RESUMO
Fungal nonribosomal peptide synthetases (NRPSs) are megasynthetases that produce cyclic and acyclic peptides. In Aspergillus nidulans, the NRPS ivoA (AN10576) has been associated with the biosynthesis of grey-brown conidiophore pigments. Another gene, ivoB (AN0231), has been demonstrated to be an N-acetyl-6-hydroxytryptophan oxidase that putatively acts downstream of IvoA. A third gene, ivoC, has also been predicted to be involved in pigment biosynthesis based on publicly available genomic and transcriptomic information. In this paper, we report the replacement of the promoters of the ivoA, ivoB, and ivoC genes with the inducible promoter alcA in a single cotransformation. Co-overexpression of the three genes resulted in the production of a dark-brown pigment in hyphae. In addition, overexpression of each of the Ivo genes, ivoA-C, individually or in combination, allowed us to isolate intermediates and confirm the function of each gene. IvoA was found to be the first known NRPS to carry out the acetylation of the amino acid, tryptophan.
Assuntos
Monofenol Mono-Oxigenase/genética , Biossíntese de Peptídeos Independentes de Ácido Nucleico/genética , Peptídeo Sintases/genética , Pigmentação/genética , Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Família Multigênica/genética , Regiões Promotoras Genéticas , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Triptofano/biossínteseRESUMO
The UvrD helicase has been implicated in the disassembly of RecA nucleoprotein filaments in vivo and in vitro. We demonstrate that UvrD utilizes an active mechanism to remove RecA from the DNA. Efficient RecA removal depends on the availability of DNA binding sites for UvrD and/or the accessibility of the RecA filament ends. The removal of RecA from DNA also requires ATP hydrolysis by the UvrD helicase but not by RecA protein. The RecA-removal activity of UvrD is slowed by RecA variants with enhanced DNA-binding properties. The ATPase rate of UvrD during RecA removal is much slower than the ATPase activity of UvrD when it is functioning either as a translocase or a helicase on DNA in the absence of RecA. Thus, in this context UvrD may operate in a specialized disassembly mode.
Assuntos
DNA Helicases/metabolismo , Proteínas de Escherichia coli/metabolismo , Recombinases Rec A/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Recombinases Rec A/antagonistas & inibidores , Recombinases Rec A/química , Recombinases Rec A/ultraestrutura , Deleção de SequênciaRESUMO
During bacterial conjugation, genetic material from one cell is transferred to another as single-stranded DNA. The introduction of single-stranded DNA into the recipient cell would ordinarily trigger a potentially deleterious transcriptional response called SOS, which is initiated by RecA protein filaments formed on the DNA. During F plasmid conjugation, however, the SOS response is suppressed by PsiB, an F-plasmid-encoded protein that binds and sequesters free RecA to prevent filament formation. Among the many characterized RecA modulator proteins, PsiB is unique in using sequestration as an inhibitory mechanism. We describe the crystal structure of PsiB from the Escherichia coli F plasmid. The stucture of PsiB is surprisingly similar to CapZ, a eukaryotic actin filament capping protein. Structure-directed neutralization of electronegative surfaces on PsiB abrogates RecA inhibition whereas neutralization of an electropositive surface element enhances PsiB inhibition of RecA. Together, these studies provide a first molecular view of PsiB and highlight its use as a reagent in studies of RecA activity.
Assuntos
Proteínas de Bactérias/química , Escherichia coli/química , Recombinases Rec A , Proteínas de Bactérias/metabolismo , Proteína de Capeamento de Actina CapZ/química , Conjugação Genética/fisiologia , Cristalografia por Raios X , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Escherichia coli/metabolismo , Fator F/química , Fator F/metabolismo , Estrutura Terciária de Proteína , Resposta SOS em Genética/fisiologia , Homologia Estrutural de ProteínaRESUMO
The process of bacterial conjugation involves the transfer of a conjugative plasmid as a single strand. The potentially deleterious SOS response, which is normally triggered by the appearance of single-stranded DNA, is suppressed in the recipient cell by a conjugative plasmid system centered on the product of the psiB gene. The F plasmid PsiB protein inhibits all activities of the RecA protein, including DNA binding, DNA strand exchange, and LexA protein cleavage. The proteins known to negatively regulate recombinases, such as RecA or Rad51, generally work at the level of dismantling the nucleoprotein filament. However, PsiB binds to RecA protein that is free in solution. The RecA-PsiB complex impedes formation of RecA nucleoprotein filaments on DNA.
