Analysis of gene control signals by DNA fusion and cloning in Escherichia coli

Casadaban, Malcolm J.; Cohen, Stanley N.

doi:10.1016/0022-2836(80)90283-1

Cited by 2,419 publications

(747 citation statements)

References 42 publications

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“…S. Typhimurium SL5338 (15), a strain deficient in all three restriction systems, was the mediator to transform plasmid DNA into strain S. Typhimurium SL1344 (16), used for all the in vitro experiments. The Escherichia coli strains used were DH5␣ (17) and MC1061 (18) and clinical isolate NF7 (19). Genes bla and bla VIM-2 were obtained from Acinetobacter baumannii strain 39AT (20) and Pseudomonas aeruginosa strain I42, respectively (21); the latter strain was kindly provided by Juan Ayala (Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Spain).…”

Section: Methodsmentioning

confidence: 99%

Synthesis of Metallo-β-Lactamase VIM-2 Is Associated with a Fitness Reduction in Salmonella enterica Serovar Typhimurium

Cordeiro

Chabalgoity

Yim

et al. 2014

Antimicrob Agents Chemother

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Antibiotic resistance, especially due to ␤-lactamases, has become one of the main obstacles in the correct treatment of Salmonella infections; furthermore, antibiotic resistance determines a gain of function that may encompass a biological cost, or fitness reduction, to the resistant bacteria. The aim of this work was to determine in vitro if the production of the class B ␤-lactamase VIM-2 determined a fitness cost for Salmonella enterica serovar Typhimurium. To that end the gene bla VIM-2 was cloned into the virulent strain S. Typhimurium SL1344, using both the tightly regulated pBAD22 vector and the natural plasmid pST12, for inducible and constitutive expression, respectively. Fitness studies were performed by means of motility, growth rate, invasiveness in epithelial cells, and plasmid stability. The expression of bla VIM-2 was accompanied by alterations in micro-and macroscopic morphology and reduced growth rate and motility, as well as diminished invasiveness in epithelial cells. These results suggest that VIM-2 production entails a substantial fitness cost for S. Typhimurium, which in turn may account for the extremely low number of reports of metallo-␤-lactamase-producing Salmonella spp.

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Section: Methodsmentioning

confidence: 99%

Synthesis of Metallo-β-Lactamase VIM-2 Is Associated with a Fitness Reduction in Salmonella enterica Serovar Typhimurium

Cordeiro

Chabalgoity

Yim

et al. 2014

Antimicrob Agents Chemother

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“…Murray, ED8641 (recA56 hsdR hsdMk A(trpE-B)9 trpR) from Prof. W.J. Brammar, and MC1000 (araD139 A(ara-leu)7697 AlacX74 galU galK strA) from Dr. M.J. Casadaban (9). The plasmid vector used, was pMC81, also from Dr. Casadaban (9).…”

Section: Methodsmentioning

confidence: 99%

“…In particular, when general RNA synthesis in E.coli is partially blocked by the inhibitor of initiation, rifampicin, rpoBC-transcription is paradoxically stimulated, while transcription of the rpl genes is only weakly affected (8, and other papers cited in 5). We have attempted to define the transcriptional signal(s) mediating stimulation of rpoBC expression by rifampicin, by fusing the strong shared promoters of the rplKAJL and rpo genes (PLll and/or PLlO) to lacZ in plasmids derived from pMC81 (9). We have similarly fused to lacZ the weak promoter (P) and the partial terminator or "attenuator" (tl) which lie between the rpl and rpo genes in the rpoBC operon.…”

Section: Introductionmentioning

confidence: 99%

Effect of rifampicin on expression oflacZfused to promoters or terminators of theE.coli rpoBCoperon

et al. 1982

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The genes encoding the f and a' subunits of RNA polymerase in E.coli lie downstream of at least two ribosomal protein genes in a single unit of transcription. Treatment of E.coli with rifampicin, under conditions producing partial inhibition of general RNA synthesis, can strongly stimulate transcription of the polymerase genes, while activating the neighbouring ribosomal genes only slightly. We have investigated the mechanism of this effect by fusing strong promoters, a weak internal promoter, and an attenuator of the polymerase operon to the lacZ gene, in derivatives of plasmid pMC81. Studies of these fusions confirm our conclusion, based on similar fusions to galK, that rifampicin can foster readthrough of transcriptional terminators. They also suggest the existence of extra terminatois and anti-termination elements in the above transcription unit.

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“…The plasmid vector used here was pMC1396 which lost eight codons of the NH, terminus of the P-galactosidase gene of lactose operon [12]. Therefore, the incomplete lacZ gene, l a Z , is not expressed until a foreign DNA fragment containing the promoter region and the 5'-coding sequence is inserted just before IacZ in the same reading frame (Fig.…”

Section: Construction Of Gene Fusionsmentioning

confidence: 99%

“…W3110 was of our fabolatory stock, and MCIOOO [araDl39, A(ara, leu)7697, AlacX74, gulU, galK, rpsL] was sent from M. J. Casadaban [12]. The plasmids used wer ColEZ, pMC1396 [I31 and pACYC177 [14].…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

Factors necessary for the export process of colicin E1 across cytoplasmic membrane of Escherichia coli

Yamada

Nakazawa

1984

Eur J Biochem

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Factors necessary for the export process of colicin E1 across the cytoplasmic membrane of Escherichia coli were investigated. β‐Galactosidase activities from gene fusions between the colicin E1 and lacZ genes were recovered in the inner membrane fraction of E. coli when the region containing the internal signal‐like sequence of colicin E1 [M. Yamada et al. (1982) Proc. Natl Acad. Sci. USA 79, 2827–2831] was present, but were found in the soluble fraction when the region was eliminated. The colicin E1 export was reduced upon insertion mutation in a gene that is located downstream from the colicin E1 gene in the same operon and responsible for mitomycin‐C‐induced killing of the host cell. A frame shift mutation of the colicin E1 plasmid was constructed to direct the protein which had lost the COOH‐terminal 13 residues of original colicin E1 and was altered in 6 residues of the new COOH‐terminal portion. The aberrant colicin E1 that was inducibly synthesized remained inside the cells. These results indicate that colicin E1 is exported with the aid of a product of the downstream gene and that the COOH‐terminal portion is necessary for the export. The binding of colicin E1 to the cytoplasmic membrane through the internal signal‐like sequence may be a step in the protein export process.

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Analysis of gene control signals by DNA fusion and cloning in Escherichia coli

Cited by 2,419 publications

References 42 publications

Synthesis of Metallo-β-Lactamase VIM-2 Is Associated with a Fitness Reduction in Salmonella enterica Serovar Typhimurium

Synthesis of Metallo-β-Lactamase VIM-2 Is Associated with a Fitness Reduction in Salmonella enterica Serovar Typhimurium

Effect of rifampicin on expression oflacZfused to promoters or terminators of theE.coli rpoBCoperon

Factors necessary for the export process of colicin E1 across cytoplasmic membrane of Escherichia coli

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