detection of genetically engineered traits among bacteria in the environment

TIBTECH- NOVEMBER 1990 [Vol. 8] 329

S., Cosman, D. and Sims, J. E. (1989) J. Immunol. 142, 4314-4320

12 Maliszewski, C. R., Sato, T. A., VandenBos, T., Waugh, S., Dower, S. K., Slack J., Beckmann, M. P. and Grabstein, K. H. (1990) J. Immunoi. 144, 3028-3033

13 Fanslow, W. C., Sims, J. E., Sassenfeld, H., Morrissey, P. J., Gillis, S., Dower, S. K. and Widmer, M. B. (1990) Science 248, 739-742

14 Bunning, R., Crawford, A., Richardson, H., Opendakker, G., Van Damme, J. and Russell, R. (1987) Biochim. Biophys. Acta 924, 473-482

15 Leizer, T., Clarris, B. J., Ash, P. E., Van Damme, J., Saklatvala, J. and Hamilton, J. A. (1987) Arthritis Rheum. 30, 562-566

16 Jacobs, C., Young, D., Tyler, S., Callis, S., Gillis, S. and Conlon, P. (1988) J. lmmunol. 141, 2967-2974

17 Paul, W. E. and Ohara, J. (1987) Annu. Rev. Immunol. 5,429-459

18 Park, L. S., Tushinski, R. J., Mochizuki, D. Y. and Urda], D. L. (1988) J. Cell. Biochem. Suppl. 12A, 11

19 Mosley, B., Beckmann, M. P., March, C. J., Idzerda, R. L., Gimpel, S. D., VandenBos, T., Friend, D., Alpert, A.,

Anderson, D., Jackson, J., Wignall, J. M., Smith, C., Gallis, B., Sims, J. E., Urdal, D., Widmer, M. B., Cosman, D. and Park, L. S. (1989) Cell 59, 335-348

20 Idzerda, R. L., March, C. J., Mosley, B., Lyman, S. D., VandenBos, T., Gimpel, S. D., Din, W. S., Grabstein, K. H., Widmer, M. B., Park, L. S., Cosman, D. and Beckmann, M. P. (1990) J. Exp. Med. 171,861-873

21 Noelle, R. J., Krammer, P. H., Ohara, J., Uhr, J. W. and Vitetta, E. S. (1984) Proc. Nat] Acad. Sci. USA 81, 6149-6153

22 Hudak, S. A., Gollnick, S. O., Conrad, D. H. and Kehry, M. R. (1987) Proc. Natl Acad. Sci. USA 84, 4606-4610

23 Coffman, R. L., Ohara, J., Bond, M. W., Carry, J., Zlotnick, A. and Paul, W. E. (1986) J. Immunol. 136, 4538-4541

24 Snapper, C. M., Finkelman, F. D. and Paul, W. E. (1988) J. Exp. Med. 167, 183-196

25 Finkelman, F. D., Katona, I. M., Urban, J. F., Holmes, J., Ohara, J., Tung, A. S., Sample, J. V. G. and Paul, W. E. (1988) J. Immunol. 141, 2335-2341

26 Goodwin, R. G., Friend, D., Ziegler, S. F., Jerzy, R., Falk, B. A., Gimpel, S.,

[] [] [] [] [] [] [] [] []

Detection of genetically engineered traits among

bacteria in the environment R. W. Pickup and J. R. Saunders

The release of genetically engineered microorganisms (GEMs) into the environment has, as its main aims, the benefits of improved agricultural yield and control of environmental pollution. However, effective and safe release programmes necessitate the development of sensitive, selective detection methods to monitor the environ-

mental impact of released organisms.

The release of genetical ly engineered microorganisms (GEMs) and other genet ical ly modif ied organisms (GMOs) is subject to legislative re- str ict ions in most countr ies , and permiss ion for their release must be

R. W. Pickup is at the Institute of Freshwater Ecology, Windermere Labora- tory, Ambleside, Cumbria LA22 OLP, UK. J. R. Saunders is at the Department of Genetics and Microbiology, University of Liverpool, Liverpool L69 3BX, UK.

granted by the appropr ia te regu- latory authori ty . Consequent ly , the release of GMOs requires that m u c h data be obta ined regarding the possible fate of the organism and its r ecombinan t DNA in the environment.

The need to monitor GEM release Most del iberate releases into soil

for agricultural purposes , and acci- dental spillages from conta ined

~) 1990, Elsevier Science Publishers Ltd (UK) 0167 - 9430/90/$2.00

Cosman, D., Dower, S. K., March, C. J., Namen, A. E. and Park, L. S. (1990) Cell 60, 941-951

27 Novick, D., Engelmann, H., Wallach, D. and Rubinstein, M. (1989) J. Exp. Med. 170, 1409-1414

28 Lenng, D. W., Spencer, S. A., Cachianes, G., Hammonds, R. G., Collins, C., Henzel, W. J., Barnar, R., Water, M. J. and Wood, W. I. (1987) Nature 330, 537-543

29 Marcon, L., Ritz, M. E., Kurman, C. C., Jensen, J. C. and Nelson, D. L. (1988) Clin. Exp. Immuno]. 73, 29-33

30 Peetre, C., Thysel], H., Grubb, A. and Olsson, I. (1988) Eur. J. Haematol. 41, 414-419

31 Gershoni, J. M. and Aronheim, A. (1988) Proc. Nat] Acad. Sci. USA 85, 4087-4089

32 Fisher, R. A., Bertonis, J. M., Werner, M., Johnson, V. A., Costopoulos, D. S., Lin, T., Tizard, R., Walker, B. D., Hirsch, M. S., Schooley, R. T. and Flavel, R. A. (1988) Nature 33, 76-78

33 Tranecker, A., Luke, W. and Karjalainen, K. (1988) Nature 331, 84-86

34 Risen, F. S., Steiner, L. A. and Unanue, E. R. (1989) Dictionary of Immunology, Stockton Press

[] [] []

laboratory or b io technology plant facilities will, probably, lead ulti- mate ly to some GEMs becoming dis- persed th rough the envi ronment . Dis- persal is l ikely to occur by w ind and/or animal vectors and through run-off water into lakes and streams. It would , therefore, be impruden t to release a novel, man ipu la ted microorganism wi thout knowing some- thing of its chances of survival and the potent ia l for d isseminat ion of its r ecombinan t DNA to indigenous organisms. Informat ion is also re- qui red on the possible impac t of GEMs, and any indigenous microorganisms that acquire recombinant DNA traits, on natural ecosystems. A prerequis i te for a release p rogramme is to have sensi t ive and select ive systems available to detect the GEM and its r ecombinan t DNA, and for quant i fy ing microbial act ivi ty in open envi ronments . Therefore, the abil i ty to moni to r survival and per- s is tence of GEMs accura te ly is criti- cal in de te rmining their potent ia l impact on a given envi ronment .

Any moni tor ing programme must be able to dis t inguish the GEM from the indigenous microflora present in

330 TIBTECH - NOVEMBER 1990 [Vol. 8]

~Fig. 1

an environment. Methods for the isolation of bacteria from environmental samples are well established but are limited by their non-selec- tivity and the inability to culture more than 1-10% of the total bacteria present 1,2. It is becoming apparent that conventional culture methods are unable to cope with the task of tracing specific bacteria (including GEMs) in the environment. However, by extending the traditional techniques of microbial ecology and combining them with those of molecular biology, the survival and spread of recombinant microorganisms can be monitored with a certain degree of confidence.

Techniques for GEM detection A simple method of identification

is to incorporate into the microorganism genetic marker(s) that are unlikely to be widely distributed in the natural environment. Depending on the type of marker employed, the GEM may be tracked phenotypically, relying on the expression of the marker gene, and/or genotypically where the organism is identified by the presence of that gene by methods that do not depend on its expression 3. In general, detection methods can be classified as either quantitat- ive (permitting the direct enumer- ation of the GEM), or qualitative (where assessment is based on presence or absence of the GEM) 4. What- ever the method used to detect and monitor GEMs, success is likely to be compromised by a variety of factors. These include sampling strategy, sensitivity, specificity, reliability and practicability. The detection of bacteria in the environment 1, and GEMS in particular 3,4, have been the subject of comprehensive re- views. This review describes some techniques that have been developed to increase the sensitivity of methods employed to detect GEMs in the environment.

Marker gene systems Many monitoring systems are

based on genetic marker systems that distinguish the released GEM from the natural population. In many cases, the genetically engineered trait itself is the characteristic on which the detection system may be based. Genetic markers may be categorized into three groups:

(1) Functional genetic systems.

Identification of bacteria carrying a xy lE colorimetric marker in lakewater; (a) bacteria isolated on agar plate at 20°C, (b) incubation temperature is raised to 37°C for I h and the substrate, catechol, is applied by aerosol. Bacteria containing xy lE are identified by the formation of the yellow product, 2-hydroxymuconic semialdehyde.

These may provide a selective characteristic (e.g. antibiotic resistance or the ability to metabolize an un- usual substrate), or a non-selective characteristic in the form of a unique biomarker (e.g. a cell-wall protein). (2) Chromogenic markers which provide a colorimetric change in the appearance of bacterial colonies. Such marker systems contain one or more genes that encode enzyme(s) that produce a colour change in a substrate. Isolation of bacteria on media supplemented with chromogenic substrates has been used successfully to distinguish bacteria carrying xylE (catechol 2,3 dioxygenase) 5 and lacYZ (~-galactosidase) 6 markers, from indigenous bacteria. Other potential bacterial markers include GUS ([~-glucuronidase) 7 which produces a fluorogenic product that can be detected at very low levels, and the lux system in which the bacteria are identified by their ability to bioluminesce 8. (3) Short, but unique oligonucleotide markers. Such markers are detected, not by their translational product, but by nucleic acid hybridization probes.

Insertion of marker genes Marker genes may be inserted into

a plasmid which is introduced into the release host. However, there is increasing evidence that some plasmid marker systems are inherently unstable due to the segregation of the plasmid at cell division once released into the environmenr 3,~°. In addition, it may not be good practice to use plasmid systems that have

enhanced potential to disseminate recombinant DNA to natural microbial populations. Preferably, such DNA may be inserted into the genome of the organism. The stability of the recombinant DNA may be increased but expression reduced (in comparison to plasmid-encoded systems) as a result of a reduction in gene copy n u m b e r . One further advantage would be that chromo- somally located genes are inherently less likely to be readily disseminated in the environment.

It is possible that introduction of new genetic material will reduce the viability of the GEM in the environment. Firstly, it is possible that maintenance of the marker system may impart a metabolic burden to

.the cell. Secondly, the continual expression of the marker gene may reduce the ability of the cell to survive. Locating the marker system on the chromosome may reduce the maintenance budget and controlling the expression of the gene(s} will remove any disadvantages from over-expression. Such a system has been developed for xflE 5. The marker gene xylE is expressed from the bacteriophage lambda pL or pR promoter under the control of the temperature-sensitive lambda re- pressor, ci857. The potentially deleterious metabolic burden im- posed on the host cell is controlled by incubation temperature. Bacteria containing this marker system can be isolated on solid medium with the xylE gene repressed. Its activation, and subsequent identification of target organisms, requires only that

TIBTECH - NOVEMBER 1990 [Vol. 8]

- -Tab le 1

331

Detection limits of various methods

Detect ion Method l im i ts a Target Med ium Ref.

Viable plating 10 3 Plasmid analysis/Total DNA 2 x 107 ELISA 103 DNA hybridization 103 Solution hybridization 102-103

DNA hybridization-MPN

Polymerase chain reaction (PCR)

Fluorescent antibodies Fluorescent oligonucleotides

101-102

102 (1009)

2 x 101 nd b

xylE lake water 9 RP4 soil xylE lake water 9 xylE lake water 9 2,4,5,T soil 23

plasmid Tn5 soil 37

transposon 2,4,5,T soil 25

plasmid Flavobacterium soil 29 16S RNA nd 36

a in cells per mill i l i tre, or cells per gram, of medium. bAbbreviations: nd = not detectable.

the temperature be raised above 37°C for one hour followed by the application of the substrate in the form of an aerosol. These cells are identified by the immediate formation of an intense yellow colour (Fig. 1). This type of system has the advantage that it does not require selective media which have been shown to reduce the viability of organisms isolated from the environment al. Further- more, the presence of cells carrying the xylE marker system can also be confirmed directly from lake water by assaying for the presence of catechol 2,3 dioxygenase enzyme. Its activity, and its sensitivity to oxygen once released from the cell by lysis, is a direct indication of the presence of viable (unlysed) cells 9.

Many detection methods share one common strategy: that the microorganisms can be cultured and the relevant marker genes tran- scribed, imply ing that they are metabolically active. The type of sample preparation method is deter- mined by the monitoring strategy employed. Detection of viable bacteria can be achieved by direct plating methods on both selective and non-selective media. Selective en- richment before plating may be required to increase the relative numbers of target organisms above th:e detection limit. Viable plating methods are limited to detecting only those microorganisms that are culturable. However, a large number of dormant or non-culturable cells maintain their viability in the environment (for example, Vibrio species) 12. It is possible that some

organisms that are chosen for release may enter this state once released into the environment, thus rendering these types of isolation techniques ineffective. In addition, the expression of the marker gene may be inefficient in non-laboratory growth conditions. It is therefore essential that techniques are available which do not rely directly on the expression of the marker gene but allow the organism to be monitored genotypically.

Detection systems not requiring culturability

Detection methods that do not rely on culturability but use immunological and hybridization techniques can employ strategies which either extract the total cell or total DNA from a sample 13. DNA isolation can be performed with bacterial cells isolated by a bulk method 14 or by direct lysis of the cells followed by DNA recovery 15,16. These methods circumvent the need for culturing the organisms yet have limitations in the range of organisms and the size range of DNA which can be isolated.

Detection of the gene using nucleic acid probes

DNA hybridization technology is based on the reannealing of two complementary, denaturated DNA strands (single-stranded DNA). The target DNA can either be transferred to a filter directly after extraction and purification, or transferred fol- lowing restriction analysis and elec- trophoretic separation. Both methods are labour-intensive and so the num-

ber of bacterial isolates that can be screened is limited. Colony hybridization, where cells are cultured and lysed directly on to the filter, can screen up to several thousand colonies at a time 17. The probe DNA is labelled (either radioactively or non-isotopically) and in its single- stranded form is applied to the target DNA. Excess probe DNA is washed from the filter leaving only the complementary sequences attached. The presence of the probe is visualized either using X-ray film or by chemical colour production.

The application of nucleic acid probes to identify and detect bacteria in the environment shows great promise TM. If suitable probes are available it is possible to use hybridization to detect the presence of specific nucleic acid sequences, from oligonucleotides to functional recombinant genes, not only in cultures but also direct ly in environmental samples without first having to culture target bacteria. There are several hybridization strategies available. Colony hybridization, in which bacterial colonies grown on a filter are probed for a particular trait, has been successfully used to detect a range of organisms carrying specific traits. Examples of the types of populations monitored by colony hybridization include toluene-degrading bacteria 19,2°, polychlorinated biphenyl-degrading bacteria 21 and mercury-resistant bacteria 22. The requirement for culturable cells makes it possible to assess quantitatively the extent to which the trait has been transferred within the indigenous population. In contrast, total DNA extr.action and subsequent probing for a particular trait can be Used to monitor that characteristic on a presence or absence basis. The problem is, however, that such samples often do not contain enough of either the target microorganism or its nucleic acid to make detection possible. This might be circum- vented in a number of ways using methods designed to increase the detection sensitivity.

• High specific-activity probes can be used in dot blot procedures (DNA is spotted and fixed to a filter before the probe is applied) to detect sub- picogram levels of DNA 13,16. How- ever, this approach is limited when the target DNA comprises a very small fraction of the total DNA.

332

iFig. 2


o target cells

• non-target cells

• magnetic beads

1 2

• o

O ° •

• o

•% o •

n O o

• o

•11•

e S • e • e

"'l 4. • q l

I i I-- LIJ Z (_9 <

Immunomagnetic capture of bacteria from lakewater. Step 1, a lakewater sample is taken containing both target and indigenous bacteria. Step 2, magnetic beads coated with antibodies specific to the target organism are introduced. Step 3, after a short incubation period, a magnet is applied to the side of the tube. Bacterial cells binding to the antibodies on the beads and free beads are attracted to the magnet. Step 4, the remaining lakewater sample containing unattached bacteria is removed and the bead celi mixture is washed several times. Step 5, the isolated bacterial cells are resuspended in an appropriate medium for analysis or further manipulation. The photograph shows the target organism, Pseudomonas putida, attached to a lOym bead.

• Solution hybridization is another strategy which permits large amounts of total DNA from a sample to be screened 23. This method relies on the specific formation of hybrids between target DNA and radiolabelled single-stranded RNA probe molecules. After hybridization, non- hybridized probe is removed along with unincorporated nucleotides and the amount of double-stranded radiolabelled hybrid is measured. Using this method between 100 and 1000 cells per gram of sample can be detected 23. Previous detection limits using a variety of methods are usu- ally an order of magnitude less sensitive (Table 1). • Polymerase chain reaction (PCR) can be used to enrich the target sequences in order to increase the relative concentration of target DNA in the sample 24. The target nucleic acid sequences extracted with total DNA from the sample are hybridized to complementary oligonucleotide primers. The target sequence is then amplified in a series of cycles in which a primer-directed enzymatic polymerizing step is alternated with one which separates the complementary strands. This step generates

more template to which more prim- ing oligonucleotide can anneal to the sequences flanking the target and permit the next round of polymer- ization. The resulting chain reaction permits the amplification, in a few hours, of desired nucleic acids from essentially undetectable levels to quantities which permit detection of a target sequence from which the presence of the target organism may be inferred. This method will detect the trait but will not allow any interpretation on whether the trait was in the original organism or existed in others as well. Stephan and Atlas 25 showed that they could detect as little as 0.3 pg of target DNA which was equivalent to 100 target organisms in 100g of soil against a background of 10 ~1 non-target microorganisms.

PCR may also be combined with solution hybridization and high specific activity probes to incorporate the advantages of each method, permitting highly sensitive detection techniques for target organisms that occur in extremely low numbers 23. A further refinement would be to use ribosomal RNA (rRNA) as the target sequence as it is

amplified naturally within the cell during protein synthesis, and it con- tains sequences that may be specific to the target organism.

I m m u n o l o g i c a l de tec t ion The use of either polyclonal or

monoclonal antibodies offers a potentially sensitive and specific means of identifying GEMs. Anti- bodies of either type can be used to identify specific: marker gene prod- ucts or even intact microorganisms. that express the appropriate antigen. Antisera have been commonly used for the detection of clinically important bacteria and medical diag- nosis. There is now increasing interest in raising monoclonal and polyclonal antisera against ecologically important microorganisms, particularly pathogens 26,27 and subsequently for GEMs 9. Enzyme linked immunosor- bant assay (ELISA) has been used for the detection of specific strains (e.g. Rh]zobium) TM and recombinant bacteria (e.g. Pseudomonas putida) 9 in the presence of mixed populations. Experience with strains marked with xylE showed that the detection limit was around 103 cells per millilitre of lake water using ELISA techniques

T I B T E C H - NOVEMBER 1990 [Vol. 8] 333

and polysera specific to catechol 2,3 dioxygenase (Table 1).

Direct counting using epifluor- escence microscopy is one of the most reliable methods for determin- ing numbers and biomass of bacteria in natural samplesL The use of stains commonly associated with this technique such as acridine orange and DAPI are non-specific and would not distinguish a GEM from indigenous bacteria. However, specificity can be achieved using antibodies coupled chemically to a fluorochrome (fluorescent antibodies). There are several problems associated with the use of fluorescent antibodies to detect cells in the environment 3.

(1) Detection of marked strains will rely on the presence of an antigen specifically introduced into tile GEM. It is likely to be difficult to ensure that the antigen will be expressed as a cell-surface component which could therefore be detected. It is important that the identifying antigen is a unique cell-surface protein and therefore would have to be host-specific. (2) Assuming that a unique cell- surface antigen is available to distinguish the GEM, then much would have to be known about its stability once the organism encounters environmental conditions. If the anti: gen-encoding gene is lost, or not expressed, the survival of the GEM cannot be monitored. (3) Reduction in specificity of the antibody may occur due to inter- ference from particulate matter, natural autofluorescence of co-exist- ing material, crossreactivity with other antigens and failure to reach the target organism due to the presence of extracellular material 4.

A careful choice of release host may circumvent these problems. Mono- clonal antibodies raised against Flavobacterium P25, originally isolated from soil, have been shown to be active even when the organism encountered starvation conditions and cell densities as low as 20 bacteria per gram of soil could be detected 29. Fluorescent monoclonal antibodies specific for the 01 antigen of Vibrio cholerae were used in con- junction with fluorescence microscopy. This procedure was shown to be a more sensitive method of assess- ing water quality than standard culture methods 26.

Detecting the transfer of recombinant traits

A variety of detection strategies and probes have been developed: some specifically recognize a marker gene, and others monitor the host cell independently of the marker system. These two approaches can be used in parallel; probes specific to the marker will detect the gene in the release host and indigenous bacteria that have received the DNA through gene transfer processes. Conversely, the host-specific probe will monitor the release host, which may persist in the environment after deletion or lass of the recombinant marker. Detection of gene-transfer events would rely on the genetic trait being detected in bacteria other than the release host. When the marker en- codes a chromogenic or metabolic function then the presence of transconjugants can be detected in culturable cells on solid media under either selective or non-selective conditions depending on the detection system in operation. How- ever, considerable care must be taken when selective media are used. It is possible that gene transfer through processes such as conjugation may occur during the isolation process leading to the appearance of false positive events. Incorporation of agents (such as nalidixic acid) that inhibit conjugal transfer has been applied in a number of studies in- volving the assessment of gene-transfer frequency 3°. Furthermore, transconjugants carrying recombinant DNA traits can be distinguished from the original release strain by their differing nutritional requirements, colony morphology and chromosomal DNA fingerprint pattern as judged by restriction profile. In addition, transconjugants can be distinguished further by their inability to react with nucleic acid or immunological probes specific to the release host. Techniques that rely on direct DNA extraction or lysis of total bacterial cells will preclude the detection of transconjugants as they assess the presence or absence of the recombinant marker gene and do not distinguish different bacterial hosts in which the gene might be carried.

Future techniques for the detection of GEMs in the environment

Organisms which are present in low numbers are often difficult to

enumerate. The level of sensitivity for many of the methods available may not be sufficient to detect low numbers of recombinant organisms in the environment. Selective en- richment techniques included as an integral part of any monitoring strategy would increase the proportion of viable target organisms within the total population. One such method has been developed using monoclonal antibodies raised against the flagella of a model recombinant pseudomonad have been shown to be specific only to this strain 31. Their specificity provides a highly selective tool for identifying this strain. Fur- thermore, antibodies can be coupled to the surface of magnetized poly- styrene beads. The antibody-antigen reaction can then be used to capture the specific bacteria from environmental samples using magnets (Fig. 2). The ability to process large numbers of cells is impractical using many of these techniques. Flow cytometry and fluorescence-activated cell sorting may provide a rapid means of identification, sorting and enumer- ation of specific microorganisms 32,33. In this technique a suspension of particles (microorganisms in this instance), that have been labelled with a fluorochrome which fluor- esces at a particular wavelength of light, is passed through a fine laser beam in such a way that only one cell passes through the beam at a time. By using a range of fluorochromes it is possible to analyse several different cell types at once. Coupling fluorochromes to GEM- specific antibodies permits the detection of recombinant organisms. The cell-sorting function allows the recovery of ceils with defined cellular characteristics including those which show positive binding to fluorescent antibodies. Sorting allows the recovered cells to be enumerated or subjected to further analysis (Fig. 3). Fluorochromes can also be attached to oligonuc|eotide probes for the identification of bacteria 34,35. Probes specific to the 16S RNA coupled to ~luorochromes has permitted the single-cell identification of Fibrobacter succinogenes and Methanosarcina acetivorans in mixed ruminant populations. This labelling m e t h o d has potential in flow-cytometric applications be- cause of its specificity 36. This method involves fixing the probe

334

--Fig. 3

Soil sample I I Cells extracted from soil into

aqueous phase

Probe ,'•• t cMixandstainnGIIsuspensio \ / / Non-target cell

Z ~ Wash

,! I ~ I '

Cellular rameters

Water sample Cells in aqueous phase

Nozzet li Cell size Cell density T E

I LASER Red fluorescence Green fluorescence Blue fluorescence

Continuous stre

Each cell is analysed and the data stored

C O M P

"=' U T E R

Droplet break-off 0

©

© - 0

+/o.OOo 0 ~ ~ I I Target cell s

enumerated and sorted for further analysis

Flow cytometer Application of flow cytometry and cell sorting for monitoring or recovering GEMs from the environment.


into the target cell in a similar way to tradit ional microscopic staining procedures. The f luorescent signal can be further increased by applying mul t ip le f luorescent ol igonucleot ide probes. Its invasive nature precludes isolating viable cells but will a l low enumerat ion. However, it is specu- lated that the specificity of the probes should al low changes in specific microbial populat ions in the environment , from k ingdom through genera to species, to be moni tored 36.

Conclusions The economic benefits of releasing

GEMs are likely to be great in m a n y countries, part icularly in the im- provement of agriculture and in the management of envi ronmenta l pollution. Success and safe application of genetically engineered microorganisms will require the development of rapid and sensit ive monitor ing methods, part icular ly those which can be automated. It is unlikely, due to the inherent limitations in the methods so far developed, that any one system will be suitable for moni tor ing GEMs in all environmental locations.

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7 Jefferson, R. A. (1989) Nature 342, 837-838

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30 O'Morchoe, S. B., Ogenseitan, O.,

Sayler, G. S. and Miller, R. V. (1988) Appl. Environ. Microbiol. 54, 1923-1929

31 Saunders, J. R., Morgan, J. A. W., Winstanley, C., Raitt, F. C., Carter, J. P., Pickup, R. W., Jones, J. G. and Saunders, V. A. (1990) in Bacterial Genetics in Natural Environments (Fry, J. C. and Day, M. J., eds), pp. 3-21, Chapman and Hall

32 Li, W. K. W. and Wood, M. (1988) Deep-Sea Res. 35, 1615-1638

33 Robertson, B. R. and Button, D. K. (1989) Cflometry 10, 70-76

34 Giovannoni, S. J., Delong, E. F., Olsen, G. J. and Pace, N. R. (1988) J. Bacteriol. 170, 720-726

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i! :i i' i I I I I i i i i i i i i i A valuable bench companion

PCR PROTOCOLS -- A GUIDE TO

METHODS AND APPLICATIONS

edi ted by M. A. Innis, D. H. Garfield, J. J. Sn insky and T. ]. White, Academic Press, 1990. $39.95 (xviii + 482 pages) ISBN 0 12 372181 4

This col lec t ion of exper imenta l procedures describes various aspects of the me thodo logy of po lymerase chain react ion (PCR) as well as the appl ica t ions of PCR to different problems in molecular biology and medic ine . Each article consists of a brief in t roduc t ion giving some theor- etical background as well as the pr inc ip le of the methods or applicat ions to be discussed, fo l lowed by a deta i led descr ip t ion of the practi- cal procedures . In many cases ad- di t ional notes are given on potent ial difficult ies or mistakes, and indica- t ions on for thcoming developments . The articles are accompan ied by ci tat ions of the most per t inent lit- erature.

This vo lume owes its value to the exper t ise of its contr ibutors, all

specialists of in ternat ional r e n o w n in the deve loping field of PCR. This col lec t ion of protocols, rather loosely grouped into five sect ions and in te r twined by a brief preface, demonst ra tes the almost unbel iev- able var ie ty of scientific forth- comings from a basic deve lopmen t wh ich or iginated only a few years ago.

This book is an excel lent compi-

lat ion for all who, having a general background in molecu la r biology, wish to practise PCR. Those invo lved in developing new method- ology will f ind suggestions for op- t imizat ion. Likewise, it will p rov ide an or ienta t ion for all those who are working on new applicat ions.

In short, this is a book meant for the laboratory bench, and no user of the PCR techn ique could w i s h to miss it.

H. SELIGER

Sektion Polymere, University of Ulm, Albert Einstein Allee 11, D-7900 Ulm, FRG.

[ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]

A missed opportunity POSTHARVEST BIOTECHNOLOGY OF

FLOWERS AND ORNAMENTAL PLANTS

by D. H. Salunkhe, N. R. Bhat and B. B. Desai, Springer-Verlag, 1990. DM 129.00 (xii + 192 pages) ISBN 3 54O 194O6 1

The wor ld p roduc t ion of cut flowers, pot ted plants and foliage plants is s teadi ly growing and of considerable commerc ia l value. Due to improper handl ing, about 20% of all floral

crops are unsaleable. This est imate does not inc lude the quant i ty of crop that was never harves ted and the post-harvest losses that occur along the market ing channel . Salunkhe, Bhat and Desai have made an inven- tory of the factors invo lved in postharvest losses of f lowers and or- namentals .

In an in t roduc tory chapter they descr ibe the ul trastructural , bio- chemica l and metabol ic changes

detection of genetically engineered traits among bacteria in the environment

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