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.

https://www.pnas.org/content/pnas/113/9/2502.full.pdf

A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species.

Magnetotactic bacteria (MTB) biomineralize intracellular, membrane-bounded crystals of magnetite (Fe3 O4 ) and/or greigite (Fe3 S4 ) called magnetosomes. MTB play important roles in the geochemical cycling of iron, sulfur, nitrogen and carbon. Significantly, they also represent an intriguing model system not just for the study of microbial biomineralization but also for magnetoreception, prokaryotic organelle formation and microbial biogeography. Here we review current knowledge on the ecology of and biomineralization by MTB, with an emphasis on more recent reports of unexpected ecological and phylogenetic findings regarding MTB. In this study, we conducted a search of public metagenomic databases and identified six novel magnetosome gene cluster-containing genomic fragments affiliated with the Deltaproteobacteria and Gammaproteobacteria classes of the Proteobacteria phylum, the Nitrospirae phylum and the Planctomycetes phylum from the deep subseafloor, marine oxygen minimum zone, groundwater biofilm and estuary sediment, thereby extending our knowledge on the diversity and distribution of MTB as well deriving important information as to their ecophysiology. We point out that the increasing availability of sequence data will facilitate researchers to systematically explore the ecology and biomineralization of MTB even further.

Diversity and ecology of and biomineralization by magnetotactic bacteria

Microbial α-amylase: A biomolecular overview

The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.

λ Recombination and Recombineering.

Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we introduce a high-throughput method for SSAP discovery that we call "serial enrichment for efficient recombineering" (SEER). By performing SEER in Escherichia coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold in E. coli, while PapRecT enables efficient recombineering in Pseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.

https://www.pnas.org/content/pnas/early/2020/05/27/2001588117.full.pdf

Improved bacterial recombineering by parallelized protein discovery

Bacillus subtilis and its closely related species are important strains for industry, agriculture, and medicine However, it is difficult to perform genetic manipulations using the endogenous recombination machinery In many bacteria, phage recombineering systems have been employed to improve recombineering frequencies To date, an efficient phage recombineering system for B subtilis has not been reported Here, we, for the first time, identified that GP35 from the native phage SPP1 exhibited a high recombination activity in B subtilis On this basis, we developed a high-efficiency GP35-meditated recombineering system Taking single-stranded DNA (ssDNA) as a recombineering substrate, ten recombinases from diverse sources were investigated in B subtilis W168 GP35 showed the highest recombineering frequency (171 ± 015 × 10−1) Besides targeting the purine nucleoside phosphorylase gene (deoD), we also demonstrated the utility of GP35 and Beta from Escherichia coli lambda phage by deleting the alpha-amylase gene (amyE) and uracil phosphoribosyltransferase gene (upp) In all three genetic loci, GP35 exhibited a higher frequency than Beta Moreover, a phylogenetic tree comparing the kinship of different recombinase hosts with B subtilis was constructed, and the relationship between the recombineering frequency and the kinship of the host was further analyzed The results suggested that closer kinship to B subtilis resulted in higher frequency in B subtilis In conclusion, the recombinase from native phage or prophage can significantly promote the genetic recombineering frequency in its host, providing an effective genetic tool for constructing genetically engineered strains and investigating bacterial physiology

A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35

Manipulating the bacterial genomes in an efficient manner is essential to biological and biotechnological research. Here, we reprogrammed the bacterial TA systems as the toxin counter-selectable cassette regulated by an antitoxin switch (TCCRAS) for genetic modifications in the extensively studied and utilized Gram-positive bacteria, B. subtilis and Corynebacterium glutamicum. In the five characterized type II TA systems, the RelBE complex can specifically and efficiently regulate cell growth and death by the conditionally controlled antitoxin RelB switch, thereby serving as a novel counter-selectable cassette to establish the TCCRAS system. Using a single vector, such a system has been employed to perform in-frame deletion, functional knock-in, gene replacement, precise point mutation, large-scale insertion, and especially, deletion of the fragments up to 194.9 kb in B. subtilis. In addition, the biosynthesis of lycopene was first achieved in B. subtilis using TCCRAS to integrate a 5.4-kb fusion cluster ...

Bacterial Genome Editing via a Designed Toxin-Antitoxin Cassette.

Magnetotactic bacteria (MTB), a group of phylogenetically diverse organisms that use their unique intracellular magnetosome organelles to swim along the Earth’s magnetic field, play important roles in the biogeochemical cycles of iron and sulfur. Previous studies have revealed that the bacterial actin protein MamK plays essential roles in the linear arrangement of magnetosomes in MTB cells belonging to the Proteobacteria phylum. However, the molecular mechanisms of multiple-magnetosome-chain arrangements in MTB remain largely unknown. Here, we report that the MamK filaments from the uncultivated ‘Candidatus Magnetobacterium casensis’ (Mcas) within the phylum Nitrospirae polymerized in the presence of ATP alone and were stable without obvious ATP hydrolysis-mediated disassembly. MamK in Mcas can convert NTP to NDP and NDP to NMP, showing the highest preference to ATP. Unlike its Magnetospirillum counterparts, which form a single magnetosome chain, or other bacterial actins such as MreB and ParM, the polymerized MamK from Mcas is independent of metal ions and nucleotides except for ATP, and is assembled into well-ordered filamentous bundles consisted of multiple filaments. Our results suggest a dynamically stable assembly of MamK from the uncultivated Nitrospirae MTB that synthesizes multiple magnetosome chains per cell. These findings further improve the current knowledge of biomineralization and organelle biogenesis in prokaryotic systems.

/pdf/in-vitro-assembly-of-the-bacterial-actin-protein-mamk-from-2wz95abzco.pdf

In vitro assembly of the bacterial actin protein MamK from ‘Candidatus Magnetobacterium casensis’ in the phylum Nitrospirae

The invention relates to a recombinant bacterium for generating inosine. Compared with an original strain, the recombinant bacterium has the weakened phosphopentose mutase activityor inactivated transphosphorylase. The original strain is a strain capable of accumulating inosine. The invention finds an improving target for increasing the fermenting yield of inosine and establishing the corresponding recombinant bacterium. A test proves that the inosine yield of the recombinant bacterium can be obviously increased and the accumulation volume of the side product hypoxanthine can be reduced.

Qinyun Sun

Papers

A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35

Bacterial Genome Editing via a Designed Toxin-Antitoxin Cassette.

In vitro assembly of the bacterial actin protein MamK from ‘Candidatus Magnetobacterium casensis’ in the phylum Nitrospirae

Recombinant bacterium for generating inosine, preparation method and application thereof