Bacteriophage growth on stationary phase achromabacter strains
- Authors: Robb, Susan Mary
- Date: 1980
- Subjects: Bacteriophages , Strains and stresses
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4125 , http://hdl.handle.net/10962/d1014131
- Description: Achromobacter w.t. and strain 14 both support phage α3a growth in stationary phase, but unlike the w.t. strain, exponential phase cultures of strain 14 block phage development. A standard method was developed for determining phage growth in stationary phase cultures. Lyophilised cells were used to eliminate variations due to the unstable phenotype of Achromobacter strain 14 cells. Phage α3a growth in stationary phase was characterized by a long and variable latent period of 6 to 9 h and an increased burst size of 709 p.f.u. per cell as compared with 153 p.f.u. per cell in exponential wild type cells. During the latent period the infected cells were very sensitive to changes in growth conditions and in particular, dilution. Pre-conditioning of the bacterial cells by allowing them to stand for 24 h after shaking for 3 days was an important aspect of the stationary phase phage growth system. Cells which had been allowed to stand retained the ability to be infected and to support phage growth for at least 16 days. Shaking cultures gradually lost the ability to support phage growth but the phage could persist in the host cell for 10 days until removal from shaking when the lytic cycle could proceed after allowing the cultures to stand. In comparison the latent period and burst size in Achromobacter w.t. stationary phase cells were reduced to less than 2 h and less than 200 respectively. Stationary phase cultures differed physiologically and morphologically depending on the aeration conditions. In comparison with non-aerated standing cultures, vigorously aerated cultures showed a decrease in viability, RNA synthesis, membrane transport, intracellular ATP levels, UV resistance and heat resistance but had markedly higher protein synthesis levels. Aerated cells were small non-motile rods which did not support phage growth. They developed into large motile rods under conditions of limited aeration and were able to propagate phage. It was proposed that changes in the host control mechanisms for macromolecular synthesis may be instrumental in either blocking or permitting phage development. A spontaneous mutant of Achromobacter strain 14 (14x) which liberated phage and was resistant to superinfection was isolated. The phage-host relationship was unstable and similar to the phage carrier state. The liberated phage were able to grow in exponential strain 14 cells. It was proposed that strain 14 was a defective lysogen and that an immunity phase shift model may account for the differential phage growth in exponential and stationary phase cells. Host transcriptional control appears to be implicated in control of phage development in exponential and stationary phase cells. Achromobacter Lp only supported phage in exponential phase but a rifampicin resistant mutant of this strain was able to propagate phage in stationary phase. In vitro RNA synthesis assays showed that the rifampicin resistance was caused by an alteration in the RNA polymerase. Preliminary experiments to determine intracellular phage macromolecular synthesis were carried out using exponential Achromobacter w.t. cells which had been irradiated with UV prior to infection. In irradiated cells, infection with phage resulted in stimulation of DNA synthesis but no stimulation of protein synthesis. Phage production was drastically reduced in cells which had been treated with very low UV doses. It was proposed that α3a development may rely heavily on host cell functions which are destroyed by UV. Achromobacter mutants with defective leucine transport systems were isolated. Mutants which lost the leucine uptake system completely were totally resistant to phage infection and were unable to adsorb phage α3a. This is the first report to implicate an amino-acid transport system in phage adsorption.
- Full Text:
- Authors: Robb, Susan Mary
- Date: 1980
- Subjects: Bacteriophages , Strains and stresses
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4125 , http://hdl.handle.net/10962/d1014131
- Description: Achromobacter w.t. and strain 14 both support phage α3a growth in stationary phase, but unlike the w.t. strain, exponential phase cultures of strain 14 block phage development. A standard method was developed for determining phage growth in stationary phase cultures. Lyophilised cells were used to eliminate variations due to the unstable phenotype of Achromobacter strain 14 cells. Phage α3a growth in stationary phase was characterized by a long and variable latent period of 6 to 9 h and an increased burst size of 709 p.f.u. per cell as compared with 153 p.f.u. per cell in exponential wild type cells. During the latent period the infected cells were very sensitive to changes in growth conditions and in particular, dilution. Pre-conditioning of the bacterial cells by allowing them to stand for 24 h after shaking for 3 days was an important aspect of the stationary phase phage growth system. Cells which had been allowed to stand retained the ability to be infected and to support phage growth for at least 16 days. Shaking cultures gradually lost the ability to support phage growth but the phage could persist in the host cell for 10 days until removal from shaking when the lytic cycle could proceed after allowing the cultures to stand. In comparison the latent period and burst size in Achromobacter w.t. stationary phase cells were reduced to less than 2 h and less than 200 respectively. Stationary phase cultures differed physiologically and morphologically depending on the aeration conditions. In comparison with non-aerated standing cultures, vigorously aerated cultures showed a decrease in viability, RNA synthesis, membrane transport, intracellular ATP levels, UV resistance and heat resistance but had markedly higher protein synthesis levels. Aerated cells were small non-motile rods which did not support phage growth. They developed into large motile rods under conditions of limited aeration and were able to propagate phage. It was proposed that changes in the host control mechanisms for macromolecular synthesis may be instrumental in either blocking or permitting phage development. A spontaneous mutant of Achromobacter strain 14 (14x) which liberated phage and was resistant to superinfection was isolated. The phage-host relationship was unstable and similar to the phage carrier state. The liberated phage were able to grow in exponential strain 14 cells. It was proposed that strain 14 was a defective lysogen and that an immunity phase shift model may account for the differential phage growth in exponential and stationary phase cells. Host transcriptional control appears to be implicated in control of phage development in exponential and stationary phase cells. Achromobacter Lp only supported phage in exponential phase but a rifampicin resistant mutant of this strain was able to propagate phage in stationary phase. In vitro RNA synthesis assays showed that the rifampicin resistance was caused by an alteration in the RNA polymerase. Preliminary experiments to determine intracellular phage macromolecular synthesis were carried out using exponential Achromobacter w.t. cells which had been irradiated with UV prior to infection. In irradiated cells, infection with phage resulted in stimulation of DNA synthesis but no stimulation of protein synthesis. Phage production was drastically reduced in cells which had been treated with very low UV doses. It was proposed that α3a development may rely heavily on host cell functions which are destroyed by UV. Achromobacter mutants with defective leucine transport systems were isolated. Mutants which lost the leucine uptake system completely were totally resistant to phage infection and were unable to adsorb phage α3a. This is the first report to implicate an amino-acid transport system in phage adsorption.
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Genetic studies and physiological responses to ultraviolet radiation in the Bacteroides fragilis group
- Authors: Jones, David Todman
- Date: 1980
- Subjects: Bacteroides Ultraviolet radiation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4072 , http://hdl.handle.net/10962/d1007047
- Description: The contents of this thesis have been divided into 2 parts . The first part deals with genetic studies carried out on 36 strains belonging to the Bacteroides fragilis group. A number of mutants were isolated from several of the strains. A notable feature of the methods used was the low yield of mutants obtained and the marked sensitivity of these organisms to the mutagenic agents. Variations in colonial morphology was found to be a common feature amongst these organisms. In a few strains this phenomenon was clearly visible, in the remainder it was much weaker, and often could only be seen with the aid of a microscope . Colonial variation was found to be due to the ability of a proporti on of the cells to pruduce capsules or slime layers. The variants were found to segregate at high frequency and different growth conditions were found to have little effect on the segregation frequency or capsule formation . A number of phages specific for B. fragilis and B. t hetaiotaomicron were isol ated. All these phages were virulent and attempts to induce lysogenic phages were unsuccesful . The use of these phages in attempts to obtain transduction proved unsuccessful. A phage carrier state was found to occur in the majority of the phage-host cell systems, which seemed to be due to the presence of phage-resistant encapsulated cells in the population. Bacteriocins were produced by about half the strains, these inhibited the growth of a high proportion of the 36 strains tested. The bacteriocins were released into the growth media at the end of the growth period in the 2 bacteriocins tested. A link between the mode of action of one bacteriocin and rifampicin-resistance was investigated. All the bacter iocins tested were found to be inactive against some rifampicin-resistant mutants of a susceptible strain, suggesting a common mode of action. The presence of capsules in some cells appeared to confer bacteriocin-resistance on these variants. The second part of the thesis deals with a study of the physiological responses of a single strain of B.fragilis to ultraviolet radiation. This strain was found to be more sensitive to ultraviolet radiation under aerobic conditions. The amount of pyrimidine dimers formed after irradiation under anaerobic and aerobic conditions, was not found to differ significantly, indicating that the increase in sensitivity under aerobic conditions was not due to an increase in DNA damage. The use of repair inhibitors and the survival characteristics indicate that this difference was due to decreased repair capabilities under aerobic conditions. Liquid holding recovery in B.fragiZis was found to occur under aerobic conditions . This process was brought about by excision repair and appeared to be due to a decrease in repair efficiency under aerobic conditions. Under anaerobic conditions, where full repair capabilities were present, liquid holding recovery was inhibited. Both minimal medium recovery and fluence dependent filament formation were found to occur in irradiated B.fragiZis cells. The survival kinetics of a number of irradiated B.fragiZis phages were determined and a number of phage reactivation processes were investigated. Little or no host cell reactivation appeared to occur in the strains investigated, however, some ultraviolet reactivation and multiplicity reactivation was found to occur, but only under anaerobic conditions. Photoreactivation was found to be absent in this organism, but an excision repair system was present . The excision repair system was partially characterized and was found to resemble short patch excision repair in E.coli. Evidence was found which suggested that a second mode of repair which was sensitive to oxygen, also occurred in this strain. This repair system which appeared to be responsible for error-prone repair, and the systems which were responsible for ultraviolet reactivation and multiplicity reactivation, seemed to be dependent on a recombination function' which was inhibited by oxygen. The significance of this finding for future genetic studies was discussed.
- Full Text:
- Authors: Jones, David Todman
- Date: 1980
- Subjects: Bacteroides Ultraviolet radiation
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4072 , http://hdl.handle.net/10962/d1007047
- Description: The contents of this thesis have been divided into 2 parts . The first part deals with genetic studies carried out on 36 strains belonging to the Bacteroides fragilis group. A number of mutants were isolated from several of the strains. A notable feature of the methods used was the low yield of mutants obtained and the marked sensitivity of these organisms to the mutagenic agents. Variations in colonial morphology was found to be a common feature amongst these organisms. In a few strains this phenomenon was clearly visible, in the remainder it was much weaker, and often could only be seen with the aid of a microscope . Colonial variation was found to be due to the ability of a proporti on of the cells to pruduce capsules or slime layers. The variants were found to segregate at high frequency and different growth conditions were found to have little effect on the segregation frequency or capsule formation . A number of phages specific for B. fragilis and B. t hetaiotaomicron were isol ated. All these phages were virulent and attempts to induce lysogenic phages were unsuccesful . The use of these phages in attempts to obtain transduction proved unsuccessful. A phage carrier state was found to occur in the majority of the phage-host cell systems, which seemed to be due to the presence of phage-resistant encapsulated cells in the population. Bacteriocins were produced by about half the strains, these inhibited the growth of a high proportion of the 36 strains tested. The bacteriocins were released into the growth media at the end of the growth period in the 2 bacteriocins tested. A link between the mode of action of one bacteriocin and rifampicin-resistance was investigated. All the bacter iocins tested were found to be inactive against some rifampicin-resistant mutants of a susceptible strain, suggesting a common mode of action. The presence of capsules in some cells appeared to confer bacteriocin-resistance on these variants. The second part of the thesis deals with a study of the physiological responses of a single strain of B.fragilis to ultraviolet radiation. This strain was found to be more sensitive to ultraviolet radiation under aerobic conditions. The amount of pyrimidine dimers formed after irradiation under anaerobic and aerobic conditions, was not found to differ significantly, indicating that the increase in sensitivity under aerobic conditions was not due to an increase in DNA damage. The use of repair inhibitors and the survival characteristics indicate that this difference was due to decreased repair capabilities under aerobic conditions. Liquid holding recovery in B.fragiZis was found to occur under aerobic conditions . This process was brought about by excision repair and appeared to be due to a decrease in repair efficiency under aerobic conditions. Under anaerobic conditions, where full repair capabilities were present, liquid holding recovery was inhibited. Both minimal medium recovery and fluence dependent filament formation were found to occur in irradiated B.fragiZis cells. The survival kinetics of a number of irradiated B.fragiZis phages were determined and a number of phage reactivation processes were investigated. Little or no host cell reactivation appeared to occur in the strains investigated, however, some ultraviolet reactivation and multiplicity reactivation was found to occur, but only under anaerobic conditions. Photoreactivation was found to be absent in this organism, but an excision repair system was present . The excision repair system was partially characterized and was found to resemble short patch excision repair in E.coli. Evidence was found which suggested that a second mode of repair which was sensitive to oxygen, also occurred in this strain. This repair system which appeared to be responsible for error-prone repair, and the systems which were responsible for ultraviolet reactivation and multiplicity reactivation, seemed to be dependent on a recombination function' which was inhibited by oxygen. The significance of this finding for future genetic studies was discussed.
- Full Text:
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