Use of real-time quantitative PCR for the analysis of phiLC3 prophage stability in lactococci

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage phi LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six phi LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30 degrees C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the phi LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the phi LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.     

Prophage-Cured Derivatives of Streptococcus lactis and Streptococcus cremoris

Prophage curing was achieved in Streptococcus lactis and Streptococcus cremoris, and the cured derivatives were shown to be indicators for their temperate bacteriophages. Relysogenization of these cured derivatives completed the first formal demonstration of the lysogenic state in lactic streptococci.     

The bacteriophages of lactic acid bacteria with emphasis on genetic aspects of group N lactic streptococci

The paper reviews the bacteriophages of the group N lactic streptococci centering on isolation, ultrastructure and morphology, phage receptors, the structure of the genome, protein components, the phenomenon of the lysogenic state, restriction-modification systems and genetic exchange by transfection and transduction. The resulting consequences on industrial fermentations are briefly discussed.     

Transformation and Transfection in Lysogenic Strains of Bacillus subtilis 168

Strains of Bacillus subtilis lysogenic for either temperate bacteriophage phi105 or SPO2 were reduced to less than 1.0% of the level of transformation of the nonlysogenic strains. Strains lysogenic for both phi105 and SPO2 are virtually nontransformable, indicating that the effect of lysogeny is additive. Lysogenic cultures transfected at essentially wild-type levels with deoxyribonucleic acid (DNA) isolated from bacteriophages phi29 and SPO1. The residual transformation and transfection achieved by the lysogenic cultures changed dramatically during growth in SPII medium, whereas nonlysogenic strains remained competent for 5 hr in SPII medium. Despite a marked reduction in transformation, lysogenic cultures initially irreversibly bound as much DNA as nonlysogenic cultures. After 60 min in SPII medium, there was a rapid decrease in the capacity of lysogenic cells to bind DNA irreversibly. These results, as discussed, indicate that the inhibition of transformation is probably due to an alteration of the cell surface or a differential inactivation of bacterial genes after lysogenic conversion.     

Properties of Rhizobium trifolii isolates surviving exposure to specific bacteriophage

Six strains of Rhizobium trifolii were exposed to specific bacteriophages and the properties of 10 surviving clones of each studied. Two temperate bacteriophages produced clones which were lysogenic but showed no changes in colony form, symbiotic properties, or somatic antigens. Of forty clones selected by exposure to four other bacteriophages, none were lysogenic although there was some indication of unusually long association of phage with bacteria in infected cultures. Seventeen of these clones were changed in symbiotic properties, 15 in colony morphology, and 13 in somatic cross-reaction with the parent. Unexpectedly, despite stringent selection conditions, 28 were still either partially or completely susceptible to the selecting phage.     

Lysogeny by phages specific to Yersinia pestis in bacteria of the microbiocenoses of wild plague-carrying rodents

For the first time lysogenic bacteria of the genus Escherichia, spontaneously producing bacteriophages specific to the infective agent of plague, have been detected in the microbiocenoses of midday gerbils, plague carrier in the Volga-Urals natural focus. The morphological, cultural and biochemical properties of 22 lysogenic Escherichia strains isolated from wild rodents, as well as the spectra of the lytic activity of temperate bacteriophages produced by the above-mentioned microorganisms have been studied. Proceeding from the findings obtained in this research, all the strains have been divided into 3 lysotypes.     

Effect of lysogeny on transfection and transfection enhancement in Bacillus subtilis.

Strains of Bacillus subtilis 168 lysogenic for bacteriophage phi105 transfer with deoxyribonucleic acid (DNA) isolated from bacteriophage SPO2 at a higher efficiency than non-lysogenic strains. This enhancement of transfection was not the result of recombination between bacteriophages SPO2 and phi105. Superinfection marker rescue increased transfection with DNA from bacteriophage phi105 occurred simultaneously with the addition of the transfecting DNA. Again, this enhancement of transfection was not the result of recombination but rather a protection of the transfecting DNA by the superinfecting bacteriophage. The ability of the superinfecting bacteriophage to protect the transfecting DNA from inactivation was maximal when the bacteria were just becoming competent. Bacteriophage phi1 cannot replicate after the transfection of competent bacteria lacking a functional DNA replication system, whereas bacteriophage phi1 was able to replicate after infection of competent bacteria grown under comparable conditions. These observations support the hypothesis that GAPase and an inducible repair system play an important role in the development of competence.     

Improved Rapid Plate Method for the Isolation of Bacteriophages from Lysogenic Bacteria

A modification of a recently reported rapid plate method for the isolation of bacteriophages from lysogenic bacteria is described. The velveteen replica plate technique was used for inoculation of mitomycin C-induced colonies onto agar plates, and tetrazolium chloride was used to enhance detection of phage activity on replicated indicator plates.     

Development of a New Method for Detection and Identification of Oenococcus oeni Bacteriophages Based on Endolysin Gene Sequence and Randomly Amplified Polymorphic DNA

Malolactic fermentation (MLF) is a biochemical transformation conducted by lactic acid bacteria (LAB) that occurs in wine at the end of alcoholic fermentation. Oenococcus oeni is the main species responsible for MLF in most wines. As in other fermented foods, where bacteriophages represent a potential risk for the fermentative process, O. oeni bacteriophages have been reported to be a possible cause of unsuccessful MLF in wine. Thus, preparation of commercial starters that take into account the different sensitivities of O. oeni strains to different phages would be advisable. However, currently, no methods have been described to identify phages infecting O. oeni. In this study, two factors are addressed: detection and typing of bacteriophages. First, a simple PCR method was devised targeting a conserved region of the endolysin (lys) gene to detect temperate O. oeni bacteriophages. For this purpose, 37 O. oeni strains isolated from Italian wines during different phases of the vinification process were analyzed by PCR for the presence of the lys gene, and 25 strains gave a band of the expected size (1,160 bp). This is the first method to be developed that allows identification of lysogenic O. oeni strains without the need for time-consuming phage bacterial-lysis induction methods. Moreover, a phylogenetic analysis was conducted to type bacteriophages. After the treatment of bacteria with UV light, lysis was obtained for 15 strains, and the 15 phage DNAs isolated were subjected to two randomly amplified polymorphic DNA (RAPD)-PCRs. By combining the RAPD profiles and lys sequences, 12 different O. oeni phages were clearly distinguished.     

Effect of nucleases on bacteria infected with bacteriophages

The effect of bacterial nucleases on bacteria infected by DNA- or RNA-containing bacteriophages with different serogroups was studied. Bacillary RNases have a strong inhibitory effect on RNA-containing bacteriophages. It was shown that nucleases suppressed the infection process of bacteria by bacteriophages M12, f2, PP7, and QB. The minimal inhibitory concentration ranged from 0.6 to 6 μg/mL. Bacterial ribonucleases have no impact on the development of DNA-containing bacteriophages PZ-A, PZ-B, P₃k, P118, and a lysogenic culture of Escherichia coli (λ) and Bacillus subtilis 168 (phi105). RNase from Bacillus pumilus did not inactivate bacteriophages Qβ and f2 in vitro and did not influence the adsorption on bacteriophages on the cell wall of the bacteria host E. coli AB301. The enzyme effect was shown at the level of bacteriophage infection of the host bacteria. Presumably, the phase between the adsorption and penetration of phage RNA into bacterial pili is the most sensitive to the effect of RNases.     

Bacteriophages in Arctic and Antarctic low-temperature systems

Comparative analysis of the presence of bacteriophages was carried out for the water column of a permanently ice-covered, extremely oligotrophic Lake Untersee (East Antarctica) and the ancient ice wedge of the Mamontova Gora outcrop (Aldan River, Central Yakutia). Microscopy revealed bacteriophages in the Mamontova Gora ice samples and in the lysates of the pure cultures of phage-sensitive bacteria isolated from the same samples. Bacteriophages isolated from these cultures were filamentous and interacted with bacteria as moderate (lysogenic) phages. A similar filamentous bacteriophage was isolated from the Lake Untersee water column. The highest morphological diversity of bacteriophages was revealed by microscopy in the oxic Lake Untersee water column in the chemocline zone (70–76 m) and in the sulfide layer (85 m). Detection of similar filamentous bacteriophages in a relic ice sample and in the samples from Antarctic Lake Untersee indicate wide occurrence of bacteriophages and lysogeny in microbial communities of low-temperature ecosystems.     

Bacteriophage in polar inland waters

Bacteriophages are found wherever microbial life is present and play a significant role in aquatic ecosystems. They mediate microbial abundance, production, respiration, diversity, genetic transfer, nutrient cycling and particle size distribution. Most studies of bacteriophage ecology have been undertaken at temperate latitudes. Data on bacteriophages in polar inland waters are scant but the indications are that they play an active and dynamic role in these microbially dominated polar ecosystems. This review summarises what is presently known about polar inland bacteriophages, ranging from subglacial Antarctic lakes to glacial ecosystems in the Arctic. The review examines interactions between bacteriophages and their hosts and the abiotic and biotic variables that influence these interactions in polar inland waters. In addition, we consider the proportion of the bacteria in Arctic and Antarctic lake and glacial waters that are lysogenic and visibly infected with viruses. We assess the relevance of bacteriophages in the microbial loop in the extreme environments of Antarctic and Arctic inland waters with an emphasis on carbon cycling.     

Removal and inactivation of indicator bacteriophages in fresh waters

AIMS: The removal and inactivation of faecal coliform (FC) bacteria, enterococci (ENT), sulphite-reducing clostridia (SRC), somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis in fresh waters. METHODS AND RESULTS: Removal was studied in two areas of a river. The results showed different removal of each group of microbes. Faecal coliform bacteria were removed faster than any other, whereas SRC and bacteriophages infecting Bact. fragilis were the most persistent. Inactivation was measured by 'in situ' experiments, which showed significant differences in survival of the different groups of bacterial and bacteriophage indicators. The SRC and bacteriophages were more resistant than faecal coliforms and enterococci, with the exception of F-specific RNA bacteriophages in the summer. Inactivation experiments with pure cultures of bacteriophages confirmed that phage B40-8 of Bact. fragilis was the most resistant. CONCLUSIONS: Bacteria and bacteriophages show different resistance to natural inactivation. The use of phages allows information to be obtained in addition to that provided by bacterial indicators. Somatic coliphages and phages infecting Bact. fragilis might supply that indicator function. SIGNIFICANCE AND IMPACT OF THE STUDY: Confirmation was obtained that bacteriophages provided additional information to that provided by bacterial indicators to monitor the natural inactivation of viruses and/or pathogens.     

New waterborne bacteriophages active on Yersinia enterocolitica.

Seven bacteriophages active on Yersinia enterocolitica (YE) were isolated from surface water samples collected in Granada, Spain. A comparison of the respective host ranges of these new phages and of reference phages used for YE phage typing showed that YE strains belonging to various phage types, grown at either 37 or 25 degrees C, expressed susceptibility to reference sewage water phages whereas susceptibility to new waterborne phages, as well as to reference phages from lysogenic YE, was only demonstrated in YE strains grown at 25 degrees C. A YE strain isolated by stool culture from a pig was lysogenic for a bacteriophage which behaved like waterborne phages and reference phages from lysogenic YE strains. The possibility that the isolation of waterborne bacteriophages might, in certain circumstances, reflect the presence of lysogenic YE was raised.     

Lytic systems in lactic acid bacteria and their bacteriophages

Summary Lytic systems of lactic acid bacteria and their bacteriophages are reviewed with an emphasis on molecular characterization. Details of enzyme biochemistry and the cloning and analysis of lytic genes are presented, with coverage of lactococcal prolate headed bacteriophages, lactococcal isometric bacteriophages, Lactobacillus bacteriophages and lactococcal autolysins. Some comments on the importance of autolysis in cheese ripening are included and the biotechological exploitation of cloned and characterized lytic genes is presented.     

Phage-plasmid interactions in bacteria]

A review is devoted to consideration of the basic types of interaction of moderate and virulent bacteriophages and extrachromosomal elements in bacterial cells. Various experimental data confirm diversity of such interactions as well as resistance of bacteria to productive phage infection. Role of plasmids in such processes as abortion infection, lysogenic state, reparation of phage DNA as well as the effect of plasmids on the basic stages of phage development in the cells are analyzed. Possible mechanisms for limiting development of phages as a result of plasmids' presence in bacterial cells are considered.     

Usefulness of different groups of bacteriophages as model micro-organisms for evaluating chlorination

AIMS: To assess the usefulness of bacterial and viral indicators in chlorination processes and to collect quantitative information necessary for risk assessment analysis in water disinfection processes based on chlorination. METHODS AND RESULTS: Naturally occurring bacterial indicators, bacteriophages and enteroviruses were determined to evaluate the effect of chlorination in groundwater and secondary sewage effluents. Additionally, the effect of chlorinating on selected bacteriophages, enteroviruses and Escherichia coli was also tested in spiked samples of bottled water and sewage effluents. Results indicate that chlorination inactivates more efficiently bacteria than phages and enteroviruses. Among the human viruses, phages infecting Bacteroides fragilis and selected somatic coliphages belonging to the Siphoviridae family were the most persistent to chlorination. CONCLUSIONS: The three groups of bacteriophages studied were all more resistant to chlorination than bacteria and some of the phages were more resistant than enteroviruses. Results presented here indicate that it is very risky to generalize from information obtained with inactivation experiments done with single isolates of any phage or virus. If possible, inactivation studies should be done with naturally occurring populations. Phages offer a good opportunity for studying naturally occurring populations. Thus, the bacteriophages offer a range of resistance to chlorination that may represent most of the viruses that can be found in water. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported in this study support the inclusion of bacteriophages as additional indicators of the efficiency of water chlorination processes and water quality.     

Biotechnology of lactic acid bacteria with special reference to bacteriophage resistance

Lactic acid bacteria play an important role in many food and feed fermentations. In recent years major advances have been made in unravelling the genetic and molecular basis of significant industrial traits of lactic acid bacteria. Bacteriophages which can infect and destroy lactic acid bacteria pose a particularly serious threat to dairy fermentations that can result in serious economic losses. Consequently, these organisms and the mechanisms by which they interact with their hosts have received much research attention. This paper reviews some of the key discoveries over the years that have led us to our current understanding of bacteriophages themselves and the means by which their disruptive influence may be minimized.     

Inactivation of bacteriophages with cis-platinum(II) diamminedichloride.

The ability of the cis-plantinum(II) diamminedichloride and its hydrolytic products to inactive DNA bacteriophages was examined on the models T2, T4, T4BO1, T3, and lambda. The inactivation of all bacteriophages under study increases gradually during the first 40-90 min of the action of neutral cis-Pt(II) and later passes into an exponential phase. The extent of the region of slower inactviation is larger for osmotically sensitive strains T2 and T4. Inactivation with the hydrolytic products for cis-Pt(II) proceeds exponentially starting from the very beginning and their inactivating effect is higher by 40-80 times than for a comparable concentration of the original complex. The extent of inactivation is not affected with the HCR marker of the host bacteria. The sensitivity to cis-Pt(II) is higher for bacteriophages with a head permeable to salts. An additional inactivation ("after-effect") was observed after dilution of the complex; it can be removed by adding S-aminoisothiuronium dihydrobromide (AET). The results obtained are in good accord with the assumption that inactivation is due to the hydrolytic products arising in the head of bacteriophage.