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PubMed2002

Overexpression of peptidases in Lactococcus and evaluation of their release from leaky cells.

Walker and Klaenhammer (2001) developed a novel expression system in Lactococcus lactis that facilitated the release of beta-galactosidase (117 kDa monomer) without the need for secretion or export signals. The system is based on the controlled expression of integrated prophage holin and lysin cassettes via a lactococcal bacteriophage phi31 transcriptional activator (Tac31A) that resides on a high-copy plasmid. Approximately 85% of beta-galactosidase activity was detected in the supernatant of leaky lactococci without evidence of hindered growth, cell lysis, or membrane damage. The objective of this study was to determine if intracellular peptidases were externalized from leaky lactococci. Five L. lactis peptidases (PepA, PepC, PepN, PepO and PepXP) and two Lactobacillus helveticus peptidases (PepN and PepO) were cloned and overexpressed on two high-copy vectors. The lactococcal peptidases were also cloned into the high-copy vector that contained the Tac31A transcriptional activator to determine if they were externalized from the leaky prophage-containing L. lactis subsp. lactis strain NCK203. Two of the lactococcal peptidases (PepA and PepO) required an additional strong promoter (Lactobacillus paracasei P144) and optimized assay conditions to detect enzyme activity. Results showed different levels of enzymatic overexpression associated with the cellular fraction (2 to 250-fold increases in activity) and negligible amounts of activity present within the supernatant fraction (0 to 6% of total peptidase activity). The lactococcal phage-based protein release mechanism did not facilitate the externalization of the lactococcal peptidases investigated in this study.

Aminopeptidases↗
PubMed2003

Reemergence of epidemic Vibrio cholerae O139, Bangladesh.

During March and April 2002, a resurgence of Vibrio cholerae O139 occurred in Dhaka and adjoining areas of Bangladesh with an estimated 30,000 cases of cholera. Patients infected with O139 strains were much older than those infected with O1 strains (p<0.001). The reemerged O139 strains belong to a single ribotype corresponding to one of two ribotypes that caused the initial O139 outbreak in 1993. Unlike the strains of 1993, the recent strains are susceptible to trimethoprim, sulphamethoxazole, and streptomycin but resistant to nalidixic acid. The new O139 strains carry a copy of the Calcutta type CTX(Calc) prophage in addition to the CTX(ET) prophage carried by the previous strains. Thus, the O139 strains continue to evolve, and the adult population continues to be more susceptible to O139 cholera, which suggests a lack of adequate immunity against this serogroup. These findings emphasize the need for continuous monitoring of the new epidemic strains.

Adolescent↗
PubMedDate not supplied

[Virus, provirus and cancer].

Our purpose here is to retrace the history of the concept of prophage, to show how it expands into that of the episome and provirus. Its prehistory is that of lysogeny. We stress the relations between heredity and infection, the discovery of a non infectioius phase of the phage. André Lwoff is responsible for the concept of prophage. We then examine the research, discoveries and interpretations of François Jacob, Elie Wollman, William Hayes and Joshua Lederberg.

History, 20th Century↗
PubMed1976

[New lambdoid phages of Escherichia coli. II. Comparison of several genetic characteristics with lambda phages].

Functions of some newly isolated lambdoid phages and phage lambda genes were compared by their ability to interact with unrelated phages and the product of the bacterial gene gro P. 19 of 23 lambdoid phages studied interfere with prophage P2, that points out the presence of functionally active genes, essential for spi+ phenotype in their genomes. The development of 4 lambdoid phages with spi- phenotype is independent on the prophage P2 presence. Most of lambdoid phages show the reduced growth ability on the C600 groP- bacterial lawn. This indicates that they have a function similar to the gene P of phage lambda. The development of phage phi M417 in bacterial mutants groP- is not disturbed, which indicates that the gene P of phage phi M417 is different from that of the phage lambda. Newly isolated phages, that are homoimmune to phage lambda, restrict the development of T4 rII phage. The rest lambdoid phages have no rex function. The growth efficiency of lampodid phages on E. coli C cells, carrying Eco R1 plasmid, varies from 10(-6) to 10(-8) that presumbaly indicates on different amounts of restionci strites in DNA of these phages.

Coliphages↗
PubMed1976

Value of phage typing of Vibrio cholerae biotype eltor in West Africa.

The epidemiological value of phage typing of El Tor vibrios in West Africa was assessed by testing 211 representative strains from all outbreaks of the cholera epidemic in Togo (1970-73) with the prophage typing method of Nicolle et al. and the lytic El Tor phages of Basu & Mukerjee. Prophage typing proved to be of limited epidemiological value in Togo since 203 of the 211 strains tested belonged to the same phage type-namely, type 2 of Nicolle et al., which corresponds to the Celebes type of Takeya. Six strains belonged to type 1, 1 strain to type 4, and 1 strain was untypable.Tested by the lytic El Tor phages, 175 strains were found to belong to type 4 of Basu & Mukerjee, 35 strains to type 1, and 1 strain was untypable. All but two strains of type 1 were isolated from 3 outbreaks in which this phage type was found almost exclusively. The results are in good agreement with epidemiological data collected during the epidemic. El Tor phage V reacted with a specificity of over 99% when tested against 601 strains of El Tor and NCV vibrios from Togo and may be recommended for the rapid identification of El Tor vibrios.

Africa, Western↗
PubMed2000

[Properties of natural interspecific hybrids of transposable phages of Pseudomonas aeruginosa: specific characteristics of phage PL24 transposition].

Properties of natural hybrid transposable phages (TP) of Pseudomonas aeruginosa, including phage PL24 and lysogens for this phage, were studied. PL24 possesses the properties of TP from two previously described groups, B3 and D3112. Its genome, unlike the genome of D3112, contains many sites susceptible to the SalGI restriction endonuclease and possesses no more than 100 nucleotides of bacterial origin located at the left genome end. However, unlike B3, phage PL24 failed to induce auxotrophic mutants upon integration in the bacterial genome. This phage differed from both B3 and D3112 in sensitivity to chloroform treatment. A more detailed examination of a group containing 25 randomly isolated lysogens for phage PL24 revealed previously unknown processes occurring at early stages of bacterial lysogenization. There are at least two different modes of cell lysogenization with phage PL24. In the first case, the emerging lysogens contained a single prophage genome located (in each lysogen) at individual sites. In the second case, polylysogenic bacteria appeared, and, after primary integration of a phage genome, replicative transposition occurred at new sites (often accompanied by the appearance of prophage clusters at these sites). The choice of the mode of lysogenization can be determined both by differences in the physiological state of bacteria and by specific features of phage PL24, which possibly affect the time of repressor accumulation to the concentration sufficient for blocking phage growth or the stability of the lysogenic state.

DNA Transposable Elements↗
PubMed1975

[Influence of the recA- mutation on spontaneous filamentation and the titer of the lambda phage in ylonB- mutants of E. coli K12 (lambda)].

A study was made of the effect of recA- mutation on the spontaneous filament formation and the spontaneous induction of the lambda prophage in the E. coli K12 strain bearing lonB- mutation. It was revealed that in the absence of a functionally-active product of the recA- gene the lonB- strain of E. coli K12 displayed but a partial depression of spontaneous formation of filamentous forms and of spontaneous induction of the lambda prophage.

Coliphages↗
PubMed1992

[Isolation, characterization and mapping of the N15 phasmid insertion mutations].

Prophage of N15 temperate bacteriophage is stably maintained in Escherichia coli lysogens as a 46.33 kb linear plasmid. Using different transposons we obtained 18 insertion mutants of the N15 plasmid prophage. They were analysed for plaque formation ability, stability of the plasmid state and lysogenic conversion. Restriction mapping of the insertions allowed us to localize on the map the regions necessary for lytic growth and to map the lysogenic conversion gene. A recombinant phage encoding two antibiotic resistance genes was obtained. The phage contains an additional 4.77 kb DNA fragment (over 10% of the N15 genome).

Bacteriophages↗
PubMed2004

[Expression regulation of the protelomerase gene of the bacteriophage N15].

The N15 bacteriophage, when in the lysogenic state, does not integrate into the chromosome; in fact, it exists as a linear plasmid with the covalently closed ends. Upon infection, the phage DNA circularizes via its cohesive ends, after which a specific enzyme, the N15 protelomerase, cuts the circular molecule thus generating a linear plasmid with the covalently closed telomeres. Protelomerase generates, as the replication of plasmid prophage proceeds, the hairpin telomeres in replicated molecules. We identified the promoter of the protelomerase gene and demonstrated that it could be repressed presumably due to its binding with 3 tosL sites overlapping the promoter. We also found the transformation efficiency of E. coli cells of linear DNA with hairpin telomeres to be approximately 100-fold lower versus the circular DNA of the same size. At the same time, presence of the N15 prophage or of the protelomerase-expressing vector enhances, in a strain being transformed, the efficiency of its transformation by linear DNA up to a level ensured in transformation by circular plasmids. We believe that protelomerase, while binding with the hairpin telomeres, protects the latter from degradation by cellular nucleases.

Bacteriophages↗
PubMed1990

[Interactions of the phytopathogenic bacteria Erwinia with phage P1 CRL100 CML].

The bacteriophage P1 Cm clr100 lysogenises the bacteria E. chrysanthemi, E. aroideae, E. atroseptica being localized in the cytoplasm, replicating but causing no cell lysis. The prophage induction results in transformation of the lysogenic bacteria E. chrysanthemi into nonviable filamentous cells. However, a portion of cells gets rid of the prophage and gives rise to normal heritage inheritors permitting to use the bacteriophage as an efficient vehicle for introducing the transposons into the chromosome of E. chrysanthemi. The transposon Tn9 has been found to insert into the different chromosomal sites causing no inactivation of the genes, while the transposition of Tn5 from the bacteriophage P1::Tn5Cmclr100 induces the different mutations with the frequency up to 3%. The bacteriophage P1Cmclr100 may also serve a tool for construction of the homology regions in the chromosome of E. chrysanthemi and Flac+ plasmid for further construction of Hfr-type donors.

Bacteriophages↗
PubMed1990

Toxicity and mutagenicity of a mixture of 25 chemicals found in contaminated groundwater.

A defined mixture of 25 chemicals that are often found in contaminated groundwater was prepared as an aqueous solution and studied for mutagenicity in bacteria, for prophage induction in bacteria, for palatability and effect on weight-gain in rats and mice, and for cytogenetic effects in bone marrow cells of rats and mice. The bacterial mutation and prophage induction tests were negative. Exposure to the mixture in drinking water for two weeks resulted in a concentration-related decrease in water consumption in male and female rats and mice. Concentration-related decreases in weight gain were observed in male and female mice; in rats, only the high-dose groups showed decreased weight gains. A small but significant increase in sister chromatid exchanges was seen in male mice and a similar weak effect on micronucleated polychromatic erythrocytes (PCE) in the bone marrow of males and females. Also in bone marrow of male and female mice, an increase in mitotic index and a decrease in average cell generation time was observed. The %PCE in bone marrow was decreased in female mice only, while the %PCE in peripheral blood was increased in both sexes. In rats, the only effects observed in the cytogenetic studies were increased PCE frequencies in the peripheral blood of males and in the bone marrow of males and females. These results indicate that the 25-chemical mixture studied is not genotoxic in bacteria and that a concentration-dependent effect on its palatability to rodents leads to reduced water consumption, food consumption and weight gain. Although the bone marrow effects may be associated with disruptions of normal erythropoiesis that, in turn, alter the cytogenetic end-points reported, elevations of SCE and MN-PCE in mice suggest the 25-chemical mixture, under the conditions of administration, leads to cytogenetic damage in the bone marrow. Such damage may indicate a potential health hazard.

Animals↗
PubMed1985

Mutagenic activity of radiosensitizers.

Seven radiosensitizers, six derivatives of nitroimidazole (coded P1 to P5 and one imidazole derivative--P6 were investigated for mutagenicity using 3 short-term tests: Ames test, prophage lambda induction and tryptophan reversion test. Out of seven investigated compounds five were not mutagenic. Only P1 derivative induces base pair substitutions. Another derivative of nitroimidazole: metronidazole induces base pair substitution and frameshift mutations. Its positive response in the prophage lambda induction test suggests that metronidazole provokes also epigenetic changes.

Animals↗
PubMedDate not supplied

[Possibilities of using bacterial cultures for the biological indication of relatively small doses of gamma radiation].

Some approaches to using bacterial cultures as test objects in biological indication of relatively low gamma-radiation doses have been considered. The survival rate, mutability and induction of prophages in the lysogenic strains have been investigate. A model for the prophage induction in Pseudomonas aeruginosa is proposed to estimate the biological effect of gamma-irradiation with doses of 0.25 to 10 Gy.

Bacteria↗
PubMed1989

[Bacteriophage conversion as a factor modifying the intensity of phagocytosis of Staphylococcus aureus by human leukocytes].

Staphylococcus aureus NCTC 8325-4 and its eight variants lysogenized with phages responsible for the synthesis of staphylococcal staphylokinase were used for the study. Influence of phage conversion of S. aureus on its interaction with human leucocytes and influence of prophage on strain susceptibility to intracellular killing by human granulocytes without opsonins were evaluated. It was found that lysogenization of the strain with the bacteriophages decreased in each case reactivity of human leucocytes for staphylococcal strain what was expressed by lower bioluminescence values and by lower percentage of intracellular killing of bacterial cells carrying prophage.

Culture Media↗
PubMed1985

[Large deletions in the region of the replication termination of Escherichia coli K-12 chromosome].

The Escherichia coli strain PLK1427 (Henson, Kopp, Kuempel, 1984) was used in this work. It carries a deletion of 60 thousand pairs of nucleotides in the chromosomal region 30-31 min and a partially deleted prophage lambda rev cI875Sam7 integrated into the 30 min region, instead of the rac prophage. Among the mutants of PLK1427 strain selected for resistance to 42 degrees C, deletions extending about 4 min and affecting the loci nirR (29.3 min), zdc235::Tn10 (32.3 min) and zdd230::Tn9 (33.3 min) were found. Although the deletion mutants obtained affect the region of replication termination (terC) of the chromosome, they have no alterations in the growth rate. It was demonstrated that some deletions may be transferred and are capable of recombination, giving the wild type in transductional experiments with the mutant phage T4.

Bacteriophage lambda↗
PubMed1988

[Transactivation of p'R promoter of phage lambda].

The host-vector system for efficient expression of the cloned genes under the control of transactivated promoter p'R of bacteriophage lambda has been elaborated. The Q protein activating p'R promoter is coded by the defective prophage constructed in vitro by means of excision of the late phage genes between the distant sites of the restriction endonuclease MluI and change of the central SalI fragment carrying the kill gene for the kanamycin resistance gene. The general recombination system is impaired during the change, thus the bacteriophage DNA can be obtained from the induced RecA cells as a plasmid DNA. The induction of the prophage results in a sharp increase of beta-lactamase synthesis (30% of soluble cell protein) under the control of p'R promoter in a plasmid derived of pBR322.

Bacteriophage lambda↗
PubMed1986

[Role of temperate phage in bacterial dissociation].

The analysis of literary and own data testifies that the dissociants may appear in bacteria population from spontaneous mutations and transfer of genetic material (conjugation, transformation, transduction). The phage conversion and different DNA reorganizations within a cell where prophage plays an active role, probably introduce the largest contribution into the dissociative transitions of variants which occur with high frequency (about 10(-2)-10(-4). The dissociation of various bacteria has been studied with different degree. The role of temperate phage has been shown in splitting of bacteria into variants in the genera Mycobacterium, Corynebacterium, some Bacillus, Clostridium, Staphylococcus, some enterobacteria, Yersinia, Vibrio Pseudomonas, Rhizobium, Nostoc; the participation of prophage in dissociation of bacteria of the genera Xanthomonas, Erwinia, Bacteroides is proposed. A method for obtaining the nondissociating S-variants for stability of biologically active substances synthesized by cells has been suggested.

Bacteria↗
PubMed1986

[Phasmids. Properties and the use in genetic engineering. II. Packing in vitro. Lysogeny of Escherichia coli K-12].

Circular monomeric lambda DNA molecules were used as a substrate for packaging reaction in vitro. For obtaining lambda DNA in circular monomeric form only, Escherichia coli recA plasmid bearing cells were used. This hybrid DNA molecule which we designated phasmid lambda pMYF11, is the pBR322 plasmid in which lambda 47.1 DNA was introduced in vitro. The phasmid can exist in the plasmid form or as a non-defective phage. The efficiency of packaging reaction in vitro proved to be similar for monomeric circular and linear form of phasmid DNA molecules. The cI- variant of the phasmid is not able to exist as a plasmid even in the cells containing homoimmune prophage. Still, cI+ phasmid variants capable of lysogenizing arise with low frequency, as a result of recombination between the resident cI+ prophage and infecting cI- phasmid.

Bacteriophage lambda↗