Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1973-1982 ◽  
Author(s):  
Takuji Oka ◽  
Tetsu Hamaguchi ◽  
Yuka Sameshima ◽  
Masatoshi Goto ◽  
Kensuke Furukawa
Keyword(s):  
Cell Wall ◽  
Aspergillus Nidulans ◽  
Protein Modification ◽  
Wild Type Strain ◽  
Wild Type ◽  
Gene Encoding ◽  
And Function ◽  
Encoding Protein

Protein O-glycosylation is essential for protein modification and plays important roles in eukaryotic cells. O-Mannosylation of proteins occurs in the filamentous fungus Aspergillus. The structure and function of the pmtA gene, encoding protein O-d-mannosyltransferase, which is responsible for the initial O-mannosylation reaction in Aspergillus nidulans, was characterized. Disruption of the pmtA gene resulted in the reduction of in vitro protein O-d-mannosyltransferase activity to 6 % of that of the wild-type strain and led to underglycosylation of an extracellular glucoamylase. The pmtA disruptant exhibited abnormal cell morphology and alteration in carbohydrate composition, particularly reduction in the skeletal polysaccharides in the cell wall. The results indicate that PmtA is required for the formation of a normal cell wall in A. nidulans.

scholarly journals Recovery of Fusarium oxysporum Fo47 Mutants Affected in Their Biocontrol Activity After Transposition of the Fot1 Element

2002 ◽  
Vol 92 (9) ◽  
pp. 936-945 ◽  
Author(s):  
Sophie Trouvelot ◽  
Chantal Olivain ◽  
Ghislaine Recorbet ◽  
Quirico Migheli ◽  
Claude Alabouvette
Keyword(s):  
Fusarium Oxysporum ◽  
Insertional Mutagenesis ◽  
Wild Type Strain ◽  
Growth Habit ◽  
Antagonistic Activity ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Biocontrol Activity

To investigate the biocontrol mechanisms by which the antagonistic Fusarium oxysporum strain Fo47 is active against Fusarium wilt, a Fot1 transposon-mediated insertional mutagenesis approach was adopted to generate mutants affected in their antagonistic activity. Ninety strains in which an active Fot1 copy had transposed were identified with a phenotypic assay for excision and tested for their biocontrol activity against F. oxysporum f. sp. lini on flax in greenhouse experiments. Sixteen strains were affected in their capacity to protect flax plants, either positively (more antagonistic than Fo47) or negatively (less antagonistic). The molecular characterization of these mutants confirms the excision of Fot1 and its reinsertion in most of the cases. Moreover, we demonstrate that other transposable elements such as Fot2, impala, and Hop have no transposition activity in the mutant genomes. The phenotypic characterization of these mutants shows that they are affected neither in their in vitro growth habit nor in their competitiveness in soil compared with wild-type strain Fo47. These results show that mutants are not impaired in their saprophytic phase and suggest that the altered biocontrol phenotype should likely be expressed during the interaction with the host plant.

scholarly journals Identification and Characterization of the Bacillus thuringiensis phaZ Gene, Encoding New Intracellular Poly-3-Hydroxybutyrate Depolymerase

2006 ◽  
Vol 188 (21) ◽  
pp. 7592-7599 ◽  
Author(s):  
Chi-Ling Tseng ◽  
Hui-Ju Chen ◽  
Gwo-Chyuan Shaw
Keyword(s):  
Bacillus Thuringiensis ◽  
Wild Type ◽  
Gene Encoding ◽  
Significant Similarity ◽  
Phb Depolymerase ◽  
New Type ◽  
Identification And Characterization

ABSTRACTA gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome ofBacillus thuringiensissubsp.israelensisATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designatedphaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD ofPseudomonas putidawas unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. TheB. thuringiensisPhaZ was inactive againstp-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-typeB. thuringiensiscells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from aphaZmutant lost this activity. ThephaZmutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S102-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate thatB. thuringiensisharbors a new type of intracellular PHB depolymerase.

scholarly journals Isolation and Characterization of Burkholderia cenocepacia Mutants Deficient in Pyochelin Production: Pyochelin Biosynthesis Is Sensitive to Sulfur Availability

2004 ◽  
Vol 186 (2) ◽  
pp. 270-277 ◽  
Author(s):  
Kate L. Farmer ◽  
Mark S. Thomas
Keyword(s):  
Wild Type Strain ◽  
Opportunistic Pathogen ◽  
Burkholderia Cenocepacia ◽  
Wild Type ◽  
Iron Starvation ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Putative Operon ◽  
Green Fluorescent

ABSTRACT The opportunistic pathogen Burkholderia cenocepacia produces the yellow-green fluorescent siderophore, pyochelin. To isolate mutants which do not produce this siderophore, we mutagenized B. cenocepacia with the transposon mini-Tn5Tp. Two nonfluorescent mutants were identified which were unable to produce pyochelin. In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter. The cysW gene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl. Sulfate uptake assays confirmed that both mutants were defective in sulfate transport. Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin. Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth. We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis. These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.

scholarly journals Isolation and molecular characterization of the Aspergillus nidulans wA gene.

Genetics ◽  
1990 ◽  
Vol 126 (1) ◽  
pp. 73-79 ◽  
Author(s):  
M E Mayorga ◽  
W E Timberlake
Keyword(s):  
Aspergillus Nidulans ◽  
Wild Type Strain ◽  
Nuclear Dna ◽  
Cosmid Library ◽  
Wild Type ◽  
Green Pigment ◽  
Mrna Accumulation ◽  
Pigment Synthesis ◽  
Regulatory Loci

Abstract The walls of Aspergillus nidulans conidia contain a green pigment that protects the spores from damage by ultraviolet light. At least two genes, wA and yA, are required for pigment synthesis: yA mutants produce yellow spores, wA mutants produce white spores, and wA mutations are epistatic to yA mutations. We cloned wA by genetic complementation of the wA3 mutation with a cosmid library containing nuclear DNA inserts from the wild-type strain. The wA locus was mapped to an 8.5-10.5-kilobase region by gene disruption analysis. DNA fragments from this region hybridized to a 7500 nucleotide polyadenylated transcript that is absent from hyphae and mature conidia but accumulates during conidiation beginning when pigmented spores first appear. Mutations in the developmental regulatory loci brlA, abaA, wetA and apsA prevent wA mRNA accumulation. By contrast, yA mRNA fails to accumulate only in the brlA- and apsA- mutants. Thus, the level of wA transcript is regulated during conidiophore development and wA activation requires genes within the central pathway regulating conidiation.

Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2931-2939 ◽  
Author(s):  
Alex K. Jaques ◽  
Tamo Fukamizo ◽  
Diana Hall ◽  
Richard C. Barton ◽  
Gemma M. Escott ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Wild Type Strain ◽  
Chitinase Activity ◽  
Gene Replacement ◽  
Wild Type ◽  
Gene Encoding ◽  
Batch Cultures ◽  
Putative Active Site ◽  
Hydrolysis Of

The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1. Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action. That is, the β-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases. Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc4, GlcNAc5 and GlcNAc6 indicated that the predominant reaction involved hydrolysis of GlcNAc2 from the non-reducing end of each substrate. Products of transglycosylation were also identified in each incubation. Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical. However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type. The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database (http://www.tigr.org/).

scholarly journals Involvement of the SppA1 Peptidase in Acclimation to Saturating Light Intensities in Synechocystis sp. Strain PCC 6803

2004 ◽  
Vol 186 (12) ◽  
pp. 3991-3999 ◽  
Author(s):  
E. Pojidaeva ◽  
V. Zinchenko ◽  
S. V. Shestakov ◽  
A. Sokolenko
Keyword(s):  
Light Intensity ◽  
Wild Type Strain ◽  
Wild Type ◽  
Additional Mechanism ◽  
Gene Encoding ◽  
Light Intensities ◽  
In The Wild ◽  
Pcc 6803

ABSTRACT The sll1703 gene, encoding an Arabidopsis homologue of the thylakoid membrane-associated SppA peptidase, was inactivated by interposon mutagenesis in Synechocystis sp. strain PCC 6803. Upon acclimation from a light intensity of 50 to 150 μE m−2 s−1, the mutant preserved most of its phycobilisome content, whereas the wild-type strain developed a bleaching phenotype due to the loss of about 40% of its phycobiliproteins. Using in vivo and in vitro experiments, we demonstrate that the ΔsppA1 strain does not undergo the cleavage of the LR 33 and LCM 99 linker proteins that develops in the wild type exposed to increasing light intensities. We conclude that a major contribution to light acclimation under a moderate light regime in cyanobacteria originates from an SppA1-mediated cleavage of phycobilisome linker proteins. Together with changes in gene expression of the major phycobiliproteins, it contributes an additional mechanism aimed at reducing the content in phycobilisome antennae upon acclimation to a higher light intensity.

scholarly journals Characterization of the Phthalate Permease OphD from Burkholderia cepacia ATCC 17616

1999 ◽  
Vol 181 (19) ◽  
pp. 6197-6199 ◽  
Author(s):  
Hung-Kuang Chang ◽  
Gerben J. Zylstra
Keyword(s):  
Burkholderia Cepacia ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Uptake ◽  
Uptake System ◽  
Wild Type ◽  
Knockout Mutant ◽  
Gene Encoding ◽  
Uptake Inhibition

ABSTRACT The ophD gene, encoding a permease for phthalate transport, was cloned from Burkholderia cepacia ATCC 17616. Expression of the gene in Escherichia coli results in the ability to transport phthalate rapidly into the cell. Uptake inhibition experiments show that 4-hydroxyphthalate, 4-chlorophthalate, 4-methylphthalate, and cinchomeronate compete for the phthalate permease. An ophD knockout mutant of 17616 grows slightly more slowly on phthalate but is still able to take up phthalate at rates equivalent to that of the wild-type strain. This means that 17616 must have a second phthalate-inducible phthalate uptake system.

scholarly journals Disruption of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition.

2008 ◽  
Vol 55 (2) ◽  
pp. 251-259 ◽  
Author(s):  
Wioletta Górka-Nieć ◽  
Michał Pniewski ◽  
Anna Kania ◽  
Urszula Perlińska-Lenart ◽  
Grazyna Palamarczyk ◽  
...  
Keyword(s):  
Cell Wall ◽  
Essential Role ◽  
Unit Length ◽  
Total Activity ◽  
Cell Wall Composition ◽  
Wild Type ◽  
Protein Activity ◽  
Gene Encoding ◽  
Encoding Protein

In fungi transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases encoded by pmt genes. Disruption of the pmt1 gene in Trichoderma caused a significant decrease in the total activity of protein O-mannosyltransferases. Moreover, disruption of the pmt1 gene also led to osmotic sensitivity of the strain, indicating an essential role of the PMTI protein activity for cell wall synthesis. At the same time, the strain was defective in septa formation, producing only half the number of septa per unit length of hypha compared with the wild type. Disruption of the pmt1 gene decreased protein secretion but had no effect on glycosylation of secreted proteins, which suggests that PMTI protein O-mannosyltranferase does not take part in glycosylation of these proteins.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

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