Mesophilic cellulolytic clostridia play an important role in the global carbon cycle because they are able to degrade abundantly-produced cellulosic material. Many strains may also take part in the nitrogen cycle inasmuch as they are able to satisfy their nitrogen requirement for growth by fixing dinitrogen (N$\sb2).$ The first objective of this research was to characterize nitrogenase systems of mesophilic cellulolytic clostridial species using physiological and molecular approaches. DNA probes, nifDK, vnfDGK, and anfDGK, constructed from structural genes representing three different nitrogenase systems in Azotobacter sp., were used to detect and clone potential nitrogenase genes from cellulolytic clostridia. A 7-kb genomic DNA fragment, which hybridized the nifDK probe, was cloned from Clostridium cellobioparum ATCC 18532, and a 2.1-kb subfragment was found to contain a nifH sequence. Northern analyses suggested this sequence was expressed under nitrogen-fixing conditions. Based on phylogenetic analyses involving 34 known nifH gene sequences, nifH from C. cellobioparum was most closely related to the multiple nifH genes of Clostridium pasteurianum. In addition, a 3-kb genomic DNA fragment was cloned from Clostridium hungatei B3B and found to have a high sequence identity to anfDGK from Azotobacter vinelandii. However, in studies of nitrogen fixation by C. hungatei B3B under Mo-deficient conditions, ethane was not detected during acetylene reduction assays, suggesting that the putative anfDGK genes were, if expressed in C. hungatei, part of a nitrogenase system that differed from that of A. vinelandii. The second objective of this research was to isolate plasmids indigenous to cellulolytic clostridia that may be used as vectors in strain modification. Several antibiotic resistant strains were screened for the presence of plasmids and a small plasmid (2450 bp), designated pMCF1, was isolated from strain MCF1 and ligated to the Hind III site of the vector pBluescript II KS. DNA sequence analyses identified an open reading frame encoding a peptide with homology to plasmid replication proteins. It is proposed that pMCF1 belongs to a subfamily of the single-strand DNA plasmids, which replicate via a rolling circle mechanism.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-6913 |
| Date | 01 January 1995 |
| Creators | Chen, Tsute |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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