Unusual radioresistance of nitrogen-fixing cultures of <Emphasis Type="Italic">Anabaena</Emphasis> strains

Nitrogen-fixing cultures of two species of the filamentous, heterocystous cyanobacterium Anabaena, namely Anabaena sp. strain L-31 and Anabaena torulosa were found to be highly tolerant to ⁶⁰Co gamma radiation. No adverse effect on diazotrophic growth and metabolism were observed up to a dose of 5 kGy. At higher doses, radiation tolerance showed a correspondence with the inherent osmotolerance, with Anabaena L-31 being the more radiation tolerant as well as osmotolerant strain. In Anabaena L-31, exposure to 6 kGy of gamma rays resulted in genome disintegration, but did not reduce viability. Irradiation delayed heterocyst differentiation and nitrogen fixation, and marginally affected diazotrophic growth. All the affected parameters recovered after a short lag, without any discernible postirradiation phenotype. The radiation tolerance of these Gram-negative photoautodiazotrophs is comparable with that of the adiazotrophic photoautotrophic cyanobacterium Chroococcidiopsis or adiazotrophic heterotroph Deinococcus radiodurans. This is the first report of extreme radioresistance in nitrogen-fixing Anabaena cultures.     

Modulation of fungicidal potential of <Emphasis Type="Italic">Anabaena</Emphasis> strains by light and temperature

The regulation of fungicidal and hydrolytic enzyme activity was investigated in a set of cyanobacterial strains belonging to the genus Anabaena (Anabaena laxa RPAN8, Anabaena iyengarii RPAN9, Anabaena variabilis RPAN59 and Anabaena oscillarioides RPAN69), with A. variabilis RPAN16 serving as negative control. Time course studies undertaken with cultures incubated under different light and temperature conditions revealed enhancement in growth and fungicidal activity under continuous light (CL) and light dark (LD, 16:8) conditions and temperature of 30 °C and 40 °C. A significant increase of 3–18 % in chitosanase activity was recorded in all the 4-week-old cultures under CL condition and at 40 °C. Endoglucanase activity of RPAN8 and 9 was twofolds higher than the other strains under all light/dark conditions and temperature in the 4-week-old cultures, while continuous dark (CD) enhanced CMCase activity in RPAN69. This study provided useful information regarding the most suitable conditions of light and temperature for maximizing hydrolytic enzyme activity and fungicidal activity, as a prelude to their effective use as biocontrol agents.     

Morphological characteristics and phylogenetic relationship of <Emphasis Type="Italic">Anabaena</Emphasis> species from Lakes Dianchi and Erhai,China

Although Anabaena is one of the most prevalent planktonic freshwater genus in China, there are few taxonomic reports of Anabaena strains by morphology and genetics. In this study, morphological characteristics and phylogenetic relationships of seven Anabaena strains isolated from two plateau lakes, Lakes Dianchi and Erhai, were investigated. Morphological characteristics such as morphology of filament, cellular shapes and sizes, relative position of heterocytes and akinetes, and presence or absence of aerotopes, were described for these seven strains. Phylogenetic relationships were determined by constructing 16S rRNA gene tree using the neighbor-joining algorithm. The seven strains were morphologically identified as three groups, and phylogenetic analysis based on 16S rRNA gene sequences also showed that these seven strains were in three groups. Strains EH-2, EH-3, and EH-4 were in group A belonging to the Anabaena circinalis and A. crassa group, and strains DC-1, DC-2, and EH-1 were in group B and identified as A. flos-aquae. Strain DC-3 without aerotopes was significantly different from the other isolated strains and was determined as A. cylindrica. Handling editor: J. Padisak     

Modulation of biocidal activity of <Emphasis Type="Italic">Calothrix</Emphasis> sp. and <Emphasis Type="Italic">Anabaena</Emphasis> sp. by environmental factors

An investigation was undertaken to evaluate a set of cyanobacterial strains in terms of production of biocidal compounds exhibiting allelochemical and fungicidal properties. Two cyanobacterial strains — Anabaena sp. and Calothrix sp. were selected for further investigation, on the basis of their larger inhibition zones on the lawn of Synechocystis and Synechococcus sp. and two phytopathogenic fungi — Rhizoctonia bataticola and Pythium debaryanum. The diameter of the inhibition zone was largest when extracellular filtrates of the two cultures incubated at high light intensity (90–100 μmol photons m⁻² s⁻¹) and temperature (40 ± 2 °C) or grown in medium containing two-folds higher P (1.4 mg/L, as compared to 0.7 mg/L in BG 11 medium) were taken. A pH of 8 was the most optimal for both strains, in terms of growth and biocidal activity. Partial purification of ethyl acetate extract using TLC, followed by GLC revealed a single peak. This study highlights the importance of environmental factors in aggravating or reducing the toxic effects of these harmful cyanobacteria and their potential as a biocontrol agent.     

Protection from UV-B Damage of Mosquito Larvicidal Toxins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">israelensis</Emphasis> Expressed in <Emphasis Type="Italic">Anabaena</Emphasis> PCC 7120

A transgenic strain of the nitrogen-fixing filamentous cyanobacterium Anabaena PCC 7120 protected expressed δ-endotoxin proteins of Bacillus thuringiensis subsp. israelensis from damage inflicted by UV-B, a sunlight component that penetrates Earth's ozone layer. This organism, which serves as a food source to mosquito larvae and could multiply in their breeding sites, may solve the environment-imposed limitations of B. thuringiensis subsp. israelensis as a mosquito biological control agent. Received: 20 November 2001 / Accepted: 31 December 2001     

Fatty Acid Composition of Planktonic Species of <Emphasis Type="Italic">Anabaena</Emphasis> (Cyanobacteria) with Coiled Trichomes Exhibited a Significant Taxonomic Value

Twenty-six axenic strains of planktonic Anabaena with coiled trichomes belonging to 13 species were investigated by analyzing the pattern and content of their fatty acid composition, and by comparing their fatty acid composition with their morphological properties. In general, the planktonic Anabaena with coiled trichomes contained 14:0, 16:0, 16:1(cis-), 18:0, 18:1, 18:2, and 18:3() as their major fatty acid component, and were classified as Type 2 according to the Kenyon-Murata System. The Type 2 was further divided into two subtypes: Type 2A with 16:2 and 16:3, and Type 2B without 16:2 and 16:3. Among these Anabaena strains with coiled form, A. oumiana (NIES-73 and Ana Kas1) and A. eucompacta (Ana Chiba) contained Type 2B fatty acid composition, and other strains contained Type 2A. Among the strains with the latter type, A. circinalis (Ana Da) and A. curva (Ana Ao) had low levels of 18:3(). Most Anabaena strains with coiled trichomes showed a strong correlation between morphological characteristics and fatty acid composition.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected. An erratum to this article can be found at     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Genetic variability in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> strains nodulating soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill]

Brazil has succeeded in sustaining production of soybean [Glycine max (L.) Merrill] by relying mainly on symbiotic N₂ fixation, thanks to the selection and use in inoculants of very effective strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. It is desirable that rhizobial strains used in inoculants have stable genetic and physiological traits, but experience confirms that rhizobial strains nodulating soybean often lose competitiveness in the field. In this study, soybean cultivar BR 16 was single-inoculated with four B. japonicum strains (CIAT 88, CIAT 89, CIAT 104 and CIAT 105) under aseptic conditions. Forty colonies were isolated from nodules produced by each strain. The progenitor strains, the isolates and four other commercially recommended strains were applied separately to the same cultivar under controlled greenhouse conditions. We observed significant variability in nodulation, shoot dry weight, shoot total N, nodule efficiency (total N mass over nodule mass) and BOX-PCR fingerprinting profiles between variant and progenitor strains. Some variant strains resulted in significantly larger responses in terms of shoot total N, dry weight and nodule efficiency, when compared to their progenitor strain. These results highlight the need for intermittent evaluation of stock bacterial cultures to guarantee effective symbiosis after inoculation. Most importantly, it indicates that it is possible to improve symbiotic effectiveness by screening rhizobial strains for higher N₂ fixation capacity within the natural variability that can be found within each progenitor strain.     

Overexpression of <Emphasis Type="Italic">ADH1</Emphasis> and <Emphasis Type="Italic">HXT1</Emphasis> genes in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves the fermentative efficiency during tequila elaboration

This work assessed the effect of the overexpression of ADH1 and HXT1 genes in the Saccharomyces cerevisiae AR5 strain during fermentation of Agave tequilana Weber blue variety must. Both genes were cloned individually and simultaneously into a yeast centromere plasmid. Two transformant strains overexpressing ADH1 and HXT1 individually and one strain overexpressing both genes were randomly selected and named A1, A3 and A5 respectively. Overexpression effect on growth and ethanol production of the A1, A3 and A5 strains was evaluated in fermentative conditions in A. tequilana Weber blue variety must and YPD medium. During growth in YPD and Agave media, all the recombinant strains showed lower cell mass formation than the wild type AR5 strain. Adh enzymatic activity in the recombinant strains A1 and A5 cultivated in A. tequilana and YPD medium was higher than in the wild type. The overexpression of both genes individually and simultaneously had no significant effect on ethanol formation; however, the fermentative efficiency of the A5 strain increased from 80.33% to 84.57% and 89.40% to 94.29% in YPD and Agave medium respectively.     

Morphological,biochemical and molecular characterization of <Emphasis Type="Italic">Anabaena</Emphasis>, <Emphasis Type="Italic">Aphanizomenon</Emphasis> and <Emphasis Type="Italic">Nostoc</Emphasis> strains (Cyanobacteria,Nostocales) isolated from Portuguese freshwater habitats

Studies of cyanobacterial nostocacean taxa are important to the global scientific community, mainly because a significant number of beneficial strains that belong to the order Nostocales fix atmospheric nitrogen, thus contributing to the fertility of agricultural soils worldwide, while others behave as nuisance microorganisms in aquatic ecosystems due to their involvement in toxic bloom events. However, in spite of their ecological importance and environmental concerns, their identification and taxonomy are still problematic and doubtful, often being based on current morphological and physiological studies, which generate confusing classification systems and usually vary under different conditions. Therefore, the present research aimed to investigate through a polyphasic approach differences in morphological, biochemical and genotypic features of three nostocacean cyanobacterial strains isolated from central-western Portuguese shallow freshwater bodies. Morphometric, genetic (16S rRNA, nifH and hetR fragments) and biochemical (fatty acid methyl ester; FAME profiles) data were used to characterize the strains. Morphological analysis and sequencing of 16S rRNA fragments showed that the strains belonged to Anabaena cylindrica (UTAD_A212), Aphanizomenon gracile (UADFA16) and Nostoc muscorum (UTAD_N213) species. These strains showed clear distinct morphological and genetic features, allowing easy allocation to their respective genera. The same happened by using partial sequences of hetR and nifH genes, in spite of the scarcity of deposited sequences. Biochemical characterization showed that the FAME profiles obtained were consistent with both morphological and molecular analyses. It was suggested that the ratio of monounsaturated to polyunsaturated FAMEs, together with the unsaturation index, could be used as genus-specific chemotaxonomic biomarkers.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

<Emphasis Type="Italic">Wolbachia</Emphasis> Strains Typing in Different Geographic Population Spider, <Emphasis Type="Italic">Hylyphantes Graminicola</Emphasis> (Linyphiidae)

The Wolbachia endosymbiont of spiders has not been extensively examined. In order to investigate the distribution, evolutionary history, and reproductive phenotype of Wolbachia in spiders in China, we tested 11 geographic populations of Hylyphantes graminicola. Wolbachia infection has been detected in each population. 10 Wolbachia strains have been characterized by multilocus sequence typing (MLST). Phylogenetic analyses indicated that eight Wolbachia strains in H. graminicola belonged to supergroup B, and two belonged to supergroup A. No correlation existed between Wolbachia diversity and host’s geographic distance. The significant correlation was observed between pairwise distance of H. graminicola COI and genetic divergence of associated Wolbachia strains. We also found that Wolbachia infection frequencies in hosts varied over geographic space.     

<Emphasis Type="Italic">In vitro</Emphasis> colonization of hydrophilic contact lenses by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

In vitro colonization of hydrophilic contact lenses by Aspergillus niger was investigated. Five strains of the fungus, four polymers, two culture media and four incubation periods were considered for analysis. Only the 2700 strain colonized the lenses. The degrees of adhesion and invasion varied significantly according to the characteristics of the culture under investigation. Journal of Industrial Microbiology & Biotechnology (2002) 29, 6–9 doi:10.1038/sj.jim.7000255 Received 06 August 2001/ Accepted in revised form 23 March 2002     

Variability in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and <Emphasis Type="Italic">B. elkanii</Emphasis> Seven Years after Introduction of both the Exotic Microsymbiont and the Soybean Host in a Cerrados Soil

The plasticity of rhizobial genomes is far greater than previously thought, with complex genomic recombination events that may be accelerated by the often stressful environmental conditions of the tropics. This study aimed at evaluating changes in soybean rhizobia due to adaptation to inhospitable environmental conditions (high temperatures, drought, and acid soils) in the Brazilian Cerrados. Both the host plant and combinations of four strains of soybean Bradyrhizobium were introduced in an uncropped soil devoid of rhizobia capable of nodulating soybean. After the third year, seeds were not reinoculated. Two hundred and sixty-three isolates were obtained from nodules of field-grown soybean after the seventh year, and their morphological, physiological, serological, and symbiotic properties determined, followed by genetic analysis of conserved and symbiotic genes. B. japonicum strain CPAC 15 (same serogroup as USDA 123) was characterized as having high saprophytic capacity and competitiveness and by the seventh year represented up to 70% of the cultivable population, in contrast to the poor survival and competitiveness of B. japonicum strain CPAC 7 (same serogroup as CB 1809). In general, adapted strains had increased mucoidy, and up to 43% of the isolates showed no serological reaction. High variability, presumably resulting from the adaptation to the harsh environmental conditions, was verified in rep-PCR (polymerase chain reaction) profiles, being lower in strain CPAC 15, intermediate in B. elkanii, and higher in CPAC 7. Restriction fragment length polymorphism (RFLP)-PCR types of the 16S rDNA corresponded to the following: one type for B. elkanii species, two for B. japonicum, associated to CPAC 15 and CPAC 7, and unknown combinations of profiles. However, when nodC sequences and RFLP-PCR of the nifH region data were considered, only two clusters were observed having full congruence with B. japonicum and B. elkanii species. Combining the results, variability was such that even within a genetically more stable group (such as that of CPAC 15), only 6.4% of the isolates showed high similarity to the inoculant strain, whereas none was similar to CPAC 7. The genetic variability in our study seems to result from a variety and combination of events including strain dispersion, genomic recombination, and horizontal gene transfer. Furthermore, the genetic variability appears to be mainly associated with adaptation, saprophytic capacity, and competitiveness, and not with symbiotic effectiveness, as the similarity of symbiotic genes was higher than that of conserved regions of the DNA.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Effects of macrophyte leachate on <Emphasis Type="Italic">Anabaena</Emphasis> sp. and <Emphasis Type="Italic">Chlamydomonas moewusii</Emphasis> growth in freshwater tropical ecosystems

This study reports on the effects of dissolved organic matter (DOM) derived from the aquatic macrophyte Pistia stratiotes (collected from a tropical reservoir) on the mixotrophic growth of two phytoplankton species (Chlamydomonas moewusii and Anabaena sp.). The DOM from P. stratiotes had a mainly aliphatic structure, low molecular weight, low cellulose and lignin content and high carbon content. The addition of DOM (5% v/v) significantly decreased the growth rate of Anabaena sp. but increased the chlorophyll a concentration of C. moewusii. Higher light intensity (100 versus 30 µmol m^?2 s^?1) was important for Anabaena sp., increasing its growth rate and chlorophyll content. The use of DOM from P. stratiotes to mitigate cyanobacterial blooms should be further explored in future studies.     

Autofluorescence of the fungus <Emphasis Type="Italic">Morchella conica</Emphasis> var. <Emphasis Type="Italic">rigida</Emphasis>

Autofluorescence (primary fluorescence (AF)) of fruiting bodies and stems of the fungus Morchella conica var. rigida was studied by fluorescence microscopy including sporangia and ascospores. The ascospores were characterized by a weak green–yellow AF at blue excitation. Using a green excitation, no AF was observed. The hyphae located under the layer of asci with ascospores exhibited a higher primary fluorescence, namely their walls that had green-yellow color at blue excitation. Also, their red AF observed when a green excitation was used was significant. Similarly, the hyphae located in the fungal stem exhibited a significant AF, especially their walls when the blue light was used for excitation. In addition, large, yellow-to-yellow/green, oval-to-round bodies with strong fluorescence were detected whose morphological equivalents were not clearly visible in the white halogen light. The AF of the fungus M. conica var. rigida was lower compared with the other higher fungi studied so far.