Biomedical subjects

P Ritzenthaler

Publications and source records attributed to P Ritzenthaler.

At least 37 records · Page 2Linked to original sources

PubMed1990

Taxonomic differentiation of 101 lactococcal bacteriophages and characterization of bacteriophages with unusually large genomes.

Sixty-three virulent bacteriophages of Lactococcus lactis were differentiated by DNA-DNA hybridization. The results, including those of a previous classification of 38 phages of the same bacterial species (P. Relano, M. Mata, M. Bonneau, and P. Ritzenthaler, J. Gen. Microbiol. 133:3053-3063, 1987) show that 48% of the phages analyzed belong to a unique DNA homology group (group III). Phages of this most abundant group had small isometric heads. Group I comprised 29% of the phages analyzed and was characterized by a small phage genome (19 to 22 kilobases) and a particular morphology with a prolate head. Like group III, this group contained representative phages of other classifications. Group II (21%) included virulent and temperate phages with small isometric heads. Two large isometric-headed phages, phi 109 and phi 111, were not related to the three DNA homology groups I, II, and III. The genome of phi 111 was unusually large (134 kilobases) and revealed partial DNA homology with another large isometric phage, 1289, described by Jarvis (type e) (A. W. Jarvis, Appl. Environ. Microbiol. 47:343-349, 1984). The protein compositions of phi 111 and 1289 were similar (three common major proteins of 21, 28, and 32 kilodaltons).

Bacteriophages↗

PubMed1989

Genome comparison of Lactococcus strains by pulsed-field gel electrophoresis.

The two restriction enzymes SmaI and ApaI were found to produce distributions of DNA fragments useful for genome analysis of some lactic acid bacteria (Lactococcus lactis and Streptococcus salivarius subsp. thermophilus) by pulsed-field gel electrophoresis. The genome size was estimated to be 1750 to 2500 kb depending on the species. Each strain displayed unique restriction patterns; nevertheless, the percentage of the comigrating fragments of two isogenic or closely related strains was about 80% and fell to 20-40% when the patterns of two non-related strains of the same species or two strains belonging to different species were compared.

DNA, Bacterial↗

PubMed1988

Present state of lactic acid bacteria phage taxonomy.

Recent developments in molecular taxonomy of bacteriophages of lactobacilli and lactic streptococci are presented. DNA homology appears to be the most valid criterion in studying phage taxonomy. For each bacterial species, phages can be classified in a few families according to this criterion. A fair correspondence is observed between the groups differentiated by DNA-DNA hybridization and those differenciated by protein composition. In 2 bacterial species, a close relatedness between some virulent and temperate phages has been demonstrated. The presence of homologous sequences in the genome of various phages may play an important role in their evolution. For future taxonomic studies, some phages representative of DNA homology groups are proposed as reference phages.

Bacteriophages↗

PubMed1988

Molecular taxonomy of Lactobacillus phages.

Forty-eight strains of lactobacilli used as starter strains in the dairy industry were examined for lysogeny after treatment with mitomycin C. Two strains of L. delbrueckii subsp. bulgaricus were able to produce active phages. These temperate phages as well as 4 virulent phages isolated during abnormal fermentations were compared to a previously characterized phage mv4 which is temperate. All these phages were shown to be partially homologous by DNA-DNA hybridization. Genes that code for viral proteins seem to be well conserved since 2 major virion polypeptides of 18 (or 19) kD and 34 kD could be detected in the protein composition of each phage. Immunoblotting studies of the 7 phages using serum raised against phage mv4 confirmed that the proteins of the different phages were related. All these phages can be classified in the previously constituted group a, which now comprises 4 temperate and 15 virulent phages. These results show that some virulent phages appearing during abnormal fermentations and some temperate phages isolated by appearing during abnormal fermentations and some temperate phages isolated by induction of starter strains can be closely related genetically. Five virulent phages of L. helveticus were also compared according to their restriction pattern and their DNA homology. They were shown to be related to one another, but unrelated to phages of other lactic acid bacteria species.

Bacteriophages↗

PubMed1987

Molecular characterization and comparison of 38 virulent and temperate bacteriophages of Streptococcus lactis.

Thirty-three virulent and five temperate phages of Streptococcus lactis and Streptococcus cremoris were differentiated into three groups by DNA homology. A complete lack of DNA homology was demonstrated between the phage groups. Within each group, large parts of the phage genomes were homologous except for a few phages. One group consisted of five temperate and two virulent phages suggesting that virulent phages isolated during abnormal fermentations and temperate phages isolated after induction from lactic streptococcal starter cultures may be related to one another. We observed a good correlation between the grouping of phages by DNA homology and by their protein composition since within the same DNA homology group, the protein composition of a phage exhibited some similarities with that of the other phages of the group. Therefore, the DNA homologies seemed to be located, at least, in the region coding for the structural proteins. By immunoblotting, we confirmed the relatedness between the proteins of the phages belonging to the same DNA homology group. The important host range factor in bacterium-phage interactions appeared to be an unreliable criterion in determining phage taxonomy.

Bacteriophages↗

PubMed1986

The regulatory region of the uxuAB operon in Escherichia coli K12.

The uxuAB operon is composed of two genes coding for enzymes involved in hexuronate degradation. This operon is negatively controlled by the uxuR and exuR regulatory gene products. Starting from uxu hybrid plasmids, a 300 bp Sau3A restriction fragment was isolated and shown to contain the entire uxu regulatory region using in vitro gene fusions that brought lac gene expression under the control of the transcriptional and translational signals of the uxuA gene. The nucleotide sequence of this fragment was established. The start of the uxu mRNA was localized by S1 mapping experiments (the presence of a CAP binding site upstream of the promoter was shown by DNAse I footprinting and in vitro titration). The N-terminal amino acid sequence of the purified uxuA-lacZ gene product allowed the mapping of the uxuA initiation codon at 115 bp from the start of transcription.

Base Sequence↗

PubMed1986

Negative dominant mutations of the uidR gene in Escherichia coli: genetic proof for a cooperative regulation of uidA expression.

The uidA gene is the first gene involved in the hexuronide-hexuronate pathway in Escherichia coli K-12 and is under the dual control of the uidR and uxuR encoded repressors. Point mutations affecting the uidR regulatory gene were sought to investigate the regulation of uidA. When the uidR mutant allele was on a multicopy plasmid and the wild-type allele was on the chromosome, some of the mutant phenotypes were dominant to the wild-type phenotype, indicating that the active form of the UidR repressor is multimeric. We have demonstrated that expression of the mutant phenotype is dependent on gene dosage. The dominance of the uidR allele was also sensitive to the presence of the wild-type uxuR allele in the cell. This behavior probably results from UidR-UxuR repressor interactions. A mechanism is proposed: we suggest that the UidR and UxuR repressors interact after their binding to the operator site of uidA; the binding of one regulatory molecule may facilitate the binding of the other one in a cooperative process.

Enzyme Induction↗

PubMed1986

Thirteen Virulent and Temperate Bacteriophages of Lactobacillus bulgaricus and Lactobacillus lactis Belong to a Single DNA Homology Group.

Thirteen virulent phages and two temperate phages of two closely related bacterial species (Lactobacillus lactis and L. bulgaricus) were compared for their protein composition, their antigenic properties, their restriction endonuclease patterns, and their DNA homology. The immunoblotting studies and the DNA-DNA hybridizations showed that the phages could be differentiated into two groups. One group contained 2 temperate phages of L. bulgaricus and 11 virulent phages of L. lactis. Inside each group, at least two common proteins of identical sizes could be detected for each phage. These proteins were able to cross-react in immunoblotting experiments with an antiserum raised against one phage of the same group. Temperate phage DNAs showed partial homology with DNAs from some virulent phages. These homologies seem to be located on the region coding for the structural proteins since recombinant plasmids coding for one of the major phage proteins of one phage were able to hybridize with the DNAs from phages of the same group. These results suggest that temperate and virulent phages may be related to one another.

Journal Article↗

PubMed1986

Physical and genetic characterization of the genome of Lactobacillus lactis bacteriophage LL-H.

Bacteriophage LL-H is a virulent phage of Lactobacillus lactis LL23. A restriction map of the phage genome was constructed with various restriction endonucleases. This chromosome has a 34-kilobase size and seems to be circularly permuted. We used a bank of LL-H restriction fragments to study the expression of five of the seven main phage particle proteins. Immunoblotting experiments permitted the mapping on the chromosome of several genes coding for phage particle proteins. We also show that the gene of the main capsid protein is expressed from its own promoter in an Escherichia coli strain.

Bacteriolysis↗

PubMed1985

Nucleotide sequence of a regulatory region of the uidA gene in Escherichia coli K12.

Multiple regulatory events are involved in the expression of the uidA gene. A regulatory region of this gene has been located on a 460 base pair Sau3A-EcoRI fragment and its nucleotide sequence was determined by the dideoxy method using pEMBL plasmids. A preliminary analysis of this sequence revealed the presence of numerous palindromic structures with some overlaps.

Base Sequence↗

PubMed1985

Genetic analysis of uxuR and exuR genes: evidence for ExuR and UxuR monomer repressors interactions.

The uxuAB operon is under the dual control of uxuR- and exuR-encoded repressors whereas the exu regulon genes are regulated by the sole ExuR repressor. Mutations affecting the two exuR and uxuR regulatory genes were selected to investigate the relationship between the two repressors. The isolation of exuR and uxuR negative dominant mutations on a multicopy plasmid indicated that the active form of the two repressors was multimeric. The introduction of a uxuR negative dominant allele into a wild-type strain resulted in a significant increase in exu gene expression. This unexpected effect may have been the consequence of the formation of hybrid repressor molecules. This protein must be composed of native ExuR+ subunits aggregated with altered UxuR subunits. The same interference was observed for the exuR negative dominant allele on uxu gene derepression. The hypothesis given here implies that the two regions of the ExuR and UxuR repressors involved in the subunit aggregation present enough homologies to allow the formation of hybrid repressor molecules.

Carbohydrate Dehydrogenases↗

PubMed1985

The use of gene fusions to study the expression of uidR, a negative regulatory gene of Escherichia coli K-12.

The uidR regulatory gene of Escherichia coli codes for a repressor molecule that negatively controls the expression of the uidA gene. The uidR gene was fused in front of the lacZ gene in vitro on plasmid cloning vectors developed by Casadaban et al. [J. Bacteriol. 143 (1980) 971-980. The transcriptional direction of uidR was deduced from the restriction pattern and the phenotypic properties of the uidR-lacZ fusion plasmids. The gene is transcribed counterclockwise on the standard E. coli map, as is the uidA gene. The uidR-lacZ fusions were also used to examine the regulation of expression from the uidR promoter. It was observed that the uidR gene expression is repressed by its own product and is sensitive to catabolite control. The uidR-lacZ-encoded proteins of various sizes were isolated but attempts to obtain hybrid molecules possessing, in a single polypeptide, both the beta-galactosidase and UidR repressor activities were unsuccessful.

Chimera↗

PubMed1984

Construction and expression of hybrid plasmids containing the structural gene of the Escherichia coli K-12 3-deoxy-2-oxo-D-gluconate transport system.

The kdgT gene of Escherichia coli, which encodes for the 3-deoxy-2-oxo-D-gluconate transport system, was isolated as a ColE1-kdgT hybrid plasmid from the Clarke and Carbon bank. A restriction and genetic map of the min 88 region of the chromosome was established; by subcloning the restriction fragments into the plasmid vector pBR322, the kdgT gene was localized on a 1.4-megadalton PstI DNA fragment, and the direction of transcription of the gene was determined by making use of an in vitro gene fusion between kdgT and lacZ genes. Amplification of the gene product of kdgT was up to 14-fold the level found in a haploid strain. When plasmids bearing kdgT were expressed in an in vivo maxicell system, a specific polypeptide of 28,000 daltons appeared that was found to be associated with the membrane fraction.

Carrier Proteins↗

PubMed1983

Interchangeability of repressors for the control of the uxu and uid operons in E. coli K12.

The uidA and uxuAB operons are each under the dual control of two repressor molecules: the uidR and uxuR encoded repressors negatively control the uidA operon whereas the uxuAB operon is regulated by the uxuR and exuR gene products. Plasmids overproducing the regulatory molecules encoded by exuR, uxuR, or uidR were used to investigate the regulation of these two operons. Large amounts of either exuR or uxuR repressor caused a complete repression of the uxuAB operon in exuR and uxuR double-deleted mutants, suggesting that the two repressors, when they are overproduced, are totally interchangeable for the control of the uxuAB operon. In contrast, the UxuR molecule has no effect on the other exu regulon operons controlled by the exuR gene product. The uidR and uxuR gene products appear to be partially interchangeable for the regulation of the uidA gene since addition of multicopy plasmids bearing uidR+, in uxuR deleted mutant strains, only partially suppresses the derepression of the uidA gene. Inversely, multicopies of uidR weakly reduced the synthesis of the uxuB gene product in uxuR derepressed mutants. The restrictions placed by these phenomena on the formulation of the mechanism of cooperation between the two repressor molecules for repressing the uxuAB and uidA operons are discussed.

Escherichia coli↗

PubMed1983

Multiple regulation involved in the expression of the uxuR regulatory gene in Escherichia coli K-12.

We have isolated a strain carrying a fusion of the beta-galactosidase structural gene to the promoter of the uxuR regulatory gene with the aid of the Casadaban Mud (Aprlac) phage. Analysis of mutants with deletions that were derived from the uxuR::Mud1 insertion strain confirmed the counterclockwise transcription direction of the uxuR gene. The uxuR-lacZ fusion strain was also used to examine the regulation of expression from the uxuR promoter. It was observed that an increase in the copy number of the uxuR gene results in an increased repression of beta-galactosidase synthesis. Overproduction of the exuR repressor also caused a decrease of the beta-galactosidase level. In all cases, the repression of beta-galactosidase synthesis was accompanied by a stronger repression of uxuB gene product synthesis. These results indicate that the expression of the uxuR gene is repressed by its own product but also by the exuR repressor. The different types of regulation of the two uxu operons are thus identical.

Coliphages↗

PubMed1983

Physical mapping of mutations in the structural gene encoding for the Escherichia coli aldohexuronate transport system.

We used a convenient plasmid recombination procedure to map within the exuT gene, the structural gene for the aldohexuronate transport system, the regional locations of ten independent mutations which affect the transport activity. A set of six plasmids containing various portions of the exuT gene was constructed which allowed us to divide this gene into five parts.

Biological Transport, Active↗

PubMed1983

Isolation of a functional exuR-repressor-beta-galactosidase hybrid protein by use of in vitro gene fusions.

The exuR regulatory gene of Escherichia coli codes for a repressor molecule that controls negatively the expression of the exu regulon genes involved in the hexuronate degradation. The exuR gene was fused to the lacZ gene in vitro on plasmid cloning vectors developed by Casadaban et al. [J. Bacteriol. 143 (1980) 971-980]. The exuR-lacZ fusion gene allows the production of hybrid molecules possessing in a single polypeptide beta-galactosidase activity as well as the capacity to bind in vivo specifically to the operators of the exu operons. However, the affinity of ExuR hybrid protein for the exu operators seems to be reduced in comparison with the native repressor since, even overproduced in the cell, the chimeric polypeptide was not able to completely repress the expression of the exu operons. The other functions of the ExuR repressor, i.e., repression of the uxuAB operon and inactivation by the inducers, were not retained in the chimeric protein. This particular chimaera of Mr about 120 000 was purified by chromatography on phosphocellulose from strains carrying the fusion plasmids. The purified polypeptide was able to repress in vitro the synthesis of an exu gene product in a cell-free transcription and translation system. The implications of the repressor activity in the chimeric protein are discussed. The NH2-terminus of ExuR repressor probably recognizes the exu operators specifically, and this region represents less than 20% of the entire ExuR molecule.

Bacterial Proteins↗

PubMed1983

Characterization of the operator sites of the exu regulon in Escherichia coli K-12 by operator-constitutive mutations and repressor titration.

In Escherichia coli, the exu regulon of the hexuronate system involves the three exuT, uxaCA and uxaB operons and is under the negative control of the exuR regulatory gene product. The technique developed by Casadaban, Chou and Cohen was employed to construct two plasmids containing operon fusions in which the lactose genes were fused to the uxaCA and exuT operons. These fusions were transferred into the chromosome by a reciprocal recombination event, and the resulting strains were used for isolation of mutants defective in repression. Two types of operator-constitutive mutants were obtained: one specific for the uxaCA operon expression and the other affecting the exuT gene expression. This genetic evidence confirms that these two operons which are divergently transcribed each possess their own operator site.--The derepressed expression of the two exuT-lac and uxaCA-lac operons and the uxaB gene was also examined upon introduction of plasmids bearing various operators of the exu regulon. The results of testing exuR repressor titration by multiple copies of the exu operators allowed us to show a gradation in the affinity degrees for the three exu operators: uxaBo has the strongest affinity for the exuR repressor and uxaCo the weakest, although that of exuTo seems to be just slightly greater. This gradation may play a role in the control of the exu regulon expression.

Chromosomes, Bacterial↗