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A Lazdunski

Publications and source records attributed to A Lazdunski.

At least 55 records · Page 3Linked to original sources
PubMed1989

Cloning of xcp genes located at the 55 min region of the chromosome and involved in protein secretion in Pseudomonas aeruginosa.

Pleiotropic mutations (xcp) affecting secretion of proteins in Pseudomonas aeruginosa have been previously characterized and mapped at 0 min, 55 min and 65 min. Genomic libraries of this organism have been constructed and the genes xcp-5 and xcp-54, located at the 55 min region, were cloned using the adjacent met allele as a marker, and complementation of xcp strains. From our linkage and cloning analysis, the most probable gene order in this region appears to be pyrD... xcp-5/xcp-54/met-9011/oru-314/trpF/leu-10. Restriction mapping and transposon (Tn1725) insertion mutagenesis demonstrated that: (i) the overall size of DNA necessary for xcp expression was 9kb, (ii) the two loci are not adjacent on the chromosome, and (iii) the two loci are expressed independently. The xcp-5 gene has been subcloned on a 4kb EcoRI fragment.

Bacterial Proteins↗
PubMed1989

Protein secretion in Pseudomonas aeruginosa: molecular cloning and characterization of the xcp-1 gene.

Pleiotropic mutations (xcp) affecting the secretion of proteins in Pseudomonas aeruginosa have been previously characterized and mapped at three different loci on the chromosome. The xcp-1 gene, which is located at the 0-min region, was isolated from a genomic bank containing DNA from P. aeruginosa PAO1. The recombinant cosmid pLX25 complemented the xcp::Tn5-751 insertion mutation previously described. The xcp-1 gene was located on pLX25 by mapping the insertion point of transposon Tn5-751 and by deletion and subcloning analysis. The xcp-1 gene was expressed when transcription was initiated from a tac promoter, and a 26,000-dalton protein was identified in Escherichia coli minicells. The Xcp-1 protein was associated with the membrane fraction of E. coli. A 30-kilodalton outer membrane protein was also affected by the xcp::Tn5-751 mutation in P. aeruginosa. The possible correlation between Xcp-1 and this protein is discussed.

Bacterial Proteins↗
PubMed1988

Phosphate regulation in Pseudomonas aeruginosa: cloning of the alkaline phosphatase gene and identification of phoB- and phoR-like genes.

In Pseudomonas aeruginosa, phosphate limitation results in the synthesis of several protein species. We report the cloning of the P. aeruginosa alkaline phosphatase structural gene, phoA, and we show that this gene is regulated normally in Escherichia coli. We have also identified and cloned two P. aeruginosa genes which can complement phoB and phoR mutations in E. coli. This suggests that a pho regulon system similar to that in E. coli may exist in P. aeruginosa, using at least two similar regulatory factors.

Alkaline Phosphatase↗
PubMed1987

Deletion analysis of the promoter region of the Escherichia coli pepN gene, a gene subject in vivo to multiple global controls.

The pepN gene codes for aminopeptidase N whose expression is regulated at the transcriptional level by anaerobiosis and phosphate starvation. To define and characterize the functional region of the pepN promoter (pepNp), various promoter fragments were fused to the malPQ operon of Escherichia coli and transferred to the chromosome. The expression of the single copy operon fusion was measured by assaying the amylomaltase activity. Sequences upstream from the canonical promoter elements, located 110 to 60 bp preceding the transcription start site, are important for promoter functioning. This region plays a role in the expression of the two divergent promoters pepNp and pncBp. The regulation of pepNp under phosphate starvation was conserved in the various constructs in which pepNp is functional. However, no particular sequence specific for phosphate regulation was detected. In addition, the regulation of pncBp by Pi starvation was demonstrated.

Aminopeptidases↗
PubMed1986

Nucleotide sequence of the promoter and amino-terminal encoding region of the Escherichia coli pepN gene.

The nucleotide sequence of the region probably responsible for regulation of pepN expression and of the region encoding the amino-terminal part of aminopeptidase N, has been determined. The transcription start site was identified by S1 nuclease mapping. All features of the promoter are those of a weak promoter and no obvious structure responsible for regulation was identified, although a possible Pho box is located 63 base pairs upstream from the Pribnow box. The reading frame was unambiguously determined by purifying the protein and by sequencing the first 21 NH2-terminal residues. The NH2-terminal region of aminopeptidase N does not contain any fragment resembling signal sequence and the protein is not produced in a precursor form. A divergent promoter, which might be that of pncB, encoding the nicotinic acid phosphoribosyltransferase (P. Terpstra, personal communication), has also been identified, which allows the assignment of the gene organization in this chromosomal region as ompF asnS pncB pepN.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of the pepN gene encoding aminopeptidase N of Escherichia coli.

We have sequenced a 3.3-kb fragment of the Escherichia coli chromosome that contains pepN gene encoding aminopeptidase N. This gene codes for a protein of 870 amino acid residues. From the size of the pepN transcript and the presence of inverted repeats in the nucleotide (nt) sequence, a putative transcription terminator has been identified. The N-terminal amino acid sequence deduced from the pepN nt sequence corresponds to the N-terminal sequence of the purified protein; the amino acid composition of the protein is also in good agreement with that deduced from the gene sequence. No obvious homology with previously sequenced peptidases has been detected.

Amino Acid Sequence↗
PubMed1985

Multiple controls exerted on in vivo expression of the pepN gene in Escherichia coli: studies with pepN-lacZ operon and protein fusion strains.

Three physiological conditions were shown to promote transcriptional regulation of pepN expression: phosphate limitation, the nature of the source of carbon and energy, and anaerobiosis. The transcriptional level of regulation can be deduced from the observation of these effects in strains carrying operon fusion pepN-lacZ. Mutations in the various genes phoB, phoM, phoR, crp, and fnr (oxrA) did not affect pepN expression.

Aminopeptidases↗
PubMed1984

Physical mapping of the gene for aminopeptidase N in Escherichia coli K12.

The pepN gene has been cloned into the multicopy plasmid pBR322. The restriction map of the insert was established and the gene was localized. By comparison with the restriction map of the plasmid pJP30 bearing the ompF region, it has been possible to order the ompF, asnS, and pepN genes. The ompF and asnS genes are contiguous and pepN is separated from asnS by a DNA fragment of about 1.6 kb.

Aminopeptidases↗
PubMed1984

Cloning and orientation of the gene encoding aminopeptidase N in Escherichia coli.

The pepN gene, that encodes aminopeptidase N in Escherichia coli, has been cloned into the multicopy plasmid pBR322. Expression of the cloned pepN gene results in overproduction of the enzyme. The restriction map of the 6.7 Kb insert was established and the gene was further localized by analysis of the in vitro constructed delection plasmid and mutant plasmids generated by Tn5 insertions. Chromosome mobilization experiments, using pep-N-lac fusion strains allowed us to infer a clockwise direction of transcription for the pepN gene.

Aminopeptidases↗
PubMed1982

The receptor of bacteriophage lambda: evidence for a biosynthesis dependent on lipid synthesis.

Inhibition of lipid synthesis in cerulenin-treated cells or in a mutant strain defective in sn-glycerol-3-phosphate acyltransferase after glycerol deprivation, results in a marked decrease of insertion of lamB protein into the outer membrane. No lambda receptor was found in any other cell compartment or in the medium under these conditions. The LamB protein synthesis was inhibited by about 70% in the absence of lipid synthesis. The residual 30% protein produced during inhibition of fatty-acid or phospholipid synthesis, was probably incorporated into the outer membrane since no further incorporation was observed after resumption of these syntheses. Besides OmpF and OmpC protein [Bocquet-Pagès, C., Lazdunski, C., and Lazdunski, A. (1981) Eur. J. Biochem. 118, 105-111], at least four other proteins of the outer membrane are also subject to alteration of levels in the absence of lipid synthesis. Under these conditions the uptake of maltose, like the uptake of 5'AMP [Bocquet-Pagès, C., Lazdunski, C., and Lazdunski, A. (1981) Eur. J. Biochem. 118, 105-111], was inhibited as much as 60%. These results are discussed with regard to the biosynthesis and assembly of the outer membrane proteins.

Bacterial Outer Membrane Proteins↗
PubMed1981

Lipid-synthesis-dependent biosynthesis (or assembly) of major outer-membrane proteins of Escherichia coli.

Upon inhibition of fatty acid synthesis in the presence of cerulenin, the uptake of 5' AMP and of other nutrients using similar pore systems can be inhibited as much as 70%. The same effect was observed upon inhibition of phospholipid synthesis after glycerol deprivation in a mutant strain defective in sn-glycerol-3-phosphate acyltransferase. Resumption of both fatty acid synthesis and phospholipid synthesis restores a normal uptake of 5' AMP. The protein composition of the outer membranes, analyzed after pulse radiolabelling by [35S]methionine, was mainly altered in OmpF and OmpC proteins. These proteins are the main porins used by most nutrients like 5' AMP. Whereas the synthesis or the assembly of OmpF protein seems to be more inhibited that that of OmpC protein after inhibition of fatty acid synthesis, the reverse case was observed after inhibition of phospholipid synthesis. No protein produced during inhibition of fatty-acid or phospholipid synthesis is reincorporated into the outer membrane after resumption of these syntheses. These results are discussed with regard to the biosynthesis and assembly of these proteins.

Adenosine Monophosphate↗
PubMed1977

Aminopeptidase N from Escherichia coli. Unusual interactions with the cell surface.

The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30-40%) by osmotic shock or by converting Escherichia coli K10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75-85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N-ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I-labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and beta-galactosidase to fluorescamine in intact cells. Only 16% of the total beta-galactosidase was labeled with this fluorescent reagent whereas 44-45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places. Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.

Alkaline Phosphatase↗
PubMed1976

Isolation and genetic mapping of Escherichia coli aminopeptidase mutants.

Many mutant strains devoid of aminopeptidase activity have been isolated in Escherichia coli. All of them produce cross-reacting material when tested against specific antiaminopeptidase antibody. The map position of the locus specifying this enzyme has been determined by three conjugations and two P1 mediated transduction experiments. By analogy with Salmonella typhimurium this locus has been called pepN (Miller, 1975). Mutations in pepN are jointly transduced with fabA and pyrD at high frequency. These data and conjugation results suggest a location between 20.5 and 22.5 minutes on E. coli genetic map.

Aminopeptidases↗
PubMed1976

Existence, localization and regulation of the biosynthesis of aminoendopeptidase in gram-negative bacteria.

An enzyme capable of hydrolyzing the substrate L-alanine p-nitroanilide has been found in 18 gram-negative Enterobacteriaceae and Pseudomonas that we tested. This enzyme might be located near the cell surface. Contrastingly we have not detected this activity on colonies of the 8 gram-positive Bacillus that we tested, which suggest a taxonomic value for this test. The aminoendopeptidases found in the various gram-negative bacteria showed similar electrophoretic mobilities and immunological cross-reactivity when tested again Escherichia coli anti-aminoendopeptidase antiserum. The regulation of aminoendopeptidase biosynthesis by the endogenous level of inorganic phosphate that we previously demonstrated in E. coli has been found to exist in all gram-negative organisms tested.

Alanine↗
PubMed1975

Evidence for an aminoendopeptidase localized near the cell surface of Escherichia coli. Regulation of synthesis by inorganic phosphate.

An enzyme capable of hydrolyzing the substrate L-alanine p-nitroanilide has been found in the various Escherichia coli strains tested. This enzyme has been called aminoendopeptidase since it shows both activities (see accompanying paper). It is released from the cells by osmotic shock and by lysozyme -- EDTA spheroplasting treatment, and 50% of the total activity is directly detectable with suspensions of intact cells. However, the release by osmotic shock or spheroplasting is not as efficient as it is for alkaline phosphatase. This periplasmic aminoendopeptidase is constitutively produced but the differential rate of synthesis is increased 4-fold when the cell growth is limited by Pi. The occurrence of this 'derepression' is simultaneous with that of alkaline phosphatase. Increasing the concentration of inorganic phosphate in the medium has no effect on the constitutive aminoendopeptidase synthesis. The effect of phosphate starvation is specific since starvation for neither nitrogen nor carbon and energy source are effective in derepressing aminoendopeptidase.

Alkaline Phosphatase↗