Efficient expression of a promoter-controlled gene: Tandem promoters of lambda PR and PL functional in enteric bacteria

Murooka, Yoshikatsu; Mitani, Isao

doi:10.1016/0168-1656(85)90032-x

Cited by 17 publications

(5 citation statements)

References 15 publications

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“…confirmed that both promoters are able to direct gene expression in a range of gram-negative bacteria. Our results also confirmed reported findings that expression from PL and PR is tightly regulated by cI857 in Klebsiella and Serratia species and the natural host E. coli (25,28). We found, however, that uninduced C230 activity in both pL-xylE-c1857 and PR-XYIE-cI857 systems was much higher in Pseudomonas and Acinetobacter hosts than in enteric bacteria, probably because of Pseudomonas promoter regions associated with xylE on the constructs.…”

Section: Discussionsupporting

confidence: 92%

Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system

Winstanley

Morgan

Pickup

et al. 1989

Appl Environ Microbiol

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Plasmid systems with unique markers were constructed to assess the fate of recombinant DNA and genetically manipulated bacteria in soil and freshwater model environments. On such constructs the marker gene, xylE (for catechol 2,3-dioxygenase), is expressed from the lambda promoter PL or PR, each of which is controlled by the temperature-sensitive lambda repressor c1857. Combinations of these elements were cloned into the broad-host-range plasmid pKT230 to form pLV1010 (PL-xylE), pLV1011 (PL-xylE-cI857), and pLV1013 (PR-xylE-cI857). The recombinant plasmids were introduced into different gram-negative bacteria. The thermoregulated system of pLV1013 functioned well in a range of species, with xylE induction being readily achieved by elevation of the temperature from 28 to 3rC. There was a difference in the induction of catechol 2,3-dioxygenase activity, depending on whether xylE was expressed from PL (pLV1011) or PR (pLV1013). Our observations on testing the different systems in a number of hosts suggest that genes carried by the DNA of genetically engineered microorganisms may not be expressed in a predictable manner following transfer from the release host to other species.

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Section: Discussionsupporting

confidence: 92%

Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system

Winstanley

Morgan

Pickup

et al. 1989

Appl Environ Microbiol

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“…We did not investigate which of these factors contribute to the observed differences between the genera. In this respect, notice that relative promoter strength seems to differ even among different E. coli strains (29). Because several strains of Erwinia are known to be sensitive to the mutator phage Mu (15), it is perhaps not surprising that the ner ribosome binding site that is used to direct the synthesis of IL-2 is functional in this strain.…”

Section: Discussionmentioning

confidence: 99%

“…To these we hereby add E. chrysanthemi and E. carotovora. Murooka and Mitani (29) used a vector based on a ColEl-derived replicon that has a limited host range. The vector presented here is derived from an RSF1010 replicon and should have retained the property of replicating in a wide variety of gram-negative hosts, including Pseudomonas, Acetobacter, Rhizobium, Agrobacterium, and other species (2).…”

Section: Discussionmentioning

confidence: 99%

A broad-host-range expression vector based on the pL promoter of coliphage lambda: regulated synthesis of human interleukin 2 in Erwinia and Serratia species

Leemans¹,

Remaut²,

Fiers³

1987

J Bacteriol

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We report the construction of a broad-host-range expression vector based on an RSF1010-derived replicon.The vector carries the strong leftward promoter (pL) of coliphage A as well as the c1857 allele, which codes for a thermolabile repressor protein. The coding region of mature human interleukin 2, which is preceded by the ner ribosome binding site of phage Mu, was cloned downstream from the pL promoter. The plasmid was introduced into Erwinia and Serratia species by means of mobilization. Heat-inducible synthesis of interleukin 2 protein was obtained, showing that the pL promoter is functional in these genera. As in Escherichia coli, the bulk of the overproduced protein was present in an insoluble form.In recent years much attention has been paid to the development of gene cloning and expression systems in bacteria other than the widely used Escherichia coli (13,22,35). Many gram-negative bacteria, some of which are of medical or agricuiLural importance (27, 38), possess a broad diversity of metabolic activities and might therefore be promising hosts for cloning and expression studies. Indeed, some genes can only be functionally expressed in a particular physiological background, e.g., when an enzyme is integrated into a specific biochemical pathway (21, 33) or when the base composition of the foreign gene differs widely from that of the host genome. It has been suggested that the weak expression of Pseudomonas genes in E. coli may be attributed to the unusual codon preference of Pseudomonas species (28). Therefore, there is a need for expression vectors that are adapted to the host of interest. The most versatile of these are likely to be based on broad-host-range plasmids, which can be stably inherited over a wide range of gram-negative species (2).We describe the cloning of the strong leftward promoter (PL) of coliphage X, together with the cI repressor gene, onto a derivative of the broad-host-range plasmid RSF1010 (19). This plasmid was introduced into three other enteric bacteria: Serratia marcescens, Erwinia chrysanthemi, and Erwinia carotovora subsp. carotovora. By obtaining controlled expression of cloned human interleukin 2 (IL-2), we demonstrate that the PL promoter is functional in these organisms and that its activity is regulated by the cI repressor as in the natural host E. coli.MATERUILS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Tables 1 and 2. Unless otherwise mentioned, bacterial cultures were grown at 28°C in LB medium (1% tryptone [Difco Laboratories, Detroit, Mich.], 0.5% yeast extract [Difco], 0.5% NaCl). Antibiotics were used at the following concentrations: kanamycin, 50 ,ug/ml; carbenicillin, 100 ,ug/ml; tetracycline, 10 ,Lg/ml. * Corresponding author.Transformation and conjugation procedures. E. coli strains were transformed as described previously (31). Plasmid DNA was introduced into Serratia and Erwinia species by means of conjugational transfer by using pRK2013 (16) or pRK2073 (26) as mobilizing plasmids. Conjuga...

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“…2, Table 1). Fusion protein synthesis was regulated by the Pl~oUv~ and p~ promoter (Murooka and Mitani 1985), which can be induced by adding isopropyl-fl-D-thiogalactopyranoside (IPTG) to the medium or by a temperature shift, respectively. Besides this, the system contained a second plasmid, pEcoR4 or pLBM4420 (Bougueleret et al 1985), the aim of which was to reduce the toxicity of overproduced endonuclease by constitutive expression of the corresponding methylase.…”

Section: Methodsmentioning

confidence: 99%

Continuous production of restriction endonucleases: continuous two-stage cultivation with E. coli JM103; continuous cell disintegration and purification by affinity chromatography

Beer

Maschke

Schügerl

1992

Appl Microbiol Biotechnol

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The optimization of the production of recombinant DNA-derived proteins in Escherichia coli was investigated. We chose restriction endonucleases EcoRI and EcoRV from E. coli as model proteins, despite the observation that overproduction can result in a toxic effect to the cells. The enzymes were expressed as fusion proteins consisting of protein A from Staphylococcus aureus and the desired enzyme in order to facilitate purification. The expression of the fusion protein was induced by a temperature shift using the pR promoter of phage lambda regulated by the repressor plasmid pRK248cI. Data from batch fermentations provided the basis for planning a continuous two-stage fermentation. The EcoRI enzyme activity was investigated as a function of the induction time after cell disintegration and allowed an estimation of yield of the continuous culture. Plasmid instability, which was only observed under continuous conditions, could be prevented by adding tetracycline (resistance of the repressor plasmid) to the medium. We established a continuous cell disintegration system and purified the fusion protein semicontinuously by affinity chromatography. The biological activity of the fusion protein was the same as the native endonuclease so there was no need for cleavage of the fusion protein and the product could be used without further processing.

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Efficient expression of a promoter-controlled gene: Tandem promoters of lambda PR and PL functional in enteric bacteria

Cited by 17 publications

References 15 publications

Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system

Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system

A broad-host-range expression vector based on the pL promoter of coliphage lambda: regulated synthesis of human interleukin 2 in Erwinia and Serratia species

Continuous production of restriction endonucleases: continuous two-stage cultivation with E. coli JM103; continuous cell disintegration and purification by affinity chromatography

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