Synthesis of Messenger Ribonucleic Acid After Bacteriophage T1 Infection

Synthesis of host-specific and phage-specific messenger ribonucleic acid (mRNA) was studied in bacteria infected by unmodified (T1 . B) or modified [T1 . B(P1)] bacteriophage T1. In a "standard" infection of Escherichia coli B by T1 . B (no host-controlled modification involved), the rate and amount of T1 mRNA synthesis was intermediate between those values reported for infections by a virulent phage such as T4 or a temperate phage such as lambda. The initial rate of mRNA synthesis was slightly increased after T1 . B(P1) infection of E. coli B in comparison with T1 . B infection of the same host. Little or no phage mRNA synthesis could be detected in T1 . B infection of E. coli B(P1). Phage mRNA synthesis in T1 . B(P1)-infected E. coli B(P1) cells was approximately the same in amount as that seen in T1 . B(P1) infection of E. coli B. Synthesis of host-specific mRNA continued throughout the latent period in all infections studied. However, the enzyme beta-galactosidase could not be induced, except after T1 . B infection of E. coli B(P1). In an attempt to understand the apparent differences in mRNA synthesis after infection of E. coli B by phages T1 . B or T1 . B(P1), the effect of altered T1 deoxyribonucleic acid (DNA) methylation on mRNA synthesis was studied. Methyl-deficient T1 DNA, made in cells infected with ultraviolet-irradiated phage T3, inhibited (14)C-uridine incorporation more strongly than normal T1. One passage of methyl-deficient T1 through E. coli B restored uracil incorporation rates to those seen with ordinary T1. This suggests that methylation of T1 DNA can influence the rate of phage mRNA synthesis. However, attempts to relate the difference in mRNA synthesis seen between T1 . B and T1 . B(P1) in E. coli B to the activity of the P1 modification gene were not conclusive.     

Differences in the allele state of genes controlling the specificity of adsorption in transposable phages of Pseudomonas aeruginosa

To reveal possible differences in adsorptional specificities of transposable phages of Pseudomonas aeruginosa and to study the genetical control of this character, we isolated a group of phage-resistant P. aeruginosa mutants using some temperate and virulent phages. The study of resistance of the mutants to all the phages permitted us to find some types of mutants and to build a formal scheme of distribution of adsorptional receptors on the surface of P. aeurginosa cell. According to the results obtained, there are two main "receptor chains", where the receptors for all phages under study are grouped. For the majority of phages, just a single adsorptional receptor is obligatory, and at least two essential receptors are needed for adsorption of virulent phage E79. Two receptors were found also for another virulent phage, phi 11, one of them only being essential. Transposable phages can be grouped into three types, according to their adsorptional specificities. No correlations of adsorptional specificity types and all other characteristics of transposable phages studied (including the sub-groups of transposable phages belonging to different DNA homology types) were found. Genes of natural transposable phages controlling the differences in adsorptional specificities revealed can recombine in phage crosses.     

Erratum: Causes and Consequences of Bacteriophage Diversification via Genetic Exchanges across Lifestyles and Bacterial Taxa

Bacteriophages (phages) evolve rapidly by acquiring genes from other phages. This results in mosaic genomes. Here, we identify numerous genetic transfers between distantly related phages and aim at understanding their frequency, consequences, and the conditions favoring them. Gene flow tends to occur between phages that are enriched for recombinases, transposases, and nonhomologous end joining, suggesting that both homologous and illegitimate recombination contribute to gene flow. Phage family and host phyla are strong barriers to gene exchange, but phage lifestyle is not. Even if we observe four times more recent transfers between temperate phages than between other pairs, there is extensive gene flow between temperate and virulent phages, and between the latter. These predominantly involve virulent phages with large genomes previously classed as low gene flux, and lead to the preferential transfer of genes encoding functions involved in cell energetics, nucleotide metabolism, DNA packaging and injection, and virion assembly. Such exchanges may contribute to the observed twice larger genomes of virulent phages. We used genetic transfers, which occur upon coinfection of a host, to compare phage host range. We found that virulent phages have broader host ranges and can mediate genetic exchanges between narrow host range temperate phages infecting distant bacterial hosts, thus contributing to gene flow between virulent phages, as well as between temperate phages. This gene flow drastically expands the gene repertoires available for phage and bacterial evolution, including the transfer of functional innovations across taxa.     

Molecular characterization and comparison of 38 virulent and temperate bacteriophages of Streptococcus lactis

Thirty-three virulent and five temperate phages of Streptococcus lactis and Streptococcus cremoris were differentiated into three groups by DNA homology. A complete lack of DNA homology was demonstrated between the phage groups. Within each group, large parts of the phage genomes were homologous except for a few phages. One group consisted of five temperate and two virulent phages suggesting that virulent phages isolated during abnormal fermentations and temperate phages isolated after induction from lactic streptococcal starter cultures may be related to one another. We observed a good correlation between the grouping of phages by DNA homology and by their protein composition since within the same DNA homology group, the protein composition of a phage exhibited some similarities with that of the other phages of the group. Therefore, the DNA homologies seemed to be located, at least, in the region coding for the structural proteins. By immunoblotting, we confirmed the relatedness between the proteins of the phages belonging to the same DNA homology group. The important host range factor in bacterium-phage interactions appeared to be an unreliable criterion in determining phage taxonomy.     

Effectiveness of phages in treating experimental Escherichia coli diarrhoea in calves, piglets and lambs

A mixture of two phages, B44/1 and B44/2, protected calves against a potentially lethal oral infection with an O9:K30,99 enteropathogenic strain of Escherichia coli, called B44, when given before, but not after, the onset of diarrhoea; a mixture in which phage B44/3 was replaced by phage B44/3 was effective after the onset of diarrhoea. Calves that responded to phage treatment had much lower numbers of E. coli B44 in their alimentary tract than untreated calves. Usually, high numbers of phage B44/1 and rather lower numbers of phage B44/2 or B44/3 were present in the alimentary tract of these animals. At death, most calves that had not responded to treatment with phages B44/1 and B44/2 had high numbers of mutants of E. coli B44 resistant to phage B44/1 in their small intestine. Phage-treated calves that survived E. coli infection continued to excrete phage in their faeces, at least until the numbers of E. coli B44 also excreted were low. The phages survived longer than E. coli B44 in faecal samples taken from phage-treated calves and exposed to the atmosphere in an unheated animal house. Calves inoculated orally with faecal samples from phage-treated calves that contained sufficient E. coli B44 to cause a lethal infection remained healthy. A mixture of two phages, P433/1 and P433/2, and phage P433/1 alone cured diarrhoea in piglets caused by an O20:K101,987P strain of E. coli called P433. The numbers of the infecting bacteria and phages in the alimentary tract of the piglets resembled those in the calves. Another phage given to lambs 8 h after they were infected with an O8:K85,99 enteropathogenic strain of E. coli, called S13, reduced the numbers of these organisms in the alimentary tract and had an ameliorating effect on the course of the disease. No phage-resistant mutants of E. coli S13 were isolated from the lambs. The only mutants of E. coli B44 and P433 that emerged in the calves and piglets were K30- or K101- and resistant to phage B44/1 or P433/1 respectively; those tested were much less virulent than their parent strains.     

Rapid and sensitive detection method of a bacterium by using a GFP reporter phage

A rapid, sensitive, and convenient method for detecting a specific bacterium was developed by using a GFP phage. Here we describe a model system that utilizes the temperate Escherichia coli-restricted bacteriophage lambda, which was genetically modified to express a reporter gene for GFP to identify the colon bacillus E. coli in the specimen. E. coli infected with GFP phage was detected by GFP fluorescence after 4-6 hr of incubation. The results show that a few bacteria in a specimen can be detected under fluorescence microscopy equipped with a sensitive cooled CCD camera. When E. coli and Mycobacterium smegmatis were mixed in a solution containing GFP phage, only E. coli was infected, indicating the specificity of this method. The method has the following advantages: 1) Bacteria from biological samples need not be purified unless they contain fluorescent impurities; 2) The infection of GFP phage to bacteria is specific; 3) The fluorescence of GFP within infected bacteria enables highly sensitive detection; 4) Exogenous substrates and cofactors are not required for fluorescence. Therefore this method is suitable for any phage-bacterium system when bacteria-specific phages are available.     

Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7

Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages. To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage. Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E. coli K12 cells. The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E. coli O157:H7 and related species. On the other hand, T2ppD1 could not infect E. coli K12, the original host of T2, or its derivatives. The host range of T2ppD1 was the same as that of PP01. Infection of T2ppD1 produced turbid plaques on a lawn of E. coli O157:H7 cells. The binding affinity of T2ppD1 to E. coli O157:H7 was weaker than that of PP01. The adsorption rate constant (ka) of T2ppD1 (0.17 x 10(-9)(ml CFU(-1) min(-1)) was almost 1/6 that of PP01 (1.10 x 10(-9)(ml CFU(-1) min(-1))). In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage.     

Search for host specificity systems in Shigella using DD-series phages]

A total of 712 Shigella strains were studied with the use of dysentery diagnostic phages DD II, DD VI and DD VII in order to reveal the systems of host DNA specificity. The study comprised 4 tests: mass screening by the intensity of phagolytic reaction of phages in various strains; and the determination of the parameters of adsorption. As a result, an effective modification and restriction systems were revealed in Sh. sonnei 311 with the use of phage DD II. Bacteriophage DD VII was effectively restricted in E. coli CK, BB and BB/T4. Cross titration showed that the modification and restriction systems of E. coli BB and BB/T4 differed from the specificity system of E. coli CK. Phage DD VI had an exceptionally broad spectrum of activity and was not sensitive to any known restriction system.     

The control of experimental Escherichia coli diarrhoea in calves by means of bacteriophages

Seven phages highly active in vitro and in vivo against one or other of seven bovine enteropathogenic strains of Escherichia coli belonging to six different serotypes were isolated from sewage. Severe experimentally induced E. coli diarrhoea in calves could be cured by a single dose of 10(5) phage organisms. It could be prevented by doses as low as 10(2), by spraying the litter in the calf rooms with aqueous phage suspensions or simply by keeping the calves in uncleaned rooms previously occupied by calves whose E. coli infections had been treated with phage. Microbiological examinations of calves used in these experiments revealed that the phage organisms multiplied rapidly and profusely after gaining entry to the E. coli-infected small intestine, quickly reducing the E. coli to numbers that were virtually harmless. The only phage-resistant E. coli that emerged in the studies on calves infected with one or other of the seven E. coli strains were K-. These organisms were much less virulent than the K+ organisms from which they were derived and did not present a serious problem in calves given adequate amounts of colostrum. Infections produced by oral inoculation of a mixture of six strains of the E. coli could be controlled by administration of a pool of the six phages that were active against them but, in general, the control was less complete than that observed in the single-strain infections. K+ phage-resistant bacteria emerged in some of the calves used in these mixed infections and they were as virulent as their parent organisms; evidence from in vitro studies suggested that they might have arisen by genetic transfer between organisms of the different infecting strains. Infections produced by these K+ mutants and their parents could be controlled by the use of mutant phages derived from phages that were active on their parents. During the experiments with mixed E. coli infection, an extraneous phage active against one of the six E. coli strains suddenly appeared in calves kept in the same rooms. Microbiological examinations revealed that this phage was effectively controlling the multiplication of organisms of that particular strain of E. coli in the small intestines of the calves.     

Defective site-specific integration elements are present in the genome of virulent bacteriophage LL-H of Lactobacillus delbrueckii.

The phage attachment site, attP, and the integrase-encoding gene, int, are sufficient to promote site-specific integration of the temperate phage mv4 genome into the chromosome of the Lactobacillus delbrueckii host (L. Dupont, B. Boizet-Bonhoure, M. Coddeville, F. Auvray, and P. Ritzenthaler, J. Bacteriol. 177:586--595, 1995). The mv4 genome region containing these elements was compared at the nucleotide and amino acid levels with that of the closely related virulent phage LL-H. Complex DNA rearrangements were identified; a truncated integrase gene and two sites homologous to the mv4 attP site were detected in the genome of the virulent phage LL-H. These observations suggest that the two phages derive from a common temperate ancestor.     

Effect of ozone on bacteriophages in the phage--host-cell system

The antiviral action of ozone was studied using Escherichia coli K-12 AB1157 virulent phage T4 and Pseudomonas aeruginosa PAO1 temperate phage SM as models depending on the phage state during the action: a free phage, a phage in the presence of sensitive host cells, or a vegetative phage. Bacteriophages T4 and SM were found to be much more sensitive to ozone as compared to the bacterial strains AB1157 and PAO1. The latter protected phage particles against the activation by ozone at a concentration which effectively inactivated these phages in the absence of the bacteria. Ozone also exerted an inhibiting effect on vegetative phage SM, and the degree of inhibition decreased with the termination of intracellular growth stages.     

Identification of Site-Specific Recombination Genes int and xis of the Rhizobium Temperate Phage 16-3

Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP. Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis (prophage excision). We concluded that Int protein of phage 16-3 belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E. coli. The application of the 16-3 site-specific recombination system in biotechnology is discussed.     

Luria effect in bacteriophages and the role of the products of recA+ and lexA+ genes in its induction

An analysis of UV-damages accumulation in the phages as revealed by delay of intracellular growth is represented using temperate lambda phage. The maximum of growth delay of phage lambda at given UV-dose was found with lambda red+, infecting Escherichia coli AB1886 uvrA strain. The growth delay was absent, when a strain RH-1 uvrA-recA- was infected with UV-irradiated phage lambda red3. A moderate growth delay was obtained with the phages lambda red+, infecting E. coli RH-1 uvrA-recA- or phage lambda red3, infecting E. coli AB1886 uvrA-. THe growth delay was also absent when wild type, recA- and uvrA mutants of E. coli were infected with phage lambda after 8-metnoxypsoralen + light (lambda > 310 nm) treatment. It is known that the crosslinks appear to be the DNA defects which give rise to the observed biological inactivation following psoralen + light treatment. However, a considerable growth delay of phage lambda, treated by 8-metnoxypsoralen + light, was only found under condition of crosslinks repair (W-reactivation and prophage-reactivation). The results obtained are best explained by the assumption that the growth delay reflects the time required for the postreplication repair (RecA, LexA, Red) of any lethal UV-lesion.     

Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sps. strains

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bungori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperate phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.     

Comparative study of nine Lactobacillus fermentum bacteriophages

AIMS: To investigate the basic properties of six temperate and three virulent phages, active on Lactobacillus fermentum, on the basis of morphology, host ranges, protein composition and genome characterization. METHODS AND RESULTS: All phages belonged to the Siphoviridae family; two of them showed prolate heads. The host ranges of seven phages contained a common group of strains. SDS-PAGE protein profiles, restriction analysis of DNA and Southern blot hybridization revealed a high degree of homology between four temperate phages; partial homologies were also detected among virulent and temperate phages. Clustering derived from host range analysis was not related to the results of the DNA hybridizations. CONCLUSION: The phages investigated have common characteristics with other known phages active on the genus Lactobacillus. Sensitivity to viral infection is apparently enhanced by the presence of a resident prophage. SIGNIFICANCE AND IMPACT OF THE STUDY: These relationships contribute to the explanation for the origin of phage infection in food processes where Lact. fermentum is involved, such as sourdough fermentation.     

Comparison of synonymous codon distribution patterns of bacteriophage and host genomes.

Synonymous codon usage patterns of bacteriophage and host genomes were compared. Two indexes, G + C base composition of a gene (fgc) and fraction of translationally optimal codons of the gene (fop), were used in the comparison. Synonymous codon usage data of all the coding sequences on a genome are represented as a cloud of points in the plane of fop vs. fgc. The Escherichia coli coding sequences appear to exhibit two phases, "rising" and "flat" phases. Genes that are essential for survival and are thought to be native are located in the flat phase, while foreign-type genes from prophages and transposons are found in the rising phase with a slope of nearly unity in the fgc vs. fop plot. Synonymous codon distribution patterns of genes from temperate phages P4, P2, N15 and lambda are similar to the pattern of E. coli rising phase genes. In contrast, genes from the virulent phage T7 or T4, for which a phage-encoded DNA polymerase is identified, fall in a linear curve with a slope of nearly zero in the fop vs. fgc plane. These results may suggest that the G + C contents for T7, T4 and E. coli flat phase genes are subject to the directional mutation pressure and are determined by the DNA polymerase used in the replication. There is significant variation in the fop values of the phage genes, suggesting an adjustment to gene expression level. Similar analyses of codon distribution patterns were carried out for Haemophilus influenzae, Bacillus subtilis, Mycobacterium tuberculosis and their phages with complete genomic sequences available.     

A visualization method of filamentous phage infection and phage-derived proteins in Escherichia coli using biotinylated phages.

Direct visualization of filamentous phage infection in Escherichia coli (E. coli) was attempted using biotinylated phages (BIO-phages). The biotinylation of the phages did not influence their infectivity into E. coli. E. coli infected with BIO-phages could be detected by using fluorescein-conjugated avidin with confocal laser scanning microscopy, and BIO-phages and BIO-phage-derived proteins in E. coli could be directly observed by using the avidin-biotin-peroxidase complex method with electron microscopy. This is the first report of direct visualization of phage infection and phage-derived proteins in the host cell using a biotin-avidin interaction. This simple and powerful method is applicable to the study of infection by various viruses.     

Temperate coliphages: classification and correlation with habitats

Temperate coliphages were recovered from sewage, mammalian feces, and lysogenic strains of Escherichia coli. A total of 32 phages of independent origin were divided into six groups by applying the criteria of host range, antigenic homology, and the ultraviolet inducibility of the prophage. The demonstration of genetic interactions in some cases has confirmed the classification scheme. Nine phages were assigned to the P2 family and 19 to the lambda family. The remaining four isolates may represent some novel phylogenetic types. Phages recovered from the lysogenic strains of E. coli were all found to be P2 related, whereas a majority of the phages recovered as cell-free plaque-forming units were assignable to the lambda family. It is proposed that the biological attributes of the phages belonging to the two principal families are reflected in the distribution patterns observed. The virions of phage HK256 show multiple tail fibers and may thus represent a "new" virion form among the temperate coliphages.     

Thirteen Virulent and Temperate Bacteriophages of Lactobacillus bulgaricus and Lactobacillus lactis Belong to a Single DNA Homology Group

Thirteen virulent phages and two temperate phages of two closely related bacterial species (Lactobacillus lactis and L. bulgaricus) were compared for their protein composition, their antigenic properties, their restriction endonuclease patterns, and their DNA homology. The immunoblotting studies and the DNA-DNA hybridizations showed that the phages could be differentiated into two groups. One group contained 2 temperate phages of L. bulgaricus and 11 virulent phages of L. lactis. Inside each group, at least two common proteins of identical sizes could be detected for each phage. These proteins were able to cross-react in immunoblotting experiments with an antiserum raised against one phage of the same group. Temperate phage DNAs showed partial homology with DNAs from some virulent phages. These homologies seem to be located on the region coding for the structural proteins since recombinant plasmids coding for one of the major phage proteins of one phage were able to hybridize with the DNAs from phages of the same group. These results suggest that temperate and virulent phages may be related to one another.