Novobiocin-induced elimination of F'lac and mini-F plasmids from Escherichia coli.

Novobiocin eliminated (cured) F'lac and three low-copy-number mini-F plasmids (pML31, pMF21, and pMF45) from Escherichia coli to different extents. F'lac was cured 0 to 3%. pML31, whose replication region is contained on the 9-kilobase f5 EcoRI restriction enzyme fragment of F, was eliminated 10 to 92%. pMF21, deleted of the origin of mini-F replication at 42.6 kilobases on the F map and known to initiate from an origin at 45.1 kilobases, and its closely related derivative pMF45 were cured to the greatest extent (greater than 97%). pMF45 was eliminated from a wild-type bacterial strain but not from an isogenic novobiocin-resistant gyrB mutant strain, indicating involvement of the B subunit of DNA gyrase in the curing phenomenon. The number of bacteria containing pMF45 halved with each generation of growth in the presence of novobiocin, as is predicted for complete inhibition of plasmid DNA replication.     

Replication Region Fragments Cloned from Flac+ Are Identical to EcoRI Fragment f5 of F

The replication region fragments from Flac⁺ cloned in plasmids pSC138 and pML31 are identical with each other and with EcoRI fragment 5 of plasmid F.     

Plasmid Copy Number Underlies Adaptive Mutability in Bacteria

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac⁺ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac⁺ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac⁺ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac⁺ revertant colonies are initiated by preexisting cells with multiple copies of the F′lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F′lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F′lac copies and consequently the number of Lac⁺ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F′lac copies divide very little under selection but have enough energy to replicate their F′lac plasmids repeatedly until reversion initiates a stable Lac⁺ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac⁺ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced.     

Cotransformation of lactococcin-producing, 2.0-mega dalton and erythromycin-resistant pGB 301 plasmids toLactococcus lactis subsp.lactis protoplast

Protoplasts of plasmid-freeLactococcus lactis subsp.lactis LM 0230 and PC₄ strains were cotransformed successfully with the plasmid pools ofL. lactis subsp.lactis 484, a lactosefermenting (Lac⁺), lactococcin-producing (Lap⁺), lactococcin-resistant (Lap^r), sucrosefermenting (Suc⁺) wild strain, its derivatives, and pGB 301 erythromycin resistance plasmid (Ery^r) at the frequencies of 10⁴ transformants/g of DNA. PC₄ protoplasts were transformed at slightly lower frequencies that LM 0230 protoplasts when the same plasmid combinations were used for transformation. Agarose gel electrophoresis of plasmids from three groups of transformants, namely, Lac^–Lap^–Ery^r, Lac⁺Suc⁺Lap⁺Lap^rEry^r, and Lac^–Suc⁺Lap⁺ Lap^rLap^r, confirmed that 2.0 and 65.0 megadalton (MDa) plasmids carried genes for Suc⁺Lap⁺Lap^r and Lac⁺ phenotypes respectively. The protoplasts could be transformed with low-molecular-weight 2.0 MDa Lap plasmid at a relatively higher frequency than those with high-molecular-weight 65.0 MDa Lac plasmid. All the transformants resembled parent culture 484 in terms of lactic acid production (0.810–0.840%), milk curdling time (6 h), and lactococcin activity (7–12 mm, zone of inhibition) againstListeria monocytogenes, Salmonella typhi, andStaphylococcus aureus. The plasmids and their respective phenotypes in PC₄ transformants were genetically more stable than those of LM 0230 protoplasts. The marker plasmid pGB 301 disappeared more frequently from the transformants when present in association with the lowmolecular-weight, high-copy-number 2.0 MDa plasmid, thereby suggesting the incompatibility of these two plasmids.     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

pHG276: A multiple cloning site pBR322 copy number vector expressing a functional lacα peptide from the bacteriophage lambda PR promoter

The bacteriophage lambda early promoter P_R has been used to direct the synthesis of lacα in a plasmid containing the multiple cloning site of pUC8. The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI⁸⁵⁷ gene for temperature regulation of lacα expression. Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lacα and consequently, a Lac⁻ phenotype in a host carrying the M15 deletion of lac. The potential of pHG276 as a fully regulated expression vector is examined.     

Growth and reactivation of single stranded DNA phage phiX174 in E. coli undergoing "thymineless death".

Summary Flac maintenance was aberrant at permissive temperature in a temperature-sensitive dnaC mutant of Salmonella typhimurium when the normally resident pLT2 plasmid was present. Flac was, however, efficiently transferred into the dnaC pLT2⁺ strain and the resulting Flac derivative was almost as efficient in transferring Flac as were dnaC ⁺ pLT2⁺ Flac strains indicating that aberrant Flac maintenance was not associated with appreciable inhibition of transfer replication. A range of F-like plasmids behaved like pLT2 in causing aberrant Flac maintenance when present in the dnaC pLT2^- strain. Flac was, however, stably maintained in the dnaC strain in the absence of other plasmids. Although the F-like plasmids destabilized Flac, each was stably maintained when introduced into strain 11G dnaC pLT2⁺ and pLT2 was also apparently stable under these conditions. The destabilizing effect of pLT2 and other fi ⁺ plasmids was not consequent upon their inhibiting the formation of a repressible F transfer component needed for Flac replication in the dnaC strain. Incompatibility between Flac and the other plasmids induced by the dnaC lesion also appeared unlikely to be a cause of the aberrant Flac maintenance. The possibility is discussed that the initiation of Flac replication differs from that of pLT2 and the F-like plasmids with F competing less effectively than the others for the DnaC gene product.     

The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac⁺) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac⁺ triggers exponential cell growth leading to a stable Lac⁺ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac⁺ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac⁺ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions.     

Stabilization of Lactose Metabolism in Streptococcus lactis C2

The integration of the lactose plasmid from lactic streptococci into the host chromosome could stabilize this trait for dairy fermentations. Sixty lactose-positive (Lac⁺) transductants of lactose- and proteinase-negative (Lac⁻ Prt⁻) LM0220 were induced for temperature phage by UV irradiation or mitomycin C. Four of the transductants, designated KB18, KB21, KB54, and KB58, yielded lysates demonstrating less than one Lac⁺ transductant per 0.2 ml of phage lysate. Successive transferring in the presence of acriflavine did not yield Lac⁻ segregants from KB18, KB21, KB54, or KB58, whereas Streptococcus lactis C2 (parent culture) and three other Lac⁺ transductants showed 12 to 88% conversion from Lac⁺ to Lac⁻ within 6 to 10 repetitive transfers. When grown in continuous culture, KB21 did not show any Lac⁻ variants in 168 h, while S. lactis C2 had 96% conversion from Lac⁺ to Lac⁻ in 144 h. Agarose gel electrophoresis of plasmid DNA isolated from KB18, KB21, KB54, and KB58 revealed that the lactose plasmid, pLM2103, normally present in Lac⁺ transductants, was missing. This suggested integration of the transferred lactose plasmid into the chromosome. In contrast to phage lysates induced from S. lactis C2, which exhibited an exponential decrease in the number of Lac⁺ transductants after exposure to small doses of UV irradiation, the transduction frequency for lactose metabolism was stimulated by UV irradiation of lysates from KB58. The latter indicated chromosomal linkage for lac and that integration of the lactose genes plasmid into the chromosome had occurred.     

Transposon-mediated conjugational transmission of nonconjugative plasmids.

When coresident with conjugative plasmid pNC21, the nonconjugative deletion F-prime pJC59, which retains the F transfer origin oriT, was transmitted to transconjugants at a frequency comparable to that of pNC21. In addition, pJC59 was transmitted as an independent plasmid, physically separate from pNC21, an example of plasmid donation. In contrast, two plasmids that are derived from F and deleted for the oriT site, pJC61 and pML31, were transmitted at frequencies 10(4) lower than that of pNC21. This low-frequency transmission was associated with the appearance of a new plasmid in the transconjugants. In the case of pML31, we determined that this new plasmid was a recombinant composed of pNC21 and pML31, the latter flanked by two copies of transposable element Tn3. We believe that this recombinant plasmid was formed as an intermediate in the transposition of Tn3 from pNC21 to pML31 and was the vehicle for conjugational transmission of pML31 genes by a process known as plasmid conduction.     

Restriction endonuclease mapping and mutagenesis of the F sex factor replication region

Summary The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of -lactamase production.By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that 1.9x10⁶ daltons of the 6.0x10⁶ dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

Carbohydrate metabolism and transport in Bacillus subtilis. A study of ctr mutations

Summary Indirect ultraviolet induction of prophage occurs when lysogenic E. coli K12 cells are mated with ultraviolet-irradiated donor strains carrying a transmissible episome such as F lac ⁺. Indirect induction occurs in wild type, uvrA, or recB recipient lysogens, but not in recA lysogens. When nonpermissive lysogens carrying prophages susO or susP are similarly mated, the defective prophages are induced and indirect curing takes place.Although indirect induction is independent of the capacity of the lysogen for repair by pyrimidine dimer excision, indirect curing (and hence indirect induction) is subject to photoreactivation when the recipient lysogen is exposed to visible light after mating. This confirms that the structure initiating indirect ultraviolet induction is a damaged transferred episome consisting of one DNA strand containing ultraviolet photoproducts and a newly synthesized discontinuous DNA strand such that pyrimidine dimers remain in single-stranded regions.F^- lac ⁺ recombinants are formed in either nonlysogenic or lysogenic Lac^- cells receiving damaged F lac ⁺ episomes from ultraviolet irradiated F lac ⁺ donors. prophage induction occurs more frequently in zygotes that form Lac⁺ recombinants than in zygotes that remain Lac^-. In contrast, cells receiving intact (undamaged) episomes are converted to F lac ⁺ secondary donors, but are rarely induced or cured.     

Isolation and characterisation of Hfr strains from a recombination-deficient strain of Escherichia coli

Summary A T6^s RecA^- strain carrying a lac proB deletion and F_ts lac ⁺ was challenged with phage T6 and survivors which were both T6^r and Lac⁺ at 42° were tested for fertility. Among these were a number of Hfr strains which had their points of origin at or near the tsx locus and which still carried the recA allele. These arose in comparable frequencies in the RecA^- strain and in a Rec⁺ analogue. We conclude that such integration does not require the RecA function. The rate of chromosome transfer was similar in one such RecA^- Hfr and its Rec⁺ derivative; the yield of recombinants from the RecA^- strain was slightly lower than from its Rec⁺ derivative.     

Interspecific plasmid transfer between Streptococcus pneumoniae and Bacillus subtilis

Summary The streptococcal plasmids pMV158 and pLS1, grown in Streptococcus pneumoniae, were transferred to Bacillus subtilis by DNA-mediated transformation. The plasmids were unchanged in the new host; no deletions were observed in 80 instances of transfer. Their copy number was similar to that in S. pneumoniae. Two B. subtilis plasmids, pUB110 and pBD6, could not be transferred to S. pneumoniae. Hybrid plasmids were produced by recombining the EcoRI fragment of pBD6 that confers Km^r with EcoRI-cut pLS1, which confers Tc^r. The simple hybrid, pMP2, was transferable to both species and expressed Tc^r and Km^r in both. A derivative, pMP5, which contained an insertion in the pBD6 component, expressed a higher level of kanomycin resistance and was more easily selected in S. pneumoniae. Another derivative, pMP3, which contained an additional EcoRI fragment, presumably of pneumococcal chromosomal DNA, could not be transferred to B. subtilis. Previous findings that monomeric plasmid forms could transform S. pneumoniae but not B. subtilis were confirmed using single plasmid preparations. Although plasmids extracted from either species were readily transferred to S. pneumoniae, successive passage in B. subtilis increased the ability of plasmid extracts to transfer the plasmid to a B. subtilis recipient. This adaptation was tentatively ascribed to an enrichment of multimeric forms in extracts of B. subtilis as compared to S. pneumoniae. A review of host ranges exhibited by plasmids of Gram-positive bacteria suggested differences in their ability to use particular host replication functions. The pMP5 plasmid, with readily selectable Km^r and Tc^r markers in both hosts, and with the potential for inactivation of Km^r by insertion in the Bg/II site, could be a useful shuttle vector for cloning in S. pneumoniae and B. subtilis.     

Nonintegrated plasmid-folded chromosome complexes: genetic studies on formation and possible relationship to plasmid replication.

When folded chromosomes are purified from plasmid-containing bacteria, a reproducible fraction of the host's covalently closed, circular (CCC) plasmid DNA copurifies with the chromosomes. From this copurification, we infer the existence of nonintegrative plasmid-chromosome (NPC) complexes. Previously, we noted that plasmids dependent on DNA polymerase III and with stringent control of replication complex to a greater extent than plasmids dependent on DNA polymerase I and with relaxed control of replication. We have examined this subject in more depth and find that: (i) The composite plasmids formed by in vitro recombination of a “stringent” with a “relaxed” replicon complex to chromosomes at the frequency of the component replicon which directs replication; (ii) all of the detectable replicative intermediates, but only 25% of the CCC forms, of plasmid ColE1 complex to chromosome; and (iii) when a mini-F plasmid is deleted for the DNA sequences which include the primary origin of replication, the complexing frequency decreases 30 to 40%. We conclude from these findings that NPC complexes either indirectly or directly relate to plasmid replication. Further, we find that the EcoRI kan⁺ fragments of pML31 and the ampicillin resistance transposon, Tn3, promote complexing of both ColE1 and mini-F plasmids to host chromosomes. The biological significance of this latter complexing is unknown. However, we conclude from these studies and from point (iii) that complexing is determined in part by unique plasmid sequences.     

Tn1000 insertional mutagenesis of cloned repressor gene of the phage L: Plasmid oligomerization in the presence of F’lac

An ampicillin resistance plasmid carrying the cloned repressor gene c_II of the L phage (Salmonella lyphimurium) was conducted by F’lac into an F^- recipient. Two types of plaamids were isolated from Ap^r transconjugants. The majority of plasmids were dimers with one copy of Tn1000 inserted, the minority being monomers with one copy of Tn1000. This proportion remained unaltered when we used the F’lac strain transformed with a monomeric form of the recombinant plasmid as a donor. An extensive oligomerization of pBR322-originating plasmids was proved in the presence of F’lac; its presumable relationship to transposition-related processes is suggested.     

Involvement of a Plasmid in Production of Ropiness (Mucoidness) in Milk Cultures by Streptococcus cremoris MS

Curing and genetic transfer experiments showed that lactose-fermenting ability (Lac⁺) and the ability to produce mucoidness in milk cultures (Muc⁺) in Streptococcus cremoris MS were coded on plasmids. The Lac⁺ phenotype was associated with a 75.8-megadalton plasmid, pSRQ2201. The Muc⁺ phenotype was associated with a 18.5-megadalton plasmid, pSRQ2202. The Lac plasmid, pSRQ2201, was first conjugatively transferred from S. cremoris MS to Lac⁻S. lactis ML-3/2.2. Later, the Muc plasmid, pSRQ2202, was conjugatively transferred from Lac⁻ Muc⁺S. cremoris MS04 to Lac⁺ nonmucoid S. lactis transconjugant ML-3/2.201. Subsequently, pSRQ2201 and pSRQ2202 were cotransferred from Lac⁺ Muc⁺S. lactis transconjugant ML-3/2.202 to Lac⁻, nonmucoid, malty S. lactis 4/4.2 and S. lactis subsp. diacetylactis SLA3.25. Transconjugants showing pSRQ2201 were Lac⁺; those containing pSRQ2202 were Muc⁺. With the transfer of pSRQ2202, the transconjugants S. lactis ML-3/2.202 and S. lactis subsp. diacetylactis SLA3.2501 not only acquired the Muc⁺ phenotype but also resistance to bacteriophages, which were lytic to the respective parent strains S. lactis ML-3/2.201 and S. lactis subsp. diacetylactis SLA3.25.     

The maintenance affinities of KLF-1 proximal F merogenotes inEscherichia coli

Summary In conjugation with donor strains carrying proximal F merogenotes of KLF-1 type about 100-fold lower frequency ofLeu ⁺ orLac ⁺ recombinants was found. The determination of the level of β-galactosidase synthesis during the initial period of mating indicated that the transfer process of plasmid DNA was not impaired. Among the recombinants selected a large fraction have not expressed the plasmic fertility functions. This phenomenon was found to be replicon specific and was observed only with proximal F merogenotes but not with classical F'lac and F'ORF-1 elements or RI-19 plasmid. The expression of KLF-1 plasmid functions in the cell seems to be affected by a chromosomal gene of the proximal F merogenote closely linked toleu marker.     

Construction of Streptococcus lactis subsp. lactis Strains with a Single Plasmid Associated with Mucoid Phenotype

Lactose-fermenting mucoid (Lac⁺ Muc⁺) variants of plasmid-free Streptococcus lactis subsp. lactis MG1614 were obtained by protoplast transformation with total plasmid DNA from Muc⁺S. lactis subsp. cremoris ARH87. By using plasmid DNA from these variants for further transformations followed by novobiocininduced plasmid curing, Lac⁻ Muc⁺ MG1614 strains containing only a single 30-megadalton plasmid could be constructed. This plasmid, designated pVS5, appeared to be associated with the Muc⁺ phenotype.