The Sinorhizobium fredii HH103 MucR1 Global Regulator Is Connected With the nod Regulon and Is Required for Efficient Symbiosis With Lotus burttii and Glycine max cv. Williams

Sinorhizobium fredii HH103 is a rhizobial strain showing a broad host range of nodulation. In addition to the induction of bacterial nodulation genes, transition from a free-living to a symbiotic state requires complex genetic expression changes with the participation of global regulators. We have analyzed the role of the zinc-finger transcriptional regulator MucR1 from S. fredii HH103 under both free-living conditions and symbiosis with two HH103 host plants, Glycine max and Lotus burttii. Inactivation of HH103 mucR1 led to a severe decrease in exopolysaccharide (EPS) biosynthesis but enhanced production of external cyclic glucans (CG). This mutant also showed increased cell aggregation capacity as well as a drastic reduction in nitrogen-fixation capacity with G. max and L. burttii. However, in these two legumes, the number of nodules induced by the mucR1 mutant was significantly increased and decreased, respectively, with respect to the wild-type strain, indicating that MucR1 can differently affect nodulation depending on the host plant. RNA-Seq analysis carried out in the absence and the presence of flavonoids showed that MucR1 controls the expression of hundreds of genes (including some related to EPS production and CG transport), some of them being related to the nod regulon.

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Coordinated Regulation of the Size and Number of Polyhydroxybutyrate Granules by Core and Accessory Phasins in the Facultative Microsymbiont Sinorhizobium fredii NGR234

Applied and Environmental Microbiology ◽

10.1128/aem.00717-19 ◽

2019 ◽

Vol 85 (19) ◽

Cited By ~ 4

Author(s):

Yan-Wei Sun ◽

Yan Li ◽

Yue Hu ◽

Wen-Xin Chen ◽

Chang-Fu Tian

Keyword(s):

Broad Host Range ◽

Metabolic Profiles ◽

Free Living ◽

Sinorhizobium Fredii ◽

Content Type ◽

Cellular Processes ◽

Size Number ◽

Associated Proteins ◽

Auxiliary Gene

ABSTRACT The exact roles of various granule-associated proteins (GAPs) of polyhydroxybutyrate (PHB) are poorly investigated, particularly for bacteria associated with plants. In this study, four structural GAPs, named phasins PhaP1 to PhaP4, were identified and demonstrated as true phasins colocalized with PHB granules in Sinorhizobium fredii NGR234, a facultative microsymbiont of Vigna unguiculata and many other legumes. The conserved PhaP2 dominated in regulation of granule size under both free-living and symbiotic conditions. PhaP1, another conserved phasin, made a higher contribution than accessory phasins PhaP4 and PhaP3 to PHB biosynthesis at stationary phase. PhaP3, with limited phyletic distribution on the symbiosis plasmid of Sinorhizobium, was more important than PhaP1 in regulating PHB biosynthesis in V. unguiculata nodules. Under the test conditions, no significant symbiotic defects were observed for mutants lacking individual or multiple phaP genes. The mutant lacking two PHB synthases showed impaired symbiotic performance, while mutations in individual PHB synthases or a PHB depolymerase yielded no symbiotic defects. This phenomenon is not related to either the number or size of PHB granules in test mutants within nodules. Distinct metabolic profiles and cocktail pools of GAPs of different phaP mutants imply that core and accessory phasins can be differentially involved in regulating other cellular processes in the facultative microsymbiont S. fredii NGR234. IMPORTANCE Polyhydroxybutyrate (PHB) granules are a store of carbon and energy in bacteria and archaea and play an important role in stress adaptation. Recent studies have highlighted distinct roles of several granule-associated proteins (GAPs) in regulating the size, number, and localization of PHB granules in free-living bacteria, though our knowledge of the role of GAPs in bacteria associated with plants is still limited. Here we report distinct roles of core and accessory phasins associated with PHB granules of Sinorhizobium fredii NGR234, a broad-host-range microsymbiont of diverse legumes. Core phasins PhaP2 and PhaP1 are conserved major phasins in free-living cells. PhaP2 and accessory phasin PhaP3, encoded by an auxiliary gene on the symbiosis plasmid, are major phasins in nitrogen-fixing bacteroids in cowpea nodules. GAPs and metabolic profiles can vary in different phaP mutants. Contrasting symbiotic performances between mutants lacking PHB synthases, depolymerase, or phasins were revealed.

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The rkpU gene of Sinorhizobium fredii HH103 is required for bacterial K-antigen polysaccharide production and for efficient nodulation with soybean but not with cowpea

Microbiology ◽

10.1099/mic.0.042499-0 ◽

2010 ◽

Vol 156 (11) ◽

pp. 3398-3411 ◽

Cited By ~ 20

Author(s):

Ángeles Hidalgo ◽

Isabel Margaret ◽

Juan C. Crespo-Rivas ◽

Maribel Parada ◽

Piedad del Socorro Murdoch ◽

...

Keyword(s):

Escherichia Coli ◽

Glycine Max ◽

Nitrogen Starvation ◽

Broad Host Range ◽

Rhizobial Strain ◽

Sinorhizobium Fredii ◽

Symbiotic Interaction ◽

First Time ◽

K Antigen

In this work, the role of the rkpU and rkpJ genes in the production of the K-antigen polysaccharides (KPS) and in the symbiotic capacity of Sinorhizobium fredii HH103, a broad host-range rhizobial strain able to nodulate soybean and many other legumes, was studied. The rkpJ- and rkpU-encoded products are orthologous to Escherichia coli proteins involved in capsule export. S. fredii HH103 mutant derivatives were contructed in both genes. To our knowledge, this is the first time that the role of rkpU in KPS production has been studied in rhizobia. Both rkpJ and rkpU mutants were unable to produce KPS. The rkpU derivative also showed alterations in its lipopolysaccharide (LPS). Neither KPS production nor rkpJ and rkpU expression was affected by the presence of the flavonoid genistein. Soybean (Glycine max) plants inoculated with the S. fredii HH103 rkpU and rkpJ mutants showed reduced nodulation and clear symptoms of nitrogen starvation. However, neither the rkpJ nor the rkpU mutants were significantly impaired in their symbiotic interaction with cowpea (Vigna unguiculata). Thus, we demonstrate for the first time to our knowledge the involvement of the rkpU gene in rhizobial KPS production and also show that the symbiotic relevance of the S. fredii HH103 KPS depends on the specific bacterium–legume interaction.

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RNA Sequencing Analysis of the Broad-Host-Range Strain Sinorhizobium fredii NGR234 Identifies a Large Set of Genes Linked to Quorum Sensing-Dependent Regulation in the Background of atraIandngrIDeletion Mutant

Applied and Environmental Microbiology ◽

10.1128/aem.01835-14 ◽

2014 ◽

Vol 80 (18) ◽

pp. 5655-5671 ◽

Cited By ~ 23

Author(s):

Dagmar Krysciak ◽

Jessica Grote ◽

Mariita Rodriguez Orbegoso ◽

Christian Utpatel ◽

Konrad U. Förstner ◽

...

Keyword(s):

Quorum Sensing ◽

Host Range ◽

Rna Sequencing ◽

Homoserine Lactone ◽

Differentially Expressed ◽

Broad Host Range ◽

Large Set ◽

Sequencing Analysis ◽

Rna Seq ◽

Sinorhizobium Fredii

ABSTRACTThe alphaproteobacteriumSinorhizobium frediiNGR234 has an exceptionally wide host range, as it forms nitrogen-fixing nodules with more legumes than any other known microsymbiont. Within its 6.9-Mbp genome, it encodes twoN-acyl-homoserine-lactone synthase genes (i.e.,traIandngrI) involved in the biosynthesis of two distinct autoinducer I-type molecules. Here, we report on the construction of an NGR234-ΔtraIand an NGR234-ΔngrImutant and their genome-wide transcriptome analysis. A high-resolution RNA sequencing (RNA-seq) analysis of early-stationary-phase cultures in the NGR234-ΔtraIbackground suggested that up to 316 genes were differentially expressed in the NGR234-ΔtraImutant versus the parent strain. Similarly, in the background of NGR234-ΔngrI466 differentially regulated genes were identified. Accordingly, a common set of 186 genes was regulated by the TraI/R and NgrI/R regulon. Coregulated genes included 42 flagellar biosynthesis genes and 22 genes linked to exopolysaccharide (EPS) biosynthesis. Among the genes and open reading frames (ORFs) that were differentially regulated in NGR234-ΔtraIwere those linked to replication of the pNGR234asymbiotic plasmid and cytochromecoxidases. Biotin and pyrroloquinoline quinone biosynthesis genes were differentially expressed in the NGR234-ΔngrImutant as well as the entire cluster of 21 genes linked to assembly of the NGR234 type III secretion system (T3SS-II). Further, we also discovered that genes responsible for rhizopine catabolism in NGR234 were strongly repressed in the presence of high levels ofN-acyl-homoserine-lactones. Together with nodulation assays, the RNA-seq-based findings suggested that quorum sensing (QS)-dependent gene regulation appears to be of higher relevance during nonsymbiotic growth rather than for life within root nodules.

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Activation of cytochrome c to a peroxidase compound I-type intermediate by H2O2: relevance to redox signalling in apoptosis

Biochemical Society Symposium ◽

10.1042/bss0710097 ◽

2004 ◽

Vol 71 ◽

pp. 97-106 ◽

Cited By ~ 10

Author(s):

Mark Burkitt ◽

Clare Jones ◽

Andrew Lawrence ◽

Peter Wardman

Keyword(s):

Cytochrome C ◽

Hydroxyl Radical ◽

Redox Regulation ◽

Chemotherapeutic Agents ◽

Tyrosyl Radical ◽

Compound I ◽

Definitive Evidence ◽

Enhanced Production ◽

Physico Chemical

The release of cytochrome c from mitochondria during apoptosis results in the enhanced production of superoxide radicals, which are converted to H2O2 by Mn-superoxide dismutase. We have been concerned with the role of cytochrome c/H2O2 in the induction of oxidative stress during apoptosis. Our initial studies showed that cytochrome c is a potent catalyst of 2′,7′-dichlorofluorescin oxidation, thereby explaining the increased rate of production of the fluorophore 2′,7′-dichlorofluorescein in apoptotic cells. Although it has been speculated that the oxidizing species may be a ferryl-haem intermediate, no definitive evidence for the formation of such a species has been reported. Alternatively, it is possible that the hydroxyl radical may be generated, as seen in the reaction of certain iron chelates with H2O2. By examining the effects of radical scavengers on 2′,7′-dichlorofluorescin oxidation by cytochrome c/H2O2, together with complementary EPR studies, we have demonstrated that the hydroxyl radical is not generated. Our findings point, instead, to the formation of a peroxidase compound I species, with one oxidizing equivalent present as an oxo-ferryl haem intermediate and the other as the tyrosyl radical identified by Barr and colleagues [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498-15503]. Studies with spin traps indicated that the oxo-ferryl haem is the active oxidant. These findings provide a physico-chemical basis for the redox changes that occur during apoptosis. Excessive changes (possibly catalysed by cytochrome c) may have implications for the redox regulation of cell death, including the sensitivity of tumour cells to chemotherapeutic agents.

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Safety and Performance of the Omnipod®Hybrid Closed-Loop System in Adolescents with Type 1 Diabetes over Five Days Under Free-Living Conditions

Diabetes ◽

10.2337/db18-1376-p ◽

2018 ◽

Vol 67 (Supplement 1) ◽

pp. 1376-P

Author(s):

GREGORY P. FORLENZA ◽

BRUCE BUCKINGHAM ◽

JENNIFER SHERR ◽

THOMAS A. PEYSER ◽

JOON BOK LEE ◽

...

Keyword(s):

Type 1 Diabetes ◽

Closed Loop ◽

Living Conditions ◽

Loop System ◽

Closed Loop System ◽

Free Living ◽

And Performance ◽

Free Living Conditions

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Safety and Performance of the Omnipod® Hybrid Closed-Loop System in Adults with Type 1 Diabetes over Five Days Under Free-Living Conditions

Diabetes ◽

10.2337/db18-207-or ◽

2018 ◽

Vol 67 (Supplement 1) ◽

pp. 207-OR

Author(s):

BRUCE A. BUCKINGHAM ◽

JENNIFER SHERR ◽

GREGORY P. FORLENZA ◽

THOMAS A. PEYSER ◽

JOON BOK LEE ◽

...

Keyword(s):

Type 1 Diabetes ◽

Closed Loop ◽

Living Conditions ◽

Loop System ◽

Closed Loop System ◽

Free Living ◽

And Performance ◽

Free Living Conditions

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Enhanced Production of the Mical Redox Domain for Enzymology and F-actin Disassembly Assays

International Journal of Molecular Sciences ◽

10.3390/ijms22041991 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1991

Author(s):

Jimok Yoon ◽

Heng Wu ◽

Ruei-Jiun Hung ◽

Jonathan R. Terman

Keyword(s):

Nicotinamide Adenine Dinucleotide Phosphate ◽

Actin Dynamics ◽

Specific Substrate ◽

Actin Assembly ◽

Redox Enzymes ◽

Enhanced Production ◽

Reversible Redox ◽

Future Work ◽

Signaling Process

To change their behaviors, cells require actin proteins to assemble together into long polymers/filaments—and so a critical goal is to understand the factors that control this actin filament (F-actin) assembly and stability. We have identified a family of unusual actin regulators, the MICALs, which are flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in Redox reactions. F-actin is a specific substrate for these MICAL Redox enzymes, which oxidize specific amino acids within actin to destabilize actin filaments. Furthermore, this MICAL-catalyzed reaction is reversed by another family of Redox enzymes (SelR/MsrB enzymes)—thereby revealing a reversible Redox signaling process and biochemical mechanism regulating actin dynamics. Interestingly, in addition to the MICALs’ Redox enzymatic portion through which MICALs covalently modify and affect actin, MICALs have multiple other domains. Less is known about the roles of these other MICAL domains. Here we provide approaches for obtaining high levels of recombinant protein for the Redox only portion of Mical and demonstrate its catalytic and F-actin disassembly activity. These results provide a ground state for future work aimed at defining the role of the other domains of Mical — including characterizing their effects on Mical’s Redox enzymatic and F-actin disassembly activity.

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Data-driven modelling approach to circadian temperature rhythm profiles in free-living conditions

Scientific Reports ◽

10.1038/s41598-021-94522-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jari Lipsanen ◽

Liisa Kuula ◽

Marko Elovainio ◽

Timo Partonen ◽

Anu-Katriina Pesonen

Keyword(s):

Skin Surface ◽

Living Conditions ◽

Data Driven ◽

Physiological Data ◽

Free Living ◽

Physiological Level ◽

Temperature Rhythm ◽

Time Series Modelling ◽

Circadian Preference ◽

Free Living Conditions

AbstractThe individual variation in the circadian rhythms at the physiological level is not well understood. Albeit self-reported circadian preference profiles have been consolidated, their premises are grounded on human experience, not on physiology. We used data-driven, unsupervised time series modelling to characterize distinct profiles of the circadian rhythm measured from skin surface temperature in free-living conditions. We demonstrate the existence of three distinct clusters of individuals which differed in their circadian temperature profiles. The cluster with the highest temperature amplitude and the lowest midline estimating statistic of rhythm, or rhythm-adjusted mean, had the most regular and early-timed sleep–wake rhythm, and was the least probable for those with a concurrent delayed sleep phase, or eveningness chronotype. While the clusters associated with the observed sleep and circadian preference patterns, the entirely unsupervised modelling of physiological data provides a novel basis for modelling and understanding the human circadian functions in free-living conditions.

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Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

Journal of Industrial Microbiology & Biotechnology ◽

10.1093/jimb/kuab004 ◽

2021 ◽

Author(s):

Chang-Hun Shin ◽

Hang Soo Cho ◽

Hyung-Jin Won ◽

Ho Jeong Kwon ◽

Chan-Wha Kim ◽

...

Keyword(s):

Clavulanic Acid ◽

Wild Type Strain ◽

Random Mutagenesis ◽

Streptomyces Clavuligerus ◽

Wild Type ◽

Mutant Selection ◽

Enhanced Production ◽

Industrial Strains ◽

Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

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Myco-Suppression Analysis of Soybean (Glycine max) Damping-Off Caused by Pythium aphanidermatum

Plants ◽

10.3390/plants10040788 ◽

2021 ◽

Vol 10 (4) ◽

pp. 788

Author(s):

Shaban R. M. Sayed ◽

Shaimaa A. M. Abdelmohsen ◽

Hani M. A. Abdelzaher ◽

Mohammed A. Elnaghy ◽

Ashraf A. Mostafa ◽

...

Keyword(s):

Glycine Max ◽

Biocontrol Agent ◽

Lateral Roots ◽

Dry Weight ◽

Pythium Aphanidermatum ◽

Damping Off ◽

Pythium Oligandrum ◽

Rdna Its ◽

Half Strength

The role of Pythium oligandrum as a biocontrol agent against Pythium aphanidermatum was investigated to avoid the harmful impacts of fungicides. Three isolates of P. oligandrum (MS15, MS19, and MS31) were assessed facing the plant pathogenic P. aphanidermatum the causal agent of Glycine max damping-off. The tested Pythium species were recognized according to their cultural and microscopic characterizations. The identification was confirmed through sequencing of rDNA-ITS regions including the 5.8 S rDNA. The biocontrol agent, P. oligandrum, isolates decreased the mycelial growth of the pathogenic P. aphanidermatum with 71.3%, 67.1%, and 68.7% through mycoparasitism on CMA plates. While the half-strength millipore sterilized filtrates of P. oligandrum isolates degrade the pathogenic mycelial linear growth by 34.1%, 32.5%, and 31.7%, and reduce the mycelial dry weight of the pathogenic P. aphanidermatum by 40.1%, 37.4%, and 36.8%, respectively. Scanning electron microscopy (SEM) of the most effective antagonistic P. oligandrum isolate (MS15) interaction showed coiling, haustorial parts of P. oligandrum to P. aphanidermatum hyphae. Furthermore, P. oligandrum isolates were proven to enhance the germination of Glycine max seedling to 93.3% in damping-off infection using agar pots and promote germination of up to 80% during soil pot assay. On the other hand, P. oligandrum isolates increase the shoot, root lengths, and the number of lateral roots.

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