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[Mechanism of genetic recombination in bacterial conjugation. VIII. The heterogeneity of the progeny of exconjugants].

It is shown on several examples that the heterogeneity of recombinant clones after conjugation is independent on the conditions of primary selection (i.e. on the yield of primary clones). Therefore the prediction of the model of autonomously multiplying donor fragments is not confirmed by experimental data. Two additional characteristics of heterogeneity are introduced. The statistical distribution of merozygotes according to segregation time is measured. It is an exponential curve with a relaxation time approximately coinsident with the third cell division. A liner function is found for the dependence of heterogeneity on the distance between the selective and non-selective markers. The highest value for non-linked markers is about 15%. Alternative models for the explanation of the heterogeneity of exconjugants are discussed.

Bacteria

Chromosomal location of genes participating in the degradation of purines in Pseudomonas aeruginosa.

Genetic mapping of the genes (puu) that encode the enzymes catalysing degradation of purines in Pseudomonas aeruginosa strain PAO has been carried out. Mutants that are deficient in adenine deaminase (puuA), guanine deaminase (puuB), xanthine dehydrogenase (puuC), uricase (puuD), allantoinase (puuE), and/or allantoicase (puuF) were isolated and used for the genetic study. Conjugation by FP5 factor and generalized transduction by phage G101 gave the following map locations of these six genes on the chromosome: hisI--puuB--hisII; trpA,B--puuA--ilv202; met9011--catA1--tyu--nar9011--(puuC, puuD, puuE)--puuF. A close linkage among the puuC, puuD and puuE was demonstrated by the transduction.

Acetates

Method for the genetic labeling of cryptic plasmids.

A recently developed method for detecting transposition was employed to genetically "label" conjugative plasmids such as F and Ent P307, which do not normally exhibit a readily identifiable phenotype.

Conjugation, Genetic

Naturally occurring plasmid carrying genes for enterotoxin production and drug resistance.

Escherichia coli strain 86, isolated from a piglet with diarrhea, carries plasmid-linked genes for resistance to tetracycline, streptomycin, and sulfonamides and for production of heat-labile and heat-stable enterotoxin. Results of (i) genetic experiments involving conjugal transfer and phage P1-mediated transduction and (ii) physical experiments involving electron microscopic examination of plasmid DNA and heteroduplex analysis show that a single conjugative plasmid carries the genes for drug resistance and production of enterotoxin.

Conjugation, Genetic

Role of a lipopolysaccharide gene for immunogenicity of the enterobacterial common antigen.

It is known that only certain strains of the family of Enterobacteriaceae, notably rough (R) mutants with the type R1 or R4 core, evoked antibodies in high titers against the common enterobacterial antigen (CA) after immunization of rabbits with heated cell suspensions. The present investigation deals with genetic and immunochemical aspects of certain R1 and R4 mutants isolated from Escherichia coli 08 and various Shigella serotypes which, unexpectedly, do not induce CA antibody formation. Immunochemical and genetical (transduction and conjugation) experiments revealed that the rough phenotype of these special mutants was evoked by a mutation of pyrE-linked rfa gene, called rfaL, which is involved in translocation of O-specific polysaccharides onto the lipopolysaccharide core. The transduction of the defective rfaL, allele into appropriate rough recipients results in transductants which have simultaneously lost the ability to evoke CA antibodies. This finding suggests that a close connection exists between the function of the rfaL gene and the expression of CA immunogenicity in R1 and R4 mutants. One of the strains synthesized neither O-hapten nor CA, suggesting a mutation in a region equivalent to the rfe genes of Salmonella.

Animals

Genetic properties of the Salmonella enteritidis R404 plasmid aggregate I. Conjugational separation of different derivative plasmid aggregates and their genetic properties.

Conjugational transfer of R404 factor, found in Salmonella enteritidis strain, was examined. Six derivative forms differing in resistance pattern were isolated. The manner of conjugational separation during transfer of R404 factor from E. coli to E. coli strain was the same as from the original strain of S. enteritidis to E. coli strain. During following conjugation, all isolated forms except one, could give forms differing in a resistance pattern from the parenteral ones. This one form, from which different conjugational forms could not be separated, was assumed to be a single plasmid and was designated pCK2. All other forms, including the original R404 factor were assumed to be plasmid aggregates.

Conjugation, Genetic

Chromosome mobilization by the R plasmid R68.45: a tool in Pseudomonas genetics.

The conjugative plasmid R68.45 mobilizes the chromosome of Pseudomonas aeruginosa strain PAO from multiple sites located in different chromosome regions. In interrupted matings on the plate, selection for any single marker tested resulted in entry times of 3-5 min. When selection was imposed for two markers linked in R68.45-mediated conjugation, double recombinants appeared after a delay which corresponded approximately to the map distance between the two markers as measured by the sex factor FP2. Thus, R68.45 and FP2 appear to promote chromosome transfer at similar rates, but R68.45, unlike FP2, seems to give non-polarized transfer. R68.45 may be used to estimate map distances between linked markers located in those chromosome regions where other sex factors do not produce enough recombinants to permit accurate measurement of entry times. In R68.45 matings on the plate, most recombinants inherited short donor chromosome fragments (usually less than 10 min long) and lost the R plasmid during purification. Used like a "large" generalized transducing phage, R68.45 has proved valuable in construction of PAO strains with desired genotypes.

Chromosomes, Bacterial

[Genetic localization of a mutation rendering the growth of E coli K12 insensitive to illumination at 365 nm].

The genotype of the Nop mutant recently isolated from the E. coli K 12 strain AB 1157 has been characterized. This mutant lacks 4-thiouridine in its tRNA and is much less susceptible to near ultraviolet-induced growth delay than wild type cells. This phenotype results from a single mutation called nuv which has been localized on the E. coli genetic map. nuv is found by conjugation to lie between the origins of injection of Hfr P4X and Hfr cavalli in the vicinity of the lac gene. Cotrans-duction with bacteriophage P1 more precisely maps nuv at 0.3 min. clockwise from tsx.

Conjugation, Genetic

Activation and modulation of the host response to DNA damage by an integrative and conjugative element.

Mobile genetic elements help drive horizontal gene transfer and bacterial evolution. Conjugative elements and temperate bacteriophages can be stably maintained in host cells. They can alter host physiology and regulatory responses and typically carry genes that are beneficial to their hosts. We found that ICEBs1, an integrative and conjugative element (ICE) of Bacillus subtilis, inhibits the host response to DNA damage (the SOS response). Activation of ICEBs1 before DNA damage reduced host cell lysis that was caused by SOS-mediated activation of two resident prophages. Further, activation of ICEBs1 itself activated the SOS response in a subpopulation of cells, and this activation was attenuated by the functions of the ICEBs1 genes ydcT and yddA (now ramT and ramA; ram for RecA modulator). Double-mutant analyses indicated that RamA functions to inhibit and RamT functions to both inhibit and activate the SOS response. Both RamT and RamA caused a reduction in RecA filaments, one of the early steps in activation of the SOS response. We suspect that there are several different mechanisms by which mobile genetic elements that generate single-stranded DNA (ssDNA) during their life cycle inhibit the host SOS response and RecA function, as RamT and RamA differ from the known SOS inhibitors encoded by conjugative elements.IMPORTANCEBacterial genomes typically contain mobile genetic elements, including bacteriophages (viruses) and integrative and conjugative elements, that affect host physiology. ICEs can excise from the chromosome and undergo rolling-circle replication, producing ssDNA, a signal that indicates DNA damage and activates the host SOS response. We found that following excision and replication, ICEBs1 of B. subtilis stimulates the host SOS response and that ICEBs1 encodes two proteins that limit the extent of this response. These proteins also reduce the amount of cell killing caused by resident prophages following their activation by DNA damage. These proteins are different from those previously characterized that inhibit the host SOS response and represent a new way in which ICEs can affect their host cells.

Bacillus subtilis

[Chemiosmotic mechanism of transport of biological macromolecules through bacterial membranes].

A general mechanism of the nucleic acids transport through bacterial membranes during genetic transformation, transfection, viral infection and bacterial conjugation, has been developed. The uptake of nucleic acid occurs due to the symport with H+ ions down to an electrochemical potential gradient ("minus" inside) generated by respiration or ATP hydrolysis within recipient cells. The nucleic acid anions of non--lethal viruses are extruded from the negatively charged host cell cytoplasm by electrostatic repulsion. The difference of electrochemical potentials between the conjugating cells cytoplasms is considered as a driving force for the transport of DNA from the donor to the recipient cell.

Adenosine Triphosphatases

Transfer of drug resistance factors to the dimorphic bacterium Caulobacter crescentus.

The P-type drug resistance factors RP4, RK2, R702, R68.45, and the N-type drug resistance factor R46 are transferred to Caulobacter crescentus at high frequencies. They are stably maintained and their antibiotic resistances are expressed. Experiments with RP4 have shown that intergeneric transfer of RP4 occur at a frequency of 10(-1). C. crescentus strains maintain RP4 as a plasmid, are sensitive to RP4-specific phage, and segregate phage-resistant cells at a frequency of 10(-4) to 10(-5). The RP4 plasmid can be used in several ways: (1) the RP4 plasmid will promote chromosomal exchange between C. crescentus strains at frequencies ranging from 10(-6) to 10(-8); (2) RP4 will promote the transfer of nonconjugative colE1 plasmids from E. coli to C. crescentus; once transferred, the colE1 plasmid is stably maintained under nonselective conditions, can be transferred serially, and segregates independently from RP4; and (3) RP4 can be used to introduce transposons into the C. crescentus chromosome, providing the basis for additional genetic techniques.

Bacteria

[Sex factor F of Escherichia coli K12 and its participation in mobilizing bacterial chromosomes].

The structural and functional organization of F-factor reviewed and the physical map of F DNA, supplemented with a list of genetic markers, is presented. The DNA transfer during the conjugation is considered and especial attention is given to F-gene functions involved in this process. The special sequences of F DNA, homologous to resident insertion sequences in bacterial DNA are described and its participation in plasmido-chromosomal recombinant events both dependent and independent on recA function is discussed. The mechanism for chromosome mobilization by F-Factor are reviewed. A possibility of chromosome transfer without F DNA insertion is considered. In a latter case it proposed that spontaneous single-strand breaks may serve as the origins for initiation of chromosomal transfer.

Chemical Phenomena

[Mechanism of plasmid ColEl and pMB-9 mobilization by plasmid F'lac+ in Escherichia coli K-12].

The genetic control and mechanism of mobilization of the non-conjugative plasmids ColE1 and pMB-9 by the conjugative plasmids was orived to be recA-independent process in contrast to the mobilization of the chromosomal marker pro. Acridine orange and ethidium bromide curing data together with the results of electrophoretic analysis of plasmid DNA suggest that the plasmids F' lac+ and pMB-9 as well as F' lac+ and ColE1 remain autonomous after their contransfer to recipient cells. These data argue in favour of non-recombinational nature of the plasmid mobilization process. The possibility of transmission of a non-conjugative plasmid without transmission of a conjugative one from the donor strain carrying both plasmids was established. The results obtained are discussed with respect to the hypothesis on the effect of diffusible products encoded by the conjugative plasmid and required of the mobilization of the non-conjugative plasmid.

Conjugation, Genetic

Role of dnaB43 in F'-plasmid incompatibility.

In order to perform complementation tests with mutations of DNA replication of F'-plasmids incompatibility must overcome. We report out in ability to duplicate the results presented by Palchoudhury and Iyer (1971) and Bezanson and Iyer (1975) who have claimed to demonstrate the autonomous replication two incompatible F'-plasmids in a strain carrying the temperature sensitive dna B43 allele. In addition, we describe experiments designed to measured complementation using transient heterozygotes and compatible plasmids. Assessment of our data and those of others in the light of a recent report by Uhlin and Nordström (1975) suggests that new approaches will have to be developed for the successful employment of complementation analysis in F-plasmid genetics.

Conjugation, Genetic