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In Methanococcus maripaludis, the euryarchaeal archaellum regulator A (EarA) is required for the transcription of the fla operon, which is comprised of a series of genes which encode most of the proteins needed for the formation of the archaeal swimming organelle, the archaellum. In mutants deleted for earA (1earA), there is almost undetectable transcription of the fla operon, Fla proteins are not synthesized and the cells are non-archaellated. In this study, we have isolated a spontaneous mutant of a 1earA mutant in which the restoration of the transcription and translation of the fla operon (using flaB2, the second gene of the operon, as a reporter), archaella formation and swarming motility were all restored even in the absence of EarA. Analysis of the DNA sequence from the fla promoter of this spontaneous mutant revealed a deletion of three adenines within a string of seven adenines in the transcription factor B recognition element (BRE). When the three adenine deletion in the BRE was regenerated in a stock culture of the 1earA mutant, very similar phenotypes to that of the spontaneous mutant were observed. Deletion of the three adenines in the fla promoter BRE resulted in the mutant BRE having high sequence identity to BREs from promoters that have strong basal transcription level in Mc. maripaludis and Methanocaldococcus jannaschii. These data suggest that EarA may help recruit transcription factor B to a weak BRE in the fla promoter of wild-type cells but is not required for transcription from the fla promoter with a strong BRE, as in the three adenine deletion version in the spontaneous mutant. [ABSTRACT FROM AUTHOR]

Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody V H domain library from which we identified a high-affinity V H binder ab8. Bivalent V H , V H -Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. V H -Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of V H -Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic., Competing Interests: Declaration of Interests W.L., C.C., Z.S., J.W.M., and D.S.D. are co-inventors of a patent, filed on March 12 by the University of Pittsburgh, related to ab8 described in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Archaella are the swimming organelles in the Archaea. Recently, the first archaellum regulator in the Euryarchaeota, EarA Mma , was identified in Methanococcus maripaludis , one of the model organisms used for archaellum studies. EarA Mma binds to 6 bp consensus sequences upstream of the fla promoter to activate the transcription of the fla operon, which encodes most of the proteins required for archaella synthesis. In this study, synteny analysis showed that earA homologues are widely distributed in the phylum of Euryarchaeota, with the notable exception of extreme halophiles. We classified Euryarchaeota species containing earA homologues into five classes based on the genomic location of the earA genes relative to fla and chemotaxis operons. EarA homologues from Methanococcus vannielii , Methanothermococcus thermolithotrophicus and Methanocaldococcus jannaschii successfully complemented the function of EarA Mma in a ΔearAMma mutant, demonstrated by the restoration of FlaB2 expression in Western blot analysis and the appearance of archaella on the cell surface in complemented cells. Furthermore, the 6 bp consensus sequence was also found in the fla promoter region in these methanogens, indicating that the EarA homologues ly use a similar mechanism to activate transcription of the fla operons in their own hosts. Attempts to demonstrate complementation of the function of EarA Mma in a ΔearAMma mutant by the EarA homologue of Pyrococcus furiosus were unsuccessful, despite the presence of a copy of the 6 bp consensus EarA-binding sequence upstream of the fla promoter in the P. furiosus genome.