Transcription initiation sites within an IS<i>2</i>insertion in a Gal-constitutive mutant of<i>Escherichia coli</i>

Hinton, Deborah M.; Musso, Richard E.

doi:10.1093/nar/10.16.5015

Cited by 25 publications

(8 citation statements)

References 36 publications

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“…This implies that induction of transcription by leucine at the tdh promoter is not an activation event but involves the loss of repression. (1,22,29,38,46). In this paper we have shown that IS3 activates the tdh operon of E. coli by this mechanism.…”

Section: Methodsmentioning

confidence: 68%

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Activation of a cryptic pathway for threonine metabolism via specific IS3-mediated alteration of promoter structure in Escherichia coli

1989

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The tdh operon of Escherichia coli consists of two genes whose products catalyze sequential steps in the formation of glycine and acetyl coenzyme A from threonine. The operation of the tdh pathway can potentially confer at least two capabilities on the cell: the first is to provide a biosynthetic source of glycine, serine, or both that is an alternative to the conventional (triose phosphate) pathway; the second is to enable cells to utilize threonine as the sole carbon source. The latter capability is referred to as the Tuc+ phenotype. In wild-type E. coli, the tdh operon is expressed at levels that are too low to bestow the Tuc+ phenotype, even in leucine-supplemented media, where the operon is induced eightfold. In eight Tuc+ mutants, the expression of the tdh operon was elevated 100-fold relative to the uninduced wild-type operon. The physical state of the DNA at the tdh locus in these Tuc+ strains was analyzed by Southern blotting and by DNA sequencing. In eight independent isolates the mobile genetic element IS3 was found to lie within the tdh promoter region in identical orientations. In six cases that were examined by DNA sequencing, IS3 occupied identical sites between the -10 and -35 elements of the tdh promoter. The transcription start points for the wild-type tdh promoter and one IS3-activated tdh promoter were identical. In effect, the repeatedly observed transposition event juxtaposed an IS3-borne -35 region and the tdh-specific -10 region, generating a hybrid promoter whose utilization led to elevated, constitutive expression of the tdh operon. This is the first case of promoter activation by IS3 where the site of transcription initiation is unaltered.In Escherichia coli, the conversion of threonine to other metabolites can be initiated by at least three enzymes.

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Section: Methodsmentioning

confidence: 68%

“…P-Galactosidase was determined as described (32 (11,48,55). The (22,29,38,46). In such cases, the -10 hexamer is derived from the target DNA into which the IS element inserts, while the -35 hexamer is part of the inverted repeat sequence at the terminus of the IS element.…”

Section: Methodsmentioning

confidence: 99%

Activation of a cryptic pathway for threonine metabolism via specific IS3-mediated alteration of promoter structure in Escherichia coli

1989

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“…This study is the first demonstration of the activation of gene expression by transposable elements in a Pseudomonas species; however, this phenomenon has been well documented in both eucaryotes (15,23,31,35) and E. coli (4,5,8,16,17,20,27,29,36). Studies on E. coli have suggested mechanisms for the activation of gene expression by transposable elements.…”

Section: Discussionmentioning

confidence: 85%

Identification of transposable elements which activate gene expression in Pseudomonas cepacia

Scordilis¹,

Ree²,

Lessie³

1987

J Bacteriol

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This study demonstrated that transposable elements in Pseudomonas cepacia could be inserted upstream of a poorly expressed gene and increase its expression more than 30-fold. Five elements, TnPcl, IS402, IS403, IS404, and IS405, were isolated by their ability to increase expression of the P-lactamase gene of the broad-host-range plasmid pRP1. Increased expression resulted only from insertion of these elements, suggesting that insertional activation is an important means of elevating gene expression in this organism. Four of the elements inserted between a Pstl site within the ,B-lactamase gene and a BamHI site located 375 base pairs upstream of its promoter. The element IS403 inserted distal to the BamHI site within the coding region for the gene tnpR, suggesting that insertional activation can act over greater than expected distances. In addition, the element IS402 activated the P-lactamase genes carried on plasmids pRP1 and pMR5 (temperature-sensitive pRP1) equally well in opposite orientations, demonstrating that insertional activation by this element occurs independent of its orientation.

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“…When the fucosepositive pseudorevertants, ECL56 (from strain ECL3) and ECL459 (from strain ECL421), were probed for the presence J. BACTERIOL. of IS5 in the HindIII1-HindIII2 region, the insertion element DNA and a section of inserted DNA (19,22,23). In the case was still there (Fig.…”

Section: Resultsmentioning

confidence: 96%

Constitutive activation of the fucAO operon and silencing of the divergently transcribed fucPIK operon by an IS5 element in Escherichia coli mutants selected for growth on L-1,2-propanediol

1989

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L-1,2-Propanediol is an irretrievable end product of L-fucose fermentation by Escherichia coli. Selection for increased aerobic growth rate on propanediol results in the escalation of basal synthesis of the NAD+-linked oxidoreductase encoded by fucO, a member of thefuc regulon for the utilization of L-fucose. In general, when fucO becomes constitutively expressed, two other simultaneous changes occur: the fucA gene encoding fuculose-1-phosphate aldolase becomes constitutively expressed and the fucPIK operon encoding fucose permease, fucose isomerase, and fuculose kinase becomes noninducible. In the present study, we show that fucO and fucA form an operon which is divergently transcribed from the adjacent fucPIK operon. In propanediol-positive and fucose-negative mutants the cis-controliing region shared by the operonsfucAO and fucPIK is lengthened by 1.2 kilobases. DNA hybridization identified the insertion element to be IS5. This element, always oriented in the same direction with the left end (the BglII end) proximal to fucA, apparently causes constitutive expression offucAO and noninducibility of fucPIK. The DNA of the fucAO operon and a part of the adjacent fucP was sequenced.Following the discovery that a genetic derepression of ribitol dehydrogenase in Klebsiella pneumoniae conferred a growth ability on xylitol (27, 33), we tried to find other models of experimental evolution for assessing the importance of regulatory mutations in the acquisition of novel functions (for a review, see reference 30). During this search, we discovered that Escherichia coli can give rise to mutants that utilize L-1,2-propanediol aerobically as a sole source of carbon and energy. Serial selection on the compound resulted in the emergence of a mutant (ECL3) which grew on the novel carbon and energy source at a rate close to that on glycerol. This mutant produced constitutively an NAD+-linked oxidoreductase active on L-1,2-propanediol (46). The proximity of the locus specifying this enzyme activity and the fuc locus (at min 60) specifying the growth ability on L-fucose led us to ask whether there was a link between the metabolism of the two compounds. When the propanediol-positive mutant was tested with fucose as a carbon and energy source, growth no longer occurred (13).Previous work, together with our further studies (13), revealed the biochemical connection between the two metabolic pathways (Fig. 1). The dissimilation of fucose by wild-type E. coli requires the sequential action of fucose permease (encoded byfucP), fucose isomerase (encoded by fucf), fucose kinase (encoded by fucK), and fuculose-lphosphate aldolase (encoded by fucA). The last enzyme catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Under anaerobic conditions, the aldehyde is reduced to L-1,2-propanediol by an NAD+-linked oxidoreductase (encoded by fucO) and excreted into the medium. Under aerobic conditions, the aldehyde is oxidized to Llactate and then to pyruvate which enters the general metabolic pool. The inducer of the fucose syst...

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Transcription initiation sites within an IS2insertion in a Gal-constitutive mutant ofEscherichia coli

Cited by 25 publications

References 36 publications

Activation of a cryptic pathway for threonine metabolism via specific IS3-mediated alteration of promoter structure in Escherichia coli

Activation of a cryptic pathway for threonine metabolism via specific IS3-mediated alteration of promoter structure in Escherichia coli

Identification of transposable elements which activate gene expression in Pseudomonas cepacia

Constitutive activation of the fucAO operon and silencing of the divergently transcribed fucPIK operon by an IS5 element in Escherichia coli mutants selected for growth on L-1,2-propanediol

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