Biomedical subjects

A Toussaint

Publications and source records attributed to A Toussaint.

At least 37 records · Page 2Linked to original sources

PubMed1993

Characterization of a temperate phage hosted by Alcaligenes eutrophus strain A5.

Nineteen strains of Alcaligenes eutrophus were tested for the presence of prophages. One strain that lysed upon mitomycin C treatment produced a phage which could not form plaques on any of the strains available. DNA extracted from partially purified phage lysates was digested with various restriction enzymes which showed that the 42 kb long viral double-stranded DNA circularizes by means of cohesive ends. To our knowledge, this is the first description of a phage for the genus Alcaligenes.

Alcaligenes↗

PubMed1992

Virulence in bacteriophage Mu: a case of trans-dominant proteolysis by the Escherichia coli Clp serine protease.

The importance of proteases in gene regulation is well documented in both prokaryotic and eukaryotic systems. Here we describe the first example of genetic regulation controlled by the Escherichia coli Clp ATP-dependent serine protease. Virulent mutants of bacteriophage Mu, which carry a particular mutation in their repressor gene (vir mutation), successfully infect Mu lysogens and induce the resident Mu prophage. We show that the mutated repressors have an abnormally short half-life due to an increased susceptibility to Clp-dependent degradation. This susceptibility is communicated to the wild type repressor present in the same cell, which provides the Muvir phages with their trans-dominant phenotype. To our knowledge this is the first case where the instability of a mutant protein is shown to trigger the degradation of its wild type parent.

ATP-Dependent Proteases↗

PubMed1992

Stabilization of bacteriophage Mu repressor-operator complexes by the Escherichia coli integration host factor protein.

All of the previously described effects of integration host factor (IHF) on bacteriophage Mu development have supported the view that IHF favours transposition-replication over the alternative state of lysogenic phage growth. In this report we show that, consistent with a model in which Mu repressor binding to its operators requires a particular topology of the operator DNA, IHF stimulates repressor binding to the O1 and O2 operators and enhances Mu repression. IHF would thus be one of the keys, besides supercoiling and the H-NS protein, that lock the operator region into the appropriate topological conformation for high-affinity binding not only of the phage transposase but also of the phage repressor.

Bacterial Proteins↗

PubMed1992

Cloning and sequencing of IS1086, an Alcaligenes eutrophus insertion element related to IS30 and IS4351.

A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacillus subtilis sacB and sacR genes. The 1,106-bp IS1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame. Hybridization data suggest the presence of one copy of IS1086 in the strain CH34 heavy-metal resistance plasmid pMOL28 and at least two copies in its chromosome. Analysis of the IS1086 nucleotide sequence revealed striking homology with two other IS elements, IS30 and IS4351, suggesting that they are three close members in a family of phylogenetically related insertion sequences. One open reading frame of the Spiroplasma citri phage SpV1-R8A2 B was also found to be related to this IS family but to a lesser extent. Comparison of the G+C contents of IS30 and IS1086 revealed that they conform to their respective hosts (46 versus 50% for IS30 and Escherichia coli and 64.5% for IS1086 and A. eutrophus). The pressure on the AT/GC ratio led to a very different codon usage in these two closely related IS elements. Results suggesting that IS1086 transposition might be activated by some forms of stress are discussed.

Alcaligenes↗

PubMed1991

Simultaneous expression of a bacteriophage Mu transposase and repressor: a way of preventing killing due to mini-Mu replication.

In vitro studies of bacteriophage Mu transposition have shown that the phage-encoded transposase and repressor bind the same sequences on the phage genome. We attempted to test that prediction in vivo and found that Mu repressor directly inhibits transposition. We also found that, in the absence of repressor, constitutive expression of Mu transposition functions pA and pB is lethal in Escherichia coli strains lysogenic for a mini-Mu and that this is the result of intensive replication of the mini-Mu. These findings have important consequences where such mini-Mus are used as genetic tools. We also tested whether in Erwinia chrysanthemi the effect of transposition functions on a resident mini-Mu was the same as in E. coli. We observed that expression of pA alone was lethal in E. chrysanthemi and that a large fraction of the survivors underwent precise excision of the mini-Mu.

Bacteriophage mu↗

PubMed1991

Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain.

Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42 degrees C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47----Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of beta-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.

Amino Acid Sequence↗

PubMed1991

Frameshift mutations in the bacteriophage Mu repressor gene can confer a trans-dominant virulent phenotype to the phage.

Virulent mutations in the bacteriophage Mu repressor gene were isolated and characterized. Recombination and DNA sequence analysis have revealed that virulence is due to unusual frameshift mutations which change several C-terminal amino acids. The vir mutations are in the same repressor region as the sts amber mutations which, by eliminating several C-terminal amino acids, suppress thermosensitivity of repressor binding to the operators by its N-terminal domain (J. L. Vogel, N. P. Higgins, L. Desmet, V. Geuskens, and A. Toussaint, unpublished data). Vir repressors bind Mu operators very poorly. Thus the Mu repressor C terminus, either by itself or in conjunction with other phage or host proteins, tunes the DNA-binding properties at the repressor N terminus.

Amino Acid Sequence↗

PubMed1990

Characterisation of an IS1 induced mutation in the carboxy-terminal end of bacteriophage Mu transposase which affects several functional domains of the protein.

We show that a mutation in bacteriophage Mu transposase (pA) which was isolated as a deletion of the C-terminal end of the protein actually consists of the replacement of the last 16 amino acids (which are mostly hydrophilic) by 26 mostly hydrophobic amino acids. This change almost completely inactivates the in vivo enzyme activity as well as its capacity to bind Mu ends in vitro, although the end-binding domain of the protein resides at least 150 amino acids from the C-terminus. This sharply contrasts with the properties of a series of missense mutations and short C-terminal deletions in pA described earlier which only slightly decrease the overall transposase activity.

Amino Acid Sequence↗

PubMed1990

Instability of bacteriophage Mu transposase and the role of host Hfl protein.

The activity of the transposase of bacteriophage Mu is unstable, requiring the protein to be synthesized throughout the lytic cycle (Pato and Reich, 1982). Using Western blot analysis, we analysed the stability of the transposase protein during the lytic cycle and found that it, too, is unstable. The instability of the protein is observed both in the presence and the absence of Mu DNA replication, and is independent of other Mu-encoded proteins and the transposase binding sites at the Mu genome ends. Stability of the protein is enhanced in host strains mutated at the hfl locus; however, stability of the transposase activity is not enhanced in these strains, suggesting that functional inactivation of the protein is not simply a result of its proteolysis.

Bacteriophage mu↗

PubMed1989

Characterization of amber mutations in bacteriophage Mu transposase: a functional analysis of the protein.

We have characterized a series of amber mutations in the A gene of bacteriophage Mu encoding the phage transposase. We tested different activities of these mutant proteins either in a sup0 strain or in different sup bacteria. In conjunction with the results described in the accompanying paper by Bétermier et al. (1989) we find that the C-terminus of the protein is not absolutely essential for global transposase function, but is essential for phage growth. Specific binding to Mu ends is defined by a more central domain. Our results also reinforce the previous findings (Bétermier et al., 1987) that more than one protein may be specified by the A gene.

Amino Acid Sequence↗

PubMed1989

Multiple defects in Escherichia coli mutants lacking HU protein.

The HU protein isolated from Escherichia coli, composed of two partially homologous subunits, alpha and beta, shares some of the properties of eucaryotic histones and is a major constituent of the bacterial nucleoid. We report here the construction of double mutants totally lacking both subunits of HU protein. These mutants exhibited poor growth and a perturbation of cell division, resulting in the formation of anucleate cells. In the absence of HU, phage Mu was unable to grow, to lysogenize, or to carry out transposition.

Bacterial Proteins↗

PubMed1988

Chromosomal mapping of the pel and cel genes in Erwinia chrysanthemi strain B374.

Using the RP4::mini-Mu in vivo cloning technique, van Gijsegem et al. (1985) isolated several pel and cel genes of Erwinia chrysanthemi (Ech) B374 strain. We have localized these genes on the Ech chromosome by co-transfer mapping of MudI1734 insertion mutants and refined the map by co-transposition analysis. This analysis has enabled us to identify another cel gene.

Chromosome Mapping↗

PubMed1987

Phage Mu transposase: deletion of the carboxy-terminal end does not abolish DNA-binding activity.

We demonstrate that a specific site on the transposase protein, pA, of bacteriophage Mu is highly susceptible to proteolytic cleavage. Cleavage is observed in a minicell system on solubilisation with the non-ionic detergent Triton X-100 or following addition of a solubilised minicell preparation to pA synthesised in a cell-free coupled transcription/translation system. Cleavage occurs at the carboxy-terminal end of the protein and generates a truncated polypeptide of 64 kDa, pA*, which retains some of the DNA-binding properties of pA. These results suggest that pA may be divided into functional domains for DNA binding and for interaction with the proteins involved in phage replication.

Bacteriophage mu↗

PubMed1987

Involvement of heat shock proteins in bacteriophage Mu development.

Growth of bacteriophage Mu was severely inhibited at elevated temperature in mutants defective in the heat shock genes dnaK, groEL, and groES and in the rpoH (htpR) regulatory mutant, but not in mutants defective in the heat shock genes dnaJ or grpE; growth of a mutant of Mu deficient in functions encoded in the accessory region of the Mu genome was inhibited in the latter two host mutants. Phage production in the dnaJ mutant was restored by growth in low-salt medium. The stage in Mu development primarily affected in all except the groE mutants was phage late transcription. In contrast, the groE mutants did not support growth of Mu at any temperature; neither Mu DNA replication nor transcription was inhibited in these strains, suggesting that groE is required for phage morphogenesis as observed with several other coliphages.

Bacteriophage mu↗

PubMed1987

In vivo mutagenesis of bacteriophage Mu transposase.

We devised a method for isolating mutations in the bacteriophage Mu A gene which encodes the phage transposase. Nine new conditional defective A mutations were isolated. These, as well as eight previously isolated mutations, were mapped with a set of defined deletions which divided the gene into 13 100- to 200-base-pair segments. Phages carrying these mutations were analyzed for their ability to lysogenize and to transpose in nonpermissive hosts. One Aam mutation, Aam7110, known to retain the capacity to support lysogenization of a sup0 host (M. M. Howe, K. J. O'Day, and D. W. Shultz, Virology 93:303-319, 1979) and to map 91 base pairs from the 3' end of the gene (R. M. Harshey and S. D. Cuneo, J. Genet. 65:159-174, 1987) was shown to be able to complement other A mutations for lysogenization, although it was incapable of catalyzing either the replication of Mu DNA or the massive conservative integration required for phage growth. Four Ats mutations which map at different positions in the gene were able to catalyze lysogenization but not phage growth at the nonpermissive temperature. Phages carrying mutations located at different positions in the Mu B gene (which encodes a product necessary for efficient integration and lytic replication) were all able to lysogenize at the same frequency. These results suggest that the ability of Mu to lysogenize is not strictly correlated with its ability to perform massive conservative and replicative transposition.

Bacteriophage mu↗

PubMed1987

Characterization and virulence properties of Erwinia chrysanthemi lipopolysaccharide-defective, phi EC2-resistant mutants.

Outer membrane alterations were characterized in spontaneous mutants of the Erwinia chrysanthemi 3937jRH, which were selected for resistance to bacteriophage phi EC2. All but one of the mutants analyzed were affected in their lipopolysaccharide (LPS) structure, lacking the entire heterogeneous region of apparent high molecular weight present in the wild-type E. chrysanthemi LPS. At least two 3937jRH mutants, one selected as phi EC2 resistant (RH6065) and the other previously selected (D. Expert and A. Toussaint, J. Bacteriol. 163:221-227, 1985) as bacteriocin resistant (R1456), were cross-resistant to bacteriophage Mu and had rough LPSs with an altered core structure. Two phi EC2r mutants (RH6053 and RH6065) were most severely affected in their outer membrane integrity and also lost their virulence on saintpaulia plants, although they still possessed normal extracellular levels of pectinolytic and cellulolytic activities. The two Mur mutants RH6065 and R1456 were also able to induce systemic resistance in the challenged plant. All the other phi EC2r mutants retained the virulence of 393jRH.

Adsorption↗

PubMed1986

Transposition and replication of maxi-Mu derivatives of bacteriophage Mu.

The insertion of DNA fragments within the lac sequence of a MudI(Ap,lac) prophage resulted in the formation of a set of maxi-Mu genomes which were 39.8, 59, 85.6, and 88.2 kb long, respectively. The comparison of these maxi-Mu's with the 37.2-kb-long parental MudI(Ap,lac) indicated that the transposition frequency decreased as the length of the prophage increased. No replication of the two longest maxi-Mu's could be detected. The 59- and the 39.8-kb-long chimeric genomes were noted to replicate at approximately 1-2 and 30%, respectively, of the rate found with the MudI(Ap,lac) prophage. The length dependence of the transposition and replication could be explained by the impairment of an early step of the transposition/replication mechanism.

Coliphages↗

PubMed1985

In vivo cloning of the pectate lyase and cellulase genes of Erwinia chrysanthemi.

Using an RP4 plasmid which carries a mini-Mu prophage which allows it to integrate spontaneously random pieces of its host chromosome, we cloned in vivo at least some of the pectate lyase and cellulase genes of the Erwinia chrysanthemi strain B374. The RP4-prime plasmids were used to localize the cloned genes on the B374 chromosome by co-transposition mapping and to subclone most of the genes in a classic high copy number plasmid vector.

Journal Article↗