Teresa Thiel

An overview of the genome of Nostoc punctiforme, a multicellular, symbiotic cyanobacterium.

Nostoc punctiforme is a filamentous cyanobacterium with extensive phenotypic characteristics and a relatively large genome, approaching 10 Mb. The phenotypic characteristics include a photoautotrophic, diazotrophic mode of growth, but N. punctiforme is also facultatively heterotrophic; its vegetative cells have multiple developmental alternatives, including terminal differentiation into nitrogen-fixing heterocysts and transient differentiation into spore-like akinetes or motile filaments called hormogonia; and N. punctiforme has broad symbiotic competence with fungi and terrestrial plants, including bryophytes, gymnosperms and an angiosperm. The shotgun-sequencing phase of the N. punctiforme strain ATCC 29133 genome has been completed by the Joint Genome Institute. Annotation of an 8.9 Mb database yielded 7432 open reading frames, 45% of which encode proteins with known or probable known function and 29% of which are unique to N. punctiforme. Comparative analysis of the sequence indicates a genome that is highly plastic and in a state of flux, with numerous insertion sequences and multilocus repeats, as well as genes encoding transposases and DNA modification enzymes. The sequence also reveals the presence of genes encoding putative proteins that collectively define almost all characteristics of cyanobacteria as a group. N. punctiforme has an extensive potential to sense and respond to environmental signals as reflected by the presence of more than 400 genes encoding sensor protein kinases, response regulators and other transcriptional factors. The signal transduction systems and any of the large number of unique genes may play essential roles in the cell differentiation and symbiotic interaction properties of N. punctiforme.

Genetic Analysis of Cyanobacteria

In recent years great strides have been made in developing genetic systems for the analysis of various aspects of cyanobacterial physiology and development. Transformation, electroporation and conjugation systems provide effective means for gene transfer in diverse cyanobacterial strains. Gene transfer, combined with the ability to clone and inactivate genes in cyanobacteria, has opened the door to advanced studies of photosynthesis, nitrogen fixation, heterocyst development and metabolism in these unique and important procaryotic microorganisms.

Effect on heterocyst differentiation of nitrogen fixation in vegetative cells of the cyanobacterium Anabaena variabilis ATCC 29413.

Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions. Anabaena variabilis ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a nif1 mutant; however, that expression did not prevent differentiation of heterocysts and expression of the nif1 nitrogenase in either the nif1 mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells.

CHARACTERIZATION OF CANARYPOX-LIKE VIRUSES INFECTING ENDEMIC BIRDS IN THE GALAPAGOS ISLANDS

The presence of avian pox in endemic birds in the Galápagos Islands has led to concern that the health of these birds may be threatened by avipoxvirus introduction by domestic birds. We describe here a simple polymerase chain reaction–based method for identification and discrimination of avipoxvirus strains similar to the fowlpox or canarypox viruses. This method, in conjunction with DNA sequencing of two polymerase chain reaction–amplified loci totaling about 800 bp, was used to identify two avipoxvirus strains, Gal1 and Gal2, in pox lesions from yellow warblers (Dendroica petechia), finches (Geospiza spp.), and Galápagos mockingbirds (Nesomimus parvulus) from the inhabited islands of Santa Cruz and Isabela. Both strains were found in all three passerine taxa, and sequences from both strains were less than 5% different from each other and from canarypox virus. In contrast, chickens in Galápagos were infected with a virus that appears to be identical in sequence to the characterized fowlpox virus and about 30% different from the canarypox/Galápagos group viruses in the regions sequenced. These results indicate the presence of canarypox-like viruses in endemic passerine birds that are distinct from the fowlpox virus infecting chickens on Galápagos. Alignment of the sequence of a 5.9-kb region of the genome revealed that sequence identities among Gal1, Gal2, and canarypox viruses were clustered in discrete regions. This indicates that recombination between poxvirus strains in combination with mutation led to the canarypox-like viruses that are now prevalent in the Galápagos.

Molybdate transport and its effect on nitrogen utilization in the cyanobacterium Anabaena variabilis ATCC 29413

Molybdenum is an essential component of the cofactors of many metalloenzymes including nitrate reductase and Mo‐nitrogenase. The cyanobacterium Anabaena variabilis ATCC 29413 uses nitrate and atmospheric N2 as sources of nitrogen for growth. Two of the three nitrogenases in this strain are Mo‐dependent enzymes, as is nitrate reductase; thus, transport of molybdate is important for growth of this strain. High‐affinity transport of molybdate in A. variabilis was mediated by an ABC‐type transport system encoded by the products of modA and modBC. The modBC gene comprised a fused orf including components corresponding to modB and modC of Escherichia coli. The deduced ModC part of the fused gene lacked a recognizable molybdate‐binding domain. Expression of modA and modBC was induced by starvation for molybdate. Mutants in modA or modBC were unable to grow using nitrate or Mo‐nitrogenase. Growth using the alternative V‐nitrogenase was not impaired in the mutants. A high concentration of molybdate (10 µM) supported normal growth of the modBC mutant using the Nif1 Mo‐nitrogenase, indicating that there was a low‐affinity molybdate transport system in this strain. The modBC mutant did not detectably transport low concentrations of 99Mo (molybdate), but did transport high concentrations. However, such transport was observed only after cells were starved for sulphate, suggesting that an inducible sulphate transport system might also serve as a low‐affinity molybdate transport system in this strain.

Transport of molybdate in the cyanobacteriumAnabaena variabilis ATCC 29413

Heterocyst-forming filamentous cyanobacteria, such asAnahaena variabilis ATCC 29413, require molybdenum as a component of two essential cofactors for the enzymes nitrate reductase and nitrogenase.A. variabilis efficiently transported99Mo (molybdate) at concentrations less than 10−9 M. Competition experiments with other oxyanions suggested that the molybdate-transport system ofA. variabilis also transported tungstate but not vanadate or sulfate. Although tungstate was probably transported, tungsten did not function in place of molybdenum in the Mo-nitrogenase. Transport of99Mo required prior starvation of the cells for molybdate, suggesting that the Mo-transport system was repressed by molybdate. Starvation, which required several generations of growth for depletion of molybdate, was enhanced by growth under conditions that required synthesis of nitrate reductase or nitrogenase. These data provide evidence for a molybdate storage system inA. variabilis. NtcA, a regulatory protein that is essential for synthesis of nitrate reductase and nitrogenase, was not required for transport of molybdate. The closely related strainAnabaena sp. PCC 7120 transported99Mo in a very similar way toA. variabilis.

NEW ZWITTERIONIC BUTANESULFONIC ACIDS THAT EXTEND THE ALKALINE RANGE OF FOUR FAMILIES OF GOOD BUFFERS : EVALUATION FOR USE IN BIOLOGICAL SYSTEMS

Four new zwitterionic butanesulfonic acid buffers that are structurally related to four families of Good buffers were evaluated for use in biological systems. These buffers, with pKa values from 7.6 to 10.7, were compared with a variety of other buffers from the same family and with unrelated buffers to determine their effect on enzyme activity and on microbial growth. The activity of four enzymes with optimum pH values in the alkaline range were tested: beta-galactosidase, esterase, phosphodiesterase and alkaline phosphatase. In general, all the Good buffers, including the new butanesulfonic acid buffers, gave good activity; however, there was variation in activity of certain enzymes with certain buffers. Tris, glycine, and phosphate buffers typically showed variation in activity compared to the family of Good buffers. beta-Galactosidase, in particular, showed greater activity with Good buffers than with phosphate or Tris buffers. Similarly, growth of seven bacterial strains was consistent, with a few exceptions, for all the Good family of buffers with Tris often inhibiting growth. Quantitation of alkaline phosphatase conjugated to antibodies is an important tool in many applications in molecular biology. Several Good buffers gave good signals when compared with Tris at pH 9.5 for detection of proteins using alkaline phosphatase-conjugated antibodies.

Regulation of Fructose Transport and Its Effect on Fructose Toxicity in Anabaena spp.

Anabaena variabilis grows heterotrophically using fructose, while the close relative Anabaena sp. strain PCC 7120 does not. Introduction of a cluster of genes encoding a putative ABC transporter, herein named frtRABC, into Anabaena sp. strain PCC 7120 on a replicating plasmid allowed that strain to grow in the dark using fructose, indicating that these genes are necessary and sufficient for heterotrophic growth. FrtR, a putative LacI-like regulatory protein, was essential for heterotrophic growth of both cyanobacterial strains. Transcriptional analysis revealed that the transport system was induced by fructose and that in the absence of FrtR, frtA was very highly expressed, with or without fructose. In the frtR mutant, fructose uptake was immediate, in contrast to that in the wild-type strain, which required about 40 min for induction of transport. In the frtR mutant, high-level expression of the fructose transporter resulted in cells that were extremely sensitive to fructose. Even in the presence of the inducer, fructose, expression of frtA was low in the wild-type strain compared to that in the frtR mutant, indicating that FrtR repressed the transporter genes even in the presence of fructose. FrtR bound to the upstream region of frtA, but binding was not visibly altered by fructose, further supporting the hypothesis that fructose has only a modest effect in relieving repression of frtA by FrtR. A. variabilis grew better with increasing concentrations of fructose up to 50 mM, showing increased cell size and heterocyst frequency. Anabaena sp. strain PCC 7120 did not show any of these changes when it was grown with fructose. Thus, although Anabaena sp. strain PCC 7120 could take up fructose and use it in the dark, fructose did not improve growth in the light.

High-Affinity Vanadate Transport System in the Cyanobacterium Anabaena variabilis ATCC 29413

High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 x 10(-9) M. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri.

Transcription of hupSL in Anabaena variabilis ATCC 29413 is regulated by NtcA and not by hydrogen.

ABSTRACT Nitrogen-fixing cyanobacteria such as Anabaena variabilis ATCC 29413 use an uptake hydrogenase, encoded by hupSL, to recycle hydrogen gas that is produced as an obligate by-product of nitrogen fixation. The regulation of hupSL in A. variabilis is likely to differ from that of the closely related Anabaena sp. strain PCC 7120 because A. variabilis lacks the excision element-mediated regulation that characterizes hupSL regulation in strain PCC 7120. An analysis of the hupSL transcript in a nitrogenase mutant of A. variabilis that does not produce any detectable hydrogen indicated that neither nitrogen fixation nor hydrogen gas was required for the induction of hupSL. Furthermore, exogenous addition of hydrogen gas did not stimulate hupSL transcription. Transcriptional reporter constructs indicated that the accumulation of hupSL transcript after nitrogen step-down was restricted primarily to the microaerobic heterocysts. Anoxic conditions were not sufficient to induce hupSL transcription. The induction of hupSL after nitrogen step-down was reduced in a mutant in the global nitrogen regulator NtcA, but was not reduced in a mutant unable to form heterocysts. A consensus NtcA-binding site was identified upstream of hupSL, and NtcA was found to bind to this region. Thus, while neither hydrogen gas nor anoxia controlled the expression of hupSL, its expression was controlled by NtcA. Heterocyst differentiation was not required for hupSL induction in response to nitrogen step-down, but heterocyst-localized cues may add an additional level of regulation to hupSL.