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O Raibaud

Publications and source records attributed to O Raibaud.

At least 37 records · Page 2Linked to original sources
PubMed1986

Comparison of the malA regions of Escherichia coli and Klebsiella pneumoniae.

Using the mini-Mu-duction technique, we cloned the malA regions from Escherichia coli K-12 and Klebsiella pneumoniae. A comparison of the structures of the cloned DNAs indicated that the malT, malP, and malQ genes, encoding the transcriptional activator of the maltose regulon, maltodextrin phosphorylase, and amylomaltase, respectively, are similarly organized in both species; malP and malQ constitute an operon divergent from the malT gene. We sequenced 1,200 nucleotides encompassing the beginnings of the malT and malP genes, their promoters, and the intergenic region. The DNA sequences from the two species were very different; the levels of homology ranged from 28 to 80%, depending on the region. The sequences of the coding regions and of elements known to be important for the functions of these two promoters in E. coli were well conserved between the two bacteria, whereas the sequence of the malT-malP intergenic region had totally diverged.

Amino Acid Sequence↗
PubMed1985

A convenient technique to compare the efficiency of promoters in Escherichia coli.

We describe a technique which allows one to insert any promoter in front of the chromosomal malPQ operon. This can be done easily by using only one plasmid, one strain, and two simple selections. Properties of the final chromosomal fusion are such that the level of amylomaltase, the product of the malQ gene, measures quantitatively the efficiency of the inserted promoter. This method was utilized to compare the efficiency of four well-known promoters: lacZp, trp, tac, lambdaPR and three malT activated promoters: malPp, malkP and malEp.

Chromosomes, Bacterial↗
PubMed1985

The mac promoters: functional hybrid promoters activated by the malT product and repressed by the lacI product.

Using in vitro techniques we have fused upstream sequences from the malPp promoter (normally activated by the MalT protein) to downstream sequences from the lacZp promoter (normally repressed by the LacI protein). Several hybrid promoters were thus obtained, which were controlled by the MalT protein, but were poorly active. More efficient promoters were then isolated using in vivo selection. Three main conclusions could be derived from the analysis of all of these hybrid promoters. Firstly, the MalT protein seems able to force RNA polymerase to start transcription at any DNA sequence, albeit with a low efficiency. Secondly, the strength of the hybrid promoters is considerably increased if a Pribnow Box is positioned at a precise location with respect to the MalT binding site. Thirdly, the presence of the lac operator, even when properly positioned with respect to the transcription startpoint, does not suffice to permit full repression by the lacI product.

Base Sequence↗
PubMed1985

Essential and nonessential sequences in malPp, a positively controlled promoter in Escherichia coli.

A plasmid bearing the malPp promoter was digested with Bal31 to obtain a set of deletions with closely spaced endpoints in the upstream region of this promoter. Some of these deletions were sequenced, and their effect on malPQ expression was determined after having transferred them onto the chromosome. We found that a site which binds the cyclic AMP receptor protein in vitro and which is centered at position -93 with respect to the site of transcription initiation could be deleted without affecting malPQ expression. In contrast, the activity of the malPp promoter decreased abruptly when the deletions reached position -72. The downstream region of the promoter was analyzed by using a technique of "sequence replacement" which involved the selection of Mal+ pseudorevertants from strains which carried small deletions in the -25 region. The pseudorevertants, which expressed the malPQ operon in a manner indistinguishable from wild type, had grossly different sequences downstream from position -38, except for a few positions, some of which must be important for promoter function. By combining all presently available information, it is suggested that the malPp promoter contains three binding sites for its activator, the product of gene malT. These sites are defined by three quasi-identical hexanucleotides present in one orientation around position -37 and twice in the other orientation around positions -60 and -73.

Base Sequence↗
PubMed1985

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

Pullulanase is an extracellular starch-debranching enzyme produced by Klebsiella pneumoniae. When its structural gene, pulA, is introduced into Escherichia coli, it is controlled by malT, the positive regulator 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 pulA and malX promoters have a conserved hexanucleotide (consensus sequence, 5'-GGATGGA) 35 base pairs upstream from the transcription initiation site; and (iv) upstream from this conserved hexanucleotide the pulA and malX promoters differ from the other mal promoters in that they lack any detectable binding site for the cyclic AMP-binding protein.

Amino Acid Sequence↗
PubMed1984

Point mutations that reduce the expression of malPQ, a positively controlled operon of Escherichia coli.

malPQ is one of three operons controlled by the positive regulator gene malT. With the objective of defining DNA sequences essential for malPQ transcription, we looked for cis-dominant mutations that reduced the level of expression of this operon. We first constructed malP-lac fusion strains, selected from one of them a series of mutants resistant to p-nitrophenyl-beta-D-thiogalactopyranoside (a bacteriostatic compound that enters the cells via lac permease), and retained the clones that contained a mutation reducing the expression of the hybrid operon in a cis-dominant fashion. Nineteen such mutations were sequenced, and their effect on an otherwise wild type malPQ operon was studied. Three of them mapped in a transcribed portion of the operon, and are believed to exert their effect at the translation level. The others map upstream from the transcription startpoint (co-ordinate +1) and help define three DNA segments that must play a predominant role in transcription initiation: the Pribnow box (from positions -7 to -12); and two inverted repeats, extending from position -32 to -36, and -59 to -63, respectively, which are proposed to constitute part of the binding site for MalT protein.

Base Sequence↗
PubMed1983

Use of deletions created in vitro to map transcriptional regulatory signals in the malA region of Escherichia coli.

The malA region of Escherichia coli contains one of the three maltose operons, namely malPQ, and the positive regulatory gene, malT. Gene malT and the malPQ operon are transcribed in opposite directions, in a divergent manner. The distance separating the transcription start-points in the two directions was previously shown to be 513 base-pairs. We are now presenting a deletion analysis of this unexpectedly long intergenic region. Two sets of deletions were created in vitro, by using exonuclease BAL31. One set comprised deletions centered on a HincII restriction site located in the malPQ promoter, and extending towards gene malT. The other set was centered on an EcoRI site, which had been introduced close to the beginning of the malT cistron, and extended towards gene malP. These deletions, initially created on plasmids, were transferred onto the bacterial chromosome. By studying the phenotype resulting from the presence of these deletions, we concluded that: (1) all of the DNA sequences required for expression of malT and malPQ are within 100 base-pairs of the respective transcription start-points for these genes; (2) a sequence located more than 120 base-pairs upstream from the malT transcription start-point plays a role in limiting malT expression; and (3) a remaining DNA segment, 150 to 300 base-pairs in length, and centrally located in the inter-promoter region, seems to play no role in the expression of malT or malPQ.

Base Sequence↗
PubMed1983

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

The malPQ operon, one of the three operons of the maltose regulon, is positively controlled by the product of gene malT. The starting point for malPQ transcription was deduced from experiments which involved a hybridization of in vivo-synthesized malPQ mRNA with adequate DNA probes, followed either by a digestion of nonhybridized DNA (S1 nuclease mapping) or by an extension of the hybridized probe (reverse transcriptase mapping). In the wild-type strain, this starting point was 37 nucleotides upstream from the initiation codon for malP. This analysis was also performed on a double mutant which contained both a 13-base pair deletion and a 3-base pair insertion in the promoter region. This double mutant expressed the malPQ operon exactly as the wild-type strain did, in a maltose-inducible manner. In this strain, the starting point for malPQ transcription was shifted 11 nucleotides downstream from the wild-type location. An analysis of these results suggests that (i) the binding site for the malT product is located upstream from the region which is severely altered in the double mutant, i.e., upstream from position -31; and (ii) the 30-base pair sequence which precedes the transcription starting point contains very few positions which are essential for promoter activity.

Base Sequence↗
PubMed1982

A DNA sequence containing the control sites for gene malT and for the malPQ operon.

The order of 802 base pairs was established in a DNA segment containing the promoter for malPQ which is one of the three maltose operons, and the promoter for malT, the positive regulator gene of the maltose regulon. The determination of the amino-terminal sequence of the MalT protein allowed us to identify the beginning of the malT gene on the sequence. The position of the malP gene was deduced from the published amino-terminal sequence of maltodextrin phosphorylase. A total of 611 base pairs separate the initiation codons for these two genes, which are transcribed in opposite directions. This large intergenic region does not code for any polypeptide of significant size. The main features of this sequence are discussed in terms of the regulation known to operate on malT and malPQ expression.

Bacterial Proteins↗
PubMed1982

[Clonage of the "malA" region of "Escherichia coli" K12: nucleotide sequence of the regulatory region and the promoters, identification and purification of the MalT-activator protein (author's transl)].

A 5,800-bp (base pair) HindIII-EcoRI DNA fragment containing malT, the positive regulator gene of the maltose regulon, and most of malP, the structural gene for maltodextrin phosphorylase, was cloned into pBR322. A sequence of 802 bp was established in a DNA segment containing the promotor for malPQ and the promoter for malT. A total of 611 bp separates the initiation codons for these two genes, which are transcribed in opposite directions. The malT product was identified as a 94,000 dalton polypeptide.

Base Sequence↗
PubMed1980

Restriction map of the Escherichia coli malA region and identification of the malT product.

A series of plaque-forming lambda h80 transducing phages carrying various portions of the malA region were isolated. A 5,800-base pair HindIII-EcoRI DNA fragment from one of these phages was cloned into pBR322 and shown to contain malT, which is the positive regulator gene of the maltose regulon, and most of malP, the structural gene for maltodextrin phosphorylase. A restriction map of the HindIII-EcoRI fragment was established, and it was correlated with the genetic map of the malA region (i) by mapping deletions which had been generated in vitro on the plasmid and (ii) by locating on the restriction map a DNA insertion of known genetic position. A 600-base pair HincII-HaeII segment was shown to contain all or part of the promoters for malT and malP, which are known to be transcribed in opposite directions. Strains carrying gene malT on a plasmid synthesized a 94,000-dalton polypeptide which was not produced by identical strains carrying similar plasmids in which malT was partially deleted. Estimates of the size of the malT gene support the conclusion that the 94,000-dalton polypeptide is the malT product.

Bacterial Proteins↗
PubMed1979

Structure of the malB region in Escherichia coli K12. I. Genetic map of the malK-lamB operon.

A series of deletions, Mu insertions and point mutations affecting the malK-lamB operon have been isolated. They were used to establish a deletion map of this operon, which could be divided in 27 intervals, with 16 in malK and 11 in lamB. One interesting feature of this map is the lack of randomness in the distribution of Mu insertions in the lamB gene; by using data published elsewhere on the physical length of the deletion intervals it can be concluded that about 25% of these Mu insertions are clustered in a segment representing 2 to 8% of the gene. This map is presently being used to study the biosynthesis, structure, and function of the lamB product, which is an outer membrane protein involved in the transport of maltose and maltodextrin, and which in addition constitutes the receptor for phage lambda.

Chromosome Mapping↗
PubMed1979

Structure of the malB region in Escherichia coli K12. III. Correlation of the genetic map with the restriction map.

A correlation between the genetic and physical maps of the malB region was obtained by performing a restriction cleavage analysis of DNA's carrying various genetically characterized malB deletions. This also allowed to localize the boundaries between malF and malE, malE and malK, mal K and lamB on the restriction map. The genetic map is not grossly distorted with respect to the physical map.

Chromosome Mapping↗
PubMed1977

The reactivity of one essential cysteine as a conformational probe in Escherichia coli tryptophanase. Application to the study of the structural influence of subunit interactions.

It is first shown that the inactivation of Escherichia coli apotryptophanase byN-ethylmaleimide results from the labeling of a single particularly reactive cysteine per protomer. The reactivity of this cysteine under various conditions is investigated and the results indicate that the protein can exist under two classes of conformation: one, corresponding to inactive protein,in which the cysteine is reactive, and second, corresponding to active enzyme, where the cysteine is masked. The rate of the isomeriation step involved in this change in conformation is measured by the stopped-flow technique(tou = 0.4s). Finally, the reactivity of the cysteine is used to characterize the conformation of dimeric holotryptophanase (i.e. a dissociated form of the enzyme obtained as a transient species between dimeric apoenzyme and the natural tetrameric holoenzyme). By this criterion, it is shown that dimeric holotryptophanase falls in the class of 'inactive' conformations. These results are used to discuss the influence of the quaternary structure on the functional and conformational properties of tryptophanase and the nature of the conformational change involved in the activation of the enzyme by its cofactor and specific cations.

Apoproteins↗
PubMed1976

Structural and functional interdependence of the protomers of Escherichia coli K 12 tryptophanase during binding of pyridoxal 5'-phosphate.

It has been shown previously that the binding of pyridoxal 5'-phosphate to Escherichia coli K 12 tryptophanase brings about an important conformational change of the protein. The way in which this structural change is transmitted from holoprotomers to apoprotomers is investigated here, using hybrid molecules (between apoprotomers and irreversibly saturated holoprotomers). It is shown that the binding of two or three coenzyme molecules per tetramer stabilizes the whole molecule against thermal inactivation and cold-induced dissociation. The change in conformation induced on an apoprotomer by the proximity of three holoprotomers is described, using three structural probes: the kinetics of binding of the cofactor and of 5'-phosphopyridoxyl-tryptophan (an analog of an intermediate in the catalytic reaction), and the reactivity of the essential cysteines. The kinetic anticooperatively in the binding of pyridoxal 5'-phosphate is confirmed, some of its parameters are determined, and its mechanism is interpreted in relation to the coupling between the protomers.

Apoenzymes↗
PubMed1976

The dissociated tryptophanase subunit is inactive.

The dissociation into dimers of apotryptophanase has been studied from two points of view: the nature of the interactions which govern the dimer-tetramer equilibrium and the effect of dissociation on the functional properties of the enzyme. It is shown that the order in which different anions are able to shift the dimer-tetramer equilibrium is that of the Hofmeister series, thus showing that the main contribution to the interaction between two dimers is of hydrophobic nature. It is also shown that, when dimeric apotryptophanase is incubated in the presence of its cofactor and substrate, the kinetics of appearance of active molecules is of second order in enzyme and is independent of the pyridoxal-P concentration; its rate constant has been determined (5-10(4) M-1 S-1). These results indicate that the reassociation of dimers into tetramers is the rate-limiting step in the appearance of enzymatic activity, and that the tryptophanase dimer is not functional.

Anions↗
PubMed1975

Kinetic and equilibrium studies on the activation of Escherichia coli K12 tryptophanase by pyridoxal 5'-phosphate and monovalent cations.

An improved purification of Escherichia coli K12 tryptophanase is presented. It is shown that the apoenzyme crystals, oxidized by exposure to air, can be reactivated by treatment with a reducing agent. The titration of sulfhydryl groups shows that four --SH groups are exposed and two are masked per protomer. The influence of two effectors, monovalent cations and the coenzyme pyridoxal 5'-phosphate, on the reactivity of --SH groups and the enzymatic activity was investigated. The --SH groups react more slowly in holo- than in apoenzyme in the presence of potassium ions. If these ions are replaced by sodium ions, the reactivity becomes the same. Potassium and ammonium ions, both activators, give sigmoidal activation curves. The sodium ion is a Michaelian inhibitor of potassium activation. The binding of pyridoxal 5'-phosphate was examined by kinetics and at equilibrium. The kinetics are shown to be very slow; the rate constants of the forward and reverse reactions have been measured. The binding equilibrium, examined with 3H-labeled pyridoxal 5'-phosphate, gives one site per protomer with a K-D value of (3.2 plus or minus 0.8) times 10-7 M. The K-m for pyridoxal-P was determined by activity measurements. The binding equilibrium is attained after several hours, giving a value of 4.2 times 10-7 M, being nearly identical with the dissociation constant and 5 times smaller than previously reported.

Apoenzymes↗