Although adenoviruses (AdVs) have been found in a wide variety of reptiles, including numerous squamate species, turtles, and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (Heloderma horridum). Here we report the full genomic and proteomic characterization of the latter, designated lizard adenovirus 2 (LAdV-2). The double-stranded DNA (dsDNA) genome of LAdV-2 is 32,965 bp long, with an average G؉C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome were largely concordant with those in other atadenoviruses, except for four novel open reading frames (ORFs) at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits shared with SnAdV-1 further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein compositions of the virions. The two fiber genes produce two fiber proteins of different sizes that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers nor a mixed number of fibers per vertex had previously been reported for adenoviruses or any other virus. IMPORTANCEHere we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins-one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton base. This observation raises new intriguing questions on virus structure. How can the triple fiber attach to a pentameric vertex? What determines the number and location of each vertex type in the icosahedral particle? Since fibers are responsible for primary attachment to the host, this novel architecture also suggests a novel mode of cell entry for LAdV-2. Adenoviruses have a recognized potential in nanobiomedicine, but only a few of the more than 200 types found so far in nature have been characterized in detail. Exploring the taxonomic wealth of adenoviruses should improve our chances to successfully use them as therapeutic tools.A denoviruses (AdVs) occur commonly in humans and in a large spectrum of animals representing every major class of vertebrates (1). Individual AdV types are generally restricted to a single host species, suggesting a long common evolutionary history. Nonetheless, numerous examples of hypothesized host switches have also been described (2). The family Adenoviridae is currently divided into five officially approved genera (1). The...

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 ssDNA 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 to maintain genome integrity. Through its interaction with SsbA and with a preprimosomal component, RarA might impede the assembly of the replicative helicase, to prevent that recombination intermediates contribute to pathological DNA replication restart.

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.

Natural transformation and viral-mediated transduction are the main avenues of horizontal gene transfer in Firmicutes. Bacillus subtilis SPP1 is a generalized transducing bacteriophage. Using this lytic phage as a model, we have analyzed how viral replication and recombination systems contribute to the transfer of plasmid-borne antibiotic resistances. Phage SPP1 DNA replication relies on essential phage-encoded replisome organizer (G38P), helicase loader (G39P), hexameric replicative helicase (G40P), recombinase (G35P) and in less extent on the partially dispensable 5′→3′ exonuclease (G34.1P), the single-stranded DNA binding protein (G36P) and the Holliday junction resolvase (G44P). Correspondingly, the accumulation of linear concatemeric plasmid DNA, and the formation of transducing particles were blocked in the absence of G35P, G38P, G39P, and G40P, greatly reduced in the G34.1P, G36P mutants, and slightly reduced in G44P mutants. In contrast, establishment of injected linear plasmid DNA in the recipient host was independent of viral-encoded functions. DNA homology between SPP1 and the plasmid, rather than a viral packaging signal, enhanced the accumulation of packagable plasmid DNA. The transfer efficiency was also dependent on plasmid copy number, and rolling-circle plasmids were encapsidated at higher frequencies than theta-type replicating plasmids.