Regensburg, Univ., Diss., 2001. / Computerdatei im Fernzugriff.

Regensburg, Universiẗat, Diss., 2001.

The cyanobacterium Anabaena sp. (PCC #6411) is known to produce the siderophore, schizokinen, in response to iron limitation. Environmental factors which influence schizokinen production have been examined utilizing a bioassay method based upon the stimulation of growth of the siderophore-requiring bacterium Arthrobacter flavescens JG-9. Schizokinen production by Anabaena increased in low-iron media and was stimulated approximately five-fold media containing one millimolar citric acid. Growth of Anabaena under nitrogen-fixing conditions reduced extracellular schizokinen concentrations 3-4 fold, in late growth phase, compared to cells grown on nitrate. Cells grown in ammonium-containing medium yielded intermediate concentrations of schizokinen. The Csaky assay, which is often used to detect hydroxamate siderophores, was found to be subject to interferences that can yield erroneously high values. The problems associated with detection and quantitation of cyanobacterial siderophores are discussed. Anabaena was found to utilize schizokinen to accumulate > 90% of the (('55)Fe)-ferric iron added to the medium. Iron transport capability was increased in iron-starved cells. The transport system appears to be fairly specific for schizokinen, in that an acetylated derivitive of schizokinen, also supported iron transport, but the structurally related siderophore, aerobactin, and the trihydroxamate siderophore, ferrioxamine B, did not support iron uptake by Anabaena. The uptake of ferric schizokinen displayed saturation-type kinetics with an apparent K(,M) of 35 nM, and required the input of metabolic energy. Lightdriven transport was blocked by uncouplers and ATPase inhibitors. Transport in dark-adapted cells was additionally blocked by inhibitors of respiration. We conclude that ATP serves as an energy source for the cellular uptake of schizokinen. Two other kinds of Anabaena were examined for siderophore production. Anabaena sp. (PCC #7120) was found to produce a substance which stimulates the growth of the Arthrobacter JG-9. This substance binds iron and has a similar absorbance maximum to schizokinen in ferric-perchlorate solutions. Anabaena 7120 also utilized ferric schizokinen and ferric acetyl-schizokinen for iron uptake, but not aerobactin or ferrioxamine B. These results suggest that the iron transports systems of Anabaena 6411 and 7120 may be the same. Anabaena cylindrica Lemm. apparently does not produce or utilize a schizokinen-type siderophore. No Arthrobacter JG-9 stimulating material could be detected in low-iron filtrates, even if concentrated 25-fold.

The purpose of this study was as follows: 1. Elucidate the laboratory conditions under which Anabaena circinalis produces the noxious odor component or components in maximum concentration, 2. Isolate the major noxious odorous metabolite(s), 3. Structurally define the odorous metabolite(s), 4. Quantitate the amount of metabolite(s) that a given amount of the organism produced.

Dissertation (doctoral)--Universität Münster, 1934. / Cover title. "Botanik." Bibliography: p. 23-[24].

This research investigated the factors influencing the structure of the phytoplanktori community in Chaffey Dam, which is located in sub-tropical Australia. In particular, the research aimed to determine the influence of light at time scales ranging from seconds to seasons, on the growth and photophysiology of the cyanobacterium Anabaena circinalis. On a large scale, field monitoring programs between 1987 and 1997 indicated that the phytoplankton community of Chaffey Dam was dominated by colonial or relatively large phytoplankton that move either with the aid of flagella or can be positively buoyant. Diatoms contributed only a minor component, which may be the result of the reservoir being stratified for much of the year. Several of the dominant taxa bloomed in each of the seasons during the eleven year period, with some blooms lasting >9 months, indicating that environmental variability between seasons can be low. In contrast to other studies, A. circinalis was more likely to grow and bloom during the cooler months (March-October). A two-year intensive monitoring program (1995-1997) identified a seasonal progression that was similar in both years. Chlorophytes occurred in spring, Ceratium in mid summer, a relatively clear period in February, A. circinalis in March and cryptomonads in winter. On a smaller scale, short-term (2-3 day) in-situ and laboratory enclosure experiments found that the light and nutrient requirements of the dominant taxa varied. In comparison to most other phytoplankton, A. circinalis cells disappeared at very rapid rates when supplied irradiances <10 (umol photons m-2 s-1. Over several days of darkness, the filaments broke apart and the cell numbers declined. The experiments also showed that at certain times, field populations of A. circinalis were subject to high losses at all irradiances. Laboratory studies investigating the influence of inter- and intra-daily changes in light availability showed that the growth rate of A. circinalis was not affected by the frequency of daytime light:dark cycles, indicating that the rate of water mixing will not have major influence on its growth if the total daily light dose is maintained. It was also found that A. circinalis cultures did not accumulate large reserves of energy in the form of carbohydrate, other than that required for one night. This strategy may enable the colonies to have a high level of buoyancy each morning so that they float quickly to the surface waters and obtain sufficient light each day to minimise losses. However, this strategy limits the ability of A. circinalis to grow and maintain vital cell processes during extended periods of low irradiances and may be a factor causing them to be susceptible to cell breakdown. Weekly measurements of algal growth rates in Chaffey Dam identified two factors that may have acted singly or simultaneously to influence the development of A. circinalis blooms during 1996 and 1997. The blooms developed during a 4-6 week period when the mean irradiance in the surface mixed layer (SML) was sufficient to prevent high losses. Secondly, the blooms developed when soluble phosphorus in the epilimnion was relatively high but soluble nitrogen was low. This may have favoured A. circinalis, which has the potential to fix atmospheric nitrogen. The decline of A. circinalis blooms was correlated with a deepening of the SML and a reduction of the mean daytime irradiance within the SML. Their decline did not appear to be related to nutrient limitation or to changes in zooplankton concentrations. This research also developed a physiological technique for tracking daily changes in the mean daytime irradiance of A. circinalis and for estimating cell growth rate. This method is based on chlorophyll-a fluorescence quenching analysis of the state transition mechanism, which regulates light availability between the photosystems. The mean daytime irradiance of A. circinalis showed a strong relationship with the degree of non-photochemical quenching (qn), whereas the relative change to the maximum fluorescence showed a strong relationship with cell growth. It is anticipated that this method will provide a useful research tool for determining the relative importance of light and other factors on the net growth of A. circinalis and other cyanobacteria.

The release of the neurotoxic alkaloid saxitoxin by cyanobacterial cells was previously thought to occur primarily after cell lysis, yet recent evidence also suggests active toxin export by membrane transporters. Transporter proteins associated with STX biosynthesis in Cylindrospermopsis raciborskii T3 (sxtF and sxtM) and Anabaena circinalis 131C (naDt) were predicted to be involved in the export of STX from cyanobacterial cells. The main aim of this project was to characterise the transporters associated with STX biosynthesis, by investigation of their genetic prevalence, functional substrates and specific regulation. An sxtM homologue was discovered in A. circinalis 131C, as part of an sxt cluster, and found to be uniquely associated with STX-producing strains. Bioinformatic and phylogenetic analysis showed that the translated sxt transporters clustered with the NorM prokaryotic MATE sub-family and membrane topology analysis predicted 12 membrane-spanning regions. To characterise the functional substrates of the putative STX-transporters, they were heterologously expressed in the antibiotic-sensitive E. coli strain KAM32. Expression of the sxt MATES complemented host sensitivity to the cationic fluroquinolone antibiotics, ciprofloxacin and ofloxacin. Disruption of gene homologues of naDt and the sxt MATE genes in Synechocystis sp. PCC6803 yielded mutant strains with increased sensitivity to the toxic organic cations, methyl viologen and acriflavine. Transcription of the putative STX transporters, and the putative STX biosynthesis gene sxtA, was studied in C. raciborskii T3 and A. circinalis 131C under alkali and Na+ stress. Alkali stress (pH 9) decreased total STX levels in A. circinalis 131C and was correlated with a down-regulation of the putative transport and biosynthetic genes. In C. raciborskii T3, alkali stress promoted higher extracellular but lower intracellular STX levels, which also correlated with large increases in transcription of the putative STX transport genes.

"January 2003" / Amendments in pocket inside back cover. / Bibliography: leaves 212-229. / xi, 229 leaves : ill. (some col.), map, plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Earth and Environmental Sciences, Discipline of Environmental Biology, 2003

Many multicellular cyanobacteria produce specialized nitrogenfixing heterocysts. During diazotrophic growth of Anabaena (Nostoc) sp. strain PCC 7120, a regulated developmental pattern of single heterocysts separated by about 10 to 20 photosynthetic vegetative cells is maintained along filaments. Heterocyst structure and metabolic activity function together to accommodate oxygensensitive nitrogen fixation, catalyzed by nitrogenase. In this work, we show that the promoter of the nifHDK genes that encode nitrogenase, lies upstream from the intergenic region between nifH and nifU. Excision of the fdxN element is required for transcription of the nifHDK genes. Fluorescence microscopy of reporter strain PnifHDgfp, in the chromosomal nif locus indicated that expression of nifHDK is blocked in mutants that are unable to excise the fdxN element after nitrogen deprivation. We proposed that a promoter upstream of the element, likely PnifB, is required for transcription of the nifHDK genes. Indeed, the PnifHDgfp reporter at an ectopic site did not show GFP fluorescence. A PnifBgfp reporter was expressed specifically in heterocysts indicating that a promoter for the nifB gene lies in the intergenic region upstream of nifB. A stem loop structure located in the intergenic region between nifH and nifU may act as a processing site for production of nifHDK transcripts. We also provide evidence that DevH, a transcriptional regulator, is involved in regulating the nifBfdxNnifSUHDK genes. DevH is a protein belonging to the cAMP receptor protein (CRP) family of proteins that are widespread in bacteria and regulate genes in response to a gamut of physiological conditions. We show that DevH binds specifically to the nifB upstream region but not to the immediate upstream region of nifH. We predict that DevH binds to an NtcAlike binding site upstream of nifB and functions as an activator of the nifBfdxNnifSUHDK genes. Finally, we show that sigE, which is expressed at 16 hours after nitrogen deprivation, is required for normal expression of some heterocyst specific genes, including nifHDK. A sigE mutant shows delayed and reduced expression of nifHDK and some middle and late genes. We hypothesize that DevH in concert with SigE upregulates the expression of nifHDK in heterocysts after nitrogen deprivation.

The release of the neurotoxic alkaloid saxitoxin by cyanobacterial cells was previously thought to occur primarily after cell lysis, yet recent evidence also suggests active toxin export by membrane transporters. Transporter proteins associated with STX biosynthesis in Cylindrospermopsis raciborskii T3 (sxtF and sxtM) and Anabaena circinalis 131C (naDt) were predicted to be involved in the export of STX from cyanobacterial cells. The main aim of this project was to characterise the transporters associated with STX biosynthesis, by investigation of their genetic prevalence, functional substrates and specific regulation. An sxtM homologue was discovered in A. circinalis 131C, as part of an sxt cluster, and found to be uniquely associated with STX-producing strains. Bioinformatic and phylogenetic analysis showed that the translated sxt transporters clustered with the NorM prokaryotic MATE sub-family and membrane topology analysis predicted 12 membrane-spanning regions. To characterise the functional substrates of the putative STX-transporters, they were heterologously expressed in the antibiotic-sensitive E. coli strain KAM32. Expression of the sxt MATES complemented host sensitivity to the cationic fluroquinolone antibiotics, ciprofloxacin and ofloxacin. Disruption of gene homologues of naDt and the sxt MATE genes in Synechocystis sp. PCC6803 yielded mutant strains with increased sensitivity to the toxic organic cations, methyl viologen and acriflavine. Transcription of the putative STX transporters, and the putative STX biosynthesis gene sxtA, was studied in C. raciborskii T3 and A. circinalis 131C under alkali and Na+ stress. Alkali stress (pH 9) decreased total STX levels in A. circinalis 131C and was correlated with a down-regulation of the putative transport and biosynthetic genes. In C. raciborskii T3, alkali stress promoted higher extracellular but lower intracellular STX levels, which also correlated with large increases in transcription of the putative STX transport genes.