Summary The process of bacterial conjugation involves the transfer of a conjugative plasmid as a single strand. The potentially deleterious SOS response, 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.

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.

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.

No abstract

Multiple bacterial species exist that can survive the extensive DNA damage caused by ionizing radiation, yet the exact mechanisms of this resistance are not entirely understood. Using a directed evolution protocol, our lab has generated several strains of E. coli that are extremely radiation‐resistant. The genomes of multiple isolates from the evolved populations have been sequenced to identify mutations responsible for this phenotype. Two alleles were identified which encode mutations in RecA (A289S and D276A), a central protein in DNA repair. Inserting these alleles into wildtype E. coli resulted in small increases in survival after exposure to radiation. Thus, understanding the altered activity of these mutants can provide insight into DNA repair mechanisms and how an organism can survive ionizing radiation.RecA D276A displays an increased ability to bind to DNA coated with the single stranded DNA binding protein (SSB). This suggests that faster activation of RecA may aid in cell survival after DNA damage. Conversely, the RecA A289S protein has a diminished capability to bind to SSB‐coated DNA, and thus, the RecF, RecO, and RecR loading proteins may have a greater role in facilitating RecA binding to SSB‐coated DNA with this mutant. Also, RecA A289S filaments disassemble faster when exposed to the inhibitory RecX protein. This data suggests that tighter regulation of RecA A289S activity may produce a filament form that enhances certain types of RecA‐mediated recombination events. This work was supported by a grant from the National Institutes of Health, GM32335.