scholarly journals Bacillus licheniformisglobal nitrogen homeostatic regulator TnrA is a direct repressor ofpgsBCAAtranscription in Poly-γ-glutamic acid synthesis

2018 ◽  
Author(s):  
Dongbo Cai ◽  
Yaozhong Chen ◽  
Shiyi Wang ◽  
Fei Mo ◽  
Xin Ma ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Binding Site ◽  
Computer Analysis ◽  
Regulatory Mechanism ◽  
Acid Synthesis ◽  
Transcriptional Level ◽  
Mobility Shift ◽  
New Approach ◽  
Software Analysis ◽  
Regulation Network

AbstractPoly-γ-glutamic acid (γ-PGA) is a multifunctional and naturally occurring biopolymer made from D- and L-glutamate as monomers, which is mainly produced byBacillus. Few reports have been focused on the regulation network of γ-PGA synthesis in recent years. In this study, we have demonstrated thatBacillus licheniformisglobal nitrogen homeostatic regulator TnrA is a direct repressor of γ-PGA synthase PgsBCAA in γ-PGA synthesis. First, our results confirmed that TnrA repressed γ-PGA synthesis, deficiency oftnrAled to a 22.03% increase of γ-PGA production, and the γ-PGA yield was decreased by 19.02% in the TnrA overexpression strain. Transcriptional level assay illustrated that the γ-PGA synthase gene clusterpgsBCAAtranscriptional level were increased in thetnrAdeficient strain WXΔtnrA, indicating that γ-PGA synthase PgsBCAA was negatively regulated by TnrA. Furthermore, electrophoretic mobility shift assay (EMSA) and enzyme expression assays confirmed that TnrA directly repressedpgsBCAAexpression by binding topgsBCAApromoter, and the TnrA-binding site “CGTCGTCTTCTGTTACA” in thepgsBCAApromoter was identified by sequence and software analysis. Finally, computer analysis confirmed that the transcription regulations of γ-PGA synthase PgsBCAA by TnrA were highly conserved in other well-studiedBacillusspecies (B.licheniformis,Bacillus subtilisandBacillus amyloliquefaciens). Collectively, our results implied that TnrA was a direct repressor forpgsBCAAexpression in γ-PGA synthesis, and this research provided a novel regulatory mechanism underlying γ-PGA synthesis, and a new approach that deficiency oftnrAincreases γ-PGA production.Importanceγ-PGA is an important biopolymer with many applications, which is mainly produced byBacillusspecies. Glutamic acid is the precursor for γ-PGA synthesis, which is catalyzed by the γ-PGA synthase PgsBCAA. Previously, the expression of PgsBCAA was reported to be regulated by ComA-ComP and DegS-DegU, DegQ and SwrA systems, however, few researches were focused on the regulation network of γ-PGA synthesis in recent years. In our research, the γ-PGA synthase PgsBCAA was confirmed to be negatively regulated by the nitrogen metabolism regulator TnrA, and the TnrA binding site in thepgsBCAApromoter was identified inB. licheniformisWX-02. Furthermore, computer analysis implied that TnrA-mediated regulation effect onpgsBCAAexpression was highly conserved inBacillus. Collectively, our research provided a novel regulatory mechanism underlying γ-PGA synthesis, and a new approach that deficiency oftnrAincreases γ-PGA production.

scholarly journals Mycobacterium tuberculosisRequires Regulation of ESX-5 Secretion for Virulence in Irgm1-Deficient Mice

2018 ◽  
Vol 87 (2) ◽  
Author(s):  
Sarah R. Elliott ◽  
Dylan W. White ◽  
Anna D. Tischler
Keyword(s):  
Binding Site ◽  
Response Regulator ◽  
Phosphate Limitation ◽  
Infection Model ◽  
Transcriptional Level ◽  
Mobility Shift ◽  
Content Type ◽  
Constitutive Activation ◽  
Type Vii Secretion ◽  
Mouse Infection Model

ABSTRACTTheMycobacterium tuberculosistype VII secretion system ESX-5, which has been implicated in virulence, is activated at the transcriptional level by the phosphate starvation-responsive Pst/SenX3-RegX3 signal transduction system. Deletion ofpstA1, which encodes a Pst phosphate transporter component, causes constitutive activation of the response regulator RegX3, hypersecretion of ESX-5 substrates and attenuation in the mouse infection model. We hypothesized that constitutive activation of ESX-5 secretion causes attenuation of the ΔpstA1mutant. To test this, we uncoupled ESX-5 from regulation by RegX3. Using electrophoretic mobility shift assays, we defined a RegX3 binding site in theesx-5locus. Deletion or mutation of the RegX3 binding site reversed hypersecretion of the ESX-5 substrate EsxN by the ΔpstA1mutant and abrogated induction of EsxN secretion in response to phosphate limitation by wild-typeM. tuberculosis. Theesx-5RegX3 binding site deletion (ΔBS) also suppressed attenuation of the ΔpstA1mutant in Irgm1−/−mice. These data suggest that constitutive ESX-5 secretion sensitizesM. tuberculosisto an immune response that still occurs in Irgm1−/−mice. However, the ΔpstA1ΔBSmutant remained attenuated in both NOS2−/−and C57BL/6 mice, suggesting that factors other than ESX-5 secretion also contribute to attenuation of the ΔpstA1mutant. In addition, a ΔpstA1ΔesxNmutant lacking the hypersecreted ESX-5 substrate EsxN remained attenuated in Irgm1−/−mice, suggesting that ESX-5 substrates other than EsxN cause increased susceptibility to host immunity. Our data indicate that whileM. tuberculosisrequires ESX-5 for virulence, it tightly controls secretion of ESX-5 substrates to avoid elimination by host immune responses.

scholarly journals McbG, a LysR Family Transcriptional Regulator, Activates the mcbBCDEF Gene Cluster Involved in the Upstream Pathway of Carbaryl Degradation in Pseudomonas sp. Strain XWY-1

2021 ◽  
Vol 87 (9) ◽  
Author(s):  
Zhijian Ke ◽  
Yidong Zhou ◽  
Wankui Jiang ◽  
Mingliang Zhang ◽  
Hui Wang ◽  
...  
Keyword(s):  
Binding Site ◽  
Gene Cluster ◽  
Transcriptional Activation ◽  
Regulatory Mechanism ◽  
Hydrolysis Product ◽  
Transcriptional Regulator ◽  
Transcriptional Regulators ◽  
Palindromic Sequence ◽  
Mobility Shift ◽  
Lysr Family

ABSTRACT Although enzyme-encoding genes involved in the degradation of carbaryl have been reported in Pseudomonas sp. strain XWY-1, no regulator has been identified yet. In the mcbABCDEF cluster responsible for the upstream pathway of carbaryl degradation (from carbaryl to salicylate), the mcbA gene is constitutively expressed, while mcbBCDEF is induced by 1-naphthol, the hydrolysis product of carbaryl by McbA. In this study, we identified McbG, a transcriptional activator of the mcbBCDEF cluster. McbG is a 315-amino-acid protein with a molecular mass of 35.7 kDa. It belongs to the LysR family of transcriptional regulators and shows 28.48% identity to the pentachlorophenol (PCP) degradation transcriptional activation protein PcpR from Sphingobium chlorophenolicum ATCC 39723. Gene disruption and complementation studies reveal that mcbG is essential for transcription of the mcbBCDEF cluster in response to 1-naphthol in strain XWY-1. The results of the electrophoretic mobility shift assay (EMSA) and DNase I footprinting show that McbG binds to the 25-bp motif in the mcbBCDEF promoter area. The palindromic sequence TATCGATA within the motif is essential for McbG binding. The binding site is located between the –10 box and the transcription start site. In addition, McbG can repress its own transcription. The EMSA results show that a 25-bp motif in the mcbG promoter area plays an important role in McbG binding to the promoter of mcbG. This study reveals the regulatory mechanism for the upstream pathway of carbaryl degradation in strain XWY-1. The identification of McbG increases the variety of regulatory models within the LysR family of transcriptional regulators. IMPORTANCE Pseudomonas sp. strain XWY-1 is a carbaryl-degrading strain that utilizes carbaryl as the sole carbon and energy source for growth. The functional genes involved in the degradation of carbaryl have already been reported. However, the regulatory mechanism has not been investigated yet. Previous studies demonstrated that the mcbA gene, responsible for hydrolysis of carbaryl to 1-naphthol, is constitutively expressed in strain XWY-1. In this study, we identified a LysR-type transcriptional regulator, McbG, which activates the mcbBCDEF gene cluster responsible for the degradation of 1-naphthol to salicylate and represses its own transcription. The DNA binding site of McbG in the mcbBCDEF promoter area contains a palindromic sequence, which affects the binding of McbG to DNA. These findings enhance our understanding of the mechanism of microbial degradation of carbaryl.

scholarly journals The Mycobacterium tuberculosis Phosphate-Sensing Pst/SenX3-RegX3 System Regulates ESX-5 Secretion to Evade Host Immunity

2018 ◽  
Author(s):  
Sarah R. Elliott ◽  
Dylan W. White ◽  
Anna D. Tischler
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Immune Responses ◽  
Binding Site ◽  
Host Immunity ◽  
Phosphate Limitation ◽  
Infection Model ◽  
Transcriptional Level ◽  
Mobility Shift ◽  
Constitutive Activation ◽  
Host Immune Responses

ABSTRACTThe Mycobacterium tuberculosis Type VII secretion system ESX-5, which has been implicated in virulence, is activated at the transcriptional level by the phosphate starvation responsive Pst/SenX3-RegX3 signal transduction system. Deletion of pstA1, which encodes a Pst phosphate transporter component, causes constitutive activation of the response regulator RegX3, hyper-secretion of ESX-5 substrates and attenuation in the mouse infection model. We hypothesized that constitutive activation of ESX-5 secretion causes attenuation of the ΔpstA1 mutant. To test this, we uncoupled ESX-5 from regulation by RegX3. Using electrophoretic mobility shift assays, we defined a RegX3 binding site in the esx-5 locus. Deletion or mutation of the RegX3 binding site reversed hyper-secretion of the ESX-5 substrate EsxN by the ΔpstA1 mutant and abrogated induction of EsxN secretion in response to phosphate limitation by wild-type M. tuberculosis. Deletion of the esx-5 RegX3 binding site (ΔBS) suppressed attenuation of the ΔpstA1 mutant in Irgm1-/- mice, suggesting that constitutive ESX-5 secretion limits M. tuberculosis evasion of host immune responses that are independent of Irgm1. However, the ΔpstA1ΔBS mutant remained attenuated in both NOS2-/- and C57BL/6 mice, suggesting that factors other than ESX-5 secretion also contribute to attenuation of the ΔpstA1 mutant. In addition, a ΔpstA1ΔesxN mutant lacking the hyper-secreted ESX-5 substrate EsxN remained attenuated in Irgm1-/- mice, suggesting that ESX-5 substrates other than EsxN cause increased susceptibility to host immunity. Our data indicate that while M. tuberculosis requires ESX-5 for virulence, it tightly controls secretion of ESX-5 substrates to avoid elimination by host immune responses.

scholarly journals Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens

2008 ◽  
Vol 190 (7) ◽  
pp. 2496-2504 ◽  
Author(s):  
Po-Chi Soo ◽  
Yu-Tze Horng ◽  
Jun-Rong Wei ◽  
Jwu-Ching Shu ◽  
Chia-Chen Lu ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Binding Site ◽  
Promoter Region ◽  
Transcriptional Start Site ◽  
Direct Interaction ◽  
Underlying Mechanism ◽  
Inhibitory Effect ◽  
Start Codon ◽  
Mobility Shift

ABSTRACT Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDCSm and hagSm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB∼P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB∼P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB∼P binding site is located between base pair positions −341 and −364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “−35” promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB∼P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

scholarly journals The Regulatory Mechanism of Water Activities on Aflatoxins Biosynthesis and Conidia Development, and Transcription Factor AtfB Is Involved in This Regulation

Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 431
Author(s):  
Longxue Ma ◽  
Xu Li ◽  
Xiaoyun Ma ◽  
Qiang Yu ◽  
Xiaohua Yu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Environmental Factors ◽  
Health Risks ◽  
Sexual Development ◽  
Regulatory Mechanism ◽  
Food Storage ◽  
Economic Losses ◽  
Transcriptional Level ◽  
Conidia Production

Peanuts are frequently infected by Aspergillus strains and then contaminated by aflatoxins (AF), which brings out economic losses and health risks. AF production is affected by diverse environmental factors, especially water activity (aw). In this study, A. flavus was inoculated into peanuts with different aw (0.90, 0.95, and 0.99). Both AFB1 yield and conidia production showed the highest level in aw 0.90 treatment. Transcriptional level analyses indicated that AF biosynthesis genes, especially the middle- and later-stage genes, were significantly up-regulated in aw 0.90 than aw 0.95 and 0.99. AtfB could be the pivotal regulator response to aw variations, and could further regulate downstream genes, especially AF biosynthesis genes. The expressions of conidia genes and relevant regulators were also more up-regulated at aw 0.90 than aw 0.95 and 0.99, suggesting that the relative lower aw could increase A. flavus conidia development. Furthermore, transcription factors involved in sexual development and nitrogen metabolism were also modulated by different aw. This research partly clarified the regulatory mechanism of aw on AF biosynthesis and A. flavus development and it would supply some advice for AF prevention in food storage.

scholarly journals Silencing of the Gene for the α-Subunit of Human Chorionic Gonadotropin by the Embryonic Transcription Factor Oct-3/4

1997 ◽  
Vol 11 (11) ◽  
pp. 1651-1658 ◽  
Author(s):  
Limin Liu ◽  
Douglas Leaman ◽  
Michel Villalta ◽  
R. Michael Roberts
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Α Subunit ◽  
Choriocarcinoma Cells ◽  
Human Choriocarcinoma Cells ◽  
Electrophoretic Mobility Shift Assays ◽  
Jar Cells

Abstract CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG β-subunit (hCGβ) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGβ gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG α-subunit (hCGα) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the −170 hCGα promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the −148 hCGα or the −99 hCGα promoter. Unexpectedly, no Oct-3/4-binding site was identified within the −170 to −148 region of the hCGα promoter, although one was found around position −115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGα gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGα mRNA and protein by 70–80%. Oct-3/4 is therefore capable of silencing both hCGα and hCGβ gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGα and hCGβ genes.

scholarly journals FarRRegulates the farAB-Encoded Efflux Pump of Neisseriagonorrhoeae via an MtrR RegulatoryMechanism

2003 ◽  
Vol 185 (24) ◽  
pp. 7145-7152 ◽  
Author(s):  
E.-H. Lee ◽  
C. Rouquette-Loughlin ◽  
J. P. Folster ◽  
W. M. Shafer
Keyword(s):  
Regulatory Mechanism ◽  
Efflux Pump ◽  
Regulatory Protein ◽  
Transcriptional Repressors ◽  
Sequence Database ◽  
Mobility Shift ◽  
Pump System ◽  
Regulatory Cascade ◽  
Genome Sequence Database ◽  
Gel Mobility Shift

ABSTRACT The farAB operon of Neisseria gonorrhoeae encodes an efflux pump which mediates gonococcal resistance to antibacterial fatty acids. It was previously observed that expression of the farAB operon was positively regulated by MtrR, which is a repressor of the mtrCDE-encoded efflux pump system (E.-H. Lee and W. M. Shafer, Mol. Microbiol. 33:839-845, 1999). This regulation was believed to be indirect since MtrR did not bind to the farAB promoter. In this study, computer analysis of the gonococcal genome sequence database, lacZ reporter fusions, and gel mobility shift assays were used to elucidate the regulatory mechanism by which expression of the farAB operon is modulated by MtrR in gonococci. We identified a regulatory protein belonging to the MarR family of transcriptional repressors and found that it negatively controls expression of farAB by directly binding to the farAB promoter. We designated this regulator FarR to signify its role in regulating the farAB operon. We found that MtrR binds to the farR promoter, thereby repressing farR expression. Hence, MtrR regulates farAB in a positive fashion by modulating farR expression. This MtrR regulatory cascade seems to play an important role in adjusting levels of the FarAB and MtrCDE efflux pumps to prevent their excess expression in gonococci.

Does Hepatic Hepcidin Play an Important Role in Exercise-Associated Anemia in Rats?

2011 ◽  
Vol 21 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Yu-Qian Liu ◽  
Yan-Zhong Chang ◽  
Bin Zhao ◽  
Hai-Tao Wang ◽  
Xiang-Lin Duan
Keyword(s):  
Regulatory Mechanism ◽  
Iron Absorption ◽  
White Blood Cells ◽  
Strenuous Exercise ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Transcriptional Level ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Sports Anemia

Some athletes are diagnosed as suffering from sports anemia because of iron deficiency, but the regulatory mechanism remains poorly understood. It is reported that hepcidin may provide a way to illuminate the regulatory mechanism of exercise-associated anemia. Here the authors investigate the hepcidin-involved iron absorption in exercise-associated anemia. Twelve male Wistar rats (300 ± 10 g) were randomly divided into 2 groups, 6 in a control group (CG) and 6 in an exercise group (EG, 5 wk treadmill exercise of different intensities with progressive loading). Serum samples were analyzed for circulating levels of IL-6 by means of enzyme-linked immunosorbent assay (ELISA). The expression of hepatic hepcidin mRNA was examined by real-time polymerase chain reaction analysis. The protein levels of divalent metal transporter 1 (DMT1), ferroportin1 (FPN1), and heme-carrier protein 1 (HCP1) of duodenum epithelium were examined by Western blot. The results showed that the amount of iron and ferritin in serum were lower in EG than in CG (p < .05). The levels of IL-6 and white blood cells were greater in EG than in CG (p < .01). The expression of DMT1, HCP1, and FPN1 was significantly lower in EG than in CG (p < .01). The mRNA expressions of hepatic hepcidin and hemojuvelin in skeletal muscle were remarkably higher in EG than in CG. The data indicated that inflammation was induced by strenuous exercise, and as a result, the transcriptional level of the hepatic hepcidin gene was increased, which further inhibited the expression of iron-absorption proteins and led to exercise-associated anemia.

Sign in / Sign up

Export Citation Format

Share Document