scholarly journals Stable Genetic Transformation and Heterologous Expression in the Nitrogen-fixing Plant Endosymbiont Frankia alni ACN14a

2018 ◽  
Author(s):  
Isaac Gifford ◽  
Summer Vance ◽  
Giang Nguyen ◽  
Alison M Berry
Keyword(s):  
Genetic Transformation ◽  
Root Nodule ◽  
Cell Types ◽  
Nitrogen Fixing ◽  
Chloramphenicol Resistance ◽  
Type Iv ◽  
Frankia Alni ◽  
Limited Culture ◽  
Nif Gene Cluster ◽  
Common Plant

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes, which are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. However the corresponding endosymbionts, Frankia and the rhizobia, are distantly related groups of bacteria, leading to questions of their symbiotic mechanisms and evolutionary history. To date, a stable system of genetic transformation has been lacking in Frankia. Here, we report the successful electrotransformation of Frankia alni ACN14a, by means of replicating plasmids expressing chloramphenicol-resistance for selection, and the use of GFP as a marker of gene expression. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation and circumvented this barrier by using unmethylated plasmids, which allowed the transformation of F. alni as well as the maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the plasmid transformation system was used with expression of egfp under the control of the nif gene cluster promoter, it was possible to demonstrate by fluorescence imaging the expression of nitrogen fixation in vesicles but not hyphae in nitrogen-limited culture.

scholarly journals Casuarina glauca Prenodule Cells Display the Same Differentiation as the Corresponding Nodule Cells

2000 ◽  
Vol 13 (1) ◽  
pp. 107-112 ◽  
Author(s):  
Laurent Laplaze ◽  
Emile Duhoux ◽  
Claudine Franche ◽  
Thierry Frutz ◽  
Sergio Svistoonoff ◽  
...  
Keyword(s):  
Root Nodule ◽  
Common Ancestor ◽  
Cortical Cell ◽  
Cell Types ◽  
Nitrogen Fixing ◽  
Nitrogen Fixing Bacteria ◽  
Casuarina Glauca ◽  
Cell Divisions ◽  
Phylogenetic Studies ◽  
Infected Cells

Recent phylogenetic studies have implied that all plants able to enter root nodule symbioses with nitrogen-fixing bacteria go back to a common ancestor (D. E. Soltis, P. S. Soltis, D. R. Morgan, S. M. Swensen, B. C. Mullin, J. M. Dowd, and P. G. Martin, Proc. Natl. Acad. Sci. USA, 92:2647–2651, 1995). However, nodules formed by plants from different groups are distinct in nodule organogenesis and structure. In most groups, nodule organogenesis involves the induction of cortical cell divisions. In legumes these divisions lead to the formation of a nodule primordium, while in non-legumes they lead to the formation of a so-called prenodule consisting of infected and un-infected cells. Nodule primordium formation does not involve prenodule cells, and the function of prenodules is not known. Here, we examine the differentiation of actinorhizal prenodule cells in comparison to nodule cells with regard to both symbionts. Our findings indicate that prenodules represent primitive symbiotic organs whose cell types display the same characteristics as their nodule counterparts. The results are discussed in the context of the evolution of root nodule symbioses.

scholarly journals A Genome-Scale Metabolic Model of Anabaena 33047 to Guide Genetic Modifications to Overproduce Nylon Monomers

Metabolites ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 168
Author(s):  
John I. Hendry ◽  
Hoang V. Dinh ◽  
Debolina Sarkar ◽  
Lin Wang ◽  
Anindita Bandyopadhyay ◽  
...  
Keyword(s):  
Electron Transport ◽  
Pyruvate Decarboxylase ◽  
Metabolic Model ◽  
Cell Types ◽  
Economic Feasibility ◽  
Nitrogen Fixing ◽  
Design Algorithm ◽  
A Genome ◽  
Anabaena Sp ◽  
Genome Scale

Nitrogen fixing-cyanobacteria can significantly improve the economic feasibility of cyanobacterial production processes by eliminating the requirement for reduced nitrogen. Anabaena sp. ATCC 33047 is a marine, heterocyst forming, nitrogen fixing cyanobacteria with a very short doubling time of 3.8 h. We developed a comprehensive genome-scale metabolic (GSM) model, iAnC892, for this organism using annotations and content obtained from multiple databases. iAnC892 describes both the vegetative and heterocyst cell types found in the filaments of Anabaena sp. ATCC 33047. iAnC892 includes 953 unique reactions and accounts for the annotation of 892 genes. Comparison of iAnC892 reaction content with the GSM of Anabaena sp. PCC 7120 revealed that there are 109 reactions including uptake hydrogenase, pyruvate decarboxylase, and pyruvate-formate lyase unique to iAnC892. iAnC892 enabled the analysis of energy production pathways in the heterocyst by allowing the cell specific deactivation of light dependent electron transport chain and glucose-6-phosphate metabolizing pathways. The analysis revealed the importance of light dependent electron transport in generating ATP and NADPH at the required ratio for optimal N2 fixation. When used alongside the strain design algorithm, OptForce, iAnC892 recapitulated several of the experimentally successful genetic intervention strategies that over produced valerolactam and caprolactam precursors.

Differential expression of type-specific c-abl mRNAs in mouse tissues and cell lines

1988 ◽  
Vol 8 (10) ◽  
pp. 4547-4551
Author(s):  
M W Renshaw ◽  
M A Capozza ◽  
J Y Wang
Keyword(s):  
Alternative Splicing ◽  
Differential Expression ◽  
Cell Lines ◽  
Relative Abundance ◽  
Cell Types ◽  
Type I ◽  
Rnase Protection ◽  
Type Iv ◽  
Mouse Tissues ◽  
Different Tissues

The mammalian c-abl proto-oncogene produces mRNAs with 5' heterogeneity from two distinct promoters and the alternative splicing of variable 5' exons. By using quantitative RNase protection assays, the relative abundance of two major c-abl mRNAs, type I and type IV, in several mouse tissues and cell lines has been determined. Our results demonstrate that the level of type IV c-abl mRNA is rather constant, whereas that of the type I mRNA varies over a 10-fold range in different tissues and cell types. This finding has interesting implications for the function of the two c-abl proteins.

Role of integrins in melanocyte attachment and dendricity

1994 ◽  
Vol 107 (10) ◽  
pp. 2739-2748 ◽  
Author(s):  
M. Hara ◽  
M. Yaar ◽  
A. Tang ◽  
M.S. Eller ◽  
W. Reenstra ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Biology ◽  
Type Iv Collagen ◽  
Cell Attachment ◽  
Pigment Cell ◽  
Cell Types ◽  
Dependent Manner ◽  
Flow Cytometry Analysis ◽  
Type Iv ◽  
Alpha V Beta 3

Integrins are a family of proteins known to mediate attachment of cells to extracellular matrix materials. The substratum specificity and cation dependence of specific integrin heterodimers have been extensively characterized, and to a lesser degree specialized roles in cell attachment versus dendricity have been defined in some cell types. In the past decade, melanocyte attachment rate and morphology have been found to have strong substratum dependence, suggesting a major role for integrins in these processes. In order to investigate this aspect of pigment cell biology, human newborn melanocytes were subjected to flow cytometry analysis and plated on a variety of substrata under conditions known to promote or block the binding of specific integrin pairs. Melanocyte attachment to laminin and type IV collagen was promoted by Mg2+ and Mn2+ but not by Ca2+, in the range of concentrations examined. However, dendrite outgrowth from melanocytes already attached on laminin or type IV collagen was promoted by Ca2+ to a far greater degree than by Mg2+, and Mn2+ had no effect on dendrite outgrowth. Flow cytometry analysis revealed that melanocytes expressed beta 1, alpha 2, alpha 3, alpha 5, alpha 6 and alpha v integrin subunits as well as the alpha v beta 3 heterodimer. The influence of substratum on the profile of integrin expression was minimal, but alpha 6 and beta 1 integrins were observed by confocal microscopy to be expressed over the entire cell surface, while alpha 2, alpha 5 and alpha v beta 3 integrins localized along dendritic processes or at their tips. In accordance with the implications of these distribution patterns, anti-beta 1 and anti-alpha 6 integrin monoclonal antibodies blocked melanocyte attachment to laminin, while anti-alpha 2, anti-alpha 5 and anti-alpha v beta 3 inhibited dendrite outgrowth but did not block substratum attachment on either laminin or type IV collagen. On the basis of these data and the known characteristics of integrin molecules, we conclude that melanocyte attachment to laminin is mediated primarily by alpha 6 beta 1 integrin in a Ca(2+)-independent, Mg(2+)- and/or Mn(2+)-dependent manner, while dendrite outgrowth on laminin and type IV collagen requires extracellular Ca2+ and is mediated by alpha v beta 3 as well as alpha 2 and alpha 5 integrins.

scholarly journals A New Species of Devosia That Forms a Unique Nitrogen-Fixing Root-Nodule Symbiosis with the Aquatic Legume Neptunia natans (L.f.) Druce

2002 ◽  
Vol 68 (11) ◽  
pp. 5217-5222 ◽  
Author(s):  
Raul Rivas ◽  
Encarna Velázquez ◽  
Anne Willems ◽  
Nieves Vizcaíno ◽  
Nanjappa S. Subba-Rao ◽  
...  
Keyword(s):  
Root Nodule ◽  
Root Nodules ◽  
Infection Process ◽  
Nitrogen Fixing ◽  
Rhizobium Tropici ◽  
Rdna Sequences ◽  
Root Nodule Symbiosis ◽  
16S Rdna Sequences ◽  
The Common ◽  
Nodule Symbiosis

ABSTRACT Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants. Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola. This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N. natans and found that they are phylogenetically located in the genus Devosia, not Allorhizobium or Rhizobium. The 16S rDNA sequences of these two Neptunia-nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina. From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici, suggesting that this newly described Neptunia-nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.

Distribution, frequency of occurrence and symbiotic properties of the Australian native legume Trigonella suavissima Lindl. and its associated root-nodule bacteria

2010 ◽  
Vol 32 (4) ◽  
pp. 395 ◽  
Author(s):  
J. Brockwell ◽  
Catherine M. Evans ◽  
Alison M. Bowman ◽  
Alison McInnes
Keyword(s):  
Root Nodule ◽  
Clay Soils ◽  
Frequency Of Occurrence ◽  
Nitrogen Fixing ◽  
Nodule Bacteria ◽  
River Systems ◽  
Root Nodule Bacteria ◽  
North East ◽  
Inland River ◽  
Native Legume

Trigonella suavissima Lindl. is an Australian native legume belonging to the tribe Trifolieae. It is an ephemeral species that is widely distributed in the arid interior of the continent where it occurs, following periodic inundation, on clay soils of the watercourse country of the Channel Country (far-western Queensland, north-east South Australia and north-western New South Wales). T. suavissima is the only member of its tribe that is endemic to Australia. Likewise, its root-nodule bacteria (Sinorhizobium sp.) may be the only member of its taxonomic group (S. meliloti, S. medicae) that is an Australian native. The distribution and frequency of occurrence of T. suavissima and the size of soil populations (density) of Sinorhizobium were monitored at 64 locations along inland river systems of the Channel Country. Measurements were made of (i) the nitrogen-fixing effectiveness of the symbioses between T. suavissima and strains of its homologous Sinorhizobium and (ii) the nitrogen-fixing effectiveness of the symbioses between legumes symbiotically related to T. suavissima and diverse strains of Sinorhizobium. It was concluded that the distribution and frequency of occurrence of T. suavissima is soil related. The species is most widespread on fine-textured clay soils with deep, self-mulching surfaces and high moisture-holding capacity. By contrast, the occurrence of T. suavissima is sporadic in the upper reaches of the inland river systems where the soils are poorly structured clays with lower moisture-holding capacity. Sinorhizobium is most abundant where the plant is most common. The nitrogen-fixing symbioses between T. suavissima and strains of Sinorhizobium isolated from soils across the region were consistently effective and often highly effective. Some of these strains fixed a little nitrogen with lucerne (Medicago sativa L.). T. suavissima also had some symbiotic (nitrogen-fixing) affinity with an exotic Trigonella (T. arabica Del.). The economic value of T. suavissima (and its symbiosis with Sinorhizobium) to the beef industry in the Channel Country is discussed.

Clinical, Pathological, and Molecular Correlates in Ferroportin Disease. A Study of Two Novel Mutations.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 706-706
Author(s):  
Domenico Girelli ◽  
Ivana De Domenico ◽  
Claudia Bozzini ◽  
Ilaria Tenuti ◽  
Nadia Soriani ◽  
...  
Keyword(s):  
Iron Overload ◽  
Cellular Localization ◽  
Transferrin Saturation ◽  
Cell Types ◽  
Mouse Bone Marrow ◽  
Pathological Features ◽  
Functional Studies ◽  
Type Iv

Abstract Background: Mutations in the iron exporter Ferroportin (Fpn) lead to type IV hemochromatosis (Ferroportin Disease, FD), a dominantly inherited disorder with heterogeneous clinical and biochemical patterns. Some patients present with predominant macrophage iron overload (M), marked elevation of serum ferritin, normal-to-low transferrin saturation (TS), and, possibly, iron restricted erythropoiesis. Others present with a phenotype resembling classical HFE-related hemochromatosis, i.e. characterized by high TS and predominant hepatocyte iron overload (H). These differences are thought to reflect heterogeneity in the functional behaviour of Fpn mutant proteins. Methods: Two unrelated probands referring to the Centre for Iron Overload Disorders in Verona because of non-HFE hemochromatosis were screened for Fpn mutations by DHPLC (Cremonesi L, Br J Haematol 2005). The functional behaviour of mutants Fpn was studied by generating Fpn-GFP constructs transfected into different cell types (HEK293T, Cos7, and mouse bone marrow macrophages), and analyzing their cellular localization, as well as their capabilities to bind hepcidin and export iron (De Domenico I, PNAS 2005). The two mutations were also expressed in zebrafish, to evaluate their impact on iron-dependent erythropoiesis. Results: Patient 1, a 59 year old male, had clinical, biochemical (TS 74.8%, ferritin 9,000 μg/l), and pathological features (marked iron overload in either macrophages and hepatocytes, absence of overt cirrhosis) somewhat ambiguous, possibly suggesting a type M Fpn variant with late secondary hepatocyte overload. He was found to be heterozygous for the new L233P mutation. Functional studies revealed that Fpn L233P does not appropriately traffic to the cell surface, resulting in inappropriate inhibition by hepcidin. Fpn L233P expression in vivo in zebrafish resulted in iron limited erythropoiesis, consistent with a type M mutation leading to macrophage iron retention. Patient 2, a 59 year old female, had features more clearly suggesting a type M Fpn variant (TS 22.7%, ferritin 1,771 μg/l, macrophage iron load), but tolerated very well phlebotomies without developing signs of anemia. She was found to be heterozygous for the new I152F mutation. Functional studies revealed a unique pattern (never observed until now), since Fpn I152F localized appropriately on cell membrane, bound near normally to hepcidin, but showed a “primary” deficit of iron export capability. I152F expression in zebrafish resulted in a trend towards iron limited erythropoiesis, though quantitatively less clear than L223P. Conclusions: FD is a heterogeneous disease caused by generally “private” mutations in Fpn. The clinical, biochemical, and pathological features vary depending on the different behaviour of mutant Fpn. In vitro and in vivo molecular expression studies are very useful to clarify the pathophysiogical spectrum of this disease.

scholarly journals FrankiaDiversity in Host Plant Root Nodules Is Independent of Abundance or Relative Diversity ofFrankiaPopulations in Corresponding Rhizosphere Soils

2017 ◽  
Vol 84 (5) ◽  
Author(s):  
Seifeddine Ben Tekaya ◽  
Trina Guerra ◽  
David Rodriguez ◽  
Jeffrey O. Dawson ◽  
Dittmar Hahn
Keyword(s):  
Host Plant ◽  
Root Nodule ◽  
Root Nodules ◽  
Actinorhizal Plants ◽  
Nodule Formation ◽  
Nitrogen Fixing ◽  
Content Type ◽  
Rhizosphere Soils ◽  
Root Nodule Formation ◽  
Host Infection

ABSTRACTActinorhizal plants form nitrogen-fixing root nodules in symbiosis with soil-dwelling actinobacteria within the genusFrankia, and specificFrankiataxonomic clusters nodulate plants in corresponding host infection groups. In same-soil microcosms, we observed that some host species were nodulated (Alnus glutinosa,Alnus cordata,Shepherdia argentea,Casuarina equisetifolia) while others were not (Alnus viridis,Hippophaë rhamnoides). Nodule populations were represented by eight different sequences ofnifHgene fragments. Two of these sequences characterized frankiae inS. argenteanodules, and three others characterized frankiae inA. glutinosanodules. Frankiae inA. cordatanodules were represented by five sequences, one of which was also found in nodules fromA. glutinosaandC. equisetifolia, while another was detected in nodules fromA. glutinosa. Quantitative PCR assays showed that vegetation generally increased the abundance of frankiae in soil, independently of the target gene (i.e.,nifHor the 23S rRNA gene). Targeted Illumina sequencing ofFrankia-specificnifHgene fragments detected 24 unique sequences from rhizosphere soils, 4 of which were also found in nodules, while the remaining 4 sequences in nodules were not found in soils. Seven of the 24 sequences from soils represented >90% of the reads obtained in most samples; the 2 most abundant sequences from soils were not found in root nodules, and only 2 of the sequences from soils were detected in nodules. These results demonstrate large differences between detectableFrankiapopulations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specificFrankiapopulations in soils.IMPORTANCEThe nitrogen-fixing actinobacteriumFrankiaforms root nodules on actinorhizal plants, with members of specificFrankiataxonomic clusters nodulating plants in corresponding host infection groups. We assessedFrankiadiversity in root nodules of different host plant species, and we related specific populations to the abundance and relative distribution of indigenous frankiae in rhizosphere soils. Large differences were observed between detectableFrankiapopulations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specificFrankiapopulations in soils but rather results from plants potentially selecting frankiae from the soil for root nodule formation. These data also highlight the necessity of using a combination of different assessment tools so as to adequately address methodological constraints that could produce contradictory data sets.

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