Cold-Sensitive Pseudomonas RNA Polymerase I. Characterization of the Host Dependent Cold-Sensitive Restriction of Phage CB3

Analysis of bacteriophage CB3 infection of Pseudomonas aeruginosa strain PAT2 establishes that phage induced changes in net macromolecular synthesis are absent at nonpermissive phage growth temperatures (32 C). Alterations which are evident in the PAT2 strain at 37 C or in the fully permissive strain, PAO1C, at either warm or cold temperatures do not occur in PAT2 at low temperatures. CB3 DNA synthesis and the degradation of host DNA to approximately 78S components occur at 37 C, but are absent in PAT2 at 20 C. Nevertheless, attachment of phage DNA to host cytoplasmic material occurs under permissive and nonpermissive conditions. This binding of phage DNA at 20 C is identical in nature to phage DNA bound at 37 C. Thus, the conditional cold-sensitive PAT2 host function in the growth of CB3 is expressed subsequent to membrane binding of the infecting genomes but prior to the onset of the initiation of CB3 DNA synthesis, the inhibition of host DNA synthesis, and the transient depression in RNA synthesis which occurs in permissive cells.     

The Influence of <Emphasis Type="Italic">P. fluorescens</Emphasis> Cell Morphology on the Lytic Performance and Production of Phage ϕIBB-PF7A

This study aims at assessing the influence of Pseudomonas fluorescence cell morphology on the effectiveness and production of the lytic bacteriophage ϕIBB-PF7A. P. fluorescens were cultured as rods or as elongated cells by varying the temperature and rotary agitation conditions. Cells presented rod shape when grown at temperatures up to 25°C and also at 30°C under static conditions, and elongated morphology only at 30°C when cultures were grown under agitation. Elongated cells were 0.4 up to 27.9 μm longer than rod cells. Rod-shaped hosts were best infected by phages at 25°C which resulted in an 82% cell density reduction. Phage infection of elongated cells was successful, and the cell density reductions achieved was statistically similar (P > 0.05) to those obtained at the optimum growth temperature of P. fluorescens. Phage burst size varied with the cell growth conditions and was approximately 58 and 153 PFU per infected rod and elongated cells, grown at 160 rpm, at 25°C (the optimal temperature) and 30°C, respectively. Phage adsorption was faster to elongated cells, most likely due to the longer length of the host. The surface composition of rod and elongated cells is similar in terms of outer membrane proteins and lipopolysaccharide profiles. The results of this study suggest that the change of rod cells to an elongated morphology does not prevent cells from being attacked by phages and also does not impair the phage infection.     

Factors influencing the replication of somatic coliphages in the water environment

The potential replication of somatic coliphages in the environment has been considered a drawback for their use as viral indicators, although the extent to which this affects their numbers in environmental samples has not been assessed. In this study, the replication of somatic coliphages in various conditions was assayed using suspensions containing naturally occurring somatic coliphages and Escherichia coli WG5, which is a host strain recommended for detecting somatic coliphages. The effects on phage replication of exposing strain WG5 and phages to a range of physiological conditions and the effects of the presence of suspended particles or other bacteria were also assayed. Phage replication was further tested using a strain of Klebsiella terrigena and naturally occurring E. coli cells as hosts. Our results indicate that threshold densities of both host bacterium and phages should occur simultaneously to ensure appreciable phage replication. Host cells originating from a culture in the exponential growth phase and incubation at 37 degrees C were the best conditions for phage replication in E. coli WG5. In these conditions the threshold densities required to ensure phage replication were about 10(4) host cells/ml and 10(3) phages/ml, or 10(3) host cells/ml and 10(4) phages/ml, or intermediate values of both. The threshold densities needed for phage replication were higher when the cells proceeded from a culture in the stationary growth phase or when suspended particles or other bacteria were present. Furthermore E. coli WG5 was more efficient in supporting phage replication than either K. terrigenae or E. coli cells naturally occurring in sewage. Our results indicate that the phage and bacterium densities and the bacterial physiological conditions needed for phage replication are rarely expected to be found in the natural water environments.     

Evidence for the existence of a restriction-modification system common to several species of the family Vibrionaceae

A broad-host-range vibriophage KVP40 originally isolated on Vibrio parahaemolyticus 1010 was restricted and modified by strains of at least five Vibrio and one Photobacterium species. 1010 was a non-restricting host. An anti-restriction mutant KVP40 aar1 was isolated after propagating the phage on a restricting host, V. anguillarum VIB36. KVP40 aar1 grown on either 1010 or VIB36, as well as the parental phage grown on VIB36, showed much higher efficiencies of plating on all the restricting hosts as compared with the parental phage grown on 1010, indicating that these restricting hosts probably share a common restriction-modification system active in vivo on KVP40.     

Role of Microcolony Formation in the Protistan Grazing Defense of the Aquatic Bacterium Pseudomonas sp. MWH1

A BSTRACTThe defense strategy of the aquatic bacterium Pseudomonas sp. MWH1 against flagellate grazing was investigated in chemostat and batch experiments. The influence of predation on the Pseudomonas population was studied in the absence and presence of a potential competitor ( Vibrio sp. CB5), as well as under starvation conditions and in a situation of unlimited growth. In the competition experiment the two bacterial strains were distinguished by immunofluorescence microscopy. When the Pseudomonas strain was cultured in the absence of the predator Ochromonas sp. DS, only mobile single cells were detectable. Grazing by this bacterivorous flagellate resulted in all experiments in the occurrence of a Pseudomonas subpopulation, which grew as floclike, suspended microcolonies. These microcolonies consisted of up to approximately 1,000 cells and were, because of their large size, protected against flagellate grazing. The microcolony subpopulation dominated the total Pseudomonas population in situations of high grazing pressure at a wide range of bacterial growth conditions. Thus, the formation of the microcolonies is interpreted as a successful grazing-defense strategy, which is effective under several growth conditions, allowing for the survival of the strain even when substrate depletion is combined with strong grazing pressure. Batch culture experiments demonstrated that the change in morphology of Pseudomonas sp. MWH1 is not controlled by growth rate, although no formation of microcolonies was observed after the addition of 0.2-&mgr;m-filtered flagellate cultures to Pseudomonas cultures, indicating that a chemical trigger released by the flagellate is not involved in the control of this defense mechanism.     

The effect of the IncP-2-group plasmid on the growth of Pseudomonas aeruginosa bacteriophages

The influence of plasmids of the IncP-2 group on development of bacteriophages of Pseudomonas aeruginosa was studied. Six different types of phage growth inhibition conferred by natural plasmids of the IncP-2 group were found. All these plasmids were shown to have no effect on adsorption and injection of phage DNA into cells, only blocking intracellular phage development. The differences between phage inhibition mechanisms were shown by comparison of efficiency of colony formation by cells containing different plasmids, in the presence of different phages. The presence of the RpL11 plasmid reduces the frequency of lysogenization with G101 phage but not with B3 phage. The mutants of pMG53 plasmid having modified phage inhibition spectrum were obtained. It was inferred that inhibition of different phages is under control of different loci of this plasmid. The mutants of phage B3 overcoming inhibition by plasmids were obtained. It was supposed that the plasmids act at least at three different sites of the phage B3 genome.     

Bacteriophage Phi S1 infection of Pseudomonas fluorescens planktonic cells versus biofilms

This communication focuses on the efficacy of a specific lytic phage, phage Phi S1, as a control agent of Pseudomonas fluorescens biofilms. The effect of phage infection temperature and the host growth temperature were evaluated. The results obtained showed that the phage infection process was temperature dependent and that the optimum temperature of infection of planktonic cells and biofilms was 26 degrees C. At this temperature, bacteriophage Phi S1, at a multiplicity of infection (MOI) of 0.5 infected both planktonic cells and biofilms causing a biomass reduction of about 85% in both cases.     

Temperature-sensitive mutants of bacteriophage SH-133 specific for the hydrogen bacterium Pseudomonas facilis: isolation, complementation, and partial characterization.

Seventeen temperature-sensitive mutants of bacteriophage SH-133 have been isolated following mutagenesis with UV-light, nitrosoguanidine, and ethyl methanesulfonate. The mutants were classified into 15 complementation groups according to their ability to complement each other at 32 degrees C, the nonpermissive temperature. Each mutant was studied with regard to the relationship between its ability to multiply in heterotrophically (H-) and autotrophically (A-) grown Pseudomonas facilis cells. At 27 degrees C, the permissive temperature, the plaque-forming ability of the 17 mutants and wild-type phage was reduced 10-fold in A-grown cells. At 32 degrees C, mutants belonging to 10 groups exhibited identical levels of multiplicity-dependent leak under both modes of growth. However, the infection of A-grown cells by mutants belonging to the remaining five groups resulted in as much as 500-fold inhibition of multiplicity-dependent leak when contrasted with the infection of cells grown heterotrophically. These observations indicate that the expression of five SH-133 phage cistrons is defective when multiplication proceeds under autotrophic metabolism. Seven mutants were found to differ from the wild-type phage with regard to thermal stability at 56 degrees C which suggests that they possess altered structural proteins. Four of the seven thermosensitive mutants exhibited reduced levels of multiplicity-dependent leak in A-grown cells. The data suggest that the reduction in plaque-forming ability of SH-133 in A-grown cells is caused by a defect in the expression of specific phage structural components.     

Transfer of plasmids fromEscherichia coli andPseudomonas aeruginosa to methylotrophic bacteria and their detection in the new hosts

Several plasmids of incompatibility group P were transferred fromEscherichia coli andPseudomonas aeruginosa strains toMethylophilus methylotrophus and two other methylotrophs to test their recipient ability. The presence of plasmids in transconjugants was confirmed by electrophoretic analysis. Optimal conditions for detection of plasmid DNA in the strains tested based on alkaline lysis of cells at elevated temperature were established. Special behaviour of plasmids carrying the Mu phage in methylotrophic hosts is described.     

Influence of infected cell growth state on bacteriophage reactivation levels

Reactivation of UV-C-inactivated Pseudomonas aeruginosa bacteriophages D3C3, F116, G101, and UNL-1 was quantified in host cells infected during the exponential phase, during the stationary phase, and after starvation (1 day, 1 and 5 weeks) under conditions designed to detect dark repair and photoreactivation. Our experiments revealed that while the photoreactivation capacity of stationary-phase or starved cells remained about the same as that of exponential-phase cells, in some cases their capacity to support dark repair of UV-inactivated bacteriophages increased over 10-fold. This enhanced reactivation capacity was correlated with the ca. 30-fold-greater UV-C resistance of P. aeruginosa host cells that were in the stationary phase or exposed to starvation conditions prior to irradiation. The dark repair capacity of P. aeruginosa cells that were infected while they were starved for prolonged periods depended on the bacteriophage examined. For bacteriophage D3C3 this dark repair capacity declined with prolonged starvation, while for bacteriophage G101 the dark repair capacity continued to increase when cells were starved for 24 h or 1 week prior to infection. For G101, the reactivation potentials were 16-, 18-, 10-, and 3-fold at starvation intervals of 1 day, 1 week, 5 weeks, and 1. 5 years, respectively. Exclusive use of exponential-phase cells to quantify bacteriophage reactivation should detect only a fraction of the true phage reactivation potential.     

Bacteriophage Φ S1 Infection of Pseudomonas fluorescens Planktonic Cells versus Biofilms

This communication focuses on the efficacy of a specific lytic phage, phage F S1, as a control agent of Pseudomonas fluorescens biofilms. The effect of phage infection temperature and the host growth temperature were evaluated. The results obtained showed that the phage infection process was temperature dependent and that the optimum temperature of infection of planktonic cells and biofilms was 26°C. At this temperature, bacteriophage F S1, at a multiplicity of infection (MOI) of 0.5 infected both planktonic cells and biofilms causing a biomass reduction of about 85% in both cases.     

Regulation of polar surface structures in Caulobacter crescentus: Pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors

Summary A large number of Caulobacter mutants resistant to DNA or RNA phages were isolated. These phage-resistant mutants exhibited phenotypic variations with respect to cell motility and sensitivity to other phages.The majority of the mutants was resistant to both DNA and RNA phages tested. In addition, these mutants were either motile or non-motile. The analysis of spontaneous revertants from these mutants indicated that a single mutation is involved in these phenotypic variations. Other mutants were resistant to RNA phages and only to a certain DNA phage tested, and were also motile or non-motile.Several temperature-sensitive phage-resistant mutants were also isolated. One of them, CB13 ple-801, exhibited the wild type phenotype when grown at 25°C. However, at a higher temperature (35°C), the mutant cells became non-motile and resistant to both DNA and RNA phages. These phenotypes seem to be attributed to the concommitant loss of flagella, pili and phage receptors. In other respects (cell growth and morphology, and asymmetric stalk formation), CB13 ple-801 was normal at 35°C. The spontaneous revertants from CB13 ple-801 simultaneously regained the wild type phenotypes in all respects.It is suggested that a single mutation pleiotropically affects the formation of flagella, pili and phage receptors.     

The cost of phage resistance in a plant pathogenic bacterium is context‐dependent

Parasites are ubiquitous features of living systems and many parasites severely reduce the fecundity or longevity of their hosts. This parasite‐imposed selection on host populations should strongly favor the evolution of host resistance, but hosts typically face a trade‐off between investment in reproductive fitness and investment in defense against parasites. The magnitude of such a trade‐off is likely to be context‐dependent, and accordingly costs that are key in shaping evolution in nature may not be easily observable in an artificial environment. We set out to assess the costs of phage resistance for a plant pathogenic bacterium in its natural plant host versus in a nutrient‐rich, artificial medium. We demonstrate that mutants of Pseudomonas syringae that have evolved resistance via a single mutational step pay a substantial cost for this resistance when grown on their tomato plant hosts, but do not realize any measurable growth rate costs in nutrient‐rich media. This work demonstrates that resistance to phage can significantly alter bacterial growth within plant hosts, and therefore that phage‐mediated selection in nature is likely to be an important component of bacterial pathogenicity.     

Effective, plasmid RP4-dependent replication-transposition of DNA of the transposable phage D3112 of Pseudomonas aeruginosa in a heterologous Escherichia coli system

The processes of replication and transposition of Pseudomonas aeruginosa transposable phage D3112 in cells of Escherichia coli (D3112) and E. coli (RP4::D3112) were studied. D3112 genome is a "silent cassette" ("conex-phage"--conditionally expressible) in E. coli cells incubated at 42 degrees C. Two compulsory conditions for D3112 genome expression are incubation at 30 degrees C and the presence in cells of RP4 plasmid. Processes of replication and transposition in E. coli are coupled. RP4 plasmid stimulates D3112 DNA synthesis in E. coli at least by two order of magnitude. In correspondence with this observation is the fact that when Mg2+ is present in high concentration (0.1 M) in a cultural medium, the production of mature phage is enhanced by two order of magnitude in E. coli (RP4::D3112) or in E. coli (D3112, RP4) cells, and is approx. 10(-1)-10(-2) phage per cell. No influence of Mg on phage production is observed in E. coli (D3112) cells.     

TOL plasmid pWW0 in constructed halobenzoate-degrading Pseudomonas strains: prevention of meta pathway.

The hybrid pathway for chlorobenzoate metabolism was studied in WR211 and WR216, which were derived from Pseudomonas sp. B13 by acquisition of TOL plasmid pWW0 from Pseudomonas putida mt-2. Chlorobenzoates are utilized readily by these strains when meta cleavage of chlorocatechols is suppressed. When WR211 utilizes 3-chlorobenzoate (3CB), the expression of catechol 2,3-dioxygenase (C23O) and the catabolic activities for chloroaromatics via the ortho pathway coexist as a consequence of inactivation of the meta cleavage activity by 3-chlorocatechol. Utilization of 4-chlorobenzoate (4CB) by WR216 presupposes the suppression of C23O by a spontaneous mutation in the structural gene, so that 4-chlorocatechol is not misrouted into the meta pathway. Such C23O- mutants were also selected when WR211 was grown continuously on 3CB. Our data explain why the phenotypic characters 3CB+ and Mtol+ (m-toluate) are compatible, whereas 4CB+ and Mtol+ are incompatible.     

Growth Inhibition and Appearance of the Membranous Structure in Escherichia coli Infected with Bacteriophage fd

Thirty-six mutants of fd, a virus that infects but does not kill Escherichia coli, were isolated; 35 mutants were categorized into six complementation groups. Abortive infection with mutants in genes 1, 3, 4, 5, and 6, but not in gene 2, produced a cessation of host cell growth, generally linked to low burst size and to the formation of aberrant intracytoplasmic membranous structures. The membranous structure was studied during infection with various phage and hosts. Appearance of the membranous structure was linked specifically to incomplete phage maturation at the cell membrane, rather than solely to the inhibition of host cell growth or to infection with mutant phage, since (i) in one host, cell growth was inhibited, but no membranous structure developed; and (ii) when antibody against virus was added to cells infected with wild-type phage, phage extrusion was inhibited, cell growth stopped, and the membranous structure once again developed.     

Loss of Host-controlled Restriction of λ Bacteriophage in Escherichia coli Following Methionine Deprivation

lambda Bacteriophages produced in Escherichia coli C (designated as lambda . C) are restricted in their ability to grow in E. coli K-12. The rare successful infections that arise in the K-12 population occur in "special" cells which have lost their capacity to restrict lambda . C. These infections yield modified progeny phage (designated as lambda . K) which, unlike lambda . C, plate equally well on E. coli C and E. coli K-12. When methionine, but no other amino acid, was removed from the growth medium of a mutant strain of E. coli K-12, the number of special cells rapidly increased 500- to 3,000-fold. These new special cells retain their capacity to produce modified lambda . K progeny. This conversion of restricting cells into special cells does not require the synthesis of new protein. The special cells formed when methionine was removed from the culture did not revert into restricting cells when methionine was restored. Such cells have also lost the ability to divide for at least 4 hr after methionine supplementation. When methionine was restored, the remaining restricting cells, but not the special cells, immediately resumed growth. Removing methionine from cultures of E. coli B caused a similar increase in the number of special cells able to support the growth of lambda . C and lambda . K. However, when E. coli K-12 (P1) cultures were deprived of methionine, the number of special cells increased for lambda . C but not for lambda . K. Thus, retention of the P1-restriction system, unlike the B- and the K-12-systems, does not require the presence of methionine.     

Transduction and transformation of plasmid DNA in Streptomyces fradiae strains that express different levels of restriction.

We constructed nonrestricting strains of Streptomyces fradiae blocked in different steps in tylosin biosynthesis. Plasmid transformation frequencies were 10(3)- to 10(4)-fold higher and bacteriophage plating efficiencies were 10(4)- to 10(8)-fold higher in the nonrestricting strains than in the restricting strains. The efficiencies of transduction of plasmid pRHB101 in S. fradiae strains varied by over 1,000-fold, depending on growth conditions, and optimum transduction frequencies were obtained when cells were grown to mid-exponential phase at 39 degrees C. Under these conditions, restricting and nonrestricting strains were transduced at frequencies that differed by only two- to fivefold.