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Biomedical subjects

P Cossart

Publications and source records attributed to P Cossart.

At least 127 records · Page 7Linked to original sources
PubMed1989

Reacquisition of virulence of haemolysin-negative Listeria monocytogenes mutants by complementation with a plasmid carrying the hlyA gene.

An haemolysin negative mutant of Listeria monocytogenes LO28 produced by insertion of Tn917 resulted in an avirulent derivative. The hlyA gene of the same strain was previously cloned in Escherichia coli and retransfered by transformation to the hly- derivative, after subcloning in the shuttle vector pMK4. The transformant strain (L828) reacquired an haemolytic activity at a similar level than the wild strain. The virulence of this hly+ transformant was estimated by determining the LD50 in Swiss mice infected intravenously. With increasing doses of bacteria a hly- control strain (transformant with only pMK4) appeared to be totally avirulent; however, no significant difference in virulence was found between the hly+ transformants and the wild strain. Seriol viable counts in the liver and spleen of infected mice demonstrated an increase in number of L828 hly+ transformants at 48 h, but the hly- control transformants were rapidly eliminated. These results confirm that the production of haemolysin is a major factor in the pathogenic capabilities of L. monocytogenes.

Bacterial Toxins↗
PubMed1989

A genetic approach to demonstrate the role of listeriolysin O in the virulence of Listeria monocytogenes.

The locus of insertion of a transposon previously used to obtain a non-haemolytic avirulent mutant was identified: it is the structural gene encoding lisreriolysin O, the thiol-dependent haemolysin, now called hlyA. The gene was completely sequenced. The preliminary structural and functional study of the chromosomal region containing the gene indicates that hlyA belongs to a monocistronic transcriptional unit. If it is the case, the transposon insertion would have no major polar effect on downstream genes and would only affect hlyA expression. These results emphasize the importance of the haemolysin in the virulence of Listeria monocytogenes.

Amino Acid Sequence↗
PubMed1988

The listeriolysin O gene: a chromosomal locus crucial for the virulence of Listeria monocytogenes.

In culture supernatants of a Tn 1545-induced non-hemolytic mutant of Listeria monocytogenes, by immunoblotting with an anti-serum raised against purified listeriolysin O, we have detected the presence of a truncated protein of 52,000D (the secreted listeriolysin O is 60,000D). The region of insertion of the transposon has been cloned and sequenced. The transposon had inserted in an open reading frame. The homologies detected between this ORF, streptolysin O and pneumolysin demonstrate the the transposon had indeed inserted in the listeriolysin O gene. As the non-hemolytic mutant was non-virulent, our work demonstrated that a genetic determinant essential for virulence is the listeriolysin O gene or its adjacent region.

Amino Acid Sequence↗
PubMed1988

Expression in Escherichia coli and sequence analysis of the listeriolysin O determinant of Listeria monocytogenes.

To evaluate the role of hemolysin production in the virulence of Listeria monocytogenes, we have undertaken the analysis of the chromosomal region containing hlyA, the gene coding for listeriolysin O. A recombinant cosmid, conferring a hemolytic phenotype to Escherichia coli, was shown to express listeriolysin O, by immunoblotting with a specific antiserum against listeriolysin O. The presence of hlyA on the cosmid was demonstrated by DNA hybridization with a probe previously shown to contain part of hlyA. The complete nucleotide sequence of hlyA has been determined. The deduced protein sequence reveals the presence of a putative 25-amino-acid signal sequence: the secreted form of listeriolysin O would have 504 amino acids, in agreement with the molecular weight of purified listeriolysin O (58,000). The protein sequence is highly homologous to those of streptolysin O and pneumolysin. A peptide of 11 amino acids conserved in the three proteins contains the unique cysteine known to be essential for lytic activity. By DNA-DNA hybridization, the listeriolysin O gene was detected in all L. monocytogenes strains tested, even in the nonhemolytic type strain. The gene was absent in other species of the genus Listeria.

Amino Acid Sequence↗
PubMed1987

Identification of the structural gene encoding the SH-activated hemolysin of Listeria monocytogenes: listeriolysin O is homologous to streptolysin O and pneumolysin.

By immunoblotting with an antiserum raised against purified listeriolysin O, we have detected the presence of a truncated protein of 52 kilodaltons in culture supernatants of a Tn1545-induced nonhemolytic mutant of Listeria monocytogenes (J.L. Gaillard, P. Berche, and P. Sansonetti, Infect. Immun. 52:50-55, 1986). The region of insertion of the transposon has been cloned and sequenced. The transposon had inserted in an open reading frame the listeriolysin O gene. The deduced amino acid sequence of this open reading frame revealed that listeriolysin O is homologous to streptolysin O and pneumolysin, although homologies were not detectable at the DNA level.

Bacterial Proteins↗
PubMed1986

crp genes of Shigella flexneri, Salmonella typhimurium, and Escherichia coli.

The complete nucleotide sequences of the Salmonella typhimurium LT2 and Shigella flexneri 2B crp genes were determined and compared with those of the Escherichia coli K-12 crp gene. The Shigella flexneri gene was almost like the E. coli crp gene, with only four silent base pair changes. The S. typhimurium and E. coli crp genes presented a higher degree of divergence in their nucleotide sequence with 77 changes, but the corresponding amino acid sequences presented only one amino acid difference. The nucleotide sequences of the crp genes diverged to the same extent as in the other genes, trp, ompA, metJ, and araC, which are structural or regulatory genes. An analysis of the amino acid divergence, however, revealed that the catabolite gene activator protein, the crp gene product, is the most conserved protein observed so far. Comparison of codon usage in S. typhimurium and E. coli for all genes sequenced in both organisms showed that their patterns were similar. Comparison of the regulatory regions of the S. typhimurium and E. coli crp genes showed that the most conserved sequences were those known to be essential for the expression of E. coli crp.

Amino Acid Sequence↗
PubMed1985

Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes.

The DNA sequence of the region located downstream from the kanamycin resistance gene of Tn5 up to the right inverted repeat IS50R has been determined. This completes the determination of the sequence of Tn5 which is 5818 bp long. The 2.7 Kb central region contains three resistance genes: the kanamycin-neomycin resistance gene, a gene coding for resistance to CL990 an antimitotic-antibiotic compound of the bleomycin family and a third gene that confers streptomycin resistance in some bacterial species but is cryptic in E. coli. A Tn5* mutant able to express streptomycin resistance in E. coli was isolated. With this mutant, it was demonstrated that in E. coli the expression of the three resistance genes is coordinated in a single operon.

Amino Acid Sequence↗
PubMed1985

Regulation of expression of the crp gene of Escherichia coli K-12: in vivo study.

Expression of the crp gene was studied in vivo by use of a crp-lacZ gene fusion first constructed on a plasmid and then transferred onto the chromosome. Our in vivo data confirm the in vitro findings that crp is negatively autoregulated via the cyclic AMP-catabolite gene activator protein complex. We present evidence that gene crp is repressed by glucose.

Cyclic AMP↗
PubMed1984

Molecular basis of DNA sequence recognition by the catabolite gene activator protein: detailed inferences from three mutations that alter DNA sequence specificity.

Previously, we reported that substitution of Glu-181 of the catabolite gene activator protein (CAP) by lysine, leucine, or valine results in a protein that has specificity for A X T base pairs at positions 7 and 16 of the DNA recognition site, rather than G X C base pairs as is the case with the wild-type CAP. In this paper, we deduce from these genetic data both (i) the specific chemical interactions by which amino acid side chains at position 181 interact with base pairs 7 and 16 and (ii) the precise alignment between the structures of the CAP and DNA in the intermolecular CAP-DNA complex. Our analysis supports the idea that the two symmetry-related F alpha-helices of the CAP dimer interact with successive major grooves of right-handed B-type DNA [Pabo, C. & Lewis, M. (1982) Nature (London) 298, 443-447; and Steitz, T., Weber, I. & Matthew, J. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 419-426].

Base Composition↗
PubMed1983

Nucleotide sequence of thrC and of the transcription termination region of the threonine operon in Escherichia coli K12.

The entire threonine operon (thrABC) of Escherichia coli K12 was cloned, and the nucleotide sequence of the thrC gene and its 3' flanking region was determined. The translation initiation codon was identified by sequencing the N-terminal part of threonine synthase, the thrC gene product. Analysis of the deduced protein sequence (428 amino acid residues) revealed a region of homology, 35 amino acids long, between the three enzymes encoded by the threonine operon. During examination of the nucleotide sequence of the 1045 base pair fragments following the thrC gene, we detected some potential rho-independent and rho-dependent transcription termination signals.

Amino Acid Sequence↗
PubMed1982

Cloning and sequence of the crp gene of Escherichia coli K 12.

We have determined the nucleotide sequence of the crp gene of Escherichia coli K 12. From a lambda transducing phage, the crp region was subcloned into pBR322. The gene was localized on the cloned fragment by determining the length of deletions which affect its expression. Its nucleotide sequence was established by using the technique of Maxam and Gilbert. The deduced amino-acid sequence is in agreement with the previously published amino acid composition of the protein (1, 2). Analysis of the sequence confirms that the DNA binding domain is located in the C-terminal portion of the protein.

Bacteriophage lambda↗
PubMed1982

Homologies between different procaryotic DNA-binding regulatory proteins and between their sites of action.

Comparison of the amino acid sequences of 13 procaryotic regulatory proteins, including the products of genes crp (catabolite activator protein; CAP), lacI, galR , lexA, lysR, araC, trpR, and tnpR of Escherichia coli, of genes cI, cII and cro of phage lambda, cro of phage 434, and c2 of phage P22, has revealed two regions of homology. The sites of action of these proteins also share common features in their DNA sequence. Taking into account the models proposed for the lambda repressors, cro and cI, and for CAP, a general type of DNA-protein interaction is suggested.

Amino Acid 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↗
PubMed1981

Nucleotide sequence of the thrB gene of E. coli, and its two adjacent regions; the thrAB and thrBC junctions.

We have sequenced a DNA fragment containing the Escherichia coli thrA-thrB junction, the complete thrB gene and the thrB-thrC junction. The intergenic sequence thrA and thrB is only one base pair. The coding region for homoserine kinase is 927 base pairs long. It is followed by 114 base pair segment in an open reading frame predicting that thrC begins just after non-sense codon of thrB. The presence at the end of thrA and of thrB of sequences that can pair with the 3' end of the 16 S ribosomal RNA suggests that reinitiation of translation occurs at the end of the two genes. The deduced aminoacid sequence for homoserine kinase shows no striking homology with aspartokinase I homoserine dehydrogenase I.

Amino Acid Sequence↗
PubMed1980

Nucleotide sequence of the thrA gene of Escherichia coli.

The thrA gene of Escherichia coli codes for a single polypeptide chain having two enzymatic activities required for the biosynthesis of threonine, aspartokinase I and homoserine dehydrogenase I. This gene was cloned in a bacterial plasmid and its complete nucleotide sequence was established. It contains 2460 base pairs that encode for a polypeptide chain of 820 amino acids. The previously determined partial amino acid sequence of this protein is in good agreement with that predicted from the nucleotide sequence. The gene contains an internal sequence that resembles the structure of bacterial ribosome-binding sites, with an AUG preceded by four triplets, each of which can be converted to a nonsense codon by a single mutation. This suggests that the single polypeptide chain was formed by the fusion of two genes and that initiation of translation may occur inside the gene to give a protein fragment having only the homoserine dehydrogenase activity.

Aspartokinase Homoserine Dehydrogenase↗