Weakening of bacteriophage lambda EcoK DNA restriction in the presence of plasmid pKM101 ard+. I. General characteristics and genetic localization

The host-controlled K-restriction of unmodified phage lambda is ten to hundred-fold alleviated in the E. coli K12 strain, carring plasmid pKM101 of N-incompatibility group. By restriction mapping Tn5 insertion in pKM101, which reduced pKM101-mediated alleviation of K-restriction, was shown to by located within BglII-B-fragment approximately 9 kb anticlockwise from the EcoRI-site of pKM101. We have termed the gene(s) promoting the alleviation of K-restriction ARD (Alleviation of Restriction of DNA). It was shown that (i) plasmid pKM101-mediated alleviation of K-restriction did not depend on bacterial genes LexA, RecBC, umuC and plasmid gene muc; (ii) ard gene did not mediate EcoK type modification of DNA and did not enhance the modification activity of EcoK system in a way similar to that observed with RAL gene of phage lambda. Action of Ard gene of plasmid pKM101 is highly specific: alleviation of restriction of DNA lambda takes place only in K-strains of E. coli and is practically absent in B-strains and also in E. coli strains which have restricting enzymes of 11 type, EcoRI and EcoRIII.     

Alleviation of type I restriction in the presence of plasmids of group inc I. General characteristics and molecular cloning of the ard gene

The capability of a number of plasmids of incN and incI groups to alleviate an action of type I EcoK, EcoB, EcoD, and EcoA restriction endonucleases on the unmodified DNA was revealed. The efficiency of EcoK action on lambda 0 DNA is alleviated about 10 divided by 100 fold in E. coli K12 AB 1157 bacteria containing the plasmid of incN group (pKM101, N3, pJA4733) or incI group (R144, R648; R621a; ColIb-P9). We have cloned ard gene of ColIb-P9 plasmid (SalI-C fragment) in pBR322 multicopying vector. A hybrid clone abolishing the EcoK restriction has been received. Ard gene activity is independent of the recA, recBc, recF, lexA, umuC, lon bacterial genes activity. Ard gene's product does not inhibit the EcoK restriction endonuclease action as well as ocr protein (phage T7) and does not increase the process of methylation of DNA as well as ral protein of phage lambda.     

Nucleotide sequence of an OXA-2 beta-lactamase gene from the R-plasmid R1767 derived plasmid pBP11 and comparison to closely related resistance determinants found in R46 and Tn2603

Plasmid pBP11 contains a sequence homologous to Tn21-like element Tn2410 encoding dihydropteroate synthetase and beta-lactamase OXA-2. The nucleotide sequence of a 1.5 kb segment of this region has been determined including the bla gene. It reveals strong sequence homology with the OXA-2 operon of plasmid R46. The implications of an additional 319 bp segment in pBP11 for the different evolution of R46/pKM101 and pBP11 are discussed.     

Restriction endonuclease cleavage map of pKM101: Relationship to parental plasmid R46

Summary A detailed restriction endonuclease cleavage map of the plasmid pKM101 has been constructed. pKM101 plasmids containing individual Tn5 insertions were used to facilitate the ordering of restriction fragments generated by enzymes cleaving pKM101 at multiple sites. By restriction enzyme analysis, pKM101 (35.4 kilobases) appears to have arisen from its clinically-isolated parent by deletion of a single DNA region which codes for three of the four drug resistances carried by R46.     

Partial characterization of a small, multiple-copy plasmid from Streptomyces espinosus and the derivation of a high copy-number deletion mutant

An organism classified as Streptomyces espinosus was found to carry an approx. 9.2-kb plasmid. This plasmid, designated pUC6, has a copy number of 30-40 per host genome equivalent. Plasmid pUC1061, a copy-number mutant of pUC6, was isolated after in vitro deletion of an approx. 2.0-kb XhoI restriction fragment. Plasmid pUC1061 has a copy number of 500-600. Plasmid pUC1061 appears to be incompatible with pUC6 and will transform a pUC6-containing culture at a frequency of approx. 1%. The sizes, restriction maps and copy numbers of plasmids pUC6 and pUC1061 indicate these may be valuable vectors for gene cloning Streptomyces.     

Molecular cloning of the L-phenylalanine transaminase gene from Paracoccus denitrificans in Escherichia coli K-12

The L-phenylalanine transaminase gene of Paracoccus denitrificans was cloned by a shotgun method using the Escherichia coli K-12 mutant DG30, which lacks three distinct transaminase genes. Plasmid pPAP142 was constructed by inserting a 2.2-kb fragment carrying the transaminase gene into pUC18. Strain E. coli K-12 HB101 cells harboring the plasmid produced 20-fold to 30-fold more transaminase than wild type P. denitrificans cells. The nucleotide sequence of the 2.2-kb fragment was determined, revealing that the deduced amino acid sequence of the transaminase of P. denitrificans is similar to that of other transaminases.     

Evasion of type I and type II DNA restriction systems by Incl1 plasmid Collb-P9 during transfer by bacterial conjugation

Transmission of unmodified plasmid CoIIb-P9 by bacterial conjugation is markedly resistant to restriction compared with transfer by transformation. One process allowing evasion of type I and II restriction systems involves conjugative transfer of multiple copies of the plasmid. A more specialized evasion mechanism requires the Ard (alleviation of restriction of DNA) system encoded by CoIIb. The ard gene is transferred early in conjugation and specifically alleviates DNA restriction by all known families of type I enzyme, including EcoK. CoIIb has no effect on EcoK modification but this activity is impaired by multicopy recombinant plasmids supporting overexpression of ard. Genetic evidence shows that Ard protects CoIIb from EcoK restriction following conjugative transfer and that this protection requires expression of the gene on the immigrant plasmid. It is proposed that carriage of ard facilitates transfer of CoIIb between its natural enterobacterial hosts and that the route of DNA entry is important to the restriction-evasion mechanism.     

Repair promoted by plasmid pKM101 is different from SOS repair.

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid pKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions.     

The trimethoprim resistance transposon Tn7 contains a cryptic streptothricin resistance gene

The transposon Tn7 codes for a trimethoprim resistance and for a streptomycin/spectinomycin resistance function of the bacterial host cells. Cloning of a restriction fragment of Tn7 into the vector plasmid pUC19 reveals the presence in Tn7 of an additional potential resistance determinant. A streptothricin resistance gene, which appears cryptic in the original Tn7 context becomes activated in the recombinant plasmid upon supplying the promoter function of the lacZ system of pUC19. These results together with previously published sequence data further disclose the modular character in the resistance gene regions of Tn7-like transposons.     

Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) pathway of plasmid pJP4: mapping and characterization of the TFD regulatory gene, tfdR.

Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pRO101) and some Pseudomonas transconjugants grew on TFD. Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate. A mutant of one such phenol-degrading strain, Pseudomonas putida PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101. Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101. This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA. Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols.     

Tn4556, a 6.8-kilobase-pair transposable element of Streptomyces fradiae.

A 6.8-kilobase-pair (kbp) transposable element (Tn4556) was found in a neomycin-producing strain of Streptomyces fradiae. This element was first observed in two 30.3-kbp plasmids (pUC1123 and pUC1124) which arose when a thiostrepton resistance gene (1 kbp) was ligated with the BclI-2 fragment (22.5 kbp) that contains the origin of replication of phage SF1. The Tn4556 segment was deleted when these plasmids were transduced into another S. fradiae host with phage SF1. These deletion plasmids (pUC1210 and pUC1211) had copy numbers of less than 1 per chromosome and were unstable. In contrast, pUC1123 and pUC1124, with copy numbers of 12 to 15 per chromosome, respectively, were relatively stable. When pUC1210 and pUC1211 were reintroduced into S. fradiae by protoplast transformation, the Tn4556 element transposed again to the plasmids at numerous new locations in either of two orientations. A copy of Tn4556 was found in the S. fradiae chromosome by hybridization studies. It appears that Tn4556 originated from the chromosome, transposed into unstable pUC1210 and pUC1211, and made stable plasmids. A temperature-sensitive hybrid plasmid carrying a viomycin resistance derivative of Tn4556 (pMT660::Tn4556::vph) was constructed. When Streptomyces lividans UC8390 containing the hybrid plasmid was grown at 39 degrees C, Tn4556::vph (Tn4560) transposed to random positions in the host chromosome.     

Isolation of plasmid pKM101 in the Stocker laboratory

pKM101 is a mutagenesis-enhancing resistance transfer plasmid (R plasmid) that was introduced into several tester strains used in the Salmonella/microsome mutation assay (Ames test). Plasmid pKM101 has contributed substantially to the effectiveness of the Ames assay, which is used on a world-wide basis to detect mutagens and is required by many government regulatory agencies for approval to market new drugs and other chemical agents. Widely used since 1975, the Ames test is still regarded as one of the most sensitive genetic toxicity assays and a useful short-term test for predicting carcinogenicity in animals. Plasmid pKM101, which is a deletion derivative of plasmid R46 (also referred to as R-Brighton after its origin of isolation in Brighton, England), has also been used to elucidate molecular mechanisms of mutagenesis. It was isolated in the laboratory of Professor Bruce A.D. Stocker at Stanford University as part of my doctoral research with 20 R plasmids. Professor Stocker's phenomenal insight into the genetics of Salmonella typhimurium and plasmid behavior was a major factor that led to the isolation of pKM101. This paper includes a tribute to Bruce Stocker, together with a summary of my research with mutagenesis-enhancing R plasmids and a brief discussion of the molecular mechanisms involved in pKM101 plasmid-mediated bacterial mutagenesis.     

Cloning in Escherichia coli of the mutant gene coding the diphtheria toxin

Bacteriophage beta 45 of Corynebacterium diphtheriae was harvested. The extracted DNA of the bacteriophage was digested by the restriction endonuclease BamHI and inserted into the BamHI cleavage site of pUC19 vector plasmid. Plasmid pNVY5 containing a mutant gene crm45 of diphtheriae toxin in a 3.9 bpn fragment was isolated from the hybrid plasmids obtained. Cell free extracts of E. coli strain TG1 (pVNY5) contain the nontoxic protein crm45 possessing the specific enzymatic activity of diphtheriae toxin (ADP ribosylation on wheat elongation factor two). According to orientation of BamHI fragment in pNVY5 plasmid it is concluded that the crm45 gene is expressed using its own promoter.     

The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction

Summary The host-controlled K restriction of unmodified phage was 10-100-fold alleviated in the wild-type strain E. coli K12, carrying plasmid pKM101 of incompability group N. pKM101-mediated release of K restriction was also observed in lexA ^-, recA ^-, and recB ^- strains of E. coli K12. By restriction mapping Tn5 insertions in pKM101, which reduced pKM101-mediated alleviation of restriction, were shown to be located within the BglIIB fragment approximately 11 kb anticlockwise from the RI site of pKM101. We have termed the gene(s) promoting the alleviation of K restriction of phage ard (alleviation of restriction of DNA). It was shown (1) that ard function affected only the EcoK restriction system and not the EcoB, EcoRI, EcoRIII, or EcoPI system, (2) ard gene(s) did not mediate EcoK type modification of DNA and did not increase the modification activity of the EcoK system in a way similar to that observed with gene ral of bacteriophage .     

Functional organization of plasmid pKM101.

Tn5 insertion mutants and in vitro-generated deletion mutants of the mutagenesis-enhancing plasmid pKM101 have been used to identify several genetic regions on the pKM101 map. In clockwise order on the pKM101 map are: (i) the bla gene, coding for a beta-lactamase; (ii) the Slo region, responsible for retarding cell growth on minimal medium; (iii) the tra genes, enabling pKM101 to transfer conjugally; (iv) sensitivity to IKe phage (this function[s] maps within the tra region); (v) the muc gene(s), responsible for enhancing ultraviolet light and chemically induced mutagenesis in the cell; and (vi) the Rep region, essential for plasmid replication. The muc gene(s) and the Rep region are contained in a deoxyribonucleic acid region bounded by inverted repeated sequences.     

Plasmid pGW16, a derivative of pKM101, increases post-UV DNA synthesis, but sensitises some strains of Escherichia coli to UV

Plasmid pKM101, which carries muc genes that are analogous in function to chromosomal umu genes, protected Escherichia coli strains AB1157 uvrB+ umuC+, JC3890 uvrB umuC+, TK702 uvrB+ umuC and TK501 uvrB umuC against ultraviolet irradiation (UV). Plasmid pGW16, a derivative of pKM101 selected for its increased spontaneous mutator effect, also gave some protection to the UmuC-deficient strains, TK702 and TK501. However, it sensitised the wild-type strain AB1157 to low, but protected against high doses of UV, whilst sensitising strain JC3890 to all UV doses tested. Even though its UV-protecting effects varied, pGW16 was shown to increase both spontaneous and UV-induced mutation in all strains. Another derivative of pKM101, plasmid pGW12, was shown to have lost all spontaneous and UV-induced mutator effects and did not affect post-UV survival. Plasmids pKM101 and pGW16 increased post-UV DNA synthesis in strains AB1157 and TK702, whereas pGW12 had no effect. Similarly, the wild-type UV-protecting plasmids R46, R446b and R124 increased post-UV DNA synthesis in strain TK501, but the non-UV-protecting plasmids R1, RP4 and R6K had no effect. These results accord with the model for error-prone DNA repair that requires umu or muc gene products for chain elongation after base insertion opposite non-coding lesions. They also suggest that the UV-sensitizing effects of pGW16 on umu+ strains can be explained in terms of overactive DNA repair resulting in lethal, rather than repaired UV-induced lesions.     

Identification of the Escherichia coli K-12 cpxA locus as a single gene: construction and analysis of biologically-active cpxA gene fusions

In the accompanying communication we showed that a 2 kb EcoRI-BamHI restriction fragment from the pfkA-rha interval of the Escherichia coli K-12 chromosome fully complemented a chromosomal cpxA mutation when the fragment was cloned in pBR325. The same fragment cloned in pBR322 lacked any complementing activity. We show here that minicells containing the pBR325 derivative (pRA310) synthesized a 33 kDa polypeptide, designated phi 33, that was not synthesized in minicells containing the pBR322 derivative (pRA311) or either of the parent plasmids. Synthesis of the phi 33 polypeptide did not occur in minicells containing Tn5 insertion alleles of pRA310 that inactivated its cpxA complementing activity. These insertions mapped within the vector cat (chloramphenicol acetyltransferase gene) sequence immediately adjacent to the EcoRI site of pRA310 and within the 700-800 bp of the cloned EcoRI-BamHI fragment immediately adjacent to the EcoRI site. Tn5 insertions located within the fragment but closer to the BamHI terminus affected neither the cpxA complementing activity of pRA310 nor synthesis of the phi 33 polypeptide in minicells. Plasmid pRA311 could be converted to a plasmid with cpxA complementing activity by cloning into its EcoRI site a restriction fragment containing a hybrid trp-lacUV5 promoter, the lacZ ribosome binding site, and the first eight lacZ codons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of the replication region of an indigenous plasmid of Rhodobacter sphaeroides

Abstract The isolation of the replication region of an indigenous plasmid of 42 kb of the phototrophic bacterium Rhodobacter sphaeroides is described. This plasmid was digested with the Bgl II restriction enzyme, ligated to the 2.7 Bgl II fragment of transposon Tn 10 , which contains the tet genes conferring tetracycline resistance, and the mixture was transformed into the Escherichia coli MC1061 strain. One of several chimeric plasmids harboring the replication region of the 42-kb plasmid obtained by this process was named pUA33 and further characterized. Plasmid pUA33 is approx. 8.3 kb. A partial restriction map has been constructed. Plasmid pUA33 is stable in E. coli cells growing under non-selective conditions and is non-self-transmissible. All these data suggest that the pUA33 plasmid may be a very useful tool for gene cloning in R. spheroides .     

Cloning and expression of the transhydrogenase gene of Escherichia coli.

Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, we have isolated three clones bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclease analysis. A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into the PstI site of plasmid pUC13. Examination of several deletion derivatives of the resulting plasmid and subsequent treatment with exonuclease BAL 31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment. This segment was located at 35.4 min in the E. coli genome. Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase. The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids. Two polypeptides of molecular weights 50,000 and 47,000 were coded by the 3.05-kilobase fragment of pDC11. Both polypeptides were required for expression of transhydrogenase activity.     

Cloning of pMOL28-encoded nickel resistance genes and expression of the genes in Alcaligenes eutrophus and Pseudomonas spp.

The 163-kilobase-pair (kb) plasmid pMOL28, which determines inducible resistance to nickel, cobalt, chromate, and mercury salts in its native host Alcaligenes eutrophus CH34, was transferred to a derivative of A. eutrophus H16 and subjected to cloning procedures. After Tn5 transposon mutagenesis, restriction endonuclease analysis, and DNA-DNA hybridization, two DNA fragments, a 9.5-kb KpnI fragment and a 13.5-kb HindIII fragment (HKI), were isolated. HKI contained EK1, the KpnI fragment, as a subfragment flanked on both sides by short regions. Both fragments were ligated into the suicide vector pSUP202, the broad-host-range vector pVK101, and pUC19. Both fragments restored a nickel-sensitive Tn5 mutant to full nickel and cobalt resistance. The hybrid plasmid pVK101::HKI expressed full nickel resistance in all nickel-sensitive derivatives, either pMOL28-deficient or -defective, of the native host CH34. The hybrid plasmid pVK101::HKI also conferred nickel and cobalt resistance to A. eutrophus strains H16 and JMP222, Alcaligenes hydrogenophilus, Pseudomonas putida, and Pseudomonas oleovorans, but to a lower level of resistance. In all transconjugants the metal resistances coded by pVK101::HKI were expressed constitutively rather than inducibly. The hybrid plasmid metal resistance was not expressed in Escherichia coli. DNA sequences responsible for nickel resistance in newly isolated strains showed homology to the cloned pMOL28-encoded nickel and cobalt resistance determinant.