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.
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 Medicago truncatula, 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.
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•Modeling of CO2 hydrogenation to hydrocarbons in a three-side reactor is developed.•Tr, Ts, Tt, θ and φ are considered as main effects in determining product yields.•Statistical ...analysis is performed to study the effects of variables on the yields.•Optimized operating conditions are determined to maximize olefin yields.
Direct CO2 hydrogenation to hydrocarbons is a promising method of reducing CO2 emissions along with producing value-added products. However, reactor design and performance have remained a challenging issue because of low olefin efficiency and high water production as a by-product. Accordingly, a one-dimensional non-isothermal mathematical model is proposed to predict the membrane reactor performance and statistical analysis is used to assess the effects of important variables such as temperatures of reactor (Tr:A), shell (Ts:B) and tube (Tt:C) as well as sweep ratio (θ:D) and pressure ratio (φ:E) and their interactions on the products yields. In addition, the optimized operating conditions are also obtained to achieve maximum olefin yields. Results reveal that interacting effects comprising AB (TrTs), AC (TrTt), AE (Trφ), BC (TsTt), CE (Ttφ), CD (Ttθ) and DE (θφ) play important roles on the product yields. It is concluded that higher temperatures at low sweep and pressure ratios can maximize the yields of olefins, while simultaneously the yields of paraffins are minimized. In this regard, optimized values for Tr, Ts, Tt, θ and φ are determined as 325 °C, 306.96 °C, 325 °C, 1 and 1, respectively.
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.
As part of the effort to create a high resolution representative sequence database of the medieval Hungarian conquerors we have resequenced the entire mtDNA genome of 24 published ancient samples ...with Next Generation Sequencing, whose haplotypes had been previously determined with traditional PCR based methods. We show that PCR based methods are prone to erroneous haplotype or haplogroup determination due to ambiguous sequence reads, and many of the resequenced samples had been classified inaccurately. The SNaPshot method applied with published ancient DNA authenticity criteria is the most straightforward and cheapest PCR based approach for testing a large number of coding region SNP-s, which greatly facilitates correct haplogroup determination.
Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard ...laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms. Here, we introduce directed evolution with random genomic mutations (DIvERGE), a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments. In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Finally, we discuss potential future applications of DIvERGE in synthetic and evolutionary biology.
Before 1914 the vocabulary of anti-Semitism was already present in public discourses in Hungary, but it did not yet represent the central problem of a still ‘liberal Hungary.’ With the First World ...War, the Hungarian middle classes became the main losers in the social disruption of Hungarian society. 1916 must be seen as the turning point of the social splits and divisions. The former policy of the “Burgfrieden,” or party truce, was undermined by the profound psychological experiences of the war. In this context, old anti-Semitic stereotypes prejudices were reactivated while new ones emerged. Jews, in general, came to be treated as internal enemies, earning huge profits from the war at the expense of Christian Hungarian society that was being ruined. This paper analyzes three stages of growing anti-Semitic agitation in Hungarian society during the war: First, the attacks against the banks around 1916; second, the public debate on the Jewish question in 1917, opened by the publication of the book A zsidók útja The Path of the Jews by the sociologist Péter Ágoston and intensified by the “inquiry into the Jewish question” of the journal Huszadik Század Twentieth Century; third, the surge of anti-Semitism that began with anti-Semitic speeches in the Hungarian Diet in 1917, leading to a broad anti-Semitic campaign by predominantly Catholic newspapers, in which Otto Prohaszka and Bela Bangha were the leading figures. The thesis is that Hungarian anti-Semitism was far from being a spontaneous outburst of popular feelings. It was fairly well organized and coordinated, mainly by ecclesiastical circles. It was the First World War that proved to be the catalyst, contributing to an extreme anti-Semitism and thereby sealing the fate of “liberal Hungary.”
Although stream-dwelling gudgeons (Cyprinidae, genus: Gobio) are widespread in Central Europe, the taxonomy of this group and the distribution of its species are still unexplored in detail. The aims ...of our study are to ascertain taxonomic composition and distribution of the former Gobio gobio superspecies in the inner area of the Carpathian Basin. Since the presence of cryptic species is suspected in this area, we examined the taxonomic and phylogenetic relationships of Central European Gobio taxa by sequencing the mitochondrial DNA control region (mtCR). Additionally, we characterized the genetic structure of 27 stream-dwelling gudgeon populations of this area by Amplified Fragment Length Polymorphism (AFLP). Results of mtCR analysis proved the presence of three species already known as G. obtusirostris (dominant in NW-Hungary), G. gobio (sporadic) and G. carpathicus (sporadic). Additionally, the analysis revealed the existence of one doubtful taxon, G. sp1 (dominant in NE-Hungary), and a new isolated haplogroup (dominant in SW-Hungary). Although Network analysis showed significant detachment among haplogroups, their genetic distances were quite small. Therefore Bayesian phylogenetic analysis showed weak nodal support for the branching pattern both for newly described haplotypes, and for the already accepted species. AFLP data showed distinct population structure and a clear pattern of isolation was revealed by distance of stocks. At the same time, level of separation was not affected by the altitudinal position of sites. Moreover we found three major clusters of populations which were separated according to hydrographic regions, and corresponded to the findings of mtCR analysis. Our results suggest the on-going speciation of gudgeons in the Carpathian Basin, however the separation of haplogroups seems to only be an intermediate phase. The discovered natural pattern seems to be only slightly influenced by anthropogenic impacts. Additionally our results put into question the suitability of the recently accepted within Gobio genus taxonomy.
Two alkaliphilic and moderately halophilic bacterial strains B16-10
and Z23-18 characterized by optimal growth at pH 9.0-10.0 and 5 % (w/v) NaCl, were isolated from the rhizosphere soil of the ...bayonet grass (Bolboschoenus maritimus) in the Kiskunság National Park, Hungary. Cells of both strains stained Gram-positive, were motile straight rods, and formed terminal, ellipsoidal endospores with swollen sporangia. The isolates were facultative anaerobic, catalase positive, oxidase negative. Both strains contained meso-diaminopimelic acid as diagnostic diaminoacid of the peptidoglycan. Menaquinone-7 (MK-7) was the predominant isoprenoid quinone. Anteiso-C15 : 0, C16 : 1ω11c and iso-C14 : 0 were the major cellular fatty acids. The DNA G+C content of both strains was 35.8 mol%. The 16S rRNA gene based phylogenetic analysis revealed that the facultative anaerobic strains B16-10
and Z23-18 showed the highest similarities to the type strains of anaerobic Anaerobacillus isosaccharinicus NB2006
(98.7 and 99.1 %), A. macyae JMM-4
(98.2 and 98.4 %), A. alkalidiazotrophicus MS 6
(97.7 and 98.4 %), A. alkalilacustris Z-0521
(97.5 and 98.3 %) and A. arseniciselenatis DSM 15340
(97.5 and 98.2 %). However, the distinctive phenotypic and genetic results of this study confirmed that strains B16-10
and Z23-18 represent a novel species, for which the name Anaerobacillus alkaliphilus sp. nov. is proposed. The type strain is B16-10
(=DSM 29790
=NCAIM B 02608
).
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