High-throughput insertion mutagenesis and functional screening in the entomopathogenic fungus Beauveria bassiana

Fan, Yanhua; Zhang, Shizhu; Kruer, Nathan; Keyhani, Nemat O.

doi:10.1016/j.jip.2010.11.003

Cited by 41 publications

(37 citation statements)

References 32 publications

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“…The MIC 50 for oosporein tested against the major bacterial isolates identified (i.e., Pantoea, Staphylococcus, Stenotrophomonas, and Acinetobacter) ranged from 3 to 30 μg/mL, with an MIC 50 of 100 μg/mL for Enterococcus. Achieving >90% growth inhibition required ∼100 μg/mL of oosporein for the former group of bacteria and >200 μg/mL for Enterococcus.…”

Section: Discussionmentioning

confidence: 99%

“…The relative abundance of the bacterial species at the initial time point of infection followed the order E. faecalis (48%) > Stenotrophomonas (35%) > Pantoea (14%) > A. calcoaceticus (3.4%) > Staphylococcus (<1%), whereas at the 48-h time point, the order of abundance was E. faecalis (81%) > Stenotrophomonas (13%) > Staphylococcus (6%), with almost no Pantoea or A. calcoaceticus detected ( Table 2). The minimum inhibitory concentration (MIC 50 , for 50% inhibition of growth) for oosporein tested against Pantoea, Staphylococcus, Stenotrophomonas, Acinetobacter, and Enterococcus was 3, 5, 10, 30, and 100 μg/mL, respectively ( Table 2). The concentration required for ≥90% growth inhibition (MIC 90 ) was ∼100 μg/mL for all of the bacteria tested except for Enterococcus, which required >200 μg/mL ( Table 2).…”

Section: B Bassiana-produced Oosporein Inhibits Bacterial Growth On mentioning

confidence: 99%

“…The resultant fragment was digested with EcoRI and XbaI (unique sites engineered in the primers) and cloned into pCB1536 containing the sulfonylurea resistance gene (sur) cassette. The complementation vector was ectopically transformed into the ΔBbsmr1 strain via an LiCl-PEG-mediated blastospore transformation protocol as described previously (50). The integrity of the transformants was verified by PCR and Southern blot analysis.…”

Section: Mutant Screening and Gene Manipulationsmentioning

confidence: 99%

“…The PCR fragment was cloned upstream of a promoterless eGFP ORF in the pUC-bar vector containing the phosphinothricin resistance cassette. The integrity of the construct was verified by sequencing and transformed into the B. bassiana WT strain as described previously (50). B. bassiana spores harboring the OpS1p::eGFP construct ectopically integrated into the fungal genome were topically inoculated on G. mellonella larvae and examined over a time course of infection.…”

Section: Mutant Screening and Gene Manipulationsmentioning

confidence: 99%

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Regulatory cascade and biological activity of Beauveria bassiana oosporein that limits bacterial growth after host death

Fan

Liu

Keyhani

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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124

119

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The regulatory network and biological functions of the fungal secondary metabolite oosporein have remained obscure. Beauveria bassiana has evolved the ability to parasitize insects and outcompete microbial challengers for assimilation of host nutrients. A novel zinc finger transcription factor, BbSmr1 (B. bassiana secondary metabolite regulator 1), was identified in a screen for oosporein overproduction. Deletion of Bbsmr1 resulted in up-regulation of the oosporein biosynthetic gene cluster (OpS genes) and constitutive oosporein production. Oosporein production was abolished in double mutants of Bbsmr1 and a second transcription factor, OpS3, within the oosporein gene cluster (ΔBbsmr1ΔOpS3), indicating that BbSmr1 acts as a negative regulator of OpS3 expression. Real-time quantitative PCR and a GFP promoter fusion construct of OpS1, the oosporein polyketide synthase, indicated that OpS1 is expressed mainly in insect cadavers at 24-48 h after death. Bacterial colony analysis in B. bassiana-infected insect hosts revealed increasing counts until host death, with a dramatic decrease (∼90%) after death that correlated with oosporein production. In vitro studies verified the inhibitory activity of oosporein against bacteria derived from insect cadavers. These results suggest that oosporein acts as an antimicrobial compound to limit microbial competition on B. bassiana-killed hosts, allowing the fungus to maximally use host nutrients to grow and sporulate on infected cadavers.Beauveria bassiana | oosporein | biological role | transcription factor | fungal-bacterial competition

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Section: Discussionmentioning

confidence: 99%

Section: B Bassiana-produced Oosporein Inhibits Bacterial Growth On mentioning

confidence: 99%

Section: Mutant Screening and Gene Manipulationsmentioning

confidence: 99%

Section: Mutant Screening and Gene Manipulationsmentioning

confidence: 99%

See 2 more Smart Citations

Regulatory cascade and biological activity of Beauveria bassiana oosporein that limits bacterial growth after host death

Fan

Liu

Keyhani

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

124

119

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show abstract

“…Identification of mtrA, sequence analysis and generation of BbmtrA disruption and complementation strains Construction of a B. bassiana random insertion mutant library has been previously reported (Fan et al, 2011). A screen for clones that failed to grow after passage resulted in the identification of one clone (designated S34-1) that failed to grow upon conidial transfer to fresh plates from plates aged .60 days.…”

Section: Resultsmentioning

confidence: 99%

A putative methyltransferase, mtrA, contributes to development, spore viability, protein secretion and virulence in the entomopathogenic fungus Beauveria bassiana

2014

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The filamentous fungus, Beauveria bassiana, is a ubiquitously distributed insect pathogen, currently used as an alternative to chemical pesticides for pest control. Conidiospores are the means by which the fungus disseminates in the environment, and these cells also represent the infectious agent most commonly used in field applications. Little, however, is known concerning the molecular basis for maintenance of spore viability, a critical feature for survival and persistence. Here, we report on the role of a putative methyltransferase, BbmtrA, in conidial viability, normal fungal growth and development, and virulence, via characterization of a targeted gene knockout strain. Loss of BbmtrA resulted in pleiotropic effects including reduced germination, growth and conidiation, with growing mycelia displaying greater branching than the WT parent. Conidial viability dramatically decreased over time, with ,5 % of the cells remaining viable after 30 days as compared with .80 % of the WT. Reduced production of extracellular proteins was also observed for the DBbmtrA mutant, including protease/peptidases, glycoside hydrolases and the hyd1 hydrophobin. The latter was further confirmed by hyd1 gene expression analysis. Insect bioassays using the greater wax moth, Galleria mellonella, further revealed that the DBbmtrA strain was attenuated in virulence and failed to sporulate on host cadavers. These data support a global role for mtrA in fungal physiological processes.

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Mutation of a Conserved Alanine Residue in Transcription Factor AraR Leads to Hyperproduction of α‐l‐Arabinofuranosidases in Penicillium oxalicum

Gao

et al. 2019

Biotechnology Journal

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α‐l‐Arabinofuranosidases are important in the degradation of plant polysaccharides and are used in several industrial processes. Although the use of filamentous fungi for the production of α‐l‐arabinofuranosidases is widely reported, there are few reports on strain engineering for enhanced production of these enzymes by fungi. In this study, the function of transcription factor AraR in l‐arabinose release and catabolism by the fungus Penicillium oxalicum (P. oxalicum) is investigated. Also, a mutant of AraR, AraRA731V, is constructed to improve the production of α‐l‐arabinofuranosidases on the basis of the sequence homology between AraR and the xylanolytic gene activator XlnR. The AraRA731V‐overexpressing strain can synthesize α‐l‐arabinofuranosidase in the absence of an inducer and shows a 54.1‐fold increase in α‐l‐arabinofuranosidase production and a 7.4‐fold increase in α‐galactosidase production in the medium containing wheat bran. Determination of the transcript abundances of lignocellulolytic enzyme genes reveals significant upregulation of multiple α‐l‐arabinofuranosidase genes and downregulation of some cellulolytic and xylanolytic enzyme genes in the engineered strain relative to its parent. Taken together, the results suggest the conserved regulatory function of AraR in the family Trichocomaceae and provide a strategy for engineering fungal strains for enhanced α‐ l‐arabinofuranosidase production.

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High-throughput insertion mutagenesis and functional screening in the entomopathogenic fungus Beauveria bassiana

Cited by 41 publications

References 32 publications

Regulatory cascade and biological activity of Beauveria bassiana oosporein that limits bacterial growth after host death

Regulatory cascade and biological activity of Beauveria bassiana oosporein that limits bacterial growth after host death

A putative methyltransferase, mtrA, contributes to development, spore viability, protein secretion and virulence in the entomopathogenic fungus Beauveria bassiana

Mutation of a Conserved Alanine Residue in Transcription Factor AraR Leads to Hyperproduction of α‐l‐Arabinofuranosidases in Penicillium oxalicum

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