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PubMed1985

[Development of bacteriophage Mu in E. coli gyrBts mutant strain].

The study deals with Mu growth in cells carrying a temperature-sensitive mutation in the gene of DNA gyrase B subunit. At a nonpermissive temperature the Mu growth is shown to be blocked in the host gyrB ts mutant both on infection and on prophage induction. Mu DNA does not get integrated in the host chromosome upon the infection of mutant cells, as demonstrated by DNA-DNA hybridization experiments. In the case of prophage induction in mutant cells, as opposed to the wild type cells early mRNA synthesis is practically fully inhibited while the total RNA synthesis is three times reduced after 20 min of induction. The transcription of phage DNA associated with the changed superhelicity of DNA in the cell.

Bacteriophage mu↗

PubMed1988

Differential stability of filamentous phage genomes in Xanthomonas campestris pv citri.

Stability of carrier state in filamentous phage-infected Xanthomonas campestris pv citri varied drastically even for closely related phage types. The spontaneous curing frequency for cells infected with Cf16-12, Cf16, Cf16-v1 and Cf was 1, 5, 96 and 100%, respectively. The size of the phage replicative-form (RF) pool which built up rapidly at the onset of Cf16 infection was critical to the maintenance of the carrier state and the eventual integration of the prophage. A correlation in stability between phage carrier state in infected cells and lysogeny was found for these phages. The past history of a phage infection altered the response of the cured host to reinfection. In those cured cells which remained susceptible, the stability of the reinfecting phage genome was not altered appreciably, while a distinctive new plaque morphology appeared in greater than 50% of these cells. These alterations were not dependent on the prophage integration prior to curing, and no phage DNA was detected in cured cells by blot hybridisation. Notwithstanding these changes, the sites of phage integration in cured cells remained the same as the original infection on both the phage RF and the host chromosome.

Bacteriophages↗

PubMed1977

[Discovery of a new type of restriction and modification in a group of intestinal bacteria].

The adsorption of 23 new lambdoid bacteriophages to 547 strains which isolated from natural population of Enterobacteriaceae was studied. The frequency of positive combinations of phage-bacterium with adsorption is not more than 2%. A study of possible causes of limited growth of lambdoid phages in the bacterial strains revealed that neither homoimmune prophage nor prophage P2 are single factors of the growth limitation. It is found that in natural populations a selection of bacterial strains with the least limitation of phage takes place. Three cases of killing bacteria after infection with high multiplicity are found. The reason of the killing effect is manifestation of some functions by infecting phages. A new restriction-modification system is found which differs from restriction-modification system A, B, K, 15, P1, EcoRI, EcoRII. The most of strains, which adsorb phages but do not support their growth, are supposed to possess several mechanisms of restriction. Thus, the search of new restriction system in Escherichia coli is worthwhile.

Adsorption↗

PubMed1977

[Incorporation of phi80 phage into unusual chromosome sites and isolation of a specialized transducing bacteriophage].

The mutant strain KS713 of Escherichia coli K-12 deleted for the normal insertion site and secondary preferable one was obtained. The insertion frequency of phage phi80 into the double deletion strain is reduced about 30-fold with respect to integration into the strain H47 with deletion of the primary phi80 attachment site and about 500-fold relative to integration into wild type Escherichia coli. Analysis of the rare abnormal lysogens of KS 713 strain indicates that there are secondary sites on the chromosome, which are utilized for prophage attachment if insertion at preferable secondary att80-II site is eliminated too. The insertion of phi80 phage into the bfe locus was obtained by the appropriate selection technique. Induced prophage excision from the bfe site was rather efficient and lysates contained phi80 phage particles that could specificically transduce the argH+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harbouring both the wild-type and the mutant argH genes were isolated. These heterogenotes were used for producing high-frequency transducing lysates.

Chromosome Deletion↗

PubMedDate not supplied

[Repression of the enzyme inducible syntheses in Escherichia coli K12 mutant with a deleted ptsH gene].

The genome of lambda phage with thermosensitive repressor was integrated into the pts region of the E. coli chromosome. Such a lysogenic culture behaves as a pts mutant at 30 degrees. Heating of cells of this strain leads to the induction of lambda prophage and formation of deletions in the pts region. A mutant with a deletion covering ptsH gene was isolated after prophage induction. The deletion nature of pts mutation was confirmed in genetic and biochemical experiments. It was shown that the deletion is small and does not involve ptsI and lig genes. The isolated deltaptsH mutant possesses all characteristics of pts mutants: pleiotropic impairment of transport and utilization of a number of carbohydrates, repression of the enzyme inducible synthesis and resistance to catabolite repression with glucose. These data (together with earlier ones) allow us to conclude that the phosphorylated form of HPr is involved (in direct of indirect manner/ in activation of DNA transcription.

Chromosomes, Bacterial↗

PubMed1984

Integration of a temperate phage infecting Spiroplasma citri.

A physical map of the genome of a temperate Type 3 spiroplasma-virus, ai, has been constructed. Host DNA has been digested with restriction enzymes, and recombinant DNA clones of ai fragments in coliphage M13 vectors have been used as probes to detect viral DNA sequences integrated into spiroplasmas. All strains of Spiroplasma citri examined contained a deleted form of ai integrated as a cryptic prophage which was unable to confer resistance to ai superinfection. Stable ai lysogens also contained a complete ai genome. We propose that the infecting viral DNA circularizes and integrates adjacent to the cryptic prophage in a site-specific recombination event at unique points in both the virus and host genomes.

Bacteriophages↗

PubMed1981

Blocking by the carcinogen, L-ethionine, of SOS functions in a tif-1 mutant of Escherichia coli B/r.

In Escherichia coli, DNA damage by carcinogenic agents results in the coordinate expression of a diversity of functions (SOS functions), many of which are thermally inducible without any damage to DNA in a tif-1 mutant. These include prophage induction, filamentous growth, and an error-prone DNA repair activity, which is responsible for ultraviolet-induced mutagenesis. Ethionine causes hepatic carcinoma in rats after prolonged feeding but is not a mutagen in the Ames test. The present study shows that 10 mM ethionine prevents the thermal induction of lambda-prophage in a tif-1 derivative of E. coli. The enhancement of mutation, which normally occurs at high temperature after a low dose of ultraviolet light, is also blocked by ethionine. Ethionine does not block, to any appreciable extent, the incorporation of radioactive precursors into RNA, DNA, or protein.

DNA↗

PubMed1982

Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination.

Excision of the lambda prophage from the chromosome of its Escherichia coli host requires the products of the two viral genes int and xis. This paper reports a purification of the lambda xis gene product using a complementation assay in which functional Xis must be added to purified Int and an E. coli-derived host factor extract. Excisive recombination between a left (attL) and right (attR) prophage attachment site cloned on the same plasmid DNA substrate occurred efficiently under these conditions. Purified Int and Xis together could not carry out excision in vitro unless an extract derived from the E. coli host was added; purified integration host factor satisfied this requirement. Xis appears to have a molecular weight of 8800 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It possesses no detectable endonuclease or topoisomerase activities, does not appear to bind DNA to filters, and does not increase the ability of Int to bind DNA. The addition of Xis not only stimulated excisive recombination in vitro but also inhibited integrative recombination. Xis protected Int protein from heat inactivation, suggesting a possible interaction between the two proteins. In light of these observations, possible roles for Xis in recombination are discussed.

Bacteriophage lambda↗

PubMed1981

[Lambda H-lambda T 80 hybrid study of the DNA structural gene region in lambda and phi 80 phages].

Hybrids lambda H lambda T80 are formed due to recombination of the phage lambda att80 and phi 80 prophage partially deleted in the region of structural genes. Genetic structure of 22 independently isolated lambda H lambda T80 hybrids was determined by the restriction method and it was shown that recombination took place in the genes A, C, D and H. The frequencies of hybrid formation diminish from 1.10(-3) to 4.10(-5) for this gene order, which suggests that the polar divergence of nucleotide sequencies in the region of structural genes exists. It was found that formation of hybrids with recombination in the region of "weak" homology (gene H) was possible only when the region of "strong" homology was present in the deleted phi 80 prophage to initiate recombination.

Bacteriophage lambda↗

PubMed1983

[Independence of the processes of transposition and genome rearrangements induced by transposons].

The data on the influence of the tnm mutations affecting transposition process on the deletion formation promoted by Tn and IS elements are presented. It was shown that the tnm mutations did not affect the frequency of deletion formation. The results of genetic analysis of the tnm mutant deficient in both transposition and genomic rearrangements induced by Tn9 inserted into lambda prophage, indicated that the mutant phenotype was caused by two different but linked mutations. A mutation affecting the process of genomic rearrangements was designated gerA2. The gerA2 mutation decreased sharply the frequency of rearrangements promoted by Tn9, Tn10 or Tn601 inserted into lambda prophage. However, this mutation had no influence upon transposition of the same Tn elements. The data obtained could be interpreted as indicating the independence of the processes of transposition and genomic rearrangements or as indication of the existence of specific steps of these processes.

Bacteriophage lambda↗

PubMed1983

[Genetic approach to the virulence of bacteria].

Bacteria only possess a single chromosome, but they also contain prophages, plasmids and transposons. The genetic basis for the pathogenic power of invasive and toxigenic bacteria only represents a small part of the total genotype of these bacteria. It is curious that although the plasmids and the prophages only represent about 1 p. cent of the total genome, they are disproportionately responsible for carrying the genetic information concerning virulence. The genetic study of virulence has revealed new mechanisms of pathogenic activity, for example the interaction of siderophores, adherence factors and haemolysins in pathogenic activity. Furthermore, the understanding of the genetic basis of virulence will lead to new methods of diagnosis and a new generation of vaccines.

Bacteria↗

PubMed1980

[Lysogenic conversion of cholera vibrios].

As a result of ultraviolet irradiation and treatment with nitrosmethyl urea, mitomycin C, ascorbic acid and sarcolysin, the elimination of prophage from wild lysogenic Vibrio cholerae strains, biotype El Tor, has been achieved. The elimination of prophage was accompanied by a change in the biological properties of the vibrios, and some of these biological properties were found to be correlated with the lysogenization of Vibrio cholerae by the definite races of temperate phages. Only a few races of type II phages, whose genome was responsible for the serological characteristics of the strain, its sensitivity to cholera diagnostic phages and polymyxin, were capable of conversion.

Ascorbic Acid↗

PubMed1993

Biochemical analysis of mutant alleles of the vaccinia virus topoisomerase I carrying targeted substitutions in a highly conserved domain.

The 32-kDa topoisomerase I encoded by vaccinia virus relaxes supercoiled DNA in a manner which is mechanistically equivalent to that utilized by eucaryotic enzymes. Its amino acid sequence contains significant homology to the enzymes encoded by Saccharomyces cerevisiae, Saccharomyces pombe, human cells, and other poxviruses. The small size of the viral enzyme, and its essentiality in the viral life cycle, make it ideally suited for structural and functional analysis. In this report we present the construction and analysis of 15 mutant alleles of the topoisomerase containing amino acid substitutions in a highly conserved region. The enzymes encoded by these alleles were expressed in Escherichia coli and various parameters of their activity were examined. All of the alleles which show diminished (seven alleles) or abrogated (three alleles) DNA relaxation activity are deficient in DNA cleavage and the concomitant formation of the covalent enzyme/DNA intermediate. None are deficient in the prior step of noncovalent interaction with substrate DNA. Five of the mutant enzymes show significant temperature sensitivity in vitro. The extent of in vitro activity of the enzymes shows a good but incomplete correlation with the enzymes' abilities to lethally induce the resident lambda prophage within E. coli BL21(DE3) (via illegitimate recombination). Mutations in 1 amino acid, in particular, impair prophage induction in vivo more significantly than DNA relaxation in vitro. In sum, these studies suggest that this region of the topoisomerase (amino acids 216-225) plays a proximal role in mediating DNA cleavage and the covalent interaction between the 3'-phosphoryl of the nicked DNA and tyrosine 274 of the vaccinia topoisomerase I. The studies also provide useful reagents for the molecular genetic analysis of the role of the topoisomerase within the context of vaccinia virus infection.

Alleles↗

PubMed1977

[Isolation and study of int-mutants of pi81 bacteriophage].

Int mutants of the lambdoid bacteriophage pi81 were isolated. With mapping of deletion the location of Int gene on the genetic map of prophage pi81 was was determined. Complementation of int function of prophages lambda and pi81 was demonstrated in the formation of stable lysogenics.

Coliphages↗

PubMed1996

Effect of bisquaternary ammonium salts on Salmonella typhimurium.

The effect of 4,7-dioxo-3,8-dioxadekan-1, 1[bis(alkyl-dimethyldiammonium dibromides)] on growth, synthesis of macromolecules, respiration and induction of prophage in Salmonella typhimurium cells was studied. Only those compounds with alkyl chains of C10-C12 (octyl, decyl) in their molecules showed antimicrobial efficacy and inhibition incorporation of [14C]-adenine and [14C]-leucine as precursors of macromolecular biosynthesis. The highest inhibition of endogenous respiration was evoked by compounds with a long alkyl chain in the molecule (tetradecyl, hexadecyl). Endogenous respiration of the cells was more sensitive to a compound with hexadecyl than the respiration of various intermediates of the Krebs cycle. The sub-MICs of compounds with octyl induced a prophage of a lysogenic S. typhimurium strain.

Adenine↗

PubMed1997

Pharmacodynamic parameters of ofloxacin, tobramycin and ceftriaxone and their effect on the biological properties of salmonellae.

The postantibiotic effect and postantibiotic sub-MIC effect of ofloxacin (CAS 82419-36-1), tobramycin (CAS 32986-56-4) and ceftriaxone (CAS 73384-59-5) on two salmonella serotypes (S. typhimurium and S. enteritidis) were studied. The influence of postantibiotic effect and postantibiotic sub-MIC effect of the antibiotics on prophage induction of the lysogenic S. typhimurium strain and on Congo red binding by both serovars as indicator of their invasive ability was examined. The postantibiotic effect was induced by exposure of the bacteria to the 2x and 4x MIC concentrations of antibiotics studied for 0.5 h. The postantibiotic effects were different; ceftriaxone induced the longest postantibiotic effect against S. enteritidis, and the 4x MIC of tobramycin induced the longest postantibiotic effect against S. typhimurium. The postantibiotic sub-MIC effects lasted longer and in the case of subinhibitory concentrations of tobramycin on S. typhimurium and ceftriaxone on S. enteritidis did not allow any regrowth. The results showed that the postantibiotic effect and postantibiotic sub-MIC effect of ofloxacin induced a prophage of a lysogenic S. typhimurium strain, and the postantibiotic sub-MIC effects of tobramycin influenced Congo red binding by S. enteritidis cells.

Anti-Bacterial Agents↗

PubMed1998

Postantibiotic effects of norfloxacin and netilmicin and their influence on the biological properties of Salmonella strains.

The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PA SME) of norfloxacin and netilmicin on two clinical strains--Salmonella typhimurium and Salmonella enteritidis was investigated. After both PAE and PA SME of antibiotics were studied, we determined their effect on the induction of a prophage in the lysogenic S. typhimurium and on Congo red binding by both serovars, as an indicator of invasive ability in vitro. The PAE was induced by 2.MIC and 4.MIC of norfloxacin and netilmicin for 0.5 h. Norfloxacin induced a longer lasting PAE on both Salmonella serovars as compared to netilmicin. Supra-subinhibitory concentrations (PA SMEs) delayed regrowth of tested strains. The PA SMEs of norfloxacin as well as of netilmicin (except 2.MIC + 0.1.MIC concentration) did not allow regrowth of S. enteritidis. The prophage-inductive ability of norfloxacin was more expressive after PA SMEs than PAE. The PA SMEs of netilmicin caused loss of Congo red binding by S. typhimurium cells and decreased this binding by S. enteritidis cells.

Anti-Bacterial Agents↗

PubMed2026

Mutations in filamentous bacteriophages spark eco-evolutionary feedbacks in Pseudomonas aeruginosa.

Microbial populations strongly shape their environment, which can re-route adaptation toward organism-generated fitness optima. However, the conditions that promote these eco-evolutionary feedbacks are unclear. Here, we used experimental evolution to test whether high population density, by strengthening niche construction, drives eco-evolutionary feedbacks in the bacterial pathogen Pseudomonas aeruginosa (Pa) MPAO1. We tested for adaptation to organism-modified environments by measuring the relative performance of ancestral and endpoint populations in filtrate generated by each evolutionary line sampled across generations. Contrary to expectations, we found that endpoint populations had higher performance than the ancestral strain in filtrate across nearly all evolutionary lines regardless of population density. This was caused by the emergence of hyperactive filamentous bacterio(phage) mutants during experimental passaging that inhibited the ancestral strain but not endpoint populations in modified media. Hyperactive phages emerged from one of two avirulent prophages in MPAO1's genome (Pf4 or Pf6). Hyperactive phages drove the evolution of phage resistance in bacterial populations via mutations in the type IV pilus (TIVP), the phage's binding receptor. In a follow-up experiment, we showed that these TIVP mutations pleiotropically reduced motility and conferred resistance to a TIVP-targeting virulent phage, both of which are important traits for Pa infection and treatment. Overall, this work suggests that filamentous phage evolution can drive eco-evolutionary feedbacks in bacterial populations, causing phenotypic and genetic changes that would not be anticipated from adaptation to the extrinsic environment alone.

Pseudomonas aeruginosa↗