A new gene responsible for an energy-transducing system in Escherichia coli

A mutant strain (ttr-3) of Escherichia coli was originally isolated as a strain resistant to tributyltin exhibiting temperature-sensitive depressions of growth and ATP synthesis on succinate plates at 42 degrees C. The ttr gene was mapped between the pyrE and dnaA genes (in the 82-83 min region) on the chromosome by P1-transduction experiments. Comparison of proline transport and oxygen uptake by membrane vesicles of the wild-type transductant and the mutant (ttr-3) transductant showed that membrane vesicles of the mutant exhibited temperature-sensitive decrease of proline transport and increase of oxygen uptake at the restrictive temperature (42 degrees C), compatible with depression of growth of the mutant at this temperature. Therefore, the ttr gene seems to code for some factor involved in the respiratory chain that is present in the inner membrane of Escherichia coli.     

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R100.1: origin of chromosome replication during exponential growth.

We have investigated the behavior, during exponential growth, of strains of Escherichia coli carrying a dnaA(Ts) mutation that has been suppressed by the integration of the F-like R plasmid R100.1. We present evidence showing that replication in these strains proceeds largely from the normal chromosome origin at 30 degrees C, a permissive temperature for the dnaA(Ts) gene product, whereas, at 42 degrees C, replication proceeds largely from the integrated plasmid. These conclusions are based on measurements made by deoxyribonucleic acid:deoxyribonucleic acid hybridization of the relative frequencies of the prophages Mu-1 and lambdaind- and R100.1 integrated at known locations on the E. coli chromosome in these Hfr strains.     

Rifampicin-induced replication of the plasmid pBR322 in Escherichia coli strains carrying dnaA mutations

The replication pattern of the plasmid pBR322 was examined in the dnaA mutants of Escherichia coli. The rate of pBR322 DNA synthesis is markedly decreased after dnaA cells are shifted to the restrictive temperature of 42 degrees C. However, addition of rifampicin (RIF) to cultures of dnaA strains incubated at 42 degrees C after a lag of 90 min results in a burst of pBR322 synthesis. This RIF-induced pBR322 replication remains dependent on DNA polymerase I activity. Efficient plasmid pBR322 replication is observed at 42 degrees C in the double mutant dnaA46cos bearing an intragenic suppressor of dnaA46. Though replication of pBR322 in dnaA46cos growing at 42 degrees C is initially sensitive to RIF plasmid synthesis is restored after 90 min incubation in the presence of the drug. RIF-induced replication of the plasmid pBR327, lacking the rriB site implicated in RIF-resistant synthesis of the L strand of ColE1-like plasmids (Nomura and Ray 1981; Zipursky and Marians 1981), was observed also in dnaA46 at 42 degrees C.     

Evidence for positive regulation of plasmid prophage P1 replication: integrative suppression by copy mutants.

Like low-copy-number plasmids including P1 wild type, multicopy P1 mutants (P1 cop, maintained at five to eight copies per chromosome) can suppress the thermosensitive phenotype of an Escherichia coli dnaA host by forming a cointegrate. At 40 degrees C in a dnaA host suppressed by P1 cop, the only copy of P1 is the one in the host chromosome. Trivial explanations of the lack of extrachromosomal copies of P1 cop have been eliminated: (i) during integrative suppression, the P1 cop plasmid does not revert to cop+; (ii) the dnaA+ function of the host is not required to maintain P1 cop at a high copy number; and (iii) integrative recombination does not occur within the region of the plasmid involved in regulation of copy number. Since there are no more copies of the chromosomal origin (now located within the integrated P1 plasmid) than in a P1 cop+-suppressed strain, the extra initiation potential of the P1 cop is not used to provide multiple initiations of the chromosome. When a P1 cop-suppressed dnaA strain was grown at 30 degrees C so that replication could initiate from the chromosomal origin as well as from the P1 origin, multicopy supercoiled P1 DNA was found in the cells. This plasmid DNA was lost again when the temperature was shifted back to 40 degrees C.     

Host cell variations resulting from F plasmid-controlled replication of the Escherichia coli chromosome.

Cell size and DNA concentration were measured in Escherichia coli K-12 ET64. This strain carries a dnaA (Ts) mutation that has been suppressed by the insertion of the F plasmid into the chromosome. ET64 can grow in a balanced steady state of exponential growth at the restrictive temperature for its dnaA allele (39 degrees C), in which chromosome replication is controlled by the F plasmid, and at the permissive temperature (30 degrees C), in which chromosome replication is controlled by dnaA-oriC. When cells grown at the indicated temperatures were compared, it was observed that at 39 degrees C, the cell mass increased and the amount of cellular DNA decreased slightly; therefore, the DNA concentration was strongly reduced. These changes can neither be explained by the reduction of the generation time (which is only 10-15%) nor from observed changes in the replication time and in the time between DNA synthesis termination and cell division. Variations were mainly due to the increase in cell mass per origin of replication, at initiation, in cells grown at 39 degrees C. Control of chromosome replication by the F plasmid appears to be the reason for the increase in the initiation mass. Other possible causes, such as the modification of growth temperature, the generation time, or both, were discarded. These observations suggest that at one growth rate, the F plasmid replicates at a particular cell mass to F particle number ratio, and that this ratio is higher than the cell mass to oriC ratio at the initiation of chromosome replication. This fact might be significant to coordinate the replication of two different replicons in the same cell.     

Chromosome-plasmid interaction in Escherichia coli K-12 carrying a thermosensitive plasmid, Rts1, in autonomous and in integrated states.

An Hfr strain of Escherichia coli K-12 was obtained by integrative suppression with a thermosensitive plasmid, Rts1. The R plasmid was integrated into the chromosome between rif and thr, and transfer of the chromosome occurred counterclockwise. The thermosensitivity of host cell growth due to the dnaA mutation was markedly but not completely reduced in this integratively suppressed Hfr strain. When the dnaA mutation was removed by transducing the dnaA+ genome to this Hfr, the thermosensitivity of cell growth due to existence of Rts1 was suppressed in contrast to strains carrying it autonomously. Thermosensitivity of cell growth appeared again when the plasmid was detached from the chromosome to exist autonomously. Contrary to the effect on cell growth, the transfer of the chromosome and the plasmid itself and the ability to "restrict" T-even phages were still thermosensitive in all of these strains carrying Rts1, irrespective of its state of existence. The detached plasmid as well as the original Rts1 were segregated upon growth at 42 C. These data are discussed in relation to chromosome-plasmid interaction. One of the most important conculusions is that some plasmid genes, related to their replication, are phenotypically suppressed by the chromosome when it is integrated.     

Two types of cold sensitivity associated with the A184-->V change in the DnaA protein

Multicopy dnaA(Ts) strains carrying the dnaA5 or dnaA46 allele are high-temperature resistant but are cold sensitive for colony formation. The DnaA5 and DnaA46 proteins both have an A184-->V change in the ATP binding motif of the protein, but they also have one additional mutation. The mutations were separated, and it was found that a plasmid carrying exclusively the A184-->V mutation conferred a phenotype virtually identical to that of the dnaA5 plasmid. Strains carrying plasmids with either of the additional mutations behaved like a strain carrying the dnaA+ plasmid. In temperature downshifts from 42 degrees C to 30 degrees C, chromosome replication was stimulated in the multicopy dnaA46 strain. The DNA per mass ratio increased threefold, and exponential growth was maintained for more than four mass doublings. Strains carrying plasmids with the dnaA(A184-->V) or the dnaA5 gene behaved differently. The temperature downshift resulted in run out of DNA synthesis and the strains eventually ceased growth. The arrest of DNA synthesis was not due to the inability to initiate chromosome replication because marker frequency analysis showed high initiation activity after temperature downshift. However, the marker frequencies indicated that most, if not all, of the newly initiated replication forks were stalled soon after the onset of chromosome replication. Thus, it appears that the multicopy dnaA(A184-->V) strains are cold sensitive because of an inability to elongate replication at low temperature. The multicopy dnaA46 strains, on the contrary, exhibit productive initiation and normal fork movement. In this case, the cold-sensitive phenotype may be due to DNA overproduction.     

Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells'' initiation potential.

The dnaA gene is essential for initiation of chromosomal replication in Escherichia coli. A gene homologous with the E. coli dnaA was found in the replication origin region of the Bacillus subtilis chromosome. We have now isolated a temperature sensitive mutant of the B. subtilis dnaA by in vitro mutagenesis of the cloned gene. At a nonpermissive temperature, 49 degrees C, DNA replication stops completely after 60% increase in a rich medium, while cell mass continues to increase exponentially at 2.5 times the rate at 30 degrees C. A ratio of gene frequency between purA (origin marker) and metB (terminus marker) changes gradually from 2.7 at 30 degrees C to 1.0 in 45 min at 49 degrees C, indicating completion of the ongoing replication cycle. Upon the temperature shift down to 30 degrees C after the incubation at 49 degrees C for 60 min, DNA replication resumes without delay, and the purA/metB ratio increases rapidly to 6, i.e. consecutive initiation of more than two rounds of replication. Addition of chloramphenicol at the time of the temperature shift down did not inhibit the increase in the purA/metB ratio, while rifampicin inhibited the re-initiation completely. The mutation is a single base change from C to T in the dnaA gene resulting in an amino acid substitution from Ser to Phe in the DnaA protein. The mutation was responsible for both temperature sensitive growth and the defect in initiation of chromosomal replication. We observed a remarkable correlation between the amount of DnaA protein and the amount of initiation potential accumulated during incubation at the non-permissive temperature.     

Transposition of the phage D3112 genome in Escherichia coli cells

It has been demonstrated that the genome of phage D3112 of Preudomonas aeruginosa can be transposed into Escherichia coli chromosome as a component of the hybrid plasmid RP4 TcrKms::D3112. Also, transposition of D3112 from E. coli (D3112) chromosome into RP4 plasmid occurs. The phage stimulates the chromosome mobilizing activity of RP4 plasmid, similar to other transposons. E. coli (RP4::D3112) cells were previously shown to form no colonies at 30 degrees C. Auxotrophic mutants and mutants incapable of utilizing different carbohydrates were found among E. coli clones survived after a long incubation at 30 degrees C (at frequencies approximately 10(-3) - 10(-4). These mutants inherited stably the capability to produce D3112 phage. E. coli auxotrophic mutants have arisen indeed as a consequence of phage integration into the E. coli chromosome, since prototrophic transductants derived from these mutants after their treatment with generalized transducing P1 phage have lost the ability to produce D3112 phage. Clones with mutations in Km or Tc genes of RP4 plasmid, occurring at high frequencies (about 3%) were found after introduction of RP4 into E. coli (D3112). These mutant RP4 plasmids carry insertions of D3112 genomes. Clones of E. coli which lost mutant plasmids still produce D3112 and retain their initial auxotrophic mutations.     

Kinetics of minichromosome replication in Escherichia coli B/r.

Replication control of the minichromosome pAL2 was found to differ from that of the chromosome in synchronously dividing populations of Escherichia coli B/r. Initiation of minichromosome replication took place at an increasing rate throughout synchronous growth. No coupling to initiation of chromosome replication was detected. Minichromosome replication was further examined in a dnaA5(Ts) temperature-sensitive initiation mutant. When cultures held at nonpermissive temperature (41 degrees C) for 60 min were shifted to permissive temperature (25 degrees C), initiation of both pAL2 and chromosome replication ensued in two waves spaced 25 to 35 min apart. Evidence is presented that minichromosomes terminate replication by passing slowly through a series of dimeric intermediate forms before reaching the closed circular monomeric form. The consequence of this slow passage as a rate-limiting step in the initiation reaction is discussed.     

Erwinia carotovora as a recipient system for the natural hybrid plasmid RP4::Mu cts62

The plasmid RP4::Mu cts62 is transferred from Escherichia coli cells into a recipient strain Erwinia carotovora 268 by conjugation with the frequency 1.5-5 x 10(-7) per donor cell. The maximal frequencies of transfer are obtained by cultivation of donor and recipient cells for 3-5 h on the filters. Structural and functional validity of the plasmid in transconjugants is expressed in preservation of all antibiotic-resistant markers of RP4, thermosensitivity to growth at 42 degrees C as well as spontaneous and thermally-induced production and zygotic induction of bacteriophage determined by the genome of Mu cts62, total length of the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid RP4::Mu cts62 in Erwinia carotovora transconjugant cells has been determined. A single bacteriophage genome has been shown to transpose into the chromosome of the cell with the elimination of RP4 fragment under the conditions of thermal induction.     

Isolation of an Hfr donor of Pseudomonas aeruginosa PAO by insertion of the plasmid RP1 into the tryptophan synthase gene

Summary A derivative of the IncP-1 plasmid RP1, temperature-sensitive for maintenance, was inserted into the Pseudomonas aeruginosa chromosome by selection for a plasmid marker (carbenicillin resistance) at nonppermissive temperature. In one strain, PAO 1000, the plasmid was stably integrated in the trpA, B gene cluster mapped at 27 min, as shown by the following evidence. (i) Trp⁺ transductants lost all plasmid markers. (ii) Cleared lysates of PAO 1000 showed no plasmid band typical of the autonomous RP1 in agarose gel electrophoresis. (iii) No transfer of carbenicillin resistance by PAO 1000 was detectable. (iv) PAO 1000 mobilised the chromosome from an origin at, or very near, the plasmid insertion site with high frequency (recovery of proximal markers 10^–3 per donor). Matings on the plate with and without interruption of conjugation showed that chromosome transfer was unidirectional. (v) Recombinants from PAO 1000-mediated crosses did not inherit plasmid markers or the trpA, B mutation. A derivative of PAO 1000 was obtained which had lost the Hfr property and all plasmid markers except carbenicillin resistance. This strain (PAO 1001), when carrying the autonomous RP1 plasmid, was capable of unidirectional chromosome mobilisation like PAO 1000, but with 50-fold lower efficiency. We propose that integration of the temperature-sensitive RP1 plasmid in PAO 1000 occurred via transposition of Tnl, the element specifying carbenicillin resistance.     

Molecular cloning and DNA sequencing of the radC gene of Escherichia coli K-12.

The radC102 mutation that sensitizes E. coli K-12 cells to ultraviolet light, ionizing radiations and alkylating agents was localized between the fpg and pyrE genes at 81.7 min on the bacterial chromosome. E. coli strain BH20 (radC+, fpg-1::KnR) has a 10.5-kb EcoRI/KpnI DNA fragment spanning the region from pyrE to the insertion mutation fpg-1::KnR. The proximity of the radC gene to this insertion mutation provided a strategy to isolate the radC+ gene based on the cloning of radC+ and fpg-1::KnR on the same DNA fragment using the resistance to kanamycin as a selector. A library of EcoRI/KpnI DNA fragments of E. coli strain BH20 was inserted into pUC19. One recombinant plasmid conferring resistance to kanamycin was selected and named pRCV10. The pRCV10 plasmid partially restores the resistance to UV-radiation when transformed into SR1187 (radC102), but sensitizes the wild-type strain to the same treatment. The radC102 complementing region was localized on a 1.2-kb BglII/BglII DNA fragment which was sequenced. The DNA sequence complementing the radC102 mutation contained an ATG translation start codon with an open reading frame of 297 base pairs which encodes a polypeptide of Mr 11,500. The order of the genes in this region of the E. coli chromosome is: fpg--rpmBG--radC--pyrE.     

The mutation that makes Escherichia coli resistant to lambda P gene-mediated host lethality is located within the DNA initiator Gene dnaA of the bacterium

Earlier, we reported that the bacteriophage lambda P gene product is lethal to Escherichia coli, and the E. coli rpl mutants are resistant to this lambda P gene-mediated lethality. In this paper, we show that under the lambda P gene-mediated lethal condition, the host DNA synthesis is inhibited at the initiation step. The rpl8 mutation maps around the 83 min position in the E. coli chromosome and is 94 % linked with the dnaA gene. The rpl8 mutant gene has been cloned in a plasmid. This plasmid clone can protect the wild-type E. coli from lambda P gene-mediated killing and complements E. coli dnaAts46 at 42 degrees C. Also, starting with the wild-type dnaA gene in a plasmid, the rpl-like mutations have been isolated by in vitro mutagenesis. DNA sequencing data show that each of the rpl8, rpl12 and rpl14 mutations has changed a single base in the dnaA gene, which translates into the amino acid changes N313T, Y200N, and S246T respectively within the DnaA protein. These results have led us to conclude that the rpl mutations, which make E. coli resistant to lambda P gene-mediated host lethality, are located within the DNA initiator gene dnaA of the host.     

An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus

A Thermus thermophilus host strain of which the leuB gene was totally deleted was constructed from a delta pyrE strain by a two step method. First, the leuB gene was replaced with the pyrE gene. Second, the inserted pyrE gene was deleted by using 5-fluoroorotic acid. A plasmid vector with the leuB marker was constructed and the plasmid complemented the leuB deficiency of the host. When the leuB gene from Escherichia coli and its derivative encoding a stabilized enzyme were expressed with the host-vector system, their growth temperature reflected the stability of the enzyme. These results suggest that the gene replacement deletion method using the pyrE gene is useful for the construction of a reliable plasmid vector system and it can be applied to the selection of stabilized enzymes.     

Features of genome expression of phage transposon D3112 of Pseudomonas aeruginosa in Escherichia coli bacteria: dependence of bacterial phenotype on copy number of D3112 genome]

Escherichia coli (RP4 :: D3112) bacteria manifest Tcs phenotype (thirty centigrade sensitivity), i.e. the cells do not divide and form colonies under conditions of lowered temperature (30 degrees C and lower), while cells grow normally at 42 degrees C. In this work it is demonstrated that replication-transposition of D3112 and the Tcs phenotype depend on no recA system of E.coli. Following events lead to the loss of the Tcs phenotype (in E.coli (RP4 :: D3112) cells survived after growing at 30 degrees C): occurrence of mutations in bacterial, phage and plasmid genomes, elimination of DNA of hybrid plasmid or RP4 DNA (a portion of DNA) as well as integration of the hybrid plasmid into bacterial chromosome. In the latter case, the E.coli (D3112) cells acquired the properties shared by the initial bacteria and those with the Tcs phenotype. Such clones are designated tcl (thirty centigrade low sensitivity), they are able to form colonies at 30 degrees C but their growth is more slow, they maintain instability at lowered temperature and continue to produce D3112 phage. The tcl clones in which replication-transposition of D3112 DNA in less effective than in the tcs clones are a suitable object for the study of genetic rearrangements caused by D3112 phage transposon. It is shown that either complete RP4 genome or its portion are comprised between direct repeats of D3112 and are built into various chromosomal sites, i.e. cointegrates are being formed. Two types of deletions are revealed: eliminating sites of RP4 plasmid adjacent to the left end of D3112 genome as well as deletions of the D3112 genome. It is demonstrated that alteration in the growth nature of E.coli, carrying D3112 DNA, at 30 degrees C depends on the copy number of D3112 per bacterial cell.     

Overinitiation of chromosome and plasmid replication in a dna Acos mutant of Escherichia coli K12. Evidence for dnaA-dnaB interactions

The dnaAcos mutations are phenotypic suppressors of dnaAts46 that are co-transduced with dnaA, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees C to 32 degrees C. We have used pulse labelling and DNA-DNA hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids pSC101, RTF-Tc, and lambda dv in such strains. After a shift of a dnaAcos strain from 42 degrees C to 32 degrees C (non-permissive temperature), initiation of the chromosome and replication of the plasmid pSC101 are stimulated, while the dnaA-independent plasmid RTF-Tc is not affected. The presence of pSC101 does not affect the level of overinitiation of the chromosome. The presence of lambda dv suppresses the cold sensitivity of dnaAcos mutants and allows the cells to grow at both 32 degrees C and 42 degrees C. The presence of lambda dv suppresses the overinitiation of chromosome and of pSC101 replication at 32 degrees C. Previous reports had shown that these suppressions involve an interaction between the dnaA product and the lambda P protein, which is also known to interact with dnaB. We show here that the mutant prophage P1 bac-crr, which produces high levels of a dnaB analogue, suppresses the dnaAcos phenotype, while wild type P1 does not. These results suggest that initiation involves interactions between the dnaA and dnaB products.     

Occurrence, transfer and mobilization in epilithic strains of Acinetobacter of mercury-resistance plasmids capable of transformation

A 7.8 kb plasmid (pQM17) encoding mercury resistance was isolated from two epilithic strains of Acinetobacter calcoaceticus. The plasmid had a broad host range when mobilized by RP1, transferring into Pseudomonas aeruginosa, P. putida, P. fluorescens, Escherichia coli, Proteus vulgaris and Chromobacterium sp. with frequencies ranging from 5.3 x 10(-9) to 4.6 x 10(-4) per recipient. The plasmid could be transferred into A. calcoaceticus BD413 using intact cells of donor and recipient bacteria (i.e. natural transformation) and there was a broad temperature optimum (14-37 degrees C) for transformation. Transformation was as efficient in liquid matings as on plates but there was no effect of pH in the range 5.6-7.9. Maximum transformation frequencies were obtained after 24 h on agar plates containing 3.5-10 g C 1-1 with donor to recipient ratios ranging from 6 to 415.     

Conjugation in Rhodopseudomonas sphaeroides mediated by R plasmids

Plasmids R68.45, RP4, RP4::Mu cts62, RP1ts::Tn10, RP1ts::Tn9, Rts1 and RP41 were transferred into cells of photosynthetic nitrogen-fixation bacterium Rhodopseudomonas sphaeroides from Escherichia coli and Pseudomonas aeruginosa. The transfer of plasmids occurred with high frequency of 10(-1) to 10(-2) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell. Bacteriophage Mu cts62 could be induced from the plasmid DNA in R. sphaeroides 2R cells and was capable of the lytic growth and producing phage progeny. It was demonstrated that an increase in the efficiency of donor chromosomal genes transfer into recipient cells could be achieved in crosses with the donor carrying RP4::Mcts62 plasmid.     

Plasmid and transposon transfer to Thiobacillus ferrooxidans.

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and pUB307 were transferred to acidophilic, obligately chemolithotrophic Thiobacillus ferrooxidans from Escherichia coli by conjugation. A genetic marker of kanamycin resistance was expressed in T. ferrooxidans. Plasmid RP4 was transferred back to E. coli from T. ferrooxidans. The broad-host-range IncQ vector pJRD215 was mobilized to T. ferrooxidans with the aid of plasmid RP4 integrated in the chromosome of E. coli SM10. pJRD215 was stable, and all genetic markers (kanamycin/neomycin and streptomycin resistance) were expressed in T. ferrooxidans. By the use of suicide vector pSUP1011, transposon Tn5 was introduced into T. ferrooxidans. The influence of some factors on plasmid transfer from E. coli to T. ferrooxidans was investigated. Results showed that the physiological state of donor cells might be important to the mobilization of plasmids. The transfer of plasmids from E. coli to T. ferrooxidans occurred in the absence of energy sources for both donor and recipient.