scholarly journals Coordinated and Distinct Functions of Velvet Proteins in Fusarium verticillioides

2014 ◽  
Vol 13 (7) ◽  
pp. 909-918 ◽  
Author(s):  
Nan Lan ◽  
Hanxing Zhang ◽  
Chengcheng Hu ◽  
Wenzhao Wang ◽  
Ana M. Calvo ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Affinity Purification ◽  
Fusarium Verticillioides ◽  
Toxin Production ◽  
Transcriptional Analysis ◽  
Phenotypic Characterization ◽  
Morphological Development ◽  
Content Type ◽  
Knockout Mutants ◽  
Fungal Kingdom

ABSTRACTVelvet-domain-containing proteins are broadly distributed within the fungal kingdom. In the corn pathogenFusarium verticillioides, previous studies showed that the velvet proteinF. verticillioidesVE1 (FvVE1) is critical for morphological development, colony hydrophobicity, toxin production, and pathogenicity. In this study, tandem affinity purification of FvVE1 revealed that FvVE1 can form a complex with the velvet proteinsF. verticillioidesVelB (FvVelB) and FvVelC. Phenotypic characterization of gene knockout mutants showed that, as in the case of FvVE1, FvVelB regulated conidial size, hyphal hydrophobicity, fumonisin production, and oxidant resistance, while FvVelC was dispensable for these biological processes. Comparative transcriptional analysis of eight genes involved in the ROS (reactive oxygen species) removal system revealed that both FvVE1 and FvVelB positively regulated the transcription of a catalase-encoding gene,F. verticillioidesCAT2(FvCAT2). Deletion ofFvCAT2resulted in reduced oxidant resistance, providing further explanation of the regulation of oxidant resistance by velvet proteins in the fungal kingdom.

scholarly journals Regulation of Sporangium Formation by BldD in the Rare Actinomycete Actinoplanes missouriensis

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Yoshihiro Mouri ◽  
Kenji Konishi ◽  
Azusa Fujita ◽  
Takeaki Tezuka ◽  
Yasuo Ohnishi
Keyword(s):  
Vegetative Growth ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Transcriptional Regulator ◽  
Saccharopolyspora Erythraea ◽  
Transcriptional Analysis ◽  
Morphological Development ◽  
Sequencing Analysis ◽  
Content Type ◽  
Biological Studies

ABSTRACT The rare actinomycete Actinoplanes missouriensis forms sporangia, including hundreds of flagellated spores that start swimming as zoospores after their release. Under conditions suitable for vegetative growth, zoospores stop swimming and germinate. A comparative proteome analysis between zoospores and germinating cells identified 15 proteins that were produced in larger amounts in germinating cells. They include an orthologue of BldD (herein named AmBldD [BldD of A. missouriensis]), which is a transcriptional regulator involved in morphological development and secondary metabolism in Streptomyces. AmBldD was detected in mycelia during vegetative growth but was barely detected in mycelia during the sporangium-forming phase, in spite of the constant transcription of AmbldD throughout growth. An AmbldD mutant started to form sporangia much earlier than the wild-type strain, and the resulting sporangia were morphologically abnormal. Recombinant AmBldD bound a palindromic sequence, the AmBldD box, located upstream from AmbldD. 3′,5′-Cyclic di-GMP significantly enhanced the in vitro DNA-binding ability of AmBldD. A chromatin immunoprecipitation-sequencing analysis and an in silico search for AmBldD boxes revealed that AmBldD bound 346 genomic loci that contained the 19-bp inverted repeat 5′-NN(G/A)TNACN(C/G)N(G/C)NGTNA(C/T)NN-3′ as the consensus AmBldD-binding sequence. The transcriptional analysis of 27 selected AmBldD target gene candidates indicated that AmBldD should repress 12 of the 27 genes, including bldM, ssgB, whiD, ddbA, and wblA orthologues. These genes are involved in morphological development in Streptomyces coelicolor A3(2). Thus, AmBldD is a global transcriptional regulator that seems to repress the transcription of tens of genes during vegetative growth, some of which are likely to be required for sporangium formation. IMPORTANCE The rare actinomycete Actinoplanes missouriensis undergoes complex morphological differentiation, including sporangium formation. However, almost no molecular biological studies have been conducted on this bacterium. BldD is a key global regulator involved in the morphological development of streptomycetes. BldD orthologues are highly conserved among sporulating actinomycetes, but no BldD orthologues, except one in Saccharopolyspora erythraea, have been studied outside the streptomycetes. Here, it was revealed that the BldD orthologue AmBldD is essential for normal developmental processes in A. missouriensis. The AmBldD regulon seems to be different from the BldD regulon in Streptomyces coelicolor A3(2), but they share four genes that are involved in morphological differentiation in S. coelicolor A3(2).

scholarly journals Fumonisin and Beauvericin Chemotypes and Genotypes of the Sister Species Fusarium subglutinans and Fusarium temperatum

2020 ◽  
Vol 86 (13) ◽  
Author(s):  
M. Veronica Fumero ◽  
Alessandra Villani ◽  
Antonia Susca ◽  
Miriam Haidukowski ◽  
Maria T. Cimmarusti ◽  
...  
Keyword(s):  
Fusarium Verticillioides ◽  
Gene Clusters ◽  
Toxin Production ◽  
Sister Species ◽  
Ear Rot ◽  
Genome Sequences ◽  
Fusarium Subglutinans ◽  
Content Type ◽  
Close Phylogenetic Relationship ◽  
Fusarium Temperatum

ABSTRACT Fusarium subglutinans and Fusarium temperatum are common maize pathogens that produce mycotoxins and cause plant disease. The ability of these species to produce beauvericin and fumonisin mycotoxins is not settled, as reports of toxin production are not concordant. Our objective was to clarify this situation by determining both the chemotypes and genotypes for strains from both species. We analyzed 25 strains from Argentina, 13 F. subglutinans and 12 F. temperatum strains, for toxin production by ultraperformance liquid chromatography mass spectrometry (UPLC-MS). We used new genome sequences from two strains of F. subglutinans and one strain of F. temperatum, plus genomes of other Fusarium species, to determine the presence of functional gene clusters for the synthesis of these toxins. None of the strains examined from either species produced fumonisins. These strains also lack Fum biosynthetic genes but retain homologs of some genes that flank the Fum cluster in Fusarium verticillioides. None of the F. subglutinans strains we examined produced beauvericin although 9 of 12 F. temperatum strains did. A complete beauvericin (Bea) gene cluster was present in all three new genome sequences. The Bea1 gene was presumably functional in F. temperatum but was not functional in F. subglutinans due to a large insertion and multiple mutations that resulted in premature stop codons. The accumulation of only a few mutations expected to disrupt Bea1 suggests that the process of its inactivation is relatively recent. Thus, none of the strains of F. subglutinans or F. temperatum we examined produce fumonisins, and the strains of F. subglutinans examined also cannot produce beauvericin. Variation in the ability of strains of F. temperatum to produce beauvericin requires further study and could reflect the recent shared ancestry of these two species. IMPORTANCE Fusarium subglutinans and F. temperatum are sister species and maize pathogens commonly isolated worldwide that can produce several mycotoxins and cause seedling disease, stalk rot, and ear rot. The ability of these species to produce beauvericin and fumonisin mycotoxins is not settled, as reports of toxin production are not concordant at the species level. Our results are consistent with previous reports that strains of F. subglutinans produce neither fumonisins nor beauvericin. The status of toxin production by F. temperatum needs further work. Our strains of F. temperatum did not produce fumonisins, while some strains produced beauvericin and others did not. These results enable more accurate risk assessments of potential mycotoxin contamination if strains of these species are present. The nature of the genetic inactivation of BEA1 is consistent with its relatively recent occurrence and the close phylogenetic relationship of the two sister species.

scholarly journals HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866

2015 ◽  
Vol 82 (2) ◽  
pp. 724-731 ◽  
Author(s):  
Hong-Jun Chao ◽  
Yan-Fei Chen ◽  
Ti Fang ◽  
Ying Xu ◽  
Wei E. Huang ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Environmental Protection Agency ◽  
High Performance ◽  
Specific Activity ◽  
Gene Knockout ◽  
Transcriptional Analysis ◽  
Ring Cleavage ◽  
Protection Agency ◽  
Content Type ◽  
Priority Pollutant

ABSTRACTIn addition to growing onp-cresol,Pseudomonas putidaNCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of thepara-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the genehipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His6was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His6catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg−1and showed apparentKmvalues of 11.40 ± 3.05 μM for 4-hydroxyisophthalate with NADPH and 11.23 ± 2.43 μM with NADH and similarKmvalues for NADPH and NADH (64.31 ± 13.16 and 72.76 ± 12.06 μM, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His6has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated thathipHis essential for 2,4-xylenol catabolism but not forp-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds.

scholarly journals Xylanase Gene Transcription in Trichoderma reesei Is Triggered by Different Inducers Representing Different Hemicellulosic Pentose Polymers

2013 ◽  
Vol 12 (3) ◽  
pp. 390-398 ◽  
Author(s):  
Silvia Herold ◽  
Robert Bischof ◽  
Benjamin Metz ◽  
Bernhard Seiboth ◽  
Christian P. Kubicek
Keyword(s):  
Trichoderma Reesei ◽  
Plant Cell Wall ◽  
Glycosyl Hydrolase ◽  
Xylose Reductase ◽  
Genome Mining ◽  
Transcriptional Analysis ◽  
Cell Wall Degrading Enzymes ◽  
Content Type ◽  
Knockout Mutants ◽  
Catabolite Repressor

ABSTRACTThe ascomyceteTrichoderma reeseiis a paradigm for the regulation and production of plant cell wall-degrading enzymes, including xylanases. Four xylanases, including XYN1 and XYN2 of glycosyl hydrolase family 11 (GH11), the GH10 XYN3, and the GH30 XYN4, were already described. By genome mining, we identified a fifth xylanase, XYN5, belonging to GH11. Transcriptional analysis reveals that the expression of all xylanases butxyn3is induced byd-xylose, dependent on the cellulase and xylanase regulator XYR1 and negatively regulated by the carbon catabolite repressor CRE1. Impairment ofd-xylose catabolism at thed-xylose reductase and xylitol dehydrogenase step strongly enhanced induction byd-xylose. Knockout of thel-xylulose reductase-encoding genelxr3, which connects thed-xylose andl-arabinose catabolic pathways, had no effect on xylanase induction. Besides the induction byd-xylose, theT. reeseixylanases were also induced byl-arabinose, and this induction was also enhanced in knockout mutants inl-arabinose reductase (xyl1),l-arabitol dehydrogenase (lad1), andl-xylulose reductase (lxr3). Induction byl-arabinose was also XYR1 dependent. Analysis of intracellular polyols revealed accumulation of xylitol in all strains only during incubation withd-xylose and accumulation ofl-arabitol only during incubation withl-arabinose. Induction byl-arabinose could be further stimulated by addition ofd-xylose. We conclude that the expression of theT. reeseixylanases can be induced by bothd-xylose andl-arabinose, but independently of each other and by using different inducing metabolites.

scholarly journals The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

2012 ◽  
Vol 56 (6) ◽  
pp. 3216-3223 ◽  
Author(s):  
Tina Manzhu Kang ◽  
Jessica Yuan ◽  
Angelyn Nguyen ◽  
Elinne Becket ◽  
Hanjing Yang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Anthracis ◽  
Gene Knockout ◽  
Aminoglycoside Antibiotic ◽  
Double Strand Breaks ◽  
Repair Capacity ◽  
Content Type ◽  
Strand Breaks ◽  
Knockout Mutants ◽  
Increased Sensitivity

ABSTRACTThe distribution of mutants in the Keio collection ofEscherichia coligene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ,holC,holD, andpriAknockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded byrecA,recB, andrecC, among others. Additionally, caffeine partially protects cells of bothEscherichia coliandBacillus anthracisfrom killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

scholarly journals Draft Genome Sequences of Salmonella Lysozyme Gene Knockout Mutants

2017 ◽  
Vol 5 (23) ◽  
Author(s):  
Narine Arabyan ◽  
Bihua C. Huang ◽  
Bart C. Weimer
Keyword(s):  
Gene Deletion ◽  
Gene Knockout ◽  
Draft Genome ◽  
Deletion Mutants ◽  
Genome Sequences ◽  
Content Type ◽  
Knockout Mutants ◽  
Serovar Typhimurium ◽  
Encoding Gene ◽  
Glucoside Hydrolases

ABSTRACT Lysozyme enzymes hydrolyze the β-1,4-glycosidic bond in oligosaccharides. These enzymes are part of a broad group of glucoside hydrolases that are poorly characterized; however, they are important for growth and are being recognized as emerging virulence factors. This is the release of four lysozyme-encoding-gene-deletion mutants in Salmonella enterica serovar Typhimurium LT2.

scholarly journals AraC-Type Regulator Rsp Adapts Staphylococcus aureus Gene Expression to Acute Infection

2015 ◽  
Vol 84 (3) ◽  
pp. 723-734 ◽  
Author(s):  
Tianming Li ◽  
Lei He ◽  
Yan Song ◽  
Amer E. Villaruz ◽  
Hwang-Soo Joo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Regulatory Networks ◽  
Acute Infection ◽  
Toxin Production ◽  
Transcriptional Analysis ◽  
Chronic Infections ◽  
Accessory Gene ◽  
Content Type ◽  
Regulatory Impact

Staphylococcus aureusis an important human pathogen that can cause two categories of severe infections. Acute infections are characterized by pronounced toxin production, while chronic infections often involve biofilm formation. However, it is poorly understood howS. aureuscontrols the expression of genes associated with acute versus biofilm-associated virulence. We here identified an AraC-type transcriptional regulator, Rsp, that promotes the production of key toxins while repressing major biofilm-associated genes and biofilm formation. Genome-wide transcriptional analysis and modeling of regulatory networks indicated that upregulation of the accessory gene regulator (Agr) and downregulation of theicaoperon coding for the biofilm exopolysaccharide polysaccharide intercellular adhesin (PIA) were central to the regulatory impact of Rsp on virulence. Notably, the Rsp protein directly bound to theagrP2andicaADBCpromoters, resulting in strongly increased levels of the Agr-controlled toxins phenol-soluble modulins (PSMs) and alpha-toxin and reduced production of PIA. Accordingly, Rsp was essential for the development of bacteremia and skin infection, representing major types of acuteS. aureusinfection. Our findings give important insight into howS. aureusadapts the expression of its broad arsenal of virulence genes to promote different types of disease manifestations and identify the Rsp regulator as a potential target for strategies to control acuteS. aureusinfection.

scholarly journals Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

2012 ◽  
Vol 80 (10) ◽  
pp. 3521-3532 ◽  
Author(s):  
Annie Aubry ◽  
Greg Hussack ◽  
Wangxue Chen ◽  
Rhonda KuoLee ◽  
Susan M. Twine ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Parent Strain ◽  
Regulatory Networks ◽  
Toxin Production ◽  
Transcriptional Analysis ◽  
Hamster Model ◽  
Content Type ◽  
Flagellar Regulon ◽  
The Impact ◽  
Culture Supernatants

ABSTRACTWe show in this study that toxin production inClostridium difficileis altered in cells which can no longer form flagellar filaments. The impact of inactivation offliC,CD0240,fliF,fliG,fliM, andflhB-fliRflagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in afliCmutant compared to that in theC. difficile630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parentalC. difficile630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR,tcdB,tcdE, andtcdA) revealed a significant increase of all four genes in thefliCmutant strain, while transcription of all four genes was significantly reduced infliM,fliF,fliG, andflhB-fliRmutants. These results demonstrate that toxin transcription inC. difficileis modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model ofC. difficileinfection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production inC. difficileand provides evidence for elaborate regulatory networks for virulence genes inC. difficile.

scholarly journals Phosphoethanolamine Modification of Lipid A in Colistin-Resistant Variants of Acinetobacter baumannii Mediated by the pmrAB Two-Component Regulatory System

2011 ◽  
Vol 55 (7) ◽  
pp. 3370-3379 ◽  
Author(s):  
Alejandro Beceiro ◽  
Enrique Llobet ◽  
Jesús Aranda ◽  
José Antonio Bengoechea ◽  
Michel Doumith ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Clinical Isolate ◽  
Lipid A ◽  
Gene Knockout ◽  
Regulatory System ◽  
Clinical Isolates ◽  
Strain Atcc ◽  
Colistin Resistance ◽  
Content Type ◽  
Knockout Mutants

ABSTRACTColistin resistance is rare inAcinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient.pmrABsequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression ofpmrA(4- to 13-fold),pmrB(2- to 7-fold), andpmrC(1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg2+induction conditions.pmrBgene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression ofpmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance inA. baumanniirequires distinct genetic events, including (i) at least one point mutation inpmrB, (ii) upregulation ofpmrAB, and (iii) expression ofpmrC, which lead to addition of phosphoethanolamine to lipid A.

scholarly journals Sulfate Assimilation Mediates Tellurite Reduction and Toxicity in Saccharomyces cerevisiae

2010 ◽  
Vol 9 (10) ◽  
pp. 1635-1647 ◽  
Author(s):  
Lars-Göran Ottosson ◽  
Katarina Logg ◽  
Sebastian Ibstedt ◽  
Per Sunnerhagen ◽  
Mikael Käll ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Knockout ◽  
Common Mechanism ◽  
Sulfate Assimilation ◽  
Complete Collection ◽  
Content Type ◽  
Knockout Mutants ◽  
Sulfate Assimilation Pathway ◽  
Mechanistic Basis ◽  
Respiratory Mechanism

ABSTRACT Despite a century of research and increasing environmental and human health concerns, the mechanistic basis of the toxicity of derivatives of the metalloid tellurium, Te, in particular the oxyanion tellurite, Te(IV), remains unsolved. Here, we provide an unbiased view of the mechanisms of tellurium metabolism in the yeast Saccharomyces cerevisiae by measuring deviations in Te-related traits of a complete collection of gene knockout mutants. Reduction of Te(IV) and intracellular accumulation as metallic tellurium strongly correlated with loss of cellular fitness, suggesting that Te(IV) reduction and toxicity are causally linked. The sulfate assimilation pathway upstream of Met17, in particular, the sulfite reductase and its cofactor siroheme, was shown to be central to tellurite toxicity and its reduction to elemental tellurium. Gene knockout mutants with altered Te(IV) tolerance also showed a similar deviation in tolerance to both selenite and, interestingly, selenomethionine, suggesting that the toxicity of these agents stems from a common mechanism. We also show that Te(IV) reduction and toxicity in yeast is partially mediated via a mitochondrial respiratory mechanism that does not encompass the generation of substantial oxidative stress. The results reported here represent a robust base from which to attack the mechanistic details of Te(IV) toxicity and reduction in a eukaryotic organism.

Sign in / Sign up

Export Citation Format

Share Document