Heterologous Expression of Pediocin/Papa in Bacillus Subtilis

Listeria moniocytogenes is food-borne pathogen. Pediocin is group II α bacteriocin with anti-listeria activity, naturally produced by Pediococuus acidilactic and Lactobacillus plantarum. Gene pepA/papA encode for pediocin. Expression and secretion of active papA was relayed on transporter papC and accessary protein papD on the same operon in native host. The excretion machines were also necessary for pediocin protein expression in heterologous host of E. coli, Lactobacillus lactis, and Corynebacterium glutamicum. In this study, two vector carrying codon sequence of papA mature peptide was constructed, with or without His tag. Both fragments were inserted into plasmid pHT43 and transformed Bacillus subtilis WB800N. The strains were induced with IPTG to secrete recombination protein PA1 and PA2 respectively. Supernatant from both recombination strains can inhibit Listeria monocytogenes ATCC54003 directly. The fused protein possesses inhibition activity as a whole, exempting from cleavage of leading peptide. Protein PA1 can be purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. This is the first report for active pediocin expression without assistance of papCD in heterogenous host.

Sequence Recognition by a Pseudorotational Cycle of Sugar Puckers: A Base Pair Switching Model via N-E Sugar Pucker Conversion for Homologous Recombination

A triplex DNA model bound to a helical filament of homologous recombination protein, such as Escherichia coli RecA, is presented. This model suggests that a function of the nucleoprotein filament could be at least partly attributed to the lowering of transition energy of duplex DNA structure having the E-type sugar puckers. Key events during homologous recombination such as sequence recognition, base pair switching and elongation/contraction of the helical pitch may correlate with the pseudo-rotation angle of sugar puckers along the N-, E-, S- and W-types. A conformational change of sugar puckers during the reaction is resolved into two motions by introducing a pair of parameters, μ1 and ν3, which defines a swing orientation of bases and a torsional angle of phosphate backbones, respectively.

Rice (Oryza sativa) TIR1 and 5′adamantyl-IAA Significantly Improve the Auxin-Inducible Degron System in Schizosaccharomyces pombe

The auxin-inducible degron (AID) system is a powerful tool to induce targeted degradation of proteins in eukaryotic model organisms. The efficiency of the existing Schizosaccharomyces pombe AID system is limited due to the fusion of the F-box protein TIR1 protein to the SCF component, Skp1 (Skp1-TIR1). Here, we report an improved AID system for S. pombe that uses the TIR1 from Oryza sativa (OsTIR1) not fused to Skp1. Furthermore, we demonstrate that degradation efficiency can be improved by pairing an OsTIR1 auxin-binding site mutant, OsTIR1F74A, with an auxin analogue, 5′adamantyl-IAA (AID2). We provide evidence for the enhanced functionality of the OsTIR1 AID and AID2 systems by application to the essential DNA replication factor Mcm4 and to a non-essential recombination protein, Rad52. Unlike AID, no detectable auxin-independent depletion of AID-tagged proteins was observed using AID2.

foxl3, a sexual switch in germ cells, initiates two independent molecular pathways for commitment to oogenesis in medaka

Germ cells have the ability to differentiate into eggs and sperm and must determine their sexual fate. In vertebrates, the mechanism of commitment to oogenesis following the sexual fate decision in germ cells remains unknown.Forkhead-box protein L3(foxl3) is a switch gene involved in the germline sexual fate decision in the teleost fish medaka (Oryzias latipes). Here, we show thatfoxl3organizes two independent pathways of oogenesis regulated byREC8 meiotic recombination protein a(rec8a), a cohesin component, andF-box protein(FBP) 47(fbxo47), a subunit of E3 ubiquitin ligase. In mutants of either gene, germ cells failed to undergo oogenesis but developed normally into sperm in testes. Disruption ofrec8aresulted in arrest at a meiotic pachytenelike stage specifically in females, revealing a sexual difference in meiotic progression. Analyses offbxo47mutants showed that this gene regulates transcription factors that facilitate folliculogenesis:LIM homeobox 8(lhx8b),factor in the germlineα (figla), andnewborn ovary homeobox(nobox). Interestingly, we found that thefbxo47pathway ensures that germ cells do not deviate from an oogenic pathway until they reach diplotene stage. The mutant phenotypes together with the timing of their expression imply that germline feminization is established during early meiotic prophase I.

Access to PCNA by Srs2 and Elg1 Controls the Choice between Alternative Repair Pathways in Saccharomyces cerevisiae

ABSTRACT During DNA replication, stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions. Under these circumstances, the processivity factor PCNA gets ubiquitylated at lysine 164, inducing the DNA damage tolerance (DDT) mechanisms that can bypass lesions encountered during DNA replication. PCNA can also be SUMOylated at the same residue or at lysine 127. Surprisingly, pol30-K164R mutants display a higher degree of sensitivity to DNA-damaging agents than pol30-KK127,164RR strains, unable to modify any of the lysines. Here, we show that in addition to translesion synthesis and strand-transfer DDT mechanisms, an alternative repair mechanism (“salvage recombination”) that copies information from the sister chromatid is repressed by the recruitment of Srs2 to SUMOylated PCNA. Overexpression of Elg1, the PCNA unloader, or of the recombination protein Rad52 allows its activation. We dissect the genetic requirements for this pathway, as well as the interactions between Srs2 and Elg1. IMPORTANCE PCNA, the ring that encircles DNA maintaining the processivity of DNA polymerases, is modified by ubiquitin and SUMO. Whereas ubiquitin is required for bypassing lesions through the DNA damage tolerance (DDT) pathways, we show here that SUMOylation represses another pathway, salvage recombination. The Srs2 helicase is recruited to SUMOylated PCNA and prevents the salvage pathway from acting. The pathway can be induced by overexpressing the PCNA unloader Elg1, or the homologous recombination protein Rad52. Our results underscore the role of PCNA modifications in controlling the various bypass and DNA repair mechanisms.

Access to PCNA by Srs2 and Elg1 controls the choice between alternative repair pathways in yeast

ABSTRACTDuring DNA replication stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions. Under these circumstances the processivity factor PCNA gets ubiquitylated at lysine 164, inducing the DNA damage tolerance (DDT) mechanisms that can bypass lesions encountered during DNA replication. PCNA can also be SUMOylated at the same residue or at lysine 127. Surprisingly, pol30-K164R mutants display a higher degree of sensitivity to DNA damaging agents than pol30-KK127,164RR strains, unable to modify any of the lysines. Here we show that in addition to trans-lesion synthesis and strand-transfer DTT mechanisms, an alternative repair mechanism (“salvage recombination”) that copies information from the sister chromatid, is repressed by the recruitment of Srs2 to SUMOylated PCNA. Overexpression of Elg1, the PCNA unloader, or of the recombination protein Rad52 allows its activation. We dissect the genetic requirements for this pathway, as well as the interactions between Srs2 and Elg1.