Title page for ETD etd-07292002-130002


Type of Document Dissertation
Author Kasap, Murat
Author's Email Address mkasap@vt.edu
URN etd-07292002-130002
Title Nitrogen Metabolism and Solvent Production in Clostridium Beijerinckii Nrrl B593
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Chen, Jiann-Shin Committee Chair
Claus, G. William Committee Member
Gregory, Eugene M. Committee Member
Newton, William E. Committee Member
White, Robert H. Committee Member
Keywords
  • nif genes
  • Clostridium beijerinckii
  • solvent production
  • nitrogen fixation
Date of Defense 2002-07-12
Availability unrestricted
Abstract
(ABSTRACT)

The onset of solvent production by the clostridia involves regulation at the transcriptional level. The signal triggering the onset has not been identified, but redox and energetic states have been suggested as possible factors. Because several solvent-producing clostridia, including Clostridium acetobutylicum and Clostridium beijerinckii, are nitrogen-fixing organisms and both nitrogen-fixation and alcohol production (n-butanol, isopropanol and ethanol) are reductant-dependent processes, the effect of nitrogen fixation on the onset and progression of solvent production in C. beijerinckii NRRL B593 and vice versa was investigated. For this purpose, a defined growth medium containing three amino acids was developed for C. beijerinckii NRRL B593, and this medium was used for growing solvent-producing and nitrogen-fixing cultures. The nitrogen-fixing cultures produced solvents with a solventogenic shift, which appeared to coincide with a decrease in nitrogen-fixing activity. Measurement of specific activities of acetoacetate decarboxylase and aldehyde dehydrogenase and Northern blot analysis of the mRNA of the solvent-producing genes in samples harvested periodically from a nitrogen-fixing culture of C. beijerinckii showed the presence of both enzyme activities and the mRNA carrying the solvent-production genes throughout incubation. A 2.5-fold increase in the specific activity of acetoacetate decarboxylase and a 4.5-fold increase in the specific activity of aldehyde dehydrogenase were observed when the activities in the latest cell-free extract was compared with the activities in the earliest cell-free extract. When C. beijerinckii was grown in the medium containing 4 mM ammonium acetate, the onset of nitrogen fixation coincided with the onset of solvent production and prevented accumulation of solvents to high levels, which suggested competition between alcohol-producing enzymes and nitrogenase for the reductant.

Recently, a 20-kb region of the genomic DNA of C. beijerinckii NRRL B593 that contained the nif genes and ORFs with other putative functions was sequenced in our laboratory. An examination of the nif clusters of C. beijerinckii, C. acetobutylicum and C. pasteurianum revealed apparent differences in the intervening ORFs which suggested differences in the regulation of nitrogen fixation in these organisms. Transcriptional analysis of genes in the nif cluster of C. beijerinckii by Northern blotting revealed four different transcripts. The absence of mRNAs of the nif-associated ORFs in RNA samples isolated from non-nitrogen-fixing cells indicated that the nif-associated ORFs are regulated in parallel to the nif genes. By studying the effect of ammonia addition on nitrogen-fixing activities of C. beijerinckii and C. pasteurianum, significant differences in the regulation of nitrogen-fixation in the two species were observed. C. beijerinckii NRRL B593, but not C. pasteurianum, showed a rapid decrease in nitrogen-fixing activity in vivo upon ammonium acetate addition. However, measurement of nitrogen-fixing activities in vitro before and after ammonium acetate addition showed the presence of active nitrogenase throughout growth in both organisms. The results suggest that the nitrogenase activity in C. beijerinckii NRRL B593 is inhibited when ammonia is available.

A second nifH-hybridizing mRNA was detected in Northern blots during studies of the expression of nifH1 in C. pasteurianum. The mRNA was identified as that from either the nifH2 or nifH6 gene after sequencing the cDNA strands, which were generated by RT (Reverse Transcriptase)-PCR. In addition, Western blot analysis of the cell-free extracts of nitrogen-fixing cells of C. pasteurianum indicated the presence of a second NifH-related polypeptide. The two NifH-related polypeptides were separated by preparative gel electrophoresis and characterized by MALDI-TOF (Matrix-assisted Laser Desorption Ionization Time-Of-Flight) mass spectrometry. The results suggested the expression of NifH2/H6 protein in nitrogen-fixing cells of C. pasteurianum. The physiological significance of the expression of the nifH2 or nifH6 gene or both is yet to be determined.

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