Assimilation of ethylenediamine as nitrogen source by the cyanobiont Nostoc ANTH

Nostoc ANTH metabolizes ethylenediamine (EDA) as sole nitrogen source but not as a carbon source. EDA is assimilated by the glutamine synthetase-glutamate synthase pathway. EDA represses heterocyst formation and nitrogenase activity but this is reversed by l-methionine-dl-sulphoximine.The authors are with the Department of Microbiology, Barkatullah University, Bhopal 462 026, India     

Role of membrane potential in ammonium inhibition of nitrogenase activity in the cultured cyanobiont Nostoc ANTH

Ammonium at 5mM completely inhibited nitrogenase activity of Nostoc ANTH but only slightly inhibited the membrane potential, indicating that these two events are independent and that nitrogenase activity is not regulated by the latter.B.B. Singh and P.S. Bisen are with the Department of Microbiology, Barkatullah University, Bhopal-462026, India; S. Singh was with the Department of Microbiology, Barkatullah University, Bhopal, India; he is now with the Department of Microbiology, North Maharastra University, Jalgaom-425001, India.     

Localization of iron-superoxide dismutase in the cyanobiont ofAzolla filiculoides Lam

Summary Immunogold labeling and transmission electron microscopy were used to localize iron-superoxide dismutase (Fe-SOD) in the different cells of nitrogen-fixing cyanobacterial symbiont present within different leaf cavity groups ofAzolla filiculoides Lam. As evidenced by Western blotting and immunoprecipitation, Fe-SOD antibody fromAnabaena cylindrica recognized Fe-SOD in extracts of the cyanobiont and showed the same electrophoretic mobility and pattern as purifiedA. cylindrica Fe-SOD. In vegetative cells of the cyanobiont, Fe-SOD was mainly localized in the thylakoidal membranes and in the outer membrane. The labeling pattern was similar in vegetative cells of the various groups of leaf cavities examined except at the apex where a lower gold particle density was seen. In heterocysts of the leaf cavity groups containing high nitrogenase activity, Fe-SOD labeling was most pronounced and more intense than in vegetative cells. The Fe-SOD label was preferentially located throughout the heterocyst cytoplasm and in the honeycomb regions. In accordance with the decline in nitrogenase activity, the Fe-SOD gold particle density decreased significantly in heterocysts of basal leaf cavity group. The presence of Fe-SOD in regions of high nitrogenase protein levels, and the fact that the pattern of Fe-SOD label parallels that of nitrogenase activity support a role of Fe-SOD in the protection of nitrogenase against superoxide radicals.     

Common nitrogen control of caesium uptake, caesium toxicity and ammonium (methylammonium) uptake in the cyanobacterium Nostoc muscorum

Abstract Studies were carried out to examine the role of ammonium transport activity in the control of caesium uptake and toxicity in Nostoc muscorum . The results showed a definite specific role of the ammonium-repressible/derepressible ammonium transport system of the cyanobacterium in caesium uptake, accumulation and toxicity. Furthermore, the results showed that N. muscorum can acquire resistance against diazotrophically-associated caesium toxicity when supplied with ammonium as a nitrogen source. In addition, alternatively, a mutant strain was Cs-resistant in the absence of any effect on NH₄⁺-transport, suggesting that Cs⁺ resistance may be determined at more than one cellular site.     

Genetic transformation of glutamine auxotrophy to prototrophy in the cyanobacterium Nostoc muscorum

Glutamine auxotrophic (Gln ^-) and l-methionine d,l-sulfoximine (MSX) resistant (MSX ^r) mutants of N. muscorum were isolated and characterized for nitrogen nutrition, nitrogenase activity, glutamine synthetase (GS) activity and glutamine amide, -keto-glutarate amido transferase (GOGAT) activity. The glutamine auxotroph was found to the GOGAT-containing GS-defective, incapable of growth with N₂ or NH ₄ ⁺ but capable of growth with glutamine as nitrogen source, thus, suggesting GS to be the primary enzyme of both ammonia assimilation and glutamine formation in the cyanobacterium. The results of transformation and reversion studies suggests that glutamine auxotrophy is the result of a mutation in the gln A gene and that gln A gene can be transferred from one strain to another by transformation.     

Effect of cyanophage N-1 development on nitrogen metabolism of cyanobacterium Nostoc muscorum

Abstract The impact of cyanophage N-1 development on nitrogenase, glutamine synthetase (GS) and aminotransferases activities in the diazotrophic cyanobacterium Nostoc muscorum was investigated during its latent period. The nitrogenase activity was inhibited after 2 h of infection, suggesting that phage development does not require the product of nitrogenase activity. GS activity was not inhibited until 4 h of infection; however, a decline in activity was subsequently observed. Glutamate oxaloacetate transaminase was inhibited after 1 h of infection and no activity was detectable during the entire latent period. In contrast, glutamate pyruvate transaminase activity increased 2-fold by 4 h of infection and remained higher than the background level until the end of the latent period. The results suggested that under nitrogen fixing conditions, N-1 multiplication proceeds in the absence of nitrogen fixation and that the metabolism of amino acids is altered in favour of phage multiplication.     

Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue

Structural genes encoding an uptake hydrogenase of Nostoc sp. strain PCC 73102 were isolated. From partial libraries of genomic DNA, two clones (pNfo01 and pNfo02) were selected and sequenced, revealing the complete sequence of both a hupS (960 bases) and a hupL (1,593 bases) homologue in Nostoc sp. strain PCC 73102. A comparison between the deduced amino acid sequences of HupS and HupL of Nostoc sp. strain PCC 73102 and Anabaena sp. strain PCC 7120 showed that the HupS proteins are 89% identical and the HupL proteins are 91% identical. However, the noncoding region between the genes in Nostoc sp. strain PCC 73102 (192 bases) is longer than that of Anabaena sp. strain PCC 7120 and of many other microorganisms. Southern hybridizations using DNA from both N₂-fixing and non-N₂-fixing cells of Nostoc sp. strain PCC 73102 and different probes from within hupL clearly demonstrated that, in contrast to Anabaena sp. strain PCC 7120, there is no rearrangement within hupL of Nostoc sp. strain PCC 73102. Indeed, 6 nucleotides out of 16 within the potential recombination site are different from those of Anabaena sp. strain PCC 7120. Furthermore, we have recently published evidence demonstrating the absence of the bidirectional/reversible hydrogenase in Nostoc sp. strain PCC 73102. The present knowledge, in combination with the unique characteristics, makes Nostoc sp. strain PCC 73102 an interesting candidate for the study of deletion mutants lacking the uptake-type enzyme. Received: 20 August 1997 / Accepted: 24 November 1997     

Inorganic nitrogen regulation of glutamate uptake in the cyanobacterium Nostoc muscorum

In Nostoc muscorum (Anabaena ATCC 27893) glutamate was not metabolised as a fixed nitrogen source, rather it functioned as an inhibitor of growth. The latter effect was nitrogen source specific and occurred in N₂-fixing cultures but not in cultures assimilating nitrate or ammonium. NO₃^--grown cultures lacked heterocysts and nitrogenase activity and showed a nearly 50% reduction in glutamate uptake rates, as well as in the final extent of glutamate taken up, compared to N₂-fixing or nitrogen-limited control cultures. NH₄⁺-grown cultures showed a similar response, except that the reduction in glutamate uptake rates and the final exten of glutamate taken up was over 80%. The present results suggest a relation between nitrate/ammounium nitrogen-dependent inhibition of glutamate uptake, probably via repression of the glutamate transport system, and glutamate toxicity.     

Immunolocalization of the uptake hydrogenase in the marine cyanobacterium Lyngbya majuscula CCAP 1446/4 and two Nostoc strains

In N₂-fixing cyanobacteria, the reduction of N₂ to NH₃ is coupled with the production of molecular hydrogen, which is rapidly consumed by an uptake hydrogenase, an enzyme that is present in almost all diazotrophic cyanobacteria. The cellular and subcellular localization of the cyanobacterial uptake hydrogenase remains uncertain, and it is definitely strain dependent. Previous studies focused mainly on heterocystous cyanobacteria and used heterologous antisera. The present work represents the first effort to establish the subcellular localization of the uptake hydrogenase in a N₂-fixing filamentous nonheterocystous cyanobacterium, Lyngbya majuscula CCAP 1446/4, using the first antiserum produced against a cyanobacterial uptake hydrogenase. The data obtained revealed higher specific labelling associated with the thylakoid membranes of L. majuscula , reinforcing the idea that the cyanobacterial uptake hydrogenase is indeed a membrane-bound protein. For comparative purposes, the localization of the uptake hydrogenase was also investigated in two distinct heterocystous cyanobacterial strains, and while in Nostoc sp. PCC 7120 the labelling was only observed in the heterocysts, in Nostoc punctiforme , the presence of uptake hydrogenase antigens was detected in both the vegetative cells and heterocysts, corresponding most probably to an inactive and an active form of the enzyme.     

念珠藻葛仙米的藻殖段分化——信号转导参与过程的初步研究

钙调素拮抗剂W_7和FPZ均可抑制葛仙米藻殖段的分化。利用FPZ作为荧光染色剂发现 ,经过钙调素拮抗剂处理的藻体有较低的钙调素分布。在达到一定浓度后 ,金属离子螯合剂EGTA也抑制藻殖段分化。金霉素 (CTC)荧光显示 ,经EGTA处理的藻细胞其膜钙分布大大减少。但实验表明 ,藻殖段得以正常分化却是多种金属离子共同参与的结果。一氧化氮 (NO)可以大大降低葛仙米藻殖段的分化率 ,一氧化氮合成酶 (NOS)的抑制剂NNA也可以抑制藻殖段的分化。外源地加入氧自由基抑制了藻殖段的分化 ,但是氧自由基的清除物过氧化氢酶 (CA)也使藻殖段分化完全受抑制。以上结果初步表明 ,葛仙米藻殖段分化是个复杂的、信号转导参与的过程     

High cyanobiont selectivity of epiphytic lichens in old growth boreal forest of Finland

Here, cyanobiont selectivity of epiphytic lichen species was examined in an old growth forest area in Finland. Samples of the eight lichen species were collected from the same aspen (Populus tremula) and adjacent aspens in the same stand. The cyanobionts of these samples were compared with free and symbiotic Nostoc obtained from other habitats and geographic regions. Our results, based on the phylogenetic analysis of a partial small subunit of the ribosomal DNA (16S rDNA) and the rbcLX gene complex did not show any correlation with the geographic origin of the samples at any spatial scale. Instead, there was a correlation between the cyanobionts and the alleged taxonomy of their mycobionts. The results indicate that the lichen species examined are highly selective towards their cyanobiont partners. Only Lobaria pulmonaria proved to be more flexible, being able to associate with a wide range of Nostoc. A same Nostoc strain was found to form associations with taxonomically unrelated lichens indicating that the cyanobiont-mycobiont associations as a whole were not highly specific in the examined species.     

The cyanobiont in an Azolla fern is neither Anabaena nor Nostoc

The cyanobacterial symbionts in the fern Azolla have generally been ascribed to either the Anabaena or Nostoc genera. By using comparisons of the sequences of the phycocyanin intergenic spacer and a fragment of the 16S rRNA, we found that the cyanobiont from an Azolla belongs to neither of these genera.     

Nitrate metabolism in the cyanobacterium Anabaena cycadeae: Regulation of nitrate uptake and reductase by ammonia

Nitrogen regulation of nitrate uptake and nitrate reductase (EC 1.7.99.4) was studied in the cyanobacterium Anabaena cycadeae Reinke and its glutamine auxotroph. Development of the nitrate uptake system preceded, and was independent of, the development of the nitrate reductase system. The levels of both systems were several-fold higher in the glutamine auxotroph lacking glutamine synthetase (EC 6.3.1.2) than in the wild type strain having normal glutamine synthetase activity. The nitrate uptake system was found to be NH4-repressible and the nitrate reductase system NO₃⁻-inducible. NH₄⁺ was the initial repressor signal for the uptake process which was involved in the control of the NO₃⁻inducible reductase system.     

Characterization of an exopolysaccharide mutant of Nostoc spongiaeforme: Zn2+-sorption and uptake

Exposure of the exopolysaccharide (EPS)-synthesizing cyanobacterium Nostoc spongiaeforme to Zn²⁺ (20 M) transformed the biomass into white debris. However, a few blue–green pin-heads emerged after 2 weeks in the same Zn²⁺-containing medium and formed less mucoid microcolonies (1–2 mm) relative to the protruding colonies (2–4 mm) of the parent strain on nutrient agar. One of such survivors (designated as Zn₂₀) that was stable through 10 successive transfers in Zn²⁺-lacking medium has been adopted for further characterization. The parent strain retained almost 88% of the total EPS synthesized, the rest being released into the ambient medium, while for Zn₂₀, the EPS retained approximated to 74%. Although the Zn²⁺-sensitivity of the mutant was comparable with that of the parent (LD₅₀, 7 M), Zn²⁺ uptake was still 5-fold higher in the former (2 g mg^–1 biomass dry wt., 20 M, external concentration). Also, both the strains showed insignificant difference in Zn²⁺-sorption onto their isolated EPS. The mutant was characterized by having higher cell carbohydrate content (642.8 g mg^–1 dry wt.) than its parent (513.6 g). The X-ray diffraction pattern revealed Zn²⁺ deposition on EPS from the parent mainly as zinc hypophosphite monohydrate [Zn(H₂PO₂)₂·H₂O], whereas there was a lack of distinct peaks in similar samples from Zn₂₀, thus confirming the amorphous nature. There was participation in Zn²⁺ binding of only COO^–, N=O, NO₂, SO₂ groups in the parent while participation of P—O and C=O groups in mutant EPS was evident in IR spectra. The observations suggest that the mutant could be deployed to achieve sustained EPS synthesis, its release and metal sorption/desorption in repeated cycles.     

Nitrogen nutrition in the cyanobacterium Nostoc ANTH, a symbiotic isolate from Anthoceros: uptake and assimilation of inorganic-n and amino acids

Amino acid uptake and utilization of various nitrogen sources (amino acids, nitrite, nitrate and ammonia) were studied in Nostoc ANTH and i ts mu tant (Het(-)Nif(-)) isolate defective in heterocyst formation and N2-fixation. Both parent and its mutant grew at the expense of glutamine, asparagine and arginine as a source of fixed-nitrogen. Growth was better in glutamine-and asparagine-media as compared to that in arginine media. Glutamine and asparagine repressed heterocyst formation, N2-fixation and nitrate reduction in Nostoc ANTH, but arginine did so only partially. The poor growth in arginine-medium was not due to poor uptake rates, since the uptake rates were not significantly different from those for glutamine or asparagine. The glutamine synthetase activity remained unaffected during cultivation in media containing any one of the three amino acids tested. The uptake of amino acids was substrate-inducible, energy-dependent and required de novo protein synthesis. Nitrate and ammonium repressed ammonium uptake, but did not repress uptake of amino acids. In N2-medium (BG-11(0)), the uptake of ammonium and amino acids in the mutant was significantly higher than its parent strain. This was apparently due to nitrogen limitation since the mutant was unable to fix N2 and the growth medium lacked combined-N.     

念珠藻属植物hetR基因的适应性进化分析

以念珠藻属(Nostoc)及其近缘类群hetR基因的51条序列为研究对象,对hetR基因的编码蛋白进行生物信息学分析和系统发育分析,并使用分支模型、位点模型和分支-位点模型进行该基因位点的适应性进化研究。系统发育分析结果显示,51条hetR基因蛋白序列可分为4个大分支。适应性进化分析结果表明,在3种进化模型中,大多数分支及藻株都没有检测到统计学上具有显著性的正选择位点,说明检测的位点大多处于负选择压力下。但在普通念珠藻(Nostoc commune,CHAB2802)中检测到正选择位点(126T),提示念珠藻属植物hetR基因发生了适应性改变。     

地木耳规模化培养的初步研究

利用跑道式培养池规模化培养地木耳,结果显示:在一定的实验条件下,地木耳群体的直径明显增大,其生长速度最高可达445.2g·m-2·d-1,并可将其它杂藻类控制在较低的水平。这是地木耳规模化培养的首次报道。     

Ammonium-stimulated,sodium-dependent uptake of glutamine in Bacillus pasteurii

The uptake of glutamine was studied in Bacillus pasteurii DSM 33. Only one uptake system was detected in the concentration range studied (between 1 and 100 M glutamine) which exhibited Michaelis-Menten saturation kinetics, with an apparent K _t of 10.7 (±3.5) M glutamine. The uptake was sodium-dependent (apparent K _t=0.2 mM Na⁺); none of several monovalent cations tested was able to replace sodium in the uptake reaction. Ionophores interfering with proton, sodium or potassium gradients across membranes strongly inhibited uptake of glutamine. Low uptake rates correlating with low potassium content and an acidic cytoplasm were measured in cells grown at high ammonium¹ concentrations. Ammonium and other permeant amines as well as potassium stimulated the uptake reaction in these cells, leading to an increase of up to 100-fold in V _max without affecting the affinity of the uptake system. In cells grown at low concentrations of ammonium, an alkaline cytoplasm and both high glutamine uptake activities and potassium content were measured; the uptake reaction was not further stimulated by permeant amines or potassium in such cells. Growth of the strain was inhibited by Tris at high concentrations; this inhibition was relieved by the addition of increasing amounts of ammonium.Abbreviations CCCP carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide This work is dedicated to Prof. Dr. H. Kaltwasser on the occasion of his 60th birthday