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The phasmid as a tool for plasmid genetics Isolation of point mutations that affect replication of a ColEl-related plasmid

II. SUMMARY The insertion of a high-copy-number plasmid into a lambdoid phage chromosome which lacks a functional repressor gene confers on the hybrid ' phasmid' the capacity to grow on an immune lysogen. This was found to be due to titration of repressor because of plasmid replication. We have exploited this property in order to isolate mutants that affect plasmid replication. These mutants have been mapped in a region that was previously characterized as necessary for plasmid replication and incompatibility properties. Some of the mutations could revert at frequencies characteristic of single-base-pair change mutations.


INTRODUCTION
The small multicopy plasmid ColEl and its close relative pMBl do not specify any proteins required for their own replication, which depends entirely on the host replication machinery (Donoghue & Sharp, 1978;Kahn & Helinski, 1978). Faithful initiation and completion of ColEl replication has been achieved in bacterial extracts (Tomizawa, Sakakibara & Kakefuda, 1975). Itoh & Tomizawa (1978) have shown that RNA polymerase, DNA polymerase I and RNAse H are sufficient for the correct initiation of replication of this plasmid in vitro. DNA synthesis is primed by an RNA fragment transcribed by RNA polymerase and processed by RNAse H (Itoh & Tomizawa, 1981). This processing step is essential for the initiation of DNA synthesis and it is specifically inhibited by a small RNA molecule (RNA I) synthesized in a region approximately 400 nucleotides upstream from the origin of DNA replication (Tomizawa et al. 1981). The DNA sequence of the region that codes for the primer precursor (Backman et al. 1978;Ohmori & Tomizawa, 1979;Oka et al. 1979) contains particular sequences and potential secondary structures that may be relevant to the processing reaction and its regulation. Unfortunately few point mutants are available, to help correlation of functional steps in replication initiation with the DNA sequence (Naito & Uchida, 1980;Muesing et al. 1981). In this paper we describe the use of a pMBl-A hybrid (Brenner, Cesareni & Karn, 1982) in a classical genetic approach to the study of plasmid replication. Seventy-two independent mutations that affect the replication of a pMB 1 derivative have been isolated and a fine-structure genetic map of some of them has been obtained.

Media chemicals and enzymes
The composition of media and source of enzymes and chemicals have been described Castagnoli, Cesareni & Brenner, 1982).

Construction of plasmids and phages
Phages, phasmids and plasmids used in this work were constructed by standard in vivo or in vitro recombinant DNA technology. The construction of pacl29, pacl30 and pacl53 has already been described . pcl7 was constructed, inserting the EcoRl fragment carrying the 6522 deletion to the right of att site (Parkinson & Davis, 1971) into the EcoRl site of the plasmid pVH51 (Herschfield et al. 1976).

Identification of plasmid replication mutants
General microbiological techniques as well as special phasmid methods are as described previously by Brenner et al. (1982) or Castagnoli et al. (1982. Phasmids containing plasmids with functional ColEl replication origins are virulent. A colour test for phasmid virulence was devised for rapid screening of plaques of mutants or recombinants, based on a double-indicator plating technique. One of the two strains, the ' plating strain' (usually CA274) is used simply to generate plaques from every phage, virulent or not. This plating strain must itself carry a mutation in the /?-galactosidase lacZ gene. The second strain, the 'tester strain', is an immune lysogen and carries a mutation in the lacY gene which specifies /?-galactoside permease. /?-galactosidase synthesis was induced in this strain with isopropyl-/?-D-thio-galactopyranoside (IPTG), but hydrolysis of the dye 5-bromo-4-chloro-3-indolyl-/?-D-galactoside (BCIG) is very poor, since it cannot penetrate into the bacteria. However, if it is lysed in a plaque, a strong reaction is found. To prevent plaque formation by phages produced by spontaneous induction of the prophage in the lysogen, a plating strain preventing growth of the prophage was used. Generally we used a prophage with a P amber mutation and an Su~ plating strain. Wild type (vir + ) and vir phasmids when plated on the double indicator in presence of IPTG and BCIG gave blue and white plaques respectively, after overnight incubation at 37 °C. Phasmids were pre-adsorbed to 0-05 ml of a fresh stationary phase culture of CA274. After 15 min at room temperature 005 ml of Q37 + 004 ml of IPTG (20 mg/ml in water) and 0-04 ml of BCIG (20 mg/ml in dimethyl formamide) were added, and the mixture plated in 3 ml of top agar. Virulent phasmids make blue plaques while vir mutants make white plaques.
Mutagenesis, isolation of mutants and reversion tests are as described in Castagnoli et al. (1982). vir mutants were identified using the blue plaque test, and after purification and retesting, seventy-two independent mutants were finally isolated.

Assay of fl-lactamase and colicin immunity
The y?-lactamase marker was assayed in two ways as previously described (Castagnoli et al. 1982). Staining with nitrocephin could be coupled with the blue plaque test only if the plates were not very crowded (< 200 plaques), otherwise diffusion of the red colour confused the results.
The assay for colicin immunity was as previously described (Castagnoli et al. 1982). Crude extracts of colicin El were prepared from the strain JC411 (col El) according to Shafferman, Cohen & Flashner (1978), and kept at -20 °C in the presence of 50 % glycerol.

Other methods
Plasmid copy number was estimated by measuring the levels of ampicillin resistance of plasmid-containing bacteria (Uhlin & Nordstrom, 1977). Plasmid mutants were released into EQ84 by infection with the corresponding phasmid according to the method described in Castagnoli et al. (1982). Plasmid-containing bacteria were selected using a low concentration of ampicillin (50 fig/ml), and their efficiency of plating at different ampicillin concentrations was subsequently tested.
DNA synthesis of chloramphenicol-treated cells infected with wild-type or mutant phasmids was measured as described by Donoghue & Sharp (1978).

RESULTS
The isolation of plasmid mutants that are defective in replication requires the use of hybrid molecules carrying two different replication origins. In this way even mutants that are completely defective in one of the two replication systems can be identified and propagated.
Our work employed a 'phasmid', in which a pMBl derivative was contained within a lambda bacteriophage with its own replication functions Castagnoli et al. 1982). The combination has distinctive properties which allowed the isolation, maintenance and analysis of replication mutants of the plasmid.

Phasmid virulence
Insertion of a ColEl plasmid into the phage chromosome conferred on the hybrid the capacity for growth on a strain lysogenic for a prophage with the same immunity. Similar properties have recently been found for other phage-plasmid hybrids (Windass & Brammar, 1979) but since our findings are somewhat different we will describe them briefly. As shown in Table 1, this property was found in  (21) + + + + + Growth was tested by spotting ~ 10 4 and ~ 10 2 plaque-forming units on a lawn of lysogenic bacteria. +, Growth with an efficiency of plating > (M ; +, single plaques were usually not viable but killing was observed when 10 4 phages were spotted. phasmids with four different immunities. However, the strength of virulence varied; although single plaques with a plating efficiency of approximately one were found for the i21 or i81 phasmids on corresponding lysogens, the i\ or z'434 phasmids gave a weaker response, and virulence could only be observed by killing the lysogen with many phages. There are two possible explanations of phasmid virulence: either the hybrid overcomes repression because of activation of transcription of phage genes from a plasmid promoter, or the high-copy-number phasmids titrate out repressor, leading to initiation of transcription from, nonrepressed promoters. The observations described below show that the phasmid overcomes repression by the resident prophage because the plasmid replication functions support phasmid replication even in the presence of phage repressor.

Virulence depends upon the absence of phasmid repressor function
If the repressor titration hypothesis is correct, virulence should not be observed if the incoming phasmid itself synthesizes active repressor. In this situation titration of repressor would not be possible because repressor synthesis is autocatalytic (Reichardt, 1975) and because the genes for repressor and operators are equally amplified during phasmid replication. In fact, when the infecting phasmid carried a gene for a thermosensitive repressor, virulence was observed only at 37 °C, and not at 32 °C when the repressor is active. When the repressor gene was removed by the KH54 deletion (081, Fig. 1 Furthermore, the expression of phage replication functions did not depend upon the orientation of the plasmid with respect to the chromosome, as would be expected if transcription starting from a plasmid promoter were responsible for virulence. 01 and 02, which carry the same plasmid in opposite orientations, were both equally virulent.

Virulence is linked to the origin of replication of the plasmid
The region of DNA responsible for virulence could be mapped by comparing phasmids carrying different derivatives of the plasmids ColEl and pMBl. All the phasmids derived from the plasmids pcl7, pacl29, pacl30 and pacl53 ) could grow on homo-immune lysogens. Apart from 250 base pairs on the P' side of Aatt site the only fragment of DNA that they have in common is a region of about 800 base pairs, mostly upstream of the origin of DNA replication. The unlikely possibility that the presence of two Aatt sites in the phasmid were responsible for the anomalous behaviour observed was ruled out by constructing phasmids in which the plasmid ColEl was directly inserted between the two arms by in vitro methods. These showed the expected virulence.

Virulence is abolished if the infected lysogen contains a plasmid of the same compatibility group
ColEl replication is negatively regulated by a £rans-acting repressor (Shepard, Gelfand & Polinski, 1979). As a consequence, if plasmid-driven replication were responsible for the virulence of the phasmid we expected this to be abolished if the lysogenic bacteria also contained a plasmid of the same compatibility group. Fig. 2 shows that this was correct; phasmids were not produced if a lysogen Exponentially growing cultures of C600, C600 harbouring the plasmid pacl29, C600(i81<Sam7) or C600(i81Saw7) harbouring pacI29 were grown to o.D. 0-3 (600 nm), harvested and infected at a multiplicity of 0-1 with a phasmid in which the right arm of 01 had been replaced by an iHlts. The infected cells were diluted 100-fold in CY broth at 37 °C and aliquots were plated on an Suĩ ndicator at different times.
containing a ColEl derivative was infected. No difference in burst size was observed after infection of non-lysogenic bacteria with or without the same ColEl derivative. As expected, the presence in the lysogen of a compatible plasmid such as pSClOl did not affect phasmid growth (not shown).

Virulence is due to titration of repressor
To prove that virulence is due to titration of repressor and not to escape from repression we needed to show that even the resident prophage was eventually derepressed. This was proved by infecting an Su" lysogen with the phasmid <j> m61 (Fig. 1) carrying an amber mutation in the iVgene of lambda. Derepression of the prophage would be required to complement this defect in the phasmid and allow it to grow. This proved to be the case.

Isolation of vir mutants
The correlation between phasmid virulence and plasmid replication made it likely that non-virulent mutants of the phasmid would be due to mutations affecting plasmid replication functions. We are able to screen for such mutants by a technique which measures the release of /?-galactosidase in plaques. In this system the wild-type virulent phasmid made blue plaques while vir mutants made white plaques.
ColEl replication mutants were first isolated in phasmid <f>\ (Fig. 1). This is a phasmid which shows weak virulence (Table 1). We expected that this would be sensitive even to small defects in plasmid replication and that even a relatively small decrease in plasmid copy number would result in a vir phasmid. A total of 72 independent mutants were isolated using either UV (38) or NG (34) mutagenesis. None of these mutations was suppressed either by supD or supF. This is in accordance with previous evidence that no ColE 1-encoded polypeptide is required for ColEl replication (Donoghue & Sharp, 1978).

Reversion frequency
The replication mutants of ColEl isolated by Hashimoto-Gotoh & Inselburg (1979) were deletions extending into the origin or replication of ColEl. This suggested that it might be difficult to eliminate ColE 1 replication by single base-pair substitutions. With this in mind we tested the reversion frequency of some of our mutants. Most showed a spontaneous reversion frequency in the range 10~9 to 5 x 10~8 typical of single base-pair changes. The reversion frequency was increased by mutagens such as 2-aminopurine or EMS. Some of the mutagen-induced revertants proved to carry secondary mutations.

vir mutants have defects in plasmid replication
ColEl replication continues in the absence of protein synthesis in the host bacteria (Clewell, 1972). This allows the study of ColEl replication in vivo in the absence of host DNA synthesis. When exponentially growing bacteria were treated with chloramphenicol, incorporation of 3 [H]dTP into bacterial DNA decreased to a level of about 5 % of the initial incorporation. As already shown by Donoghue 6 Sharp (1978), if such bacteria were infected with a phasmid, DNA replication proceeded at levels well above the background for at least 10 h. Most of the 3 [H]dTP incorporated under these conditions was found as supercoiled circles of phasmid DNA. Fig. 3 shows that when a vir phasmid was used, 3 [H]dTP incorporation, though slightly higher than the uninfected control, was clearly below the level of that corresponding to bacteria infected with a wild-type phasmid. This experiment clearly proves that vir mutants have defects impairing ColEl-type replication.   l-5xlO" Phasmid lysates were used to infect an exponentially growing culture of the integrase-producing strain EQ84 at MOI «0-l. After 30 min growth at 32 °C, different dilutions were spread on TYE plates containing 50/ig/ml of ampicillin. The plates were incubated at 37 °C.
vir phasmids can release plasmids with a low copy number The integrated plasmid in the phasmid can be released simply by infection of a bacterial strain containing a prophage that synthesizes the int protein constitutively . Phasmids with vir mutations would be expected to release plasmids with defects in replication. In fact, when we excised these plasmids    Fig. 5A. f These results were obtained in crosses similar to the one in Fig. 5A in which the vir~ phasmid was replaced by a col" one.
looking for ampieillin transductants in the presence of repressor, very few were obtained, and these gave unusually small colonies. The few large colonies that appeared at a lower frequency were revertants (Table 2).
It has been shown that the production of /?-lactamase and the consequent level of resistance to ampieillin is proportional to the number of /9-lactamase genes available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672300019121 These results refer to crosses of the type shown in Fig. 5B and C. The cross used to map vir mutants with respect to bla-3. Phasmid recombinants carrying the M34 i* 1 " markers were selected on an Su~ host resistant to phage A. The unselected markers bla-3 and vir were tested among the recombinants as described in Methods. A similar scheme was used to map vir mutants with respect to col-356. (B and C) The crosses used to map vir mutants with respect to col-356. Selection in both crosses was carried out on an Su~ host resistant to A. The percentage of vir col or vir + col + amongst h P recombinants was obtained by testing the phasmids recombinant for the two unselected markers as described in Methods.  (Uhlin & Nordstrom, 1977). Thus a measure of the relative copy numbers of plasmids containing a /Mactamase gene can be obtained by measuring the level of ampicillin resistance. Fig. 4 shows that the wild-type plasmid conferred a level of resistance at least 40 times higher than vir-40 and approximately 20 times higher than the three other vir mutants tested. Although the exact copy number of plasmid pacl29 has not been measured it is likely to be about 50, judging from the copy number of similar ColEl derivatives. The experiment in Fig. 4 suggests that the average copy number of vir-40 is very close to one. We were therefore surprised to find that bacteria carrying the vir-40 plasmid mutant did not segregate the plasmid. This stability seems to be due to integration of the non-replicating plasmid into the bacterial chromosome via m<-mediated recombination. About 50% of the bacteria can be cured of plasmid functions by infection with a phage that provides the products of the int and xis genes.

Mapping of the vir mutations
A stretch of DNA of about 580 nucleotides upstream of the origin of replication has been shown to be sufficient for ColEl replication (Backman et al. 1978). This segment of DNA is located between the /S-lactamase gene and the colicin immunity gene in the plasmid pacl29. To test if vir mutations map in this region, a series of four point crosses between phasmids carrying various plasmid and phage markers were performed (Figs. 5 and 6). The results shown in Table 4 are consistent with a position for the vir mutations between bla-3, an amber mutant in the carboxyterminal part of the /?-lactamase gene, and col-356, a mutation that affects the colicin immunity gene of pacl29. The data that map the mutations vir-40 and vir-42 to the left of col-356 were not conclusive because of strong negative interference.
A genetic map of the /Mactamase, ori V, and colicin immunity region aligned with a physical map of pacl29 is shown in Fig. 6. We have shown previously that the frequency of recombination between two markers in the /?-lactamase gene is approximately proportional to the physical distance between the two markers up to 500 nucleotides (Castagnoli et al. 1982). If we assume that this is true also in the neighbouring region containing the origin of replication and the colicin immunity gene we can try to locate the genetic markers more precisely on the physical map. Using as a reference the physical distance between h and bla-3 it is therefore possible to assign the vir and col mutations to the regions shown in Fig. 6. These regions correspond approximately to the DNA fragment containing the origin of replication and to the sequence that has been identified as the colicin immunity gene (Oka et al. 1979).

Fine-structure genetic mapping
A fine-structure genetic map of the mutants affecting replication was determined by phasmid crosses as an aid to subsequent analysis of the mutants by DNA sequencing. Phasmid crosses of the type shown in Fig. 7 allow the relative position of the two alleles vir A and virB to be distinguished. Recombinants for the external markers h and P were selected on a double-indicator lawn and the vir* recombinants were screened for segregation of an unselected marker, bla-3. Depending on the relative positions of the vir alleles it is possible to predict that in one configuration  Fig. 6. Alignment of the fine structure map of vir mutants with the physical map of the ori col immunity region in plasmid pacl29. The physical positions of vir and col mutants were assigned using the data in Table 3, assuming that the frequency of recombination between two genetic markers is proportional to their physical distance as demonstrated in the case of the /?-lactamase gene (Castagnoli et al. 1982). The distance between h and bla-3 was used as a standard. The frequency of recombination between h and the end point of the deletion 6189 was negligible. The order of the vir mutants was inferred from the results shown in Table 5. among h-P recombinants 3 x 10" 2 < 4 x 10"" io-" 005 4xlO~2 2 x 10"" 0-4 0 1 0-2 0 3 2 x 10"" 4 x 10" 3 < 2 x 10"" % bla + among vir + 36 54 34 36 9 7 8 9 40 33 28 A434 P* recombinants carrying a vir + phasmid were selected on double-indicator plates from crosses of the type shown in Fig. 7. Dilutions of the cross were preadsorbed to CA274 A r . The tester strain Q37 A r was subsequently added together with IPTG and BCIG. Blue plaques were purified on CA274 A r and screened for the presence of bla-3 marker with nitrocephin.  basis of internal consistency, allowing the genetic map in Fig. 6 to be drawn, vir-41 and vir-47 are shown beneath vir-364 and vir-40 since no recombination was observed between these alleles. vir-49 was drawn in a different position from the group including vir-50, vir-51 and vir-52, despite the fact that no direct mapping data was obtained, because of the clear difference in the recombination frequency in the crosses with vir-364. The same is true for the position of vir-42, vir-45 and vir-48.

CONCLUSIONS
In this paper we have described the isolation, characterization and genetic mapping of mutants in the replication functions of the plasmid pMBl. This was made possible by the observation that phasmids containing integrated plasmids can grow on a lysogen containing a prophage of the same immunity provided the plasmid replication origin is functional.
We have shown that the function that confers this behaviour maps in the region of the origin of replication of the integrated plasmid and that it is inhibited by a resident plasmid of the same in compatibility group. Contrary to findings for similar hybrids (Mukai et al. 1978), in our phasmids the plasmid replication functions complemented phage 0 and P mutants very poorly, if at all. This allowed us to use P amber mutants as selective markers in mapping crosses. We proved that phasmid virulence was not due to a general insensitivity to repression, but rather to titration of the repressor present by the increased copy number of the incoming phasmid due to plasmid replication. This phenotype, together with a convenient colour test, can be used to select vir mutants which are defective in plasmid replication functions as shown by infection of chloramphenicol-treated cells. Furthermore, the mutant plasmids excised from the phage chromosome were very poorly propagated; most are probably not self-sustaining.
All the vir mutants mapped between the /Mactamase and colicin immunity genes of the plasmid pacl29, which had already been characterized as the region necessary for ColEl replication. Many of the mutations appear to be single base changes, and a fine-structure genetic map shows that they are at different sites. The genetic studies are a first step and will permit further studies of the relationship between DNA sequence and replication phenotype.
Phasmids have proved to be versatile for the study of ColEl replication. In addition to the vir mutants reported here, high-copy-number mutants and mutants in incompatibility properties have been selected and will be described in detail elsewhere.