Detection of bacteria by transduction of ice nucleation genes

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With protein pharmaceuticals, the problem of abnormal variants, such as deaminated, oxidized and proteo: lytically modified forms generated in an in-vivo system 7, does not occur in vitro because of the absence of post-translational modification systems. Therefore, protein pharmaceuticals such as insulin, growth hormones, oMnterferon, tissue plasminogen activator (TPA), and various vaccines may be produced on a preparative scale using the Spirin system, which also lacks the endotoxins and infectious agents which constitute a significant problem in in-vivo production systems. Similarly, proteins that may be cytotoxic for the host cell, for example, antibodies recognizing tumor-associated antigens at the cell surface, various proteases, immunotoxins, lectins and ribosome-inactivating proteins (RIPs), can be produced safely by

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this system. For research purposes, this system is of most interest for studies on the fidelity of protein synthesis, posttranslational modifications, protein stability, protein folding and transport. Furthermore, the possibilities of protein engineering can also be extended significantly by permitting a rapid expression and analysis of many mutant proteins on a preparative scale. It may also be possible to produce large quantities of proteins involved in the regulation of gene expression for the purposes of somatic-cell therapy of genetic diseases. It is hoped that the results of extensive studies in progress in several laboratories will soon demonstrate the feasibility and success of the Spirin system in producing a wide range of proteins with medical, industrial and agricultural value on a large scale.

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Detection of bacteria by transduction of ice nucleation genes The ideal to which quality-control bacteriologists aspire is to detect vanishingly small numbers of target bacteria, even after damage to the microorganisms by disinfectants or manufacturing processes, in the presence of much larger numbers of non-target bacteria, and rapidly enough to prevent manufacture or sale of a contaminated product 1. In addition, the ideal test should never report false negatives or false positives, employ no hazardous chemicals and produce no hazardous waste. However, all assays represent a compromise between these often conflicting goalsL A recent addition to the array of test strategies available for bacterial pathogen surveillance is the Bacterial Ice Nucleation Diagnostic (BIND TM) assay. This 1990, Elsevier Science Publishers Ltd (UK)

system combines the recognition specificity of a bacteriophage with the sensitivity of a unique reporter gene: the gene encoding a bacterial ice nucleation protein. Detection of bacterial ice nuclei Ice nucleus production appears to be restricted to certain species of Pseudomonas, Erwinia or Xanthomonas which form epiphytic communities on plant surfaces 2~. In all of the cases examined so far 5-9, the ice nucleation-positive (Ina +) phenotype is imparted by a single gene of - 3 . 6 k b . Ice nuclei are synthesized rapidly; they appear within 15 minutes of the initiation of transcription of an ice nucleation gene 1°. Genes from different species show a high degree of homology and

0167 - 9430/90/$2.00

References 1 Nirenberg, M. W. and Matthai, J. H. (1961) Proc. Natl Acad. Sci. USA 47, 1588-1607 2 Spirin, A. S., Baranov, V. I., Ryabova, L. A., et a]. (1988) Science 242, 1162-1164 3 Ryabova, L. A., Ortlepp, A. A. and Baranov, V. I. (1989) Nucl. Acids Res. 17, 4412 4 Baranov, V. I., Morozov, I. Y., Ortlepp, S. A., eta]. (1989) Gene 84,463-466 5 Goff, S. A. and Goldberg, A. L. (1985) Cell 41,587-595 6 Kane, J. F. and Hartley, D. L. (1988) Trends Biotechnol. 6, 95-101 7 Anicetti, V. R., Keyt, B. A. and Hancock, W. S. (1989) Trends Biotechnol. 7, 342-349 S U R E S H I. S. R A T T A N OLE K R I S T E N S E N

Laboratory of Cellular Ageing, Department of Chemistry, Aarhus University, DK-8000 Aarh us-C, Denmark.

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have probably diverged from a common ancestral gene 7'8. The encoded proteins all show the same modular organization: the N-terminal region (which constitutes - 1 5 % of the protein) and the C-terminal region (-5%) have homologous, nonrepetitive sequences, while the central region (-80%) is highly repetitive, containing nested repeat sequences which are 8, 16 or 48 amino acids in length. It is currently thought that this central domain binds water in a configuration which mimics the structure of ice, and thus forms a template for ice crystal formation 3'4. The periodicity of the repeat sequence also appears to be of functional significance. Such biological proteinaceous nuclei induce formation of ice in water at temperatures as high as -2°C. By contrast, most inorganic nucleators do not even begin to show activity at temperatures above -8°C, and small volumes of pure water can be supercooled to temperatures as low as -40°C. It appears that much of the ice encountered in everyday life is due either to bacterial ice nuclei, or to secondary nucleation by pre-existing ice. Very few nuclei are needed, since one nucleation event is sufficient to cause freezing of a

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--Table I

Comparison of several existing tests forSalmonella Test

Manufactu rer

Time (confirmed negative)

Method

Cost (US$ per test)

(h) 72

BAM-AOAC

Many

Gene-Trak Salmonella

Gene-Trak

46-50

Salmonella-Tek

Organon Teknika

50-56

Salmonella 1-2

BioControl Systems

Tecra Salmonella

Bioenterprises

46-52

Q-Trol Salmonella

Dynatech labs

44-50

Oxoid Salmonella Rapid Test

Oxoid

theoretically infinite volume of supercooled water. Sensitive assays for biological and inorganic ice nuclei have been known for some time 11-13. These assays measure either the temperature at which a sample containing many nuclei freezes, or the frequency with which small volumes of a dilute sample freeze. In the former case, the assay depends upon the empirical observation that, over certain temperature ranges, the highest temperature at which any ice nucleus functions is linearly related to the log of the overall nucleation frequency ~3. In the latter case, the small volumes contain, on average, no nuclei (unfrozen), or one nucleus (frozen), active at the assay temperature, and the nucleation frequency is simply calculated from the proportion 0£ samples that freeze n. In practice, assays to detect biological nuclei are carried out at approximately - 9°C to - 10°C. Above t h i s temperature, the nuclei assembled by many transduced bacteria are inactive due to insufficient size; the frequency of nucleation events w o u l d be less than one per cell and many cells would not be detected. At -9°C to -10°C every cell should have a biological nucleus and, at this temperature, inorganic nucleators would not make a significant contribution to the overall number of nuclei. An important feature of icenucleation assays is their sensitivity, (i.e. their ability to detect rare events). This was first noted during mapping of the inaW gene of P.

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Standard microbiological culture techniques DNA probe; radioisotope or colorimetric detection ELISA: colorimetric detection Immunodiffusion, immobilization; fluorescence ELISA; colorimetric detection Enzyme immunoassay, fluorescence Standard microbiology; latex agglutination

fluorescens by transposon mutagenesis 12. 'Leaky' phenotypes of certain Ina- mutants were observed, apparently caused by transposon excision and reversion to the Ina + phenotype at frequencies as low as one event per 107 cells. Since that discovery, ice nucleation genes and proteins have been used as sensitive probes of gene promoter activity 33, and as labels in conventional immunoassays 14. In addition, the detection of freezing events has been made easier by the discovery of dyes which fluoresce in supercooled water, but are quenched and change color when that water freezes 14. The BIND TM assay for Salmonella

Salmonella contamination of foods has long been a problem for the food industry and for the governmental agencies charged with regulating that industry. The genus Salmonella is a 'zero-tolerance' contaminant: detection of even one viable bacterium in 25 g of finished food is usually sufficient justification for rejection of the entire lot 1. Efforts to detect and classify Salmonella species have led to the establishment of well-accepted serotyping schemes 15, phage-typing schemes 16 and assay methods I for these pathogens. However, the standard method for detection of Salmonella (based on the US Food and Drug Administration (FDA) Bacterial Analysis Manual, and approved by the Association of Official Analytic Chemists: the 'BAM-AOAC' method) takes three to five days to complete 1. More rapid

2.00 8.75 5.00 10.00 6.25 3.00 6.00

methods based on DNA probes or monoclonal antibodies can reduce that time to slightly over two days 1. A summary of many of the currently available methods for detection of Salmonella is shown in Table 1. Most of these assays include two time-consuming steps: a 24-hour nonselective enrichment (to allow injured contaminating microorganisms to recover and multiply), followed by an 18-24 hour selective enrichment (to eliminate contaminating, non-target microorganisms). Any method which takes less than 24 hours must, therefore, detect, with great sensitivity, injured organisms in the presence of many nontarget bacteria. The BIND TM assay strategy (outlined in Fig. 1), was designed to increase the sensitivity and speed of bacterial assays, through the use of an ice nucleation reporter gene. The strategy is simple: an ice nucleation gene under the control of a strong promoter is introduced into the genome of a bacteriophage specific for a bacterial target (e.g. certain Salmonella species) by recombinant DNA techniques. The resulting transducing particles cannot themselves serve as ice nuclei: the BIND TM assay relies on infection of the bacterial host by the phage and the subsequent expression of the ice nucleation gene, for highly specific detection of viable Salmonella. Transducing particles and a freezing-indicator dye 14 are added to aliquots of a 1:10 (w/v) suspension of a sample suspected of containing the bacterial target. In prototype

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--Fig.1 assays, ten 50 ~1 samples and two negative (i.e. no phage) 50 ~1 controis are tested in one row of a 96well microtiter plate. After appropriate incubations to promote phage attachment and assembly of ice nuclei, the aliquots are chilled to -9.5°C, and examined for freezing (indicating the presence of host bacteria) or supercooling (indicating an uncontaminated sample). Because of the presence of the indicator dye, the frozen wells are orange and non-fluorescent, while the supercooled wells are green and fluorescent. The results can be recorded by eye, or by using a simple optical scanner. The frequency of freezing of aliquots may be used to calculate the degree of bacterial contamination from the relationship

d Bacterium

@

TransducingPhage

Transduction

C = V-1ln(NT/Nu) where C is the concentration of ice nuclei in the aliquots tested (proportional to the bacterial concentration), V is the aliquot volume, NT is the total number of aliquots, and Nu is the number of aliquots which remain unfrozen 11. Performance of the BIND T M assay A prototype BIND TM assay, based on the Salmonella phage P22, was tested using samples of culture m e d i u m or raw egg. A two-hour BIND TM assay was performed on i ml samples of culture medium or raw egg, to both of which Salmonella had been added, to give a range of bacterial concentrations. The number of bacterial cells per sample was evaluated by plating known volumes of sample, and numbers of ice nuclei were determined using a droplet freezing assay 11. Results with culture m e d i u m indicated that the phage carrying the ice nucleation gene (inaW) transduces the gene with high efficiency, and neither the viscosity nor the complex mixture of proteins present in egg interferes with the assay. Similar results have been obtained with other food materials such as milk, meat and gelatin. In all food samples tested to date, few or no background ice nuclei have been detected, indicating that conditions used to minimize bacterial pathogen contaminants generally render most foods ice nucleusflee. Further studies with an optimized

Expression Detection The BIND TM assay. A sample, possibly containing bacteria, is mixed with the phage, engineered to carry the ice nucleation gene, and incubated at 37°C to allow phage attachment and injection of phage DNA (transduction). The sample is then incubated at 23°C, to maximize assembly of ice nuclei from ice nucleation protein expressed from the transduced nucleation gene (expression). Finally, the sample is cooled to - 10°C, and monitored for freezing (detection).

assay protocol have shown that transducing particles constructed from phage P22 appear to have the same host specificity as the parent phage (i.e. they transduce Salmonella from serogroups A, B, and D, which display O-antigen 12 on their surfaces). The Salmonella species tested include S. typhimurium, S. paratyphi-B, S. dublin, S. enteritidis, S. gallinarum, and S. typhi; all were detected with the same high sensitivity (410 bacteria per ml). The transducing particles also detected as few as 10 S. dublin organisms per ml, in the presence of 107 non-target organisms per ml. No ice nuclei were formed when only the non-target bacteria (E. coli K12, B. subtilis, Citrobacter, or S. havana, a P22resistant Salmonella strain) were present: the need for a selective enrichment of the sample is thus obviated, since non-target organisms do not interfere with the assay.

Finally, a test of the ability of the BIND TM assay to detect Salmonella injured by heat-treatment is available. The assay detects injured, but not dead, organisms. This is very important in food contamination surveillance, since injured Salmonella may recover and cause disease, while dead Salmonella are relatively harmless. The probable reason for the ability of the BIND TM assay to detect injured organisms is the nature of the ice nucleation reporter gene. A bacterial cell need only produce 100-200 copies of the ice nucleation protein in order to assemble a nucleus active at -9°C (Ref. 10). Thus, a bacterial cell which is damaged but still alive is capable of forming an ice nucleus and being detected. Therefore, the non-selective enrichment step (which is included for recovery of injured cells) may be greatly shortened when using the BIND TM assay.

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Future prospects The specificity of the phage-host interaction confers a major advantage on the BIND TM assay. However, the application of the assay for the simultaneous detection of a range of pathogens (e.g. for food tests), will necessitate either the development of a broad-host-range phage, specific for several pathogens or, more feasibly, the combination of many specific phage in a single assay. Either approach would require optimization of the assay to establish no loss of sensitivity due to interference between components. Ice nucleation genes have been expressed in a wide variety of organisms; the BIND TM strategy should therefore be applicable to other foodborne pathogens such as Listeria, Campylobacter or E. coli, for which phage are known. In addition, certain clinical pathogens may be assayable with BIND TM. The dependence of the BIND TM assay on viable bacteria can also be used to measure the resistance of the target bacteria to one or more bactericides; samples pre-incubated with a drug lethal to

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the target never develop nuclei. Finally, the ease and rapidity of the BIND TM assay may lead to a resurgence in the use of phage-typing as a method of detailed identification of bacteria. Thus a time-honored method of bacteriology may be reborn, after being combined with the unrelated and esoteric field of bacterial ice nucleation. References 1 Veld, J. H. I., Hartog, B. and Hofstra, H. (1988) Food Rev. Int. 4, 271-329 2 Lindow, S. E. (1982) in Phytopathogenic Prokaryotes (Mount, M. S. and Lacy, G. H., eds), pp. 335-362, Academic Press 3 Warren, G. J. (1987) in Biotechnology and Genetic Engineering Reviews (Russell, G. E., ed.), pp. 107-135, Intercept 4 Wolber, P. K. and Warren, G. J. (1989) Trends Biochem. Sci. 14, 179-182 50rser, C., Staskawicz, B. J., Panopoulos, N. J., Dahlbeck, D. and Lindow, S. E., (1985) J. Bacteriol. 164, 359-366 6 Green, R. L. and Warren, G. J. (1985) Nature 317, 645-648 7 Warren, G. J., Corotto, L. and Wolber,

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P A U L K. W O L B E R R O B E R T L. GREEN

D N A Plant Technology Corporation, 6701 San Pablo A v e n u e , Oakland, CA 94608:1239, USA.

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Sense-antisense complementarity of hormone-receptor interaction sites In the June issue of TIBTECH, J. Edwin Blalock discusses the Molecular Recognition Theory 1. The theory states that not only do complementary nucleic acid sequences interact, but that in addition interacting sites in proteins are composed of complementary amino acid sequences (sense-antisense peptides). In this letter we present an interesting extension of the data. We studied sense-antisense complementarity of hormone and receptor binding sites of ten receptor-hormone combinations 2. Our ~tudy showed that hormone and receptor interaction sites share complementary sequences, e.g. the erythropoietin sequence 111-116 (ALGAQK), involved in receptor binding 3, is complementary to its

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P. K. (1986) Nucleic Acids Res. 14, 8047-8060 Warren, G. J. and Corotto, L. (1989) Gene 85, 239-242 Arai, S., Abe, K., Watabe, S., Emori, Y. and Watanabe, M. (1989) FEMS Microbio]. Lett. 61, 53-56 Watanabe, N. M., Southworth, M. W., Warren, G. J. and Wolber, P. K. Mol. Microbiol. (in press) Vali, G. (1971) J. Atmos. Sci. 28, 402-409 Corotto, L. V., Wolber, P. K. and Warren, G. J. (1986) EMBO J. 5, 231-236 Lindgren, P. B. et el. (1989) EMBO J. 8, 1291-1301 Warren, G. J. and Wolber, P. K. (1988) US Patent No. 4,784,943, US Patent Office Le Minor, L. (1981) in The Prokaryotes (Starr, M. P., Stolp, H., Trfiper, H. G., Balows, A. and Schlegel, H. G., eds), pp. 1148-1159, Springer-Verlag Gershman, M. (1977) J. Clin. Microbiol. 5, 302-314

receptor sequence 48-56 (LLCFTQR), suggested to be important in hormone binding 4, for which the antisense sequence is ALGEAQK5. A similar extensive homology has been found between interleukin-2 and its antisense receptorL However, in this case a 5' to 3' hormone sequence was compared to that of the 3' to 5' antisense receptor. Since 3' to 5' reading has not been observed in nature, the significance of the observed complementarity is not clear. In our study the natural 5' to 3' sense and antisense sequences were compared. The complementarity between hormone and receptor binding sites shows interesting features. In general, the homology of sense hormones to antisense receptors is

higher than that of receptors to antisense hormones. This might account for the negative reports in which antisense hormones do not bind to the receptor or in which no sequence homology has been found between antisense hormone sequences and sense sequences of the accompanying receptorsL Furthermore, three other interesting Observations were" made. (1) Sense-antisense complementarity between binding sites of receptors and hormones from different species is sometimes higher than within species, e.g. the antisense receptor sequence of the rat prolactin receptor shows a higher structural similarity to bovine than to rat prolactin 2. Similar results from hormone-zeceptor binding studies 6 suggest a functional significance of sense-antisense complementarity. (2) For Gprotein coupled receptors (Fig. 1), sense-antisense complementarity was found between the hormones and all four extracellular regions of the receptor. Thus, all four extracellular regions may be involved in hormone binding. (3) We found that