Date of Award

1980

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Abstract

Part I. An accurate most-probable-number enumeration method was developed for counting the number of Beggiatoa trichomes from various freshwater sediments. The medium consisted of extracted hay, diluted soil extract, 0.05% sodium acetate, and 15-35 units per ml of catalase. The same enrichment medium, but without the acetate, was the best enrichment medium from which to obtain pure cultures. A total of thirty-two strains of Beggiatoa were isolated from seven different freshwater habitats and were separated into five groups based on several preliminary characteristics. Four of the groups contained cells with trichomes of approximately the same diameter (1.5-2.7 (mu)m) and may be B. leptomitiformis or an unnamed species. The fifth group appeared to be B. alba. With the exception of three strains, all of the strains deposited sulfur in the presence of hydrogen sulfide. All of the strains grew heterotrophically and deposited poly-(beta)-hydroxybutyrate and volutin when grown on acetate supplemented with low concentrations of other organic nutrients. Thin sections of sulfur bearing trichomes indicated that the sulfur granules were external to the cytoplasmic membrane and were surrounded by an additional membrane. Part II. The assimilation and metabolism of CO(,2) by Beggiatoa alba strain B18LD was investigated. Although B. alba was shown to require CO(,2) for growth, the addition of excess CO(,2) (as NaHCO(,3)) to the medium in a closed system did not stimulate growth. The K(,m) values for the CO(,2) assimilation by heterotrophically-grown and mixotrophically-grown cells were 297 (mu)M and 666 (mu)M, respectively. The high K(,m) values for CO(,2) assimilation, coupled with acetate oxidation data suggested that the required CO(,2) was endogenously produced from acetate. The first stable fixation products from CO(,2) assimilation were aspartate and glutamate. Carbon dioxide was incorporated into cell wall material, proteins, nucleic acids, and amino acids and organic acids, but not into poly-(beta)-hydroxybutyrate. Fluoroacetate and glyoxylate inhibited CO(,2) assimilation. The CO(,2) fixation enzymes isocitrate dehydrogenase (NADP; reversed) and malate dehydrogenase (NADP; decarboxylating) were found in cell-free extracts of both mixotrophically-grown and heterotrophically-grown cells. The results suggested that an incomplete reductive tricarboxylic acid cycle might be present in B. alba for the metabolism of CO(,2). Part III. The assimilation and oxidation of ('14)C-1- and ('14)C-2- acetate by several strains of Beggiatoa were investigated. Approximately 16-28% of the ('14)C-2-acetate and 28-46% of the carboxyl labeled acetate were oxidized to ('14)CO(,2). The apparent K(,m) for the combined assimilation and oxidation of ('14)C-2-acetate by Beggiatoa alba B18LD was 1.0-1.4 (mu)M under differing growth conditions. Sixty-one to 73% of the ('14)C-1- or ('14)C-2- acetate assimilated by washed trichomes was incorporated into lipid, whereas 55% of the assimilated ('14)C-1-acetate was incorporated into poly-(beta)-hydroxybutyrate (PHB). That corresponded with chemical data showing that 56% of the cell dry weight was PHB. Fluoroacetate inhibited ('14)C-1-acetate assimilation at concentrations of 100-1000 (mu)M but inhibited ('14)C-1-acetate oxidation to ('14)CO(,2) only at 1000 (mu)M. After a 5 sec incubation of B. alba with ('14)C-1-acetate, the major products were succinate (39.7%), glutamate (11.1%), and fumarate (8.9%); after 25 sec, succinate (42.3%), glutamate (15.7%), and hydroxybutyrate (11.5%) were the major intermediates observed. The results indicated that a functional glyoxylate bypass might be present, but that citrate formation via the condensing enzyme was not present.

Pages

132

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