München, Techn. University, Diss., 2000.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 89-92). Also available via the Internet.

Filaments of the desiccation-tolerant cyanobacterium Nostoc commune are embedded within, and distributed throughout, a dense glycan sheath. Analysis of the glycan of field materials and of pure cultures of N. commune DRH1 through light and electron microscopy, immunogold-labelling and staining with dyes, revealed changes in the pattern of differentiation in glycan micro-structure, as well as localized shifts in pH, upon rehydration of desiccated field material. A Ca/Si rich external (pellicular) layer of the glycan acts as a physical barrier on the surface of N. commune colonies. A purified fraction (> 12 kDa) of an aqueous extract of the glycan from desiccated field material contained glucose, N -acetylglucosamine, glucosamine, mannose and galactosamine with ratios of 3.1 : 1.4 : 1 : 0.1 : 0.06, respectively. Ethanol extracts of N. commune contained trehalose and sucrose and the levels of both became undetectable following cell rehydration. Elemental analysis of glycan extracts showed a flux in the concentrations of salts in the glycan matrix following rehydration of desiccated colonies. Intracellular cyanobacterial trehalase was identified using immunoblotting and its synthesis was detected upon rehydration of desiccated field cultures. Water-stress proteins (Wsp; molecular masses of 33, 37, and 39 kDa are the most abundant proteins in glycan), a water soluble UV-AlB-absorbing pigment, the lipid-soluble UV-protective pigment scytonernim, as well as two unidentified cyanobacterial glycoproteins (75 kDa and 110 kDa), were found within the glycan matrix. No evidence was found for either glycosylation, phosphorylation or acylation of Wsp polypeptides. NH2-terminal sequence analysis of the three proteins of Wsp were identical: Ala-Leu-Tyr-Gly-Tyr-Thr-Ile-Gly-Glu-Gln-X-Ile-Gln- Asn-Pro-Ser-Asn-Pro-Ser-Asn-Gly-Lys-Gln. An unidentified 68-kDa protein, the second most abundant protein in aqueous extracts of the glycan, was isolated and its N-terminal sequenced was determined: Ala-Phe-lle-Phe-Gly-Thr-Ile-Ser-Pro-Asn-Asn-Leu-Ser-Gly- Thr-Ser-Gly-Asn-Ser-Gly-Ile-Val-Gly-Ser-Ala. Gene bank searches with these sequences, and an internal sequence ofWsp (Glu-Ala-Arg-Val-Thr-Gly-Pro-Thr-Thr-Pro-Ile-Asp), identified homologies with various carbohydrate-modifying enzymes. Purified Wsp polypeptides associate with 1,4-β-D-xylanxylanohydrolase activity that was inhibited specifically by Wsp antiserum. In the absence of salt, Wsp polypeptides, and the water-soluble UV -A/B-absorbing pigments, form multimeric complexes through strong ionic interactions. The role of the glycan, and the protein and pigments that reside within it, in the desiccation tolerance of N. commune is discussed with respect to structure/function relationships. / Ph. D.

The molecular biology of desiccation tolerance was investigated in the cyanobacteria with emphasis on Nostoc commune. Analysis of DNA from 41 samples of desiccated Nostoc spp. of varied age and global distribution led to the amplification of 43 independent tRNALEU(UAA) group 1 intron sequences. Phylogenetic analysis of the entire data set made it possible to define the form species Nostoc commune. The synthase (spsA) and phosphatase (sppA) genes required for the synthesis of sucrose were isolated from cyanobacterium Synechocystis sp. strain PCC 6803 and overexpressed in E. coli in two different vector constructions. Transformants had a marked increased capacity for desiccation tolerance. Sucrose synthesis was confirmed through thin layer chromatography (TLC) analysis of cell extracts from transformants. Long-term stability of DNA in desiccated Nostoc samples was demonstrated by the ability to amplify selected gene loci from samples stored dry for decades. Successful amplification in some samples was possible only after treatment with phenacylthiazolium bromide, a reagent that disrupts covalent cross-links; indicating that the DNA was modified by cross-links that occurred between reducing sugars and the primary amines on the DNA. Abundant superoxide dismutase was released following rehydration of desiccated field material N. commune CHEN after 13 years in the dry state. sodF mRNA was present in the dry material but was turned over within 15 min of rehydration. mRNA levels then rose and appeared to reach steady state levels after 3 hours and remained abundant after 24 hours of rehydration. / Master of Science

Investigations into the nitrogen fixing apparatus in cyanobacterium Nostoc commune revealed a gene encoding for a hemoprotein, known as cyanoglobin. The cyanoglobin gene was isolated and subcloned into Escherichia coli previously. The study presented here encompasses the optimization of growth conditions for the transformed F. coli, with subsequent induction of cyanoglobin synthesis. These conditions were applied to large-scale (24-1) fermentor culture, permitting purification of approximately 200 mg cyanoglobin. Structural analyses, including absorption spectroscopy and circular dichroism, are presented. These studies indicate that cyanoglobin is a cytoplasmic hemoglobin with properties quite unlike those of leghemoglobin a and sperm whale myoglobin, which are used as references of comparison. For example, the optical spectral properties of oxycyanoglobin are different from those of leghemoglobin α and sperm whale myoglobin. In addition, the met-form of cyanoglobin has characteristics of a low-spin hemoglobin, in contrast to the high-spin met-forms of sperm whale myoglobin and leghemoglobin α. Unusually, the met- form of cyanoglobin fails to coordinate the strong-field ligands, cyanide and azide, at pH 7 and pH 9. The Soret region circular dichroism (CD) spectrum of cyanoglobin is unlike that of sperm whale myoglobin, yet is very similar to leghemoglobin α, suggesting a similar heme environment in these two hemoproteins. Far-UV CD of cyanoglobin revealed alphahelical character comparable to that of sperm whale myoglobin and leghemoglobin α. Cyanoglobin is the first monomeric hemoglobin detected in a prokaryote, raising questions concerning a possible role of cyanoglobin in early globin gene evolution. / Master of Science

A desiccation-tolerant cyanobacterium <i>Nostoc commune</i> accumulates a novel group of water stress proteins (Wsp) in response to cycles of repeated drying and rehydration. Antibodies, specific for Wsp, were used to screen a lambdafix II library of <i>N. commune</i> UTEX 584 Bam H1 DNA fragments and an 8.5-kb fragment, containing a gene cluster that synthesized a 59-kDa cross-reactive protein. The cloned fragment comprised five ORF’s. The ORF’s 59, 24, 22, 36, and 70, each potentially encode products of molecular weights of 59, 24, 22, 36, and 70-kDa, respectively. The 59 and 24 ORF products were found to be expressed in <i>E. coli</i>. The 59-kDa product of this fragment gives the strongest cross-reaction with the Wsp antiserum. The 59-kDa protein was partially purified. The 24-kDa product was successfully purified to homogeneity and partially characterized. This study used <i>E. coli</i> strain DH10B transformed with the pTrc 99A plasmid. The pTre 99A contains the 8.5-kb gene cluster fragment of interest. The products of ORF 24 and 59 were isolated using an initial 40-60 % ammonium sulfate precipitation of a clarified <i>E. coli</i> cell lysate. The clarified cell lysate was then subjected to streptomycin sulfate precipitation. The cell lysate was then dialyzed extensively. The cell lysate was then applied to a Mono Q HR 5/5 anion exchange column using a 2 M KCl gradient elution procedure. The Mono Q column yielded a fraction containing both ORF products which eluted with approximately 400 mM KCl. This fraction was then applied to a Superose 12 HR 10/30 gel filtration column. The eluent fraction containing the ORF 24 product was then reapplied to the Superose 12 to yield the final fraction containing only the ORF 24 product. The final fraction of ORF 24 was purified to homogeneity as determined by SDS-PAGE analysis. Approximately 750 μg of ORF 24 was isolated. This preparation was used for characterization studies. Characterization studies of ORF 24 consisted of an amino-terminal sequence analysis, an estimation of the molecular weight using gel filtration chromatography and SDS-PAGE analysis, and an analysis of enzymatic activity as suggested by amino acid sequence homologies. The amino-terminal sequence of ORF 24 is P V E Q R S H D. The molecular weight of ORF 24 using gel-filtration chromatography and SDS-PAGE analysis is 26-kDa and 23-kDa, respectively. From gene sequence analysis, the molecular weight of ORF 24 is known to be 24,340-Da. These data indicate that ORF 24 is a monomer. ORF 24 was found to have amino acid sequence homologies with a pectate lyase (E 4.2.2.2) periplasmic precursor from <i>Erwinia caratovora</i> subspecies and a dextransucrase (EC 2.4.1.5) precursor from <i>Streptoccocus mutans</i> GS-5. However, pectate lyase activity was not detected in cellular extracts over a 24 hour period. In addition, ORF 24 was not found to interact with 10 % substrate solutions of N-acetylglucosamine, pectin, UTEX 584 sheath material, DRH1 sheath material, sucrose, or glucose using thin layer chromatography. These studies indicate that the enzymatic activities proposed from amino acid sequence homologies have not been detected. The suggestion that ORF 24 is a water stress protein with a protective function on a structural level with regards to desiccation-tolerance requires further study. / Master of Science

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008. / Title from title screen (site viewed Mar. 31, 2009). PDF text: ix, 168 p. : ill. ; 5 Mb. UMI publication number: AAT 3331442. Includes bibliographical references. Also available in microfilm and microfiche formats.