scholarly journals Cloning and Identification of a New Repressor of 3,17β-Hydroxysteroid Dehydrogenase of Comamonas Testosteroni

2021 ◽  
Author(s):  
Weiqi Xie ◽  
Qin Xia ◽  
Ling Chen ◽  
Guangming Xiong ◽  
Yuwei Gao ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Function Analysis ◽  
Gene Knockout ◽  
Carbon Sources ◽  
Hydroxysteroid Dehydrogenase ◽  
Regulation Mechanism ◽  
Comamonas Testosteroni ◽  
Loss Of Function ◽  
Mobility Shift ◽  
Environmental Signals

Abstract 3,17β-hydroxysteroid dehydrogenase (3,17β-HSD) is a key enzyme that can degrade steroid compounds in Comamonas testosteroni. Tetracycline repressor (TetR) family, repressors existing in most microorganisms, may play key roles in regulating the expression of 3,17β-HSD. Previous reports showed that three TetR genes were located in the contig58 of C. testosteroni ATCC 11996 (GenBank: AHIL01000049.1), among which the first TetR gene encoded a potential repressor of 3,17β-HSD by sensing environmental signals. However, whether the other proposed TetR genes acts as repressors of 3,17β-HSD is still unknown. In the present study, we cloned the second TetR gene and analyzed the regulation mechanism of the protein on 3,17β-HSD using electrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-based assay, and loss-of-function analysis. The results showed that the second TetR gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17β-HSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17β-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second TetR gene knockout has good biological genetic stability, and the expression of 3,17β-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction, suggesting the mutant can efficiently degrade steroids as carbon sources. The mutant generated in this study can be used to treat environmental pollution caused by steroid hormones.

scholarly journals Cloning and identification of a new repressor of 3,17β-Hydroxysteroid dehydrogenase of Comamonas testosteroni

2021 ◽  
Author(s):  
Weiqi Xie ◽  
Qin Xia ◽  
Ling Chen ◽  
Guangming Xiong ◽  
Yuwei Gao ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Function Analysis ◽  
Gene Knockout ◽  
Carbon Sources ◽  
Hydroxysteroid Dehydrogenase ◽  
Regulation Mechanism ◽  
Comamonas Testosteroni ◽  
Loss Of Function ◽  
Mobility Shift ◽  
Environmental Signals

Abstract 3,17β-hydroxysteroid dehydrogenase (3,17β-HSD) is a key enzyme that can degrade steroid compounds in Comamonas testosteroni. Tetracycline repressor (TetR) family, repressors existing in most microorganisms, may play key roles in regulating the expression of 3,17β-HSD. Previous reports showed that three TetR genes were located in the contig58 of C. testosteroni ATCC 11996 (GenBank: AHIL01000049.1), among which the first TetR gene encoded a potential repressor of 3,17β-HSD by sensing environmental signals. However, whether the other proposed TetR genes acts as repressors of 3,17β-HSD is still unknown. In the present study, we cloned the second TetR gene and analyzed the regulation mechanism of the protein on 3,17β-HSD using electrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-based assay, and loss-of-function analysis. The results showed that the second TetR gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17β-HSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17β-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second TetR gene knockout has good biological genetic stability, and the expression of 3,17β-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction, suggesting the mutant can efficiently degrade steroids as carbon sources. The mutant generated in this study can be used to treat environmental pollution caused by steroid hormones.

scholarly journals Identification and Characterization of the LysR-Type Transcriptional Regulator HsdR for Steroid-Inducible Expression of the 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase Gene in Comamonas testosteroni

2011 ◽  
Vol 78 (4) ◽  
pp. 941-950 ◽  
Author(s):  
Wenjie Gong ◽  
Guangming Xiong ◽  
Edmund Maser
Keyword(s):  
Polyclonal Antibodies ◽  
Hydroxysteroid Dehydrogenase ◽  
Carbonyl Reductase ◽  
Comamonas Testosteroni ◽  
Binding Assays ◽  
Knockout Mutant ◽  
Mobility Shift ◽  
Content Type ◽  
Key Enzyme

ABSTRACT3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) fromComamonas testosteroniis a key enzyme in steroid degradation in soil and water. 3α-HSD/CR gene (hsdA) expression can be induced by steroids like testosterone and progesterone. Previously, we have shown that the induction ofhsdAexpression by steroids is a derepression where steroidal inducers bind to two repressors, RepA and RepB, thereby preventing the blocking ofhsdAtranscription and translation, respectively (G. Xiong and E. Maser, J. Biol. Chem.276:9961-9970, 2001; G. Xiong, H. J. Martin, and E. Maser, J. Biol. Chem.278:47400–47407, 2003). In the present study, a new LysR-type transcriptional factor, HsdR, for 3α-HSD/CR expression inC. testosteronihas been identified. ThehsdRgene is located 2.58 kb downstream fromhsdAon theC. testosteroniATCC 11996 chromosome with an orientation opposite that ofhsdA. ThehsdRgene was cloned and recombinant HsdR protein was produced, as was anti-HsdR polyclonal antibodies. While heterologous transformation systems revealed that HsdR activates the expression of thehsdAgene, electrophoresis mobility shift assays showed that HsdR specifically binds to thehsdApromoter region. Interestingly, the activity of HsdR is dependent on decreased repression by RepA. Furthermore,in vitrobinding assays indicated that HsdR can come into contact with RNA polymerase. As expected, anhsdRknockout mutant expressed low levels of 3α-HSD/CR compared to that of wild-typeC. testosteroniafter testosterone induction. In conclusion, HsdR is a positive transcription factor for thehsdAgene and promotes the induction of 3α-HSD/CR expression inC. testosteroni.

scholarly journals Stablization of ACOs by NatB mediated N-terminal acetylation is required for ethylene homeostasis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Hai-qing Liu ◽  
Ya-jie Zou ◽  
Xiao-feng Li ◽  
Lei Wu ◽  
Guang-qin Guo
Keyword(s):  
Protein Modification ◽  
Normal Growth ◽  
Ethylene Biosynthesis ◽  
Regulation Mechanism ◽  
Biological Functions ◽  
Loss Of Function ◽  
Auxiliary Subunit ◽  
Ethylene Content ◽  
Endogenous Ethylene ◽  
Exogenous Ethylene

AbstractN-terminal acetylation (NTA) is a highly abundant protein modification catalyzed by N-terminal acetyltransferases (NATs) in eukaryotes. However, the plant NATs and their biological functions have been poorly explored. Here we reveal that loss of function of CKRC3 and NBC-1, the auxiliary subunit (Naa25) and catalytic subunit (Naa20) of Arabidopsis NatB, respectively, led to defects in skotomorphogenesis and triple responses of ethylene. Proteome profiling and WB test revealed that the 1-amincyclopropane-1-carboxylate oxidase (ACO, catalyzing the last step of ethylene biosynthesis pathway) activity was significantly down-regulated in natb mutants, leading to reduced endogenous ethylene content. The defective phenotypes could be fully rescued by application of exogenous ethylene, but less by its precursor ACC. The present results reveal a previously unknown regulation mechanism at the co-translational protein level for ethylene homeostasis, in which the NatB-mediated NTA of ACOs render them an intracellular stability to maintain ethylene homeostasis for normal growth and responses.

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

scholarly journals Contributions of Environmental Signals and Conserved Residues to the Functions of Carbon Storage Regulator A of Borrelia burgdorferi

2013 ◽  
Vol 81 (8) ◽  
pp. 2972-2985 ◽  
Author(s):  
S. L. Rajasekhar Karna ◽  
Rajesh G. Prabhu ◽  
Ying-Han Lin ◽  
Christine L. Miller ◽  
J. Seshu
Keyword(s):  
Borrelia Burgdorferi ◽  
Carbon Storage ◽  
Vertebrate Host ◽  
Acetyl Phosphate ◽  
Mobility Shift ◽  
Acetyl Coa ◽  
Site Specific ◽  
Content Type ◽  
Environmental Signals ◽  
Carbon Storage Regulator

ABSTRACTCarbon storage regulator A ofBorrelia burgdorferi(CsrABb) contributes to vertebrate host-specific adaptation by modulating activation of the Rrp2-RpoN-RpoS pathway and is critical for infectivity. We hypothesized that the functions of CsrABbare dependent on environmental signals and on select residues. We analyzed the phenotype ofcsrABbdeletion and site-specific mutants to determine the conserved and pathogen-specific attributes of CsrABb. Levels of phosphate acetyltransferase (Pta) involved in conversion of acetyl phosphate to acetyl-coenzyme A (acetyl-CoA) and posttranscriptionally regulated by CsrABbin thecsrABbmutant were reduced from or similar to those in the control strains under unfed- or fed-tick conditions, respectively. Increased levels of supplemental acetate restored vertebrate host-responsive determinants in thecsrABbmutant to parental levels, indicating that both the levels of CsrABband the acetyl phosphate and acetyl-CoA balance contribute to the activation of the Rrp2-RpoN-RpoS pathway. Site-specific replacement of 8 key residues of CsrABb(8S) with alanines resulted in increased levels of CsrABband reduced levels of Pta and acetyl-CoA, while levels of RpoS, BosR, and other members ofrpoSregulon were elevated. Truncation of 7 amino acids at the C terminus of CsrABb(7D) resulted in reducedcsrABbtranscripts and posttranscriptionally reduced levels of FliW located upstream of CsrABb. Electrophoretic mobility shift assays revealed increased binding of 8S mutant protein to the CsrA binding box upstream ofptacompared to the parental and 7D truncated protein. Two CsrABbbinding sites were also identified upstream offliWwithin theflgKcoding sequence. These observations reveal conserved and unique functions of CsrABbthat regulate adaptive gene expression inB. burgdorferi.

scholarly journals HMGB1 Promotes Systemic Lupus Erythematosus by Enhancing Macrophage Inflammatory Response

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Mudan Lu ◽  
Shanshan Yu ◽  
Wei Xu ◽  
Bo Gao ◽  
Sidong Xiong
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Inflammatory Response ◽  
Proinflammatory Cytokines ◽  
Lupus Erythematosus ◽  
Function Analysis ◽  
Critical Role ◽  
Loss Of Function ◽  
Systemic Lupus ◽  
Glycation End Products ◽  
Hmgb1 Expression

Background/Purpose. HMGB1, which may act as a proinflammatory mediator, has been proposed to contribute to the pathogenesis of multiple chronic inflammatory and autoimmune diseases including systemic lupus erythematosus (SLE); however, the precise mechanism of HMGB1 in the pathogenic process of SLE remains obscure.Method. The expression of HMGB1 was measured by ELISA and western blot. The ELISA was also applied to detect proinflammatory cytokines levels. Furthermore, nephritic pathology was evaluated by H&E staining of renal tissues.Results. In this study, we found that HMGB1 levels were significantly increased and correlated with SLE disease activity in both clinical patients and murine model. Furthermore, gain- and loss-of-function analysis showed that HMGB1 exacerbated the severity of SLE. Of note, the HMGB1 levels were found to be associated with the levels of proinflammatory cytokines such as TNF-αand IL-6 in SLE patients. Further study demonstrated that increased HMGB1 expression deteriorated the severity of SLE via enhancing macrophage inflammatory response. Moreover, we found that receptor of advanced glycation end products played a critical role in HMGB1-mediated macrophage inflammatory response.Conclusion. These findings suggested that HMGB1 might be a risk factor for SLE, and manipulation of HMGB1 signaling might provide a therapeutic strategy for SLE.

scholarly journals Role of PIF4 and FLC in controlling flowering time under daily variable temperature profile

2020 ◽  
Author(s):  
Jutapak Jenkitkonchai ◽  
Poppy Marriott ◽  
Weibing Yang ◽  
Napaporn Sriden ◽  
Jae-Hoon Jung ◽  
...  
Keyword(s):  
Flowering Time ◽  
Molecular Mechanisms ◽  
Transcriptional Regulatory Network ◽  
Variable Temperature ◽  
Loss Of Function ◽  
Environmental Signals ◽  
Regulatory Interactions ◽  
Flowering Genes ◽  
Gene Regulatory ◽  
Flowering Locus

ABSTRACTInitiation of flowering is a crucial developmental event that requires both internal and environmental signals to determine when floral transition should occur to maximize reproductive success. Ambient temperature is one of the key environmental signals that highly influence flowering time, not only seasonally but also in the context of drastic temperature fluctuation due to global warming. Molecular mechanisms of how high or low constant temperatures affect the flowering time have been largely characterized in the model plant Arabidopsis thaliana; however, the effect of natural daily variable temperature outside laboratories is only partly explored. Several groups of flowering genes have been shown to play important roles in temperature responses, including two temperature-responsive transcription factors (TFs), namely PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and FLOWERING LOCUS C (FLC), that act antagonistically to regulate flowering time by activating or repressing floral integrator FLOWERING LOCUS T (FT). In this study, we have demonstrated that the daily variable temperature (VAR) causes early flowering in both natural accessions Col-0, C24 and their late flowering hybrid C24xCol, which carries both functional floral repressor FLC and its activator FRIGIDA (FRI), as compared to a constant temperature (CON). The loss-of-function mutation of PIF4 exhibits later flowering in VAR, suggesting that PIF4 at least in part, contributes to acceleration of flowering in response to the daily variable temperature. We find that VAR increases PIF4 transcription at the end of the day when temperature peaks at 32 °C. The FT transcription is also elevated in VAR, as compared to CON, in agreement with earlier flowering observed in VAR. In addition, VAR causes a decrease in FLC transcription in 4-week-old plants, and we further show that overexpression of PIF4 can reduce FLC transcription, suggesting that PIF4 might also regulate FT indirectly through the repression of FLC. To further conceptualize an overall model of gene regulatory mechanisms involving PIF4 and FLC in controlling flowering in response to temperature changes, we construct a co-expression – transcriptional regulatory network by combining publicly available transcriptomic data and gene regulatory interactions of our flowering genes of interest and their partners. The network model reveals the conserved and tissue-specific regulatory functions of 62 flowering-time-relating genes, namely PIF4, PIF5, FLC, ELF3 and their immediate neighboring genes, which can be useful for confirming and predicting the functions and regulatory interactions between the key flowering genes.

scholarly journals Mechanism of PQQ and CoQ10 overproduction in Methylobacterium as revealed by comparative genomic analysis between mutant and wild-type strains

2020 ◽  
Author(s):  
Changle Zhao ◽  
Yinping Wan ◽  
Xiaojie Cao ◽  
Huili Zhang ◽  
Xin Bao
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Analysis ◽  
Comparative Genomic Analysis ◽  
Comparative Genomic ◽  
Regulation Mechanism ◽  
Whole Genome ◽  
Wild Type ◽  
Coq10 Production

Abstract Background The microbial synthesis of pyrroloquinoline quinone (PQQ) and Coenzyme Q10 (CoQ10) remains the most promising industrial production route. Methylobacterium has been used to generate PQQ and other value-added chemicals from cheap carbon feedstocks.However, the low PQQ and CoQ10 production capacity of the Methylobacterium strains is a major limitation The regulation mechanism for PQQ and CoQ10 biosynthesis in this strain has also not been fully elucidated. Results Methylobacterium sp. CLZ strain was isolated from soil contaminated with chemical wastewater, which can simultaneously produce PQQ, CoQ10, and carotenoids by using cheap methanol as carbon source. We investigated a mutant strain NI91, which increased the PQQ and CoQ10 yield by 72.44% and 59.80%, respectively. Whole-genome sequencing of NI91 and wild-type strain CLZ revealed that both contain a 5.28 Mb chromosome. The comparative genomic analysis and validation study revealed that a significant increase in biomass and PQQ production was associated with the base mutations in the methanol dehydrogenase (MDH) synthesis genes, mxaD and mxaJ. The significant increase in CoQ10 production may be associated with the base mutations in dxs gene, a key gene in the MEP/DOXP pathway. Conclusions A PQQ producing strain that simultaneously produces CoQ10 and carotenoids was selected and after ANI analysis, named as Methylobacterium sp. CLZ. After random mutagenesis of this strain, we obtained NI91 strain, which showed increased production of PQQ and CoQ10. Based on comparative genomic analysis of the whole genome of mutant strain NI91 and wild-type strain CLZ, a total of 270 SNPs and InDels events were detected, which provided a reference for subsequent research. The mutations in mxaD, mxaJ and dxs genes may be related to the high yield of PQQ and CoQ10. These findings will enhance our understanding of the PQQ and CoQ10 over-production mechanism in Methylobacterium sp. NI91 at the genomic level. It will also provide useful clues for strain engineering in order to improve the PQQ and CoQ10 production.

scholarly journals When Coupled to Natural Transformation in Acinetobacter sp. Strain ADP1, PCR Mutagenesis Is Made Less Random by Mismatch Repair

2005 ◽  
Vol 71 (11) ◽  
pp. 7610-7612 ◽  
Author(s):  
Alison Buchan ◽  
L. Nicholas Ornston
Keyword(s):  
Structure Function ◽  
Mismatch Repair ◽  
Protein Function ◽  
Natural Transformation ◽  
Function Analysis ◽  
Repair System ◽  
Loss Of Function ◽  
Nucleotide Substitutions ◽  
Full Spectrum ◽  
Mismatch Repair System

ABSTRACT Random PCR mutagenesis is a powerful tool for structure-function analysis of targeted proteins, especially when coupled with DNA integration through natural transformation followed by selection for loss of function. The technique has been applied successfully to structure-function analysis of transcriptional regulators, enzymes, and transporters in Acinetobacter sp. strain ADP1. However, the mismatch repair system prevents the full spectrum of nucleotide substitutions that may be selected at the level of protein function from being recovered. This barrier may be overcome by introducing PCR-mutagenized genes into strains in which the corresponding genes have been deleted.

Jun mediates Frizzled-induced R3/R4 cell fate distinction and planar polarity determination in the Drosophila eye

Development ◽  
2000 ◽  
Vol 127 (16) ◽  
pp. 3619-3629 ◽  
Author(s):  
U. Weber ◽  
N. Paricio ◽  
M. Mlodzik
Keyword(s):  
Cell Fate ◽  
Genetic Interaction ◽  
Function Analysis ◽  
Loss Of Function ◽  
Planar Polarity ◽  
Jnk Signaling ◽  
Drosophila Eye ◽  
Polarity Determination ◽  
Signaling Components

Jun acts as a signal-regulated transcription factor in many cellular decisions, ranging from stress response to proliferation control and cell fate induction. Genetic interaction studies have suggested that Jun and JNK signaling are involved in Frizzled (Fz)-mediated planar polarity generation in the Drosophila eye. However, simple loss-of-function analysis of JNK signaling components did not show comparable planar polarity defects. To address the role of Jun and JNK in Fz signaling, we have used a combination of loss- and gain-of-function studies. Like Fz, Jun affects the bias between the R3/R4 photoreceptor pair that is critical for ommatidial polarity establishment. Detailed analysis of jun(−) clones reveals defects in R3 induction and planar polarity determination, whereas gain of Jun function induces the R3 fate and associated polarity phenotypes. We find also that affecting the levels of JNK signaling by either reduction or overexpression leads to planar polarity defects. Similarly, hypomorphic allelic combinations and overexpression of the negative JNK regulator Puckered causes planar polarity eye phenotypes, establishing that JNK acts in planar polarity signaling. The observation that Dl transcription in the early R3/R4 precursor cells is deregulated by Jun or Hep/JNKK activation, reminiscent of the effects seen with Fz overexpression, suggests that Jun is one of the transcription factors that mediates the effects of fz in planar polarity generation.

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