High efficiency transformation by electroporation of
            <i>Pichia pastoris</i>
            pretreated with lithium acetate and dithiothreitol

Wu, Shuqi; Letchworth, Geoffrey J.

doi:10.2144/04361dd02

Cited by 309 publications

(171 citation statements)

References 8 publications

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“…This is at least 100-fold-higher efficiency than previous reports, most of which reported using 5 to 10 g plasmid DNA and recovering Ͻ100 CFU (and in most cases Ͻ10 CFU) in any T. denticola host strain (2,4,5). While a linear relationship between the amount of input DNA and the efficiency of transformation by electroporation is reported for E. coli (23), we and others have found an inverse relationship between input DNA amount and transformation efficiency in other microbes (24,32). Using the modified protocol described here, we have obtained Ͼ5,000 CFU using as little as 50 ng plasmid DNA.…”

Section: Resultssupporting

(Expert classified)

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

Godovikova

Goetting-Minesky

Shin

et al. 2015

Appl Environ Microbiol

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Oral pathogens, including Treponema denticola, initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles of T. denticola in microbe-host interactions and signaling, microbial communities, microbial physiology, and molecular evolution has been hampered by limitations in genetic methodologies. This is typified by an extremely low transformation efficiency and inability to transform the most widely studied T. denticola strain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems in T. denticola contributed to these problems. To facilitate further molecular genetic analysis of T. denticola behavior, we optimized existing protocols such that shuttle plasmid transformation efficiency was increased by >100-fold over prior reports. Here, we report routine transformation of T. denticola ATCC 35405 with shuttle plasmids, independently of both plasmid methylation status and activity of the type II restriction endonuclease encoded by TDE0911. To validate the utility of this methodological advance, we demonstrated expression and activity in T. denticola of a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to the Treponema toolset will enable more-rigorous and -detailed studies of the behavior of this organism. In vitro studies of Treponema denticola and other oral spirochetes present unique challenges and opportunities in the field of molecular and cellular microbiology. As both commensal residents and pathogens of the subgingival oral mucosa, these nutritionally fastidious anaerobes are useful targets for research into microbehost interactions and signaling, microbial communities, microbial physiology, and molecular evolution. Furthermore, due to both physiological similarities between T. denticola and T. pallidum and the absence of a workable in vitro system to culture T. pallidum, T. denticola is of high interest as a model for the study of the microbial physiology and biosynthetic pathways of T. pallidum.Transformation of T. denticola ATCC 35405 by allelic replacement mutagenesis and by shuttle plasmids was first reported over 15 years ago (1, 2), but subsequent progress in this area continues to be hindered by extremely low transformation efficiency (1, 3). While T. denticola is somewhat tractable for simple gene knockouts, basic methods such as complementation analysis and expression of heterologous genes are problematic. The available shuttle plasmid system has at best an extremely limited ability to replicate in the most widely studied T. denticola strain (3-5). Thus, while plasmid-based methodologies are straightforward in the study of many prokaryotes, such studies present major obstacles to the rigorous molecular genetic analysis of treponemes.The T. denticola 35405 genome sequence reveals a robust DNA restriction-modification repertoire, including two complete type I restriction-modification lo...

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Section: Resultssupporting

(Expert classified)

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

Godovikova

Goetting-Minesky

Shin

et al. 2015

Appl Environ Microbiol

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“…To create the fet3⌬ strain, the fet3::ADE1 cassette was obtained by digestion with BsmI and BglII and electroporated in P. pastoris JC300. Electrocompetent JC300 cells were prepared by the method of Wu and Letchworth (12). White Ade ϩ colonies were plated on MD with or without 20 M BPS, and after 48 h at 30°C, they were scored for lack of growth.…”

Section: Methodsmentioning

confidence: 99%

Role of External Loops of Human Ceruloplasmin in Copper Loading by ATP7B and Ccc2p

Maio

Polticelli

Francesco

et al. 2010

Journal of Biological Chemistry

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Ceruloplasmin is a multicopper oxidase required for correct iron homeostasis.Previously, we have identified a ceruloplasmin mutant associated with the iron overload disease aceruloplasminemia, which was unable to acquire copper from the mammalian pump ATP7B but could be produced in an enzymatically active form in yeast. Here, we report the expression of recombinant ceruloplasmin in the yeast Pichia pastoris and the study of the role of five surface-exposed loops in copper incorporation by comparing the efficiencies of mammalian ATP7B and yeast Ccc2p. The possibility to "mix and match" mammalian and yeast multicopper oxidases and copper ATPases can provide clues on the molecular features underlying the process of copper loading in multicopper oxidases.

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“…A 1094-bp-long PCR fragment was gel-purified and inserted into the BamHI and EcoRI sites of pPIC3.5K plasmid (Invitrogen). The resulting plasmid pPIC3.5KϩPGC1 was transformed into P. pastoris strain GS115 using electroporation protocol according to Wu and Letchworth (38).…”

Section: Methodsmentioning

confidence: 99%

Yeast Pgc1p (YPL206c) Controls the Amount of Phosphatidylglycerol via a Phospholipase C-type Degradation Mechanism

Simockova

Holic̆

Tahotná

et al. 2008

Journal of Biological Chemistry

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The product of the open reading frame YPL206c, Pgc1p, of the yeast Saccharomyces cerevisiae displays homology to bacterial and mammalian glycerophosphodiester phosphodiesterases. Deletion of PGC1 causes an accumulation of the anionic phospholipid, phosphatidylglycerol (PG), especially under conditions of inositol limitation. This PG accumulation was not caused by increased production of phosphatidylglycerol phosphate or by decreased consumption of PG in the formation of cardiolipin, the end product of the pathway. PG accumulation in the pgc1⌬ strain was caused rather by inactivation of the PG degradation pathway. Our data demonstrate an existence of a novel regulatory mechanism in the cardiolipin biosynthetic pathway in which Pgc1p is required for the removal of excess PG via a phospholipase C-type degradation mechanism. Cardiolipin (CL)2 is a major mitochondrial anionic phospholipid with important functions in promoting cell growth, anaerobic metabolism, mitochondrial function, and biogenesis (1, 2). Phosphatidylglycerol is not only a metabolic precursor in the biosynthetic pathway leading to the formation of cardiolipin but itself is an important phospholipid; for example it is the sole phospholipid of thylakoid membranes of prokaryotic and eukaryotic oxygenic photosynthetic organisms (3). In mammals, PG is especially important in pulmonary surfactant, an essential fluid produced by alveolar type II cells that covers the entire surface of the lung (4). Considering the importance of this anionic phospholipid, little is known about the mechanisms by which eukaryotic cells control PG membrane composition.In yeast cells, PG is a low abundance phospholipid, present at best as a few tenths of a percent of total cellular phospholipids, even under the conditions of respiratory growth (5). Therefore, PG is considered to be mainly a metabolic precursor to CL. Biosynthesis of CL starts with common intermediates of phospholipid biosynthesis, phosphatidic acid and CDP-diacylglycerol (DAG) (Fig. 1). Phosphatidylglycerol phosphate (PGP) is formed from CDP-DAG and glycerol 3-phosphate. This step is catalyzed by PGP synthase, product of the PGS1/PEL1 gene (6, 7). PGP is subsequently dephosphorylated to form PG. Finally, CL is synthesized in yeast and other eukaryotic organisms from CDP-DAG and PG via a reaction catalyzed by cardiolipin synthase (CRD1) (5,8,9). It is important to note that both CL and PG are subject to intense remodeling subsequent to their de novo synthesis (10).Mitochondrial phospholipid biosynthetic activities in general respond to factors affecting mitochondrial development and function, such as carbon source, growth phase, availability of oxygen, and mutations affecting mitochondrial development and function (11,12). In fact, both enzymatic activities of the CL biosynthetic pathway, PGP synthase and CL synthase, respond to mitochondrial development factors in such a way as to increase the production of CL when cells switch from fermentative to respiratory growth (13-15). A typical feature of yeast phosphol...

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High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol

Cited by 309 publications

References 8 publications

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

Role of External Loops of Human Ceruloplasmin in Copper Loading by ATP7B and Ccc2p

Yeast Pgc1p (YPL206c) Controls the Amount of Phosphatidylglycerol via a Phospholipase C-type Degradation Mechanism

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