974). Phenol was utilized with this medium at a concentration of 2.5 mM. LB broth contains, per liter, 5 g of NaC1,5 g of yeast extract, and 10 g of tryptone, at pH 7.4. Catechol inhibition was determined utilizing viable counts following24 h of growth in DP medium containing varied amounts of catechol. Growth and catechol production experiments utilized 50mL cultures shaken in 250-mL flasks at 55 "C. Plasmidsand Plasmid Isolation. pAM12O is Escherichia coli plasmid pGLlOl containing transposon Tn916 as an insert (Gawron-Burke and Clewell, 1984). Plasmid pGGOl is a low copy number plasmid of BR219 bearing
* Corresponding author.
the phenol degradation pathway (Gurujeyalakshmi and Oriel, 198913). Plasmid DNA was isolated from exponential cultures in LB broth using the procedure of Birnboim and Doly (1979). Phenol and Catechol Determination. Phenol and catechol were determined colorimetrically using methods previously described (Gurujeyalakshmi and Oriel, 1989). Electroporation. B. stearothermophilus BR219 was transformed using a modification of the electroporation method of Brigidi et al. (1990). One milliliter of an overnight culture of BR219 in LB browth was inoculated into 50 mL of LB broth and incubated at 55 "Cwith shaking to an optical density of 0.8 at 540 nm. Cells were chilled on ice for 1 h, washed in 1 mM pH 7.0 HEPES buffer (N-(2-hydroxyethyl)piperazine-N-ethanesulfonic acid), and resuspended in 0.1 volume of electroporation buffer. A cell suspension of 0.8 mL was combined with 20 pL of plasmid DNA (0.5 pg) and incubated on ice for 10 min. The mixture was transferred to a 0.4-cm cuvette for the Bio-Rad Gene Pulser and electroporated 3000 V/cm at 25 pF capacitance. Immediately following electroporation, the mixture was diluted 5-fold with LB buffer, incubated at 55 "C with shaking for 2 h, and plated on LB agar containing 5 pg/mL tetracycline at 55 "C.
Results Development of Transpositional Mutant BR321. Since we were able to find no reports of successful electroporation of B. stearothermophilus, buffer and electric field conditions were varied to determine optimal conditions for transformation of transposon-bearing plasmid pAM12O. Since pAM12O cannot replicate in this host, tetracycline-resistant transformants arise by combined transformation and transposition events. The highest rate of Tn916 transfer observed was 4 X lo3 colony-forming unitslpg of DNA using PEB buffer (0.25 M sucrose, 1m M MgClz, and 7 mM potassium phosphate, pH 7.4), 3000 V/cm field strength, and 25 pF capacitance. Following patching of BR219 transformants to DP plates containing 5 mM phenol and incubation at 55 "C, infrequent colonies were observed with brown coloration arising from catechol oxidation products. These mutants were stable on repeated restreaking on DP/phenol plates in the absence of tetracycline. One transformant designated BR321, carrying a plasmid designated pN0102, was selected for further study.
8756-7938/92/3008-0078$03.00/0 0 1992 American Chemical Society and American Institute of Chemical Engineers
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Figure 1. Restriction endonuclease digestion profiles of plasmid DNA pGG01 and pON2. The DNAs were digested with selected restriction enzymes and electrophoresed on a 0.8% agarose gel and strained with EtBr. Plasmid pGGOl (lanes 2-4): lane 2, BamHI; lane 3, RamHI plus XhoI; lane 4, BamHI plus SalI. Plasmid pON2 (lanes 5-7): lane 5, RamHI; lane 6, RamHI plus XhoI; lane 7, RamHI plus SalI. Lane 1 contains Hind111 fragments of X DNA.
Plasmid Characterization of BR321. Comparison of restriction enzyme digestions of plasmid pN0102 from the BR321 transformant with those of pGGOl from the parental organism BR219 indicated an increase in size of 17 kb (Figure 1)and hybridization with the tetM gene of Tn916, which was not observed with pGGOl (data not shown), indicating the presence of one copy of 17.6-kb Tn916 in pN0102. Addition of 0.1 76 catechol to BR321 colonies grown in DP medium with 2.5 mM phenol did not show the characteristic yellow color of 2-hydroxymuconic semialdehyde observed with BR219, suggesting that the transposon integration site was within the catechol 2,3dioxygenase gene on the plasmid. Catechol Production by BR321. Mutant 321 was tested for catechol production in DP medium containing 2.5 mM phenol. As seen in Figure 2, catechol production by BR321 was initiated soon after growth and reached a peak at approximately 20 h. Lack of exact correspondence between phenol utilization and catechol accumulation is attributed to some spontaneous catechol oxidation evidenced by increasing brown color in the BR321 cultures with time (see Gurujeyalakshmi and Oriel, 198913). No catechol accumulation or broth coloration was observed in parallel experiments with the wild-type BR219. Viable cells for both BR219 and BR321 increased up to 8 h of incubation, then decreased, with BR321 showing a greater subsequent decrease than BR219 after 8 h (Figure 3). Catechol Toxicity. In order to determine whether the enhanced loss in BR321 viability compared to that of BR219 was due to toxicity of catechol rather than catechol oxidation products, the toxicity of exogenous catechol on BR219 and BR321 was determined. As seen in
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Figure 2. Phenol utilization and catechol production by Bacillus stearothermophilus BR219 (panel A) and BR321 (panel B). (A) 0. phenol utilization: catechol production. (B) 0. Dhenol utihzation; A, catecholA, production..
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Figure 3. Cell viability of Bacillus stearothermophilus BR219 (0) and BR321 ( 0 )during growth in DP medium with 2.5 mM phenol.
Figure 4, both organisms demonstrated similar catechol toxicity a t levels above 0.1 mM. Since the decline in BR321 viability ensued soon after the extracellular production level exceededthis amount, we attributed the loss of BR321 viability to catechol toxicity.
Discussion
Thisreport demonstrates successfultranspositional mutagenesis of B. steurothermophilus BR321 using electroporation for creation of stable catechol-accumulating mutants. To our knowledge, this is the first successful electroporation and transposon mutagenesis of this thermophile. Although plasmid genetic mapping studies are required for verification, the lack of catechol conversion when added exogenously, the endogenous catechol accumulation, and the plasmid location of the integrated transposon suggest that the Tn916 insertion occurred in the catechol 2,3-dioxygenase gene of the plasmid-encoded phenol pathway. The BR321 mutant demonstrated cat-
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State University Center for Microbial Ecology. We appreciate the assistance of Dr. G. Gurujeyalakshmi and Dr. Trevor D'Souza.
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Literature Cited
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Birnboim, H. C.; Doly, J. A rapid alkaline extraction procedure for screening of recombinant plasmid DNA. Nucleic Acids Res. 1979, 7, 1513-1523. Brigidi, P.; De Rossi, E.; Bertarini, M. L.; Riccardi, G.; Mat-
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Figure 4. Effect of exogenous catechol on growth of Bacillus stearothermophilus BR219 (0) and BR321 ( 0 ) .
echolproduction to levels of 0.75 mM without requirement for tetracycline addition and stationary-phase cells, providing an improved approach for thermophilic bioproduction of this specialty chemical. Although BR219 is stable to phenol at concentrations to 10 mM, significant loss of BR321 viability was observed during catechol production, which was attributed to toxicity by catechol. Attempts to further improve the process by continuous removal of catechol during bioproduction to reduce problems of spontaneous oxidation and product toxicity are underway.
Acknowledgment We gratefully acknowledge support by the State of Michigan Research Excellence fund and the Michigan
--- ---. Buswell, J. A. Metabolism of phenols and cresols by Bacillus stearothermophilus. J. Bacteriol. 1974, 124, 1077-1083. Gawron-Burke, C.; Clewell, D. B. Regeneration of insertionally inactivated streptococcalDNA fragment after excisionof transposon Tn916 in Escherichia coli. J.Bacteriol. 1984,159,214221. Gurujeyalakshmi, G.; Oriel, P. Isolation of a phenol-degrading Bacillus stearothermophilus and partial characterization of the phenol hydroxylase. Appl. Environ. Microbiol. 1989a,55, 500-502. Gurujeyalakshmi, G.; Oriel, P. A thermophilic process for catechol production from phenol. Biotechnol. Lett. 1989b, 11, 689-694. Tombs, M. P. Stability of proteins. J. Appl. Biochem. 1985,7, 3-24. Accepted October 14, 1991. Registry No. Phenol, 108-95-2; catechol, 120-80-9.
