Lysogenic Strains Research Articles

Evidence for Dependence of Resonant Frequency of Millimeter Wave Interaction withEscherichia coliK12 Cells on Haploid Genome Length

The method of anomalous viscosity time dependence (AVTD) was used to study the influence of millimeter electromagnetic radiation (EMR) on the genome conformational state (GCS) of Escherichiu coli K12 cells. Strain N99 of wild-type, lysogenic strains N99(A) and N99(λ,λimm434bio10) were used. In the 41.28-41.37 GHz and 51.73-51.79 GHz ranges the resonance effect of low-intensity EMR (10-10 W/cm2) on the GCS of N99 cells was shown. The resonance frequencies were 41.324 ± 0.001 GHz and 51.765 ± 0.002 GHz, respectively. The insertion in bacterial chromosome of prophage λ [strain N99(λ)] and prophage λimm434bio10 [strain N99(λ, λimm434bio10)] reduced both resonance frequencies considerably. The decrease of both resonance frequencies was proportionate to the increase of the haploid genome length. Shifts of the resonance frequency were not accompanied by change in the sign of effective circular EMR polarization. The results indicate that the frequencies of resonance interaction of E. coli cells with low-intensity...

Bioluminescence detection system of mutagen using firefly luciferase genes introduced in Escherichia coli lysogenic strain.

A rapid and convenient microbial sensing system for mutagens was developed based upon the induction of prophage from Escherichia coli lysogenic strain and bioluminescence. The system consisted of lysogenic E. coli encoding firefly luciferase genes and a photodetection system. Measurement of mutagen mitomycin C was achieved by measuring the luminescence intensity emitted from E. coli lysogenic strain for the recombinant phage in the presence of luminescence substrates. Approximately 1 h after addition of mitomycin C, the luminescence began to be observed, and 3 h after, it attained a level of 2 times greater than that of 1 h. Irradiation with ultraviolet light also produced light based on induction of phage from the E. coli lysogenic strain for the recombinant phage. On the other hand, when nonmutagenic toxic compounds like sodium azide were added to the reaction medium, luminescence was not observed. Mitomycin C could be detected within 1 h with this sensing system, at concentrations down to 10(2) ng/assay.

Attachment sites for bacteriophage P2 on the Escherichia coli chromosome: DNA sequences, localization on the physical map, and detection of a P2-like remnant in E. coli K-12 derivatives.

Integration of bacteriophage P2 into the Escherichia coli genome involves recombination between two attachment sites, attP and attB, one on the phage and one on the host genome, respectively. At least 10 different attB sites have been identified over the years. In E. coli C, one site, called locI, is preferred, being occupied before any of the others. In E. coli K-12, no such preference is seen (reviewed in L. E. Bertani and E. W. Six, p. 73-143, in R. Calendar, ed., The Bacteriophages, vol. 2, 1988). The DNA sequence of locI has been determined, and it shows a core sequence of 27 nucleotides identical to attP (A. Yu, L. E. Bertani, and E. Haggård-Ljungquist, Gene 80:1-12, 1989). By inverse polymerase chain reactions, the prophage-host junctions of DNA extracted from P2 lysogenic strains have been amplified, cloned, and sequenced. By combining the attL and attR sequences, the attB sequences of locations II, III, and H have been deduced. The core sequence of location II had 20 matches to the 27-nucleotide core sequence of attP; the sequences of locations III and H had 17 matches. Thus, the P2 integrase accepts at least up to 37% mismatches within the core sequence. The E. coli K-12 strains examined all contain a 639-nucleotide-long cryptic remnant of P2 at a site with a sequence similar to that of locI but that may have a different map position. The P2 remnant consists of the C-terminal part of gene D, all of gene ogr, and attR. Locations II, III, and H have been located on Kohara's physical map to positions 3670, 1570 to 1575, and 2085, respectively.

Expression of Erythrina corallodendron lectin in Escherichia coli.

The cDNA of the Erythrina corallodendron lectin (ECorL) has been expressed in Escherichia coli. For this purpose, an NcoI site was inserted into the cDNA coding for the lectin precursor [Arango, R., Rozenblatt, S. & Sharon, N. (1990) FEBS Lett. 264, 109-112] immediately before the codon GTG (103-105) which codes for the N-terminal valine of the mature lectin. This introduced an ATG codon for a methionine preceding the valine. The mutated cDNA was ligated into pUC-8, then subcloned into the expression vector pET-3d, which carries a strong promoter derived from gene 10 of the phage T7. The recombinant plasmid was introduced into the E. coli lysogenic strain BL21(DE3). Recombinant ECorL was expressed by growing the bacteria in the presence of isopropyl beta-D-thiogalactopyranoside. Most of the recombinant lectin was found in an insoluble aggregated form as inclusion bodies and only a small part was in the culture medium in a soluble active form. Functional recombinant lectin was recovered from the inclusion bodies by solubilization with 6 M urea in cyclohexylaminopropane sulfonate pH 10.5, renaturation by 10-fold dilution in the same buffer and further adjustment of the pH to 8.0. The recombinant lectin, obtained at a yield of 4-7 mg/l culture, had, by gel filtration, a slightly lower molecular mass (56 kDa) than the native lectin, and was devoid of covalently linked carbohydrate; it was, however, essentially indistinguishable from native ECorL by other criteria, including its dimeric structure, Western blot analysis with anti-ECorL polyclonal and monoclonal antibodies, and Ouchterlony double-diffusion analysis with polyclonal antibodies, as well as hemagglutinating activity and specificity for mono- or disaccharides.

Comparative Study of 35 Bacteriophages of Lactobacillus helveticus: Morphology and Host Range

This survey included 23 phages isolated from cheese whey and 12 temperate phages induced with mitomycin from their lysogenic host strains. All of the phages had an isometric head and a tail with a contractile sheath. In addition, short-tailed (160-nm-long) and long-tailed (260-nm-long) phages were distinguished. Short-tailed phages were by far the most widespread in French cheese factories (32 of the 35 phages studied). The study of phage relationships enabled two large groups of strains to be distinguished: those not or slightly sensitive to phages and those very sensitive to phages. There was an obvious relationship in the first group between phage sensitivity (or resistance) and the geographic origin of the strains. The second group contained primarily strains from large international collections and those isolated from commercial starters. The relationships among short-tailed phages, either temperate or isolated as lytic, suggest that lysogenic strains could be the major source of phages in French cheese factories.

Evidence and characterization of temperate bacteriophage in Streptococcus salivarius subsp. thermophilus St18.

Lysogenic strains of Streptococcus salivarius subsp. thermophilus were studied using induction with mitomycin C (MC). The induction and presence of temperate phage were investigated carrying out tests on sensitive strains, electron microscopy and phage DNA analysis. Forty-five Str. salivarius subsp. thermophilus strains were subjected to induction with MC and growth of the various cultures was evaluated. Only one strain of those tested showed lysis after adding MC, thus indicating a possible lysogenic state, 0.5 micrograms MC/ml being the optimal dose. Two phi 18 phage-sensitive strains out of 45 were isolated in which this phage behaved as virulent, causing lysis of the culture in broth, but no lysis plaques on agar medium were detected. The St18 strain was cured by u.v. irradiation but no mutants sensitive to the phi 18 phage were found among the clones non-inducible by MC. The presence of phages having a hexagonal isometric head and a long non-contractile tail in the lysate obtained after inducing the St18 strain was confirmed by examination under the electron microscope. Study of the phage DNA showed a genome size of 40.9 +/- 0.5 kb without cohesive end fragments. In addition, the restriction map of the phage genome was constructed. This study has demonstrated lysogeny in Str. salivarius subsp. thermophilus and also that several phage infections of Str. salivarius subsp. thermophilus starters may have an 'endogenous' origin.

Diphtheria toxin and Pseudomonas aeruginosa exotoxin A: active-site structure and enzymic mechanism.

Diphtheria toxin (DT), secreted by lysogenic strains of Corynebacterium diphtheriae carrying the phage-encoded DT gene, was the first ADP-ribosylating toxin for which the molecular mechanism of action was elucidated (Collier 1975; Pappenheimer 1977), and for many years DT has served as an important model system for studying the pathogenesis of bacterial exotoxins (Collier 1982; Jacobson and Jacobson 1989; Moss and Vaughan 1990). DT and the closely related exotoxin A from Pseudomonas aeruginosa(ETA) both catalyze the ADP-ribosylation of a post-translationally modified histidine (diphthamide) on elongation factor 2 (EF-2) (Honjo et al. 1968, 1969; Gill et al. 1969; Iglewski and Kabat 1975; Iglewski et al. 1977). EF-2 is a GTP-binding protein involved in protein biosynthesis by eukaryotic cells. ADP-ribosylated EF-2 is no longer able to mediate polypeptide chain elongation, and consequently, toxin-treated cells lose the ability to synthesize protein and ultimately die. Although toxins have not yet been described in detail, recent studies have yielded relevant information. This chapter will focus on some of the more recent studies and what the findings tell us about the structure of the active site and’the nature of the reaction catalyzed by these toxins.

Two Generalized Transducing Phages in Vibrio parahaemolyticus and Vibrio alginolyticus

Two bacteriophages named phi VP253 and phi VP143 isolated after ultraviolet induction from lysogenic strains of Vibrio parahaemolyticus have been shown to be generalized transducing phages. So far, seven different auxotrophic markers of a V. parahaemolyticus strain could be transduced at the frequencies ranging from 2.2 x 10(-7) to 7.5 x 10(-5) per infected cell at the m.o.i. of approximately 1.0. The phage phi VP143, but not phi VP253, lysed 20 of the 28 strains of V. alginolyticus and the occurrence of generalized transduction by this phage in this Vibrio species has been confirmed. Molecular size of the genomes of both phages were estimated to be approximately 48 kb as judged from electrophoretic mobilities of the DNAs digested with HindIII endonuclease. The results and similarity of the two phages in morphology and other properties suggest very close relatedness of the phages.

Biological characterization of induced phages from Saccharopolyspora hirsuta 367 and comparison with phage JHJ-1

Phages JHJ-2 and JHJ-3 were isolated from Saccharopolyspora hirsuta 367 UC 8106 following induction with mitomycin C and amplified on S. hirsuta NRRL B-5792. Their properties were compared with those of phage JHJ-1, isolated previously from S. hirsuta 367 NRRL 12045. The DNA restriction patterns appeared to be identical. One-step growth experiments showed no differences between the replication cycles. Burst sizes ranged from 100 to 110 p.f.u. per cell. However, the three phages showed some differences in their behaviour in different hosts. The host range of phage JHJ-1, on non-lysogenic strains, was emended to include all of the Saccharopolyspora strains tested; the host range of phage JHJ-2 was shown to be identical to JHJ-1. Phage JHJ-3 did not form detectable plaques on strains of S. rectivirgula or S. erythraea except S. erythraea NRRL 2359. Neither phage JHJ-2 nor JHJ-3 formed plaques on any lysogenic strains, while JHJ-1 formed plaques on all such strains except S. hirsuta 367 UC8106. Phage JHJ-3 was characterized as a temperate bacteriophage because it formed turbid, self-limiting plaques and lysogenized S. hirsuta NRRL B-5792. It was spontaneously released from UC8106. Both JHJ-1 and JHJ-2 formed clear and invasive (Inv+ phenotype: the property to grow on old mycelium) plaques on some Saccharopolyspora strains but clear and self-limiting plaques on others. Thus, the expression of the Inv+ phenotype encoded by JHJ-1 and JHJ-2 appears to be modulated by the host cell.

Heat shock induces thermotolerance and inhibition of lysis in a lysogenic strain of Lactococcus lactis

In this preliminary work, the heat shock response of lactic acid bacteria was investigated and characterized. Log-phase Lactococcus lactis cells pre-incubated at 40°C before heat challenge at 52°C for 30 min demonstrated increased thermotolerance as compared with cells pre-incubated at 30°C. The response persisted for at least 60 min. Additionally, we demonstrated that: (i) the physiological expression of the heat shock response is temperature dependent; (ii) ethanol 4.0% (v/v) caused, to a lesser extent, a response similar to the heat shock; and (iii) hydrogen peroxyde failed to induce a detectable response. Furthermore, we suggest that the induction of the heat shock response increases the resistance of a lysogenic strain of L. lactis, treated by mitomycin C (1.25 μg/ml), to lysis by the bacteriophage.

Lysogeny in Leuconostoc oenos

Thirty strains of Leuconostoc oenos were exposed to mitomycin C to induce lysogenic bacteriophages. Lysis curves typical for lysogenic strains were obtained with 19 strains. Indicator strans were found for 17 of these phages. Five were characterized by electron microscopy, lytic spectrum, molecular masses of the proteins, sequencing of five N-terminal amino acids of the two major proteins and DNA analysis (restriction patterns, cross hybridization). The results revealed a very close relationship between the phages. Hybridization experiments between the DNAs of the temperate phages and the appropriate lysogenic strains revealed phage-related sequences in the DNA of the lysogenic strain.

Cyclic AMP inhibits and putrescine represses expression of the speA gene encoding biosynthetic arginine decarboxylase in Escherichia coli.

The speA gene of Escherichia coli encodes biosynthetic arginine decarboxylase (ADC), the first of two enzymes in a putrescine biosynthetic pathway. The activity of ADC is negatively regulated by mechanisms requiring cyclic AMP (cAMP) and cAMP receptor protein (CRP) or putrescine. A 2.1-kb BamHI fragment containing the speA-metK intergenic region, speA promoter, and 1,389 bp of the 5' end of the speA coding sequence was used to construct transcriptional and translational speA-lacZ fusion plasmids. A single copy of either type of speA-lacZ fusion was transferred into the chromosomes of Escherichia coli KC14-1, CB806, and MC4100, using bacteriophage lambda. The speA gene in lysogenized strains remained intact and served as a control. Addition of 5 mM cAMP to lysogenic strains resulted in 10 to 37% inhibition of ADC activity, depending on the strain used. In contrast, the addition of 5 or 10 mM cAMP to these strains did not inhibit the activity of beta-galactosidase (i.e., ADC::beta-galactosidase). Addition of 10 mM putrescine to lysogenized strains resulted in 24 to 31% repression of ADC activity and 41 to 47% repression of beta-galactosidase activity. E. coli strains grown in 5 mM cAMP and 10 mM putrescine produced 46 to 61% less ADC activity and 41 to 52% less beta-galactosidase activity. cAMP (0.1 to 10 mM) did not inhibit ADC activity assayed in vitro. The effects of cAMP and putrescine on ADC activity were additive, indicating the use of independent regulatory mechanisms. These results show that cAMP acts indirectly to inhibit ADC activity and that putrescine causes repression of speA transcription.

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.

Comparative inducibility of bacteriophage in naturally lysogenic and lysogenized strains of Listeria spp. by u.v. light and Mitomycin C

A total of 34 listeria lysogens, which are either natural phage carriers or artificially lysogenized strains, were tested for production of prophage upon treatment with u.v. light and Mitomycin C (MC). Most strains were readily inducible, in particular the generated prophage carriers tested. The yield of free phage could be increased up to 1600-fold. With respect to the listeria lysogens investigated, MC was found to be a more powerful inducing agent than u.v. light. Thus, it should be further applied in screening for new phages.

Global changes in gene expression inEscherichia coli K12 induced by bacteriophage Mu Gem protein

We have studied the growth properties of some Mu lysogens with respect to the non-lysogenic strain and have observed that the division time in minimal medium was increased over 4-fold when the bacteria carried the prophage mutated in the gem gene (Mu gem3). Since this phage gene has previously been shown to be involved in modulation of expression of host genes, we have analysed the proteins extracted from lysogens and non-lysogens as a rapid assay of global gene expression. The pattern of proteins extracted showed marked quantitative variations between non-lysogens, lysogens for wild-type Mu and lysogens for phage Mu gem3. These effects were no longer as evident when the strains were grown in rich medium. This dramatic change in the physiological state of the lysogenic strain versus the non-lysogenic in particular growth conditions extends the concept of lysogeny. For many years, the prophage has been considered only as a potentially lethal factor, while here it also appears as a genetic element capable of profoundly modifying host biology.

Bacteriophage typing of Listeria species

A bacteriophage typing scheme for differentiating Listeria isolates from dairy products and various other foodstuffs was developed. Sixteen selected phages isolated from both environmental sources and lysogenic strains were used for typing and, according to their lytic spectra, divided into four groups. Thus far, 41 distinct patterns of lysis were seen when this set was used in typing 57 defined reference strains, representing all five confirmed species and 16 serotypes in addition to 454 Listeria isolates of primarily foodborne origin. Overall, typability was 84.5%; i.e., a strain was lysed by at least one phage at 100x routine test dilution. Strains belonging to serovar 3 were mostly resistant to lysis by the phages employed. The results were highly reproducible, as determined in retyping trials several weeks later. Some phages isolated from environmental sources showed a wider lytic spectrum than did those isolated from lysogenic strains. In accordance with this, the phages were found in different clusters within a computer-generated linkage map. Species specificity and serovar specificity of the lytic reaction were not found. None of the phages was able to lyse strains of Listeria grayi, Listeria murrayi or Jonesia denitrificans. This phage typing system may provide important information for a means of recognizing and eliminating sources of contamination by Listeria spp. within dairy plant equipment.

Transduction of linked chromosomal genes between Pseudomonas aeruginosa strains during incubation in situ in a freshwater habitat

Both transduction of single chromosomal loci and cotransduction of closely linked loci were observed between lysogenic and nonlysogenic strains of Pseudomonas aeruginosa in a freshwater habitat. Transductants were recovered at frequencies of 10(-6) to 10(-5) transductants per CFU. Transductants of lysogenized strains were recovered 10- to 100-fold more frequently than were transductants of nonlysogenic parents. Lysogens are thus capable of introducing phages which mediate generalized transduction into the natural microbial community and serving as recipients of transduced DNA. It would appear that lysogeny has the potential of increasing the size and flexibility of the gene pool available to natural populations of bacteria. The ability to generate and select new genetic combinations through phage-mediated exchange can be significant in the face of a continually changing environment and may contribute to the apparent fitness of the lysogenic state in natural ecosystems.

Bacteriophages of Lactic Acid Bacteria

Bacteriophages of mesophilic and thermophilic lactic streptococci and Lactobacilli have been the subject of numerous investigations. The most extensive studies have been carried out on phages of mesophilic lactic streptococci. Phages have been differentiated on the basis of morphology, serology, and DNA characterization. Morphologically distinct phages belong to different DNA homology groups, related phages being found in such widely separated locations as Ireland, Germany, and New Zealand. Surveys of thermophilic lactic streptococcal phages to date have indicated that the majority of phages belong to one morphological type. Lactobacillus phages belong to several morphological groups, including phages with contractile and noncontractile tails.The role of lysogenic strains of lactic acid bacteria as a source of lytic phages is unclear. In general, lytic and temperate phages of lactic streptococci are not closely related by DNA homology. However, some DNA homology groups contain both temperate and lytic phages, suggesting that these phages may have a common ancestor. Heteroduplex DNA studies indicated that in some instances, lytic phages evolve from one another, but it is possible that DNA from temperate phages contributes to the evolution of phage. DNA studies on Lactobacillus phages have shown a closer relationship between temperate and lytic phages.A number of recent studies have provided restriction endonuclease maps of phage genomes. Several genes, such as structural genes, and sites for attachment and packaging initiation in temperate phage DNA, have been located. Extensive genetic studies of lactic phages are required to elucidate relationships between phage and host, particularly as they relate to phage resistance in lactic acid bacteria.

Phage Induction of Lysogenic Rhizobium leguminosarum biovar trifolii in both the Free-living and the Symbiotic Form

A lysogenic strain, Rhizobium leguminosarum biovar trifolii UK-1: φU, was isolated from a wild white clover nodule. It was symbiotically effective on white clover. A lysogenic phage (U-mole) was induced from this strain by treatment with either UV irradiation or mitomycin C. Phage U-mole had an icosahedral head (40 nm wide), a short tail (9 nm long) and tail fibres (15 nm long). Phage U-mole was induced with mitomycin C both from bacterial cells in infection threads and from bacteroids in nodules. Southern hybridization using EcoRI fragments of phage U-mole as probe indicated that phage U-mole DNA was integrated into the chromosome of strain UK-1: øU at a site involving the 60 kb EcoRI fragment of phage U-mole. Phage U-mole also lysogenized the wild-type strain R. leguminosarum biovar trifolii 4S. The resulting lysogenized strain, 4S:øU, had lost its 315 kb Sym plasmid and its nodulation ability.

Capsular antigenic variations by lysogenic conversion inKlebsiella pneumoniae: Relationship with virulence

Klebsiella pneumoniae lysogenization by phages FR2 and AP3 induces modifications in capsular antigens as evidenced by utilization of K-antisera raised against lysogenic and nonlysogenic strains. After serum absorption, it was found thatK. pneumoniae K59 derivatives lysogenic forKlebsiella phages FR2 and AP3 lack a capsular antigenic determinant present in the nonlysogenic parent and carry a new one. The new antigen, codified by AP3 genes, is different from that codified by FR2. Moreover, strains lysogenic for AP3 phage are less virulent for mice than the FR2 lysogens and the nonlysogenic parent. Phages similar to AP3 for host range and plaque morphology were isolated from 3 of 80 wild-typeKlebsiella strains. In theKlebsiella strain K59, these phages cause the same antigenic modifications observed with AP3.