[23] Analysis of recombinant DNA using Escherichia coli minicells

Clark‐Curtiss, Josephine E.; Curtiss, Roy

doi:10.1016/0076-6879(83)01026-5

Cited by 59 publications

(18 citation statements)

References 28 publications

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“…MTAHC, MAAHC Minicell preparation and labeling. Plasmid-specified proteins were synthesized and labeled in minicells (8), using the E. coli P678-54 minicell-producing strain (1). Several steps in the usual procedure were modified as described in Results.…”

Section: Methodsmentioning

confidence: 99%

Sequence of the Pseudomonas aeruginosa trpI activator gene and relatedness of trpI to other procaryotic regulatory genes

1989

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In Pseudomonas aeruginosa, the trpI gene product regulates the expression of the trpBA gene pair encoding tryptophan synthase. trpI and trpBA are transcribed divergently. The trpI DNA sequence and deduced amino acid sequence were determined. The trpI start codon was found to be 103 base pairs from that of trpB. trpI encodes a 293-residue protein and the size of the trpI gene product, measured on sodium dodecyl sulfatepolyacrylamide gels, was close to that calculated from the amino acid sequence. The amino acid sequence of trpI resembles that of Enterobacter cloacae ampR, the regulatory gene for the ampC cephalosporinase. The N-terminal portions of trpI and ampR resemble corresponding portions of ilvY, metR, and lysR in Escherichia coli and nodD in Rhizobium meliloti. This resemblance may help to define a trpI-related family of activator proteins sharing a common structural plan.

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Section: Methodsmentioning

confidence: 99%

Sequence of the Pseudomonas aeruginosa trpI activator gene and relatedness of trpI to other procaryotic regulatory genes

1989

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“…Minicells were purified from 200 ml of a late-log-phase culture (A600 of 1) of the minicell-producing strain X1411 grown in L broth as described by Clark-Curtiss and Curtiss (2 (Fig. 1).…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization of the tdc operon of Escherichia coli K-12

1988

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The nucleotide sequence of a 2-kilobase DNA fragment of the tdc region of Escherichia coli K-12, previously cloned in this laboratory, revealed two open reading frames, tdcC and ORFX, downstream from the tdcB gene (formerly designated tdc) encoding biodegradative threonine dehydratase. A 24-base-pair sequence separated tdcC from the dehydratase coding region, and an untranslated region of 60 nucleotides, which contains a recognizable -10 consensus sequence, was found between tdcC and ORFX. The deduced amino acid sequence of tdcC showed it to be a large hydrophobic polypeptide of 431 amino acid residues, whereas ORFX coded for a small 135-residue polypeptide lacking glutamine and tryptophan. A computer-assisted sequence analysis revealed no similarity among the tdcB, tdcC, and ORFX polypeptides, and a search of the GenBank database failed to detect similarity with any other known proteins. The tdc genes and ORFX showed similar codon usage and, in analogy with other bacterial genes, showed codon usage typical for genes expressed at an intermediate level. Transcriptional analysis with S1 nuclease indicated two distinct transcription start sites upstream of the tdcB gene in regions previously identified as promoterlike elements P1 and P2. Interestingly, expression of tdcB and tdcC, but not ORFX, was contingent upon the presence of P1. These results taken together tend to suggest that the biodegradative threonine dehydratase is the second gene in a polycistronic transcription unit constituting a novel operon (tdcABC) in E. coli implicated in anaerobic threonine metabolism.Biodegradative threonine dehydratase (EC 4.2.1.16) of Escherichia coli catalyzes the pyridoxal phosphate-dependent dehydration of L-threonine and L-serine to ammonia and to a-ketobutyrate and pyruvate, respectively (22). The enzyme is normally induced under anaerobic culture conditions in tryptone-yeast extract medium lacking fermentable carbohydrates. Recently, Hobert and Datta (11) devised a synthetic medium consisting of four amino acids, threonine, serine, valine, and isoleucine, plus cyclic AMP and fumarate, which supported higher levels of dehydratase synthesis as compared with that found in the rich medium with or without cyclic AMP and fumarate. Because the requirements for cyclic AMP and fumarate were absolute in terms of enzyme synthesis, it was proposed that not the amino acids themselves but some metabolite(s) derived from these amino acids during anaerobic metabolism was needed for dehydratase induction. In addition, Merberg and Datta (17) reported that various strains of E. coli with unrelated genotypes produced various levels of the enzyme in the same medium and that mutations at multiple loci on the chromosome influenced enzyme expression in vivo. These cumulative findings led to the notion that a complex network of regulatory systems controls the synthesis of dehydratase in anaerobic culture conditions.To understand the mechanism of dehydratase induction at the DNA level, we cloned a 6.2-kilobase-pair (kb) DNA fragment of E. coli ...

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“…Minicells were isolated from E. coli M2142 containing pUC19‫,ء‬ pES6-1, pV25, or pV108 and labeled with [ 35 S]methionine (0.25 mCi/ml) from New England Nuclear Corp. (10). Samples containing 40,000 trichloroacetic acid-precipitable cpm were analyzed by one-dimensional electrophoresis in SDS-12% (wt/vol) acrylamide PAGE gels (25).…”

Section: Isolation Of Y Pestis Lcrv Deletion Mutantsmentioning

confidence: 99%

Differential effects of deletions in lcrV on secretion of V antigen, regulation of the low-Ca2+ response, and virulence of Yersinia pestis

1995

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The Yersinia pestis V antigen is necessary for full induction of low-calcium response (LCR) stimulon virulence gene transcription, and it also is a secreted protein believed to have a direct antihost function. We made four nonpolar deletions in lcrV of Y. pestis to determine if secretion, regulation, and virulence functions could be localized within the V antigen (LcrV). Deletion of amino acids 25 to 40 caused secretion of LcrV to be decreased in efficiency; however, removal of residues 108 to 125 essentially abolished LcrV secretion. Neither mutation had a significant effect on LCR regulation. This showed that LcrV does not have to be secreted to have its regulatory effect and that the internal structure of V antigen is necessary for its secretion. Both mutants were avirulent in mice, showing that the regulatory effect of LcrV could be separated genetically from its virulence role and raising the possibility that residues 25 to 40 are essential for the virulence function. This study provides the best genetic evidence available that LcrV per se is necessary for the virulence of Y. pestis. The repressed LCR phenotype of a mutant lacking amino acids 188 to 207 of LcrV raised the possibility that the deleted region is necessary for regulation of LCR induction; however, this mutant LcrV was weakly expressed and may not have been present in sufficient amounts to have its regulatory effect. In double mutants containing this mutant lcrV and also lacking expression of known LCR negative regulators (LcrG, LcrE, and LcrH), full induction of the LCR occurred in the absence of functional LcrV, indicating that LcrV promotes induction not as an activator per se but rather by inhibiting negative regulators.

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[23] Analysis of recombinant DNA using Escherichia coli minicells

Cited by 59 publications

References 28 publications

Sequence of the Pseudomonas aeruginosa trpI activator gene and relatedness of trpI to other procaryotic regulatory genes

Sequence of the Pseudomonas aeruginosa trpI activator gene and relatedness of trpI to other procaryotic regulatory genes

Molecular characterization of the tdc operon of Escherichia coli K-12

Differential effects of deletions in lcrV on secretion of V antigen, regulation of the low-Ca2+ response, and virulence of Yersinia pestis

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