mobilist - Europe PMC

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1983, P. 1163-1168

0099-2240/83/111163-06$02.00/0 Copyright C 1983, American Society for Microbiology

Vol. 46, No. 5

Expression of a Lactose Transposon (Tn951) in Zymomonas

mobilist V. C. CAREY,1 S. K. WALIA,1 AND L. 0. INGRAM' 2* Department of Microbiology and Cell Science' and Department of Immunology and Medical Microbiology,2 University of Florida, Gainesville, Florida 32611 Received 6 June 1983/Accepted 12 September 1983

The potential utility of Zymomonas mobilis as an organism for the commercial production of ethanol would be greatly enhanced by the addition of foreign genes which expand its range offermentable substrates. We tested various plasmids and mobilizing factors for their ability to act as vectors and introduce foreign genes into Z. mobilis CP4. Plasmid pGC91.14, a derivative of RP1, was found to be transferred from Escherichia coli to Z. mobilis at a higher frequency than previously reported for any other plasmids. Both tetracycline resistance and the lactose operon from this plasmid were expressed in Z. mobilis CP4. Plasmid pGC91.14 was stably maintained in Z. mobilis at 30°C but rapidly lost at 37°C. Zymomonas mobilis is a gram-negative bacterium which produces ethanol as a primary end product of fermentation (16). This organism is potentially useful for the commercial production of ethanol (20) and is reported to exhibit higher ethanol tolerance, higher thermotolerance, and more rapid fermentation than Saccharomyces, the organism commonly used for commercial ethanol production in the United States (26, 31). The carbohydrate substrate range of Z. mobilis is limited to only glucose, fructose, and sucrose (31) and does not include lactose, an abundant sugar in dairy waste (15). Genetic engineering offers one approach to increasing the substrate range of Z. mobilis. In this study, we investigated a variety of plasmids as potential vectors for the introduction of foreign genes into Z. mobilis. In addition, we introduced a lactose operon into Z. mobilis using a derivative of plasmid RP1. MATERIALS AND METHODS Strains, plasnids, and growth conditions. Table 1 summarizes the bacterial strains used in this stvdy. Z. mobilis was cultivated at 30°C in the complex medium described by Skotnicki et al. (27) with 20 g of glucose per liter. Escherichia coli was grown at 30 or 37°C in Luria broth (22) or in the medium described for Z. mobilis (27) with glucose (20 g/liter) or lactose (20 g/liter). Agar (15 g/liter) was added for solid media. 5Bromo-4-chloro-3-indolyl-p-D-galactoside (Xgal; 40 mg/liter) was added to plates to screen for P-galactosidase production (24). MacConkey agar plates con-

t Florida Agricultural Experiment Station publication no. 4763.

taining lactose (2 g/liter) and tetracycline (10 p.g/ml) were used to screen E. coli for tetracycline resistance and lactose utilization. Isopropyl-3-D-thiogalactopyranoside (IPTG) was included in some media at a final concentration of 1 mM. Strains L12 and L13 are spontaneous mutants of Z. mobilis strains CP4 and L12, respectively. These strains were isolated by spreading 108 cells on solid medium containing either rifampin (50 ,ug/ml) or sodium azide (3 mM). Conjugation experiments. Conjugation experiments were performed by both liquid and filter matings. Z. mobilis broth (27) was used for liquid matings. Donor and recipient cultures were grown separately to exponential phase and mixed with a donor to a recipient ratio of 3:1 for E. coli donors and 2:1 for Z. mobilis donors. The mixtures were then allowed to incubate without shaking for 1 to 3 h and plated on selective media. For filter matings, 0.45 F.M membrane filters (Millipore Corp., Bedford, Mass.) were used to collect cell mixtures, followed by incubation at 30°C on plates containing Z. mobilis medium. After the cells were washed from the filter, they were spread on plates containing tetracycline (10 p.g/ml) and incubated at 30°C until colonies developed. Characteristic Z. mobilis colonies were selected against a background of E. coli cells as described by Skotnicki et al. (29) and streaked for further purification. Alternatively, rifampin and azide were included in plates to eliminate the donor, E. coli, during matings with strain L13. Analysis of plasmid DNA. Bacteria were grown overnight in broth. DNA was extracted by a modification of the procedure described by Clewell and Helinski (5). Glucose was used instead of sucrose to resuspend cells after harvesting and washing. Cells were incubated at 37°C in the presence of lysozyme for 2 h. Pronase was added (100 ,ug/ml) after the addition of EDTA, and samples were incubated at 37°C for an additional 1 h. Plasmids were analyzed on 0.8% agarose gels (4 V/cm for 16 h) with Tris-borate buffer (89 mM Tris, 89 mM

1163

1164

APPL. ENVIRON. MICROBIOL.

CAREY, WALIA, AND INGRAM

Species and strain

TABLE 1. Bacterial strains and plasmids used Relevant propertiesa

Z. mobilis CP4 LI2 L13 LI4

Wild type Rpr Rpr Azr Contains pGC91.14

Reference or source

A. Ben-Bassat (17) This paper This paper This paper

E. coli

Alac X74 Smr JC3272 S. Kaplan (25); G. Cornelis (1, 7) Amr Kmr Tcr lac+ S. Kaplan (25); G. Cornelis (7) JC3272(pGC91.14) Amr Kmr Tcr K. T. Shanmugam (10) BE280(RP4) Amr Kmr Tcr Cbr his nif K. T. Shanmugam (12) JC5466(pRD1) Amr Kmr Tcr CR34(R68.45) K. T. Shanmugam (19) Kmr K. T. Shanmugam (11) HB101(pRK2013) Tcr K. T. Shanmugam (11) HB101(pRK290) Amr Tcr HB101(pBR322) D. H. Duckworth (4) ColIbr D. H. Duckworth (33) RM43(ColIb) BM21(A factor) K. B. Sharma (2) 42R327(F lac) lac+ K. B. Sharma (18) Cmr Smr Sur Tcr lac+ RG192(pJT2) J. F. Timoney (32) a Am, Ampicillin; Az, azide; Cb, carbenicillin; Cm, chloramphenicol; Km, kanamycin; Rp, rifampin; Sm, streptomycin; ColIb, colicin lb. boric acid, and 2.5 mM EDTA, pH 8.0). Where indicated, plasmid DNA was digested with HindlIl and PstI restriction enzymes according to the instructions of the manufacturer (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). DNA fragments were visualized by UV illumination after staining with ethidium bromide (0.5 jig/ml). Enzymatic assays. For ,-galactosidase assays, cells were permeabilized with chloroform-0.1% sodium dodecyl sulfate and assayed as described by Miller (24), except an extinction coefficient of 7,500 was used for o-nitrophenol. Both E. coli and Z. mobilis were grown without shaking ovemight at 30°C in the glucose broth (20 g/liter) described for Z. mobilis (27) in screwcapped tubes. In addition, E. coli cultures were grown in the medium described for Z. mobilis but containing lactose. Lactose permease activity was measured by using nonpermeabilized cells and expressed as the difference in o-nitrophenol produced in the presence of o-nitrophenyl-,3-D-galactopyranoside (ONPG) alone and ONPG with 10-fold excess methyl-p-D-thiogalactoside (TMG), a nonchromogenic competitive inhibitor of ONPG transport. Protein was measured by the method of Lowry et al. (21) after the cells were washed in buffer. Chemicals. Tetracycline, rifampin, ethidium bromide, ONPG, methyl-3-D-thiogalactoside, Xgal, and IPTG were obtained from Sigma Chemical Co., Saint Louis, Mo. Agarose and restriction enzymes were obtained from Bethesda Research Laboratories, Gaithersburg, Md. Cesium chloride was obtained from Gallard Schlesinger Chemical Manufacturing Corp., Carle Place, N.Y. RESULTS

Conjugal transfer of R plasmids into Z. mobilis CP4. Table 2 shows a list of the R plasmids and various mobilizing agents tried in conjugation experiments. Many of these were unsuc-

cessful. However, tetracycline resistance was transferred to Z. mobilis CP4 from E. coli strains BE280(RP4), JC5466(pRD1), JC3272 (pGC91.14), CR34(R68.45), and HBlOlpRK290) plus HB101(pRK2013). Plasmid pGC91.14 was transferred to Z. mobilis at the highest frequency. This plasmid is an RP1 derivative which encodes multiple antibiotic resistance and carries a lactose transposon. In liquid matings in which plasmid pGC91.14 (carried by E. coli JC3272) is used, tetracycline resistance was suc-

TABLE 2. Transfer of plasmids to Z. mobilis CP4a

Selection

E. coli donor

of marker'

Frequency of

Tcr Tcr

marker transfer' 4.4 x 10-4 6.8 x 10-4 9.6 x 10-3 8.7 x 1o-7

HB101(pBR322) + HB101(pRK2013)

Tcr

None

HB101(pRK290)

Tcr

1.2 x 10-7

BE280(RP4) JC5466(pRD1) JC3272(pGC91.14) CR34(R68.45) +

Tcr Tcr

HB101(pRK2013) Tcr HB101(pBR322) + None BM21(A& factor) Tcr ColIbr HB101(pBR322) + None RM43(ColIb) Tcr HB101(pBR322) + None 42R327(F lac) Tcr RG192(pJT2) None a Filter matings were performed for all conjugation experiments. bTc, Tetracycline; Collb, colicin lb. c Frequency of transfer is expressed per recipient

cell.

VOL. 46, 1983

cessfully transferred to Z. mobilis CP4 with a frequency of about 10-4 (2-h mating). This frequency of transfer could be further increased to 10-2 with filter disk matings (Table 2). Z. mobilis colonies were distinct and easily picked and purified against a background of E. coli donor cells on tetracycline plates (29). Matings were also performed with rifampin-resistant, azideresistant mutants of Z. mobilis (designated strain L13). Selection was made on plates containing rifampin (50 p.g/ml), tetracycline (10 jxg/ml), and sodium azide (3 mM) to allow selection for recombinants of Z. mobilis in the absence of a background of E. coli. Strains LI2 and L13 (rifampin resistant and rifampin and azide resistant, respectively) were used as markers for the positive identification of the recipient. The presence of plasmid pGC91.14 containing the lac operon in Z. mobilis (designated strain LI4) was also confirmed with X-gal plates. Bluecolored colonies were observed after 24 to 48 h of incubation, indicating the production of galactosidase. Secondary transfers of plasmid pGC91.14 from Z. mobilis L14 to E. coli JC3272 were performed in the broth described for Z. mobilis (27). Transconjugants of E. coli JC3272 were obtained which expressed both the tetracyclineresistant and lactose-positive characters at a frequency of 10-6 per recipient cell. Characterization of plasmid pGC91.14 in Z. mobiis. The plasmids in tetracycline-resistant transconjugants of Z. mobilis CP4 were examined by agarose gel electrophoresis (0.8% agarose) by a miniscreening procedure (10-ml cultures). Plasmid pGC91.14 and native plasmid CP4 (pRUT41) (30) were found to overlap on the gel and thus could not be easily distinguished. To establish the transfer of plasmid pGC91.14 into Z. mobilis CP4, plasmids were purified from strains LI4 and CP4 with cesium chloride-ethidium bromide gradients and their restriction fragments were compared (Fig. 1A). Restriction enzyme digests performed with HindIll on plasmid pGC91.14 alone resulted in the appearance of three distinct bands (Fig. 1A, lane 2) which were identical to those reported previously by Cornelis et al. (8). These bands were not observed in pRUT41 (Fig. 1A, lane 5). The plasmids isolated from Z. mobilis LJ4 (Fig. 1A, lane 3) showed a digestion pattern identical to that of a mixture of plasmids pGC91.14 and pRUT41 (lane 4) and distinct from plasmid pRUT41 alone (lane 5). Similar results were observed with restriction enzyme digests with PstI. Digests of pGC91.14 resulted in five bands (Fig. 1 B, lane 2), as reported previously (8). Z. mobilis L14 was found to have a digestion pattern identical to that of pGC91.14 mixed with plasmid pRUT41

Tn9OM IN Z. MOBILIS

1165

--t6l

-$A, .4*3

FIG. 1. Restriction enzyme digests. (A) HindIll endonuclease digestion of phage lambda (lane 1), pGC91.14 (lane 2), covalently closed circular plasmid DNA isolated from Z. mobilis L14 (lane 3), mixture of pGC91.14 and the native plasmid from Z. mobilis CP4, pRUT41 (37) (lane 4), and pRUT41 (lane 5). (B) Lane 1, HindlIl endonuclease digest of phage lambda; lane

2, PstI digest of pGC91.14; lane 3, PstI digest of covalently closed circular plasmid DNA isolated from strain L14; lane 4, PstI digest of mixture of pGC91.14 and pRUT41; lane 5, PstI digest of pRUT41.

(Fig. 1B, lane 3 and 4). The pRUT41 plasmid alone had a distinctly different pattern (Fig. 1B, lane 5). In the PstI digests of pRUT41, the large band which is always observed at the top of the gel may represent a second plasmid which lacks PstI sites or, more likely, a large fragment of plasmid pRUT41. We also examined the stability of plasmid pGC91.14 in Z. mobilis L14 at both 30°C (the optimum growth temperature for Z. mobilis) and 37°C. Serial transfers were made every 48 h, and samples were diluted and plated onto nonselective medium at various times. The presence of plasmid pGC91.14 was determined by picking colonies onto plates containing Xgal and plates containing tetracycline. Both markers were stable over an 8-day period at 30°C in the absence of tetracycline but were coordinately lost during growth at 37°C. Expression of (-galactosidase and lac permease genes in Z. mobilis L14. The P-galactosidase activity of both Z. mobilis LI4 and E. coli JC3272 (containing pGC91.14) grown in the glucose medium described for Z. mobilis (27) were similar in the absence of inducer (Table 3). During growth in the presence of IPTG as an inducer, P-galactosidase activity increased 3-

1166

CAREY, WALIA, AND INGRAM

TABLE 3. P-Galactosidase and lactose permease activities ,8Lactose Straina Galactosidase permease ,s/pd

activity'

Z. mobilis L14 No inducer Plus inducer

10.53 (1.79) 35.80 (3.68)

activityc

0.48 (0.09) 21.9 2.03 (0.28) 17.6

E. coli JC3272 (pGC91.14) No inducer 10.14 (1.06) 0.51 (0.09) 19.9 Plus inducer 345.60 (9.30) 18.88 (7.34) 18.3 Lactose mediume 439.90 (9.05) Z. mobilis CP4 No inducer 0 0 Plus inducer 0 0 a Both Z. mobilis and E. coli were grown in the glucose (20 g/liter) broth described for Z. mobilis (27) at 30°C in screw-capped tubes without agitation. IPTG as inducer was used at a final concentration of 1 mM. b P-Galactosidase activity and lactose permease activity were computed as nanomoles of ONPG cleaved per milligram of protein per minute at 30°C in permeabilized cells. The induction (induced/uninduced) ratio was 3.4 for Z. mobilis L14 and 34.1 for E. coli JC3272(pGC91.14). Numbers in parentheses represent standard deviations. c The induction (induced/uninduced) ratio was 4.2 for Z. mobilis L14 and 37.0 for E. coli JC3272 (pGC91.14). Numbers in parentheses represent standard deviations. d f/P, Ratio of P-galactosidase activity to lactose permease activity. e Lactose was substituted for glucose, without ONPG.

fold in Z. mobilis and 34-fold in E. coli. E. coli cells grown in the medium described for Z. mobilis with lactose (instead of glucose) exhibited even higher levels of activity. The lactose permease activity in Z. mobilis was also similar to that of E. coli in uninduced cells (Table 3). In the presence of IPTG, a 4-fold increase in permease activity was observed in Z. mobilis and a 37-fold increase was observed in E. coli. Effects of lactose and galactose on growth of Z. mobilis L14. No growth was observed with Z. mobilis L14 in medium in which lactose or galactose was used to replace glucose. Both lactose (13) and galactose (14, 23) have been reported to be toxic to bacterial growth under some conditions. However, the growth of Z. mobilis strains CP4 and L14 was not inhibited in media containing glucose (2 g/liter) with added lactose (20 g/liter) or galactose (20 g/liter). DISCUSSION The potential utility of Z. mobilis as an alcohol-producing microorganism can be improved

APPL. ENVIRON. MICROBIOL.

through the introduction of genes to expand its substrate range. We screened a variety of plasmids for their ability to be conjugally transferred into Z. mobilis by E. coli and serve as suitable vectors. The RP1 derivative, pGC91.14, was more readily transferred into Z. mobilis than any plasmid previously reported (9, 28, 29). This plasmid was stably maintained in Z. mobilis CP4 at 30°C in the absence of selective pressure from antibiotics and carried a functional lactose operon. This lactose operon was expressed in Z. mobilis with the coordinate synthesis of the Pgalactosidase enzyme and a functional lactose permease. Based upon these studies, plasmid RP1 appears to be a useful vector to introduce foreign genes into Z. mobilis. Although the lactose genes were carried on a transposon integrated into RP1 in this study, the single EcoRI cleavage site of the parent plasmid (8) may also be useful for the genetic engineering of Z. mobilis. Small plasmids such as pBR322 and pRK290 have been widely used as cloning vectors for the isolation of specific genes but lack the tra genes necessary for conjugal transfer (4). Most frequently, these have been introduced into cells via transformation. However, no suitable transformation system has been developed for Z. mobilis. Plasmids ColIb, pRK2013, and F lac and A factor have been used previously to mobilize the conjugal transfer of plasmids lacking tra genes in triparental matings with other organisms but were unsuccessful in introducing pBR322 into Z. mobilis. The transfer of pRK290 into Z. mobilis was facilitated by pRK2013, but at a very low frequency. The carbohydrate substrate range of Z. mobilis is limited to glucose, fructose, and sucrose (31) and does not include lactose, an abundant sugar in dairy waste and potential feedstock for commercial alcohol production. Our insertion of a functional lactose operon into Z. mobilis provides a first step towards expanding the substrate range of this organism. The levels of Pgalactosidase and lactose permease expressed in Z. mobilis were equivalent to that of E. coli during growth under glucose-repressed conditions (Table 3). The addition of IPTG as an inducer resulted in a fourfold increase in expression in Z. mobilis, indicating that the lactose operon was functional and responsive. Although this induction ratio is similar to that observed with foreign lactose operons inserted into Proteus mirabilis, Pseudomonas aeruginosa, and Pseudomonas putida (3), it is considerably lower than that observed in E. coli (Table 3) (3, 25). With Rhodopseudomonas sphaeroides, however, the addition of IPTG had no effect on the level of expression of P-galactosidase (29). Plasmid pGC91.14 (RP1::Tn951) was particu-

VOL . 46, 1983

larly stable in Z. mobilis at 30°C. However, this plasmid was readily lost during growth at 370C. Neither RP1 nor pGC91.14 have been reported to be unstable at 370C in E. coli (6, 7). Since this temperature is well above the optimal growth temperature of Z. mobilis, the rapid loss of pGC91.14 from Z. mobilis may be related to thermal stress of the organism rather than an inherent thermolability of the plasmid replication system. Although we have inserted a functional lactose operon into Z. mobilis, this new strain, LI4, does not grow on lactose as the sole fermentable carbohydrate. We have eliminated trivial problems such as lactose toxicity or galactose toxicity as potential reasons for its failure to grow. Lactose permease appears to be functioning within the membrane, and its level of expression was coordinated with that of P-galactosidase. The most probable reason for the failure of strain LI4 to grow on lactose is insufficient expression of the lactose gene products. The level of activity expressed by Z. mobilis LI4 was only 1/12 that of E. coli JC3272(pGC91.14) during growth on lactose. Nano and Kaplan (25) have reported that low enzyme levels equivalent to those observed in strain L14 did not support the growth of R. sphaeroides on lactose. With mutagenesis, 10-fold-higher levels of expression, which allowed growth on lactose, were achieved in R. sphaeroides (25). This approach is currently being pursued with Z. mobilis to better define the problems associated with foreign gene expression. ACKNOWLEDGMENTS We thank A. Ben-Bassat, D. H. Duckworth, G. Cornelis, S. Kaplan, K. T. Shanmugam, K. B. Sharma, and J. F. Timoney for their generous gifts of strains and plasmids. This investigation was supported by grant PCM-8204928 from the National Science Foundation. L.O.I. is the recipient of Career Development award K02 00036 from the National Institute of Alcohol Abuse and Alcoholism. LITERATURE CITED 1. Achtman, M. A., N. S. Willetts, and A. J. Clark. 1971.

Beginning a genetic analysis of conjugational transfer determined by the F factor in Escherichia coli by isolation and characterization of transfer deficient mutants. J. Bacteriol. 106:529-538. 2. Anderson, E. S. 1965. A rapid screening test for transfer factors in drug sensitive enterobacteriaceae. Nature (London) 208:1016-1017. 3. Baumberg, S., G. Cornelis, M. Panagiolakopoulos, and M. Roberts. 1980. Expression of the lactose transposon Tn951 in Escherichia coli, Proteus and Pseudomonas. J. Gen. Microbiol. 119:257-262. 4. Bolivar, F., R. L. Rodriguez, M. C. Betlach, and H. W. Boyer. 1977. Construction and characterization of new cloning vehicles. I. Ampicillin resistant derivatives of the plasmid pMB9. Gene 2:75-93. 5. Clewell, D. B., and D. R. Helinski. 1969.

Supercoiled DNA-protein complex in Escherichia coli: purification and induced conversion to open circular DNA form. Proc. Natl. Acad. Sci. U.S.A. 62:1159-1166.

Tn951 IN Z. MOBILIS

1167

6. Cornelis, G., P. M. Bennett, and J. Grinsted. 1976. Properties of pGC1, a lac plasmid originating in Yersinia enterocolitica 842. J. Bacteriol. 127:1058-1062. 7. Cornelis, G., D. Ghosal, and H. Saedler. 1978. Tn951: a new transposon carrying a lactose operon. Mol. Gen. Genet. 160:215-224. 8. Cornelis, G., D. Ghosal, and H. Saedler. 1979. Multiple integration sites for the lactose transposon Tn951 on plasmid RP1 and establishment of a coordinate system for Tn951. Mol. Gen. Genet. 168:61-67. 9. Daily, E. L., H. W. Stokes, and D. E. Eveleigh. 1982. A genetic comparison of strains of Zymomonas mobilis by analysis of plasmid DNA. Biotechnol. Lett. 4:91-96. 10. Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H. Richmond. 1971. Properties of an R-factor from Pseudomonas aeruginosa. J. Bacteriol. 108:1244-1249. 11. Ditta, G., S. Stanfield, D. Corbin, and D. R. Helinski. 1980. Broad host range DNA cloning system for gram negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. U.S.A. 77:7347-7351. 12. Dixon, R., F. Cannon, and A. Kondorosi. 1976. Construction of a P plasmid carrying nitrogen fixation genes from Klebsiella pneumoniae. Nature (London) 260:268-271. 13. Dykhuizen, D., and D. Hartl. 1978. Transport by the lactose permease of Escherichia coli as the basis of lactose killing. J. Bacteriol. 135:876-882. 14. Fietta, A., G. Grandi, M. Malcovati, G. Valentini, V. SgarameUla, and A. G. Siccardi. 1981. R-factor-mediated suppression of the galactose-sensitive phenotype of Escherichia coli K-12 galE mutants. Plasmid 6:78-85. 15. Gabler, E., R. Kalter, R. N. Boisvert, L. P. Walker, R. A. Pellerin, A. M. Rao, and Y. D. Huang. 1982. Feasibility of ethanol production from cheese whey and fruit pomace in New York, p. 13-22. In R. A. Parsons (ed.), Feed and fuel from ethanol production. Northeast Regional Agricultural Engineering Service, Cornell University, Ithica, N.Y. 16. Gibbs, M., and R. D. DeMoss. 1951. Ethanol formation in Pseudomonas lindneri. Arch. Biochem. Biophys. 43:478479. 17. Goncalves de Lima, O., I. E. Schumacher, and J. M. De Araujo. 1968. Novas observacoes sobre e acao antagoniste de Zymomonas mobilis (Lindner) (1928), Kluyver and van Niel (1936). Rev. Inst. Antibiot. Univ. Recife 8:1948. 18. Grindley, N. D. F., J. N. Grindley, and E. S. Anderson. 1972. R factor compatibility groups. Mol. Gen. Genet. 119:287-297. 19. Haas, D., and B. W. Holloway. 1976. R-factor variants with enhanced sex-factor activity in Pseudomonas aeruginosa. Mol. Gen. Genet. 144:243-251. 20. Lawford, G. R., B. H. Lavers, D. Good, R. Charley, and J. Fein. 1983. Zymomonas ethanol fermentations: biochemistry and bioengineering, p. 482-507. In H. E. Duckworth and E. A. Thompson (ed.), Proceedings of the Royal Society of Canada, International Symposium on Ethanol from Biomass. The Royal Society of Canada, Ottawa, Ontario, Canada. 21. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 22. Luria, S. E., and M. Delbruck. 1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28:491-511. 23. McKinney, K., H. Shimatake, D. Court, U. Schmeissner, C. Brady, and M. Rosenberg. 1981. A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase, p. 383-415. In J. G. Chirikjian and T. S. Papas (ed.), Gene amplification and analysis, vol. 2. Elsevier, New York. 24. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 25. Nano, F. E., and S. Kaplan. 1982. Expression of the transposable lac operon Tn95I in Rhodopseudomonas sphaeroides. J. Bacteriol. 152:924-927.

1168

CAREY, WALIA, AND INGRAM

26. Rogers, P. L., K. J. Lee, M. L. Skotnicki, and D. E. Tribe. 1982. Ethanol production by Zymomonas mobilis. Adv. Biochem. Eng. 23:37-84. 27. Skotnicki, M. L., K. J. Lee, D. E. Tribe, and P. L. Rogers. 1981. Comparison of ethanol production by different Zymomonas strains. Appl. Environ. Microbiol. 41:889893. 28. Skotnicki, M. L., K. J. Lee, D. E. Tribe, and P. L. Rogers. 1981. Genetic alteration of Zymomonas mobilis for ethanol production, p. 271-290. In A. Hollaender, R. D. DeMoss, S. Kaplan, J. Konisky, D. Savage, and R. S. Wolfe (ed.), Genetic engineering of microorganisms for chemicals. Plenum Publishing Corp., New York. 29. Skotnicki, M. L., D. E. Tribe, and P. L. Rogers. 1980. Rplasmid transfer in Zymomonas mobilis. Appi. Environ.

APPL. ENVIRON. MICROBIOL. Microbiol. 40:7-12. 30. Stokes, H. W., E. L. Dally, M. D. Yablonsky, and D. E. Eveleigh. 1983. Comparison of plasmids in strains of Zymomonas mobilis. Plasmid 9:138-146. 31. Swings, J., and J. DeLey. 1977. The biology of Zymomonas mobilis. Agr. Biol. Chem. 46:2851-2853. 32. Timoney, J. F., D. E. Taylor, S. Shin, and P. McDonough. 1980. pJT2: unusual Hi plasmid in a highly virulent lactose-positive and chloramphenicol-resistant Salmonella typhimurium strain from calves. Antimicrob. Agents Chemother. 18:480-482. 33. Walia, S. K., G. B. Dunn, K. Rose, T. Pinkerton, N. Muzycka, and D. H. Duckworth. 1982. Restriction endonuclease mapping of the colicin Ib plasmid. Mol. Gen. Genet. 185:37-42.