Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.genome engineering | synthetic biology | recombineering | off-target effects | methyl-directed mismatch repair

In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongated-branched, depending on the host plant. In , this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.

SignificanceAntibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. By building on multiplex automated genome engineering, we developed a method that enables precise mutagenesis of multiple, long genomic segments in multiple species without off-target modifications. Thereby, it enables the exploration of vast numbers of combinatorial genetic alterations in their native genomic context. This method is especially well-suited to screen the resistance profiles of antibiotic compounds. It allowed us to predict the evolution of resistance against antibiotics currently in clinical trials. We anticipate that it will be a useful tool to identify resistance-proof antibiotics at an early stage of drug development.

It has been widely accepted that the Finno-Ugric Hungarian language, originated from proto Uralic people, was brought into the Carpathian Basin by the conquering Hungarians. From the middle of the 19th century this view prevailed against the deep-rooted Hungarian Hun tradition, maintained in folk memory as well as in Hungarian and foreign written medieval sources, which claimed that Hungarians were kinsfolk of the Huns. In order to shed light on the genetic origin of the Conquerors we sequenced 102 mitogenomes from early Conqueror cemeteries and compared them to sequences of all available databases. We applied novel population genetic algorithms, named Shared Haplogroup Distance and MITOMIX, to reveal past admixture of maternal lineages. Our results show that the Conquerors assembled from various nomadic groups of the Eurasian steppe. Population genetic results indicate that they had closest connection to the Onogur-Bulgar ancestors of Volga Tatars. Phylogenetic results reveal that more than one third of the Conqueror maternal lineages were derived from Central-Inner Asia and their most probable ultimate sources were the Asian Scythians and Asian Huns, giving support to the Hungarian Hun tradition. The rest of the lineages most likely originated from the Bronze Age Potapovka-Poltavka-Srubnaya cultures of the Pontic-Caspian steppe. Available data imply that the Conquerors did not have a major contribution to the gene pool of the Carpathian Basin.

The NERC and CEH trade marks and logos ('the Trademarks') are registered trademarks of NERC in the UK and other countries, and may not be used without the prior written consent of the Trademark owner. We examine phylogenetic relationships within the Synergus complex of herbivorous 50 inquiline gallwasps associated with (Hymenoptera; Cynipidae; Synergini) associated with cynipid host galls on oak, a biologically diverse group whose genus-level morphological taxonomy has long been considered stable but whose species level taxonomy is problematic.We incorporate data for over 80% of recognised Western Palaearctic species in 5 morphology-based genera (Ceroptres, Saphonecrus, Synergus, Synophrus, Ufo), comprising 55 sequence for two mitochondrial two mitochondrial loci (coxI, cytb) and one nuclear locus (28S D2). In particular, we assess the evidence for monophyly of two long-established, morphology-defined sections within the genus Synergus that differ in a range of biological traits between-generation polymorphism and impact on the host gall inducer (lethal versus non-lethal). To aid analyses of ecological interactions within oak cynipid communities, we 60 also consider the utility of cytochrome oxidase I (coxI) DNA barcodes in the oak inquilines.In this assessment, we do not assume that species are delineated at a single threshold value of sequence divergence for a single gene, but examine concordance in the composition of molecular operational Taxonomic units (MOTUs) across a range of sequence divergences in each gene and across genes. We also assess the impact of sampling effort on MOTU stability. 65Phylogenetic reconstructions for all three loci support monophyly for Synergus and Synophrus, but reject monophyly for Saphonecrus and for the two sections within Synergus.The suites of traits associated with the two sections of the genus Synergus are thus homoplasious. All three loci also reject monophyly for three Synergus species (S. hayneanus, S. pallipes, S. umbraculus). Sequences for each locus identify robust MOTUs that are largely 70 concordant across loci for a range of cut-off values. Though many MOTU's correspond to recognised Linnean species, there is significant, multigene disagreement between groupings supported by morphology and sequence data, with both allocation of different morphospecies to the same MOTU and allocation of the same morphospecies to multiple MOTUs, regardless 3 of cutoff value. Our results imply that while DNA barcoding has considerable utility within 75 this group, morphology-based identification needs major revision at both genus and species levels. Further, lifehistory traits currently attributed to single morphospecies probably confound attributes of multiple lineages. Revealing patterns of character state evolution in Synergus requires collection of new host association and life history data explicitly linked to DNA barcode data for the specimens concerned. 80