Free-living Cultures Research Articles

Development of free-living sporangial filaments regulated by light and culture density in Neopyropia yezoensis

Neopyropia yezoensis, an important marine crop, reproduces via sporangial filaments. This special sporophyte stage, which directly releases conchospores—the “seed” of gametophytes (thalli)—is poorly understood. Under free-living culture, we found sporangial filaments varied between hollow cells (at high density) and content-enriched bipartite cells (at low density), released as conchospores. Serious intracellular degradation, which might through fusion of a double-membrane coated body with vacuole, was observed in hollow cells but disappeared in newly-formed bipartite cells and reappeared in their subsequent vegetative growth through transmission electron microscope. The bipartite cells can also return to vegetative growth when not released and the conchospores release was obviously inhibited by some growth promoting conditions such as high light (40–100 μmol photons m−2 s−1) and treatment with ampicillin and streptomycin. Based on these results, we summarized that conchopsore release was inhibited by the vegetative growth of bipartite cells and the cell hollowing caused by vacuole-based intracellular degradation in sporangial filaments. These results would increase understanding of sporangial filament development and conchospores release in N. yezoensis.

The First Record of the Brown Endophytic Alga Laminariocolax aecidioides (Rosenvinge) A.F. Peters, 1998 in the Russian Far-Eastern Seas

The brown endophytic alga Laminariocolax aecidioides (Ectocarpales: Chordariaceae) growing in Undaria pinnatifida was found in Ussuriisky Bay (Peter the Great Bay, Sea of Japan) for the first time. This finding is the first record of the species in the Russian Far-Eastern seas. This paper presents the morphological features of L. aecidioides in nature and in a free-living culture, as well as some data on its biology in Peter the Great Bay.

EDUCAÇÃO TERRRITORIALIDADE E LUTA CIGANA

O presente artigo é fruto de uma investigação social, iniciada no mestrado e continuada por meio do Doutorado em Educação na Universidade de Brasília (UnB) o qual procura trazer à tona a realidade em que vivem os Ciganos Calon em Sobradinho DF no seu processo de territorialização e luta pela posse da terra. Comunidade que passa por um processo de mudanças culturais e transita de uma situação de nomadismo para uma de sedentarismo. Devido a sua cultura de vida livre, encontram inúmeras dificuldades de adaptação ao modelo de sociedade proposta. Sendo assim, a moradia fixa e a posse da terra ainda se tratam de espaços antagônicos aos seus modos históricos de vida. Desse modo, esta pesquisa abordará o percurso histórico-cultural dos povos ciganos, contribuindo para a análise e a compreensão das associações entre etnicidade cigana e a luta, buscando identificar uma política intercultural de educação amparada pelos Direitos Humanos, a qual permita a efetiva inclusão social e educacional dos povos ciganos no Distrito Federal. De caráter qualitativo, temos como base teórica o materialismo histórico dialético, o qual investiga as representações sociais acerca do povo cigano e como essas representações, por sua vez, influenciam nos seus conflitos étnicos e territoriais

Transcriptome of Thalassicolla nucleata Holobiont Reveals Details of a Radiolarian Symbiotic Relationship

Radiolarians are a group of ubiquitous, yet poorly understood, large protists that often harbor photosymbionts. We studied the solitary radiolarian Thalassicolla nucleata by analyzing the transcriptome of its holobiont. We found that T. nucleata contained two dinoflagellate symbionts, one photosymbiont Brandtodinium sp., and one putative Peridiniales parasite. Through comparisons of gene expressions of Brandtodinium sp. and those of a close relative from a free-living culture, we found that the Brandtodinium sp. maintained its photosynthetic activities, but altered its carbon metabolism dramatically in hospite. Gene expression data also suggested carbon and nitrogen exchange between the host and photosymbiont and that lectin-glycan interaction might play an important role in host-symbiont recognition.

Exo-Metabolites of Phaseolus vulgaris-Nodulating Rhizobial Strains.

Rhizobia are able to convert dinitrogen into biologically available forms of nitrogen through their symbiotic association with leguminous plants. This results in plant growth promotion, and also in conferring host resistance to different types of stress. These bacteria can interact with other organisms and survive in a wide range of environments, such as soil, rhizosphere, and inside roots. As most of these processes are molecularly mediated, the aim of this research was to identify and quantify the exo-metabolites produced by Rhizobium etli CFN42, Rhizobium leucaenae CFN299, Rhizobium tropici CIAT899, Rhizobium phaseoli Ch24-10, and Sinorhizobium americanum CFNEI156, by nuclear magnetic resonance (NMR). Bacteria were grown in free-living cultures using minimal medium containing sucrose and glutamate. Interestingly, we found that even when these bacteria belong to the same family (Rhizobiaceae) and all form nitrogen-fixing nodules on Phaseolus vulgaris roots, they exhibited different patterns and concentrations of chemical species produced by them.

MOLECULAR REGULATION OF PSEUDOMONAS AERUGINOSA BIOFILMS

Nowadays healthcare-associated infections caused Pseudomonas aeruginosa remain actual problem due to P. aeruginosa resistance to a wide range of antimicrobials and its ability to form biofilms that are 1000 times more resistant to antibiotics than free-living (plankton) cultures. P. aeruginosa biofilms forming is regulated by Quorum Sensing (QS) communication system which is controlled by inhibitors (Quorum sensing inhibitors, QSIs). An essential role in stabilization of biofilms belongs to extracellular DNA (eDNA) as a structural polyanionic polymer whereas its genetic function is not applicable. In addition, internal signal molecules c-di-GMP, cascade Gac/Rsm also participate in formation of P. aeruginosa biofilms. A review provides a detailed description of the biofilm molecular regulation by means of QS inhibitors and QS modulators of signaling molecules QS.

Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa.

The stringent response, mediated by the (p)ppGpp synthetase RelA and the RNA polymerase-binding protein DksA, is triggered by limiting nutrient conditions. For some bacteria, it is involved in regulation of virulence. We investigated the role of two DksA-like proteins from the Gram-negative nitrogen-fixing symbiont Sinorhizobium meliloti in free-living culture and in interaction with its host plant Medicago sativa The two paralogs, encoded by the genes SMc00469 and SMc00049, differ in the constitution of two major domains required for function in canonical DksA: the DXXDXA motif at the tip of a coiled-coil domain and a zinc finger domain. Using mutant analyses of single, double, and triple deletions for SMc00469(designated dksA),SMc00049, and relA, we found that the ΔdksA mutant but not the ΔSMc00049 mutant showed impaired growth on minimal medium, reduced nodulation on the host plant, and lower nitrogen fixation activity in early nodules, while its nod gene expression was normal. The ΔrelA mutant showed severe pleiotropic phenotypes under all conditions tested. Only S. meliloti dksA complemented the metabolic defects of an Escherichia coli dksA mutant. Modifications of the DXXDXA motif in SMc00049 failed to establish DksA function. Our results imply a role for transcriptional regulator DksA in the S. meliloti-M. sativa symbiosis. The stringent response is a bacterial transcription regulation process triggered upon nutritional stress.Sinorhizobium meliloti, a soil bacterium establishing agriculturally important root nodule symbioses with legume plants, undergoes constant molecular adjustment during host interaction. Analyzing the components of the stringent response in this alphaproteobacterium helps understand molecular control regarding the development of plant interaction. Using mutant analyses, we describe how the lack of DksA influences symbiosis with Medicago sativa and show that a second paralogous S. meliloti protein cannot substitute for this missing function. This work contributes to the field by showing the similarities and differences of S. meliloti DksA-like proteins to orthologs from other species, adding information to the diversity of the stringent response regulatory system.

The effects of gibberellins and mepiquat chloride on nitrogenase activity in Bradyrhizobium japonicum

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA3) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA3 or PIX. GA3 treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA3-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA3 and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA3 treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA3, respectively, in B. japonicum.

Perfil inicial del proteoma intracelular de biopelículas de Aspergillus niger

An initial profiling of the intracellular proteome of Aspergillus niger ATCC 10864 biofilm cultures developed on polyester cloth was carried out by using 2D-PAGE and MS-TOF analysis and it was compared to the proteome of conventionally grown free-living submerged cultures. A number of 2D-PAGE protein spots from both types of cultures were subjected to MS-TOF analysis and data interrogation of the NCBI nr database available for this species. Proteomic maps showed different expression patterns in both culture systems with differentially expressed proteins in each case. In biofilm cultures, 19% and 32% of the selected protein spots were over- expressed and differentially expressed, respectively. On the contrary, in free-living cultures, 44% and 7% of the selected protein spots were over-expressed and differentially expressed, respectively. Although preliminary, results presented in this paper show that there are significant differences between the proteomes of A. niger biofilm and free-living mycelia. It seems that cell adhesion is the most important stimulus responsible for biofilm development which is the basis of Surface Adhesion Fermentation.

Borate promotes the formation of a complex between legume AGP-extensin and Rhamnogalacturonan II and enhances production of Rhizobium capsular polysaccharide during infection thread development in Pisum sativum symbiotic root nodules.

The capacity to bind to biomolecules is considered to be the basis for any physiological role of boron (B). Legume arabinogalactan protein-extensin (AGPE), a major component of the infection thread matrix of legume nodules is a potential B-ligand. Therefore, its role in infection threads development was investigated in Pisum sativum grown under B deficiency. Using the AGPE-specific antibody MAC265, immunochemical analysis revealed that a 175 kDa MAC265 antigen was abundant in +B but much weaker in -B nodule extracts. A B-dependent complex involving AGPE and rhamnogalacturonan II (RGII) could be co-purified using anti-RGII antiserum. Following fractionation of -B nodules, MAC265 antigens were mostly associated with the bacterial pellet. Immunogold staining confirmed that AGPE was closely associated with the surface of rhizobia in the lumen of threads in -B nodules whereas in +B nodules, AGPE was separated from the bacterial surface by a sheath of capsular polysaccharide. Interestingly, colonies of rhizobia grown in free-living culture without B developed low capsule production. Therefore, we propose that B could be important for apical growth of infection threads by strengthening thread wall through a B-dependent AGPE-RGII interaction and by promoting bacterial advance through a B-dependent production of a stable rhizobial capsule that prevents AGPE attachment.

BacA is essential for bacteroid development in nodules of galegoid, but not phaseoloid, legumes.

BacA is an integral membrane protein, the mutation of which leads to increased resistance to the antimicrobial peptides bleomycin and Bac7(1-35) and a greater sensitivity to SDS and vancomycin in Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. phaseoli, and Rhizobium etli. The growth of Rhizobium strains on dicarboxylates as a sole carbon source was impaired in bacA mutants but was overcome by elevating the calcium level. While bacA mutants elicited indeterminate nodule formation on peas, which belong to the galegoid tribe of legumes, bacteria lysed after release from infection threads and mature bacteroids were not formed. Microarray analysis revealed almost no change in a bacA mutant of R. leguminosarum bv. viciae in free-living culture. In contrast, 45 genes were more-than 3-fold upregulated in a bacA mutant isolated from pea nodules. Almost half of these genes code for cell membrane components, suggesting that BacA is crucial to alterations that occur in the cell envelope during bacteroid development. In stark contrast, bacA mutants of R. leguminosarum bv. phaseoli and R. etli elicited the formation of normal determinate nodules on their bean host, which belongs to the phaseoloid tribe of legumes. Bacteroids from these nodules were indistinguishable from the wild type in morphology and nitrogen fixation. Thus, while bacA mutants of bacteria that infect galegoid or phaseoloid legumes have similar phenotypes in free-living culture, BacA is essential only for bacteroid development in indeterminate galegoid nodules.

Promotion of metal accumulation in nodule of Astragalus sinicus by the expression of the iron-regulated transporter gene in Mesorhizobium huakuii subsp. rengei B3

Toxic metal contamination in agricultural fields is an important worldwide problem. In previous studies, we developed a bioremediation system based on the symbiosis between Astragalus sinicus and the recombinant rhizobium, Mesorhizobium huakuii subsp. rengei B3 developed by overexpressing a synthetic tetrameric metallothionein gene (MTL4) and cDNA encoding the phytochelatin synthase from Arabidopsis thaliana (AtPCS). To promote the transport of metals into the nodules of the rhizobium and the accumulation of metals, the iron-regulated transporter 1 gene from A. thaliana (AtIRT1) was introduced into recombinant strain B3 containing MTL4 or AtPCS in its chromosome. The fused AtIRT1-alkaline phosphatase was expressed in the free-living recombinant rhizobium and the nodule of A. sinicus. The recombinant strain B3 carrying AtIRT1 showed a higher Cd sensitivity and a higher amount of Cd accumulated in free-living culture than the wild-type strain B3. When the recombinant strain B3 established symbiosis with A. sinicus, the introduction of AtIRT1 in the recombinant strain B3 advantaged the accumulation of Cu and As in the nodules of A. sinicus, compared with that of Cd and Zn.

Burkholderia phymatum is a highly effective nitrogen‐fixing symbiont of Mimosa spp. and fixes nitrogen ex planta

* The ability of Burkholderia phymatum STM815 to effectively nodulate Mimosa spp., and to fix nitrogen ex planta, was compared with that of the known Mimosa symbiont Cupriavidus taiwanensis LMG19424. * Both strains were equally effective symbionts of M. pudica, but nodules formed by STM815 had greater nitrogenase activity. STM815 was shown to have a broader host range across the genus Mimosa than LMG19424, nodulating 30 out of 31 species, 21 of these effectively. LMG19424 effectively nodulated only nine species. GFP-marked variants were used to visualise symbiont presence within nodules. * STM815 gave significant acetylene reduction assay (ARA) activity in semisolid JMV medium ex planta, but no ARA activity was detected with LMG19424. 16S rDNA sequences of two isolates originally from Mimosa nodules in Papua New Guinea (NGR114 and NGR195A) identified them as Burkholderia phymatum also, with nodA, nodC and nifH genes of NGR195A identical to those of STM815. * B. phymatum is therefore an effective Mimosa symbiont with a broad host range, and is the first reported beta-rhizobial strain to fix nitrogen in free-living culture.

Gene Products of the hupGHIJ Operon Are Involved in Maturation of the Iron-Sulfur Subunit of the [NiFe] Hydrogenase from Rhizobium leguminosarum bv. viciae

In the present study, we investigate the functions of the hupGHIJ operon in the synthesis of an active [NiFe] hydrogenase in the legume endosymbiont Rhizobium leguminosarum bv. viciae. These genes are clustered with 14 other genes including the hydrogenase structural genes hupSL. A set of isogenic mutants with in-frame deletions (deltahupG, deltahupH, deltahupI, and deltahupJ) was generated and tested for hydrogenase activity in cultures grown at different oxygen concentrations (0.2 to 2.0%) and in symbiosis with peas. In free-living cultures, deletions in these genes severely reduced hydrogenase activity. The deltahupH mutant was totally devoid of hydrogenase activity at any of the O2 concentration tested, whereas the requirement of hupGIJ for hydrogenase activity varied with the O2 concentration, being more crucial at higher pO2. Pea bacteroids from the mutant strains affected in hupH, hupI, and hupJ exhibited reduced (20 to 50%) rates of hydrogenase activity compared to the wild type, whereas rates were not affected in the deltahupG mutant. Immunoblot experiments with HupL- and HupS-specific antisera showed that free-living cultures from deltahupH, deltahupI, and deltahupJ mutants synthesized a fully processed mature HupL protein and accumulated an unprocessed form of HupS (pre-HupS). Both the mature HupL and the pre-HupS forms were located in the cytoplasmic fraction of cultures from the deltahupH mutant. Affinity chromatography experiments revealed that cytoplasmic pre-HupS binds to the HupH protein before the pre-HupS-HupL complex is formed. From these results we propose that hupGHIJ gene products are involved in the maturation of the HupS hydrogenase subunit.

DNA amplification fingerprinting as a tool for checking genetic purity of strains in the cyanobacterial inoculum

The electrophoretic patterns for 17 different cyanobacterial cultures derived from 6 different decamer primers were analysed to provide diagnostic fingerprints for each culture and their genetic distances based on RAPD markers.The primer OPB 09 produced a maximum of 24 amplified products. The primers OPB 09, OPG 04 and OPAH 02 generated markers specific for Nostoc cultures. Westiellopsis was found to be distinct from other cyanobacterial cultures in the RAPD profile obtained with the primer OPAH 02. The primer OPF 03 generated specific markers for Tolypothrix tenuis. Fischerella cultures could be identified with the primers OPB 09, OPAG 03 and OPF 05. The study revealed that these RAPD markers could be further used to identify and establish the genetic purity of the strains in the cyanobacterial inoculum. There was a similarity of 60–90% within Westiellopsis cultures. Nostoc cultures shared 50–80% similarity with Westiellopsis cultures. Anabaenacultures were similar to Westiellopsiscultures by 60–70. The markers produced for each culture were also applied to phylogenetic analysis to infer genetic relatedness in this group of prokaryotes. The dendrogram analysis clearly revealed that free-living cyanobacterial cultures are closely related to each other and are distinct from the symbiotic forms.

Growth and survival potentials of immobilized diazotrophic cyanobacterial isolates exposed to common ricefield herbicides

The effect of graded concentrations of four common ricefield herbicides (Arozin, Butachlor, Alachlor, 2,4-D) on diazotrophic growth, macromolecular contents, heterocyst frequency and tolerance potentials of Ca-alginate immobilized diazotrophic cyanobacterial isolates Nostoc punctiforme, N. calcicola, Anabaena variabilis, Gloeocapsasp., Aphanocapsa sp. and laboratory strain N. muscorum ISU (Anabaena ATCC 27893) was studied and compared with free-living cultures. Cyanobacterial isolates showed progressive inhibition of growth with increasing dosage of herbicides in both free and immobilized states. There were significant differences in the relative toxicity of the four herbicides. Arozin proved to be more growth toxic in comparison to Alachlor, Butachlor and 2,4-D. Growth performance of the immobilized cyanobacterial isolates under herbicide stress showed a similar diazotrophic growth pattern to free cells with no difference in lethal and sub-lethal dosages. However, at lethal concentrations of herbicides, the immobilized cells exhibited prolonged survivability of 14–16 days as compared to their free-living counterparts (8–12 days). The decline in growth, macromolecular contents and heterocyst frequency was found to be similar in both the states in graded dosages of herbicides. Of the test organisms, A. variabilis showed maximum natural tolerance towards all the four herbicides tested. Evidently immobilization by Ca-alginate seems to provide protection to the diazotrophic cyanobacterial inoculants to a certain extent against the growth-toxic action of herbicides.

Rhizobium leguminosarum methyl-accepting chemotaxis protein genes are down-regulated in the pea nodule.

Regulation of methyl-accepting chemotaxis protein (MCP) genes of Rhizobium leguminosarum was studied under symbiotic conditions. Transcriptional fusions using both beta-galactosidase and beta-glucuronidase genes within two different mcp genes demonstrated that mcp expression decreased significantly during nodulation. Immunoblots using an anti-MCP antibody detected MCPs in free-living cells but not in bacteroids. Down-regulation during nodulation was not dependent upon known regulatory proteins involved in induction of expression of genes involved in nitrogen fixation. Environmental conditions found in the bacteroid that may trigger down-regulation were investigated by growing free-living cultures under a variety of growth conditions. Growth under low oxygen concentration or using succinate as a sole carbon source did not lower expression of the mcp gene fusions.

Cell surface interactions of Rhizobium bacteroids and other bacterial strains with symbiosomal and peribacteroid membrane components from pea nodules.

Samples of Rhizobium bacteroids isolated from pea nodule symbiosomes reacted positively with a monoclonal antibody recognizing N-linked glycan epitopes on plant glycoproteins associated with the peribacteroid membrane and peribacteroid fluid. An antiserum recognizing the symbiosomal lectin-like glycoprotein PsNLEC-1 also reacted positively. Samples of isolated bacteroids also reacted with an antibody recognizing a glycolipid component of the peribacteroid membrane and plasma membrane. Bacterial cells derived from free-living cultures then were immobilized on nitrocellulose sheets and tested for their ability to associate with components of plant extracts derived from nodule fractionation. A positive antibody-staining reaction indicated that both PsNLEC-1 and membrane glycolipid had become associated with the bacterial surface. A range of rhizobial strains with mutants affecting cell surface polysaccharides all showed similar interactions with PsNLEC-1 and associated plant membranes, with the exception of strain B659 (a deep-rough lipopolysaccharide mutant of Rhizobium leguminosarum). However, the presence of a capsule of extracellular polysaccharide apparently prevented interactions between rhizobial cells and these plant components. The importance of a close association between peribacteroid membranes, PsNLEC-1, and the bacterial surface is discussed in the context of symbiosome development.

Regulation of L-alanine dehydrogenase in Rhizobium leguminosarum bv. viciae and its role in pea nodules.

Alanine dehydrogenase (AldA) is the principal enzyme with which pea bacteroids synthesize alanine de novo. In free-living culture, AldA activity is induced by carboxylic acids (succinate, malate, and pyruvate), although the best inducer is alanine. Measurement of the intracellular concentration of alanine showed that AldA contributes to net alanine synthesis in laboratory cultures. Divergently transcribed from aldA is an AsnC type regulator, aldR. Mutation of aldR prevents induction of AldA activity. Plasmid-borne gusA fusions showed that aldR is required for transcription of both aldA and aldR; hence, AldR is autoregulatory. However, plasmid fusions containing the aldA-aldR intergenic region could apparently titrate out AldR, sometimes resulting in a complete loss of AldA enzyme activity. Therefore, integrated aldR::gusA and aldA::gusA fusions, as well as Northern blotting, were used to confirm the induction of aldA activity. Both aldA and aldR were expressed in the II/III interzone and zone III of pea nodules. Overexpression of aldA in bacteroids did not alter the ability of pea plants to fix nitrogen, as measured by acetylene reduction, but caused a large reduction in the size and dry weight of plants. This suggests that overexpression of aldA impairs the ability of bacteroids to donate fixed nitrogen that the plant can productively assimilate. We propose that the role of AldA may be to balance the alanine level for optimal functioning of bacteroid metabolism rather than to synthesize alanine as the sole product of N(2) reduction.

Disparate oxygen responsiveness of two regulatory cascades that control expression of symbiotic genes in Bradyrhizobium japonicum.

Two oxygen-responsive regulatory systems controlling numerous symbiotic genes in Bradyrhizobium japonicum were assayed in free-living cultures for their capacity to activate target genes under different oxygen conditions. NifA- and FixLJ-controlled target genes showed disparate relative expression patterns. Induction of NifA-dependent genes was observed only at oxygen concentrations below 2% in the gas phase, whereas that of FixLJ-controlled targets progressively increased when the oxygen concentration was lowered from 21 to 5, 2, or 0.5%. We propose that this reflects a response to a gradient of increasing oxygen deprivation as bacteria invade their host during root nodule development.