scholarly journals Virulence, Host-Selective Toxin Production, and Development of Three Cochliobolus Phytopathogens Lacking the Sfp-Type 4′-Phosphopantetheinyl Transferase Ppt1

2015 ◽  
Vol 28 (10) ◽  
pp. 1130-1141 ◽  
Author(s):  
Nur Ain Izzati Mohd Zainudin ◽  
Bradford Condon ◽  
Lieselotte De Bruyne ◽  
Christof Van Poucke ◽  
Qing Bi ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Toxin Production ◽  
Lysine Biosynthesis ◽  
Phosphopantetheinyl Transferase ◽  
Wild Type ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Asexual Sporulation ◽  
Peptide Synthetase ◽  
Sexual Crosses

The Sfp-type 4′-phosphopantetheinyl transferase Ppt1 is required for activation of nonribosomal peptide synthetases, including α-aminoadipate reductase (AAR) for lysine biosynthesis and polyketide synthases, enzymes that biosynthesize peptide and polyketide secondary metabolites, respectively. Deletion of the PPT1 gene, from the maize pathogen Cochliobolus heterostrophus and the rice pathogen Cochliobolus miyabeanus, yielded strains that were significantly reduced in virulence to their hosts. In addition, ppt1 mutants of C. heterostrophus race T and Cochliobolus victoriae were unable to biosynthesize the host-selective toxins (HST) T-toxin and victorin, respectively, as judged by bioassays. Interestingly, ppt1 mutants of C. miyabeanus were shown to produce tenfold higher levels of the sesterterpene-type non-HST ophiobolin A, as compared with the wild-type strain. The ppt1 strains of all species were also reduced in tolerance to oxidative stress and iron depletion; both phenotypes are associated with inability to produce extracellular siderophores biosynthesized by the nonribosomal peptide synthetase Nps6. Colony surfaces were hydrophilic, a trait previously associated with absence of C. heterostrophus Nps4. Mutants were decreased in asexual sporulation and C. heterostrophus strains were female-sterile in sexual crosses; the latter phenotype was observed previously with mutants lacking Nps2, which produces an intracellular siderophore. As expected, mutants were albino, since they cannot produce the polyketide melanin and were auxotrophic for lysine because they lack an AAR.

scholarly journals Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase

2016 ◽  
Vol 29 (2) ◽  
pp. 109-118 ◽  
Author(s):  
Desen Zheng ◽  
Thomas J. Burr
Keyword(s):  
Crown Gall ◽  
Polyketide Synthase ◽  
Wild Type ◽  
Tissue Necrosis ◽  
Agrobacterium Vitis ◽  
Nonribosomal Peptide ◽  
Crown Gall Disease ◽  
Peptide Synthetases ◽  
Peptide Synthetase ◽  
Polyketide Synthase Gene

Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730+) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730+ indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

scholarly journals VdNPS, a Nonribosomal Peptide Synthetase, Is Involved in Regulating Virulence in Verticillium dahliae

2020 ◽  
Vol 110 (8) ◽  
pp. 1398-1409
Author(s):  
Xiumei Luo ◽  
Tingting Tian ◽  
Xue Tan ◽  
Yixuan Zheng ◽  
Chengjian Xie ◽  
...  
Keyword(s):  
Verticillium Dahliae ◽  
Durable Resistance ◽  
Functional Characterization ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Peptide Synthetase ◽  
Pathogenesis Related ◽  
Pathogenesis Related Gene ◽  
Type V ◽  
Species Production

Nonribosomal peptide synthetases (NPS) are known for the biosynthesis of antibiotics, toxins, and siderophore production. They are also a virulence determinant in different phytopathogens. However, until now, the functional characterization of NPS in Verticillium dahliae has not been reported. Deletion of the NPS gene in V. dahliae led to the decrease of conidia, microsclerotia, and pathogenicity. ΔVdNPS strains were tolerant to H2O2, and the genes involved in H2O2 detoxification, iron/copper transport, and cytoskeleton were differentially expressed in ΔVdNPS. Interestingly, ΔVdNPS strains exhibited hypersensitivity to salicylic acid (SA), and the genes involved in SA hydroxylation were up-regulated in ΔVdNPS compared with wild-type V. dahliae under SA stress. Additionally, during infection, ΔVdNPS induced more pathogenesis-related gene expression, higher reactive oxygen species production, and stronger SA-mediated signaling transduction in host to overcome pathogen. Uncovering the function of VdNPS in pathogenicity could provide a reliable theoretical basis for the development of cultivars with durable resistance against V. dahliae-associated diseases.

scholarly journals A Nonribosomal Peptide Synthetase Gene (mgoA) of Pseudomonas syringae pv. syringae Is Involved in Mangotoxin Biosynthesis and Is Required for Full Virulence

2007 ◽  
Vol 20 (5) ◽  
pp. 500-509 ◽  
Author(s):  
Eva Arrebola ◽  
Francisco M. Cazorla ◽  
Diego Romero ◽  
Alejandro Pérez-García ◽  
Antonio de Vicente
Keyword(s):  
Pseudomonas Syringae ◽  
Chromosomal Region ◽  
Open Reading Frames ◽  
Modular Architecture ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Large Gene ◽  
Peptide Synthetase ◽  
Apical Necrosis ◽  
Reading Frames

Pseudomonas syringae pv. syringae, which causes the bacterial apical necrosis of mango, produces the antimetabolite mangotoxin. We report here the cloning, sequencing, and identity analysis of a chromosomal region of 11.1 kb from strain P. syringae pv. syringae UMAF0158, which is involved in mangotoxin biosynthesis. This chromosomal region contains six complete open reading frames (ORFs), including a large gene (ORF5) with a modular architecture characteristic of nonribosomal peptide synthetases (NRPS) named mgoA. A Tn 5 mutant disrupted in mgoA was defective in mangotoxin production, revealing the involvement of the putative NRPS gene in the biosynthesis of mangotoxin. This derivative strain impaired in mangotoxin production also showed a reduction in virulence as measured by necrotic symptoms on tomato leaflets. Mangotoxin production and virulence were restored fully in the NRPS mutant by complementation with plasmid pCG2-6, which contains an 11,103-bp chromosomal region cloned from the wildtype strain P. syringae pv. syringae UMAF0158 that includes the putative NPRS gene (mgoA). The results demonstrate that mgoA has a role in the virulence of P. syringae pv. syringae. The involvement of an NRPS in the production of an antimetabolite toxin from P. syringae inhibiting ornithine acetyltransferase activity is proposed.

Presence of peptide synthetase gene transcripts and accumulation of ergopeptines in Claviceps purpurea and Neotyphodium coenophialum

1998 ◽  
Vol 44 (1) ◽  
pp. 80-86 ◽  
Author(s):  
Seanna L Annis ◽  
Daniel G Panaccione
Keyword(s):  
Tall Fescue ◽  
Claviceps Purpurea ◽  
Neotyphodium Coenophialum ◽  
Experimental Conditions ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Peptide Synthetase ◽  
Different Ages ◽  
Gene Transcripts ◽  
Functional Analyses

The production of toxic ergopeptine alkaloids by the fungi Claviceps purpurea and Neotyphodium coenophialum involves the activity of one or more nonribosomal peptide synthetases. Claviceps purpurea and N. coenophialum each have several different peptide synthetase genes, fragments of which have been cloned previously. An additional Claviceps purpurea peptide synthetase gene was cloned by hybridization with one of the N. coenophialum peptide synthetase gene fragments. We detected the presence of mRNA from the peptide synthetase genes in cultures of different ages grown under conditions favorable or unfavorable for ergopeptine production. All four peptide synthetase genes from Claviceps purpurea were transcribed under at least some of the experimental conditions. Transcripts from three of the four genes were detected under conditions consistent with their potential involvement in ergopeptine biosynthesis. All three peptide synthetase genes previously identified in N. coenophialum were transcribed during symbiotic growth of this fungus with tall fescue, as well as in ergopeptine-producing cultures. The data show that all of the peptide synthetase genes are transcribed, that one of the peptide synthetase genes is dissociated from ergopeptine biosynthesis, and, as a result, prioritize the remaining genes for functional analyses by transformation-mediated gene disruption.Key words: Claviceps purpurea, endophyte, ergot, ergotamine, Neotyphodium coenophialum.

scholarly journals Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis

2020 ◽  
Author(s):  
Maliheh Vahidinasab ◽  
Lars Lilge ◽  
Aline Reinfurt ◽  
Jens Pfannstiel ◽  
Marius Henkel ◽  
...  
Keyword(s):  
Metabolic Flux ◽  
Plant Pathogens ◽  
Phosphopantetheinyl Transferase ◽  
Peptide Synthetases ◽  
Promising Strategy ◽  
Study Results ◽  
Peptide Synthetase ◽  
Regulation Of Biosynthesis ◽  
Stimulation Of ◽  
Producer Strains

Abstract Background: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production.Results: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain ensuring stimulation of DegU-P regulon (strain BMV10). Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition.Conclusions: This study provides evidence that degQ stimulates the native plipastatin production. Moreover, it seems that surfactin synthetase or one of its subunits positively regulates plipastatin production by an unknown mechanism. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

scholarly journals Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Maliheh Vahidinasab ◽  
Lars Lilge ◽  
Aline Reinfurt ◽  
Jens Pfannstiel ◽  
Marius Henkel ◽  
...  
Keyword(s):  
Metabolic Flux ◽  
Plant Pathogens ◽  
Phosphopantetheinyl Transferase ◽  
Peptide Synthetases ◽  
Promising Strategy ◽  
Study Results ◽  
Peptide Synthetase ◽  
Regulation Of Biosynthesis ◽  
Stimulation Of ◽  
Producer Strains

Abstract Background Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production. Results The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition. Conclusions This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

scholarly journals Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases

2015 ◽  
Vol 51 (12) ◽  
pp. 2262-2265 ◽  
Author(s):  
Sho Konno ◽  
Fumihiro Ishikawa ◽  
Takehiro Suzuki ◽  
Naoshi Dohmae ◽  
Michael D. Burkart ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Nonribosomal Peptide Synthetase ◽  
Protein Labeling ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases ◽  
Peptide Synthetase ◽  
A Domains

Active site-directed proteomic probes coupled to the 5′-O-N-(aminoacyl)sulfamoyladenosine (AMS) scaffold with a clickable benzophenone functionality selectively target nonribosomal peptide synthetase (NRPS) adenylation (A) domains in natural product producer proteomes by ligand-directed protein labeling.

scholarly journals The Role of graRS in Regulating Virulence and Antimicrobial Resistance in Methicillin-Resistant Staphylococcus aureus

2021 ◽  
Vol 12 ◽  
Author(s):  
Le Chen ◽  
Zihui Wang ◽  
Tao Xu ◽  
Hongfei Ge ◽  
Fangyue Zhou ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Type Strain ◽  
Bacterial Resistance ◽  
Wild Type Strain ◽  
Lysine Biosynthesis ◽  
Infection Model ◽  
Wild Type ◽  
Methicillin Resistant

Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of both community- and hospital-associated infections. The antibiotic resistance and virulence characteristics of MRSA are largely regulated by two-component signal transduction systems (TCS) including the graRS TCS. To make a relatively comprehensive insight into graRS TCS in MRSA, the bioinformatics analysis of dataset GSE26016 (a S. aureus HG001 WT strain vs. the ΔgraRS mutant) from Gene Expression Omnibus (GEO) database was performed, and a total of 563 differentially expressed genes (DEGs) were identified. GO analysis revealed that the DEGs were mainly enriched in the “de novo” IMP biosynthetic process, lysine biosynthetic process via diaminopimelate, and pathogenesis; and they were mainly enriched in purine metabolism, lysine biosynthesis, and monobactam biosynthesis in KEGG analysis. WGCNA suggested that the turquoise module was related to the blue module, and the genes in these two modules were associated with S. aureus virulence and infection. To investigate the role of graRS in bacterial virulence, a graRS knockout mutant (ΔgraRS) was constructed using MRSA USA500 2,395 strain as a parent strain. Compared to the wild-type strain, the USA500ΔgraRS showed reduced staphyloxanthin production, retarded coagulation, weaker hemolysis on blood agar plates, and a decreased biofilm formation. These altered phenotypes were restored by the complementation of a plasmid-expressed graRS. Meanwhile, an expression of the virulence-associated genes (coa, hla, hlb, agrA, and mgrA) was downregulated in the ΔgraRS mutant. Consistently, the A549 epithelial cells invasion of the ΔgraRS mutant was 4-fold lower than that of the USA500 wild-type strain. Moreover, on the Galleria mellonella infection model, the survival rate at day 5 post infection in the USA500ΔgraRS group (55%) was obviously higher than that in the USA500 group (20%), indicating graRS knockout leads to a decreased virulence in vivo. In addition, the deletion of the graRS in the MRSA USA500 strain resulted in its increased susceptibilities to ampicillin, oxacillin, vancomycin, and gentamicin. Our work suggests that the graRS TCS plays an important role in regulating S. aureus virulence in vitro and in vivo and modulate bacterial resistance to various antibiotics.

scholarly journals Reductive Iron Assimilation and Intracellular Siderophores Assist Extracellular Siderophore-Driven Iron Homeostasis and Virulence

2014 ◽  
Vol 27 (8) ◽  
pp. 793-808 ◽  
Author(s):  
Bradford J. Condon ◽  
Shinichi Oide ◽  
Donna M. Gibson ◽  
Stuart B. Krasnoff ◽  
B. Gillian Turgeon
Keyword(s):  
Iron Homeostasis ◽  
Iron Acquisition ◽  
Wild Type ◽  
Peptide Synthetases ◽  
Asexual Sporulation ◽  
High Affinity ◽  
Iron Assimilation ◽  
Genes Encoding ◽  
Essential Nutrient

Iron is an essential nutrient and prudent iron acquisition and management are key traits of a successful pathogen. Fungi use nonribosomally synthesized secreted iron chelators (siderophores) or reductive iron assimilation (RIA) mechanisms to acquire iron in a high affinity manner. Previous studies with the maize pathogen Cochliobolus heterostrophus identified two genes, NPS2 and NPS6, encoding different nonribosomal peptide synthetases responsible for biosynthesis of intra- and extracellular siderophores, respectively. Deletion of NPS6 results in loss of extracellular siderophore biosynthesis, attenuated virulence, hypersensitivity to oxidative and iron-depletion stress, and reduced asexual sporulation, while nps2 mutants are phenotypically wild type in all of these traits but defective in sexual spore development when NPS2 is missing from both mating partners. Here, it is reported that nps2nps6 mutants have more severe phenotypes than both nps2 and nps6 single mutants. In contrast, mutants lacking the FTR1 or FET3 genes encoding the permease and ferroxidase components, respectively, of the alternate RIA system, are like wild type in all of the above phenotypes. However, without supplemental iron, combinatorial nps6ftr1 and nps2nps6ftr1 mutants are less virulent, are reduced in growth, and are less able to combat oxidative stress and to sporulate asexually, compared with nps6 mutants alone. These findings demonstrate that, while the role of RIA in metabolism and virulence is overshadowed by that of extracellular siderophores as a high-affinity iron acquisition mechanism in C. heterostrophus, it functions as a critical backup for the fungus.

Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility

Science ◽  
2019 ◽  
Vol 366 (6466) ◽  
pp. eaaw4388 ◽  
Author(s):  
Janice M. Reimer ◽  
Maximilian Eivaskhani ◽  
Ingrid Harb ◽  
Alba Guarné ◽  
Martin Weigt ◽  
...  
Keyword(s):  
Condensation Reaction ◽  
Conformational Flexibility ◽  
Scattering Data ◽  
Nonribosomal Peptide ◽  
X Ray ◽  
Peptide Synthetases ◽  
X Ray Scattering ◽  
Peptide Synthetase ◽  
Covariation Analysis ◽  
Full Core

Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that synthesize natural product therapeutics using a modular synthetic logic, whereby each module adds one aminoacyl substrate to the nascent peptide. We have determined five x-ray crystal structures of large constructs of the NRPS linear gramicidin synthetase, including a structure of a full core dimodule in conformations organized for the condensation reaction and intermodular peptidyl substrate delivery. The structures reveal differences in the relative positions of adjacent modules, which are not strictly coupled to the catalytic cycle and are consistent with small-angle x-ray scattering data. The structures and covariation analysis of homologs allowed us to create mutants that improve the yield of a peptide from a module-swapped dimodular NRPS.

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