The ACLAME database is dedicated to the collection, analysis and classification of sequenced mobi... more The ACLAME database is dedicated to the collection, analysis and classification of sequenced mobile genetic elements (MGEs, in particular phages and plasmids). In addition to providing information on the MGEs content, classifications are available at various levels of organization. At the gene/protein level, families group similar sequences that are expected to share the same function. Families of four or more proteins are manually assigned with a functional annotation using the GeneOntology and the locally developed ontology MeGO dedicated to MGEs. At the genome level, evolutionary cohesive modules group sets of protein families shared among MGEs. At the population level, networks display the reticulate evolutionary relationships among MGEs. To increase the coverage of the phage sequence space, ACLAME version 0.4 incorporates 760 high-quality predicted prophages selected from the Prophinder database. Most of the data can be downloaded from the freely accessible ACLAME web site (http://aclame.ulb.ac .be). The BLAST interface for querying the database has been extended and numerous tools for in-depth analysis of the results have been added.
A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by... more A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacilus 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 citi phage SpVl-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%Yo 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 cin was used at a concentration of 1,000 ,ug/ml. Restriction enzymes and T4 ligase were purchased from Bethesda Research Laboratories, Inc., Gaithersburg, Md. Nitrocellulose membranes (GeneScreen; NEN, Boston, Mass.) were used in Southern blotting experiments (29). Construction of pJV240. The pLS306 plasmid contains the
Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause tempera... more Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q) and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup degrees hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup degrees hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179-V196), binds Mu operator DNA more stably at 42 degrees in vitro compared to its full-length counterpart (cts62 repre...
As previously shown for mutator phage Mu-1, to which it is closely related, temperate bacteriopha... more As previously shown for mutator phage Mu-1, to which it is closely related, temperate bacteriophage D108 induces chromosomal rearrangements (replicon fusion and transposition of chromosomal segments) in its host genome.
Ralstonia metallidurans CH34 can use biphenyl as carbon and energy source when provided with the ... more Ralstonia metallidurans CH34 can use biphenyl as carbon and energy source when provided with the catabolic transposon Tn4371. Previous results suggested that this property was dependent on the RNA polymerase subunit sigma(54). The authors sequenced the CH34 rpoN gene and flanking DNA and isolated a CH34 rpoN-deficient strain. Analysis of the sequence revealed a set of features conserved in all rpoN genes and flanking DNA regions previously analysed in other bacterial species. Nevertheless, despite this conservation, CH34 differed even from the closely related strain R. eutropha H16 by one particular ORF. The rpoN null mutation did not affect expression of the Tn4371 bph operon although it did alter the ability of the Tn4371 host strain to grow on biphenyl. The CH34 rpoN mutant had lost the capacity for autotrophic growth and for responding to poor nitrogen sources by a decrease in urease and proline oxidase activity. CH34 RNA polymerase sigma(54) thus positively controls autotrophy ...
Like several other Escherichia coli bacteriophages, transposable phage Mu does not develop normal... more Like several other Escherichia coli bacteriophages, transposable phage Mu does not develop normally in groE hosts (M. Pato, M. Banerjee, L. Desmet, and A. Toussaint, J. Bacteriol. 169:5504-5509, 1987). We show here that lysates obtained upon induction of groE Mu lysogens contain free inactive tails and empty heads. GroEL and GroES are thus essential for the correct assembly of both Mu heads and Mu tails. Evidence is presented that groE mutations inhibit processing of the phage head protein gpH as well as the formation of a 25S complex suspected to be an early Mu head assembly intermediate.
Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive ... more Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive to its Escherichia coli host ATP-dependent ClpXP protease. We further investigated the determinants of the mutant repressor's sensitivity to Clp. We show the crucial importance of a C-terminal, seven amino acid long sequence in which a single change is sufficient to decrease the rate of degradation of the protein. The sequence was fused at the C-terminal end of the CcdB and CcdA proteins encoded by plasmid F. CcdB, which is naturally stable, was unaffected, while CcdA, which is normally degraded by the Lon protease, became a substrate for ClpXP while remaining a substrate for Lon. In agreement with the current hypothesis on the mechanism of recognition of their substrates by energy- dependent proteases, these results support the existence, on the substrate polypeptides, of separate motifs responsible for recognition and cleavage by the protease.
We have located Mu delta 26 sequences straddling the forks in DNA structures which appear during ... more We have located Mu delta 26 sequences straddling the forks in DNA structures which appear during Mu delta 26 replication, i.e., keys, pending keys, dumb- bells , partially fused circles, and asymmetrical forks. This brings additional evidence that these structures are mini-Mu replication intermediates. The possible relationship between these structures and those predicted by the different models formulated to explain transposition in procaryotes is discussed.
From the histogram of the partial denaturation of a mini-Mu DNA, we constructed its standard dena... more From the histogram of the partial denaturation of a mini-Mu DNA, we constructed its standard denaturation map. We devised a computer analysis which, by using this map, allows the localization of the mini-Mu DNA in a bacterial DNA population.
From strains carrying two different F-prime factors, we recovered F' derivatives that acquire... more From strains carrying two different F-prime factors, we recovered F' derivatives that acquired the trp chromosomal region. These F'trp plasmids can be isolated at a frequency of 10(-5) to 10(-6). They were characterized genetically by looking at the size of the trp segment they acquired and at the location of that segment in the parental F' plasmid. Results are discussed in relationship to possible transposition mechanisms.
Abstract The entire DNA of an induced Escherichia coli strain lysogenic for a mini-Mu able to rep... more Abstract The entire DNA of an induced Escherichia coli strain lysogenic for a mini-Mu able to replicate has been extracted after 50 min growth at 42 and examined with the electron microscope. Several unusual DNA configurations were identified: circles, keys, pending ...
Induction of the Mu prophage of a lysogenic HfrP4X strongly stimulates the early transfer of the ... more Induction of the Mu prophage of a lysogenic HfrP4X strongly stimulates the early transfer of the purE gene, which is located far from the origin of transfer. By using a rec- Mu cts62 X lysogenic donor, it was established that this process reflects the inversion of the origin of transfer in part of the Hfr population. Hfr's with inverted polarity of gene transfer were isolated; their analysis suggests that two Mu genomes in opposite orientation surround the inverted DNA fragment. Due to the presence of the Mu genome of the invertible G segment, homologous regions in the same orientation can appear in Mu genomes in opposite orientation. In a Rec+ background, Hfr's with inverted polarity (i) return to their original polarity of transfer by recomination between the two inverted Mu and (ii) produce new F' strains by recombination between the two similarly oriented G segments.
Defective lysogens were isolated after treatment of 434hy lysogens with the acridine derivative I... more Defective lysogens were isolated after treatment of 434hy lysogens with the acridine derivative ICR-I9I-A. These were presumably frame-shift mutants. In complementation tests, a fair proportion of these mutants behaved in a polar fashion.
A series of bacteriocin-resistant mutants of Erwinia chrysanthemi 3937JRH were unable to elicit s... more A series of bacteriocin-resistant mutants of Erwinia chrysanthemi 3937JRH were unable to elicit soft-rot symptoms on saintpaulia plants. The loss of pathogenicity was correlated with the disappearance of one to three outer membrane polypeptides (molecular weights, about 80,000 to 90,000) whose production in wild-type strains was greatly enhanced under iron-limited growth conditions. The mutants did not exhibit altered extracellular pectinolytic or cellulolytic activities.
The importance of proteases in gene regulation is well documented in both prokaryotic and eukaryo... more 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.
1. Virology. 1978 Aug;89(1):146-61. Relationships between temperate phages Mu and P1. Toussaint A... more 1. Virology. 1978 Aug;89(1):146-61. Relationships between temperate phages Mu and P1. Toussaint A, Lefebvre N, Scott JR, Cowan JA, de Bruijn F, Bukhari AI. PMID: 685175 [PubMed - indexed for MEDLINE]. Publication Types: ...
Thirty-seven amber and 3 thermosensitive mutants of the temperate phage Mu-1 were isolated and as... more Thirty-seven amber and 3 thermosensitive mutants of the temperate phage Mu-1 were isolated and assigned to 15 different cistrons. The markers were ordered by deletion mapping in two independently isolated Mu lysogens. The same order was found in both cases. This order is the same as the order found by other authors (see a('('onpanying papers), indicating that Mu has a unique attachment site on it,s I)NA.
... VIROLOGY 153, 70-79 (1986) Transposition and Replication of Maxi-Mu Derivatives of Bacterioph... more ... VIROLOGY 153, 70-79 (1986) Transposition and Replication of Maxi-Mu Derivatives of Bacteriophage Mu MICHEL FAELEN,*-1 ARIANE TOUSSAINT,* BARBARA WAGGONER,+ LUCIE DESMET,* AND MARTIN PATO+ ... The steps in the isolation procedure are illustrated in Figl. ...
Nineteen strains of Alcaligenes eutrophus were tested for the presence of propha~ies. One strain ... more Nineteen strains of Alcaligenes eutrophus were tested for the presence of propha~ies. 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.
The ACLAME database is dedicated to the collection, analysis and classification of sequenced mobi... more The ACLAME database is dedicated to the collection, analysis and classification of sequenced mobile genetic elements (MGEs, in particular phages and plasmids). In addition to providing information on the MGEs content, classifications are available at various levels of organization. At the gene/protein level, families group similar sequences that are expected to share the same function. Families of four or more proteins are manually assigned with a functional annotation using the GeneOntology and the locally developed ontology MeGO dedicated to MGEs. At the genome level, evolutionary cohesive modules group sets of protein families shared among MGEs. At the population level, networks display the reticulate evolutionary relationships among MGEs. To increase the coverage of the phage sequence space, ACLAME version 0.4 incorporates 760 high-quality predicted prophages selected from the Prophinder database. Most of the data can be downloaded from the freely accessible ACLAME web site (http://aclame.ulb.ac .be). The BLAST interface for querying the database has been extended and numerous tools for in-depth analysis of the results have been added.
A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by... more A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacilus 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 citi phage SpVl-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%Yo 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 cin was used at a concentration of 1,000 ,ug/ml. Restriction enzymes and T4 ligase were purchased from Bethesda Research Laboratories, Inc., Gaithersburg, Md. Nitrocellulose membranes (GeneScreen; NEN, Boston, Mass.) were used in Southern blotting experiments (29). Construction of pJV240. The pLS306 plasmid contains the
Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause tempera... more Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q) and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup degrees hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup degrees hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179-V196), binds Mu operator DNA more stably at 42 degrees in vitro compared to its full-length counterpart (cts62 repre...
As previously shown for mutator phage Mu-1, to which it is closely related, temperate bacteriopha... more As previously shown for mutator phage Mu-1, to which it is closely related, temperate bacteriophage D108 induces chromosomal rearrangements (replicon fusion and transposition of chromosomal segments) in its host genome.
Ralstonia metallidurans CH34 can use biphenyl as carbon and energy source when provided with the ... more Ralstonia metallidurans CH34 can use biphenyl as carbon and energy source when provided with the catabolic transposon Tn4371. Previous results suggested that this property was dependent on the RNA polymerase subunit sigma(54). The authors sequenced the CH34 rpoN gene and flanking DNA and isolated a CH34 rpoN-deficient strain. Analysis of the sequence revealed a set of features conserved in all rpoN genes and flanking DNA regions previously analysed in other bacterial species. Nevertheless, despite this conservation, CH34 differed even from the closely related strain R. eutropha H16 by one particular ORF. The rpoN null mutation did not affect expression of the Tn4371 bph operon although it did alter the ability of the Tn4371 host strain to grow on biphenyl. The CH34 rpoN mutant had lost the capacity for autotrophic growth and for responding to poor nitrogen sources by a decrease in urease and proline oxidase activity. CH34 RNA polymerase sigma(54) thus positively controls autotrophy ...
Like several other Escherichia coli bacteriophages, transposable phage Mu does not develop normal... more Like several other Escherichia coli bacteriophages, transposable phage Mu does not develop normally in groE hosts (M. Pato, M. Banerjee, L. Desmet, and A. Toussaint, J. Bacteriol. 169:5504-5509, 1987). We show here that lysates obtained upon induction of groE Mu lysogens contain free inactive tails and empty heads. GroEL and GroES are thus essential for the correct assembly of both Mu heads and Mu tails. Evidence is presented that groE mutations inhibit processing of the phage head protein gpH as well as the formation of a 25S complex suspected to be an early Mu head assembly intermediate.
Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive ... more Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive to its Escherichia coli host ATP-dependent ClpXP protease. We further investigated the determinants of the mutant repressor's sensitivity to Clp. We show the crucial importance of a C-terminal, seven amino acid long sequence in which a single change is sufficient to decrease the rate of degradation of the protein. The sequence was fused at the C-terminal end of the CcdB and CcdA proteins encoded by plasmid F. CcdB, which is naturally stable, was unaffected, while CcdA, which is normally degraded by the Lon protease, became a substrate for ClpXP while remaining a substrate for Lon. In agreement with the current hypothesis on the mechanism of recognition of their substrates by energy- dependent proteases, these results support the existence, on the substrate polypeptides, of separate motifs responsible for recognition and cleavage by the protease.
We have located Mu delta 26 sequences straddling the forks in DNA structures which appear during ... more We have located Mu delta 26 sequences straddling the forks in DNA structures which appear during Mu delta 26 replication, i.e., keys, pending keys, dumb- bells , partially fused circles, and asymmetrical forks. This brings additional evidence that these structures are mini-Mu replication intermediates. The possible relationship between these structures and those predicted by the different models formulated to explain transposition in procaryotes is discussed.
From the histogram of the partial denaturation of a mini-Mu DNA, we constructed its standard dena... more From the histogram of the partial denaturation of a mini-Mu DNA, we constructed its standard denaturation map. We devised a computer analysis which, by using this map, allows the localization of the mini-Mu DNA in a bacterial DNA population.
From strains carrying two different F-prime factors, we recovered F' derivatives that acquire... more From strains carrying two different F-prime factors, we recovered F' derivatives that acquired the trp chromosomal region. These F'trp plasmids can be isolated at a frequency of 10(-5) to 10(-6). They were characterized genetically by looking at the size of the trp segment they acquired and at the location of that segment in the parental F' plasmid. Results are discussed in relationship to possible transposition mechanisms.
Abstract The entire DNA of an induced Escherichia coli strain lysogenic for a mini-Mu able to rep... more Abstract The entire DNA of an induced Escherichia coli strain lysogenic for a mini-Mu able to replicate has been extracted after 50 min growth at 42 and examined with the electron microscope. Several unusual DNA configurations were identified: circles, keys, pending ...
Induction of the Mu prophage of a lysogenic HfrP4X strongly stimulates the early transfer of the ... more Induction of the Mu prophage of a lysogenic HfrP4X strongly stimulates the early transfer of the purE gene, which is located far from the origin of transfer. By using a rec- Mu cts62 X lysogenic donor, it was established that this process reflects the inversion of the origin of transfer in part of the Hfr population. Hfr's with inverted polarity of gene transfer were isolated; their analysis suggests that two Mu genomes in opposite orientation surround the inverted DNA fragment. Due to the presence of the Mu genome of the invertible G segment, homologous regions in the same orientation can appear in Mu genomes in opposite orientation. In a Rec+ background, Hfr's with inverted polarity (i) return to their original polarity of transfer by recomination between the two inverted Mu and (ii) produce new F' strains by recombination between the two similarly oriented G segments.
Defective lysogens were isolated after treatment of 434hy lysogens with the acridine derivative I... more Defective lysogens were isolated after treatment of 434hy lysogens with the acridine derivative ICR-I9I-A. These were presumably frame-shift mutants. In complementation tests, a fair proportion of these mutants behaved in a polar fashion.
A series of bacteriocin-resistant mutants of Erwinia chrysanthemi 3937JRH were unable to elicit s... more A series of bacteriocin-resistant mutants of Erwinia chrysanthemi 3937JRH were unable to elicit soft-rot symptoms on saintpaulia plants. The loss of pathogenicity was correlated with the disappearance of one to three outer membrane polypeptides (molecular weights, about 80,000 to 90,000) whose production in wild-type strains was greatly enhanced under iron-limited growth conditions. The mutants did not exhibit altered extracellular pectinolytic or cellulolytic activities.
The importance of proteases in gene regulation is well documented in both prokaryotic and eukaryo... more 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.
1. Virology. 1978 Aug;89(1):146-61. Relationships between temperate phages Mu and P1. Toussaint A... more 1. Virology. 1978 Aug;89(1):146-61. Relationships between temperate phages Mu and P1. Toussaint A, Lefebvre N, Scott JR, Cowan JA, de Bruijn F, Bukhari AI. PMID: 685175 [PubMed - indexed for MEDLINE]. Publication Types: ...
Thirty-seven amber and 3 thermosensitive mutants of the temperate phage Mu-1 were isolated and as... more Thirty-seven amber and 3 thermosensitive mutants of the temperate phage Mu-1 were isolated and assigned to 15 different cistrons. The markers were ordered by deletion mapping in two independently isolated Mu lysogens. The same order was found in both cases. This order is the same as the order found by other authors (see a('('onpanying papers), indicating that Mu has a unique attachment site on it,s I)NA.
... VIROLOGY 153, 70-79 (1986) Transposition and Replication of Maxi-Mu Derivatives of Bacterioph... more ... VIROLOGY 153, 70-79 (1986) Transposition and Replication of Maxi-Mu Derivatives of Bacteriophage Mu MICHEL FAELEN,*-1 ARIANE TOUSSAINT,* BARBARA WAGGONER,+ LUCIE DESMET,* AND MARTIN PATO+ ... The steps in the isolation procedure are illustrated in Figl. ...
Nineteen strains of Alcaligenes eutrophus were tested for the presence of propha~ies. One strain ... more Nineteen strains of Alcaligenes eutrophus were tested for the presence of propha~ies. 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.
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Papers by A. Toussaint