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Background Pantoea ananatis, a member of the Enterobacteriacea family, is a new and promising subject for biotechnological research. Over recent years, impressive progress in its application to L-glutamate production has been achieved. Nevertheless, genetic and biotechnological studies of Pantoea ananatis have been impeded because of the absence of genetic tools for rapid construction of direct mutations in this bacterium. The λ Red-recombineering technique previously developed in E. coli and used for gene inactivation in several other bacteria is a high-performance tool for rapid construction of precise genome modifications. Results In this study, the expression of λ Red genes in P. ananatis was found to be highly toxic. A screening was performed to select mutants of P. ananatis that were resistant to the toxic affects of λ Red. A mutant strain, SC17(0) was identified that grew well under conditions of simultaneous expression of λ gam, bet, and exo genes. Using this strain, procedures for fast introduction of multiple rearrangements to the Pantoea ananatis genome based on the λ Red-dependent integration of the PCR-generated DNA fragments with as short as 40 bp flanking homologies have been demonstrated. Conclusion The λ Red-recombineering technology was successfully used for rapid generation of chromosomal modifications in the specially selected P. ananatis recipient strain. The procedure of electro-transformation with chromosomal DNA has been developed for transfer of the marked mutation between different P. ananatis strains. Combination of these techniques with λ Int/Xis-dependent excision of selective markers significantly accelerates basic research and construction of producing strains.

Serving a robust platform for reverse genetics enabling the in vivo study of gene functions primarily in enterobacteriaceae, recombineering -or recombination-mediated genetic engineering-represents a powerful and relative straightforward genetic engineering tool. Catalyzed by components of bacteriophage-encoded homologous recombination systems and only requiring short ∼40–50 base homologies, the targeted and precise introduction of modifications (e.g., deletions, knockouts, insertions and point mutations) into the chromosome and other episomal replicons is empowered. Furthermore, by its ability to make use of both double- and single-stranded linear DNA editing substrates (e.g., PCR products or oligonucleotides, respectively), lengthy subcloning of specific DNA sequences is circumvented. Further, the more recent implementation of CRISPR-associated endonucleases has allowed for more efficient screening of successful recombinants by the selective purging of non-edited cells, as well as the creation of markerless and scarless mutants. In this review we discuss various recombineering strategies to promote different types of gene modifications, how they are best applied, and their possible pitfalls.

Lambda Red recombineering is a powerful technique for making targeted genetic changes in bacteria. However, many applications are limited by the frequency of recombination. Previous studies have suggested that endogenous nucleases may hinder recombination by degrading the exogenous DNA used for recombineering. In this work, we identify ExoVII as a nuclease which degrades the ends of single-stranded DNA (ssDNA) oligonucleotides and double-stranded DNA (dsDNA) cassettes. Removing this nuclease improves both recombination frequency and the inheritance of mutations at the 3′ ends of ssDNA and dsDNA. Extending this approach, we show that removing a set of five exonucleases (RecJ, ExoI, ExoVII, ExoX, and Lambda Exo) substantially improves the performance of co-selection multiplex automatable genome engineering (CoS-MAGE). In a given round of CoS-MAGE with ten ssDNA oligonucleotides, the five nuclease knockout strain has on average 46% more alleles converted per clone, 200% more clones with five or more allele conversions, and 35% fewer clones without any allele conversions. Finally, we use these nuclease knockout strains to investigate and clarify the effects of oligonucleotide phosphorothioation on recombination frequency. The results described in this work provide further mechanistic insight into recombineering, and substantially improve recombineering performance.

To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and “scar”-less.

Scientific Reports - A new recombineering system for precise genome-editing in

Shewanella oneidensis strain MR-1 can respire using carbon electrodes and metal oxyhydroxides as electron acceptors, requiring mechanisms for transferring electrons from the cell interior to surfaces located beyond the cell. Although purified outer membrane cytochromes will reduce both electrodes and metals, S. oneidensis also secretes flavins, which accelerate electron transfer to metals and electrodes. We developed techniques for detecting direct electron transfer by intact cells, using turnover and single turnover voltammetry.

Shewanella species are renowned for their respiratory versatility, including their ability to respire poorly soluble substrates by using enzymatic machinery that is localized to the outside of the cell. The ability to engage in “extracellular respiration” to date has focused primarily on respiration of minerals. Here, we identify two gene clusters in Shewanella oneidensis strain MR-1 that each contain homologs of genes required for metal reduction and genes that are predicted to encode dimethyl sulfoxide (DMSO) reductase subunits. Molecular and genetic analyses of these clusters indicate that one (SO1427–SO1432) is required for anaerobic respiration of DMSO. We show that DMSO respiration is an extracellular respiratory process through the analysis of mutants defective in type II secretion, which is required for transporting proteins to the outer membrane in Shewanella . Moreover, immunogold labeling of DMSO reductase subunits reveals that they reside on the outer leaflet of the outer membrane under anaerobic conditions. The extracellular localization of the DMSO reductase in S. oneidensis suggests these organisms may perceive DMSO in the environment as an insoluble compound.

Four distinct pathways predicted to facilitate electron flow for respiration of externally located substrates are encoded in the genome of Shewanella oneidensis strain MR-1. Although the pathways share a suite of similar proteins, the activity of only two of these pathways has been described. Respir …

Here we demonstrate that elimination of ClaI restriction site from the sequence of a plasmid DNA increases the efficiency of transformation of Shewane…

Electron-shuttling compounds (electron shuttles [ESs], or redox mediators) are essential components in intracellular electron transfer, while microbes…

The expression and distribution of ferric reductase activity was examined in Shewanella putrefaciens MR-1. Formate-dependent ferric reductase was not …

Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribo-nucleotide (oligo) primers and the polymerase chain r…

The pBAD/His Kit provides all of the necessary reagents to express your protein in a tightly regulated fashion. The vector pBAD/His allows you to express your protein with an N-terminal tag. The vector provides: The araBAD promoter for tightly regulated expression Translation initiation signals opti

Abstract. In vivo induction of the Escherichia coli lactose operon as a function of inducer concentration generates a sigmoidal curve, indicating a non-linear r

Nature Genetics - A new logic for DNA engineering using recombination in

We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda (λ) bet or Escherichia coli recT . Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To our knowledge, this is the first study showing single-strand recombinase activity from diverse bacteria. Similar to bet and recT , most of these other recombinases were found to be associated with putative exonuclease genes. Beta and RecT in conjunction with their cognate exonucleases carry out recombination of linear double-strand DNA. Among four of these foreign recombinase/exonuclease pairs tested for recombination with double-strand DNA, three had activity, albeit barely detectable. Thus, although these recombinases can function in E. coli to catalyze oligonucleotide recombination, the double-strand DNA recombination activities with their exonuclease partners were inefficient. This study also demonstrated that Gam, by inhibiting host RecBCD nuclease activity, helps to improve the efficiency of λ Red-mediated recombination with linear double-strand DNA, but Gam is not absolutely essential. Thus, in other bacterial species where Gam analogs have not been identified, double-strand DNA recombination may still work in the absence of a Gam-like function. We anticipate that at least some of the recombineering systems studied here will potentiate oligonucleotide and double-strand DNA-mediated recombineering in their native or related bacteria.

Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage λ generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms. * oligo(s), : oligonucleotide(s); dsDNA, : double-stranded DNA; ssDNA, : single-stranded DNA