Simple method for identification of plasmid-coded proteins

Sancar, Aziz; Hack, Adelle M.; Rupp, W. Dean

doi:10.1128/jb.137.1.692-693.1979

Cited by 1,040 publications

(254 citation statements)

References 10 publications

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“…Analysis of the polypeptides encoded by the plasmid pTK806-1 and its deletion derivatives was by the maxicell technique (C). E.coli XL1 cells harboring the respective plasmids were converted to maxicells according to Sancar et al (1979). After labelling with L-[35S]methionine the polypeptides were characterized by 0.1 % SDS-12% PAGE (Laemmli, 1970), and the labelled proteins were visualized by fluorography of the stained and dried gel.…”

Section: Resultsmentioning

confidence: 99%

A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora.

et al. 1993

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Virulence of the plant pathogen Erwinia carotovora subsp. carotovora is dependent on the production and secretion of a complex arsenal of plant cell wall‐degrading enzymes. Production of these exoenzymes is controlled by a global regulatory mechanism. A virulent mutants in one of the regulatory loci, expI, show a pleiotropic defect in the growth phase‐dependent transcriptional activation of exoenzyme gene expression. The expI gene encodes a 26 kDa polypeptide that is structurally and functionally related to the luxI gene product of Vibrio fischeri. Functional similarity of expI and luxI has been demonstrated by reciprocal genetic complementation experiments. LuxI controls bioluminescence in V.fischeri in a growth phase‐dependent manner by directing the synthesis of the diffusible autoinducer, N‐(3‐oxohexanoyl) homoserine lactone. E.c. subsp. carotovora expI+ strains or Escherichia coli harboring the cloned expI gene excrete a small diffusible signal molecule that complements the expI mutation of Erwinia as well as a luxI mutation of V.fischeri. This extracellular complementation can also be achieved by E.coli harboring the luxI gene from V.fischeri or by adding the synthetic V.fischeri autoinducer. Both the production of the plant tissue‐macerating exoenzymes and the ability of the bacteria to propagate in planta are restored in expI mutants by autoinducer addition. These data suggest that the same signal molecule is employed in control of such diverse processes as virulence in a plant pathogen and bioluminescence in a marine bacterium, and may represent a general mechanism by which bacteria modulate gene expression in response to changing environmental conditions.

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Section: Resultsmentioning

confidence: 99%

A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora.

et al. 1993

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“…This suggests that the permease is making use of the existing proton gradient in accordance with the chemiosmotic theory of Mitchell (1970). In Escherichia coli, it was found that the uracil permease gene of similar plasmids is transcribed into RNA, but no translation product was detected in the maxicell system described by Sancar et al (1979). Therefore, we could not test the functioning of the eucaryotic permease in a procaryotic membrane.…”

Section: Discussionmentioning

confidence: 94%

Expression of the cloned uracil permease gene of Saccharomyces cerevisiae in a heterologous membrane.

Chevallier¹,

Lacroute²

1982

The EMBO Journal

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A piece of DNA of the yeast Saccharomyces cerevisiae complementing the uracil permease gene was introduced into a plasmid able to replicate autonomously in Schizosaccharomyces pombe. A strain of S. pombe lacking uracil transport activity was transformed with this new plasmid carrying the gene of S. cerevisiae. The behaviour of the transformant shows not only an expression of the uracil permease gene in the heterologous membrane but also that the transport of uracil is active and coupled to the energy furnishing system of the heterologous host.

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“…In ASA-dehydrogenase, Cys135 might play a similar role, even though the amino-acid sequences around the reactive cysteine residue in GPDH and ASA-dehydrogenase show no homology. Expression of the asd gene is very efficient in the cell: the protein can already be detected in crude extracts after electrophoresis in polyacrylamide gels (using a deletion strain as a control; data not shown); using the maxicell technique with strain CSR603 (Sancar et al, 1979) harboring pAD20, the band corresponding to ASA-dehydrogenase is even more intense that the one due to ,3-lactamase (data not shown). This high level of expression can be accounted for by the following observations obtained from the sequence data: (1) a preferential use of the frequent codons as discussed above; (ii) a convenient sequence of ribosome binding, AGGA, though somewhat distant (11 bp) from the initiation ATG; (iii) the existence, at positon 168, of a sequence TATGGTG very similar to the consensus Pribnow box, preceded by a TTG region, in good agreement with the canonical -35 sequence (Rosenberg and Court, 1980).…”

Section: Discussionmentioning

confidence: 99%

Nucleotide sequence of the asd gene of Escherichia coli: absence of a typical attenuation signal.

Haziza¹,

Stragier²,

Patte³

1982

The EMBO Journal

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The asd gene of escherichia coli encodes aspartic semialdehyde dehydrogenase, an enzyme involved in lysine, threonine, and methionine biosynthesis; its synthesis is controlled by a multivalent repression mechanism. It was cloned in plasmid pBR322 and its complete nucleotide sequence determined. The sequence predicts a polypeptide chain of 367 amino acids, in good agreement with results obtained for the purified protein (Biellmann et al., 1980a). Our data indicate a Cys residue instead of a His residue, which was proposed after covalent labeling of the active center of the enzyme; this is more in line with the catalytic site of glyceraldehyde‐3‐phosphate dehydrogenase, an enzyme which carries out a similar reaction. The nucleotide sequence that precedes the translational start does not display any of the characteristic features of an attenuation signal. Hence the expression of the asd gene is probably not controlled in the same way as other multivalently repressed operons such as ilva and thr.

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Simple method for identification of plasmid-coded proteins

Abstract: Proteins encoded by plasmid DNA are specifically labeled in UV-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA.

Cited by 1,040 publications

References 10 publications

A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora.

A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora.

Expression of the cloned uracil permease gene of Saccharomyces cerevisiae in a heterologous membrane.

Nucleotide sequence of the asd gene of Escherichia coli: absence of a typical attenuation signal.

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