scholarly journals The Serine Biosynthesis of Paenibacillus polymyxa WLY78 Is Regulated by the T-Box Riboswitch

2021 ◽  
Vol 22 (6) ◽  
pp. 3033
Author(s):  
Haowei Zhang ◽  
Qin Li ◽  
Yongbin Li ◽  
Sanfeng Chen
Keyword(s):  
Nitrogen Fixation ◽  
Noncoding Rna ◽  
Nitrogenase Activity ◽  
Constitutive Expression ◽  
Paenibacillus Polymyxa ◽  
Leader Region ◽  
Coding Region ◽  
Transcriptional Terminator ◽  
Stem Loop ◽  
T Box

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.

scholarly journals NfiR, a New Regulatory Noncoding RNA (ncRNA), Is Required in Concert with the NfiS ncRNA for Optimal Expression of Nitrogenase Genes in Pseudomonas stutzeri A1501

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Yuhua Zhan ◽  
Zhiping Deng ◽  
Yongliang Yan ◽  
Hongyang Zhang ◽  
Chao Lu ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Stress Responses ◽  
Posttranscriptional Regulation ◽  
Noncoding Rna ◽  
Nitrogenase Activity ◽  
Pseudomonas Stutzeri ◽  
Fine Tuning ◽  
Coding Region ◽  
Content Type ◽  
Nitrogenase Genes

ABSTRACT Expression of nitrogenase genes (nifHDK) is strictly regulated at both transcriptional and posttranscriptional levels. Efficient nitrogenase activity requires maintaining sufficient levels of nif mRNAs, yet the underlying mechanism is not fully understood due to its complexity. We have previously shown that a novel regulatory noncoding RNA (ncRNA), NfiS, optimizes nitrogen fixation through targeting nifK mRNA in Pseudomonas stutzeri A1501. Here, we report the identification and characterization of a second ncRNA inducible under nitrogen fixation conditions (nitrogen-free and microaerobic conditions), termed NfiR (for nitrogen fixation condition-inducible ncRNA), the expression of which is dependent on two global regulators, NtrC and Hfq. Comparative phenotypic and proteomic analyses of an nfiR mutant identify a role of NfiR in regulating the expression of nitrogenase genes. Further microscale thermophoresis and genetic complementation showed that an 11-nucleotide (nt) sequence in the stem-loop structure of NfiR (nucleotides 12 to 22) pairs with its counterpart in the coding region of nifD mRNA (nucleotides 1194 to 1207) by eight nucleotides. Significantly, deletion of nfiR caused a 60% reduction of nitrogenase activity, and the half-life of nifD mRNA was reduced from 20 min for the wild type to 15 min for the ΔnfiR mutant. With regard to nitrogenase activity and stability of the nifD and nifK transcripts, phenotypes were more severe for the double deletion mutant lacking nfiR and nfiS, suggesting that NfiR, in concert with NfiS, optimizes nitrogenase production at the posttranscriptional level. IMPORTANCE Biological nitrogen fixation is an energy-expensive process requiring the hydrolysis of 16 ATPs. Consequently, the expression of nif genes is highly regulated at both transcriptional and posttranscriptional levels through complex regulatory networks. Global regulation involves a number of regulatory proteins, such as the nif-specific activator NifA and the global nitrogen regulator NtrC, as well as various regulatory ncRNAs. We show that the two P. stutzeri ncRNAs, namely NfiS and NfiR (for nitrogen fixation condition-inducible ncRNA), optimize nitrogen fixation and environmental stress responses. NfiS and NfiR respond differently to various environmental signals and differ in their secondary structures. In addition, the two ncRNAs target the mRNAs of nifK and nifD, respectively. Such ncRNA-based posttranscriptional regulation of nitrogenase expression might be an evolved survival strategy, particularly in nitrogen-limiting environments. This study not only highlights the significant roles of regulatory ncRNAs in the coordination and fine tuning of various physiological processes but also provides a new paradigm for posttranscriptional regulation in nitrogen-fixing bacteria.

scholarly journals The role of Fnr paralogs for controlling anaerobic metabolism in diazotroph Paenibacillus polymyxa WLY78

2020 ◽  
Author(s):  
Haowen Shi ◽  
Yongbin Li ◽  
Tianyi Hao ◽  
Xiaomeng Liu ◽  
Xiyun Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Fixation ◽  
Bacillus Cereus ◽  
Anaerobic Condition ◽  
Anaerobic Metabolism ◽  
Nitrogenase Activity ◽  
Paenibacillus Polymyxa ◽  
Transcription Analysis ◽  
Double Deletion ◽  
Expression Of Genes

ABSTRACTFnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes (fnr1, fnr3, fnr5 and fnr7) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on the growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to 50% reduction in nitrogenase activity and double deletion of fnr1 and fnr3 resulted to 90% reduction in activity. Both of the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic condition. Fnr1 and Fnr3 inhibited expression of the genes involved in aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes.IMPORTANCEPaenibacillus is a genus of Gram-positive, facultative anaerobic and endospore-forming bacteria. The members of nitrogen-fixing Paenibacillus have great potential use as a bacterial fertilizer in agriculture. However, the functions of fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we revealed that copy numbers vary largely among different Paenibacillus species and strains. Deletion and complementation analysis demonstrated that fnr1 and fnr3 have significant impacts on the growth and nitrogenase activity. Both of the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 purified in Escherichia coli could bind to the specific DNA promoter as Bacillus cereus Fnr did. Fnr1 and Fnr3 indirectly activated nif expression under anaerobic condition. Fnr1 and Fnr3 directly or indirectly activated or inhibited expression of many important genes involved in respiration, energy metabolism, Fe uptake and potentially specific electron transport for nitrogenase under anaerobic condition. This study not only reveals the roles of fnr genes in nitrogen fixation and anaerobic metabolism, but also provides insight into the evolution and regulatory mechanisms of fnr in Paenibacillus.

scholarly journals Transcriptional Termination Control of a Novel ABC Transporter Gene Involved in Antibiotic Resistance in Bacillus subtilis

2005 ◽  
Vol 187 (17) ◽  
pp. 5946-5954 ◽  
Author(s):  
Reiko Ohki ◽  
Kozue Tateno ◽  
Teruaki Takizawa ◽  
Toshiko Aiso ◽  
Makiko Murata
Keyword(s):  
Antibiotic Resistance ◽  
Bacillus Subtilis ◽  
Constitutive Expression ◽  
Efflux Transporters ◽  
Coding Region ◽  
Transcriptional Terminator ◽  
Binding Domains ◽  
Transcriptional Termination ◽  
Membrane Spanning ◽  
Extension Analysis

ABSTRACT In members of one of the subfamilies of the bacterial ATP binding cassette (ABC) transporters, the two nucleotide binding domains are fused as a single peptide and the proteins have no membrane-spanning domain partners. Most of the ABC efflux transporters of this subfamily have been characterized in actinomycetes, producing macrolide, lincosamide, and streptogramin antibiotics. Among 40 ABC efflux transporters of Bacillus subtilis, five proteins belong to this subfamily. None of these proteins has been functionally characterized. We examined macrolide, lincosamide, and streptogramin antibiotic resistance in insertional disruptants of the genes that encode these proteins. It was found that only a disruptant of vmlR (formerly named expZ) showed hypersensitivity to virginiamycin M and lincomycin. Expression of the vmlR gene was induced by the addition of these antibiotics in growth medium. Primer extension analysis revealed that transcription of the vmlR gene initiates at an adenosine residue located 225 bp upstream of the initiation codon. From the analysis of the vmlR and lacZ fusion genes, a 52-bp deletion from +159 to +211 resulted in constitutive expression of the vmlR gene. In this region, a typical ρ-independent transcriptional terminator was found. It was suggested that the majority of transcription ends at this termination signal in the absence of antibiotics, whereas under induced conditions, RNA polymerase reads through the terminator, and transcription continues to the downstream vmlR coding region, resulting in an increase in vmlR expression. No stabilization of vmlR mRNA occurred under the induced conditions.

scholarly journals The Role of Fnr Paralogs in Controlling Anaerobic Metabolism in the Diazotroph Paenibacillus polymyxa WLY78

2020 ◽  
Vol 86 (10) ◽  
Author(s):  
Haowen Shi ◽  
Yongbin Li ◽  
Tianyi Hao ◽  
Xiaomeng Liu ◽  
Xiyun Zhao ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Paenibacillus Polymyxa ◽  
Transcription Analysis ◽  
Content Type ◽  
Double Deletion ◽  
Expression Of Genes ◽  
Genome Wide ◽  
Promoter Deletion Analysis ◽  
Single Deletion

ABSTRACT Fnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes (fnr1, fnr3, fnr5, and fnr7) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Both the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to a 50% reduction in nitrogenase activity, and double deletion of fnr1 and fnr3 resulted to a 90% reduction in activity. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic conditions. Fnr1 and Fnr3 inhibited expression of the genes involved in the aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes. IMPORTANCE The members of the nitrogen-fixing Paenibacillus spp. have great potential to be used as a bacterial fertilizer in agriculture. However, the functions of the fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we found that in P. polymyxa WLY78, Fnr1 and Fnr3 were responsible for regulation of numerous genes in response to changes in oxygen levels, but Fnr5 and Fnr7 exhibited little effect. Fnr1 and Fnr3 indirectly or directly regulated many types of important metabolism, such as nitrogen fixation, Fe uptake, respiration, and electron transport. This study not only reveals the function of the fnr genes of P. polymyxa WLY78 in nitrogen fixation and other metabolisms but also will provide insight into the evolution and regulatory mechanisms of fnr in Paenibacillus.

scholarly journals Suppression ofhesAmutation on nitrogenase activity inPaenibacillus polymyxaWLY78 with the addition of high levels of molybdate or cystine

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6294 ◽  
Author(s):  
Xiaomeng Liu ◽  
Xiyun Zhao ◽  
Xiaohan Li ◽  
Sanfeng Chen
Keyword(s):  
Nitrogen Fixation ◽  
Gene Cluster ◽  
Nitrogenase Activity ◽  
Reductase Activity ◽  
Klebsiella Oxytoca ◽  
Paenibacillus Polymyxa ◽  
Gene Clusters ◽  
Nucleotide Binding Domain ◽  
Diazotrophic Cyanobacteria ◽  
Nitrogen Fixation Gene

The diazotrophicPaenibacillus polymyxaWLY78 possesses a minimal nitrogen fixation gene cluster consisting of nine genes (nifB nifH nifD nifK nifE nifN nifX hesAandnifV). Notably, thehesAgene contained within thenifgene cluster is also found withinnifgene clusters among diazotrophic cyanobacteria andFrankia. The predicted product HesA is a member of the ThiF-MoeB-HesA family containing an N-terminal nucleotide binding domain and a C-terminal MoeZ/MoeB-like domain. However, the function ofhesAgene in nitrogen fixation is unknown. In this study, we demonstrate that thehesAmutation ofP. polymyxaWLY78 leads to nearly complete loss of nitrogenase activity. The effect of the mutation can be partially suppressed by the addition of high levels of molybdate or cystine. However, the nitrogenase activity of thehesAmutant could not be restored byKlebsiella oxytoca nifQorEscherichia coli moeBcompletely. In addition, thehesAmutation does not affect nitrate reductase activity ofP. polymyxaWLY78. Our results demonstratehesAis a novel gene specially required for nitrogen fixation and its role is related to introduction of S and Mo into the FeMo-co of nitrogenase.

scholarly journals A novel hypoxic long noncoding RNA KB-1980E6.3 maintains breast cancer stem cell stemness via interacting with IGF2BP1 to facilitate c-Myc mRNA stability

Oncogene ◽  
2021 ◽  
Author(s):  
Pengpeng Zhu ◽  
Fang He ◽  
Yixuan Hou ◽  
Gang Tu ◽  
Qiao Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Underlying Mechanism ◽  
Rapid Expansion ◽  
Breast Cancer Patients ◽  
Coding Region ◽  
Signaling Axis

AbstractThe hostile hypoxic microenvironment takes primary responsibility for the rapid expansion of breast cancer tumors. However, the underlying mechanism is not fully understood. Here, using RNA sequencing (RNA-seq) analysis, we identified a hypoxia-induced long noncoding RNA (lncRNA) KB-1980E6.3, which is aberrantly upregulated in clinical breast cancer tissues and closely correlated with poor prognosis of breast cancer patients. The enhanced lncRNA KB-1980E6.3 facilitates breast cancer stem cells (BCSCs) self-renewal and tumorigenesis under hypoxic microenvironment both in vitro and in vivo. Mechanistically, lncRNA KB-1980E6.3 recruited insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) to form a lncRNA KB-1980E6.3/IGF2BP1/c-Myc signaling axis that retained the stability of c-Myc mRNA through increasing binding of IGF2BP1 with m6A-modified c-Myc coding region instability determinant (CRD) mRNA. In conclusion, we confirm that lncRNA KB-1980E6.3 maintains the stemness of BCSCs through lncRNA KB-1980E6.3/IGF2BP1/c-Myc axis and suggest that disrupting this axis might provide a new therapeutic target for refractory hypoxic tumors.

Studies on compatibility of nitrogen fixation by high-temperature-adapted Rhizobium strains and Vigna radiata genotypes at two moisture levels in calcareous soil

1983 ◽  
Vol 101 (2) ◽  
pp. 377-381 ◽  
Author(s):  
R. Rai ◽  
V. Prasad
Keyword(s):  
Nitrogen Fixation ◽  
High Temperature ◽  
Grain Yield ◽  
Vigna Radiata ◽  
Calcareous Soil ◽  
Nitrogenase Activity ◽  
Summer Season ◽  
Green Gram ◽  
Rhizobium Strains

SUMMARYRhizobium strains adapted to high temperature, and genotypes of green gram, were used to study the symbiotic N2-fixation in a summer season at two moisture levels in calcareous soil. Different interactions between strains and genotypes were observedatthe two moisture levels. At both moisture levels, strain S4 with the green gram genotype S8 showed the greatest grain yield, nitrogenase activity, leghaemoglobin and ethanolsoluble carbohydrate of nodules.

High-temperature-adaptedAzospirillum brasilensestrains: growth and interaction response on associative nitrogen fixation, mineral uptake and yield of cheena (Panicum miliaceumL.) genotypes in calcareous soil

1988 ◽  
Vol 110 (2) ◽  
pp. 321-329 ◽  
Author(s):  
R. Rai
Keyword(s):  
Nitrogen Fixation ◽  
High Temperature ◽  
Calcareous Soil ◽  
Nitrogenase Activity ◽  
Dry Weight ◽  
Cross Resistance ◽  
Mineral Uptake ◽  
Associative Nitrogen Fixation ◽  
Seed Inoculation ◽  
Resistance To Penicillin

SummaryHigh-temperature-adapted strains RAU 1, RAU 2 and RAU 3 ofAzospirillum brasilenseC 7 were isolated from stepwise transfer to higher temperature (30 to 42 °C). One of the strains (RAU 1) showed more growth, greater nitrogenase and hydrogenase activities at 30 and 42 °C than parental and other temperature-adapted strains. This strain also showed growth and more nitrogenase activity from pH 6·5 to 8·0. Strain RAU 1 showed cross-resistance to penicillin (300/µg/ml) but not to streptomycin, kanamycin, viomycin and polymixin B at 30 and 42 °C. It was demonstrated in field plots in calcareous soil that seed inoculation with RAU 1 enhanced mineral uptake of cheena. Inoculation with RAU 1 led to a significant increase in associative nitrogen fixation, dry weight of roots, grain and straw yield of cheena compared with the uninoculated control with or without applied N, but the effect of seed inoculation with high-temperature-adapted strains was variable with different genotypes of cheena.

key genetic factors in the metabolic syndrome predisposition which may be a therapeutic options by gene therapy

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Metabolic Syndrome ◽  
Noncoding Rna ◽  
Insulin Signalling ◽  
Therapeutic Option ◽  
Apoe Genotype ◽  
The United States ◽  
Acid Oxidation ◽  
Therapeutic Effects ◽  
Coding Region ◽  
The Metabolic Syndrome

Metabolic syndrome affects 24–42 percent of individuals over the age of 50 in the United States. Dietary treatments tailored to the APOE genotype may help patients with MeS. Both mitotic and meiotic epigenetics are heritable. SREBP 1 and 2 genes overexpress in response to low cholesterol, statins, and insulin tolerance, resulting in decreased fatty acid oxidation, insulin signalling, and HDL-c levels. FTO is a key gene in the metabolic syndrome predisposition which may be a therapeutic option. MicroRNA repression of the VEGF 62 reference gene is prevented by the LncRNA MIAT/miR-150-5p complex. MEG3, a maternally expressed three-letter noncoding RNA, has been related to endothelial cell angiogenesis. Phosphorylation of p38 and JNK improved in the absence of (SRA) lncRNAs, resulting in decreased insulin signaling.Mipomersen is a 20-mer oligonucleotide that binds to the APOB mRNA's coding region. Volanersorsen has finished phase II and III clinical trials, making it the first APOC3 ASO to do so. ASO targeting APOB3 based on next-generation ligands is also in the early stages of research. Angptl3 is another successful genome editing target. Given the role of ANGPTL3 in TRL metabolism, Dr. David Seres suggests that this editing may have additional or synergistic therapeutic effects. He claims that defects in the LDLR gene cause the most common genetic form of hypercholesterolemia, FH.

Endowing plants with the capacity for autogenic nitrogen fixation

2021 ◽  
Author(s):  
Quan-hong Yao ◽  
Ri-he Peng ◽  
Bo Wang ◽  
Yong-Sheng Tian ◽  
Yan-man Zhu ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Higher Plants ◽  
Paenibacillus Polymyxa ◽  
Isotopic Labeling ◽  
Crop Productivity ◽  
Available Nitrogen ◽  
Chlorophyll Contents ◽  
Modern Agriculture ◽  
Liquid Chromatography Tandem Mass ◽  
Self Fertilization

Abstract Biologically available nitrogen is a common limitation to crop productivity in modern agriculture. The endowment of higher plants with the ability to produce their own nitrogenous fertilizers has been attempted for nearly half a century1–4. Here we report that a minimal nitrogen fixation system from Paenibacillus polymyxa5–8 can be used to create an autogenic nitrogen-fixing plant through synthetic biology. We found that the genetically modified Arabidopsis containing the cassette of all nine nif genes (nifBHDKENXhesAnifV) showed some activity of nitrogenase and caused higher biomasses and chlorophyll contents than wild-type plants grown in low-nitrogen or nitrogen-free medium. Then we found that the engineered Arabidopsis displayed resistance to KCN and NaN3, two substrates of nitrogenase9. Furthermore, overexpression of electron transfer component10 in the engineered nif gene-carrying plants resulted in higher nitrogen fixation efficiency. Isotopic labeling analysis using liquid chromatography-tandem mass spectrometry showed that the fixed nitrogen can flow to amino acids and chlorophyll11, 12. This study represents a milestone toward realizing the goal of endowing plants with the capacity for self-fertilization.

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