On the mechanism of conjugation in Escherichia coli K 12

Summary The phenomenon of conjugation consists of many stages. The most important are: the formation of contacts between mating cells, the transfer of DNA from the donor to the recipient, and the integration of the transfered DNA fragments into the chromosome of the recipient. Only after completion of all these stages are recombinants formed. With the aid of specific inhibitiors (nalidixic acid, FUDR), thymine starvation, and use of special thermosensitive mutants it is possible to study the role of DNA synthesis during every stage of conjugation. It was demonstrated that the genetic transfer is due to semiconservative DNA-replication in the donor cell. The fragments of DNA transfered are synthesized in the period of mating by a special replication system (F-replicon). In case of T _DNA ^S mutants unable to grow at 41°, the ability to synthesize DNA during conjugation is preserved.The inhibition of the DNA synthesis in the donor cell by poisons leads to complete inhibition of genetic transfer. The third stage — formation of recombinants requires DNA synthesis in the recipient cell and is inhibited by poisoning, thymine starvation or T _DNA ^S mutations in the recipient. In cases where recombination is not involved (i.e. sexduction) the inhibition of DNA synthesis in the recipient has no significant effect.     

On the role of the recipient cell during conjugation in Escherichia coli

To study the role of the E. coli recipient cell in conjugation recipient cell mutants deficient in conjugation (Con^-) were isolated. Mutants specific for F-type E. coli donor cells (ConF^-) and mutants specific deficient in conjugation with I-type donor cells (ConI^-) were isolated.Both ConF^- and ConI^- mutants were blocked in stable mating pair formation. Biochemical analysis of the mutants suggests that the outer membrane protein coded by the ompA gene and LPS are important for recipient activity in F-type conjugation while LPS is important for recipient activity in I-type conjugation.     

Temperature-Sensitive Mutants for the Replication of Plasmids in ESCHERICHIA COLI II. Properties of Host and Plasmid Mutations

Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E(1) (ColE(1)) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43 degrees C required for complete cessation of ColE(1) DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE(1) DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE(1) plasmid resulted in only a gradual cessation of ColE(1) DNA synthesis over several generations of cell growth at 43 degrees C. Conjugal transfer of the ColE(1) and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43 degrees C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE(1) or ColV in the mutant recipient cell upon conjugation with a normal donor at 43 degrees C. Various host ColE(1) replication mutants carrying either ColE(1) or ColE(2) were also defective in the mitomycin C-induced production of colicin E(1) or colicin E(2) at 43 degrees C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.     

On the ability of Salmonella typhimurium cells to form deoxycytidine nucleotides.

Summary The fate of the donor DNA after conjugation in Escherichia coli was studied through crosses with a Hfr lacZ5 donor and several F^- lacZ22 recipients. The fate of the donor allele was studied by assaying the -galactosidase activity formed by complementation between the lacZ5 allele and the lacZ22 allele. We used continuous cultures of the recipient in order to be able to study the fate of the donor DNA during many generations under constant physiological conditions. We could show that the donor DNA allele is inactivated in Rec⁺, recA171 and recB21 recipient cells. The inactivation rate depends on the nature of the recipient, Rec⁺ or recombination deficient, and especially in the case of the recombination deficient mutants on the growth rate of the recipient.     

Genetic defects in DNA repair system and enhancement of intergenote transformation efficiency in Bacillus subtilis Marburg.

Summary Mechanisms of inefficiency in heterospecies transformation were studied with a transformation system consisting of Bacillus subtilis 168TI (trpC2 thy) as recipient and of DNA prepared from partially hybrid strains of B. subtilis which had incorporated trp ⁺ DNA of B.amyloliquefaciens 203 (formerly, B.megaterium 203) in the chromosome (termed intergenote). The intergenote transformation was not so efficient as the corresponding homospecies transformation and the efficiency appeared to relate inversely with the length of heterologous portion in the intergenote. When a variety of ultraviolet light (UV) sensitive mutants, deficient in host-cell reactivation capacity, were used as recipients for the intergenote transformation, 2 out of 16 mutants exhibited significantly enhanced transformation efficiency of the trpC marker. Genetic studies by transformation showed that the trait relating to the enhancement of intergenote-transformation efficiency was always associated with the UV sensitivity, suggesting that these two traits are determined by a single gene. The efficiency of intergenote transformation was highly affected also by DNAconcentration; the lower the concentration, the less the efficiency. When, however, the UV sensitive mutant was used as recipient, the effect of DNA concentration was largely diminished, suggesting the reduction of DNA-inactivating activity in the UV sensitive recipient. These results were discussed in relation to a possible excision-repair system selectively correcting the mismatched DNA in in the course of intergenote transformation.This work was supported by a Grant-in-Aid for scientific research from the Ministry of Education, Japan.     

Quorum‐sensing regulation of a capsular polysaccharide receptor for the Rhodobacter capsulatus gene transfer agent (RcGTA)

The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) resembles a small tailed bacteriophage that packages almost random genomic DNA segments that may be transferred to other R. capsulatus cells. Gene transfer agents are produced by a number of prokaryotes; however, no receptors have been identified. We investigated the RcGTA recipient capability of wild‐type R. capsulatus cells at different culture growth phases, and found that the frequency of RcGTA‐dependent acquisition of an allele increases as cultures enter the stationary phase. We also found that RcGTA adsorption to cells follows a similar trend. RcGTA recipient capability and adsorption were found to be dependent on the GtaR/I quorum‐sensing (QS) system. Production of an extracellular polysaccharide was found to be regulated by GtaR/I QS, as was production of the cell capsule. A number of QS‐regulated putative polysaccharide biosynthesis genes were identified, and mutagenesis of two of these genes, rcc01081 and rcc01932, yielded strains that lack a capsule. Furthermore, these mutants were impaired in RcGTA recipient capability and adsorption, as was a non‐encapsulated wild‐type isolate of R. capsulatus. Overall, our results indicate that capsular polysaccharide is a receptor for the gene transfer agent of R. capsulatus, RcGTA.     

The lipopolysaccharide of recipient cells is a specific receptor for PilV proteins, selected by shufflon DNA rearrangement, in liquid matings with donors bearing the R64 plasmid

Shufflon DNA rearrangement selects one of seven PilV proteins with different C-terminal segments, which then becomes a minor component of the thin pili of Escherichia coli strains bearing the plasmid R64. The PilV proteins determine the recipient specificity in liquid matings. A recipient Escherichia coli K-12 strain was specifically recognized by the PilVA′, -C, and -C′ proteins, while E. coli B was recognized only by the PilVA′ protein. To identify specific PilV receptors in the recipient bacterial cells, R64 liquid matings were performed using various E. coli K-12 waa (rfa) mutants and E. coli B transformants as recipient cells. E. coli K-12 waa mutants lack receptors for specific PilV proteins. E. coli B cells carrying waaJ or waaJKL genes of E. coli K-12 were recognized by donors expressing the PilVC′ protein or the PilVC and -C′ proteins, respectively, in addition to the PilVA′ protein. Addition of E. coli K-12 or B lipopolysaccharide (LPS) specifically inhibited liquid matings. We conclude that the PilV proteins of the thin pili of R64-bearing donors recognize LPS molecules located on the surface of various recipient bacterial cells in liquid matings. Received: 2 September 1999 / Accepted: 18 November 1999     

DNA transfer and DNA synthesis during bacterial conjugation

Summary Mutant strains ofEscherichia coli, which were thermosensitive with respect to DNA replication, were used for conjugation experiments at 37°C and 42°C. Inhibition of DNA synthesis in the donor strain has no influence on the yield of recombinats. Inhibition of DNA synthesis in the recipient strain is accompanied by a complete loss of recombinant formation. Both are also true for homosexual crosses. Temporary inhibition of DNA synthesis in the recipient cell during conjugation effects reversible inhibition of DNA transfer.It is concluded that DNA transfer depends on DNA synthesis in the recipient strain, whereas DNA synthesis in the donor strain seems to be unnecessary.     

Single-stranded conjugation in Escherichia coli K-12

Summary The mutation BT43 in the gene dnaB leads to the inhibition of vegetative and conjugational DNA synthesis at 42°. The consequences in case of conjugation are very unusual. The fragment of donor DNA tramsmitted to the recipient cell remains single-stranded and is integrated as such into the recipient chromosome similar to the main events during transformation. We call this process single-stranded (SS) conjugation.The evidence for this statement comes from the measurement of the time of expression of the gene tsx, containing the genetic information for the receptor of phage T6. The gene tsx is introduced into a dnaBT43 recipient cell alternatively by two different donors Hfr H and Hfr C, which are characterized by opposite directions of transfer. Therefore both donors introduce into the recipient cell alternatively the informational or noninformational DNA strand. If conjugation is performed at a nonpermissive temperature, the transferred DNA piece remains single-stranded and is integrated as such into the recipient chromosome. If it is the informational strand (case of Hfr H), it is transcribed very fast and yields the protein in question in about 20 min. If the noninformational strand is integrated (Hfr C) about 40 min additional time is required to effect cell division.SS-conjugation is very sensitive to the action of exonucleases Exo I and Exo V and is much enhanced in the absence of both nucleases in the recipient.The exogenous DNA pieces are integrated as short insertions, this leads to the disjoining of linked markers and to a very short scale of the genetic map. Because the donor DNA undergoes recombination in the single-stranded state heteroduplex regions originate which are subsequently corrected by the enzymes of the recipient cell. The situation leads to a very special but predictable heterogeneity of the progeny of transconjugants.The fact of the existence of this special process, SS-conjugation, drastically different from common conjugation in many respects, suggests that common conjugation leads to the integration of double-stranded DNA pieces into the recipient chromosome.     

Conditions for quantitative transformation inAcinetobacter calcoaceticus

A transformation procedure that yielded high efficiencies was developed forAcinetobacter calcoaceticus. Strain BD413 Ura⁻ trpE was transformed to tryptophan prototrophy using highly purified DNA. Experimental parameters studied were: (i) recipient cell concentration, (ii) DNA concentration, (iii) growth phase of the recipient cell population, (iv) composition of the growth and transforming medium, and (v) time of incubation of recipient cells with donor DNA. Strain BD413 was competent for transformation throught the growth cycle, with highest competence occurring early in the exponential phase of growth. Maximal transformation efficiencies of 0.5% to 0.7% were obtained in media supporting rapid growth. Recipient cell concentrations of 1×10⁶ to 6×10⁶ cells/ml yielded the highest transformation frequencies, regardless of DNA concentration.     

Effect of the pSV2-neo Plasmid on NIH 3T3 Cell Motion Detected Electrically

Advantage was taken of DNA transfection techniques to investigate the effect of the pSV2-neo plasmid and its derivatives on recipient NIH 3T3 cell motion. Cell spreading and motion were followed by a newly developed electrical method to monitor cell morphology, referred to as electric cell-substrate impedance sensing. Using this method, we found that the eukaryotic--prokaryotic shuttle vector pSV2-neo had a strong effect on the recipient NIH 3T3 cell spreading and cell motion. However, two new neo plasmids, pSK-neo and pSP-neo, which were constructed by modifying the pSV2-neo plasmid, did not have a significant effect on the recipient cell activities. The results suggest that there may be some sequences in pSV2-neo which affect recipient cell behavior.     

Competence Mutant of Haemophilus influenzae with Abnormal Ratios of Marker Efficiencies in Transformation

In studies of competence-deficient mutants of Haemophilus influenzae which absorb deoxyribonucleic acid (DNA) but fail to produce transformants, it was observed that in some mutants the residual transforming activity for different markers varied widely, i.e., produced a ratio effect. One of these mutants, com⁻⁵⁶, was studied intensively to determine the cause of the residual efficiency of transformation and the reason for the ratio effect. The residual frequency of transformation was higher for markers considered single-site mutations (like naladixic acid resistance), whereas the least efficient markers tested were those conferring resistance to high levels of streptomycin or novobiocin which are more complex than single-site mutations. Measurement of frequencies of cotransformation indicated that overall genetic linkage was reduced. Transfection was fairly efficient with phage S2 DNA, but not prophage DNA. Donor marker activity could be detected in transformed cell lysates, but not linked to recipient markers in recombinant molecules. Sucrose gradient analysis of such lysates revealed that donor material was associated with recipient DNA in at least normal quantities, but lacked detectable genetic activity. Material from donor DNA labeled with heavy isotopes was incorporated into recipient chromosomal fragments having a density indistinguishable from normal density, unlike the hybrid density recombinant material found in normal cells. No excessive solubilization or nicking of unincorporated donor was detected. It is postulated that this strain contains a hyperactive nuclease, which reduces the effective size of the input DNA during the integration process.     

Regulation of polar surface structures in Caulobacter crescentus: Pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors

Summary A large number of Caulobacter mutants resistant to DNA or RNA phages were isolated. These phage-resistant mutants exhibited phenotypic variations with respect to cell motility and sensitivity to other phages.The majority of the mutants was resistant to both DNA and RNA phages tested. In addition, these mutants were either motile or non-motile. The analysis of spontaneous revertants from these mutants indicated that a single mutation is involved in these phenotypic variations. Other mutants were resistant to RNA phages and only to a certain DNA phage tested, and were also motile or non-motile.Several temperature-sensitive phage-resistant mutants were also isolated. One of them, CB13 ple-801, exhibited the wild type phenotype when grown at 25°C. However, at a higher temperature (35°C), the mutant cells became non-motile and resistant to both DNA and RNA phages. These phenotypes seem to be attributed to the concommitant loss of flagella, pili and phage receptors. In other respects (cell growth and morphology, and asymmetric stalk formation), CB13 ple-801 was normal at 35°C. The spontaneous revertants from CB13 ple-801 simultaneously regained the wild type phenotypes in all respects.It is suggested that a single mutation pleiotropically affects the formation of flagella, pili and phage receptors.     

Cell-cell interactions in conjugating Escherichia coli: Con? mutants and stabilization of mating aggregates

Summary Conjugation-deficient (Con^-) mutants of Escherichia coli K-12 have been previously described which were defective in recipient ability. Such Con^- mutants were obtained from several laboratories and retested by a standardized set of procedures. Many of the mutants did not satisfy minimal criteria for conjugation-deficiency and were discarded. The remaining mutants included 11 ConF^- mutants mutated in or near the ompA cistron, 3 ConF^- mutants synthesizing a heptose-deficient lipopolysaccharide and 1 ConI^- mutants synthesizing a defective lipopolysaccharide. This set of mutants was tested for resistance to a variety of bacteriophages and colicins; the only phenotype fully correlated with the ConF^- phenotype was that of resistance to colicin L. No simple correlation existed between the protein profile (on SDS polyacrylamide gel electrophoresis) of cell envelope outer membrane preparations and conjugation deficiency. However, many ConF^- mutants did not synthesize detectable levels of outer membrane protein II^* and protein II^* may have been nonfunctional in the remainder. All the ConF^- mutants were conjugation-deficient when matings were conducted in liquid but (with one exception) were conjugation-proficient on the surfaces of membrane filters. None of the ConF^- mutants formed stable mating aggregates in liquid with (Flac)⁺ donor cells although all bound purified F pili. The ConF^- phenotype associated with a II^*-deficient recipient could be mimicked by the addition of purified protein II^* (solubilized with lipopolysaccharide). In both cases, the formation of stable mating aggregates (analyzed with an improved Coulter counter technique) was inhibited whereas unstable mating aggregates were detected by electron microscopy. F pilus and wall to wall contacts were both observed under these conditions by electron microscopy. These results were used to define a stage in F-promoted conjugation, the stabilization stage, which requires the functional interaction of protein II^* and lipopolysaccharide in the outer membrane of the recipient cell.     

Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA-modifying property

A selection scheme was devised to isolate Paracoccus denitrificans mutants with increased recipient qualities in transfer experiments, using broad host range plasmids. In some of the mutants obtained, a DNA modifying activity that prevents the activity of the restriction endonucleases BamHI and BglII on isolated P. denitrificans DNA had simultaneously been lost. From a detailed analysis of the restriction properties of the enzymes SAU3 AI, MboI and DpnI, it was concluded that a subset of GATC sequences in P. denitrificans DNA may be methylated at an unusual position. It was concluded that P. denitrificans possesses at least one potent host-dependent restriction/modification system which affects conjugation. In addition to the class of restriction-defective mutants, at least one other class of enhanced transfer mutants with unknown defect(s) was isolated. Strains, in which the two mutant classes were combined, exhibited transfer frequencies which were significantly higher than strains containing either mutation alone. Such double mutant strains appeared to be well suited for future experiments like complementation analysis, transposon mutagenesis and gene replacement by homologous recombination.     

Phage P22-mutants with increased or decreased transduction abilities

Summary The properties of mutants of the Salmonella phage P22 are described which show decreased or increased frequencies for generalized transduction. It is shown that not only the markers used for detection of the mutants are affected by the mutation but also other markers tested. Evidence is presented that the altered T/P-ratios reflect changes in the actual numbers of transducing particles and that other factors like integration in the recipient cells cannot account for these alterations. Quantitative estimations of the amount of bacterial DNA converted to transducing fragments are shown for several phage mutants. Difficulties in the isolation procedure are discussed.     

Temperature-Sensitive Mutants for the Replication of Plasmids in Escherichia coli: Requirement for Deoxyribonucleic Acid Polymerase I in the Replication of the Plasmid ColE1

An Escherichia coli mutant (polA1), defective in deoxyribonucleic acid (DNA) polymerase I, (EC 2.7.7.7) is unable to maintain colicinogenic factor E1 (ColE1), whereas several sex factor plasmids are maintained normally in this strain. polA1 mutant strains containing these sex factor plasmids do not exhibit a readily detectable plasmid-induced polymerase activity. A series of E. coli mutants that are temperature sensitive for ColE1 maintenance, but able to maintain other plasmids, were isolated and shown to fall into two phenotypic groups. Mutants in one group are defective specifically in ColE1 maintenance at 43 C, but exhibit normal DNA polymerase I activity. Mutations in the second group map in the polA gene of E. coli, and bacteria carrying these mutations are sensitive to methylmethanesulfonate (MMS). Revertants that were selected either for MMS resistance or the ability to maintain ColE1 were normal for both properties. The DNA polymerase I enzyme of two of these mutants shows a pronounced temperature sensitivity when compared to the wild-type enzyme. An examination of the role of DNA polymerase I in ColE1 maintenance indicates that it is essential for normal replication of the plasmid. In addition, the presence of a functional DNA polymerase I in both the donor and recipient cell is required for the ColV-promoted conjugal transfer of ColE1 and establishment of the plasmid in the recipient cell.     

Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe

Summary Twenty seven recessive temperature sensitive mutants have been isolated in Schizosaccharomyces pombe which are unable to complete the cell division cycle at the restrictive temperature. These mutants define 14 unlinked genes which are involved in DNA synthesis, nuclear division and cell plate formation. The products from most of these genes complete their function just before the cell cycle event in which they are involved. Physiological characterisation of the mutants has shown that DNA synthesis and nuclear division form a cycle of mutually dependent events which can operate in the absence of cell plate formation. Cell plate formation itself is usually dependent upon the completion of nuclear division.     

Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes.

A genetic determinant essential for hemolysin production by Listeria monocytogenes has been inactivated by insertion of transposon Tn916 into L. monocytogenes DNA. The transposon was transferred by means of conjugation of a streptomycin-resistant L. monocytogenes recipient strain with Streptococcus faecalis CG110 on membrane filters. Among the tetracycline-resistant transconjugants, mutants were detected which had lost hemolytic activity. When tested in a mouse model, these mutants appeared to have lost the virulence that characterizes the parental strain. An extracellular protein of 58,000 apparent molecular weight was eliminated in the nonhemolytic mutants. In some of the mutants, the decrease in the production of the 58,000-dalton protein was accompanied by the production of a new protein of 49,000 apparent molecular weight. Hemolytic revertants regained the hemolytic phenotype and virulence and produced the extracellular protein that characterizes the recipient strain. Hybridization studies with Tn916 DNA indicated that the transposon is present in EcoRI and HindIII fragments of the nonhemolytic mutants. Single copies of Tn916 were detected in the chromosomal DNA of two of the three nonhemolytic mutants that were studied in detail. In hemolytic, tetracycline-sensitive revertants Tn916 appeared to be completely excised from the chromosome.     

Transduction of multi-copy plasmid pBR322 by bacteriophage Mu

Summary The temperate bacteriophage Mu transduces the 4363 bp multi-copy plasmid pBR322 at frequencies similar to those of chromosomal markers. Plasmid transducing particles contain DNA molecules of Mu DNA length. Plasmid DNA is transduced as a head-to-tail oligomer that becomes circularized in the recipient cell. The rec system of the donor strain participates in oligomer formation and the rec system of the recipient strain is required for oligomer circularization. Possible mechanisms that may explain the origin of plasmid transducing particles are discussed.