Expression of the Escherichia coli malPQ operon remains unaffected after drastic alteration of its promoter

Débarbouillé, Michel; Raibaud, Olivier

doi:10.1128/jb.153.3.1221-1227.1983

Cited by 76 publications

(16 citation statements)

References 12 publications

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“…of labelled DNA probe and lyophilized. The freeze-dried nucleic acids were dissolved in 30 Al of Hepes buffer (Debarbouille and Raibaud, 1983), heated at 90°C for 10 min and incubated at 42°C for 3 h. The mixture was then diluted 10-fold with S1 nuclease buffer and 500 units of SI nuclease were added. After 1 h of incubation at 37°C, the DNA was extracted twice with phenolchloroform, ethanol precipitated and electrophoresed.…”

Section: Transformation Of B Subtilis and E Colimentioning

confidence: 99%

In vivo transfer of genetic information between gram-positive and gram-negative bacteria.

Trieu‐Cuot¹,

Gerbaud²,

Lambert³

et al. 1985

The EMBO Journal

196

126

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A 1427‐bp DNA fragment containing the kanamycin resistance gene, aphA‐3, of plasmid pIP1433 from Campylobacter coli was inserted into a shuttle vector. Full expression of aphA‐3 was obtained in Bacillus subtilis and in Escherichia coli. This DNA fragment was sequenced in its entirety and the starting point for aphA‐3 transcription in B. subtilis, C. coli and E. coli was determined by S1 nuclease mapping. The sequence of the promoter consists of the hexanucleotides TTGACA and TATAAT, with a spacing of 17 bp. The nucleotide sequence of the aphA‐3 gene from C. coli and from the streptococcal plasmid pJH1 are identical whereas they differ by two substitutions and deletion of a codon from that cloned from the staphylococcal plasmid pSH2. These results indicate a recent extension of the resistant gene pool of Gram‐positive cocci to Gram‐negative bacilli. From an analysis of the DNA sequences surrounding the promoter region, we concluded that the DNA fragment containing the aphA‐3 gene in plasmid pJH1 has evolved by deletions from a sequence similar to that found in plasmid pIP1433.

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Section: Transformation Of B Subtilis and E Colimentioning

confidence: 99%

In vivo transfer of genetic information between gram-positive and gram-negative bacteria.

Trieu‐Cuot¹,

Gerbaud²,

Lambert³

et al. 1985

The EMBO Journal

196

126

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“…The radioactive single-stranded DNA primers were then purified by polyacrylamide gel electrophoresis in the presence of urea (28). These primers were coprecipitated with 25 ,ug of the mRNA-containing preparations, obtained as described previously (8). The pellets were dissolved in 50% formamide-1 mM EDTA-0.4 M NaCl-20 mM HEPES (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 6.5), heated for 10 min at 75°C, and incubated for 16 h at 420C in an oven to allow hybridization.…”

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confidence: 99%

“…The pellets were dissolved in 50% formamide-1 mM EDTA-0.4 M NaCl-20 mM HEPES (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 6.5), heated for 10 min at 75°C, and incubated for 16 h at 420C in an oven to allow hybridization. Elongation of the DNA primer by reverse transcriptase and analysis of the products were performed as described previously (8).…”

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confidence: 99%

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Structure of two divergent promoters located in front of the gene encoding pullulanase in Klebsiella pneumoniae and positively regulated by the malT product

Chapon¹,

Raibaud²

1985

J Bacteriol

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Pullulanase ig ph extraceliular starch-debranching enzyme produced by Klebsiella pneumoniae. When its structural gene, pulA, is introduced into Escherichia coli, it is controlled by malT, the positive reguilator gene of the maltose regulon. Characterization of the region 5' to pulA and of the beginning of the gene described herein demonstrate that (i) pullulanase is probably a lipoprotein; (ii) an additional malT-controlled promoter (the malX promoter) lies adjacent to the pulA promoter and is oriented in the opposite direction; (iii) in common with the three previously described malT-controlled promoters~the puUl and maiX promoters have a conserved hexanucleotide (consensus sequence, 5'-GGAGGA) 35 base pairs upstream from the transcription initiation site; and (iv) upstream from this conserved hexanucleotide the pulA and maiX promoters differ from the other mal promoters in that they lack any detectable binding site for the cyclic AMP-binding protein.Kiebsiella pneumoniae secretes an enzyme, pullulanase, which cleaves a(1-6) bonds in branched a(1-4), oa(1-6) glucans such as amylopectin and glycogen, producing linear ox(1-4) polymers (3). Pullulanase also converts pullulan, which is a linear polymer composed of maltotriose units linked by a(1-6) bonds, into maltotriose (3). The synthesis of pullulanase is induced when cells are grown in the presence of maltose, maltodextrin, or pullulan and is sensitive to catabolite repression (17). As an extension of our studies on the maltose regulon of Escherichia coli, we recently started analyzing the regulation of pullulanase expression in K. pneumoniae. We have cloned pulA, the structural gene for pullulanase, and introduced it into E. coli, which does not normally produce this enzyme (10,18). In E. coli, pulA was still maltose inducible and was controlled by the product ot malT, the positive regulator gene of the maltose regulon (9,18). This result provided a strong indication that (i) a gene homologous to malT exists in K. pneumoniae, (ii) the product of this gene controls the expression ofpulA, and (iii) the malT products of the two species are at least partly interchangeable. In the present work, we analyzed the structure of the promoter which controls pulA expression, with the aim of comparing it with that of the three previously described malT-controlled promoters in E. coli, i.e., the promoters of the malPQ, malEFG, and malK-lamB operons

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“…Primer extension. Primer extension was performed as described by DebarbouillC et al [29]. Thermus RNA was isolated and precipitated with the labelled primer 5'-CAAGAAGGGCC-TGAAGCTCC-3'.…”

Section: Methodsmentioning

confidence: 99%

Ornithine Carbamoyltransferase from the Extreme Thermophile Thermus Thermophilus Analysis of the Gene and Characterisation of the Protein

Sanchez

Baetens

Casteele³

et al. 1997

European Journal of Biochemistry

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The ornithine carbamoyltransferase (OTC) gene from Thermus thermophilus was cloned from a ).-ZAP genomic library. An ORF of 903 bp was found coding for a protein of M , 33200. The coding region has a very high overall G + C content of 68.0%. 7: thermophilus OTC displays 38-48% amino acid identity with other OTC, the most closely related proteins being OTC from the archaeon Pyrococcus jiiriosus and from Bacillus subtilis. The enzyme was expressed in Escherichia coli and purified to homogeneity using a thermoshock followed by affinity chromatography on d-N-phosphonoacetyl-L-ornithineSepharose. The native enzyme has an M, of about 110000, suggesting a trimeric structure, as for most anabolic OTC from various organisms. 7: thermophilus OTC exhibits Michaelis-Menten kinetics for carbamoyl phosphate and ornithine with a K:Yf" of 0.10 mM for both substrates. The pH optimum was dependent on ornithine concentration with an optimum at pH 8 for ornithine concentrations around K,, values. Higher concentrations shift the optimum towards lower pH. The optimal temperature was above 65°C and the activation energy 39.1 W/mol. The enzyme is highly thermostable. In the presence of its substrates the half-life time was several hours at 85 "C. Ionic and hydrophobic interactions contribute to the stability. The expression of 7: thermophilus OTC was negatively regulated by arginine.

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Expression of the Escherichia coli malPQ operon remains unaffected after drastic alteration of its promoter

Cited by 76 publications

References 12 publications

In vivo transfer of genetic information between gram-positive and gram-negative bacteria.

In vivo transfer of genetic information between gram-positive and gram-negative bacteria.

Structure of two divergent promoters located in front of the gene encoding pullulanase in Klebsiella pneumoniae and positively regulated by the malT product

Ornithine Carbamoyltransferase from the Extreme Thermophile Thermus Thermophilus Analysis of the Gene and Characterisation of the Protein

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