Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

Thomason, Lynn C.; Sawitzke, James A.; Li, Xintian; Costantino, Nina; Court, Donald L.

doi:10.1002/0471142727.mb0116s106

Cited by 236 publications

(130 citation statements)

References 58 publications

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“…Clearly, a complete deletion of the pssA gene, e.g. by an antibiotic resistance cassette using the lambda phage recombination system (Thomason et al 2014), would facilitate adaptation to minimal medium without the potential for regain of PE synthesis. However, it is unlikely that a PE-lacking strain can be propagated in the absence of divalent metal ion supplementation since extensive attempts have been made to eliminate this requirement (P. Heacock and W. Dowhan, unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Biosynthetic preparation of selectively deuterated phosphatidylcholine in genetically modified Escherichia coli

Maric

Thygesen

Schiller

et al. 2014

Appl Microbiol Biotechnol

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Phosphatidylcholine (PC) is a major component of eukaryotic cell membranes and one of the most commonly used phospholipids for reconstitution of membrane proteins into carrier systems such as lipid vesicles, micelles and nanodiscs. Selectively deuterated versions of this lipid have many applications, especially in structural studies using techniques such as NMR, neutron reflectivity and small-angle neutron scattering. Here we present a comprehensive study of selective deuteration of phosphatidylcholine through biosynthesis in a genetically modified strain of Escherichia coli. By carefully tuning the deuteration level in E. coli growth media and varying the deuteration of supplemented carbon sources, we show that it is possible to achieve a controlled deuteration for three distinct parts of the PC lipid molecule, namely the (a) lipid head group, (b) glycerol backbone and (c) fatty acyl tail. This biosynthetic approach paves the way for the synthesis of specifically deuterated, physiologically relevant phospholipid species which remain difficult to obtain through standard chemical synthesis.

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Section: Discussionmentioning

confidence: 99%

Biosynthetic preparation of selectively deuterated phosphatidylcholine in genetically modified Escherichia coli

Maric

Thygesen

Schiller

et al. 2014

Appl Microbiol Biotechnol

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“…E. coli strain DH5␣ (Gibco-BRL) served as a host for plasmid constructions, and E. coli strain BL21(DE3) (Novagen) was used for protein production and purification. Strain HME45 [W3110 lac ϩ pgl⌬8 gal490 cI857⌬(cro-bioA)] has a defective prophage that supplies the Red recombination functions necessary for recombineering (25). Strain MS411 carrying plasmids of interest served as the donor for mating experiments (26).…”

Section: Methodsmentioning

confidence: 99%

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

Whitaker¹,

Berry²,

Rosenthal³

et al. 2016

J Bacteriol

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Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis. A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4 At ) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4 At and the TraJ/VirD4 At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ⌬CTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4 At 's CTD to the VirE2 effector, although other VirD4 At domains bound this substrate in vitro. We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains. IMPORTANCEBacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.T he type IV secretion systems (T4SSs) display striking versatility among the known bacterial translocation systems in their capacity to translocate DNA and protein substrates to bacterial or eukaryotic target cells (1, 2). Members of one major T4SS subfamily, the conjugation machines, mediate transfer of mobile DNA elements and are responsible for widespr...

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“…The osmY promoter-lacZ transcriptional fusion reporter is encoded by a single-copy prophage (7). Gene deletions and allelic replacements were made by Red-mediated recombination (17) or were obtained from the Keio collection (18). Mutant alleles were transferred to strains by transduction with P1vir (19).…”

Section: Methodsmentioning

confidence: 99%

Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S

et al. 2014

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bBacteria utilize multiple sigma factors that associate with core RNA polymerase (RNAP) to control transcription in response to changes in environmental conditions. In Escherichia coli and Salmonella enterica, Crl positively regulates the S regulon by binding to S to promote its association with core RNAP. We recently characterized the determinants in S responsible for specific binding to Crl. However, little is known about the determinants in Crl required for this interaction. Here, we present the X-ray crystal structure of a Crl homolog from Proteus mirabilis in conjunction with in vivo and in vitro approaches that probe the Crl-S interaction in E. coli. We show that the P. mirabilis, Vibrio harveyi, and E. coli Crl homologs function similarly in E. coli, indicating that Crl structure and function are likely conserved throughout gammaproteobacteria. We utilize phylogenetic conservation and bacterial two-hybrid analyses to predict residues in Crl important for the interaction with S . The results of p-benzoylphenylalanine (BPA)-mediated UV cross-linking studies further support the model in which an evolutionarily conserved central cleft is the surface on Crl that binds to S . Within this conserved binding surface, we identify a key residue in Crl that is critical for activation of E S -dependent transcription in vivo and in vitro. Our study provides a physical basis for understanding the S -Crl interaction.

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Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

Cited by 236 publications

References 58 publications

Biosynthetic preparation of selectively deuterated phosphatidylcholine in genetically modified Escherichia coli

Biosynthetic preparation of selectively deuterated phosphatidylcholine in genetically modified Escherichia coli

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S

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