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We studied the effects of visibility, female and male distribution, microhabitat and distance to human infrastructure on display site selection in a ground-dwelling bird, the Canarian houbara bustard. Using a very high-resolution digital elevation model based on LIDAR technology, and a complete census of the breeding population, we compared 98 display sites with randomly generated sites through generalized linear models. Univariate analyses showed that males displayed at locations that increased their visibility, both at short and long distances. Interestingly, although numbers of females and males around sites did not differ between display and random locations, from display locations males could see more females and males at both distance ranges. The absence of vegetation and stones was also critical as it allowed males to perform display runs on a ground free of obstacles. The amount of trophic resources did not correlate with the selection of the display site itself, though an appropriate vegetation cover seemed to be important at a wider habitat scale. Finally, display sites were farther away than random sites from sources of human disturbance, such as urban nuclei, buildings and tracks. Logistic regression analyses confirmed the importance of viewshed, low stone and vegetation cover, and distance to urban centres and tracks, and model averaging identified short-range visibility and females visible in the long range as the most important visibility variables. These results are compatible with the sexual advertisement and predator avoidance hypotheses. We provide recommendations to ensure an appropriate management of the breeding habitat of this endangered subspecies.

During natural transformation Bacillus subtilis RecA, polymerized onto the incoming single-stranded (ss) DNA, catalyses DNA strand invasion resulting in a displacement loop (D-loop) intermediate. A null radA mutation impairs chromosomal transformation, and RadA/Sms unwinds forked DNA in the 5'→3' direction. We show that in the absence of RadA/Sms competent cells require the RecG translocase for natural chromosomal transformation. RadA/Sms tetracysteine motif (C13A and C13R) variants, which fail to interact with RecA, are also deficient in plasmid transformation, but this defect is suppressed by inactivating recA. The RadA/Sms C13A and C13R variants bind ssDNA, and this interaction stimulates their ATPase activity. Wild-type (wt) RadA/Sms interacts with and inhibits the ATPase activity of RecA, but RadA/Sms C13A fails to do it. RadA/Sms and its variants, C13A and C13R, bound to the 5'-tail of a DNA substrate, unwind DNA in the 5'→3' direction. RecA interacts with and loads wt RadA/Sms to promote unwinding of a non-cognate 3'-tailed or 5'-fork DNA substrate, but RadA/Sms C13A or C13R fail to do it. We propose that wt RadA/Sms interaction with RecA is crucial to recruit the former onto D-loop DNA, and both proteins in concert catalyse D-loop extension to favour integration of ssDNA during chromosomal transformation.

Bacillus subtilis diadenylate cyclase DisA converts two ATPs into c-di-AMP, but this activity is suppressed upon interaction with sites of DNA damage. DisA forms a rapid moving focus that pauses upon induction of DNA damage during spore development. We report that DisA pausing, however, was not observed in the absence of the RecO mediator or of the RecA recombinase, suggesting that DisA binds to recombination intermediates formed by RecA in concert with RecO. DisA, which physically interacts with RecA, was found to reduce its ATPase activity without competing for nucleotides or ssDNA. Furthermore, increasing DisA concentrations inhibit RecA-mediated DNA strand exchange, but this inhibition failed to occur when RecA was added prior to DisA, and was independent of RecA-mediated nucleotide hydrolysis or increasing concentrations of c-di-AMP. We propose that DisA may preserve genome integrity by downregulating RecA activities at several steps of the DNA damage tolerance pathway, allowing time for the repair machineries to restore genome stability. DisA might reduce RecA-mediated template switching by binding to a stalled or reversed fork.

The ubiquitous RarA/Mgs1/WRNIP protein plays a crucial, but poorly understood role in genome maintenance. We show that Bacillus subtilis RarA, in the apo form, preferentially binds single-stranded (ss) over double-stranded (ds) DNA. SsbA bound to ss-DNA loads RarA, and for such recruitment the amphipathic C-terminal domain of SsbA is required. RarA is a DNA-dependent ATPase strongly stimulated by ssDNA–dsDNA junctions and SsbA, or by dsDNA ends. RarA, which may interact with PriA, does not stimulate PriA DNA unwinding. In a reconstituted PriA-dependent DNA replication system, RarA inhibited initiation, but not chain elongation. The RarA effect was not observed in the absence of SsbA, or when the host-encoded preprimosome and the DNA helicase are replaced by proteins from the SPP1 phage with similar function. We propose that RarA assembles at blocked forks tomaintain genome integrity. Through its interaction with SsbA and with a preprimosomal component, RarAmight impede the assembly of the replicative helicase, to prevent that recombination intermediates contribute to pathological DNA replication restart.

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.

Intra-specific coloration polymorphism coupled with an ancient process of lineage differentiation in Berberomeloe majalis (Linnaeus, 1758) offers the opportunity to analyse the temporal scenario in which the correlation between toxicity and coloration might have evolved. Based on phylogenetic and phylogeographic analyses, we identified the timing for the split between red-striped and entirely black morphotypes of B. majalis. To evaluate whether coloration patterns and toxicity are related in this species, we quantified the concentration of cantharidin across morphotypes and phylogeographic lineages. Phylogenetic analyses based on cox1 mitochondrial DNA sequences recovered three major clades where both morphotypes were intermingled, indicating a multiple homoplastic origin for the entirely black coloration. Our analyses showed that cantharidin content did not differ between morphs of B. majalis; however, it significantly increased in haemolymph in females kept isolated from males, which reveals the females’ ability either to concentrate cantharidin towards haemolymph or to synthesize cantharidin themselves. Lack of monophyly and absence of genetic isolation in both morphotypes favour the hypothesis of a recent homoplastic phenomenon to explain the loss of the striped pattern. Our phylogenetic and phylogeographic analyses show that changes in coloration are recent, suggesting that the ancient pressures that fixed and maintained red-striped colorations are no longer acting today on B. majalis. The absence of change in cantharidin content (i.e. entirely black and red-striped specimens are equally poisonous) suggests that the evolution of colour polymorphisms in B. majalis is probably decoupled from toxicity.

In hot environments, where many species seek shade to decrease their heat load at midday, sexual differences in heat sensitivity could lead to different patterns of shelter use in males and females, constituting a sexual segregation mechanism. In sexually size-dimorphic species living in these areas, the larger males have more difficulties in dissipating body heat and reducing evaporative water loss. This occurs in the great bustard (Otis tarda), the most sexually size-dimorphic bird. A previous study showed a correlation between summer temperature at breeding areas and northward migration to cooler areas only in males, providing strong evidence for their lower heat tolerance compared with females. However, not all males migrate, and here, we investigate the behavioral adaptations that help sedentary males to withstand summer heat. Males showed a stronger dependence on vegetation providing shade than females at midday. All males searched for shade after the morning foraging period, whereas only two-thirds of the females did it, entering shade 1h later than males. Males foraged less intensively, moved shorter distances between foraging and resting locations, and flew less than females. The use of shade is the main cause of the observed sexual differences in microhabitat selection in summer, whereas lower food intake, and reduced locomotor and flight activity represent additive factors affecting spatial and social segregation. These patterns are probably of strong adaptive value for males, and can be interpreted as mechanisms to prevent overheating during summer, the season when males reach lowest body weights and highest mortality values.

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.