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Detection of bacteria using foreign DNA: the development of a bacteriophage reagent for Salmonella
International Journal of Food Microbiology 74 (2002) 229 – 238 www.elsevier.com/locate/ijfoodmicro
Detection of bacteria using foreign DNA: the development of a bacteriophage reagent for Salmonella Jonathan Kuhn a,*, Mordechai Suissa b, Joseph Wyse b, Ilana Cohen a, Irit Weiser b, Sarah Reznick b, Sharon Lubinsky-Mink a, Gordon Stewart b,c, Shimon Ulitzur d a
Department of Biology, Israel Institute of Technology, Technion, Haifa, 32000, Israel b Biolume Ltd., Haifa, Israel c Department of Applied Biochemistry and Food Science, University of Nottingham, Loughborough, UK d Department of Food Engineering, Israel Institute of Technology, Technion, Haifa, 32000, Israel Accepted 1 July 2001
Abstract A phage-based reagent was developed for the detection of Salmonella in food samples. The parental phage was Felix 01, which lyses practically all Salmonella. Using data obtained about the molecular biology of the phage, a recombinant phage that carried the bacterial genes specifying luciferase was produced. The method involved the isolation of amber nonsense mutations and subsequent crosses to render doubly mutant phage with a very low reversion rate on strains lacking an amber suppressor. A plasmid was constructed that contained a segment of Felix 01 DNA with two adjacent genes, one dispensable and the other essential, and their flanking sequences. Recombinant DNA technology was used to remove the two genes and the luxA and luxB genes for luciferase, and a gene specifying a tRNA that recognizes amber codons (supF = tyrT) was put in their stead. This region could be transferred into the genome of the phage by homologous recombination. The recombinant phage cannot grow because it lacks an essential gene. However, it can grow in a host that synthesizes the missing protein. This technique allows the construction of ‘‘locked’’ recombinant phages that carry foreign DNA but which cannot propagate themselves in nature. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Salmonella detection; Bacteriophage reagent; Felix 01
1. Introduction The rapid, sensitive and specific identification of pathogenic organisms is of great interest to the food microbiologist and a great deal of effort has been invested to this end. Classical techniques based on amplification of cell numbers and enrichment of
*
Corresponding author.
specific types with selective media are generally quite good with regard to sensitivity and specificity but lack the desired speed. The main obstacle to reducing the amount of time required for identification is the need to obtain a pure culture prior to definitive tests. Therefore, the main goal for improving this technology is to develop methods that do not require pure cultures and that can identify the target bacterium in raw samples. Concomitant with the ascendancy of molecular biology, attempts have been made to apply
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 6 8 3 - 3
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it to the rapid identification of various microbes of importance to the food industry. Among the techniques applied to this goal are labeled antibodies, PCR and nucleic acid hybridization. Techniques using specific antibodies generally suffer from a lack of sensitivity, while those employing PCR (polymerase chain reaction) or nucleic acid hybridization detect both alive and dead organisms. A very different approach was proposed by Ulitzur and Kuhn (1989) in which the detecting reagent is foreign DNA containing a reporter gene. Naked Neisseria DNA had previously been suggested for detecting Neisseria gonococcus (Bawdon et al., 1977). The three ways to introduce DNA into a bacterial cell are transformation, conjugation and transduction. The entry of foreign DNA via transformation or transfection is expected to lack specificity since many organisms can accept extracellular DNA. Thus, there is a good chance that besides the desired organism, related organisms which are of no interest will also be detected. In addition, restriction systems and the efficiency of transformation often differ between strains within a species. This means that the detection of some strains will be favored. Conjugation as a method for introducing DNA suffers from similar problems. In addition, some strains fail to act as recipients. These difficulties can be partly overcome by using bacteriophage as vectors of transmission (Ulitzur et al., 1989). Bacteriophage generally adsorbs specifically to their host and adsorption is very efficient. Many host-phage systems have an efficiency of plating approaching unity. This means that the entry of foreign DNA connected to a phage genome (i.e. transduction) should readily occur and this can form the basis of a test for a specific organism. The host range of many phage types is restricted to a single species and often to a subset of strains within a given species. To obtain species-wide coverage, it may be necessary to use more than one phage type. Conveniently, some phages carry anti-restriction systems which make the phage wholly or partially immune to restriction systems specified by the host. For these reasons, phages are the obvious choice when designing a species specific test based on the introduction of foreign DNA. Many bacteriophage have an obligatory lytic cycle while others have, in addition, an alternate pathway
(lysogeny) in which their DNA can become part of the bacterial complement. Phages of the latter type are somewhat easier to manipulate with respect to making them carriers of foreign reporter genes. However, as a result of lysogeny, their ability to escape the laboratory or in the field is much greater. In contrast, infection of bacterial cells by lytic phages leads to cell death unless the phage has been disabled by mutation. No stable phage – bacterial cell complex can be formed and their dispersal via bacterial cells does not occur. When this is coupled with deletion of an essential gene, the recombinant phage can be ‘‘locked’’ and its dispersal in nature entirely prevented.
2. Choice of a reporter gene Reporter genes can be isolated from a wide range of organisms. It is rarely desirable to use a gene from the test species because it is usually very difficult to reduce the preinfection background to a level that will allow the determination of low levels of organisms. The same can be said about reporter genes that might be found in organisms whose presence in a sample is irrelevant. For example, b-galactosidase is not synthesized by Salmonella but is synthesized by Escherichia coli. When E. coli is present, a test for Salmonella based on the synthesis of this enzyme from a lacZ reporter gene might give a positive result when there are no Salmonellae. It is therefore best to use a gene whose product is unlikely to be present in any sample. The green flourescent protein, the ice nucleation protein and bacterial luciferase (Meighen, 1994) fit this category. We have used the latter of these which is only found in a few aquatic organisms. Other bacteria are dark and emit no light. Since bacteriophages lack the ability to synthesize proteins on their own and have no metabolism, they are also dark. However, when a bacteriophage carrying the genes for luciferase infects a host in the presence of aldehyde, light will be emitted (Fig. 1). Present technology allows light to be measured with great sensitivity. In the presence of oxygen, bacterial luciferase converts FMNH2 and long chain aldehyde to FMN and a fatty acid while generating visible light. FMNH2 is a normal cellular constituent and aldehyde can be
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Fig. 1. Detection of specific bacteria by bacteriophage carrying the lux genes. The figure outlines the basic rationale for using a bacteriophage reagent to detect Salmonella in mixed cultures. In the uppermost part of the figure, the phage reagent (far left) carrying the lux genes for bacterial luciferase is mixed with a sample in which there are no Salmonellae. The phage fails to adsorb to the nonSalmonella cells and there is no expression of the lux genes and hence no light. In the middle section, the same reagent recognizes Salmonella but not Citrobacter, adsorbs and injects its DNA, which is then expressed. The luciferase that results leads to light emission in the presence of a long chain aldehyde. The bottom of the figure illustrates the manner in which the antibiotic sensitivities of a specific bacterial type can be determined using the same sample and phage reagent as that of the middle part of the figure. When the target bacterium, known to be present from the test in which light was emitted, is infected in the presence of an antibiotic to which it is sensitive, light output is reduced or abolished.
added extracellularly and readily diffuses into bacterial cells (Meighen, 1991).
3. Phage selection One of the main, if not the main, obstacles in producing a phage reagent carrying a reporter gene is finding a phage or phages that will cover a specific group or species without also detecting related nonpathogenic strains. A wide-range of phage types can be isolated against most species. Since recombinant DNA technology is to be employed, it is important that the phage genetic material be DNA containing the normal bases. Evaluation of the phage reagent will involve determining the number of false positives and negatives and this will be a function, in part, of the host range of the phage or phages included. Under ideal conditions, less than 50 bacteria per milliliter can be detected but poor adsorption may reduce this sensitivity. The obvious phage candidate for a Salmonella test is Felix 01, a virulent phage originally isolated by
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Felix and Callow (1943). The phage has also been called Felix 0-1 (Lindberg, 1967), Felix O (Subbaiah and Stocker, 1964), FO (Lindberg and Hellerqvist, 1971), O-1 (Fey et al., 1978), O1 (Takacs and Nagy, 1973), 0-I (He and Pan, 1992) and Sapphire (Bennett et al., 1997; Wolber and Green, 1990). At high titer, this phage lyses a high percentage of Salmonella and a few other enteric bacterial strains. Cherry et al. (1954) were the first to demonstrate the wide host range of Felix 01, hereafter referred to as F01, and to propose its use in diagnosing Salmonella in clinical microbiology. Subsequently a number of laboratories studied the efficacy of this method (Table 1) and improved it. Using a simple spot test, Kallings (1967) found that 98% of more than 2000 strains of Salmonella were lysed when a high titer phage stock was used while only a low percentage of nonSalmonellae were sensitive. At low titer, the percentage fell but the specificity increased. 51% of the strains plaqued F01 while the rest were killed but failed to propagate the phage. However, different strains were killed by different dilutions. When taken with the finding that phage-free lysates caused no lysis, this fact seems to indicate that killing of host cells is not the result of lysis from without (Kallings, 1967). In another study, Welkos et al. (1974) found that 98% of their sample of Salmonellae were lysed in spot tests while only 5.9% of E. coli were sensitive. Some Arizona strains were also lysed. In a similar manner, Gunnarsson et al. (1977) found 98% of 5287 Salmonella strains to be sensitive. In a series of studies, Fey et al. (Fey et al., 1961, 1971, 1978; Gudel and Fey, 1981) tested 22,880 strains and found 96% to be sensitive. Most resistant strains belonged to the E group of Salmonella. They developed a comprehensive test for Salmonella in which the use of F01 played a major role. In order to get Table 1 Sensitivity of Salmonella to F01 Strains examined
Sensitive strains
%
Reference
427 2996 652 5287 22,880 32,242
415 2935 640 5185 21,977 31,152
97.2 98.0 98.2 98.1 96.0 96.6
Cherry et al., 1954 Kallings and Lindberg, 1967 Welkos et al., 1974 Gunnarsson et al., 1977 Fey et al., 1978 Totals
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better coverage of the E group, a second phage, G47, was added to their test (Gudel and Fey, 1981). Thus, in a number of European countries, diagnosis of Salmonella by F01 has been routinely employed and this use has been extended to China by He and Pan (1992). Several groups (Bockemuhl, 1972; Takacs and Nagy, 1973) reported a lower percentage of lysis but this was apparently due to the lower titer of their lysates. Kallings and Lindberg (1967) examined the basis for resistance in certain Salmonella and found that, at least in some strains, this is due to the presence of prophages. On the basis of the high specificity of F01 for Salmonella, several other tests for this organism have been developed. Hirsch and Martin (1983a,b, 1984) used charged-modified filters to trap Salmonella and then selective culturing to enrich for these organisms in milk. Subsequently, they infected them with F01 and analyzed their samples for phage propagation by HPLC. More recently, Bennett et al. (1997) have prepared a biosorbent with immobilized F01 and used it to separate and concentrate Salmonella from foodstuffs. In the present communication, we describe the construction of a modified F01 phage carrying the genes for luciferase using data from the accompanying article. A method of preventing accidental release of this genetically modified phage into the environment is also described.
Because Salmonella is particularly difficult to transform, the restriction problem was solved by using a derivative of LT2 that is hsdR hsdM (r m ). This strains was received from the Salmonella Stock Center. Much of the DNA cloning work was done with E. coli strain TG1, which is r m Dlac-pro/ F’lacZDM15 proAB.
5. Mutagenesis Logarithmic cultures of K772 (sup + ) were grown in LB. N-nitroso-N0-nitro-N-methylguanidine (nitroso guanidine) was added to a final concentration of 50 mg/ml and then phage were added at an MOI of 5. After 20 –30 min, the infected culture was centrifuged in the cold at 10,000 rpm and washed twice with cold 10 mM MgSO4 and then resuspended in LB and incubation continued until lysis. After lysis, chloroform was added and the culture centrifuged to remove cellular debris. The lysate served as a source of mutagenized phage. About 1 amber mutant was found per 1000 plaques examined. Because F01 plaques are quite small, the method of visual identification of amber mutants on a mixed lawn of sup + and sup bacteria (Parkinson, 1968) was not used.
6. In vitro genetic manipulations 4. Methods 4.1. Medium Luria broth (10 g tryptone, 5 g yeast extract, 5 g NaCl, adjusted to pH 7.0 with 1 N NaOH) was used to culture bacterial strains (Maniatis et al., 1982). For the isolation of colonies, the same media contained 15 g of agar; when required, the plates were supplemented with 50 mg/ml of ampicillin. Plates for phage had 10 g agar while top-layer agar contained 7 g agar. 4.2. Bacterial strains LT2 is a standard laboratory strain of S. typhimurium and K772 is a Salmonella strain received from Fey that is particularly suitable for plating F01.
F01 DNA was isolated from liquid lysates on strain K772. After removal of cell debris by centrifugation at 10,000 rpm for 10 min in an SS34 rotor in a Sorvall RC5B centrifuge, the phages were pelleted by centrifugation at 20,000 rpm for 1 h. The pellet was resuspended in SM buffer (Maniatis et al., 1982) and 0.8 g of CsCl were added per milliliter of phage suspension. The solution was centrifuged overnight in an ultracentrifuge at 34,000 rpm in SW50 swinging buckets to form a CsCl equilibrium density gradient. The phage band was removed with a syringe, dialyzed against buffer and the DNA extracted with phenol. The DNA was precipitated with ethanol, washed and resuspended in TE buffer (Maniatis et al., 1982). The supF gene was subcloned from pIAN7 (Seed, 1983).
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7. Results 7.1. Attempts to create a F01-lux derivative by transportation Transposition as a method of producing recombinant phage is rapid but rather hit or miss. When successful, a transposed phage can be used as is or its properties as a detecting reagent can be assessed while construction of in vitro recombinants is still in progress. Both virulent and temperate phages are targets for transposition. Transposition of temperate phages using a transposon with a selectable marker such as an antibiotic resistance is usually fairly straightforward. Most phages contain non-essential genes or regions and the rare transposed phage are isolated as lysogens with selective methods. If the packaging limits of the phage head is very strict and the packaged phage genome has set ends, then the addition of several kilobases may lead to inviability. Another drawback to this method is that the transposed phage can propagate themselves outside the laboratory. The isolation of transposed virulent phages is more difficult since the host-phage association is transitory. The use of antibiotic resistance as a selective marker is very tricky because there may be only a small window between the time in which the cell has become resistant and its lysis. It is possible that other bacterial markers may prove better in this regard. For example, l bio carries the biotin genes but is unable to lysogenize because it lacks its int gene. When this phage is used to infect a biotin requiring host on plates, the result is that there is little background growth of cells on the plate but some haloes are formed. These haloes are due to cells which grow at the edge of the clear plaques on the biotin that was synthesized within the plaque and that has been released by cell lysis. An easier method may consist of a nonsense suppressor as the selective marker (Seed, 1983) in combination with phage amber mutants. Many transposons were constructed using the Kleckner plasmids (Way et al., 1984) which contain the ends of Tn10 and the Tn10 transposase under lac control. The regions transposed included sup, kanR, and lux. There is no selective regimen for phages carrying lux. However, such phages can be isolated
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using an instrument with a phototube, a small slit opening and on which plates can be rotated (Ulitzur and Kuhn, 2000). Some phages other than F01 carrying lux have been isolated in this manner. No transposed F01 were ever isolated eventhough very extensive searches were made. While this could be the result of either a lack of non-essential genes or failure to package DNA of increased length, neither of these possibilities is particularly attractive as an explanation for this failure. As described below, at least one gene of F01 is non-essential and there are intergenic regions that should serve as targets. Even though F01 DNA does have fixed ends, it is hard to imagine that addition of several kilobases would have such a dramatic effect since its genome is quite large (80 kb) and its virions are resistant to chelating agents.
8. Amber mutants of F01 Strain K772 suppresses amber mutations while LT2 does not. These type of mutations could be isolated after mutagenesis with nitrosoguanidine. The mutagenized phages were plated on K772 and plaques were picked to LT2 and K772. LT2 carried a plasmid conferring resistance to ampicillin which was used to prevent the carryover of K772 cells. Recombination and complementation between amber mutants was similar to that found with other phage. However, mutational analysis of this large genome will require a much more extensive set of mutants than what exists at present. Attempts to isolate deletions using nitrous acid mutagenesis or incubation with the chelating agents such as pyrophosphate and EDTA combined with various temperatures (Parkinson and Huskey, 1971) were unsuccessful. This phage is not sensitive to chelating agents.
9. Double amber mutants The reversion index of lysates of single amber mutants of F01 on LT2 (sup ) is 1 per 104 – 105. Since the incorporation of sup into the phage genome seems the best selective procedure for virulent phage, the relatively high rate of reversion of single mutants prevents the use of sup. However, the reversion of double mutants is the product of their reversion
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indexes and the resultant lack of reversion should allow the detection of rare recombinational events. Double mutants were prepared by crossing single mutants with one another. The progeny were plated on sup + to assess total progeny and on sup for wild type recombinants. The percentage of wild type phage gives an estimate of the frequency of double mutants. After crossing, plaques were picked from the plates with sup + bacteria and put in 1 ml of 10 mM MgSO4. A plaque usually contains 106 – 107 phage particles. After elution, half of the suspension was added to 2 ml of an early log phage culture of LT2. Wild type recombinants cause clearing of the culture quite rapidly while single mutants take 3 to 4 h longer since the number of revertants inoculated is quite small. Cultures that did not lyse had possibly been infected by double ambers in which there were no wild type revertants. In such cases, phages from the original elution tube were amplified on sup + bacteria and then tested for reversion and recombination against both of the parental mutations. Several combinations of double amber mutations were made and these had reversion indexes in the order of 10 8 – 10 9.
and in the correct orientation for transcription – translation. The cloned piece of F01 was checked for its ability to complement a deletion mutant of the E. coli phage l missing its exo, bet and gam genes. This l mutant is unable to grow on recA hosts which lack a normal recombination pathway (Zissler et al., 1971). This particular clone of F01 allowed this l to grow on a recA host. Therefore, the F01 segment is supplying the function missing in l, which in this case is known to be needed for normal replication. Subclones of this piece of F01 DNA showed that the complementing protein is encoded by ORF1. ORF1 has been renamed red because this region of l is often called by this name. In wild type recA + hosts, l can grow without these genes which are thereby termed non-essential. It seemed likely that the red gene of F01 would also be non-essential in wild type Salmonella hosts and, as shown below, this is in fact the case. A subclone of the 1 –5853 piece was made using XbaI that cleaves at base pair 4072 and EcoRI whose site is in the plasmid vector near the SmaI site (Fig. 2). This piece was cloned into a derivative of plasmid pBR322 cut with EcoRI and NheI and contains bases 1– 4072 of F01 and genes for ORF1– 5 and part of ORF6. The pBR322 derivative lacked the NdeI site of
10. Selection of region for substitution The F01 genome is approximately 80 kb and only part of it has been cloned and sequenced. The sequenced region shows the genes to be fairly tightly packed. There is no extended region that is noncoding and likely to be dispensable. In addition, the function of most of the genes is unknown. The luxA and luxB genes comprise 2.2 kb of DNA and the sup gene used (supF) is in a 244-bp segment. As shown in the accompanying article, the ends of F01 DNA are constant and a judgment about the strictness of packaging cannot be made. It is therefore desirable to avoid any packaging problems and this can be accomplished by deleting a region of F01 of the same length as luxAB –supF which is about 2.5 kb. A clone derived from F01 by cleavage with restriction endonuclease HaeIII contains bases 1 – 5853 of the sequenced region and the genes for ORF1 to ORF8. This segment was put into plasmid pHG165 (Stewart et al., 1986) via the SmaI site and the F01 genes are under control of the lac promoter
Fig. 2. Restriction enzyme cleavage map of F01. The DNA from F01 is represented by the thinner line; that of the plasmid vector by the thicker. Cleavage sites mentioned in the text are indicated. The EcoRI and SalI sites of the vector backbone are only a few base pairs distant from the SmaI site used in cloning. The coordinates of the open reading frames in this region is also presented and the direction of transcription – translation is from the lower to higher number.
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pBR322, which had been removed by filling in the site with E. coli DNA polymerase (Klenow fragment) and dNTP’s and then self-ligated. The subclone has unique sites at base 490 (BstXI) and at base 3103 (NdeI) of the F01 sequence and these sites were used to introduce the luxA – luxB genes from Vibrio fischeri and supF from E. coli (Fig. 2). In the appropriate clone, the bacterial genes are without a promoter but are in the same orientation as the phage genes and should become active when transferred to the phage chromosome.
11. Creation of F01-lux phage by recombination and selection If genetic transfer of these bacterial genes from the constructed plasmid into the phage genome is possible, it is expected to occur by homologous recombination (Fig. 3) between the flanking regions of F01 DNA in the plasmid, in this case bases 1 – 490 on one side and 3103 – 4072 on the other, and the phage chromosome. Some phages degrade the host DNA after their entry and no prediction with regard to F01 could be made as to whether recombination will take
Fig. 3. Creation of a lux carrying phage by recombination. The plasmid construct (A) contains the plasmid backbone given as a curved line, F01 DNA represented by a straight thin line, and bacterial DNA (lux and sup genes) represented by a thicker straight line. This can recombine with F01 DNA (illustrated as a long straight line) during infection by homologous recombination (x) to give a recombinant lacking some of the F01 genes but carrying the lux and sup genes. This phage can be grown on a strain containing a plasmid (B) which synthesizes the proteins of the missing phage genes.
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place. In the present example, recombinants were found but they were rare. Recombinants were also isolated when the V. fischeri lux genes were replaced by those of V. harveyi, which synthesize a luciferase with greater temperature tolerance. It remains an open question as to whether recombination could be greatly increased by lengthening the flanking regions which were relatively short in these experiments. However, it is clear that recombinants would not have been detected without a selective system. Recombinant phages carrying the lux and sup genes are missing all or part of genes red, ORF2, ORF3, ORF4 and ORF5. It is unknown whether all or some of these ORFs are essential for phage growth. Therefore, these products were supplied in trans by cloning a section of F01 DNA that contains these ORFs and attaching them to a bacterial promoter. The section of the F01 genome from bases 490 to 3103 was cloned into pHG171 (Stewart et al., 1986) to connect these genes to the lac promoter. When put in Salmonella, this plasmid should constitutively synthesize the relevant proteins because Salmonella lacks a lacI gene encoding the lac repressor. ORF5 extends from base 1524 to 3122 and probably codes for a protein of 532 amino acid residues. However, in the subclone, the DNA only extends to base 3103 and the last amino residues of the ORF5 protein are missing. This problem was solved by ligating a double stranded segment of 22 bp made from complementary synthetic oligonucleotides (one of 24 bases the other of 26) to the NdeI end before cloning. This restores the codons for the missing amino acids. The added piece creates a BamHI site at its distal end and codes for the same amino acids (TYR, GLU, GLY, LEU, ARG, ARG) that are missing in the BstXI –NdeI segment of F01. The added piece contains a stop codon at the end of the open reading frame. Since the genetic code is degenerate, all the above amino acids can be coded for by codons other than those found in the F01 sequence. The sequence of the oligonucleotides was changed so that it still coded for these amino acids but was as different as possible from the original sequence and therefore no longer homologous to F01 DNA. Therefore, eventhough this short section is outside the substituted region in the F01-lux phage and has an overlap with this phage, recombination should be prevented. This clone should synthesize
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the ORF2, 3, 4 and 5 proteins in their entirety, but not the red protein. After growth of double amber phage on the plasmid with lux and sup in place of F01 DNA, some plaques were formed on a host that lacks a suppressor gene but which contains the plasmid synthesizing the ORF proteins. After purification, some phage grow without the complementing plasmid and these are phage in which both amber mutations have reverted. Other phage fail to grow on a strain lacking both a suppressor and the complementing plasmid but do grow when the plasmid is present. These are recombinants that carry the lux and sup genes incorporated into the F01 genome. When used to infect Salmonella without the complementing plasmid, they fail to complete a life cycle but do lead to light emission! This result shows that red is not an essential gene since the recombinants lack much of this gene and grow in the absence of the red protein, which is not supplied by the complementing plasmid. At least one or more of the ORFs must be essential but we have not investigated this further. As mentioned in the accompanying article, there are no Sau3AI sites (GATC) in F01 DNA extracted from the virion and no such sites were found in the sequenced region. The lux – sup substitution contains both a BamHI site (GGATCC) and some Sau3AI sites. This indicates that F01 probably does not synthesize a restriction enzyme recognizing GATC. The extent of the deletion in F01 DNA is of the same length as the piece used to complement this missing piece. The short 21-bp overlap that was introduced at the end of the ORF5 gene is dissimilar to the original sequence. Therefore, homologous recombination between the recombinant F01 carrying lux and the complementing plasmid should be entirely prevented and should not lead to the formation of wild type recombinants. The recombinant phage is thus ‘‘locked’’ and does not need to be reisolated at frequent intervals and does not represent an ecological danger.
12. Discussion The use of foreign DNA for the detection of specific bacterial types requires a method that will give both great specificity and a high efficiency of
DNA transfer to the target bacteria. Bacteriophage are suitable on both counts. Therefore, the development of a phage reagent depends, first of all, on finding a phage or phages with the required host range. Such phages have been found for certain bacteria of industrial and/or medical importance. A second element in this approach is the selection of an appropriate reporter gene. The reporter protein should not be found among the bacteria likely to be in the sample and should be readily detectable. In the present research, bacterial luciferase was chosen but there exists a number of other possibilities in this regard, some of them arguably better. In the present research, a general method to create recombinant phage reagents was developed and it should be applicable to most or all phages. tRNAs are well conserved within the eubacteria and mutant tRNAs that allow the translation of the UAG nonsense codon (amber suppressors) have been known for many years. These suppressors can be introduced into many species by transformation and they allow the isolation of amber mutants of the phage or phages to be included in the reagent. To reduce or eliminate reversion, double amber mutants can be isolated from genetic crosses. Since the double amber mutants cannot grow in the absence of a suppressor gene, they can be used to select for rare recombinants which have incorporated a sup gene into their own genome. The sup gene can be associated with a reporter gene to concomitantly introduce this latter gene. In order to apply the method, some basic molecular biological information about the phage is necessary and the results of these studies are reported in the accompanying article. Although the genes of F01 were found to be quite tightly packed in the region sequenced, a non-essential gene was discovered and this allowed the substitution of foreign DNA in a way that neither increased the size of the phage genome nor allowed loss of the insert by recombination. In cases which lack both a convenient gene arrangement and nicely placed restriction enzyme recognition sites, appropriate sites can be introduced by site directed mutagenesis. PCR amplification can be used to isolate the exact section that has been substituted or preferably even a smaller one which encodes the missing gene products. While success is by no means guaranteed, it is likely result if several different regions are subjected to this approach.
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The method of ‘‘locking’’ the recombinant phage that is described here should prevent the loss of the foreign DNA during reagent preparation and also greatly lower the probability of the escape of such phage into nature. However, this locking method does not entirely eliminate the possibility of the emergence of a recombinant phage that can grow without the complementing plasmid. While deletion mutants cannot yield true revertants, alternate pathways or bypass mutation can sometimes occur. For example, deletion of the lacZ gene specifying bgalactosidase in E. coli leads to the loss of the ability to utilize lactose as a carbon source. Phenotypic reversion can occur by mutation at a second locus, ebg, which leads to the synthesis of an alternate enzyme with this activity (Campbell et al., 1973). Similarly, deletion mutants of phage l that lack the red region fail to grow on recA strains but pseudorevertants have been isolated. These have picked up a section of the bacterial chromosome which is a remnant of a prophage genome from a different phage (Campbell, 1996). Although N mutants of l fail to grow, bypass mutations that remove this requirement can be isolated (Court and Sato, 1969). It is therefore clear that after a phage is apparently locked, it must be exposed to different host strains to determine whether phenotypic reversion can occur by one of these mechanisms. The possibility of recombination in nature with a wild type phage of the same kind cannot be ascertained. However, normal recombination will simply remove the foreign DNA and is hence of no consequence. In contrast, illegitimate recombination with such a wild type phage or with a different phage type might allow pseudo wild type revertants to arise but this should be an exceedingly rare event. The F01 reagent developed here detected a wide range of Salmonella strains. However, it was found that different strains produce different amounts of light per cell and it appears that this may be due, in part, to variation in rate of adsorption. To overcome this, the ice nucleation gene was subsequently substituted for lux using the same method and plasmids described above (Wolber and Green, 1990). Only one molecule of the ice nucleation protein needs to be expressed for a positive response to ensue (Wolber, 1993) and this is the basis for the commercial test using F01.
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References Bawdon, R.E., Juni, E., Britt, E.M., 1977. Identification of Neisseria gonorrhoeae by genetic transformation: a clinical laboratory evaluation. Journal of Clinical Microbiology 5, 108 – 109. Bennett, A.R., Davids, F.G.C., Vlahodimou, S., Banks, J.G., Betts, R.P., 1997. The use of bacteriophage-based systems for the separation and concentration of Salmonella. Journal of Applied Microbiology 83, 259 – 265. Bockemuhl, J., 1972. Die lysosensibilitat von stammen der Salmonella subgenera I – IV gegenuber dem phagen 0-1. Medical Microbiology and Immunology 158, 44 – 53. Campbell, A.M., 1996. Cryptic prophages. In: Neidhardt, F., Curtiss III, R., Ingraham, J.L., Lin, E.C.C., Low, K.B., Magasanik, B., Reznikoff, W.S., Riley, M., Schaechter, M., Umbarger, H.E. (Eds.), Escherichia coli and Salmonella. American Society for Microbiology Press, Washington, USA, pp. 2041 – 2046. Campbell, J.H., Lengyel, J., Langridge, J., 1973. Evolution of a second gene for b-galactosidase in Escherichia coli. Proceedings of the National Academy of Science of the United States of America 70, 1841 – 1845. Cherry, W.B., Davis, B.R., Edwards, P.R., Hogan, R.B., 1954. A simple procedure for the identification of the genus Salmonella by means of a specific bacteriophage. Journal of Laboratory Clinical Medicine 44, 51 – 55. Court, D., Sato, K., 1969. Studies of novel transducing variants of lambda: dispensability of genes N and Q. Virology 39, 348 – 352. Felix, A., Callow, B.R., 1943. Typing of paratyphoid B bacilli by means of Vi bacteriophage. British Medical Journal 2, 127 – 130. Fey, H., Schweizer, R., Margadant, A., 1961. Erfahrungen mit dem polyvalenten Salmonella-phag 0-1. Roentgen- und Laboratoriumspraxis XIV, L179 – L185. Fey, H., Lozano-Gutknecht, C., Margadant, A., 1971. Eine rationelle massendiagnostik von Salmonella (Shigella). Zentrablat fuer Bakteriology, Parasitenkology, Infektionskrankheiten und Hygeine, Abteilung 1: Originale, Reihe A 218, 376 – 389. Fey, H., Burgi, E., Margadant, A., Boller, E., 1978. An economic and rapid diagnostic procedure for the detection of Salmonella/ Shigella using the polyvalent Salmonella phage 0-1. Zentrablat fuer Bakteriology, Mikrobiologie und Hygeine, Abt. I, Originale A 240, 7 – 15. Gudel, K., Fey, H., 1981. Improvement of the polyvalent Salmonella phage’s 0-1 diagnostic value by addition of a phage specific for the O groups E1 – E4. Zentrablat fuer Bakteriology, Mikrobiologie und Hygeine, Abt. I, Originale A 249, 220 – 224. Gunnarsson, A., Hurvell, B., Thal, E., 1977. Recent experiences with salmonella-0-1-phage in routine diagnostic work. Zentrablat fuer Bakteriology, Parasitenkology, Infektionskrankheiten und Hygeine, Abteilung 1: Originale, Reihe A 237, 222 – 227. He, Q.X., Pan, R.N., 1992. Bacteriophage lytic patterns for identification of salmonellae, shigellae, Escherichia coli, Citrobacter freundii, and Enterobacter cloacae. Journal of Clinical Microbiology 30, 590 – 594. Hirsch, D.C., Martin, L.D., 1983a. Rapid detection of Salmonella spp. by using Felix-01 bacteriophage and high-performance liquid chromatography. Applied and Environmental Microbiology 45, 260 – 264.
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Hirsch, D.C., Martin, L.D., 1983b. Detection of Salmonella spp. in milk by using Felix-01 bacteriophage and high-pressure liquid chromatography. Applied and Environmental Microbiology 46, 1243 – 1245. Hirsch, D.C., Martin, L.D., 1984. Rapid detection of Salmonella in certified raw mild by using charge-modified filters and Felix-01 bacteriophage. Journal of Food Protection 47, 388 – 390. Kallings, L.O., 1967. Sensitivity of various salmonella strains to Felix-0-1 phage. Acta Pathologica et Microbiologica Scandinavica 70, 446 – 454. Kallings, L.O., Lindberg, A.A., 1967. Resistance to Felix 0-1 phage in Salmonella bacteria. Acta Pathologica et Microbiologica Scandinavica 70, 455 – 460. Lindberg, A.A., 1967. Studies of a receptor for Felix 0-1 phage in Salmonella minnesota. Journal of General Microbiology 48, 225 – 233. Lindberg, A.A., Hellerqvist, C.G., 1971. Bacteriophage attachment sites, serological specificity, and chemical composition of the lipopolysaccharides of semirough and rough mutants of Salmonella typhimurium. Journal of Bacteriology 105, 57 – 64. Maniatis, T., Fritsch, E.F., Sambrook, J., 1982. Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, pp. 68 – 73. Meighen, E.A., 1991. Molecular biology of bacterial bioluminescence. Microbiological Reviews 55, 123 – 142. Meighen, E., 1994. Genetics of bacterial bioluminescence. Annual Review of Genetics 28, 117 – 139. Parkinson, J.S., 1968. Genetics of the left arm of the chromosome of bacteriophage lambda. Genetics 59, 311 – 325. Parkinson, J.S., Huskey, R.J., 1971. Deletion mutants of bacteriophage lambda: I. Isolation and initial characterization. Journal of Molecular Biology 56, 369 – 384. Seed, B., 1983. Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo. Nucleic Acids Research 11, 2427 – 2445. Stewart, G.S.A.B., Lubinsky-Mink, S., Jackson, C.G., Cassel, A.,
Kuhn, J., 1986. pHG165: a pBR322 copy number derivative of pUC8 for cloning and expression. Plasmid 15, 172 – 181. Subbaiah, T.V., Stocker, B.A.D., 1964. Rough mutants of Salmonella typhimurium. Nature 201, 1298 – 1299. Takacs, J., Nagy, G.B., 1973. The value of the 01 and R Salmonella phage tests for routine laboratory examinations. Acta Veterinaria Academiae Scientiarum Hungaricae 23, 95 – 118. Ulitzur, S., Kuhn, J., 1989. Detection and/or identification of microorganisms in a test sample using bioluminescence or other exogenous genetically introduced marker. US Patent 4,861,709. Ulitzur, S., Kuhn, J., 2000. Construction of lux bacteriophages and the determination of specific bacteria and their antibiotic sensitivities. Methods in Enzymology 305, 543 – 557. Ulitzur, S., Suissa, M., Kuhn, J.C., 1989. A new approach for the specific determination of bacteria and their antimicrobial susceptibilities. In: Balows, A., Tilton, R.C., Turano, A. (Eds.), Rapid Methods and Automation in Microbiology and Immunology. Brixia Academic Press, Brescia, Italy, pp. 235 – 240. Way, J.C., Davis, M.A., Morisato, D., Roberts, D.E., Kleckner, N., 1984. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene 32, 369 – 379. Welkos, S., Schreiber, M., Baer, H., 1974. Identification of Salmonella with the 0-1 bacteriophage. Applied Microbiology 28, 618 – 622. Wolber, P.K., 1993. Bacterial ice nucleation. Advances in Microbial Physiology 34, 203 – 237. Wolber, P.K., Green, R.L., 1990. New rapid method for the detection of Salmonella in foods. Trends in Food Science and Technology 1, 80 – 82. Zissler, J., Signer, E., Schaefer, F., 1971. The role of recombination in growth of bacteriophage lambda: I. The gamma gene. In: Hershey, A.D. (Ed.), The Bacteriophage Lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, pp. 455 – 475.
Detection of bacteria using foreign DNA: the development of a bacteriophage reagent for Salmonella Jonathan Kuhn a,*, Mordechai Suissa b, Joseph Wyse b, Ilana Cohen a, Irit Weiser b, Sarah Reznick b, Sharon Lubinsky-Mink a, Gordon Stewart b,c, Shimon Ulitzur d a
Department of Biology, Israel Institute of Technology, Technion, Haifa, 32000, Israel b Biolume Ltd., Haifa, Israel c Department of Applied Biochemistry and Food Science, University of Nottingham, Loughborough, UK d Department of Food Engineering, Israel Institute of Technology, Technion, Haifa, 32000, Israel Accepted 1 July 2001
Abstract A phage-based reagent was developed for the detection of Salmonella in food samples. The parental phage was Felix 01, which lyses practically all Salmonella. Using data obtained about the molecular biology of the phage, a recombinant phage that carried the bacterial genes specifying luciferase was produced. The method involved the isolation of amber nonsense mutations and subsequent crosses to render doubly mutant phage with a very low reversion rate on strains lacking an amber suppressor. A plasmid was constructed that contained a segment of Felix 01 DNA with two adjacent genes, one dispensable and the other essential, and their flanking sequences. Recombinant DNA technology was used to remove the two genes and the luxA and luxB genes for luciferase, and a gene specifying a tRNA that recognizes amber codons (supF = tyrT) was put in their stead. This region could be transferred into the genome of the phage by homologous recombination. The recombinant phage cannot grow because it lacks an essential gene. However, it can grow in a host that synthesizes the missing protein. This technique allows the construction of ‘‘locked’’ recombinant phages that carry foreign DNA but which cannot propagate themselves in nature. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Salmonella detection; Bacteriophage reagent; Felix 01
1. Introduction The rapid, sensitive and specific identification of pathogenic organisms is of great interest to the food microbiologist and a great deal of effort has been invested to this end. Classical techniques based on amplification of cell numbers and enrichment of
*
Corresponding author.
specific types with selective media are generally quite good with regard to sensitivity and specificity but lack the desired speed. The main obstacle to reducing the amount of time required for identification is the need to obtain a pure culture prior to definitive tests. Therefore, the main goal for improving this technology is to develop methods that do not require pure cultures and that can identify the target bacterium in raw samples. Concomitant with the ascendancy of molecular biology, attempts have been made to apply
0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 ( 0 1 ) 0 0 6 8 3 - 3
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it to the rapid identification of various microbes of importance to the food industry. Among the techniques applied to this goal are labeled antibodies, PCR and nucleic acid hybridization. Techniques using specific antibodies generally suffer from a lack of sensitivity, while those employing PCR (polymerase chain reaction) or nucleic acid hybridization detect both alive and dead organisms. A very different approach was proposed by Ulitzur and Kuhn (1989) in which the detecting reagent is foreign DNA containing a reporter gene. Naked Neisseria DNA had previously been suggested for detecting Neisseria gonococcus (Bawdon et al., 1977). The three ways to introduce DNA into a bacterial cell are transformation, conjugation and transduction. The entry of foreign DNA via transformation or transfection is expected to lack specificity since many organisms can accept extracellular DNA. Thus, there is a good chance that besides the desired organism, related organisms which are of no interest will also be detected. In addition, restriction systems and the efficiency of transformation often differ between strains within a species. This means that the detection of some strains will be favored. Conjugation as a method for introducing DNA suffers from similar problems. In addition, some strains fail to act as recipients. These difficulties can be partly overcome by using bacteriophage as vectors of transmission (Ulitzur et al., 1989). Bacteriophage generally adsorbs specifically to their host and adsorption is very efficient. Many host-phage systems have an efficiency of plating approaching unity. This means that the entry of foreign DNA connected to a phage genome (i.e. transduction) should readily occur and this can form the basis of a test for a specific organism. The host range of many phage types is restricted to a single species and often to a subset of strains within a given species. To obtain species-wide coverage, it may be necessary to use more than one phage type. Conveniently, some phages carry anti-restriction systems which make the phage wholly or partially immune to restriction systems specified by the host. For these reasons, phages are the obvious choice when designing a species specific test based on the introduction of foreign DNA. Many bacteriophage have an obligatory lytic cycle while others have, in addition, an alternate pathway
(lysogeny) in which their DNA can become part of the bacterial complement. Phages of the latter type are somewhat easier to manipulate with respect to making them carriers of foreign reporter genes. However, as a result of lysogeny, their ability to escape the laboratory or in the field is much greater. In contrast, infection of bacterial cells by lytic phages leads to cell death unless the phage has been disabled by mutation. No stable phage – bacterial cell complex can be formed and their dispersal via bacterial cells does not occur. When this is coupled with deletion of an essential gene, the recombinant phage can be ‘‘locked’’ and its dispersal in nature entirely prevented.
2. Choice of a reporter gene Reporter genes can be isolated from a wide range of organisms. It is rarely desirable to use a gene from the test species because it is usually very difficult to reduce the preinfection background to a level that will allow the determination of low levels of organisms. The same can be said about reporter genes that might be found in organisms whose presence in a sample is irrelevant. For example, b-galactosidase is not synthesized by Salmonella but is synthesized by Escherichia coli. When E. coli is present, a test for Salmonella based on the synthesis of this enzyme from a lacZ reporter gene might give a positive result when there are no Salmonellae. It is therefore best to use a gene whose product is unlikely to be present in any sample. The green flourescent protein, the ice nucleation protein and bacterial luciferase (Meighen, 1994) fit this category. We have used the latter of these which is only found in a few aquatic organisms. Other bacteria are dark and emit no light. Since bacteriophages lack the ability to synthesize proteins on their own and have no metabolism, they are also dark. However, when a bacteriophage carrying the genes for luciferase infects a host in the presence of aldehyde, light will be emitted (Fig. 1). Present technology allows light to be measured with great sensitivity. In the presence of oxygen, bacterial luciferase converts FMNH2 and long chain aldehyde to FMN and a fatty acid while generating visible light. FMNH2 is a normal cellular constituent and aldehyde can be
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Fig. 1. Detection of specific bacteria by bacteriophage carrying the lux genes. The figure outlines the basic rationale for using a bacteriophage reagent to detect Salmonella in mixed cultures. In the uppermost part of the figure, the phage reagent (far left) carrying the lux genes for bacterial luciferase is mixed with a sample in which there are no Salmonellae. The phage fails to adsorb to the nonSalmonella cells and there is no expression of the lux genes and hence no light. In the middle section, the same reagent recognizes Salmonella but not Citrobacter, adsorbs and injects its DNA, which is then expressed. The luciferase that results leads to light emission in the presence of a long chain aldehyde. The bottom of the figure illustrates the manner in which the antibiotic sensitivities of a specific bacterial type can be determined using the same sample and phage reagent as that of the middle part of the figure. When the target bacterium, known to be present from the test in which light was emitted, is infected in the presence of an antibiotic to which it is sensitive, light output is reduced or abolished.
added extracellularly and readily diffuses into bacterial cells (Meighen, 1991).
3. Phage selection One of the main, if not the main, obstacles in producing a phage reagent carrying a reporter gene is finding a phage or phages that will cover a specific group or species without also detecting related nonpathogenic strains. A wide-range of phage types can be isolated against most species. Since recombinant DNA technology is to be employed, it is important that the phage genetic material be DNA containing the normal bases. Evaluation of the phage reagent will involve determining the number of false positives and negatives and this will be a function, in part, of the host range of the phage or phages included. Under ideal conditions, less than 50 bacteria per milliliter can be detected but poor adsorption may reduce this sensitivity. The obvious phage candidate for a Salmonella test is Felix 01, a virulent phage originally isolated by
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Felix and Callow (1943). The phage has also been called Felix 0-1 (Lindberg, 1967), Felix O (Subbaiah and Stocker, 1964), FO (Lindberg and Hellerqvist, 1971), O-1 (Fey et al., 1978), O1 (Takacs and Nagy, 1973), 0-I (He and Pan, 1992) and Sapphire (Bennett et al., 1997; Wolber and Green, 1990). At high titer, this phage lyses a high percentage of Salmonella and a few other enteric bacterial strains. Cherry et al. (1954) were the first to demonstrate the wide host range of Felix 01, hereafter referred to as F01, and to propose its use in diagnosing Salmonella in clinical microbiology. Subsequently a number of laboratories studied the efficacy of this method (Table 1) and improved it. Using a simple spot test, Kallings (1967) found that 98% of more than 2000 strains of Salmonella were lysed when a high titer phage stock was used while only a low percentage of nonSalmonellae were sensitive. At low titer, the percentage fell but the specificity increased. 51% of the strains plaqued F01 while the rest were killed but failed to propagate the phage. However, different strains were killed by different dilutions. When taken with the finding that phage-free lysates caused no lysis, this fact seems to indicate that killing of host cells is not the result of lysis from without (Kallings, 1967). In another study, Welkos et al. (1974) found that 98% of their sample of Salmonellae were lysed in spot tests while only 5.9% of E. coli were sensitive. Some Arizona strains were also lysed. In a similar manner, Gunnarsson et al. (1977) found 98% of 5287 Salmonella strains to be sensitive. In a series of studies, Fey et al. (Fey et al., 1961, 1971, 1978; Gudel and Fey, 1981) tested 22,880 strains and found 96% to be sensitive. Most resistant strains belonged to the E group of Salmonella. They developed a comprehensive test for Salmonella in which the use of F01 played a major role. In order to get Table 1 Sensitivity of Salmonella to F01 Strains examined
Sensitive strains
%
Reference
427 2996 652 5287 22,880 32,242
415 2935 640 5185 21,977 31,152
97.2 98.0 98.2 98.1 96.0 96.6
Cherry et al., 1954 Kallings and Lindberg, 1967 Welkos et al., 1974 Gunnarsson et al., 1977 Fey et al., 1978 Totals
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better coverage of the E group, a second phage, G47, was added to their test (Gudel and Fey, 1981). Thus, in a number of European countries, diagnosis of Salmonella by F01 has been routinely employed and this use has been extended to China by He and Pan (1992). Several groups (Bockemuhl, 1972; Takacs and Nagy, 1973) reported a lower percentage of lysis but this was apparently due to the lower titer of their lysates. Kallings and Lindberg (1967) examined the basis for resistance in certain Salmonella and found that, at least in some strains, this is due to the presence of prophages. On the basis of the high specificity of F01 for Salmonella, several other tests for this organism have been developed. Hirsch and Martin (1983a,b, 1984) used charged-modified filters to trap Salmonella and then selective culturing to enrich for these organisms in milk. Subsequently, they infected them with F01 and analyzed their samples for phage propagation by HPLC. More recently, Bennett et al. (1997) have prepared a biosorbent with immobilized F01 and used it to separate and concentrate Salmonella from foodstuffs. In the present communication, we describe the construction of a modified F01 phage carrying the genes for luciferase using data from the accompanying article. A method of preventing accidental release of this genetically modified phage into the environment is also described.
Because Salmonella is particularly difficult to transform, the restriction problem was solved by using a derivative of LT2 that is hsdR hsdM (r m ). This strains was received from the Salmonella Stock Center. Much of the DNA cloning work was done with E. coli strain TG1, which is r m Dlac-pro/ F’lacZDM15 proAB.
5. Mutagenesis Logarithmic cultures of K772 (sup + ) were grown in LB. N-nitroso-N0-nitro-N-methylguanidine (nitroso guanidine) was added to a final concentration of 50 mg/ml and then phage were added at an MOI of 5. After 20 –30 min, the infected culture was centrifuged in the cold at 10,000 rpm and washed twice with cold 10 mM MgSO4 and then resuspended in LB and incubation continued until lysis. After lysis, chloroform was added and the culture centrifuged to remove cellular debris. The lysate served as a source of mutagenized phage. About 1 amber mutant was found per 1000 plaques examined. Because F01 plaques are quite small, the method of visual identification of amber mutants on a mixed lawn of sup + and sup bacteria (Parkinson, 1968) was not used.
6. In vitro genetic manipulations 4. Methods 4.1. Medium Luria broth (10 g tryptone, 5 g yeast extract, 5 g NaCl, adjusted to pH 7.0 with 1 N NaOH) was used to culture bacterial strains (Maniatis et al., 1982). For the isolation of colonies, the same media contained 15 g of agar; when required, the plates were supplemented with 50 mg/ml of ampicillin. Plates for phage had 10 g agar while top-layer agar contained 7 g agar. 4.2. Bacterial strains LT2 is a standard laboratory strain of S. typhimurium and K772 is a Salmonella strain received from Fey that is particularly suitable for plating F01.
F01 DNA was isolated from liquid lysates on strain K772. After removal of cell debris by centrifugation at 10,000 rpm for 10 min in an SS34 rotor in a Sorvall RC5B centrifuge, the phages were pelleted by centrifugation at 20,000 rpm for 1 h. The pellet was resuspended in SM buffer (Maniatis et al., 1982) and 0.8 g of CsCl were added per milliliter of phage suspension. The solution was centrifuged overnight in an ultracentrifuge at 34,000 rpm in SW50 swinging buckets to form a CsCl equilibrium density gradient. The phage band was removed with a syringe, dialyzed against buffer and the DNA extracted with phenol. The DNA was precipitated with ethanol, washed and resuspended in TE buffer (Maniatis et al., 1982). The supF gene was subcloned from pIAN7 (Seed, 1983).
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7. Results 7.1. Attempts to create a F01-lux derivative by transportation Transposition as a method of producing recombinant phage is rapid but rather hit or miss. When successful, a transposed phage can be used as is or its properties as a detecting reagent can be assessed while construction of in vitro recombinants is still in progress. Both virulent and temperate phages are targets for transposition. Transposition of temperate phages using a transposon with a selectable marker such as an antibiotic resistance is usually fairly straightforward. Most phages contain non-essential genes or regions and the rare transposed phage are isolated as lysogens with selective methods. If the packaging limits of the phage head is very strict and the packaged phage genome has set ends, then the addition of several kilobases may lead to inviability. Another drawback to this method is that the transposed phage can propagate themselves outside the laboratory. The isolation of transposed virulent phages is more difficult since the host-phage association is transitory. The use of antibiotic resistance as a selective marker is very tricky because there may be only a small window between the time in which the cell has become resistant and its lysis. It is possible that other bacterial markers may prove better in this regard. For example, l bio carries the biotin genes but is unable to lysogenize because it lacks its int gene. When this phage is used to infect a biotin requiring host on plates, the result is that there is little background growth of cells on the plate but some haloes are formed. These haloes are due to cells which grow at the edge of the clear plaques on the biotin that was synthesized within the plaque and that has been released by cell lysis. An easier method may consist of a nonsense suppressor as the selective marker (Seed, 1983) in combination with phage amber mutants. Many transposons were constructed using the Kleckner plasmids (Way et al., 1984) which contain the ends of Tn10 and the Tn10 transposase under lac control. The regions transposed included sup, kanR, and lux. There is no selective regimen for phages carrying lux. However, such phages can be isolated
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using an instrument with a phototube, a small slit opening and on which plates can be rotated (Ulitzur and Kuhn, 2000). Some phages other than F01 carrying lux have been isolated in this manner. No transposed F01 were ever isolated eventhough very extensive searches were made. While this could be the result of either a lack of non-essential genes or failure to package DNA of increased length, neither of these possibilities is particularly attractive as an explanation for this failure. As described below, at least one gene of F01 is non-essential and there are intergenic regions that should serve as targets. Even though F01 DNA does have fixed ends, it is hard to imagine that addition of several kilobases would have such a dramatic effect since its genome is quite large (80 kb) and its virions are resistant to chelating agents.
8. Amber mutants of F01 Strain K772 suppresses amber mutations while LT2 does not. These type of mutations could be isolated after mutagenesis with nitrosoguanidine. The mutagenized phages were plated on K772 and plaques were picked to LT2 and K772. LT2 carried a plasmid conferring resistance to ampicillin which was used to prevent the carryover of K772 cells. Recombination and complementation between amber mutants was similar to that found with other phage. However, mutational analysis of this large genome will require a much more extensive set of mutants than what exists at present. Attempts to isolate deletions using nitrous acid mutagenesis or incubation with the chelating agents such as pyrophosphate and EDTA combined with various temperatures (Parkinson and Huskey, 1971) were unsuccessful. This phage is not sensitive to chelating agents.
9. Double amber mutants The reversion index of lysates of single amber mutants of F01 on LT2 (sup ) is 1 per 104 – 105. Since the incorporation of sup into the phage genome seems the best selective procedure for virulent phage, the relatively high rate of reversion of single mutants prevents the use of sup. However, the reversion of double mutants is the product of their reversion
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indexes and the resultant lack of reversion should allow the detection of rare recombinational events. Double mutants were prepared by crossing single mutants with one another. The progeny were plated on sup + to assess total progeny and on sup for wild type recombinants. The percentage of wild type phage gives an estimate of the frequency of double mutants. After crossing, plaques were picked from the plates with sup + bacteria and put in 1 ml of 10 mM MgSO4. A plaque usually contains 106 – 107 phage particles. After elution, half of the suspension was added to 2 ml of an early log phage culture of LT2. Wild type recombinants cause clearing of the culture quite rapidly while single mutants take 3 to 4 h longer since the number of revertants inoculated is quite small. Cultures that did not lyse had possibly been infected by double ambers in which there were no wild type revertants. In such cases, phages from the original elution tube were amplified on sup + bacteria and then tested for reversion and recombination against both of the parental mutations. Several combinations of double amber mutations were made and these had reversion indexes in the order of 10 8 – 10 9.
and in the correct orientation for transcription – translation. The cloned piece of F01 was checked for its ability to complement a deletion mutant of the E. coli phage l missing its exo, bet and gam genes. This l mutant is unable to grow on recA hosts which lack a normal recombination pathway (Zissler et al., 1971). This particular clone of F01 allowed this l to grow on a recA host. Therefore, the F01 segment is supplying the function missing in l, which in this case is known to be needed for normal replication. Subclones of this piece of F01 DNA showed that the complementing protein is encoded by ORF1. ORF1 has been renamed red because this region of l is often called by this name. In wild type recA + hosts, l can grow without these genes which are thereby termed non-essential. It seemed likely that the red gene of F01 would also be non-essential in wild type Salmonella hosts and, as shown below, this is in fact the case. A subclone of the 1 –5853 piece was made using XbaI that cleaves at base pair 4072 and EcoRI whose site is in the plasmid vector near the SmaI site (Fig. 2). This piece was cloned into a derivative of plasmid pBR322 cut with EcoRI and NheI and contains bases 1– 4072 of F01 and genes for ORF1– 5 and part of ORF6. The pBR322 derivative lacked the NdeI site of
10. Selection of region for substitution The F01 genome is approximately 80 kb and only part of it has been cloned and sequenced. The sequenced region shows the genes to be fairly tightly packed. There is no extended region that is noncoding and likely to be dispensable. In addition, the function of most of the genes is unknown. The luxA and luxB genes comprise 2.2 kb of DNA and the sup gene used (supF) is in a 244-bp segment. As shown in the accompanying article, the ends of F01 DNA are constant and a judgment about the strictness of packaging cannot be made. It is therefore desirable to avoid any packaging problems and this can be accomplished by deleting a region of F01 of the same length as luxAB –supF which is about 2.5 kb. A clone derived from F01 by cleavage with restriction endonuclease HaeIII contains bases 1 – 5853 of the sequenced region and the genes for ORF1 to ORF8. This segment was put into plasmid pHG165 (Stewart et al., 1986) via the SmaI site and the F01 genes are under control of the lac promoter
Fig. 2. Restriction enzyme cleavage map of F01. The DNA from F01 is represented by the thinner line; that of the plasmid vector by the thicker. Cleavage sites mentioned in the text are indicated. The EcoRI and SalI sites of the vector backbone are only a few base pairs distant from the SmaI site used in cloning. The coordinates of the open reading frames in this region is also presented and the direction of transcription – translation is from the lower to higher number.
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pBR322, which had been removed by filling in the site with E. coli DNA polymerase (Klenow fragment) and dNTP’s and then self-ligated. The subclone has unique sites at base 490 (BstXI) and at base 3103 (NdeI) of the F01 sequence and these sites were used to introduce the luxA – luxB genes from Vibrio fischeri and supF from E. coli (Fig. 2). In the appropriate clone, the bacterial genes are without a promoter but are in the same orientation as the phage genes and should become active when transferred to the phage chromosome.
11. Creation of F01-lux phage by recombination and selection If genetic transfer of these bacterial genes from the constructed plasmid into the phage genome is possible, it is expected to occur by homologous recombination (Fig. 3) between the flanking regions of F01 DNA in the plasmid, in this case bases 1 – 490 on one side and 3103 – 4072 on the other, and the phage chromosome. Some phages degrade the host DNA after their entry and no prediction with regard to F01 could be made as to whether recombination will take
Fig. 3. Creation of a lux carrying phage by recombination. The plasmid construct (A) contains the plasmid backbone given as a curved line, F01 DNA represented by a straight thin line, and bacterial DNA (lux and sup genes) represented by a thicker straight line. This can recombine with F01 DNA (illustrated as a long straight line) during infection by homologous recombination (x) to give a recombinant lacking some of the F01 genes but carrying the lux and sup genes. This phage can be grown on a strain containing a plasmid (B) which synthesizes the proteins of the missing phage genes.
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place. In the present example, recombinants were found but they were rare. Recombinants were also isolated when the V. fischeri lux genes were replaced by those of V. harveyi, which synthesize a luciferase with greater temperature tolerance. It remains an open question as to whether recombination could be greatly increased by lengthening the flanking regions which were relatively short in these experiments. However, it is clear that recombinants would not have been detected without a selective system. Recombinant phages carrying the lux and sup genes are missing all or part of genes red, ORF2, ORF3, ORF4 and ORF5. It is unknown whether all or some of these ORFs are essential for phage growth. Therefore, these products were supplied in trans by cloning a section of F01 DNA that contains these ORFs and attaching them to a bacterial promoter. The section of the F01 genome from bases 490 to 3103 was cloned into pHG171 (Stewart et al., 1986) to connect these genes to the lac promoter. When put in Salmonella, this plasmid should constitutively synthesize the relevant proteins because Salmonella lacks a lacI gene encoding the lac repressor. ORF5 extends from base 1524 to 3122 and probably codes for a protein of 532 amino acid residues. However, in the subclone, the DNA only extends to base 3103 and the last amino residues of the ORF5 protein are missing. This problem was solved by ligating a double stranded segment of 22 bp made from complementary synthetic oligonucleotides (one of 24 bases the other of 26) to the NdeI end before cloning. This restores the codons for the missing amino acids. The added piece creates a BamHI site at its distal end and codes for the same amino acids (TYR, GLU, GLY, LEU, ARG, ARG) that are missing in the BstXI –NdeI segment of F01. The added piece contains a stop codon at the end of the open reading frame. Since the genetic code is degenerate, all the above amino acids can be coded for by codons other than those found in the F01 sequence. The sequence of the oligonucleotides was changed so that it still coded for these amino acids but was as different as possible from the original sequence and therefore no longer homologous to F01 DNA. Therefore, eventhough this short section is outside the substituted region in the F01-lux phage and has an overlap with this phage, recombination should be prevented. This clone should synthesize
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the ORF2, 3, 4 and 5 proteins in their entirety, but not the red protein. After growth of double amber phage on the plasmid with lux and sup in place of F01 DNA, some plaques were formed on a host that lacks a suppressor gene but which contains the plasmid synthesizing the ORF proteins. After purification, some phage grow without the complementing plasmid and these are phage in which both amber mutations have reverted. Other phage fail to grow on a strain lacking both a suppressor and the complementing plasmid but do grow when the plasmid is present. These are recombinants that carry the lux and sup genes incorporated into the F01 genome. When used to infect Salmonella without the complementing plasmid, they fail to complete a life cycle but do lead to light emission! This result shows that red is not an essential gene since the recombinants lack much of this gene and grow in the absence of the red protein, which is not supplied by the complementing plasmid. At least one or more of the ORFs must be essential but we have not investigated this further. As mentioned in the accompanying article, there are no Sau3AI sites (GATC) in F01 DNA extracted from the virion and no such sites were found in the sequenced region. The lux – sup substitution contains both a BamHI site (GGATCC) and some Sau3AI sites. This indicates that F01 probably does not synthesize a restriction enzyme recognizing GATC. The extent of the deletion in F01 DNA is of the same length as the piece used to complement this missing piece. The short 21-bp overlap that was introduced at the end of the ORF5 gene is dissimilar to the original sequence. Therefore, homologous recombination between the recombinant F01 carrying lux and the complementing plasmid should be entirely prevented and should not lead to the formation of wild type recombinants. The recombinant phage is thus ‘‘locked’’ and does not need to be reisolated at frequent intervals and does not represent an ecological danger.
12. Discussion The use of foreign DNA for the detection of specific bacterial types requires a method that will give both great specificity and a high efficiency of
DNA transfer to the target bacteria. Bacteriophage are suitable on both counts. Therefore, the development of a phage reagent depends, first of all, on finding a phage or phages with the required host range. Such phages have been found for certain bacteria of industrial and/or medical importance. A second element in this approach is the selection of an appropriate reporter gene. The reporter protein should not be found among the bacteria likely to be in the sample and should be readily detectable. In the present research, bacterial luciferase was chosen but there exists a number of other possibilities in this regard, some of them arguably better. In the present research, a general method to create recombinant phage reagents was developed and it should be applicable to most or all phages. tRNAs are well conserved within the eubacteria and mutant tRNAs that allow the translation of the UAG nonsense codon (amber suppressors) have been known for many years. These suppressors can be introduced into many species by transformation and they allow the isolation of amber mutants of the phage or phages to be included in the reagent. To reduce or eliminate reversion, double amber mutants can be isolated from genetic crosses. Since the double amber mutants cannot grow in the absence of a suppressor gene, they can be used to select for rare recombinants which have incorporated a sup gene into their own genome. The sup gene can be associated with a reporter gene to concomitantly introduce this latter gene. In order to apply the method, some basic molecular biological information about the phage is necessary and the results of these studies are reported in the accompanying article. Although the genes of F01 were found to be quite tightly packed in the region sequenced, a non-essential gene was discovered and this allowed the substitution of foreign DNA in a way that neither increased the size of the phage genome nor allowed loss of the insert by recombination. In cases which lack both a convenient gene arrangement and nicely placed restriction enzyme recognition sites, appropriate sites can be introduced by site directed mutagenesis. PCR amplification can be used to isolate the exact section that has been substituted or preferably even a smaller one which encodes the missing gene products. While success is by no means guaranteed, it is likely result if several different regions are subjected to this approach.
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The method of ‘‘locking’’ the recombinant phage that is described here should prevent the loss of the foreign DNA during reagent preparation and also greatly lower the probability of the escape of such phage into nature. However, this locking method does not entirely eliminate the possibility of the emergence of a recombinant phage that can grow without the complementing plasmid. While deletion mutants cannot yield true revertants, alternate pathways or bypass mutation can sometimes occur. For example, deletion of the lacZ gene specifying bgalactosidase in E. coli leads to the loss of the ability to utilize lactose as a carbon source. Phenotypic reversion can occur by mutation at a second locus, ebg, which leads to the synthesis of an alternate enzyme with this activity (Campbell et al., 1973). Similarly, deletion mutants of phage l that lack the red region fail to grow on recA strains but pseudorevertants have been isolated. These have picked up a section of the bacterial chromosome which is a remnant of a prophage genome from a different phage (Campbell, 1996). Although N mutants of l fail to grow, bypass mutations that remove this requirement can be isolated (Court and Sato, 1969). It is therefore clear that after a phage is apparently locked, it must be exposed to different host strains to determine whether phenotypic reversion can occur by one of these mechanisms. The possibility of recombination in nature with a wild type phage of the same kind cannot be ascertained. However, normal recombination will simply remove the foreign DNA and is hence of no consequence. In contrast, illegitimate recombination with such a wild type phage or with a different phage type might allow pseudo wild type revertants to arise but this should be an exceedingly rare event. The F01 reagent developed here detected a wide range of Salmonella strains. However, it was found that different strains produce different amounts of light per cell and it appears that this may be due, in part, to variation in rate of adsorption. To overcome this, the ice nucleation gene was subsequently substituted for lux using the same method and plasmids described above (Wolber and Green, 1990). Only one molecule of the ice nucleation protein needs to be expressed for a positive response to ensue (Wolber, 1993) and this is the basis for the commercial test using F01.
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