Nostoc Ellipsosporum Research Articles

Structures of the Complexes of a Potent Anti-HIV Protein Cyanovirin-N and High Mannose Oligosaccharides

The development of anti-human immunodeficiency virus (HIV) microbicides for either topical or ex vivo use is of considerable interest, mainly due to the difficulties in creating a vaccine that would be active against multiple clades of HIV. Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium (blue-green algae) Nostoc ellipsosporum with potent virucidal activity, was identified in the search for such antiviral agents. The binding of CV-N to the heavily glycosylated HIV envelope protein gp120 is carbohydrate-dependent. Since previous CV-N-dimannose structures could not fully explain CV-N-oligomannose binding, we determined the crystal structures of recombinant CV-N complexed to Man-9 and a synthetic hexamannoside, at 2.5- and 2.4-A resolution, respectively. CV-N is a three-dimensional domain-swapped dimer in the crystal structures with two primary sites near the hinge region and two secondary sites on the opposite ends of the dimer. The binding interface is constituted of three stacked alpha1-->2-linked mannose rings for Man-9 and two stacked mannose rings for hexamannoside with the rest of the saccharide molecules pointing to the solution. These structures show unequivocally the binding geometry of high mannose sugars to CV-N, permitting a better understanding of carbohydrate binding to this potential new lead for the design of drugs against AIDS.

Cyanovirin-N binds to gp120 to interfere with CD4-dependent human immunodeficiency virus type 1 virion binding, fusion, and infectivity but does not affect the CD4 binding site on gp120 or soluble CD4-induced conformational changes in gp120.

Cyanovirin-N (CV-N), an 11-kDa protein isolated from the cyanobacterium Nostoc ellipsosporum, potently inactivates diverse strains of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. While it has been well established that the viral surface envelope glycoprotein gp120 is a molecular target of CV-N, the detailed mechanism of action is of further interest. We compared matched native and CV-N-treated virus preparations in a panel of assays that measure viral replication, assessing successive stages of the viral life cycle. CV-N-treated virions failed to infect cells as detected by p24 production and quantitative PCR for HIV-1 reverse transcription products, whereas treatment of the target cells did not block infection, confirming that CV-N acts at the level of the virus, not the target cell, to abort the initial infection process. Compared to native HIV-1 preparations, CV-N-treated HIV-1 virions showed impaired CD4-dependent binding to CD4(+) T cells and did not mediate "fusion from without" of CD4(+) target cells. CV-N also blocked HIV envelope glycoprotein Env-induced, CD4-dependent cell-cell fusion. Mapping studies with monoclonal antibodies (MAbs) to defined epitopes on the HIV-1 envelope glycoprotein indicated that CV-N binds to gp120 in a manner that does not occlude or alter the CD4 binding site or V3 loop or other domains on gp120 recognized by defined MAbs and does not interfere with soluble CD4-induced conformational changes in gp120. Binding of CV-N to soluble gp120 or virions inhibited subsequent binding of the unique neutralizing MAb 2G12, which recognizes a glycosylation-dependent epitope. However, prior binding of 2G12 MAb to gp120 did not block subsequent binding by CV-N. These results help clarify the mechanism of action of CV-N and suggest that the compound may act in part by preventing essential interactions between the envelope glycoprotein and target cell receptors. This proposed mechanism is consistent with the extensive activity profile of CV-N against numerous isolates of HIV-1 and other lentiviruses and supports the potential broad utility of this protein as a microbicide to prevent the sexual transmission of HIV.

Cyanobacteria as agents for the control of pollution by pesticides and chlorinated organic compounds

Cyanobacteria are phototrophic aquatic micro-organisms that are found in a variety of environments, including polluted ones. Fifteen strains of cyanobacteria that belong to three taxonomic groups are able to degrade lindane (λ-hexachlorocyclohexane, a recalcitrant pesticide). The initial degradation pathway has been studied in two filamentous nitrogen-fixing strains of Anabaena sp. PCC 7120 and Nostoc ellipsosporum. These cyanobacteria dechlorinated loindane first to pentachlorocyclohexene and then to a mixture of trichlorobenzenes, and possibly further. Lindane dechlorination by these organisms occurred only in the presence of nitrate in the medium. Both ammonium and darkness inhibited the process. This combination of observations led us to the hypothesis that the nitrate-reduction system of cyanobacteria may be involved in dechlorination. The hypothesis was proven by the analysis of Anabaena sp. transpositional mutants in four genes of the nir operon. The mutants were unable to dechlorinate lindane. However, there was no correlation between lindane dechlorination and activities of individual proteins encoded by this operon. Genetic engineering of Anabaena sp. and N. ellipsosporum that introduced linA lindane dechlorination operon from Psuedomonas paucimobilis allowed us to uncouple dechlorination from nitrate requirement. Introduction, by genetic engineering, of fcABC (the 4-chlorobenzoate dechlorination system from Arthrobacter globiformis) to Anabaena sp and N. ellipsosporum rendered these strains newly capable of 4-chlorobenzoate dechlorination both constitutively and inducible by an environmental factor.

Isolation, Primary Sequence Determination, and Disulfide Bond Structure of Cyanovirin-N, an Anti-HIV(Human Immunodeficiency Virus) Protein from the CyanobacteriumNostoc ellipsosporum

A novel anti-HIV protein, cyanovirin-N (CV-N), was isolated from an aqueous cellular extract of the cultured cyanobacterium (blue-green alga)Nostoc ellipsosporum,purified by reverse-phase HPLC, and sequenced by N-terminal Edman degradation of the intact protein and peptide fragments produced by endoproteinase digestions. CV-N consists of a single 101 amino acid chain which exhibits significant internal sequence duplication, but no significant homology to previously described proteins or to the transcription products of known nucleotide sequences. Alignment of residues 1-50 with residues 51-101 reveals 13 conservative amino acid changes as well as direct homology between 16 amino acid residues. CV-N contains four cysteines which form two intrachain disulfide bonds. The positions of the disulfide linkages were established by fast atom bombardment mass spectral studies of peptide fragments generated by a tryptic digestion of the native protein. Reductive cleavage of these crosslinks resulted in loss of anti-HIV activity.

Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development.

We have isolated and sequenced a novel 11-kDa virucidal protein, named cyanovirin-N (CV-N), from cultures of the cyanobacterium (blue-green alga) Nostoc ellipsosporum. We also have produced CV-N recombinantly by expression of a corresponding DNA sequence in Escherichia coli. Low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. Continuous, 2-day exposures of uninfected CEM-SS cells or peripheral blood lymphocytes to high concentrations (e.g., 9,000 nM) of CV-N were not lethal to these representative host cell types. The antiviral activity of CV-N is due, at least in part, to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. The biological activity of CV-N is highly resistant to physicochemical denaturation, further enhancing its potential as an anti-HIV microbicide.

Dechlorination of lindane by the cyanobacterium Anabaena sp. strain PCC7120 depends on the function of the nir operon.

Nitrate is essential for lindane dechlorination by the cyanobacteria Anabaena sp. strain PCC7120 and Nostoc ellipsosporum, as it is for dechlorination of other organic compounds by heterotrophic microorganisms. Based on analyses of mutants and effects of environmental factors, we conclude that lindane dechlorination by Anabaena sp. requires a functional nir operon that encodes the enzymes for nitrate utilization.

Two mutations that block heterocyst differentiation have different effects on akinete differentiation in Nostoc ellipsosporum.

Evident differentiation of vegetative cells into heterocysts in Anabaena sp. strain PCC 7120 is prevented by insertions in genes hetR and hetP. Nostoc ellipsosporum possesses single copies of genes that hybridize with hetR and hetP. In mutant NE2 of N. ellipsosporum, in which hetR is interrupted by an insert, and in a double recombinant of wild-type N. ellipsosporum with a plasmid that bears an interrupted copy of hetR, neither heterocysts nor akinetes are formed. When an intact copy of hetR from Anabaena sp. strain PCC 7120 was added to NE2 the ability to form both heterocysts and akinetes was restored. In contrast to the hetR mutant, a hetP mutant of N. ellipsosporum could form akinetes, but heterocyst formation was blocked. Use of luxAB, encoding luciferase, as a reporter, and use of luxC, luxD and luxE to generate aldehyde (a substrate for the luciferase reaction), permitted visualization of the expression of hetR at the level of single cells; hetR was expressed in akinetes.

Nitrogen-fixing blue-green algae in acid forest and nursery soils.

Algal populations were studied in soils from sites representing several major forest types ranging from the spruce–fir forests in the Appalachian Mountains to the Southern pine forests along the Atlantic Coast. In addition, the nitrogen-fixing blue-green algal population of a forest tree nursery was examined.The results of this investigation indicate a general lack of nitrogen-fixing algae in acid forest soils. However, a pronounced development of these algae was observed in the acid nursery soil, especially on the soil surface. Low numbers of algae were found in some forest soils but only when the soil was above pH 5.4. No nitrogen-fixing Cyanophyceae were isolated from soils in the Appalachian Mountains regardless of soil acidity. Nostoc muscorum was the most common nitrogen-fixing species occurring under both forest and nursery conditions. Nostoc commune was isolated only from the nursery soil. Several strains of Nostoc ellipsosporum were obtained from a mixed hardwood–softwood site. An examination of the algal flora of a number of Alaskan tundra soils gave results similar to those found in the forest soils.

Algae of Quitobaquito: A Spring-Fed Impoundment in Organ Pipe Cactus National Monument

A lis of algae collected from Quitobaquito in Organ Pipe Cactus National Monument in southwestern Arizona is given along with a description of Cosmarium garrolense var. minor var. nov. The purpose of this paper is to present a list of algae identified in collections made at Quitobaquito in Organ Pipe Cactus National Monument in December, 1962 and on September 5, 1964. The only other algal collections, known to the authors, from this area were made by Cameron (1962) when he collected Nostoc ellipsosporum and N. muscorum in soil samples from a nearby hillside and a few specimens collected by Drouet and Hevly in October, 1960 (in Cameron, 1963 and 1964). Quitobaquito is a spring-fed impoundment located in Organ Pipe Cactus National Monument in southwestern Arizona (Fig. 1). It is a shallow desert watering hole surrounded by mesquite and cottonwood trees and its water supply comes via a small ditch from a spring located about 100 yards away on the side of a hill. Bottom materials consist of silt and organic debris in which is rooted an abundant growth of emergent vegetation. Heavy growths of filamentous algae also cover the bottom. Of particular interest is the presence of a subspecies of the desert minnow, Cyprinodon macularis (Cole, 1963). The principal rocks of the area are Pre-Cambrian schists and gneisses, mesozoic intrusive and extrusive rocks, and paleozoic sedimentary rocks (Bryan, 1922). The spring-waters have been analyzed only twice, once November 11, 1917, and by the authors on September 5, 1964 (see Table 1). Both values measured for total hardness are above 120 p.p.m. which indicates that this is a hard-water habitat. These data are to be expected; the Water Atlas of the United States (Miller, 1963) places Quitobaquito in the zone of 180 to 240 p.p.m. hardness as calcium carbonate. Further examination of Table 1 shows that the impound1 Contribution No. 169 from the Department of Natural Science. Field work supported by a grant from the American Philosophical Society.