Biomedical subjects

C Chapon

Publications and source records attributed to C Chapon.

27 records · Page 2Linked to original sources

PubMed1986

Extracellular pullulanase of Klebsiella pneumoniae is a lipoprotein.

Pullulanase is a starch-debranching enzyme produced by the gram-negative bacterium Klebsiella pneumoniae. In this organism, the enzyme is first exported to the outer membrane and is subsequently released into the growth medium. Evidence reported here indicates that pullulanase is a lipoprotein. It is apparently synthesized as a precursor with a 19-residue-long signal sequence and modified by the covalent attachment of palmitate to the cysteine residue which becomes the amino terminus after cleavage of the signal sequence. In this respect, pullulanase is similar to some penicillinases produced by gram-positive bacteria which are initially exported to the cell surface and subsequently released into the medium. However, pullulanase and the penicillinases differ in one important aspect, namely, that the extracellular pullulanase still carries the covalently attached fatty acyls, whereas extracellular penicillinases lack the modified amino-terminal cysteine together with a limited number of other residues from the amino terminus.

Amino Acid Sequence↗

PubMed1985

Indirect effects of the 3'-5' cyclic adenosine monophosphate binding protein (CAP) on the transcription of the malPQ operon in Escherichia coli.

Uninduced malPQ transcription, as followed by measuring beta-galactosidase expression in a strain carrying a malP-lacZ hybrid gene and grown in the absence of maltose, requires the presence of CAP. However this requirement is lost when the expression of malT, positive regulator gene of the maltose regulon, is rendered independent of CAP by a mutation in the malT promoter. This result suggests that the effect of CAP on uninduced malPQ expression is mediated through a modulation of MalT protein synthesis. The effect of CAP on the induced expression of malPQ is presumably mediated, in addition, through a modulation of the synthesis of the maltose transport system and, hence, of the entry of the inducer. Therefore the effect of CAP on malPQ expression seems to be merely indirect, and this is surprising since a CAP binding site is present at the malPQ promoter.

Carrier Proteins↗

PubMed1985

Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae.

Some strains of Klebsiella pneumonia secrete pullulanase, a debranching enzyme which produces linear molecules (maltodextrins, amylose) from amylopectin and glycogen. pulA, the structural gene for pullulanase, was introduced into Escherichia coli, either on a multiple-copy-number plasmid or as a single copy in the chromosome. When in E. coli, pulA was controlled by malT, the positive regulatory gene of the maltose regulon. Indeed, pulA expression was undetectable in a malT-negative mutant and constitutive in a malTc strain. Furthermore, the plasmid carrying pulA titrated the MalT protein. When produced in E. coli, pullulanase was not localized in the same way as in K. pneumoniae. In the latter case it was first exported to the outer membrane, with which it remained loosely associated, and was then released into the growth medium. In E. coli the enzyme was distributed both in the inner and the outer membranes and was never released into the growth medium.

Cell Membrane↗

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↗

PubMed1983

On the different binding affinities of CRP at the lac, gal and malT promoter regions.

We have determined the stoichiometry of CRP binding to various DNA fragments carrying the lac, malT or gal promoters in the presence of cAMP, using a gel electrophoresis method. In each case, one dimer of CRP binds to the functional CRP site upstream of the transcription start. At the lac promoter, a second CRP dimer can bind to the operator region. Direct binding analysis and competition experiments performed at 200 microM cAMP allow us to measure the affinity of CRP for these different sites and to correlate them with variations in the consensus sequences, already proposed. The order is lac greater than malT greater than gal greater than lac operator greater than lac L8 much greater than non specific sites. No strong coupling exists between the two lac sites when on the same fragment. Conversely, we have studied, at constant CRP concentrations, the cAMP levels required to obtain half maximal binding to a particular DNA site : the required cAMP level increases inversely as the affinity for CRP. These variations may account for the differential activation of various cAMP sensitive operons in vivo. Anomalies in the migrations of the 1:1 complexes between CRP and DNA have been analysed and related to the size and to the position of the CRP site in the fragment. The electrophoretic mobility of the complexes depends not only on the size of the fragment but on the position of the CRP site : the mobility is lower when CRP binds near the center of the fragment. This effect is due to a clear change in the persistence length of the DNA induced by CRP binding. We suggest that, upon binding, the protein introduces a local bend (or a kink) in the DNA structure.

Base Sequence↗

PubMed1983

Action of CAP on the malT promoter in vitro.

DNase I footprinting experiments demonstrated that CAP, the cyclic AMP receptor protein of Escherichia coli, binds around position -70 at the promoter of malT, the positive regulator gene of the maltose regulon. The binding of CAP in the presence of cyclic AMP favored the subsequent specific binding of RNA polymerase. Initiation of malT transcription in vitro displayed an absolute requirement for CAP at all tested RNA polymerase concentrations. However this was not the case with a mutant promoter (malTp1), which leads to CAP-independent malT expression in vivo. In that case an effect of CAP was seen only at the lower concentrations of RNA polymerase. These results, which suggest that CAP stimulates malT expression by promoting the binding of polymerase to the promoter, are compared with those obtained in other systems.

Binding Sites↗

PubMed1982

Expression of malT, the regulator gene of the maltose region in Escherichia coli, is limited both at transcription and translation.

Six mutations, which lead to an increase in malT expression, were mapped by sequencing techniques. All of them had one or other of two base changes. Determination of the transcription start point by reverse transcriptase mapping localised the two base changes with respect to the elements that control malT expression. One of the base changes ( malTp1 ) is located in the Pribnow box of the promoter, and presumably results in an increase in the rate of transcription initiation. The other ( malTp7 ) is located in the Shine and Dalgarno sequence, which precedes the malT cistron. It probably created a more favourable ribosome binding site on malT mRNA. A correlate of these observations is that the promoter and the ribosome binding site are both inefficient in a wild-type malT gene. A malTp1 malTp7 double mutant was constructed, which produced equivalent to 30 times more MalT protein than the wild-type strain.

Bacterial Proteins↗

PubMed1982

Role of the catabolite activator protein in the maltose regulon of Escherichia coli.

The maltose regulon consists of three operons controlled by a positive regulatory gene, malT. Deletions of the gene crp were introduced into strains which carried a malT-lacZ hybrid gene. From the observed reduction in beta-galactosidase activity it was concluded that the expression of malT-lacZ, and therefore of malT, is controlled by the catabolite activator protein (CAP), the product of the gene crp. Mutations were obtained which allowed a malT-lacZ hybrid gene to be expressed at a high level even in the absence of CAP. These mutations were shown to be located in or close to the promoter of the malT gene and were called malTp. The malTp mutations were transferred in the cis position to a wild-type malT gene. In the resulting strains, the expression of two of the maltose operons, malEFG and malK-lamB, still required the action of CAP, whereas that of the third operon, malPQ, was CAP independent. Therefore, in wild-type cells, CAP appears to control malPQ expression mainly, if not solely, by regulating the concentration of MalT protein in the cell. On the other hand, it controls the other two operons more stringently, both by regulating malT expression and by a more direct action, probably exerted in the promoters of these operons.

Chromosome Mapping↗

PubMed1982

Role of the catabolite activator protein in the expression of the maltose regulon of Escherichia coli.

Using malT-lacZ strains deleted for gene crp, we have shown that the expression of malT is controlled by the catabolite activator protein (CAP), the product of gene crp. malT X mutations were obtained which allowed a malT-lacZ hybrid gene to be expressed at a high level even in the absence of CAP. These mutations were shown to be located in or close to the promoter of the malT gene. We transferred the malT X mutation cis to a wild type malT gene. In the resulting strains, the study of the expression of the three operons in absence or presence of CAP led us to the following conclusion. CAP appears to control malPQ expression mainly if not only by regulating the concentration of MalT protein in the cell. On the other hand it controls the two other operons more stringently both by regulating malT expression and by a more direct action probably exerted on the promoters of these operons.

Bacterial Outer Membrane Proteins↗