scholarly journals Efficient Production of Polymyxin in the Surrogate Host Bacillus subtilis by Introducing a ForeignectBGene and Disrupting theabrBGene

2012 ◽  
Vol 78 (12) ◽  
pp. 4194-4199 ◽  
Author(s):  
Soo-Young Park ◽  
Soo-Keun Choi ◽  
Jihoon Kim ◽  
Tae-Kwang Oh ◽  
Seung-Hwan Park
Keyword(s):  
Bacillus Subtilis ◽  
Paenibacillus Polymyxa ◽  
Biosynthetic Gene Cluster ◽  
Upstream Region ◽  
Efficient Production ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Gene Encoding ◽  
Content Type ◽  
Surrogate Host

ABSTRACTIn our previous study,Bacillus subtilisstrain BSK3S, containing a polymyxin biosynthetic gene cluster fromPaenibacillus polymyxa, could produce polymyxin only in the presence of exogenously addedl-2,4-diaminobutyric acid (Dab). The dependence of polymyxin production on exogenous Dab was removed by introducing anectBgene encoding the diaminobutyrate synthase ofP. polymyxainto BSK3S (resulting in strain BSK4). We found, by observing the complete inhibition of polymyxin synthesis when thespo0Agene was knocked out (strain BSK4-0A), that Spo0A is indispensable for the production of polymyxin. Interestingly, theabrB-spo0Adouble-knockout mutant, BSK4-0A-rB, and the singleabrBmutant, BSK4-rB, showed 1.7- and 2.3-fold increases, respectively, in polymyxin production over that of BSK4. These results coincided with the transcription levels ofpmxAin the strains observed by quantitative real-time PCR (qRT-PCR). The AbrB protein was shown to bind directly to the upstream region ofpmxA, indicating that AbrB directly inhibits the transcription of polymyxin biosynthetic genes. The BSK4-rB strain, producing high levels of polymyxin, will be useful for the development and production of novel polymyxin derivatives.

scholarly journals Transcriptional Regulator PhlH Modulates 2,4-Diacetylphloroglucinol Biosynthesis in Response to the Biosynthetic Intermediate and End Product

2017 ◽  
Vol 83 (21) ◽  
Author(s):  
Xu Yan ◽  
Rui Yang ◽  
Rui-Xue Zhao ◽  
Jian-Ting Han ◽  
Wen-Juan Jia ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Gene Cluster ◽  
Plant Pathogens ◽  
Feedback Regulation ◽  
Biosynthetic Gene Cluster ◽  
Sequence Motif ◽  
Target Sequence ◽  
Gene Encoding ◽  
Negative Feedback Regulation ◽  
Content Type

ABSTRACT Certain strains of biocontrol bacterium Pseudomonas fluorescens produce the secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) to antagonize soilborne phytopathogens in the rhizosphere. The gene cluster responsible for the biosynthesis of 2,4-DAPG is named phlACBDEFGH and it is still unclear how the pathway-specific regulator phlH within this gene cluster regulates the metabolism of 2,4-DAPG. Here, we found that PhlH in Pseudomonas fluorescens strain 2P24 represses the expression of the phlG gene encoding the 2,4-DAPG hydrolase by binding to a sequence motif overlapping with the −35 site recognized by σ70 factors. Through biochemical screening of PhlH ligands we identified the end product 2,4-DAPG and its biosynthetic intermediate monoacetylphloroglucinol (MAPG), which can act as signaling molecules to modulate the binding of PhlH to the target sequence and activate the expression of phlG. Comparison of 2,4-DAPG production between the ΔphlH, ΔphlG, and ΔphlHG mutants confirmed that phlH and phlG impose negative feedback regulation over 2,4-DAPG biosynthesis. It was further demonstrated that the 2,4-DAPG degradation catalyzed by PhlG plays an insignificant role in 2,4-DAPG tolerance but contributes to bacterial growth advantages under carbon/nitrogen starvation conditions. Taken together, our data suggest that by monitoring and down-tuning in situ levels of 2,4-DAPG, the phlHG genes could dynamically modulate the metabolic loads attributed to 2,4-DAPG production and potentially contribute to rhizosphere adaptation. IMPORTANCE 2,4-DAPG, which is synthesized by biocontrol pseudomonad bacteria, is a broad-spectrum antibiotic against bacteria, fungi, oomycetes, and nematodes and plays an important role in suppressing soilborne plant pathogens. Although most of the genes in the 2,4-DAPG biosynthetic gene cluster (phl) have been characterized, it is still not clear how the pathway-specific regulator phlH is involved in 2,4-DAPG metabolism. This work revealed the role of PhlH in modulating 2,4-DAPG levels by controlling the expression of 2,4-DAPG hydrolase PhlG in response to 2,4-DAPG and MAPG. Since 2,4-DAPG biosynthesis imposes a metabolic burden on biocontrol pseudomonads, it is expected that the fine regulation of phlG by PhlH offers a way to dynamically modulate the metabolic loads attributed to 2,4-DAPG production.

scholarly journals Tracking Down Biotransformation to the Genetic Level: Identification of a Highly Flexible Glycosyltransferase from Saccharothrix espanaensis

2013 ◽  
Vol 79 (17) ◽  
pp. 5224-5232 ◽  
Author(s):  
Tina Strobel ◽  
Yvonne Schmidt ◽  
Anton Linnenbrink ◽  
Andriy Luzhetskyy ◽  
Marta Luzhetska ◽  
...  
Keyword(s):  
Natural Products ◽  
Phenolic Compounds ◽  
Natural Product ◽  
Defense Mechanism ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Gene Encoding ◽  
Content Type ◽  
Genetic Level ◽  
Level Identification

ABSTRACTSaccharothrix espanaensisis a member of the orderActinomycetales. The genome of the strain has been sequenced recently, revealing 106 glycosyltransferase genes. In this paper, we report the detection of a glycosyltransferase fromSaccharothrix espanaensiswhich is able to rhamnosylate different phenolic compounds targeting different positions of the molecules. The gene encoding the flexible glycosyltransferase is not located close to a natural product biosynthetic gene cluster. Therefore, the native function of this enzyme might be not the biosynthesis of a secondary metabolite but the glycosylation of internal and external natural products as part of a defense mechanism.

scholarly journals Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

2012 ◽  
Vol 56 (8) ◽  
pp. 4175-4183 ◽  
Author(s):  
Seung Young Kim ◽  
Kou-San Ju ◽  
William W. Metcalf ◽  
Bradley S. Evans ◽  
Tomohisa Kuzuyama ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Citrate Synthase ◽  
Wide Spectrum ◽  
The United States ◽  
Biosynthetic Gene Cluster ◽  
Gene Encoding ◽  
Acute Cystitis ◽  
Streptomyces Species ◽  
Content Type

ABSTRACTFosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production inPseudomonas syringaePB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that inStreptomyces fradiaeandStreptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome ofP. syringaePB-5123 lacks a gene encoding the PnPy decarboxylase found in theStreptomycesstrains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin inPseudomonas aeruginosafrom a fosmid encoding the fosfomycin biosynthetic cluster fromP. syringaePB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.

scholarly journals Nectrisine Biosynthesis Genes in Thelonectria discophora SANK 18292: Identification and Functional Analysis

2016 ◽  
Vol 82 (21) ◽  
pp. 6414-6422 ◽  
Author(s):  
Ryuki Miyauchi ◽  
Chiho Ono ◽  
Takashi Ohnuki ◽  
Yoichiro Shiba
Keyword(s):  
Genetic Engineering ◽  
Heterologous Expression ◽  
Production System ◽  
Therapeutic Potential ◽  
Biosynthetic Gene Cluster ◽  
Efficient Production ◽  
Biosynthetic Gene ◽  
Content Type ◽  
E Coli ◽  
Glycosidase Inhibitor

ABSTRACTThe fungusThelonectria discophoraSANK 18292 produces the iminosugar nectrisine, which has a nitrogen-containing heterocyclic 5-membered ring and acts as a glycosidase inhibitor. In our previous study, an oxidase (designated NecC) that converts 4-amino-4-deoxyarabinitol to nectrisine was purified fromT. discophoracultures. However, the genes required for nectrisine biosynthesis remained unclear. In this study, the nectrisine biosynthetic gene cluster inT. discophorawas identified from the contiguous genome sequence around thenecCgene. Gene disruption and complementation studies and heterologous expression of the gene showed thatnecA,necB, andnecCcould be involved in nectrisine biosynthesis, during which amination, dephosphorylation, and oxidation occur. It was also demonstrated that nectrisine could be produced by recombinantEscherichia colicoexpressing thenecA,necB, andnecCgenes. These findings provide the foundation to develop a bacterial production system for nectrisine or its intermediates through genetic engineering.IMPORTANCEIminosugars might have great therapeutic potential for treatment of many diseases. However, information on the genes for their biosynthesis is limited. In this study, we report the identification of genes required for biosynthesis of the iminosugar nectrisine inThelonectria discophoraSANK 18292, which was verified by disruption, complementation, and heterologous expression of the genes involved. We also demonstrate heterologous production of nectrisine by recombinantE. coli, toward developing an efficient production system for nectrisine or its intermediates through genetic engineering.

scholarly journals A Membrane-Embedded Amino Acid Couples the SpoIIQ Channel Protein to Anti-Sigma Factor Transcriptional Repression during Bacillus subtilis Sporulation

2016 ◽  
Vol 198 (9) ◽  
pp. 1451-1463 ◽  
Author(s):  
Kelly A. Flanagan ◽  
Joseph D. Comber ◽  
Elizabeth Mearls ◽  
Colleen Fenton ◽  
Anna F. Wang Erickson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Sigma Factor ◽  
Late Gene ◽  
Late Gene Expression ◽  
Gene Encoding ◽  
Content Type ◽  
Regulatory Circuit ◽  
Gene Regulatory

ABSTRACTSpoIIQ is an essential component of a channel connecting the developing forespore to the adjacent mother cell duringBacillus subtilissporulation. This channel is generally required for late gene expression in the forespore, including that directed by the late-acting sigma factor σG. Here, we present evidence that SpoIIQ also participates in a previously unknown gene regulatory circuit that specifically represses expression of the gene encoding the anti-sigma factor CsfB, a potent inhibitor of σG. ThecsfBgene is ordinarily transcribed in the forespore only by the early-acting sigma factor σF. However, in a mutant lacking the highly conserved SpoIIQ transmembrane amino acid Tyr-28,csfBwas also aberrantly transcribed later by σG, the very target of CsfB inhibition. This regulation ofcsfBby SpoIIQ Tyr-28 is specific, given that the expression of other σF-dependent genes was unaffected. Moreover, we identified a conserved element within thecsfBpromoter region that is both necessary and sufficient for SpoIIQ Tyr-28-mediated inhibition. These results indicate that SpoIIQ is a bifunctional protein that not only generally promotes σGactivity in the forespore as a channel component but also specifically maximizes σGactivity as part of a gene regulatory circuit that represses σG-dependent expression of its own inhibitor, CsfB. Finally, we demonstrate that SpoIIQ Tyr-28 is required for the proper localization and stability of the SpoIIE phosphatase, raising the possibility that these two multifunctional proteins cooperate to fine-tune developmental gene expression in the forespore at late times.IMPORTANCECellular development is orchestrated by gene regulatory networks that activate or repress developmental genes at the right time and place. Late gene expression in the developingBacillus subtilisspore is directed by the alternative sigma factor σG. The activity of σGrequires a channel apparatus through which the adjacent mother cell provides substrates that generally support gene expression. Here we report that the channel protein SpoIIQ also specifically maximizes σGactivity as part of a previously unknown regulatory circuit that prevents σGfrom activating transcription of the gene encoding its own inhibitor, the anti-sigma factor CsfB. The discovery of this regulatory circuit significantly expands our understanding of the gene regulatory network controlling late gene expression in the developingB. subtilisspore.

scholarly journals High-Level Heterologous Production of d-Cycloserine by Escherichia coli

2015 ◽  
Vol 81 (22) ◽  
pp. 7881-7887 ◽  
Author(s):  
Takanori Kumagai ◽  
Tomoki Ozawa ◽  
Momoko Tanimoto ◽  
Masafumi Noda ◽  
Yasuyuki Matoba ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Cells ◽  
Resting Cells ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Biosynthetic Genes ◽  
T7 Promoter ◽  
Α Subunit ◽  
Content Type ◽  
E Coli

ABSTRACTPreviously, we successfully cloned ad-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designateddcsAtodcsJ) fromd-CS-producingStreptomyces lavendulaeATCC 11924. In this study, we put fourd-CS biosynthetic genes (dcsC,dcsD,dcsE, anddcsG) in tandem under the control of the T7 promoter in anEscherichia colihost. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. Whenl-serine and hydroxyurea (HU), the precursors ofd-CS, were incubated together with theE. coliresting cell suspension, the cells produced significant amounts ofd-CS (350 ± 20 μM). To increase the productivity ofd-CS, thedcsJgene, which might be responsible for thed-CS excretion, was connected downstream of the four genes. TheE. coliresting cells harboring the five genes producedd-CS at 660 ± 31 μM. ThedcsDgene product, DcsD, formsO-ureido-l-serine fromO-acetyl-l-serine (OAS) and HU, which are intermediates ind-CS biosynthesis. DcsD also catalyzes the formation ofl-cysteine from OAS and H2S. To repress the side catalytic activity of DcsD, theE. colichromosomalcysJandcysKgenes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the fourd-CS biosynthetic genes, together withdcsJ, were incubated withl-serine and HU, thed-CS production was 980 ± 57 μM, which is comparable to that ofd-CS-producingS. lavendulaeATCC 11924 (930 ± 36 μM).

scholarly journals Identification of 14-α-Lanosterol Demethylase (CYP51) in Scedosporium Species

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Anne Bernhardt ◽  
Wieland Meyer ◽  
Volker Rickerts ◽  
Toni Aebischer ◽  
Kathrin Tintelnot
Keyword(s):  
Amino Acid ◽  
Resistance Mechanisms ◽  
Upstream Region ◽  
Scedosporium Apiospermum ◽  
Gene Encoding ◽  
Specific Amino Acid ◽  
Content Type ◽  
Scedosporium Dehoogii ◽  
Lanosterol Demethylase ◽  
Species Specific

ABSTRACT Scedosporium spp. cause infections (scedosporiosis) in both immunocompetent and immunocompromised individuals and may persistently colonize the respiratory tract in patients with cystic fibrosis (CF). They are less susceptible against azoles than are other molds, such as Aspergillus spp., suggesting the presence of resistance mechanisms. It can be hypothesized that the decreased susceptibility of Scedosporium spp. to azoles is also CYP51 dependent. Analysis of the Scedosporium apiospermum and Scedosporium aurantiacum genomes revealed one CYP51 gene encoding the 14-α-lanosterol demethylase. This gene from 159 clinical or environmental Scedosporium isolates and three Lomentospora prolificans isolates has been sequenced and analyzed. The Scedosporium CYP51 protein clustered with the group of known CYP51B orthologues and showed species-specific polymorphisms. A tandem repeat in the 5′ upstream region of Scedosporium CYP51 like that in Aspergillus fumigatus could not be detected. Species-specific amino acid alterations in CYP51 of Scedosporium boydii, Scedosporium ellipsoideum, Scedosporium dehoogii, and Scedosporium minutisporum isolates were located at positions that have not been described as having an impact on azole susceptibility. In contrast, two of the three S. apiospermum-specific amino acid changes (Y136F and G464S) corresponded to respective mutations in A. fumigatus CYP51A at amino acid positions 121 and 448 (Y121F and G448S, respectively) that had been linked to azole resistance.

scholarly journals Analysis of the Tunicamycin Biosynthetic Gene Cluster ofStreptomyces chartreusisReveals New Insights into Tunicamycin Production and Immunity

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
David Widdick ◽  
Sylvain F. Royer ◽  
Hua Wang ◽  
Natalia M. Vior ◽  
Juan Pablo Gomez-Escribano ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Streptomyces Coelicolor ◽  
Biosynthetic Gene Cluster ◽  
Transcriptional Analysis ◽  
Efficient Production ◽  
Primary Metabolism ◽  
Biosynthetic Gene ◽  
Content Type ◽  
High Degree ◽  
Single Operon

ABSTRACTThe tunicamycin biosynthetic gene cluster ofStreptomyces chartreusisconsists of 14 genes (tunAtotunN) with a high degree of apparent translational coupling. Transcriptional analysis revealed that all of these genes are likely to be transcribed as a single operon from two promoters,tunp1 andtunp2. In-frame deletion analysis revealed that just six of these genes (tunABCDEH) are essential for tunicamycin production in the heterologous hostStreptomyces coelicolor, while five (tunFGKLN) with likely counterparts in primary metabolism are not necessary, but presumably ensure efficient production of the antibiotic at the onset of tunicamycin biosynthesis. Three genes are implicated in immunity, namely,tunIandtunJ, which encode a two-component ABC transporter presumably required for export of the antibiotic, andtunM, which encodes a putativeS-adenosylmethionine (SAM)-dependent methyltransferase. Expression oftunIJortunMinS. coelicolorconferred resistance to exogenous tunicamycin. The results presented here provide new insights into tunicamycin biosynthesis and immunity.

scholarly journals PafR, a Novel Transcription Regulator, Is Important for Pathogenesis in Uropathogenic Escherichia coli

2014 ◽  
Vol 82 (10) ◽  
pp. 4241-4252 ◽  
Author(s):  
Mordechai Baum ◽  
Mobarak Watad ◽  
Sara N. Smith ◽  
Christopher J. Alteri ◽  
Noa Gordon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Gene Knockout ◽  
Sensory System ◽  
Phosphotransferase System ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Content Type

ABSTRACTThemetVgenomic island in the chromosome of uropathogenicEscherichia coli(UPEC) encodes a putative transcription factor and a sugar permease of the phosphotransferase system (PTS), which are predicted to compose a Bgl-like sensory system. The presence of these two genes, hereby termedpafRandpafP, respectively, has been previously shown to correlate with isolates causing clinical syndromes. We show here that deletion of both genes impairs the ability of the resulting mutant to infect the CBA/J mouse model of ascending urinary tract infection compared to that of the parent strain, CFT073. Expressing the two genes intransin the two-gene knockout mutant complemented full virulence. Deletion of either gene individually generated the same phenotype as the double knockout, indicating that bothpafRandpafPare important to pathogenesis. We screened numerous environmental conditions but failed to detect expression from the promoter that precedes thepafgenesin vitro, suggesting that they arein vivoinduced (ivi). Although PafR is shown here to be capable of functioning as a transcriptional antiterminator, its targets in the UPEC genome are not known. Using microarray analysis, we have shown that expression of PafR from a heterologous promoter in CFT073 affects expression of genes related to bacterial virulence, biofilm formation, and metabolism. Expression of PafR also inhibits biofilm formation and motility. Taken together, our results suggest that thepafgenes are implicated in pathogenesis and that PafR controls virulence genes, in particular biofilm formation genes.

scholarly journals The Bacillus subtilis tyrZ Gene Encodes a Highly Selective Tyrosyl-tRNA Synthetase and Is Regulated by a MarR Regulator and T Box Riboswitch

2015 ◽  
Vol 197 (9) ◽  
pp. 1624-1631 ◽  
Author(s):  
Rebecca N. Williams-Wagner ◽  
Frank J. Grundy ◽  
Medha Raina ◽  
Michael Ibba ◽  
Tina M. Henkin
Keyword(s):  
Bacillus Subtilis ◽  
Biofilm Formation ◽  
Normal Growth ◽  
Growth Conditions ◽  
Trna Synthetase ◽  
Cellular Proteins ◽  
Gene Encoding ◽  
Content Type ◽  
T Box ◽  
Related Compounds

ABSTRACTMisincorporation ofd-tyrosine (d-Tyr) into cellular proteins due to mischarging of tRNATyrwithd-Tyr by tyrosyl-tRNA synthetase inhibits growth and biofilm formation ofBacillus subtilis. Furthermore, manyB. subtilisstrains lack a functional gene encodingd-aminoacyl-tRNA deacylase, which prevents misincorporation ofd-Tyr in most organisms.B. subtilishas two genes that encode tyrosyl-tRNA synthetase:tyrSis expressed under normal growth conditions, andtyrZis known to be expressed only whentyrSis inactivated by mutation. We hypothesized thattyrZencodes an alternate tyrosyl-tRNA synthetase, expression of which allows the cell to grow whend-Tyr is present. We show that TyrZ is more selective forl-Tyr overd-Tyr than is TyrS; however, TyrZ is less efficient overall. We also show that expression oftyrZis required for growth and biofilm formation in the presence ofd-Tyr. BothtyrSandtyrZare preceded by a T box riboswitch, buttyrZis found in an operon withywaE, which is predicted to encode a MarR family transcriptional regulator. Expression oftyrZis repressed by YwaE and also is regulated at the level of transcription attenuation by the T box riboswitch. We conclude that expression oftyrZmay allow growth when excessd-Tyr is present.IMPORTANCEAccurate protein synthesis requires correct aminoacylation of each tRNA with the cognate amino acid and discrimination against related compounds.Bacillus subtilisproducesd-Tyr, an analog ofl-Tyr that is toxic when incorporated into protein, during stationary phase. Most organisms utilize ad-aminoacyl-tRNA deacylase to prevent misincorporation ofd-Tyr. This work demonstrates that the increased selectivity of the TyrZ form of tyrosyl-tRNA synthetase may provide a mechanism by whichB. subtilisprevents misincorporation ofd-Tyr in the absence of a functionald-aminoacyl-tRNA deacylase gene.

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