High efficiency transformation of Escherichia coli with plasmids

Inoue, Hiroaki; Nishimura, Hiroshi; Okayama, Hiroto

doi:10.1016/0378-1119(90)90336-p

Cited by 1,896 publications

(1,278 citation statements)

References 18 publications

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“…When it was used for sequencing, DNA was obtained by a modified mini alkaline lysis-polyethylene glycol precipitation procedure outlined in the instructions for a PRISM Ready Reaction DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems). Competent E. coli cells were prepared and transformed as described previously (15). Electrocompetent C. perfringens cells were prepared and transformed as described previously (36).…”

Section: Methodsmentioning

confidence: 99%

The Spatial Organization of the VirR Boxes Is Critical for VirR-Mediated Expression of the Perfringolysin O Gene, pfoA , from Clostridium perfringens

et al. 2004

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The transcriptional regulation of toxin production in the gram-positive anaerobe Clostridium perfringens involves a two-component signal transduction system that comprises the VirS sensor histidine kinase and its cognate response regulator, VirR. Previous studies showed that VirR binds independently to a pair of imperfect direct repeats, now designated VirR box 1 and VirR box 2, located immediately upstream of the promoter of the pfoA gene, which encodes the cholesterol-dependent cytolysin, perfringolysin O. For this study, we introduced mutated VirR boxes into a C. perfringens pfoA mutant and found that both VirR boxes are essential for transcriptional activation. Furthermore, the spacing between the VirR boxes and the distance between the VirR boxes and the ؊35 region are shown to be critical for perfringolysin O production. Other VirR boxes that were previously identified from the strain 13 genome sequence were also analyzed, with perfringolysin O production used as a reporter system. The results showed that placement of the different VirR boxes at the same position upstream of the pfoA promoter yields different levels of perfringolysin O activity. In all of these constructs, VirR was still capable of binding to the target DNA, indicating that DNA binding alone is not sufficient for transcriptional activation. Finally, we show that the C. perfringens RNA polymerase binds more efficiently to the pfoA promoter in the presence of VirR, indicating that interactions must occur between these proteins. We propose that these interactions are required for VirR-mediated transcriptional activation.

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Section: Methodsmentioning

confidence: 99%

The Spatial Organization of the VirR Boxes Is Critical for VirR-Mediated Expression of the Perfringolysin O Gene, pfoA , from Clostridium perfringens

et al. 2004

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“…S. aureus strains (Table 1) (14) was grown in Luria-Bertani medium (Fisher Scientific). Liquid cultures were grown with shaking at 250 rpm and 37°C.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Genetic Elements ControllingStaphylococcus aureus lrgABExpression: Potential Role of DNA Topology in SarA Regulation

2000

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Penicillin-induced killing and murein hydrolase activity in Staphylococcus aureus are dependent on a variety of regulatory elements, including the LytSR two-component regulatory system and the virulence factor regulators Agr and Sar. The LytSR effects on these processes can be explained, in part, by the recent finding that a LytSR-regulated operon, designated lrgAB, affects murein hydrolase activity and penicillin tolerance. To examine the regulation of lrgAB expression in greater detail, we performed Northern blot and promoter fusion analyses. Both methods revealed that Agr and Sar, like LytSR, positively regulate lrgAB expression. A mutation in the agr locus reduced lrgAB expression approximately sixfold, while the sar mutation reduced lrgAB expression to undetectable levels. cis-acting regulatory elements involved in lrgAB expression were identified by fusing various fragments of the lrgAB promoter region to the xylE reporter gene and integrating these constructs into the chromosome. Catechol 2,3-dioxygenase assays identified DNA sequences, including an inverted repeat and intrinsic bend sites, that contribute to maximal lrgAB expression. Confirmation of the importance of the inverted repeat was achieved by demonstrating that multiple copies of the inverted repeat reduced lrgAB promoter activity, presumably by titrating out a positive regulatory factor. The results of this study demonstrate that lrgAB expression responds to a variety of positive regulatory factors and suggest that specific DNA topology requirements are important for optimal expression.Peptidoglycan or murein hydrolases are a family of enzymes that catalyze the cleavage of specific structural components of the bacterial cell wall. These enzymes are involved in several important physiological processes, including peptidoglycan turnover and recycling, cell wall expansion during bacterial growth, septation, and daughter cell separation (13, 32). Due to the potential of these enzymes to compromise cell wall integrity, leading to cell lysis (autolysis), murein hydrolase activity must be carefully regulated. This regulation includes transcriptional control, blocked access to the specific substrate, inhibition by choline or teichoic acid, and substrate modification (13, 32).The Staphylococcus aureus lytS and lytR genes, whose products are members of the two-component regulator family of proteins, are involved in the control of murein hydrolase activity (3). A lytS mutant phenotypically displays altered murein hydrolase activity and an increased rate of penicillin-and Triton X-100-induced lysis (3, 11). Immediately downstream of lytS and lytR are two genes, lrgA and lrgB, whose transcription is dependent upon lytS and lytR. LrgA and LrgB show no sequence similarity to known murein hydrolases. Instead, LrgA has structural characteristics in common with the bacteriophage-encoded holin proteins involved in murein hydrolase export (4, 11). Recent data generated in our laboratory indicate that the LrgA and LrgB gene products inhibit extracellular murein hy...

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“…PCR products were visualised on a gel. The 0.38 kb fragment was recovered with a Qiaquick gel extraction kit (Qiagen), cloned between the NcoI and HindIII sites of pBAD/His (Invitrogen) and transformed into E. coli TOP10 using the method of Inoue et al (22) The construct was checked by sequencing from just upstream of the promoter through to the end of the insert. For the overexpression, starter cultures grown overnight in LB supplemented with ampicillin were inoculated at 1 % (v/v) into 500 ml LB in 2 L baffled flasks supplemented with ampicillin, 200 μM δ-aminolevulinic acid, and 12 μM FeCl 3 .…”

Section: Cloning and Expression Of C Jejuni Trhb In E Colimentioning

confidence: 99%

Purification and Spectroscopic Characterization of Ctb, a Group III Truncated Hemoglobin Implicated in Oxygen Metabolism in the Food-Borne Pathogen Campylobacter jejuni

et al. 2006

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Campylobacter jejuni is a foodborne bacterial pathogen that possesses two distinct hemoglobins, encoded by the ctb and cgb genes. The former codes for a truncated hemoglobin (Ctb) in group III, an assemblage of uncharacterized globins in diverse clinically-and technologically-significant bacteria. Here, we show that Ctb purifies as a monomeric, predominantly oxygenated species. Optical spectra of ferric, ferrous, O 2 -and CO-bound forms resemble those of other hemoglobins. However, resonance Raman analysis shows Ctb to have an atypical ν Fe-CO stretching mode at 514 cm -1 , compared to the other truncated hemoglobins that have been characterized so far. This implies unique roles in ligand stabilisation for TyrB10, HisE7 and TrpG8, residues highly conserved within group III truncated hemoglobins. Since C. jejuni is a microaerophile, and a ctb mutant exhibits O 2 -dependent growth defects, one of the hypothesised roles of Ctb is in the detoxification, sequestration or transfer of O 2 The midpoint potential (E h ) of Ctb was found to be −33 mV, but no evidence was obtained in vitro to support the hypothesis that Ctb is reducible by NADH or NADPH. This truncated hemoglobin may function in the facilitation of O 2 transfer to one of the terminal oxidases of C. jejuni or instead facilitate O 2 transfer to Cgb for NO detoxification.In the past three decades, our understanding of hemoglobins has grown to recognise new classes of proteins in addition to the classical vertebrate α-and β-globins, myoglobin and the symbiotic Hbs of leguminous plants. These 'new' globins -some of which may actually be amongst the oldest, or ancestral, globins in a phylogenetic context (1) -include the non-symbiotic plant Hbs, chimeric flavohemoglobins, and the trHbs. Globins may be classified functionally -into those that detoxify NO and those that are involved in O 2 transfer or detoxification -or structurally into three categories (2). The first structural category contains flavoHbs that possess a C-terminal ferredoxin-NADP + reductase-like domain and an N-terminal globin domain; bacterial (3,4) and yeast (5) examples function in the enzymic removal of NO to produce nitrate (6,7). The second category contains single domain Hbs that are very similar to the N-terminal domain of the flavoHbs; however, their functions appear more varied than those of the flavoHbs. As an example, Vgb, found in the obligate aerobe Vitreoscilla, is believed to function in the facilitation of O 2 transfer to cytochrome bo' (8), whereas the C.

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High efficiency transformation of Escherichia coli with plasmids

Cited by 1,896 publications

References 18 publications

The Spatial Organization of the VirR Boxes Is Critical for VirR-Mediated Expression of the Perfringolysin O Gene, pfoA , from Clostridium perfringens

The Spatial Organization of the VirR Boxes Is Critical for VirR-Mediated Expression of the Perfringolysin O Gene, pfoA , from Clostridium perfringens

Analysis of Genetic Elements ControllingStaphylococcus aureus lrgABExpression: Potential Role of DNA Topology in SarA Regulation

Purification and Spectroscopic Characterization of Ctb, a Group III Truncated Hemoglobin Implicated in Oxygen Metabolism in the Food-Borne Pathogen Campylobacter jejuni

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