Interrelationship between Colony Type, Phage Susceptibility and Virulence in Xanthomonas oryzae

S ummary : A colonial mutant of Xanthomonas oryzae. (tentatively designated T) had a transparent colony (associated with absence of polysaccharide), resistance to the bacteriophage Bpl and markedly attenuated virulence. Phage resistant mutants with the same colonial characteristics as the wild type (W), which is opaque because of abundant polysaccharides produced, showed various degrees of virulence. Disease symptoms were not suppressed when cells of T were mixed with W in various proportions and used to inoculate rice plants. Disease severity was dependent on the proportion of T in the inoculum and the multiplication of T in the plant was proportional to its initial number of cells in the mixture. When infected rice plants were grown in irrigation water containing phage, little or no selection of phage-resistant mutants was detected in spite of repeated and high selection pressure. This may be explained by an insufficient supply of bacterial cells from diseased leaves into the irrigation water and by the very low propotion of phage-resistant mutants that are virulent.     

Interaction of RNA-containing bacteriophages with the host cells: MS2-induced E. coli mutants and formation of DNA-containing derivatives of MS2 bacteriophage

Sensitive cells of Escherichia coli AB 259 Hfr 3000 infected with RNA-containing phage MS2 produce phage particles and continue to divide showing segregation of sensitive cells maintaining new infection cycles. Phage multiplication in sensitive cells gives rise to phage resistant forms in their progeny. The described phenomenon has been shown to be due not to pre-existing phage-resistant cell selection but is a result of interaction of the phage and the cell. In contrast to the usual spontaneous or chemically induced Escherichia coli mutants MS2-induced phage-resistant cells are genetically unstable. During their reproduction they segregate new MS2-resistant types carrying more significant changes in the region coded by the sex factor. Cells belonging to two final MS2-induced mutants also produce a new type of phages; they are DNA-containing forms neutralized, however, by anti-MS2 serum. Production of such phage proves that genetic moiety of RNA-containing phage is able to be expressed as a part of the DNA structure.     

Susceptibility of Different Coliphage Genomes to Host-controlled Variation

Twenty-eight coliphages were studied for their susceptibility to four systems of host control variation in Escherichia coli. Both temperate and virulent phages were studied, including phages with ribonucleic acid, double- and single-stranded deoxyribonucleic acid (DNA) and glucosylated DNA. The systems examined were E. coli C-K, K-B, B-K, and K-K(P1). The C-K, K-B, and B-K systems affected temperate phages and nonlysogenizing mutants derived from temperate phages. In general, these systems did not restrict virulent phages. Phage 21e, a variant of phage 21, lost the ability to undergo restriction in the C-K and B-K systems, but retained susceptibility to the K-B and K-K(P1) systems. This suggests that the genetic site(s) on the phage, as well as in the host, determines susceptibility to host-controlled variation. Both temperate and dependent virulent phages were susceptible to the host control system resulting from the presence of prophage P1. The autonomous and small virulents were not susceptible. In a given system, the various susceptible phages differed widely in their efficiency of plating on the restricting host. If the few infections that occur arise in rare special cells, then different populations of special cells are available to different phage species. For most phage types, when a susceptible phage infected a nonrestricting host, the progeny showed the specificity appropriate to that host. Behavior of T3 was exceptional, however. When T3 obtained from E. coli K infected E. coli C or B, some of the progeny phages retained K host specificity, whereas others acquired the specificity of the new host.     

Alteration of Host Specificity to Lytic Bacteriophages in Streptococcus cremoris

A mutant of Streptococcus cremoris strain ML1 was isolated based on its resistance to acriflavine. The mutant strain showed resistance to the growth of virulent bacteriophages to which the parental strain was sensitive whereas it became sensitive to a number of other virulent phages to which the parental strain was resistant. At the same time, infection of the mutant strain by another bacteriophage sc607 resulted in killing of cells without production of progeny phages. The phage adsorption appeared normal, suggesting that the killing was a postadsorption event. Such killing of bacterial cells was prevented by chloramphenicol treatment, indicating that involvement of some protein either synthesized by phage or phage-induced cellular protein. Synthesis of ribonucleic acid was abruptly terminated after infection of the mutant strain by phage sc607 but not of the parental strain. The alteration of host specificity in the mutant to different lytic bacteriophages and especially abortive infection by phage sc607 resembles the prophage-mediated interference observed in other bacteria.     

The Molecular and Genetic Basis of Repeatable Coevolution between Escherichia coli and Bacteriophage T3 in a Laboratory Microcosm

The objective of this study was to determine the genomic changes that underlie coevolution between Escherichia coli B and bacteriophage T3 when grown together in a laboratory microcosm. We also sought to evaluate the repeatability of their evolution by studying replicate coevolution experiments inoculated with the same ancestral strains. We performed the coevolution experiments by growing Escherichia coli B and the lytic bacteriophage T3 in seven parallel continuous culture devices (chemostats) for 30 days. In each of the chemostats, we observed three rounds of coevolution. First, bacteria evolved resistance to infection by the ancestral phage. Then, a new phage type evolved that was capable of infecting the resistant bacteria as well as the sensitive bacterial ancestor. Finally, we observed second-order resistant bacteria evolve that were resistant to infection by both phage types. To identify the genetic changes underlying coevolution, we isolated first- and second-order resistant bacteria as well as a host-range mutant phage from each chemostat and sequenced their genomes. We found that first-order resistant bacteria consistently evolved resistance to phage via mutations in the gene, waaG, which codes for a glucosyltransferase required for assembly of the bacterial lipopolysaccharide (LPS). Phage also showed repeatable evolution, with each chemostat producing host-range mutant phage with mutations in the phage tail fiber gene T3p48 which binds to the bacterial LPS during adsorption. Two second-order resistant bacteria evolved via mutations in different genes involved in the phage interaction. Although a wide range of mutations occurred in the bacterial waaG gene, mutations in the phage tail fiber were restricted to a single codon, and several phage showed convergent evolution at the nucleotide level. These results are consistent with previous studies in other systems that have documented repeatable evolution in bacteria at the level of pathways or genes and repeatable evolution in viruses at the nucleotide level. Our data are also consistent with the expectation that adaptation via loss-of-function mutations is less constrained than adaptation via gain-of-function mutations.     

Bacteriophage phi297, a new species of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> temperate phages with a mosaic genome: Potential use in phage therapy

The genome of halo-forming temperate Pseudomonas aeruginosa phage phi297 and lytic activity of its virulent mutant were studied. A mosaic structure was revealed for phi297 genome by its complete sequencing. The phi297 genome was partly homologous to the genomes of phages D3 and F116. High lytic activity was assumed for temperate P. aeruginosa bacteriophage phi297 on the basis of morphological features of negative colonies. Virulent mutant phi297vir, which was capable of lysing the wild-type phage bacteria, was isolated. Lytic activity was compared for phi297 and the phages from commercial mixtures of two manufacturers (facilities of Nizhnii Novgorod and Perm’). Phage phi297 caused lysis of the mutant PAO1 bacteria that were resistant to the phages from commercial preparations, but the lytic activity spectrum of phi297 was narrower that the spectra of the commercial phages. The use of nonreverting virulent mutants of certain temperate bacteriophages was proposed for the treatment of P. aeruginosa infections.     

Phages C-2 and J: IncC and IncJ plasmid-dependent phages, respectively

Phages C-2 and J were isolated from sewage. Phage C-2 was filamentous and formed plaques on Salmonella typhimurium strains carrying various C plasmids. It also plated on Proteus mirabilis and Serratia marcescens strains carrying particular C plasmids, but failed to form plaques on lines of Escherichia coli K12 strains harbouring most of these plasmids, although in all cases, phage multiplication on the strains was demonstrated. No phage increase occurred in any strain which lacked a C plasmid or contained plasmids of other incompatibility groups. The phage was sensitive to chloroform and, unlike other filamentous bacterial viruses, adsorbed to shafts of conjugative pili. It had a disc-like structure at the end which attached to the pilus. Phage C-2 had a buoyant density of 1 . 30 g cm-3 and a single-stranded circular DNA genome of 3 . 0 MDal. Phage J had an hexagonal head with an inter-apical distance of 40 nm and a short noncontractile tail. It was resistant to chloroform and diethyl ether. The phage formed plaques or propagated on E. coli strains harbouring some IncC plasmids and all IncJ and IncD plasmids tested. The phage did not form plaques but propagated on P. mirabilis and Ser. marcescens strains carrying these plasmids. It did not plate or propagate on S. typhimurium strains harbouring the plasmids. The plaques were very hazy and variable in size. The phage attached sparsely, at a site which appeared to be located at the base of the tail, to sides of conjugative pili.     

Bacteriophage phi297--the new species of temperate phages Pseudomonas aeruginosa with a mosaic genome: potential use in phagotherapy

The genome of halo-forming temperate Pseudomonas aeruginosa phage phi297 and lytic activity of its virulent mutant were studied. A mosaic structure was revealed for phi297 genome by its complete sequencing. The phi97 genome was partly homologous to the genomes of phages D3 and F116. High lytic activity was assumed for temperate P. aeruginosa bacteriophage phi297 on the basis of morphological features of negative colonies. Virulent mutant phi297vir, which was capable oflysing bacteria, while the wild-type phage induced lysogeny, was isolated. Lytic activity was compared for phi297 and the phages from commercial mixtures of two manufacturers (facilities of Nizhnii Novgorod and Perm'). Phage phi297 caused lysis of the mutant PAO1 bacteria that were resistant to the phages from commercial preparations, but the lystic activity spectrum of phi297 was narrower that the spectra of the commercial phages. The use of nonreverting virulent mutants of certain temperate bacteriophages was proposed for the treatment of P. aeruginosa infections.     

Phage Folac: an Folac plasmid-dependent bacteriophage

By enriching sewage with Escherichia coli or Salmonella typhimurium strains harbouring the plasmid EDP208, a constitutive pilus-producing derivative of plasmid F olac, a phage was isolated which plated on these two organisms but not on isogenic strains without the plasmid. The phage was named F olac; it has a hexagonal outline with a diameter of 28 nm, contained RNA, was resistant to chloroform, and probably adsorbed preferentially to the sides of EDP208 pili very near the tip. Phage multiplication could be demonstrated on E. coli or S. typhimurium strains carrying the plasmid F olac, but an increase in titre did not occur on E. coli strains carrying plasmids of the F complex. Results of phage multiplication experiments on strains carrying the depressed pilus-producing plasmids R71 or TP224-Tc, which determine pili serologically related to those of EDP208, were inconclusive. Phage F olac was found to be serologically related to phage UA-6, another isolate specific for EDP208.     

Multiplication of Pseudomonas syringae pv. phaseolicola "in planta"

Multiplication of Pseudomonas phaseolicola was determined in 17 different bean cultivars ( Phaseolus vulgaris ) and 9 other plant species, and the effect of different inoculation methods and conditions was also studied.
In susceptible leaves, a generation time of 2.1 h was determined in the early phase (2 days after inoculation). Different multiplication rates in susceptible and resistant leaves were clearly observed 4 days after inoculation. At this time the first small water-soaked spots were visible in the susceptible cultivars. Bacteria multiplied up to the 7th day after inoculation with a maximum of 10⁹ cells per cm² leaf (equal to ca. 4 × 10¹⁰ bacterial cells/cm³). At the same time, the water-soaked spots had reached their maximum size in most cases. Thus, bacterial multiplication and development of water-soaked spots paralleled each other.
In resistant leaves, no water-soaked spots appeared, and the final bacterial concentration was 1/1000–1/100 of that in susceptible leaves. Gomparison of races 1 and 2 in several bean cultivars indicated the non-existence of a gene-for-gene relationship with this disease. Old leaves were less susceptible to infection. Some bacterial multiplication was also observed in non-host plants. There was a general correlation between bacterial multiplication in the non-host plants and their botanical relation to Phaseolus vulgaris .     

Effect of the “Ribonucleic Acid Control” Locus in Escherichia coli on T4 Bacteriophage-Specific Ribonucleic Acid Synthesis

Amino acid control of ribonucleic acid (RNA) synthesis in bacteria is known to be governed genetically by the rel locus. We investigated whether the rel gene of the host would also exert its effect on the regulation of phage-specific RNA synthesis in T4 phage-infected Escherichia coli cells. Since T-even phage infection completely shuts off host macromolecular synthesis, phage RNA synthesis could be followed specifically by the cumulative incorporation of radioactivity from labeled precursors into RNA of infected cells. Labeled uracil was shown to accumulate in phage-specific RNA for 30 to 35 min after infection, a phenomenon which probably reflects an expansion of the labile phage-RNA pool. Amino acid starvation was effected by the use of auxotrophic bacterial strains or thienylalanine. The latter substance is an amino acid analogue which induces a chemical auxotrophy by inhibiting the biosynthesis of phenylalanine, tyrosine, and tryptophan. Phage RNA synthesis was strictly dependent on the presence of amino acids, whereas phage deoxyribonucleic acid synthesis was not. By the use of several pairs of bacterial strains which were isogenic except for the rel gene, it was demonstrated that amino acid dependence was related to the allelic state of this gene. If the rel gene was mutated, amino acid starvation did not restrict phage RNA synthesis.     

Influence of environmental variables in the efficiency of phage therapy in aquaculture

Aquaculture facilities worldwide continue to experience significant economic losses because of disease caused by pathogenic bacteria, including multidrug-resistant strains. This scenario drives the search for alternative methods to inactivate pathogenic bacteria. Phage therapy is currently considered as a viable alternative to antibiotics for inactivation of bacterial pathogens in aquaculture systems. While phage therapy appears to represent a useful and flexible tool for microbiological decontamination of aquaculture effluents, the effect of physical and chemical properties of culture waters on the efficiency of this technology has never been reported. The present study aimed to evaluate the effect of physical and chemical properties of aquaculture waters (e.g. pH, temperature, salinity and organic matter content) on the efficiency of phage therapy under controlled experimental conditions in order to provide a basis for the selection of the most suitable protocol for subsequent experiments. A bioluminescent genetically transformed Escherichia coli was selected as a model microorganism to monitor real-time phage therapy kinetics through the measurement of bioluminescence, thus avoiding the laborious and time-consuming conventional method of counting colony-forming units (CFU). For all experiments, a bacterial concentration of ≈ 10⁵ CFU ml⁻¹ and a phage concentration of ≈ 10^6–8 plaque forming unit ml⁻¹ were used. Phage survival was not significantly affected by the natural variability of pH (6.5–7.4), temperature (10–25°C), salinity (0–30 g NaCl l⁻¹) and organic matter concentration of aquaculture waters in a temperate climate. Nonetheless, the efficiency of phage therapy was mostly affected by the variation of salinity and organic matter content. As the effectiveness of phage therapy increases with water salt content, this approach appears to be a suitable choice for marine aquaculture systems. The success of phage therapy may also be enhanced in non-marine systems through the addition of salt, whenever this option is feasible and does not affect the survival of aquatic species being cultured.     

Transposon-induced mutations in two loci of Listeria monocytogenes serotype 1/2a result in phage resistance and lack of N-acetylglucosamine in the teichoic acid of the cell wall.

Teichoic acid-associated N-acetylglucosamine and rhamnose have been shown to serve as phage receptors in Listeria monocytogenes serotype 1/2a. We generated and characterized two single-copy Tn916DeltaE mutants which were resistant to phage A118 and several other serotype 1/2a-specific phages. In one mutant the insertion was immediately upstream of the recently identified ptsHI locus, which encodes two proteins of the phosphoenolpyruvate-dependent carbohydrate uptake system, whereas in the other the insertion was immediately upstream of an operon whose most distal gene was clpC, involved in stress responses and virulence. Transduction experiments confirmed the association of the phage-resistant phenotype of these mutants with the transposon insertion. Phage A118 resistance of the mutants could be attributed to inability of the phage to adsorb onto the mutant cells, and biochemical analysis of cell wall composition showed that the teichoic acids of both mutants were deficient in N-acetylglucosamine. Rhamnose and other teichoic acid and cell wall components were not affected.     

Pilus-dependent, double-stranded DNA bacteriophage for Caulobacter.

Caulobacter phage phi 6, previously reported to adsorb specifically to bacterial flagella, was shown here to attach to pili more frequently than to flagella. Phage phi 6 was shown to contain double-stranded DNA by circular dichroism spectroscopy and thermal denaturation accompanied by a hyperchromic shift at 260 nm. Morphologically, phage phi 6 fits group B2 (H.-W. Ackermann, in A. I. Laskin and H. A. Lechevalier, ed., Handbook of Microbiology, vol. 1, p. 638-643, 1973) with a long, noncontractile tail and an elongate head. Pilus-less mutants of the host Caulobacter vibrioides CV6 are phage phi 6 resistant, whereas flagellum-less mutants, which produce pili, are phage susceptible. Treatments of susceptible cells which remove or immobilize pili and flagella, e.g., blending or cyanide, inhibited phage phi 6 infection. Our evidence suggests that phage of phi 6 initiates infection in a manner similar to the pilus-specific phages for Pseudomonas described previously (D. E. Bradley, Virology 51:489-492, 1973; D. E. Bradley and T. L. Pitt, J. Gen. Virol. 24:1-15, 1974).     

Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.

To develop a model system for molecular genetic analysis of plant-pathogen interactions, we studied the interaction between Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae pv tomato (Pst). Pst strains were found to be virulent or avirulent on specific Arabidopsis ecotypes, and single ecotypes were resistant to some Pst strains and susceptible to others. In many plant-pathogen interactions, disease resistance is controlled by the simultaneous presence of single plant resistance genes and single pathogen avirulence genes. Therefore, we tested whether avirulence genes in Pst controlled induction of resistance in Arabidopsis. Cosmids that determine avirulence were isolated from Pst genomic libraries, and the Pst avirulence locus avrRpt2 was defined. This allowed us to construct pathogens that differed only by the presence or absence of a single putative avirulence gene. We found that Arabidopsis ecotype Col-0 was susceptible to Pst strain DC3000 but resistant to the same strain carrying avrRpt2, suggesting that a single locus in Col-0 determines resistance. As a first step toward genetically mapping the postulated resistance locus, an ecotype susceptible to infection by DC3000 carrying avrRpt2 was identified. The avrRpt2 locus from Pst was also moved into virulent strains of the soybean pathogen P. syringae pv glycinea to test whether this locus could determine avirulence on soybean. The resulting strains induced a resistant response in a cultivar-specific manner, suggesting that similar resistance mechanisms may function in Arabidopsis and soybean.     

Phage-Antibiotic Synergy (PAS): beta-lactam and quinolone antibiotics stimulate virulent phage growth

Although the multiplication of bacteriophages (phages) has a substantial impact on the biosphere, comparatively little is known about how the external environment affects phage production. Here we report that sub-lethal concentrations of certain antibiotics can substantially stimulate the host bacterial cell's production of some virulent phage. For example, a low dosage of cefotaxime, a cephalosporin, increased an uropathogenic Escherichia coli strain's production of the phage PhiMFP by more than 7-fold. We name this phenomenon Phage-Antibiotic Synergy (PAS). A related effect was observed in diverse host-phage systems, including the T4-like phages, with beta-lactam and quinolone antibiotics, as well as mitomycin C. A common characteristic of these antibiotics is that they inhibit bacterial cell division and trigger the SOS system. We therefore examined the PAS effect within the context of the bacterial SOS and filamentation responses. We found that the PAS effect appears SOS-independent and is primarily a consequence of cellular filamentation; it is mimicked by cells that constitutively filament. The fact that completely unrelated phages manifest this phenomenon suggests that it confers an important and general advantage to the phages.     

Isolation of bacteriophage infectious for Vibrio vulnificus

Nine phage isolates infectious for Vibrio vulnificus and falling into four morphological groups were isolated from estuarine waters collected in Louisiana. Of the 60 V. vulnificus strains tested, 87% were susceptible to one or more of the isolates. With the exception of V. fluvialis, Vibrio species other than vulnificus were resistant to infection. A spectrum of enteric bacterial strains were similarly resistant. Susceptibility differences were seen between opaque (virulent) V. vulnificus strains and those with translucent (nonvirulent) colony types, with the former being more susceptible. Susceptibility patterns to infection by the nine phage isolates among the V. vulnificus test strains suggest that the latter may fall into several groups. Other aspects relating to the phage isolates are presented.     

Isolation and partial characterization of a bacteriophage active on Hyphomicrobium sp. WI-926

Isolation of a Hyphomicrobium phage from raw sewage from Athens, Ohio, was achieved by a combination of differential centrifugation, filtration, enrichment in mixed Hyphomicrobium cultures, and purification on individual host strains by subculturing single plaques in soft agar overlayers. Enrichments with water from Lake Erie and Lake Beechwood (Ohio) were unsuccessful. Out of 21 Hyphomicrobium strains and 22 other Gram-negative and Gram-positive bacteria tested, only Hyphomicrobium WI-926 (isolated from a German forest pond) was susceptible. This phage had an isometric head (diameter between opposite apices, 67 nm) and a short (12 nm), noncontractile tail and belongs thus to the morphogroup C1. It contained double-stranded DNA. The single-step growth curve showed a latent period of 9 h, a rise period of 6 h, and a burst size of 35. The various differentiation stages in the host development exhibited different affinities for phage adsorption and development. While all stages allowed phage adsorption, the daughter cells were most efficient. Phage multiplication was limited to daughter cells, and the development of infected swarmer cells was arrested permanently at this stage.     

Changes in Peroxidase Activity during Potato Ring Rot Infection

The effects of the ring rot causal agent Clavibacter michiganensis subsp. sepedonicus (a virulent strain 5369) on the peroxidase activity of various tissues of potato plants grown under axenic conditions were studied. Root infection enhanced peroxidase activity in all plant tissues (roots, leaves, and stems). In the resistant cv. Lugovskoi, peroxidase activity was much higher than in the susceptible cv. Luk'yanovskii. Co-culturing of the suspension cells of these potato cultivars with the bacterial pathogen also activated peroxidase in the cells of the resistant cultivar; in the cells of the susceptible cultivar, peroxidase activation was less pronounced. Treating suspension cell with exopolysaccharides secreted by the pathogen enhanced the activity of extra- and intracellular peroxidases, and the degree of this enhancement differed in the two potato cultivars.     

Morphology and general characteristics of phages of chickpea rhizobia

Six rhizobiophages designated as RC1, RC2, RC3, RC4, RC5 and RC6, infective against six strains of chickpea Rhizobium were isolated from field soils. Seasonal incidence, morphology, host range and inactivation pattern of the phages to heat and UV-light were studied. Four investigated phages were differentiated into two morphological types; one with hexagonal head and a long flexible tail (RC1 and RC5), the other with hexagonal head and a very short tail (RC2 and RC3). Electron microscopic examination of phage RC1 infected cells revealed that phage multiplication occurred at one pole of the cell. Phage RC3 appeared to be more thermal sensitive than others and exhibited one component inactivation while relatively resistant phages (RC1 and RC2) revealed two component inactivation. The six phages could be grouped into two classes on the basis of UV sensitivity; relatively resistant (RC1, RC2 and RC5) and sensitive (RC3, RC4 and RC6).