Identification of microbial isolates by DNA fingerprinting: analysis of ATCC Zymomonas strains

Journal of Biotechnology, 13 (1990) 335-346

335

Elsevier BIOTEC 00493

Identification of microbial isolates by DNA fingerprinting: analysis of ATCC Zymomonas strains Francesco Degli-Innocenti i, Enrica Ferdani 1, Beatrice Pesenti-Barili 1, Maria Dani 1, Luciana Giovannetti 2 and Stefano Ventura 3 1Agrimont S.p.A., Centro di Biotecnologieper l'Agricoltura, Massa, ltaly," 2 Istituto di Microbiologia Agraria, Firenze, Italy," 3 Centro Studi Microorganismi Autotrofi, C.N.R., Firenze, ltaly

(Received25 September1989; accepted 23 November 1989)

Summary Nine Z y m o m o n a s strains available from the American Type Culture Collection were analyzed with a technique of restriction endonucleases fingerprinting of total D N A based on the combined use of polyacrylamide gel electrophoresis and silver staining. The pattern analysis was performed with a densitometer and a personal computer. Strains isolated independently showed very different fingerprints while strains obtained by mutagenesis had patterns identical to the parent. However, in one case it was possible to distinguish a mutant from the parent by the absence of a band in its fingerprint. The analysis of Z y m o m o n a s total protein extracts by polyacrylamide gel electrophoresis could not reveal any remarkable difference. The D N A fingerprinting similarity coefficients fitted very well with a previous taxonomical study based on classical tests, and confirmed the nomenclature problems regarding the Z y m o m o n a s CP4 strain pointed out in a recent paper (Yablonsky et al., 1988, J. Biotechnol. 9, 71-80). Furthermore a Z y m o m o n a s strain isolated in Argentina was compared with the ATCC strains showing a geographical correlation with other strains isolated in South America. On the basis of fingerprinting analysis it was possible to refer this strain to the subspecies mobilis. Zymomonas; Fingerprinting; Taxonomy; Patenting

Correspondence to: F. Degli-Innocenti,Agrimont S.p.A., Centro di Biotecnologieper rAgricoltura, via Massa-Avenza 85, 1-54100 Massa, Italy.

0168-1656/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

336

Introduction

The possibility of recognizing different isolates of the same microbial species is important for taxonomy, for clarifying the origin of new isolates, for solving cross-contamination and nomenclature problems, and for patenting. The identification of strictly correlated microorganisms is not an easy task because the characterization tests are based on phenotypic data, such as morphology, physiological properties and serological reactions. These techniques are often time consuming, usually test single genes or gene products and require expertise in specific technical assays (Devor et al., 1988). DNA base composition, DNA genome size, genome similarity (DNA-DNA and DNA-RNA homologies), amino acid sequences of proteins (e.g. cytochromes) etc. are techniques used mainly for the characterization and systematic positioning of new species, and less used for quick identification purposes (Triaper and Kr~imer, 1981). The use of restriction endonucleases has provided the microbiologist with a new tool which permits the direct exploration of the microbial genome. Such enzymes cut the DNA chain at specific sites, producing fragments of different length (restriction fragments). The number and the localization of restriction sites are specific features of each genome. The restriction fragments can be separated by electrophoresis on the basis of their molecular weight, giving rise to specific patterns called "fingerprints". The fingerprint analysis can be done on chromosomal (Bjorvatn et al., 1984; Mielenz et al., 1979) or plasmid (Yablonsky et al., 1988) DNA, electrophoresis is performed on agarose gel (Dobritsa, 1985; Chowdhury et al., 1986; Devor et al., 1988; Hookey et al., 1985; Mielenz et al., 1979) or polyacrylamide gel (Kristiansen et al., 1984), and DNA is visualized by Southern hybridization with radioactive probes (Chowdhury et al., 1986; Devor et al., 1988; Ryskov et al., 1988), by direct DNA radiolabelling before electrophoresis (Peterson and De La Maza, 1988), or by staining with ethidium bromide (Kristiansen et al., 1984; Mielenz et al., 1979; Bjorvatn et al., 1984; Dobritsa, 1985; Hookey et al., 1985). Genome fingerprinting has been achieved also by two-dimensional field inversion gel electrophoresis (Bautsch et al., 1988). The aim of this study was to gain experience with the DNA fingerprinting technology and to define its possible application for the differentiation of similar strains of Zymomonas. We have examined all the 9 available American Type Culture Collection (ATCC) Zymomonas strains using a technique of total DNA fingerprinting which exploits the good resolution properties of polyacrylamide gel electrophoresis (PAGE) and the sensibility of silver staining. Zymomonas has recently attracted considerable attention for its potential application in fuel alcohol production. Zymomonas has many advantageous features relevant for development in the industrial production of alcohol which have been extensively reviewed (Rogers et al., 1982, 1984a, b; Eveleigh et al., 1983; Skotnicki et al., 1983; Swings et al., 1984; Rogers, 1985; Buchholz et al., 1987; Baratti and Bu'Lock, 1986). Zymomonas strains have been isolated from agave sap in Mexico, from palm sap in Zaire, Nigeria, and Indonesia, from sugar cane juice in Brazil (Swings and De Ley, 1977). These bacteria have also

337

been detected in the United Kingdom as the cause of the cider sickness disease and as beer contaminant (Swings and De Ley, 1977). Swings and De Ley (1977) have performed a numerical analysis on many different isolates showing that, in spite of their greatly diverse origins, the phenotypic similarity between all the strains is very high. This stands for all the strains except for the cider sickness organisms. Therefore according to the proposal of Swings and De Ley (1977), the genus Zymomonas contains the type species Zymomonas rnobilis which contains two subspecies, namely mobilis and pomaceae (the cider sickness strains).

Materials and Methods

The Zymomonas strains studied in this work are described in Table 1. Restriction enzymes, lysozyme, proteinase K, and DNA molecular weight marker VI were obtained from Boehringer Mannheim. All the reagents for PAGE and the Silver stain kit were purchased from Bio-Rad. The protein molecular weight standard was obtained from Sigma. Yeast extract and peptone were obtained from Difco. All the other chemicals were obtained from Farmitalia-Carlo Erba. The PAGE was performed using the Protean II Slab Cell (gel size: 16 x 16 x 1.5 cm) and model 1000/500 Power Supply from Bio-Rad. Total cell DNA was extracted as follows. Cells grown stationary for 2 d at 30°C in 200 ml of YP medium (yeast extract 10 g 1-a, peptone 10 g 1-a, KH2PO 4 3 g 1-1) supplemented with 4% (w/v) glucose, were harvested by centrifugation and resuspended in 20 ml of 1 mg ml-1 lysozyme in TEN buffer (25 mM Tris-HC1, 1 mM EDTA, 10 mM NaCI, pH 8). After 2 h incubation at 37°C, 30/~1 of proteinase K (10 mg m1-1) and 1 ml of 10% (w/v) SDS were added. The mixture was incubated at 37°C with low agitation for 2 h. Then NaCI was added to the mixture to a final concentration of 0.3 M and the lysate was extracted twice with phenol-chloroform and once with chloroform. After the final extraction cold 95% ethanol was added, the DNA was spooled out with a glass rod, and dissolved in 5 ml of TE buffer (10 mM Tris-HC1, 1 mM EDTA, pH 7.5). RNase was added to a final concentration of 50 /~g ml -a and the DNA incubated 2 h at 37°C. After a phenol extraction, the DNA was ethanol precipitated and suspended in TE. DNA was digested with different restriction enzymes according to the supplier procedures (Boehringer Mannheim) using at least 5 U/~g-1 of DNA and with a long incubation time (usually overnight). Then NaC1 was added to the mixture to a final concentration of 0.3 M and DNA precipitated with two volumes of cold 95% ethanol. After an overnight incubation at - 2 0 ° C the DNA was recovered by centrifugation at 4°C, the pellet dried and carefully resuspended in a small volume of TE buffer. Finally 1/5th volume of DGE buffer (0.07 M EDTA, pH 7.5, 18% v / v glycerol, and traces of bromophenol blue) was added. About 10 /xg of DNA of each Zyrnornonas strain was loaded on a 12% discontinuous SDS-PAGE (Laemmli, 1970) run at constant current (16 mA or 19 mA; see Results). As an internal molecular weight marker 0.25 /~g of Boehringer

338

Mannheim DNA molecular weight marker VI was added in each sample. The gel was stained with the Silver Stain Bio-Rad kit. For the easiness of the subsequent analysis only enzyme digestions producing patterns with well resolved bands were chosen. Data were collected from gels with a LKB Laser Densitometer with a sensitivity of 40 #m. Scans were analyzed in the range indicated in the Results section; the starting and ending points and the peak position of each band were found with the LKB GelScan XL software using Gaussian and valley integration options. Only band positions were taken into account, irrespective of any difference in density. Patterns were then rescaled using the molecular weight marker as internal standard and compared each other looking for the common bands. Two bands, belonging to different patterns, were taken to be in the same position when the peak value of one band was included between the values of the starting and ending points of the other band. Dice similarity coefficients (SD) between each pair of strains were calculated as the ratio of twice the number of bands common to their patterns, to the sum of all bands in the two patterns (Sneath and Sokal, 1973). Rescaling, comparison and SD calculation were performed with a dedicated DBASE IV program. S o values were clustered with the UPGMA (Sneath and Sokal, 1973) using the CLUSTER and TREE procedures of the SAS package. Total protein extracts were obtained boiling for 5 min in 750/~1 of sample buffer (40 mM Tris-HC1, pH 6.8; 6% v / v glycerol; 5% w / v SDS; 3% v / v 2 /3mercaptoethanol) cells harvested from 10 ml of culture grown as described above. The mixtures were then centrifuged and supernatants collected. Ten microliters of each extract were loaded in a 12% discontinuous SDS-PAGE (Laemmli, 1970) and run at constant current (10 mA) for 16 h. Gels were stained with 0.1% Serva Blue R in methanol/water/acetic acid (5 : 5 : 1 by vol.) and destained by soaking the gel in water/methanol/acetic acid (8 : 1 : 1) for 2 h and then in methanol/water/acetic acid (5 : 5 : 1).

Results

DNA isolated from each ATCC strain was independently digested with EcoRI, EcoRV, HindlII, and BarnHI and subjected to PAGE. The fingerprints produced by each enzyme were reproducible also using DNA obtained in independent extractions (data not shown). Digestion using EcoRI and EcoRV produced a very high number of fragments, giving rise to patterns formed by scarcely resolved bands, not appropriate for the analysis (data not shown). The digestion with BamHI produced fingerprints shown in Fig. 1. This figure shows clearly that strain 1 (the parent; lane 9) and 15 (its mutant; lane 8) have identical fingerprints and, likewise, strains 2 (the parent; lane 7), 3 (a mutant from strain 2; lane 6), and 4 (another mutant from strain 2; lane 5) share the same pattern. Apart from these groupings, all the other strains exhibit peculiar fingerprints. These observations were confirmed by the HindlII fingerprints (Fig. 2). In this figure also the fingerprint of strain 19, recently isolated in Argentina (Rodriguez and Callieri, 1986), is shown.

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Fig. 1. Zymoraonas fingerprints obtained by BamHI digestion of total DNA, PAGE (19 mA for 17 h) and silver staining. Lane 1: strain 17; lane 2: strain 16; lane 3: strain 13; lane 4: strain 14; lane 5: strain 4; lane 6: strain 3; lane 7: strain 2; lane 8: strain 15; lane 9: strain 1; lane 10: molecular weight markers. The HindlII fingerprinting (Fig. 2), unlike the BamHI, distinguishes strain 4 (lane 7) from its parent (strain 2, lane 9), because a b a n d of about 360 base pairs is absent in its pattern. This difference is reproducible. The BamHI and HindlII fingerprints were subjected to analysis with a densitometer. The similarity coefficients (SD) of the HindlII fingerprints, obtained as described in Materials and Methods, are reported in Fig. 3. In the matrix the values of strain 1, which are identical to those of strain 15, are not reported. Fig. 4 shows the dendrogram obtained with the U P G M A clustering. Strains 2, 3 and 4 are almost identical; also similar to this group are the strains 13, 16 and 19. Strain 17 shows the most relevant differences to all the other strains. These indications were confirmed b y the BamHI fingerprints similarity coefficients (data not shown). The fingerprinting analysis uncovered that the Zymomonas strains differed in their restriction patterns in a remarkable manner (Figs. 1 and 2), in spite of their supposed similarity (Swings and De Ley, 1977). Therefore we wanted to check whether it would be possible to find any differences among strains at the translation product level as well. Total protein extracts of the different Zymomonas strains, subjected to P A G E and stained with Coomassie blue,

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21~ 17(

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Fig. 2. Zymomonas fingerprints obtained by HindlII digestion of total DNA, PAGE (16 mA for 17 h) and silver staining. Lane 1: strain 19; lane 2: molecular weight markers; lane 3: strain 17; lane 4: strain 16; lane 5: strain 13; lane 6: strain 14; lane 7: strain 4; lane 8: strain 3; lane 9: strain 2; lane 10: strain 15; lane 11: strain 1.

gave origin to identical patterns (Fig. 5), the only difference being in strain 13 pattern (lane 4) with a band migrating as a 49 kDa polypeptide, instead of as a 47 kDa polypeptide.

Discussion

The fingerprinting technique used in this paper is based on the combined use of PAGE and silver staining. The PAGE, already used in fingerprinting in combination with ethidium bromide (Kristiansen et al., 1984), allows a band separation superior to that achieved by agarose gel electrophoresis which only permits the identification, above a background, of poorly resolved bands (Dobritsa, 1985). The silver staining technique is applied in this work as an alternative to Southern hybridization with radioactive probes (Chowdhury et al., 1986; Devor et al., 1988; Ryskov et al., 1988), to direct

341 15

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Fig. 3. Matrix of similarity coefficients ( S D ) , calculated considering the bands of the HindllI fingerprints (Fig. 2) comprised between 1033 and 298 bp. The Zymomonas strains, indicated by their number, are faced one to the other in the matrix and the S D are reported.

DNA radiolabelling before electrophoresis (Peterson and De La Maza, 1988), or to staining with ethidium bromide (Kristiansen et al., 1984; Mielenz et al., 1979; Bjorvatn et al., 1984; Dobritsa, 1985; Hookey et al., 1985). But the use of radioactivity is time consuming and needs special facilities, and the ethidium bromide staining of double stranded DNA is 100 times less sensitive than photochemical silver stain (Walker, 1987). The method used in this work represents an

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Fig. 5. Zymomonaselectrophoretic protein patterns. Lane 1: molecular weight markers; lane 2: strain 17; lane 3: strain 16; lane 4: strain 13; lane 5: strain 14; lane 6: strain 4; lane 7: strain 3; lane 8: strain 2; lane 9: strain 15; lane 10: strain 1; lane 11: molecularweight markers. improvement of the previous techniques because the combined use of P A G E and silver staining permits easy visualization of restriction fragments as sharp bands also in the low molecular weight range ( < 1 kbp) where it is possible to have a good resolution and detect small differences. Another feature of the technique was the use of an internal molecular weight marker. This seems important not only for a correct use of the densitometer for comparing fingerprints of different strains in the same gel, but also for comparing the fingerprint of a same strain in different gels, run independently. Small unwanted differences in the accomplishment of the electrophoresis can produce patterns apparently very different and difficult to be compared without the help of an internal marker. The results indicate that the fingerprinting analysis is able to distinguish independent Zymomonas isolates and to recognize clonal groups in an unambiguous way; strains isolated independently had different fingerprints while mutants showed fingerprints identical to that of the parent. In one case it was also possible to distinguish a mutant (strain no. 4) from the parent (strain no. 2) by the absence of a single 360 bp band. This difference can be due to a point mutation which destroys one of the two HindlII restriction sites which delimits the 360 bp fragment or to a more extended rearrangement (deletion or insertion).

343 TABLE 1 Z Y M O M O N A S STRAINSUSED IN THIS WORK

Strain no. 1

Source

Taxon

Origin

ATCC 29191

Z. mobilis subsp, mobilis

Zaire (palm wine)

2

ATCC 31821

as above

3

ATCC 31823

as above

4 13

ATCC 31822 ATCC 2 9 5 0 1

as above

Brazil (sugar cane juice) via P.L. Rogers mutant from ATCC 31821 (P.L. Rogers) as above U.K. ("bad" beer)

14

ATCC 35000

15

ATCC 35001

as above

16 17

ATCC 10988 ATCC 29192

as above

19

PROIMI b

Zymomonas anaerobia a Z. mobilis subsp, mobilis

Z. mobilis subsp, pomaceae Zymomonas

subsp, not defined

mutant obtained by J. Fein from strain CP4 (formally ATCC 31821) mutant from ATCC 29191 (J. Fein) Mexico (pulque) U.K. (sick cider) Argentina (sugar cane juice)

Information obtained from Swings and De Ley (1977) and ATCC Catalogue. a Synonymfor Z. mobilis subsp, mobilis; b Proimi, Planta Piloto de Procesos Industriales Microbiolbgicos, Tucuman, Argentina.

It is obvious that different fingerprints stand for different strains, while identical fingerprints represent a good indication of very strict correlation between strains under examination. It would be very interesting, but beyond the purposes of the present study, to define a criterion to judge quantitatively the probability to have same fingerprints from different isolates. A result which was in disagreement with the other findings was observed with strain 14. This strain is a mutant obtained by J. Fein from a Z y m o m o n a s m o b i l i s strain called " C P 4". Strain CP4 was originally isolated in Brazil by Gon~alves de Lima and then distributed to many groups in the world. Strain CP4 was then deposited, along with some mutants, at the A T C C by P.L. Rogers with the number A T C C 31821 (our denomination of this strain is no. 2; see Table 1). Therefore strain 14 is formally a mutant of strain 2. However, our analysis showed that strain 14 had a pattern very different from strain 2 and its mutants (strain 3 and 4). This unexpected result is in agreement with the data presented in a recent paper by Yablonsky et al. (1988). They found that cultures of strain CP4, originally from the same source and now in independent worldwide culture collections, differed in plasmid content. Therefore they proposed to consider the culture distributed from the Recife Culture Collection (Brazil) as belonging to G r o u p I and the culture of the Rogers' laboratory (ATCC 31821) to Group II. In the present work we have analyzed total D N A and, therefore, we cannot discern whether the differences

344 shown between strains are due only to different plasmid content or whether also genomic differences are present. Our data fitted very well with the proposal of Swings and De Ley (1977) that considered the species Zymomonas mobilis as containing two subspecies, namely mobilis and pomaceae. Strain 17 (Zymomonas mobilis subsp, pomaceae) showed to be the most different strain exhibiting the lowest SD with the other strains, all belonging to the other subspecies (mobilis). In addition to the ATCC strains, we analyzed strain 19. This strain, isolated in Argentina (Rodrlguez and CaUieri, 1986), showed to be very similar to strain 16 (isolated in Mexico) and also to strain 2 (isolated in Brazil). On the other hand it showed lower values with strain 17 (isolated in U.K.) and strain 1 (isolated in Africa). The data indicate that there is a good correlation between the S o and the geographical area of isolation. The lowest S D value was with strain 17 (Zymomonas mobilis subsp, pomaceae). On the basis of this result, clearly visualized in the dendrogram of Fig. 4, we considered strain 19 as belonging to the subspecies mobilis. This identification based on D N A fingerprinting has been confirmed, during the carrying out of our work, by a characterization based on the Bergey's Manual of Determinative Bacteriology tests (Abate et al., 1989). With the analysis of Zymomonas protein patterns it was not possible to see remarkable differences among the different strains (Fig. 5) in spite of the dissimilarities revealed in the restriction fragments. Therefore, the direct examination of the genome structure by D N A fingerprinting gives the possibility to reveal subtler differences between similar strains. D N A restriction pattern and protein pattern cover two different ranges of similarity between microorganisms: the former works well when applied to strictly correlated species down to strain identification, while the latter is a powerful tool of investigation of less linked microorganisms (Hood et al., 1988). Our data, obtained analyzing different Zymomonas strains, represent a further confirmation that D N A fingerprinting is a convenient method for the strains identification as it has been shown for Frankia (Dobritsa, 1985), Leptospira (Hookey et al., 1985), Neisseria (Kristiansen et al., 1984), Rhizobium (Mielenz et al., 1979) and Chlamydia (Peterson and De La Maza, 1988). However, the method used in this work is an improvement of the previous techniques, because the combined use of P A G E and silver staining gives a better resolution and a sharper band definition. Therefore our results indicate that D N A fingerprinting by P A G E and silver staining is probably the most convenient method for the identification of very similar strains.

Acknowledgements We would like to thank Prof. Marco Bazzicalupo and Dr. Carlo Minganti for useful discussion.

345

References Abate, C.M., Santolaya, R. and Callieri, D.A.S. (1989) Ultrastructural aspects of Zymomonas mobifis during the alcoholic fermentation of sucrose-based media. MIRCEN J. 5, 87-94. Baratti, J.C. and Bu'Lock J.D. (1986) Zymomonas mobilis: a bacterium for ethanol production. Biotech. Adv. 4, 95-115. Bautsch, W., Grothues, D. and Tiimmler, B. (1988) Genome fingerprinting of Pseudomonas aeruginosa by two-dimensional field inversion gel electrophoresis. FEMS Microbiol. Lett. 52, 255-258. Bjorvatn, B., Lund, V., Kristiansen, B.E., Korsnes, L., Spanne, O. and Lindqvist B. (1984) Applications of restriction endonuclease fingerprinting of chromosomal DNA of Neisseria meningitidis. J. Clin. Microbiol. 19, 763-765. Buchholz, S.E., Dooley, M.M. and Eveleigh, D.E. (1987) Zymomonas: an alcoholic enigma. TIBTech 5, 199-204. Chowdhury, S.I., Hammerschmidt, W., Ludwig, H., Thein, P. arid Buhk, H.J. (1986) Rapid method for the identification and screening of herpesviruses by DNA fingerprinting combined with blot hybridization. J. Virol. Methods 14, 285-291. Devor, E.J., Ivanovich, A.K., Hickok, J.M. and Todd, R.D. (1988) A rapid method for confirming cell line identity: DNA "fingerprinting" with a rninisatellite probe from M13 bacteriophage. BioTechniques 6, 200-202. Dobritsa, S.V. (1985) Restriction analysis of the Frankia spp. genome. FEMS Microbiol. Lett. 29, 123-128. Eveleigh, D.E., Stokes, H.W. and Dally, E.L. (1983) Recombinant DNA approaches for enhancing the ethanol productivity of Zymornonas rnobilis. Biotechnol. Set. 4, 69-91. Hood, D.W., Dow, C.S. and Green, P.N. (1988) Electrophoretic comparison of total soluble proteins in the pink-pigmented facultative methylotrophs. J. Gen. Microbiol. 134, 2375-2383. Hookey, J.V., Waitkins, S.A. and Jackman, P.J.H. (1985) Numerical analysis of Leptospira DNA-restriction endonuclease patterns. FEMS Microbiol. Lett. 29, 185-188. Kristiansen, B.E., Bjorvatn, B., Lurid, V., Lindqvist, B. and Holten, E. (1984) Differentiation of B15 strains of Neisseria meningitidis by DNA restriction endonuclease fingerprinting. J. Infect. Dis. 150, 672-677. Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature 227, 680-685. Mielenz, J.R., Jackson, L.E., O'Gara, F. and Shanmugam, K.T. (1979) Fingerprinting bacterial chromosomal DNA with restriction endonuclease EcoRI: comparison of Rhizobium spp. and identification of mutants. Can. J. Microbiol. 25, 803-807. Peterson, E.M. and De La Maza, L.M. (1988) Restriction endonuclease analysis of DNA from Chlamydia trachomatis biovars. J. Clin. Microbiol. 26, 625-629. Rodriguez, E. and Callieri, D.A.S. (1986) High yield conversion of sucrose into ethanol by a flocculent Zymomonas sp. isolated from sugarcane juice. Biotechnol. Letters 8, 745-748. Rogers, P.L. (1985) Ethanol production and the potential of Zymomonas-based fermentations. Asean Food J. 1, 107-112. Rogers, P.L., Lee, H.J., Skotnicki, M.L. and Tribe, D.E. (1982) Ethanol production by Zyrnomonas mobilis. Adv. Biochem. Eng. 23, 37-84. Rogers, P.L., Skotnicki, M.L., Lee, K.J. and Lee, J.H. (1984a) Recent developments in the Zymomonas process for ethanol production. CRC Crit. Rev. Biotechnol. 1,273-288. Rogers, P.L., Goodman, A.E. and Heyes, R.H. (1984b) Zymomonas ethanol fermentations. Microbiol. Sci. 1, 133-136. Ryskov, A.P., Jincharadze, A.G., Prosnyak, M.I., Ivanov, P.L. and Limborska, S.A. (1988) M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms. FEBS Lett. 233, 388-392. Sheath, P.H.A. and Sokal, R.R. (1973) Numerical taxonomy. The principles and practice of numerical classification. W.H. Freeman and Co., San Francisco. Skotnicki, M.L., Warr, R.(3., Goodman, A.E., Lee, K.J. and Rogers, P.L. (1983) High-productivity alcohol fermentations using Zymomonas mobilis. Biochem. Soc. Symp. 48, 53-86.

346 Swings, J. and De Ley, J. (1977) The biology of Zymomonas. Bacteriol. Rev. 41, 1-46. Swings, J., lserentant, D. and De Ley, J. (1984) Zymomonas and other alcohol producing microorganisms. Monogr. Eur. Brew. Conv. 9, 49-71. Triiper, H.G. and Kr~mer, J. (1981) Principles of characterization and identification of prokaryotes. In: Start, M.P., Stolp, H., Tri]per, H.G., Balows, A. and Schlegel, H.G. (Eds.), The Prokaryotes. Springer-Verlag, Berlin, Heidelberg, New York, pp. 176-193. Walker, D. (1987) Silver staining in gel electrophoresis, lnternat. Biotech. Lab. 5, 12-16. Yablonsky, M.D., Goodman, A.E., Stevnsborg, N., Gon~alves de Lima, O., Falc~to de Morais, J.O., Lawford, H.G., Rogers, P.L. and Eveleigh, D.E. (1988) Zymomonas mobilis CP4: a clarification of strains via plasmid profiles. J. Biotechnol. 9, 71-80.