New high-cloning-efficiency vectors for complementation studies and recombinant protein overproduction in Escherichia coli and Salmonella enterica

VanDrisse, Chelsey M.; Escalante‐Semerena, Jorge C.

doi:10.1016/j.plasmid.2016.05.001

Cited by 39 publications

(47 citation statements)

References 20 publications

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“…Candidate constructs were confirmed via sequence analysis performed by Eton Biosciences (San Diego, CA). Plasmid derivatives of pBAD24 were created using a BspQI restriction cloning method as previously described by Galloway et al (41) with a modified vector that contained the BspQI site (pCV1) (42). P. aeruginosa competent cells were prepared by standard methods, recovered in LB, and plated to selective medium at 37°C (43).…”

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confidence: 99%

PA5339, a RidA Homolog, Is Required for Full Growth in Pseudomonas aeruginosa

2018

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The Rid protein superfamily (YjgF/YER057c/UK114) is found in all domains of life. The archetypal protein, RidA from , is a deaminase that quenches the reactive metabolite 2-aminoacrylate (2AA). 2AA deaminase activity is conserved in RidA proteins from humans, plants, yeast, archaea, and bacteria. Mutants of, , and that lack a functional RidA exhibit growth defects, suggesting that 2AA metabolic stress is similarly conserved. The PubSEED database shows (PAO1) encodes eight members of the Rid superfamily. Mutants of PAO1 lacking each of five Rid proteins were screened, and the mutant phenotypes that arose in the absence of PA5339 were dissected. A PA5339::Tn mutant has growth, motility, and biofilm defects that can all be linked to the accumulation of 2AA. Further, the PA5339 protein was demonstrably a 2AA deaminase and restored metabolic balance to a mutant The data presented here show that the RidA paradigm in had similarities to those described in other organisms but was distinct in that deleting only one of multiple homologs generated deficiencies. Based on the collective data presented here in, PA5339 was renamed RidA. RidA is a widely conserved protein that prevents endogenous metabolic stress caused by 2-aminoacrylate (2AA) damage to pyridoxal 5'-phosphate (PLP)-dependent enzymes in prokaryotes and eukaryotes. The framework for understanding the accumulation of 2AA and its consequences have largely been defined in We show here that in (PAO1), 2AA accumulation leads to reduced growth, compromised motility, and defective biofilm formation. This study expands our knowledge how the metabolic architecture of an organism contributes to the consequences of 2AA inactivation of PLP-dependent enzymes and identifies a key RidA protein in .

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confidence: 99%

PA5339, a RidA Homolog, Is Required for Full Growth in Pseudomonas aeruginosa

2018

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“…All plasmids were constructed using described protocols 34, 35 . Listeria monocytogenes 10403S eutT + and Salmonella enterica eutT + genes were amplified from genomic DNA and codon-optimized synthesized DNA (GeneScript) and cloned into the NcoI and SalI sites of vector pTAC-85 35 .…”

Section: Methodsmentioning

confidence: 99%

“…L.m. eutT + was also cloned into the expression vector pTEV18 using primers listed in Table S3 and protocols described elsewhere 34 .…”

Section: Methodsmentioning

confidence: 99%

A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity

Costa

Escalante‐Semerena

2018

Biochemistry

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ATP Co(I)rrinoid adenosyltransferases (ACATs) are involved in de novo adenosylcobamide (AdoCba) biosynthesis, and in salvaging complete and incomplete corrinoids from the environment. The ACAT enzyme family is comprised of three classes of structurally and evolutionarily distinct proteins (i.e., CobA, PduO, EutT). The structure of EutT is unknown, and an understanding of its mechanism is incomplete. The Salmonella enterica EutT (SeEutT) enzyme is the best-characterized member of its class and is known to be a ferroprotein. Here, we report the identification and initial biochemical characterization of an enzyme representative of a new class of EutTs that does not require a metal ion for activity. In vivo and in vitro evidence shows that the metal-less EutT homologue from Listeria monocytogenes (LmEutT) has ACAT activity, and that unlike other ACATs, the biologically active form of LmEutT is a tetramer. In vitro studies revealed that LmEutT was more efficient than SeEutT and displayed positive cooperativity. LmEutT adenosylated cobalamin but not cobinamide, showed specificity for ATP and 2′-deoxyATP, and released a triphosphate by-product. Bioinformatics analyses suggest that metal-less EutT ACATs are also present in other Firmicutes.

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“…Plasmid pTEV16 (22), which directs the synthesis of the protein with a cleavable N-terminal hexahistidine tag, was used for overexpression. The resulting plasmids are referred to as pDR_1057-8, pDR_1182-1, pGK0593-3, and pGK2920-1.…”

Section: Methodsmentioning

confidence: 99%

“…For complementation purposes, each gene of interest was cloned into the L-(ϩ)-arabinose-inducible vector pCV1 (22). The names and relevant genotypes of the resulting plasmids are shown in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Phosphinothricin Acetyltransferases Identified Using In Vivo , In Vitro , and Bioinformatic Analyses

VanDrisse

Hentchel

Escalante‐Semerena

2016

Appl Environ Microbiol

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Acetylation of small molecules is widespread in nature, and in some cases, cells use this process to detoxify harmful chemicals. Streptomyces species utilize a Gcn5 N-acetyltransferase (GNAT), known as Bar, to acetylate and detoxify a self-produced toxin, phosphinothricin (PPT), a glutamate analogue. Bar homologues, such as MddA from Salmonella enterica, acetylate methionine analogues such as methionine sulfoximine (MSX) and methionine sulfone (MSO), but not PPT, even though Bar homologues are annotated as PPT acetyltransferases. S. enterica was used as a heterologous host to determine whether or not putative PPT acetyltransferases from various sources could acetylate PPT, MSX, and MSO. In vitro and in vivo analyses identified substrates acetylated by putative PPT acetyltransferases from Deinococcus radiodurans (DR_1057 and DR_1182) and Geobacillus kaustophilus (GK0593 and GK2920). In vivo, synthesis of DR_1182, GK0593, and GK2920 blocked the inhibitory effects of PPT, MSX, and MSO. In contrast, DR_1057 did not detoxify any of the above substrates. Results of in vitro studies were consistent with the in vivo results. In addition, phylogenetic analyses were used to predict the functionality of annotated PPT acetyltransferases in Burkholderia xenovorans, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baylyi, and Escherichia coli. IMPORTANCEThe work reported here provides an example of the use of a heterologous system for the identification of enzyme function. Many members of this superfamily of proteins do not have a known function, or it has been annotated solely on the basis of sequence homology to previously characterized enzymes. The critical role of Gcn5 N-acetyltransferases (GNATs) in the modulation of central metabolic processes, and in controlling metabolic stress, necessitates approaches that can reveal their physiological role. The combination of in vivo, in vitro, and bioinformatics approaches reported here identified GNATs that can acetylate and detoxify phosphinothricin.

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New high-cloning-efficiency vectors for complementation studies and recombinant protein overproduction in Escherichia coli and Salmonella enterica

Cited by 39 publications

References 20 publications

PA5339, a RidA Homolog, Is Required for Full Growth in Pseudomonas aeruginosa

PA5339, a RidA Homolog, Is Required for Full Growth in Pseudomonas aeruginosa

A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity

Phosphinothricin Acetyltransferases Identified Using In Vivo , In Vitro , and Bioinformatic Analyses

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