Rac Prophage Research Articles

Unsuspected prophage-like elements in Salmonella typhimurium.

We present evidence for the existence of two large (approximately 50 kb) excisable segments in the chromosome of Salmonella typhimurium. The two elements--designated Gifsy-1 and Gifsy-2--cover, respectively, the 57 units and the 24 units of the genetic map where they contribute indicative rare restriction sites. The two elements are closely interrelated and both contain a region of sequence similarity to the recE locus of the Rac prophage of Escherichia coli. Mutations within this region of Gifsy-1 yield the classical 'Sbc' phenotype: they suppress the recombination defect of recB mutants, apparently by activating a normally silent recE-like gene. At the same time, these 'sbcE' mutations activate a Xis-type function that promotes excision of one or other of the two elements. Predictably, curing of Gifsy-1 results in the loss of recB mutant suppression. Surprisingly, the suppressor phenotype is also lost in cells cured for Gifsy-2 even though the Gifsy-1-associated sbcE mutation is still present. Moreover, the excision frequency of Gifsy-1 drops dramatically in Gifsy-2-cured cells. Thus, both elements must co-operate in the activation of recombination and excision functions. Overall, the data presented here suggest that Gifsy-1 and Gifsy-2 are cryptic prophages. They are distinct from previously described Fels prophages. Unlike Fels, they are not specific to S. typhimurium strain LT2 since they are both also found in a virulent S. typhimurium isolate (ATCC 14028s).

Identification of a new inhibitor of essential division gene ftsZ as the kil gene of defective prophage Rac.

A gene function carried by a plasmid, causing arrest of cell division in Escherichia coli, has been identified as the product of a short open reading frame of the prophage Rac, previously designated orfE, expressed only under conditions of prophage induction. Because Rac carries a killing function expressed under conditions of zygotic induction, an orfE-defective Rac+ strain was constructed. This strain had lost the killing function, indicating that orfE is kil. Division inhibition by kil was specifically relieved by overexpression of essential division gene ftsZ. The kil gene product acts independently of the min operon, and its effects are increased in conditions of high cyclic AMP (cAMP) receptor protein-cAMP complex levels in the cell. Furthermore, at high levels of expression, kil product distorts the rod shape of the cells. These features distinguish kil-encoded protein from the inhibitory product of gene dicB, which occupies a similar genetic location in Kim (Qin), another defective prophage of Escherichia coli.

Restriction by EcoKI is enhanced by co-operative interactions between target sequences and is dependent on DEAD box motifs.

One subunit of both type I and type III restriction and modification enzymes contains motifs characteristic of DEAD box proteins, which implies that these enzymes may be DNA helicases. This subunit is essential for restriction, but not modification. The current model for restriction by both types of enzyme postulates that DNA cutting is stimulated when two enzyme complexes bound to neighbouring target sequences meet as the consequence of ATP-dependent DNA translocation. For type I enzymes, this model is supported by in vitro experiments, but the predicted co-operative interactions between targets have not been detected by assays that monitor restriction in vivo. The experiments reported here clearly establish the required synergistic effect but, in contrast to earlier experiments, they use Escherichia coli K-12 strains deficient in the restriction alleviation function associated with the Rac prophage. In bacteria with elevated levels of EcoKI the co-operative interactions are obscured, consistent with co-operation between free enzyme and that bound at target sites. We have made changes in three of the motifs characteristic of DEAD box proteins, including motif III, which in RecG is implicated in the migration of Holliday junctions. Conservative changes in each of the three motifs impair restriction.

Replication of coliphage lambda DNA

A general scheme of λ phage and plasmid DNA replication in Escherichia coli is presented, and results of in vivo experiments from the authors' laboratory are superimposed. The initiator λO functions in the assembly of the replication complex (RC) at oriλ, making it a stable component of this structure. ClpP / ClpX protease-specific action on λO does not affect the regulation of replication; it only degrades the surplus of synthesized λO. The initiator λO becomes protected from proteolysis at a distinct step of the pathway of RC assembly. The host DnaA initiator-regulated transcriptional activation of oriλ seems to be coupled with RC assembly at the step of chaperone-mediated rearrangement of the pre-primosome. The once-assembled RC is inherited by one of two λ plasmid daughter copies at each round of circle-to-circle (θ) replication. The inherited, old RC-driven replication is also dependent on RNA polymerase and DnaA functions. It seems that DnaA licenses λ plasmid DNA for only one replication round, resembling the putative eukaryotic licensing factor in this respect. The λO binding to oriλ does not seem to play any role in regulation of λ plasmid replication, and the Cro-autoregulatory loop may be deleted. The emerging picture shows λ plasmid circles with RCs bound to their ori, awaiting a signal triggering initiation of replication. The host DnaA initiator-regulated transcriptional activation of oriλ may be involved in signal transmission. Inactivation of DnaA function blocks initiation of λ phage DNA replication, but the lambdoid prophage Rac compensates this defect and all parental phage DNA molecules, after one round of θ replication switch to the σ mode and produce progeny in high yield. We suspect that DnaA-regulated transcriptional activation is involved in installation and adequate positioning of two RCs, required for bidirectional replication, but in the Rac-promoted process only one RC may be installed, leading to unidirectional replication continued in the σ mode. In wild-type cells consumption of DnaA function by the rapidly replicating λ phage DNA may switch replication from bidirectional θ to unidirectional θ, and later to the σ mode; the λ circles produced earlier may play the role of Rac, which is required only when DnaA function has been inactivated prior to phage infection.

Replication of coliphage lambda DNA.

A general scheme of lambda phage and plasmid DNA replication in Escherichia coli is presented, and results of in vivo experiments from the authors' laboratory are superimposed. The initiator lambda O functions in the assembly of the replication complex (RC) at ori lambda, making it a stable component of this structure. ClpP/ClpX protease-specific action on lambda O does not affect the regulation of replication; it only degrades the surplus of synthesized lambda O. The initiator lambda O becomes protected from proteolysis at a distinct step of the pathway of RC assembly. The host DnaA initiator-regulated transcriptional activation of ori lambda seems to be coupled with RC assembly at the step of chaperone-mediated rearrangement of the pre-primosome. The once-assembled RC is inherited by one of two lambda plasmid daughter copies at each round of circle-to-circle (theta) replication. The inherited, old RC-driven replication is also dependent on RNA polymerase and DnaA functions. It seems that DnaA licenses lambda plasmid DNA for only one replication round, resembling the putative eukaryotic licensing factor in this respect. The lambda O binding to ori lambda does not seem to play any role in regulation of lambda plasmid replication, and the Cro-autoregulatory loop may be deleted. The emerging picture shows lambda plasmid circles with RCs bound to their ori, awaiting a signal triggering initiation of replication. The host DnaA initiator-regulated transcriptional activation of ori lambda may be involved in signal transmission. Inactivation of DnaA function blocks initiation of lambda phage DNA replication, but the lambdoid prophage Rac compensates this defect and all parental phage DNA molecules, after one round of theta replication switch to the sigma mode and produce progeny in high yield. We suspect that DnaA-regulated transcriptional activation is involved in installation and adequate positioning of two RCs, required for bidirectional replication, but in the Rac-promoted process only one RC may be installed, leading to unidirectional replication continued in the sigma mode. In wild-type cells consumption of DnaA function by the rapidly replicating lambda phage DNA may switch replication from bidirectional theta to unidirectional theta, and later to the sigma mode; the lambda circles produced earlier may play the role of Rac, which is required only when DnaA function has been inactivated prior to phage infection.

Restriction alleviation and modification enhancement by the Rac prophage of Escherichia coli K-12.

Bacteriophage lambda encodes an antirestriction function, RaI, which is able to modulate the activity of the Escherichia coli K-12 restriction and modification system, EcoKI. Here we report the characterization of an analogous function, Lar, expressed by E. coli sbcA mutants and the hybrid phage lambda reverse. E. coli sbcA mutants and lambda reverse both express genes of the Rac prophage, and we have located the lar gene immediately downstream of recT in this element. The lar gene has been cloned in an expression plasmid, and a combination of site-directed mutagenesis and labelling of plasmid-encoded proteins has enabled us to identify a number of translational products of lar, the smallest of which is sufficient for restriction alleviation. Lar, like RaI, is able both to alleviate restriction and to enhance modification by EcoKI. Lar, therefore, is functionally similar to RaI and the nucleotide sequences of their genes share 47% identity, indicating a common origin. A comparison of the predicted amino acid sequences of Lar and RaI shows only a 25% identity, but a few short regions do align and may indicate residues important for structure and/or function.

Modification enhancement and restriction alleviation by bacteriophage λ

The activity of EcoKI, and related restriction and modification (R-M) systems, is modulated by the bacteriophage λ ral gene product. We have identified the coding sequence for an analogous function in the Rac prophage of E. coli K-12.

Involvement of the host initiator function dnaA in the replication of coliphage lambda.

We demonstrate that the initiation of coliphage lambda DNA replication is dependent on the host initiator function dnaA, provided that the lambdoid prophage Rac is absent. Presence of Rac compensated the absence of dnaA function, causing initiation of replication. In dnaAts rac+ cells at 43 degrees, most of parental phage DNA molecules, after one round of theta replication, switched to a replication with features of the sigma mode and produced progeny at high yield. Initiation of replication of the lambda Pts1 mutant at 43 degrees was blocked by dnaA function; however, under dnaA-rac+ conditions all parental phage DNA molecules, after one round of theta replication, switched to the sigma mode and produced progeny at high yield. Taking into account our recent finding that transcriptional activation of ori lambda seems to be dnaA-regulated (to be published elsewhere), we suggest that the DnaA-lambda Pts1 incompatibility occurs at the insertion of the ori lambda-bound lambda O-lambda P-DnaB preprimosome between the complementary lambda DNA strands. The role of Rac and the mechanism of the switch from theta to sigma mode of lambda phage DNA replication are discussed.

Transcriptional activation of the origin of coliphage λ DNA replication is regulated by the host DnaA initiator function

The initiator of phage λ DNA replication, the λ O protein, is considered to be an analogue of the initiator of DNA replication (DnaA) of its host, Escherichia coli. Both specifically recognize their origins of replication, oriλ and oriC, respectively, and organize the assembly of specific replication complexes. However, DnaA has an additional activation function, acting on oriC-proximal DnaA-boxes, and regulating transcription initiated at promoters in and around oriC. Here, we demonstrate that λ plasmid replication can be synchronized by a temperature shift-down that caused renaturation of the previously denatured DnaAts protein. Moreover, we show that elimination of the activating DnaA function affects transcriptional activation at oriλ. DnaA may act by binding to DnaA-boxes, situated around the λ p R promoter; there are no such sequences in oriλ. Our results begin to explain in molecular terms why λ plasmid replication is DnaA-dependent [Kur et al., J. Mol. Biol. 198 (1987) 203–210] and why the initiation of phage λ DNA replication is blocked (in E. coli devoid of prophage Rac) after inactivation of DnaA [Wggrzyn et al., Genetics (1995) in press]

DNA sequence divergence among derivatives of Escherichia coli K-12 detected by arbitrary primer PCR (random amplified polymorphic DNA) fingerprinting.

Derivatives of Escherichia coli K-12 of known ancestry were characterized by random amplified polymorphic DNA (RAPD) fingerprinting to better understand genome evolution in this family of closely related strains. This sensitive method entails PCR amplification with arbitrary primers at low stringency and yields arrays of anonymous DNA fragments that are strain specific. Among 150 fragments scored, eight were polymorphic in that they were produced from some but not all strains. Seven polymorphic bands were chromosomal, and one was from the F-factor plasmid. Five of the six mapped polymorphic chromosomal bands came from just 7% of the genome, a 340-kb segment that includes the terminus of replication. Two of these were from the cryptic Rac prophage, and the inability to amplify them from strains was attributable to deletion (excision) or to rearrangement of Rac. Two other terminus-region segments that resulted in polymorphic bands appeared to have sustained point mutations that affected the ability to amplify them. Control experiments showed that RAPD bands from the 340-kb terminus-region segment and also from two plasmids (P1 and F) were represented in approximate proportion to their size. Optimization experiments showed that the concentration of thermostable polymerase strongly affected the arrays of RAPD products obtained. Comparison of RAPD polymorphisms and positions of strains exhibiting them in the pedigree suggests that many sequence changes occurred in these historic E. coli strains during their storage. We propose that the clustering of such mutations near the terminus reflects errors during completion of chromosome replication, possibly during slow growth in the stab cultures that were often used to store E. coli strains in the early years of bacterial genetics.

Involvement of RecE exonuclease and RecT annealing protein in DNA double-strand break repair by homologous recombination

We demonstrated that a double-stranded (ds) gap in DNA is repaired by a gene conversion mechanism in an Escherichia coli recBC sbcA23 strain, as predicted by the ds break repair models for homologous recombination. The sbc A mutation is known to induce several gene products encoded on the Rac prophage present in most strains of E. coli K-12. These include exonuclease VIII (ExoVIII), a 5′ to 3′ exonuclease working from the end of a duplex DNA, and RecT, an annealing protein. We found that a rac − strain (lacking the Rac prophage) cannot support this repair. A plasmid carrying part of the Rac prophage supported highly efficient ds gap repair activity in a rac − strain, but two ExoVIII + recT − plasmids did not. The recE159 mutation that blocks ds gap repair was found to be recT+, since these ExoVIII + recT − plasmids complemented the recE159 mutation in repair of ultraviolet light damage. From these observations, we conclude that both ExoVIII and RecT are essential for ds gap repair. We discuss their possible roles in the ds break repair reaction.

Genetic and molecular analyses of the C-terminal region of the recE gene from the Rac prophage of Escherichia coli K-12 reveal the recT gene

The nucleotide sequence of the C-terminal region of the recE gene of the Rac prophage of Escherichia coli K-12 reveals the presence of a partially overlapping reading frame we call recT. Deletion mutations show that recT is required for the RecE pathway of conjugational recombination. By cloning recT with a plasmid vector compatible with pBR322, we showed by cis-trans tests that the portion of the recE gene encoding ExoVIII DNA nuclease activity is also required for RecE pathway conjugational recombination. The recT gene can replace the redB gene of lambda for recA-independent plasmid recombination. A Tn10 insertion mutation previously thought to be in recE is located in recT and is renamed recT101::Tn10. Discrepancies between the molecular mass estimates of wild-type ExoVIII protein determined from mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and calculated from the predicted amino acid sequence are discussed. The hypothesis that wild-type ExoVIII protein results from fusion of RecE and RecT proteins is disproved genetically, thus supporting a previous hypothesis that the discrepancies are due to abnormal protein mobility in SDS-PAGE. A computer-performed scan of the bacteriophage nucleotide sequence data base of GenBank revealed substantial similarity between most of recE and a 2.5-kb portion of the b2 region of lambda. This suggests interesting speculations concerning the evolutionary relationship of lambda and Rac prophages.

Genetic analysis of potassium transport loci in Escherichia coli: evidence for three constitutive systems mediating uptake potassium

The analysis of mutants of Escherichia coli that require elevated concentrations of K+ for growth has revealed two new genes, trkG, near minute 30 within the cryptic rac prophage, and trkH, near minute 87, the products of which affect constitutive K+ transport. The analysis of these and other trk mutations suggests that high rates of transport, previously considered to represent the activity of a single system, named TrkA, appear to be the sum of two systems, here named TrkG and TrkH. Each of these two is absolutely dependent on the product of the trkA gene, a cytoplasmic protein associated with the inner membrane (D. Bossemeyer, A. Borchard, D. C. Dosch, G. C. Helmer, W. Epstein, I. R. Booth, and E. P. Bakker, J. Biol. Chem. 264:16403-16410, 1989). The TrkH system is also dependent on the products of the trkH and trkE genes, while the TrkG system is also dependent on the product of the trkG gene and partially dependent on the product of the trkE gene. It is suggested that the trkH and trkG products are membrane proteins that form the transmembrane path for the K+ movement of the respective systems. Two mutations altering the trkA product reduce the affinity for K+ of both TrkG and TrkH, indicating that changes in peripheral protein can alter the conformation of the sites at which K+ is bound prior to transport. The TrkD system has a relatively modest rate of transport, is dependent solely on the product of the trkD gene, and is the sole saturable system for Cs+ uptake in this species (D. Bossemeyer, A. Schlösser, and E. P. Bakker, J. Bacteriol. 171:2219-2221, 1989).

Physical analysis of spontaneous and mutagen-induced mutants of Escherichia coli K-12 expressing DNA exonuclease VIII activity.

We have mapped the extents of two deletion sbcA mutations which result in production of DNA exonuclease VIII (ExoVIII). One mutation, sbcA8, deletes about 140 kb of DNA which includes most of the Rac prophage and the trg gene. Western blot analysis shows that the protein produced is larger than wild type ExoVIII. The nucleotide sequence shows that a translational gene fusion has occurred. The N-terminal 294 codons of recE have been deleted and the remaining C-terminal codons have been fused to the N-terminal portion of another reading frame we call sfcA. Analysis of the protein sequence encoded by sfcA shows an 83% similarity with rat and mouse NADP-linked malic enzyme. We discuss the possibility that sfcA is identical to maeA which encodes NAD-linked malic enzyme from Escherichia coli. Restriction nuclease analysis of a second deletion, sbcA81, by Southern blot technique indicates that about 105 kb of DNA have been deleted and a transcriptional gene fusion has occurred between recE and the regulatory region of an E. coli chromosomal gene. We also examined eight other sbc mutations that result in ExoVIII production. Five have no effect on restriction nucleotide fragment sizes detected by complementarity to lambda rev as probe. These are presumed point mutations. Three seem to produce additional restriction nucleotide fragments complementary to lambda rev. The possible nature of these sbc mutations is discussed.

Recombination of bacteriophage λ in recD mutants of Escherichia coli

RecBCD enzyme is centrally important in homologous recombination in Escherichia coli and is the source of ExoV activity. Null alleles of either the recB or the recC genes, which encode the B and C subunits, respectively, manifest no recombination and none of the nuclease functions characteristic of the holoenzyme. Loss of the D subunit, by a recD mutation, likewise results in loss of ExoV activity. However, mutants lacking the D subunit are competent for homologous recombination. We report that the distribution of exchanges along the chromosome of Red-Gam-phage lambda is strikingly altered by recD null mutations in the host. When lambda DNA replication is blocked, recombination in recD mutant strains is high near lambda's right end. In contrast, recombination in isogenic recD+ strains is approximately uniform along lambda unless the lambda chromosome contains a chi sequence. Recombination in recD mutant strains is focused toward the site of action of a type II restriction enzyme acting in vivo on lambda. The distribution of exchanges in isogenic recD+ strains is scarcely altered by the restriction enzyme (unless the phage contains an otherwise silent chi). The distribution of exchanges in recD mutants is strongly affected by lambda DNA replication. The distribution of exchanges on lambda growing in rec+ cells is not influenced by DNA replication. The exchange distribution along lambda in recD mutant cells is independent of chi in a variety of conditions. Recombination in rec+ cells is chi influenced. Recombination in recD mutants depends on recC function, occurs in strains deleted for rac prophage, and is independent of recJ, which is known to be required for lambda recombination via the RecF pathway. We entertain two models for recombination in recD mutants: (i) recombination in recD mutants may proceed via double-chain break--repair, as it does in lambda's Red pathway and E. coli's RecE pathway; (ii) the RecBC enzyme, missing its D subunit, is equivalent to the wild-type, RecBCD, enzyme after that enzyme has been activated by a chi sequence.

Mutation-dependent suppression of recB21 recC22 by a region cloned from the Rac prophage of Escherichia coli K-12.

Using pBR322 as a vector, we cloned a 5.95-kilobase fragment of the Rac prophage together with 1.70 kilobases of a flanking Escherichia coli chromosome sequence. The resulting plasmid (pRAC1) was unable to suppress the mitomycin and UV sensitivity and recombination deficiency of a recB21 recC22 strain. Five spontaneous mitomycin-resistant derivatives contained deletion mutant plasmids. These plasmids also suppressed the UV sensitivity and recombination deficiency of their recB21 recC22 hosts. All five deletions were contained within a 2.45-kilobase EcoRI-to-HindIII segment of the plasmid. By substituting the corresponding 2.45-kilobase EcoRI-toHindIII fragments of Rac prophage isolated from sbcA+, sbcA6, and sbcA23 strains for the shortened segment of one of the deletion mutant plasmids, we were able to show that sbcA mutations map in this region. Also in this region is the site (or closely linked sites) at which previous studies had shown that insertion of Tn5 and IS50 leads to suppression of recB21 recC22. The sequence in this region that must be altered or circumvented to allow suppression is discussed. Also presented are data correlating the expression of nuclease activity with the degree of suppression.

OriX: a new replication origin in E. coli.

Replication of the chromosome of E. coli at 42 degrees C in an integratively suppressed dnaA mutant (dnaA46 Sin Hfr) occurs predominantly from the origin of replication of the integrated plasmid (oriV). We have carried out a detailed marker frequency analysis on such Hfrs. This analysis indicates that replication at 42 degrees C occurs not only from oriV, but also from an origin, oriX, located in the terminal region of the chromosome close to, but distinct from, the prophage rac (oriJ). In an oxa1 mutant of one of these Hfrs, we have shown that replication proceeds at 42 degrees C from all three origins: oriV, oriX, and oriC. Loss of the integrated plasmid results in a temperature- and rich-medium-sensitive strain that replicates the chromosome from oriC and oriX. Replication from oriX proceeds slowly and bidirectionally. We suggest that oriX may be involved in the coupling between replication and cell division.

Restriction nuclease and enzymatic analysis of transposon-induced mutations of the Rac prophage which affect expression and function of recE in Escherichia coli K-12.

Fourteen Tn5-generated mutations of the Rac prophage, called sbc because they suppress recB21 recC22, were found to fall into two distinct types: type I mutations, which were insertions of Tn5, and type II mutations, which were insertions of IS50. Both orientations of Tn5 and IS50 were represented among the mutants and were arbitrarily labeled A and B. All 14 of the Tn5 and IS50 insertions occurred in the same location (+/- 100 base pairs) approximately 5.6 kilobases from one of the hybrid attachment sites. Eleven of the mutants contained essentially the same amount of exonuclease VIII, the product of recE. The possibility that a promoter for recE was created by the insertion of Tn5 and IS50 was considered. Two IS50 mutants in which such a promoter could not have been created showed three to four times as much exonuclease VIII, and another showed one-half as much as the majority. The possibility was considered that a promoter internal to IS50 is responsible for this heterogeneity. Restriction alleviation was measured in all 14 mutants. An insertion of the transposon Tn10 which reduces expression of exonuclease VIII (recE101::Tn10) was located within the Rac prophage at a position 2.35 kilobases from the left hybrid attachment site. Location and orientation of the Rac prophage on the Escherichia coli genetic map are discussed in light of these results.

Genetic analysis of transposon-induced mutations of the Rac prophage in Escherichia coli K-12 which affect expression and function of recE.

Fourteen mitomycin-resistant revertants of a recB21 recC22 strain were isolated after Tn5 mutagenesis. Eight of the mutations (type I) were essentially inseparable from aphA+ (Kanr) of Tn5; six (type II) were not. We hypothesize that the former are Tn5 and that the latter are IS50 insertions. Because of their phenotypic similarity to sbcA and sbcB mutations, which also suppress recB21 recC22, we have called them sbc mutations. sbc-lll::Tn5 was cotransducible with nirR and has thereby been located at position 29.8 on the Escherichia coli map in the vicinity of the Rac prophage and sbcA mutations. A recB21 recC22 sbc-lll::Tn5 strain was subjected to Tn10 mutagenesis, and a mitomycin- and UV-sensitive mutant was isolated. tet+ of Tn10 was 85% cotransducible with aphA+ of Tn5, locating these two transposons 0.1 map unit apart. A three-point cross located the Tn10 mutation at position 29.7. We hypothesize that the Tn10 insertion is located in recE and that the Tn5 and IS50 insertions activate expression of this gene. sbc-lll::Tn5 was found to be cis acting and dominant to its wild-type allele as were two sbcA mutations (sbcA1 and sbcA6). Five other type I and type II insertion mutations were dominant to their wild-type alleles. We hypothesize that the sbc insertion and sbcA mutations affect transcription regulation of recE and discuss the possibility that they do so differently.

A third defective lambdoid prophage of Escherichia coli K12 defined by the λ derivative, λ qin111

We describe the isolation and characterization of a new Q-independent substitution mutant of lambda, lambdaqin111, which differs from other characterized Q-independent lambda phages. This mutant defines a new lambda-like prophage in the bacterial chromosome, as seen by homologous recombination between lambdaqin111 and the host DNA and by DNA/DNA hybridization methods. Genetic and electron microscopy data show that this new prophage carries, at least, genes analogous to Q-S-R of lambda and also a cos site functionally identical to lambda cos. It is located near 34 min on the Escherichia coli K12 map, i.e. in the same region but at a different site from the defective Rac prophage.