scholarly journals Creation of an inducible vector system based on the rhizobia nodA gene promoter

2021 ◽  
Vol 19 (1) ◽  
pp. 13-21
Author(s):  
Olga V. Chubukova ◽  
Zilya R. Vershinina ◽  
Rustam T. Matnyazov ◽  
Andrey K. Baymiev ◽  
Aleksey К. Baymiev
Keyword(s):  
Rhizobium Leguminosarum ◽  
Regulatory Protein ◽  
Gene Promoter ◽  
Regulatory System ◽  
Vector System ◽  
Rhizosphere Microorganisms ◽  
Gfp Gene ◽  
Urgent Task ◽  
Expression Construct ◽  
Noda Gene

Background: The possibility of changing the properties of rhizobial bacteria by giving them the ability to regulate the expression of additionally introduced genes into them is an urgent task both for fundamental science and for applied agrobiology, since this will make it possible to obtain microsymbionts with desired properties. An expression construct using the rhizobia regulatory system was created in this work. The rhizobia nodD gene encodes a regulatory protein that, in the presence of plant inducers, flavonoids, activates the transcription of nod-genes involved in the early stages of the formation of legume-rhizobium symbiosis. Materials and methods: A vector construct containing the nodD gene from Rhizobium leguminosarum bv. trifoli under the regulation of its own promoter and the gfp gene under the regulation of the nodA gene promoter from the same rhizobia was obtained. Neorhizobium galegae CIAM 0702 were transformed with the vector construct. Results: It has been shown that in recombinant strains synthetic flavonoids are capable of inducing expression of gfp gene to varying degrees. Conclusion: In the future, the results can be used to obtain rhizosphere microorganisms with a controlled synthesis of growth-stimulating and protective substances.

Regulation of apolipoprotein A-I gene expression in Hep G2 cells depleted of Cu by cupruretic tetramine

1997 ◽  
Vol 273 (4) ◽  
pp. C1362-C1370 ◽  
Author(s):  
John Y. J. Wu ◽  
Jin J. Zhang ◽  
Yenshen Wang ◽  
Scott K. Reaves ◽  
Yi Ran Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Regulatory Protein ◽  
Gene Promoter ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Nuclear Factors ◽  
Hepatocyte Nuclear Factor 4 ◽  
G2 Cells ◽  
I Gene

Studies were designed to examine the regulation of apolipoprotein (apo) A-I gene expression in Cu-depleted Hep G2 cells. The cupruretic chelator N, N′-bis(2-aminoethyl)-1,3-propanediamine ⋅ 4 HCl (2,3,2-tetramine or TETA) was used to maintain a 77% reduction in cellular Cu in Hep G2 cells. After two passages of TETA treatment, the relative abundance of apoA-I mRNA was elevated 52%. In TETA-treated cells, the rate of apoA-I mRNA decay measured by an actinomycin D chase study was accelerated 108%, and the synthesis of apoA-I mRNA determined by a nuclear runoff assay was enhanced 2.5-fold in TETA-treated cells. All of those changes could be reverted toward the control values with Cu supplementation for only 2 days. In transient transfection assays, a 26.7% increase in chloramphenicol O-acetyltransferase (CAT) activity for the reporter construct −256AI-CAT was observed in the treated cells. However, the ability of apoA-I regulatory protein 1 (ARP-1) to repress the CAT activity was not affected by the depressed Cu status. In addition, gel retardation experiments demonstrated that Cu depletion enhanced the binding of hepatocyte nuclear factor 4 (HNF-4) and other undefined nuclear factors to oligonucleotides containing site A, one of three regulatory sites of the apoA-I gene promoter. Moreover, the relative abundance of HNF-4 mRNA was increased 58% in the Cu-depleted cells. Thus the observed increase in apoA-I gene transcription may be mediated mostly by an elevated level of the regulatory factor, HNF-4. In summary, the present findings established the mechanism by which a depressed cellular Cu status can enhance apoA-I mRNA production and subsequently increase apoA-I synthesis.

scholarly journals RocA Binds CsrS To Modulate CsrRS-Mediated Gene Regulation in Group A Streptococcus

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Nicola N. Lynskey ◽  
Jorge J. Velarde ◽  
Meredith B. Finn ◽  
Simon L. Dove ◽  
Michael R. Wessels
Keyword(s):  
Gene Regulation ◽  
Target Genes ◽  
Response Regulator ◽  
Critical Role ◽  
Regulatory Protein ◽  
Regulatory System ◽  
Fine Tuning ◽  
Human Pathogen ◽  
Two Component ◽  
Group A

ABSTRACT The orphan regulator RocA plays a critical role in the colonization and pathogenesis of the obligate human pathogen group A Streptococcus. Despite multiple lines of evidence supporting a role for RocA as an auxiliary regulator of the control of virulence two-component regulatory system CsrRS (or CovRS), the mechanism of action of RocA remains unknown. Using a combination of in vitro and in vivo techniques, we now find that RocA interacts with CsrS in the streptococcal membrane via its N-terminal region, which contains seven transmembrane domains. This interaction is essential for RocA-mediated regulation of CsrRS function. Furthermore, we demonstrate that RocA forms homodimers via its cytoplasmic domain. The serotype-specific RocA truncation in M3 isolates alters this homotypic interaction, resulting in protein aggregation and impairment of RocA-mediated regulation. Taken together, our findings provide insight into the molecular requirements for functional interaction of RocA with CsrS to modulate CsrRS-mediated gene regulation. IMPORTANCE Bacterial two-component regulatory systems, comprising a membrane-bound sensor kinase and cytosolic response regulator, are critical in coordinating the bacterial response to changing environmental conditions. More recently, auxiliary regulators which act to modulate the activity of two-component systems, allowing integration of multiple signals and fine-tuning of bacterial responses, have been identified. RocA is a regulatory protein encoded by all serotypes of the important human pathogen group A Streptococcus. Although RocA is known to exert its regulatory activity via the streptococcal two-component regulatory system CsrRS, the mechanism by which it functions was unknown. Based on new experimental evidence, we propose a model whereby RocA interacts with CsrS in the streptococcal cell membrane to enhance CsrS autokinase activity and subsequent phosphotransfer to the response regulator CsrR, which mediates transcriptional repression of target genes.

Transferrin receptor hyperexpression in primary erythroblasts is lost on transformation by avian erythroblastosis virus

Blood ◽  
2002 ◽  
Vol 100 (1) ◽  
pp. 289-298 ◽  
Author(s):  
Lioba Lobmayr ◽  
Thomas Sauer ◽  
Iris Killisch ◽  
Matthias Schranzhofer ◽  
Robert B. Wilson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Messenger Rna ◽  
Large Fraction ◽  
Regulatory Protein ◽  
Regulatory System ◽  
Cell Types ◽  
Binding Activity ◽  
Chicken Cell ◽  
Avian Erythroblastosis Virus

Abstract In primary chicken erythroblasts (stem cell factor [SCF] erythroblasts), transferrin receptor (TfR) messenger RNA (mRNA) and protein were hyperexpressed as compared to nonerythroid chicken cell types. This erythroid-specific hyperexpression was abolished in transformed erythroblasts (HD3E22 cells) expressing the v-ErbA and v-ErbB oncogenes of avian erythroblastosis virus. TfR expression in HD3E22 cells could be modulated by changes in exogenous iron supply, whereas expression in SCF erythroblasts was not subject to iron regulation. Measurements of TfR mRNA half-life indicated that hyperexpression in SCF erythroblasts was due to a massive stabilization of transcripts even in the presence of high iron levels. Changes in mRNA binding activity of iron regulatory protein 1 (IRP1), the primary regulator of TfR mRNA stability in these cells, correlated well with TfR mRNA expression; IRP1 activity in HD3E22 cells and other nonerythroid cell types tested was iron dependent, whereas IRP1 activity in primary SCF erythroblasts could not be modulated by iron administration. Analysis of avian erythroblasts expressing v-ErbA alone indicated that v-ErbA was responsible for these transformation-specific alterations in the regulation of iron metabolism. In SCF erythroblasts high amounts of TfR were detected on the plasma membrane, but a large fraction was also located in early and late endosomal compartments, potentially concealing temporary iron stores from the IRP regulatory system. In contrast, TfR was almost exclusively located to the plasma membrane in HD3E22 cells. In summary, stabilization of TfR mRNA and redistribution of Fe-Tf/TfR complexes to late endosomal compartments may contribute to TfR hyperexpression in primary erythroblasts, effects that are lost on leukemic transformation.

scholarly journals Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2075-2089 ◽  
Author(s):  
Yaoping Zhang ◽  
David M. Wolfe ◽  
Edward L. Pohlmann ◽  
Mary C. Conrad ◽  
Gary P. Roberts
Keyword(s):  
Translational Regulation ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Regulatory Protein ◽  
Regulatory System ◽  
Poor Response ◽  
Photosynthetic Bacterium ◽  
Nitrogen Acquisition ◽  
Consistent Model ◽  
Energy Deprivation

The AmtB protein transports uncharged NH3 into the cell, but it also interacts with the nitrogen regulatory protein PII, which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three PII homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB 1 and amtB 2, have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB1 are defective in their response to both addition and darkness, while mutants lacking AmtB2 show little effect on the regulation of nitrogenase activity. These responses to darkness and appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB1 is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB1-PII regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by PII is proposed. Other results demonstrate a degree of specificity in interaction of AmtB1 with the different PII homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which PII proteins regulate processes involved in nitrogen acquisition and utilization.

scholarly journals Host-Dependent Expression of Rhizobium leguminosarum bv. viciae Hydrogenase Is Controlled at Transcriptional and Post-Transcriptional Levels in Legume Nodules

2008 ◽  
Vol 21 (5) ◽  
pp. 597-604 ◽  
Author(s):  
Belén Brito ◽  
Annita Toffanin ◽  
Rosa-Isabel Prieto ◽  
Juan Imperial ◽  
Tomás Ruiz-Argüeso ◽  
...  
Keyword(s):  
Rhizobium Leguminosarum ◽  
Root Nodules ◽  
Gene Promoter ◽  
Hydrogenase Activity ◽  
Transcription Analysis ◽  
Nickel Binding ◽  
Level Of Activity ◽  
Structural Subunit ◽  
Hup Genes ◽  
Related Proteins

The legume host affects the expression of Rhizobium leguminosarum hydrogenase activity in root nodules. High levels of symbiotic hydrogenase activity were detected in R. leguminosarum bacteroids from different hosts, with the exception of lentil (Lens culinaris). Transcription analysis showed that the NifA-regulated R. leguminosarum hydrogenase structural gene promoter (P1) is poorly induced in lentil root nodules. Replacement of the P1 promoter by the FnrN-dependent promoter of the fixN gene restored transcription of hup genes in lentil bacteroids, but not hydrogenase activity. In the PfixN-hupSL strain, additional copies of the hup gene cluster and nickel supplementation to lentil plants increased bacteroid hydrogenase activity. However, the level of activity in lentil still was significantly lower than in pea bacteroids, indicating that an additional factor is impairing hydrogenase expression inside lentil nodules. Immunological analysis revealed that lentil bacteroids contain reduced levels of both hydrogenase structural subunit HupL and nickel-binding protein HypB. Altogether, results indicate that hydrogenase expression is affected by the legume host at the level of both transcription of hydrogenase structural genes and biosynthesis or stability of nickel-related proteins HypB and HupL, and suggest the existence of a plant-dependent mechanism that affects hydrogenase activity during the symbiosis by limiting nickel availability to the bacteroid.

scholarly journals Cell-specific expression of the human gastrin gene: evidence for a control element located downstream of the TATA box.

1987 ◽  
Vol 7 (12) ◽  
pp. 4329-4336 ◽  
Author(s):  
L E Theill ◽  
O Wiborg ◽  
J Vuust
Keyword(s):  
Cell Line ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Tata Box ◽  
Vector System ◽  
Control Element ◽  
Gene Fragment ◽  
Early Promoter ◽  
Sv40 Enhancer ◽  
Human Gastrin

Fragments of 5'-flanking and noncoding exon I sequences of the human gastrin gene were analyzed in transient expression assays after transfection of a variety of cell lines with the pSVCAT vector system. In the presence of the simian virus 40 (SV40) enhancer, the gastrin gene fragment from nucleotides -250 to +57, relative to the cap site, was as efficient a promoter as the SV40 early promoter itself. In the absence of the SV40 enhancer, gastrin gene 5'-flanking sequences had no promoter activity except in the murine neuroblastoma cell line N18TG2. In this cell line, the fragment from -1300 to +57 stimulated transcription as actively as the SV40 early promoter with its enhancer. This cell-specific gastrin gene promoter activity was in accordance with the finding that gastrin is synthesized in certain neuronal cells. Promoter activity declined with decreasing distance from the 5' end to the cap site and disappeared after removal of the gastrin gene TATA box. In vector constructions containing short vector-linker sequences homologous to a functionally important region of the SV40 enhancer, the gastrin gene fragment from -17 to +57 showed considerable promoter activity, exclusively in N18TG2. It is concluded that the truncated gastrin gene promoter plus the first exon contains a cell-specific element that may act in collaboration with upstream elements to facilitate the accumulation of transcripts.

A multitude of CRP/FNR-like transcription proteins in Bradyrhizobium japonicum

2006 ◽  
Vol 34 (1) ◽  
pp. 156-159 ◽  
Author(s):  
S. Mesa ◽  
H. Hennecke ◽  
H.-M. Fischer
Keyword(s):  
Gene Expression ◽  
Bradyrhizobium Japonicum ◽  
Sequence Similarity ◽  
Regulatory Protein ◽  
Regulatory System ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Low Oxygen ◽  
Denitrification Gene ◽  
Camp Receptor

In Bradyrhizobium japonicum, the nitrogen-fixing soya bean endosymbiont and facultative denitrifier, three CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein)-type transcription factors [FixK1, FixK2 and NnrR (nitrite and nitric oxide reductase regulator)] have been studied previously in the context of the regulation of nitrogen fixation and denitrification. The gene expression of both fixK1 and nnrR depends on FixK2, which acts as a key distributor of the ‘low-oxygen’ signal perceived by the two-component regulatory system FixLJ. While the targets for FixK1 are not known, NnrR transduces the nitrogen oxide signal to the level of denitrification gene expression. Besides these three regulators, the complete genome sequence of this organism has revealed the existence of 13 additional CRP/FNR-type proteins whose functions have not yet been studied. Based on sequence similarity and phylogenetic analysis, we discuss in this paper the peculiarities of these additional factors.

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