Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 125-134).

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.

From Summary: A survey of collagenolytic aerobic bacteria from cured hides yielded three strains of Bacillus and eight of Achromobacter which degraded collagen at 0.4 M NaCl. Achromobacter sp. 2 was chosen for genetic studies due to its high collagenolytic activity and the lack of genetic information on Achromobacter. Four temperate bacteriophages specific for Achromobacter sp. 2 were isolated and their relationships studied. The phages caused lysogenic conversion resulting in the inability of lysogens to adsorb phage. Achromobacter sp. 2 was shown to be a cryptic lysogen as it was not immune to superinfection but had a very low rate of spontaneous induction which could be increased with mutagens. It is proposed that the cryptic lysogeny of this strain is maintained by a defective excision mechanism and the mode of prophage integration in the host chromosome. DNA extracted from phage α3a was used to transfect spheroplasts. The optimal conditions for the development of competence for transfection were determined. The presence of nuclease-attack on phage DNA under conditions of prolonged incubation of DNA and spheroplasts was proposed. A method for extracting Achromobacter DNA was devised which yielded purified, undegraded DNA, but it was not possible to transform Achromobacter sp. 2 with this DNA. The a phages were used to transduce a number of genetic markers into Achromobacter auxotrophs. The transduct ants had the ability to release the cryptic α3 prophage at a high rate while maintaining their sensitivity to homologous phage infection. It is proposed that this is due to complementation between the cryptic prophage and the residual phage functions in the transducing particles. The transductants segregated auxotrophs with a probability of 10⁻³ per cell per generation. It appears that an unusual system of generalised transduction is operating whereby the transducing particles contain both phage and bacterial DNA which is incorporated into the recipient genome by a single recombination event yielding unstable transductants. In a study on induction of Escherichia coli (λ), carcinogenic nitrosamines were shown to be inducers of phage development. This provides a screening system for potentially harmful nitrosamines.

No description available.

Seventy eight serologically distinct strains of Klebsiella bacteria are known to exist. The capsular polysaccharide surrounding the bacterial cell of these pathogenic Enterobacteria is of immunological significance. Structures of the capsular polysaccharides of nearly sixty seven strains of Klebsiella have been established, and each one found to be unique. The structures of the K antigens from Klebsiella, serotypes K67 and K80 are presented as a contribution to the continuing program of elucidation of the chemical structures of these antigens in an attempt to explain their immunological responses. Chemical methods of structural elucidation were employed and the following two structures were obtained. [formula omitted] The polysaccharide from K67 was unique among the Klebsiella K antigens in having a four-plus-two-plus-one repeating-unit (indicating a branch on a side chain), while K80 was unique as it was the first instance that a pyruvic acetal was found on a terminal rhamnose unit. The importance of bacteriophage-borne enzymes in the generation of single repeating-units containing labile substituents is demonstrated. Klebsiella K44 polysaccharide was degraded using a crude solution of Φ44 bacteriophage. The oligosaccharides obtained were crucial in the determination of the position of the 0-acetate group. In the case of the polysaccharide from Klebsiella K26, the degradation performed using a crude solution of Φ26 bacteriophage resulted in the isolation of a single repeating-unit containing a pyruvic acetal together with an oligosaccharide corresponding to a single repeating-unit devoid of its terminal pyruvate containing sugar. The structures of these compounds which are as follows, were useful in further confirmation of the structures of the original polysaccharides. [formula omitted] Escherichia coli. another pathogenic Enterobacteria possessing immunologically significant K antigens, has been found to contain capsular polysaccharides bearing a strong resemblance to those of Klebsiella. Recently it was discovered that some strains of E. coli contained K antigens comprising amino sugars. A preliminary study on the chemical behaviour of amino sugars, and chemical methods of structure elucidation of such polysaccharides have been included in an appendix as this has been a new area of research in this laboratory. An appendix containing compilations of the cross-reactions, known structures, and chemotypes of the Klebsiella K antigens has also been included. / Science, Faculty of / Chemistry, Department of / Graduate

Vibrio sp. 2 stationary phase cells are novel and interesting in that they are able to support phage growth in standing cultures, but not in shaken (aerated) cultures. Many physiological and morphological characteristics change when Vibrio sp. 2 stationary phase cells are removed from aeration: the relatively high levels of protein synthesis (Robb et al., 1977; 1978) decrease, with a concomitant increase in the levels of RNA synthesis; protein degradation rises from 1 %h⁻¹ to 2,9 %h⁻¹, and whilst the average cell length decreases, the range of cell lengths markedly increases. The magic spot nucleotides, ppGpp and pppGpp, which are present in stressed exponential phase Vibrio sp. 2 cells, are not detectable in stationary phase Vibrio cells. The specific proteolytic activity of shaking stationary phase cell-free extracts against the foreign protein [¹⁴C-me]globin was slightly higher than that of extracts from standing or exponential phase cells, while the specific proteolytic activity against [¹²⁵I]-insulin was slightly lower. On the basis of inhibitor studies and subcellular distribution, the proteolytic activities of the three types of extract differed. The addition of exogenous ATP to cell-free extracts either stimulated (Car & Woods, 1984) or depressed proteolytic activity depending on the procedure used to prepare the extracts. The proteolytic activity of fractions containing substantial amounts of membrane material, from all three types of extract, were markedly depressed by ATP. On preincubation of cell-free extracts from exponentially growing cells prior to assay of proteolytic activity, the activity was markedly stimulated (two- to four-fold). The stimulation,. however, varied, greatly between independently produced extracts. ATP had a much smaller stimulatory effect on preparations free of cell wall material from both types of stationary phase cells (the stimulation was less than two-fold), and the stimulation was not affected by preincubation of the extracts. Extracts prepared from starving cells, previously in exponential growth, were affected by the addition of ATP in a similar manner to that observed with stationary phase extracts (Car & Woods, 1984). Exponential and both types of stationary phase Vibrio sp. 2 cells have ATP-stimulated and ATP-depressed activities separable by ion-exchange chromatography, in addition to several other proteolytic activities. All types of Vibrio sp. 2 cells have a similar complement of proteolytic activities.

The present study was undertaken to define the standard conditions for growth of bacteriophage 7V on its host Pseudomonas aeruginosa strain PS-7 and to determine the factors which affect the quantity and quality of plaques in the plaque count assay. Observations from the single-step growth experiment and single-burst experiment are also included. Plaque count assays were performed under various environmental conditions. Conditions were selected as “standard” if they yielded: 1) relative maximum number of infective centers per ml of stock 7V phage, 2) clear, haloed plaques at least 2.0 mm in diameter, and 3) reproducible assays limited only by the sampling error. These conditions are: 1. fresh NBYE or NBYE agar for the growth medium 2. NYBE or buffered salts solution for diluents 3. Physiologically young cells in the log phase between 1-5 X 10⁸ bacteria/ml 4. Stock and diluted stock suspensions stored at refrigerator temperatures. Adsorption rate experiments which measured both unadsorbed phage and infective centers were performed in minimal media, minimal media supplemented with organic and and ionic cofactors, and complete media. Although overnight lysates of PS-7 in minimal media produced a high titer of phage, the rate of adsorption of phage 7V in PS-7 was extremely slow in minimal media. Addition of tryptophan caused a decrease in free phage without a corresponding increase in infective centers. Casamino acids plus tryptophan caused an increase in the velocity of the adsorption reaction which was less than the rate of adsorption of phage 7V to its host PS-7 in NBYE. In NBYE 90 percent of the initial phage were adsorbed in 5 minutes, but the recovery of phage as free phase of infective centers was not equal to the input of phage. These results suggest that this system requires a cofactor, organic, ionic, or both, in order that adsorption of phage 7V to its host PS-7 proceed at a maximum rate. And it further suggests that the incidence of abortive infection in this system is high. In this particular system under standard conditions it appears that the size of the plaques is controlled mainly by environmental factors, while the relative number of plaques is a characteristic of the system.

Six initial phage isolates with high titer lysates were obtained using Streptomyces toxytricini B-5426 as the host bacterium. These isolates were named Goby, Toma, Yosif, Yara, Deema, and Hsoos. However, upon completion of the sequencing, it was found that the Yara and Hsoos isolates were identical, as were Goby and Deema. As a result, final analysis was completed on only the four unique isolates. All of the phages mentioned above were isolated from soil samples from different locations. Also, they had different sizes of plaques, ranging from 0.3 – 0.9mm. Yosif had the largest plaque size. Yara's head diameter was 79nm with tail diameter of 94nm.

Cheng Man. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 236-256) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Call number: LD2668 .T4 1961 M58