Incidence and properties of temperate bacteriophages induced from lactic streptococci.

Sixty-three strains of lactic streptococci isolated from commercial lactic streptococcal starter cultures were examined for lysogeny by treatment with ultraviolet light or mitomycin C. After treatment with the inducing agent, all strains, whether or not they lysed, were examined for evidence of phage release by electron microscopy. Thirty-eight strains yielded intact phages or phage particles of varying morphology. All the temperate phages had isometric heads and noncontractile tails; some had collars and structurally distinctive baseplates. Indicator host strains were found for phages induced from seven different strains. Three strains that released phages spontaneously yielded titers of 10(3) to 10(4) plaque-forming units per ml. When strains that spontaneously released phages were grown in mixed culture with indicator strains, increased phage titers of 10(6) to 10(7) plaque-forming units per ml were observed. These findings indicate that lysogenic lactic streptococcal strains may serve as a reservoir for phages that attack sensitive strains in mixed- or multiple-strain lactic starter cultures.     

Lysogenic strains of lactic Acid streptococci and lytic spectra of their temperate bacteriophages

A total of 113 strains of mesophilic strains lactic streptococci of the species Streptococcus lactis, S. lactis subsp. diacetilactis, and S. cremoris, chosen from 291 strains that had been previously classified into six groups on the basis of their sensitivity to 132 virulent phages, were subjected to induction with mitomycin C. Among these strains, 43% produced phages capable of forming plaques of lysis on an indicator strain either spontaneously or after induction. There was a close correlation between the lytic spectra of temperate and virulent phages. Among the strains studied, 25% were shown to be indicator strains. These results emphasized the high probability of development of temperate phages in a starter culture containing mesophilic lactic streptococci and therefore their importance as a cause of accidents in cheese making.     

Improved medium for lactic streptococci and their bacteriophages

[This corrects the article on p. 807 in vol. 29.].     

Improved medium for lactic streptococci and their bacteriophages

Incorporation of 1.9% β-disodium glycerophosphate (GP) into a complex medium resulted in improved growth by lactic streptococci at 30 C. The medium, called M17, contained: Phytone peptone, 5.0 g; polypeptone, 5.0 g; yeast extract, 2.5 g; beef extract, 5.0 g; lactose, 5.0 g; ascorbic acid, 0.5 g; GP, 19.0 g; 1.0 M MgSO₄·7H₂O, 1.0 ml; and glass-distilled water, 1,000 ml. Based on absorbance readings and total counts, all strains of Streptococcus cremoris, S. diacetilactis, and S. lactis grew better in M17 medium than in a similar medium lacking GP or in lactic broth. Enhanced growth was probably due to the increased buffering capacity of the medium, since pH values below 5.70 were not reached after 24 h of growth at 30 C by S. lactis or S. cremoris strains. The medium also proved useful for isolation of bacterial mutants lacking the ability to ferment lactose; such mutants formed minute colonies on M17 agar plates, whereas wild-type cells formed colonies 3 to 4 mm in diameter. Incorporation of sterile GP into skim milk at 1.9% final concentration resulted in enhanced acid-producing activity by lactic streptococci when cells were inoculated from GP milk into skim milk not containing GP. M17 medium also proved superior to other media in demonstrating and distinguishing between lactic streptococcal bacteriophages. Plaques larger than 6 mm in diameter developed with some phage-host combinations, and turbid plaques, indicative of lysogeny, were also easily demonstrated for some systems.     

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.     

Functional properties of plasmids in lactic streptococci

Plasmid biology has become an important area of investigation in dairy starter cultures since it now appears that some properties, vital for successful milk fermentations, are coded by genes located on plasmid DNA. Some of these metabolic properties observed in lactic streptococci have been clearly established as being plasmid-mediated. Examples would be lactose utilization and in Streptococcus lactis subsp. diacetylactis the ability to produce a bacteriocin-like substance. Phenotypic and physical evidence for plasmid linkage has been obtained for other traits such as citrate, sucrose, galactose, glucose, mannose, and xylose utilization, proteinase activity, modification/restriction systems, as well as for nisin production. Further genetic evidence is now needed to confirm plasmid association to these properties. For some characteristics the association with plasmids is highly speculative and is solely based on the phenotypic loss of a metabolic property. In this category would be sensitivity to agglutinins, sensitivity to the lactoperoxidase-thiocyanate-hydrogen peroxide inhibitory system, arginine hydrolysis, and slime production. Other properties which appear plasmid-mediated in lactic streptococci and which will be discussed include inorganic ion resistance, drug resistance, and diplococcin production.     

Comparative study of temperate bacteriophages isolated from Yersinia

170 Yersinia strains belonging to various species were investigated for the presence of temperate bacteriophages. By induction with mitomycin C seven phages were isolated from Y. enterocolitica strains and one phage from a Y. frederiksenii strain. The phages were characterized on the basis of their morphology, host range, genome size, DNA homology, and protein composition. They belong to different phage families and reveal narrow to moderate wide host ranges. Some of the isolated phages were able to infect pathogenic as well as nonpathogenic strains of Y. enterocolitica. The genomes of all isolated phages were found to be composed of double stranded DNA ranging from about 40 to 60 kb. In addition to the analysed phages, a number of putative phages were induced in strains of Y. frederiksenii, Y. kristensenii, Y. intermedia, and Y. mollaretii. The putative phages were identified by isolation of phage DNA from cell free lysates but could not be propagated on indicator strains. Southern hybridization experiments revealed relationships between phages belonging to different families. Moreover, DNA homologies were observed between phages isolated from nonpathogenic Yersinia strains and a phage which was isolated from a pathogenic Y. enterocolitica serogroup O:3 strain.     

Arginine metabolism in lactic streptococci.

Streptococcus lactis metabolizes arginine via the arginine deiminase pathway producing ornithine, ammonia, carbon dioxide, and ATP. In the four strains of S. lactis examined, the specific activities of arginine deiminase and ornithine transcarbamylase were 5- to 10-fold higher in galactose-grown cells compared with glucose- or lactose-grown cells. The addition of arginine increased the specific activities of these two enzymes with all growth sugars. The specific activity of the third enzyme involved in arginine metabolism (carbamate kinase) was not altered by the composition of the growth medium. In continuous cultures arginine deiminase was not induced, and arginine was not metabolized, until glucose limitation occurred. In batch cultures the metabolism of glucose and arginine was sequential, whereas galactose and arginine were metabolized concurrently, and the energy derived from arginine metabolism was efficiently coupled to growth. No arginine deiminase activity was detected in the nine Streptococcus cremoris strains examined, thus accounting for their inability to metabolize arginine. All nine strains of S. cremoris had specific activities of carbamate kinase similar to those found in S. lactis, but only five S. cremoris strains had ornithine transcarbamylase activity.     

Survey of antimicrobial resistance in lactic streptococci.

A total of 26 strains of Streptococcus cremoris and 12 strains of Streptococcus lactis were challenged with 18 antimicrobial agents and with nisin in the Bauer-Kirby disk susceptibility test. All strains were susceptible to ampicillin, bacitracin, cephalothin, chloramphenicol, chlortetracycline, erythromycin, penicillin G, tetracycline, and vancomycin. All strains were resistant to trimethoprim, and almost all strains were resistant to sulfathiazole. Variability in resistance to gentamicin, kanamycin, lincomycin, nafcillin, neomycin, nisin, rifampin, and streptomycin was observed. MICs of these substances for the less susceptible strains were determined, and high-level resistance factors could not be detected, except in the case of nisin. S. lactis ATCC 7962 was resistant to at least 40-fold-higher concentrations of nisin (greater than 64 micrograms/ml) than most other strains tested. This strain was a potent nisin producer.     

Simple and rapid method for isolating large plasmid DNA from lactic streptococci.

A procedure for the rapid isolation of plasmid DNA larger than 30 megadaltons from lactic streptococci is described. This protocol can be used on a preparative scale to isolate sufficient quantities of plasmid DNA required for restriction analysis, cloning, or transformation experiments. A scaled-down protocol is very useful for rapidly screening the plasmid content of streptococcal strains. With this methodology, previously undetected large plasmids were observed.     

Preparation and storage of high-titer lactic streptococcus bacteriophages

Various techniques were employed for preparation of high-titer bacteriophage lysates of Streptococcus lactis, S. cremoris, and S. diacetilactis strains. Infection of a 4-h host culture in litmus milk at 30 C yielded the highest titers (2 × 10⁹ to 4 × 10¹¹ plaque-forming units/ml) for most phages. Host infection in lactose-containing broth produced similar virus numbers only when 0.1 M tris(hydroxymethyl)aminomethane buffer stabilized the pH. The pH at the time of infection as well as the inoculum phage titer were critical in obtaining high titers. Optimum conditions for infection in broth were coupled with a polyethylene glycol concentration procedure to routinely produce milligram quantities of phage from 1 liter of lysate. Neutralization of whey lysates, as a means of storage, offered no survival advantage over unneutralized samples. Storage of phage lysates in a 15% glycerol whey solution at -22 C yielded a high rate of survival in most cases, even with repeated freezing and thawing, over a period of 24 months.     

Optimization of the detection of bacteriophages induced from Listeria sp.

It is necessary to isolate new phages in order to improve the rate of typeability of Listeria monocytogenes strains. We propose a method which increases the detection of induced phages in the presence of inhibitory substances synthesized or liberated by the cells during phage production. Of the 29 phages isolated, 11 (38%) were detected by the spot-on-the-lawn technique and 18 (62%) were revealed by the soft-agar technique. To increase the rate of phage detection, both techniques appear useful. Listeria cultures were subjected to phage typing procedures utilizing these newly isolated phages and the French International set of phages. It appears that the newly isolated phages are good tools for the differentiation of Listeria strains. Among them, one phage seems to be complementary to the French International set.     

Isolation and characterization of temperate bacteriophages of Clostridium difficile

The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages phiC2, phiC5, phiC6, and phiC8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of phiC2 showed nucleotide homology to the sequenced C. difficile strain CD630.     

Temperate bacteriophages and lysogeny in lactic acid bacteria

Abstract Lysogeny is widespread in the lactic acid bacteria. The majority of lysogens can be induced by UV irradiation or treatment with mitomycin C, but indicator strains which allow lytic growth of the induced phage are often not easy to identify. A few temperate phages have been shown to transduce chromosomal and/or plasmid markers. Information about the molecular biology of the temperate phages from lactic acid bacteria is sparse and needs significant supplementation in order that these potentially valuable phages might be utilized more efficiently as tools for improving existing starter strains in dairy fermentations.     

Temperate bacteriophages and lysogeny in lactic acid bacteria

Lysogeny is widespread in the lactic acid bacteria. The majority of lysogens can be induced by UV irradiation or treatment with mitomycin C, but indicator strains which allow lytic growth of the induced phage are often not easy to identify. A few temperate phages have been shown to transduce chromosomal and/or plasmid markers. Information about the molecular biology of the temperate phages from lactic acid bacteria is sparse and needs significant supplementation in order that these potentially valuable phages might be utilized more efficiently as tools for improving existing starter strains in dairy fermentations.     

Within-host competition determines reproductive success of temperate bacteriophages

Within-host competition between parasites is frequently invoked as a major force for parasite evolution, yet quantitative studies on its extent in an organismal group are lacking. Temperate bacteriophages are diverse and abundant parasites of bacteria, distinguished by their ability to enter a facultative dormant state in their host. Bacteria can accumulate multiple phages that may eventually abandon dormancy in response to host stress. Host resources are then converted into phage particles, whose release requires cell death. To study within-host competition between phages, I used the bacterium Escherichia coli and 11 lambdoid phages to construct single and double lysogens. Lysogenic bacterial cultures were then induced and time to host cell lysis and productivity of phages was measured. In double lysogens, this revealed strong competitive interactions as in all cases productivity of at least one phage declined. The outcome of within-host competition was often asymmetrical, and phages were found to vary hierarchically in within-host competitive ability. In double infections, the phage with the shorter lysis time determined the timing of cell lysis, which was associated with a competitive advantage when time differences were large. The results emphasize that within-host competition greatly affects phage fitness and that multiple infections should be considered an integral part of bacteriophage ecology.