Call number: LD2668 .T4 1966 S127 / Master of Science

by Wai Ping Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1980. / Bibliography: leaves 214-216.

The secondary structure of the 33 kDa manganese-stabilizing protein from Anacystis nidulans R2 was predicted using information from a family of homologous sequences and applying the algorithms of Williams et al. and Zvelebil et al. From the sequence conservation and the predicted secondary structure, nine functionally important domains were identified. Additional algorithms predicted potential antigenic determinants and a region that may serve to bind this polypeptide to the Photosystem II core complex. One of the functionally significant regions exhibits partial sequence similarity to the Mn-binding site of the bacterial superoxide dismutase and was targeted for site-directed mutagenesis. Two aspartic acid residues in this region, which may form carboxyl bridges to stabilize Mn ions, were subjected to saturation mutagenesis. A hybrid vector which affords temperature regulated expression of a cloned gene, was constructed to introduce the various forms of the mutated gene into the cyanobacterium, A. nidulans R2. Pools of Eschericia coli and A. nidulans cells harboring mutant polypeptides have been obtained. Some mutant cyanobacteria display altered pigmentation and differences in generation time dependent upon growth temperature suggesting a functionally significant residue has indeed been altered. Future studies will be concerned with the characterization of specific mutants in order to determine the relationship between the structure and function of this polypeptide. / Department of Biology

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We demonstrate that hydrogen production can be increased by random mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and that hydrogen production can be further increased in the chemically-mutagenized strain by targeted gene deletion and overexpression of genes related to formate metabolism. Chemical mutagenesis of Escherichia coli BW25113 hyaB hybC hycE::kan/pBS(Kan)-HycE to form strain 3/86 resulted in 109 +/- 0.5- fold more hydrogen; 3/86 lacks functional hydrogen uptake hydrogenases 1 and 2, has hydrogenproducing hydrogenase 3 inactivated from the chromosome, and has constitutively active hydrogenase 3 based on expression of the large subunit of hydrogenase 3 from a high copy number plasmid. Deleting fdoG, which encodes formate dehydrogenase O, (that diverts formate from hydrogen), from chemical mutagen 3/86 increased hydrogen production 188 +/- 0.50-fold (relative to the unmutagenized strain), and deletion of hycA, which encodes the repressor of formate hydrogen lyase (FHL), increased hydrogen production 232 +/- 0.50-fold. Deleting both fdoG and hycA increased hydrogen production 257 +/- 0.50-fold, and overexpressing fhlA along with the fdoG hycA mutations increased hydrogen 308 +/- 0.52-fold. Whole-transcriptome analysis of chemical mutagen 3/86 revealed 89 genes were induced and 31 genes were repressed. In an effort to identify chromosomal mutations in chemical mutagen 3/86, we performed comparative genome sequencing and identified two chromosomal loci with mutations in coding regions of ftnA and yebJ; however, neither gene was related to the increased hydrogen production as determined by the close vial (short) hydrogen assay. In addition, transposon mutagenesis, which is one of the most efficient strategies for creating random mutations in the genomic DNA, was performed in two different strains: E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA and E. coli MG1655 to identify beneficial mutations for hydrogen production. As a result of screening 461 E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA transformants and 1000 E. coli MG1655 transformants, three interesting mutations have been discovered in E. coli BW25113 hyaB hybC hycA fdoG::kan/pCA24N-FhlA transformants (gpsA, dipZ, glgP) and 1 beneficial mutation in E. coli MG1655 transformants (malT). When any of these genes gpsA, dipZ, or glgP is disrupted by Tn5 insertion, hydrogen production decreases 17, 3 and 8-fold, respectively. Additionally, when malT gene is disrupted by Tn5 insertion, hydrogen increases 3.4-fold.

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Graduation date: 1992

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