Insect Diversity of the Lower Montane Evergreen Forest of the Western Andes Mountain Range: Cascada Chilicay and Suncamal

Biological research in the low montane evergreen forests of Ecuador focuses on ecological and botanical aspects, while knowledge of the entomofauna of these areas is almost nil. In February 2022, sampling was carried out during the dry season for 15 days, using direct and indirect capture methods (tapping, sieving, and light traps) in two waterfalls of the low montane evergreen forest of the western Andean Cordillera: Chilicay and Suncamal waterfalls, with the objective of identifying the composition of the terrestrial insect fauna at the family level. Two orders and 21 families were recorded, among which the families Carabidae and Noctuidae represented the highest percentage of the total abundance. Although preliminary, this work constitutes the first contribution to the knowledge of the entomofauna of this ecosystem. Keywords: biodiversity, conservation, entomofauna, insects. Resumen Las investigaciones biológicas en los bosques siempreverdes montanos bajos de Ecuador, se centran en aspectos ecológicos y botánicos, mientras que el conocimiento de la entomofauna de estas zonas es escaso. En febrero de 2022, en la época seca y durante 15 días, utilizando métodos de captura directa e indirecta (golpeteo, tamizado y trampas de luz), se realizaron muestreos en dos cascadas del Bosque siempreverde montano bajo de la cordillera occidental de los Andes: Cascada Chilicay y Suncamal, con el objetivo de identificar la composición de la fauna de insectos terrestres a nivel de familia. Se registraron dos órdenes y 21 familias, entre las cuales, las familias Carabidae y Noctuidae representaron el mayor porcentaje de la abundancia total. Aunque en forma preliminar, este trabajo constituye el primer aporte al conocimiento de la entomofauna de este ecosistema. Palabras Clave: Biodiversidad, conservación, entomofauna, insectos

Chromosomal transformation in Bacillus subtilis is a non-polar recombination reaction

Natural chromosomal transformation is one of the primary driving forces of bacterial evolution. This reaction involves the recombination of the internalized linear single-stranded (ss) DNA with the homologous resident duplex via RecA-mediated integration in concert with SsbA and DprA or RecO. We show that sequence divergence prevents Bacillus subtilis chromosomal transformation in a log-linear fashion, but it exerts a minor effect when the divergence is localized at a discrete end. In the nucleotide bound form, RecA shows no apparent preference to initiate recombination at the 3′- or 5′-complementary end of the linear duplex with circular ssDNA, but nucleotide hydrolysis is required when heterology is present at both ends. RecA·dATP initiates pairing of the linear 5′ and 3′ complementary ends, but only initiation at the 5′-end remains stably paired in the absence of SsbA. Our results suggest that during gene transfer RecA·ATP, in concert with SsbA and DprA or RecO, shows a moderate preference for the 3′-end of the duplex. We show that RecA-mediated recombination initiated at the 3′- or 5′-complementary end might have significant implication on the ecological diversification of bacterial species with natural transformation

Bacillus subtilisRecO and SsbA are crucial for RecA-mediated recombinational DNA repair

Genetic data have revealed that the absence of Bacillus subtilis RecO and one of the end-processing avenues (AddAB or RecJ) renders cells as sensitive to DNA damaging agents as the null recA, suggesting that both end-resection pathways require RecO for recombination. RecA, in the rATP·Mg2+ bound form (RecA·ATP), is inactive to catalyze DNA recombination between linear double-stranded (ds) DNA and naked complementary circular single-stranded (ss) DNA. We showed that RecA·ATP could not nucleate and/or polymerize on SsbA·ssDNA or SsbB·ssDNA complexes. RecA·ATP nucleates and polymerizes on RecO·ssDNA·SsbA complexes more efficiently than on RecO·ssDNA·SsbB complexes. Limiting SsbA concentrations were sufficient to stimulate RecA·ATP assembly on the RecO·ssDNA·SsbB complexes. RecO and SsbA are necessary and sufficient to ‘activate’ RecA·ATP to catalyze DNA strand exchange, whereas the AddAB complex, RecO alone or in concert with SsbB was not sufficient. In presence of AddAB, RecO and SsbA are still necessary for efficient RecA·ATP-mediated three-strand exchange recombination. Based on genetic and biochemical data, we proposed that SsbA and RecO (or SsbA, RecO and RecR in vivo) are crucial for RecA activation for both, AddAB and RecJ–RecQ (RecS) recombinational repair pathways

Roles of bacillus subtilis Dpra and Ssba in RecA-mediated genetic recombination

Bacillus subtilis competence-induced RecA, SsbA, SsbB, and DprA are required to internalize and to recombine single-stranded (ss) DNA with homologous resident duplex. RecA, in the ATP·Mg2+-bound form (RecA·ATP), can nucleate and form filament onto ssDNA but is inactive to catalyze DNA recombination. We report that SsbA or SsbB bound to ssDNA blocks the RecA filament formation and fails to activate recombination. DprA facilitates RecA filamentation; however, the filaments cannot engage in DNA recombination. When ssDNA was preincubated with SsbA, but not SsbB, DprA was able to activate DNA strand exchange dependent on RecA·ATP. This work demonstrates that RecA·ATP, in concert with SsbA and DprA, catalyzes DNA strand exchange, and SsbB is an accessory factor in the reaction. In contrast, RecA·dATP efficiently catalyzes strand exchange even in the absence of single-stranded binding proteins or DprA, and addition of the accessory factors marginally improved it. We proposed that the RecA-bound nucleotide (ATP and to a lesser extent dATP) might dictate the requirement for accessory factors

RecA Regulation by RecU and DprA During Bacillus subtilis Natural Plasmid Transformation

Natural plasmid transformation plays an important role in the dissemination of antibiotic resistance genes in bacteria. During this process, Bacillus subtilis RecA physically interacts with RecU, RecX, and DprA. These three proteins are required for plasmid transformation, but RecA is not. In vitro, DprA recruits RecA onto SsbA-coated single-stranded (ss) DNA, whereas RecX inhibits RecA filament formation, leading to net filament disassembly. We show that a null recA (ΔrecA) mutation suppresses the plasmid transformation defect of competent ΔrecU cells, and that RecU is essential for both chromosomal and plasmid transformation in the ΔrecX context. RecU inhibits RecA filament growth and facilitates RecA disassembly from preformed filaments. Increasing SsbA concentrations additively contributes to RecU-mediated inhibition of RecA filament extension. DprA is necessary and sufficient to counteract the negative effect of both RecU and SsbA on RecA filament growth onto ssDNA. DprA-SsbA activates RecA to catalyze DNA strand exchange in the presence of RecU, but this effect was not observed if RecU was added prior to RecA. We propose that DprA contributes to RecA filament growth onto any internalized SsbA-coated ssDNA. When the ssDNA is homologous to the recipient, DprA antagonizes the inhibitory effect of RecU on RecA filament growth and helps RecA to catalyze chromosomal transformation. On the contrary, RecU promotes RecA filament disassembly from a heterologous (plasmid) ssDNA, overcoming an unsuccessful homology search and favoring plasmid transformation. The DprA–DprA interaction may promote strand annealing upon binding to the complementary plasmid strands and facilitating thereby plasmid transformation rather than through a mediation of RecA filament growth

Bacillus subtilis RecA with DprA-SsbA antagonizes RecX function during natural transformation

Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal and plasmid transformation. We showed that RecA, in the rATP·Mg2+ bound form (RecA·ATP), could not compete with RecX, SsbA or SsbB for assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. RecX promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA–SsbA mediator reversed the RecX negative effect on RecA filament extension, but not DprA or DprA and SsbB. In the presence of DprA–SsbA, RecX added prior to RecA·ATP inhibited DNA strand exchange, but this inhibition was reversed when RecX was added after RecA. We propose that RecA nucleation is more sensitive to RecX action than is RecA filament growth. DprA–SsbA facilitates formation of an active RecA filament that directly antagonizes the inhibitory effects of RecX. RecX and DprA enable chromosomal transformation by altering RecA filament dynamics. DprA–SsbA and RecX proteins constitute a new regulatory network of RecA function. DprA–SsbA contributes to the formation of an active RecA filament and directly antagonizes the inhibitory effects of RecX during natural transformation

Bacillus subtilis RarA acts as a positive RecA accessory protein

Ubiquitous RarA AAA+ ATPases play crucial roles in the cellular response to blocked replication forks in pro- and eukaryotes. Here, we provide evidence that absence of RarA reduced the viability of ΔrecA, ΔrecO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H2O2- or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (ΔaddAB, ΔrecJ, ΔrecQ, ΔrecS) or branch migration (ΔruvAB, ΔrecG, ΔradA) activity. RarA contributes to RecA thread formation, that are thought to be the active forms of RecA during homology search. The absence of RarA reduced RecA accumulation, and the formation of visible RecA threads in vivo upon DNA damage. When ΔrarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in ΔrarA ΔrecU and ΔrarA ΔrecX double mutant cells, and was blocked in ΔrarA recF15 cells. These results suggest that RarA contributes to the assembly of RecA nucleoprotein filaments onto single-stranded DNA, and possibly antagonizes RecA filament disassembly

The RecD2 helicase balances RecA activities

DNA helicases of the RecD2 family are ubiquitous. Bacillus subtilis RecD2 in association with the single-stranded binding protein SsbA may contribute to replication fork progression, but its detailed action remains unknown. In this work, we explore the role of RecD2 during DNA replication and its interaction with the RecA recombinase. RecD2 inhibits replication restart, but this effect is not observed in the absence of SsbA. RecD2 slightly affects replication elongation. RecA inhibits leading and lagging strand synthesis, and RecD2, which physically interacts with RecA, counteracts this negative effect. In vivo results show that recD2 inactivation promotes RecA–ssDNA accumulation at low mitomycin C levels, and that RecA threads persist for a longer time after induction of DNA damage. In vitro, RecD2 modulates RecA-mediated DNA strand-exchange and catalyzes branch migration. These findings contribute to our understanding of how RecD2 may contribute to overcome a replicative stress, removing RecA from the ssDNA and, thus, it may act as a negative modulator of RecA filament growth

Quasar accretion disk sizes from continuum reverberation mapping in the DES standard-star fields

Measurements of the physical properties of accretion disks in active galactic nuclei are important for better understanding the growth and evolution of supermassive black holes. We present the accretion disk sizes of 22 quasars from continuum reverberation mapping with data from the Dark Energy Survey (DES) standard star fields and the supernova C fields. We construct continuum lightcurves with the \textit{griz} photometry that span five seasons of DES observations. These data sample the time variability of the quasars with a cadence as short as one day, which corresponds to a rest frame cadence that is a factor of a few higher than most previous work. We derive time lags between bands with both JAVELIN and the interpolated cross-correlation function method, and fit for accretion disk sizes using the JAVELIN Thin Disk model. These new measurements include disks around black holes with masses as small as $\sim10^7$ $M_{\odot}$, which have equivalent sizes at 2500\AA \, as small as $\sim 0.1$ light days in the rest frame. We find that most objects have accretion disk sizes consistent with the prediction of the standard thin disk model when we take disk variability into account. We have also simulated the expected yield of accretion disk measurements under various observational scenarios for the Large Synoptic Survey Telescope Deep Drilling Fields. We find that the number of disk measurements would increase significantly if the default cadence is changed from three days to two days or one day.Comment: 33 pages, 24 figure