ABSTRACT
We describe the uncommon development of leptomeningeal lipomatosis in a girl with encephalocraniocutaneous lipomatosis (ECCL). Leptomeningeal involvement had not been present at 2 years of age, but was demonstrated on CT and MRI at 10 years of age. Our case demonstrates follow-up neuroimaging features of ECCL that may be helpful to radiologists in suggesting the correct diagnosis, as ocular and cutaneous lesions may be non-specific clinically. The developmental nature of leptomeningeal involvement in our case suggests that close clinical and radiological follow-up is important in children with suspected or established ECCL.
Subject(s)
Brain Diseases/diagnosis , Lipomatosis/diagnosis , Magnetic Resonance Imaging/methods , Tomography, X-Ray Computed/methods , Adolescent , Diagnosis, Differential , Female , HumansABSTRACT
A new gene cluster, designated sepABC and a divergently transcribed sepR, was found downstream of the two-component todST phosphorelay system that regulates toluene degradation (the tod pathway) in Pseudomonas putida F1 (PpF1). The deduced amino acid sequences encoded by sepABC show a high homology to bacterial proteins known to be involved in solvent efflux or multidrug pumps. SepA, SepB and SepC are referred to be periplasmic, inner membrane and outer membrane efflux proteins respectively. Effects on growth of various PpF1 mutants compared to that of the wild type in the presence of toluene indicated a possible protective role of the solvent efflux system in a solvent-stressed environment. Growth tests with the complemented mutants confirmed the involvement of the Sep proteins in conferring solvent tolerance. The sepR gene encodes a 260-residue polypeptide that is a member of the E. coli IclR repressor protein family. The repressor role of SepR was established by conducting tests with a sep-lacZ transcriptional fusion in Escherichia coli and PpF1, expression of SepR as a maltose-binding fusion protein in a DNA binding assay, and mRNA analysis. Southern hybridization experiments and analysis of the P. putida KT2440 genome sequence indicated that sepR is a relatively rare commodity compared to homologues of the sepABC genes. We developed a whole-cell bioluminescent biosensor, PpF1G4, which contains a chromosomally based sep-lux transcriptional fusion. The biosensor showed significant induction of the sepABC genes by a wide variety of aromatic molecules, including benzene, toluene, ethylbenzene, and all three isomers of xylene (BTEX), naphthalene, and complex mixtures of aliphatic and aromatic hydrocarbons. PpF1G4 represents a second-generation biosensor that is not based on a catabolic promoter but is nonetheless inducible by aromatic pollutants and moreover functional under nutrient-rich conditions.
Subject(s)
Bacterial Proteins/genetics , Biosensing Techniques , Hydrocarbons, Aromatic/pharmacology , Membrane Transport Proteins/genetics , Pseudomonas putida/genetics , Repressor Proteins/genetics , Solvents/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , DNA, Bacterial/chemistry , DNA, Bacterial/isolation & purification , Environmental Pollutants/analysis , Environmental Pollutants/metabolism , Gene Deletion , Gene Expression Regulation, Bacterial , Genes, Bacterial , Genes, Reporter , Genetic Complementation Test , Hydrocarbons, Aromatic/analysis , Hydrocarbons, Aromatic/metabolism , Lac Operon , Luminescent Measurements , Membrane Transport Proteins/metabolism , Molecular Sequence Data , Promoter Regions, Genetic , Pseudomonas putida/metabolism , Repressor Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology , Solvents/analysis , Solvents/metabolism , Toluene/metabolism , Toluene/pharmacology , beta-Galactosidase/metabolismABSTRACT
One major function of DNA topoisomerase I in Escherichia coli is to repress R-loop formation during transcription elongation, which may otherwise inhibit cell growth. We have previously shown that the growth problems of topA mutants can be corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R-loop. The goal of the present study was to identify other potential regulators of R-loop formation. To this end, we have screened for multicopy suppressors of topA null mutations. As expected using this procedure, we cloned the rnhA gene encoding RNase H. In addition, we also identified the topB gene encoding DNA topoisomerase III as an efficient suppressor of topA null mutations and, hence, of R-loop formation. We show that DNA topoisomerase III is able to relax transcription-induced negative supercoiling both in vitro and in vivo. An R-loop is also shown to be a hot-spot for relaxation by DNA topoisomerase III, and we found that R-loop-dependent hypernegative supercoiling can be prevented by the activity of this topoisomerase in vivo. It is also shown that the topB gene can act synergistically with the rnhA gene to correct the growth defect of topA null mutants efficiently. This synergistic effect can be explained by the fact that some R-loops must not be degraded in order for the RNA to be available for protein synthesis. Topoisomerase III can presumably repress the formation of such R-loops or cause their destabilization to prevent RNA degradation. This is supported by the fact that overproduction of this topoisomerase corrects the negative effect of overexpressing RNase H activity on the growth of topA null mutants at low temperatures. Moreover, the fact that DNA topoisomerase III does not relax global supercoiling supports our previous conclusion that R-loop formation, and therefore the essential function of DNA topoisomerase I, involves local, rather than global, supercoiling.
Subject(s)
DNA Topoisomerases, Type I/genetics , Escherichia coli/genetics , Genes, Suppressor , Mutation , Base Sequence , DNA Primers , Escherichia coli/enzymology , Escherichia coli/growth & development , Protein Binding , Ribonuclease H/metabolism , Transcription, GeneticABSTRACT
Recent in vivo and in vitro studies have suggested an important role for DNA topoisomerases in regulating R-loop formation during transcription in Escherichia coli. In the present report we present genetic and biochemical evidence strongly suggesting that R-loop formation can occur during transcription of a portion of the rrnB operon and that it is regulated by DNA topoisomerase activity. We found that a multicopy plasmid (pBR322) carrying an heavily transcribed portion of the rrnB operon cannot be transformed in topA mutants unless RNase H is overproduced. Transcription of the 567-base pair HindIII fragment from the rrnB operon allows the extraction of large amount of R-looped plasmid DNAs from a topA mutant, in a manner that depends on the intracellular level of RNase H activity. When DNA gyrase is sufficiently active, hypernegatively supercoiled plasmid DNA is produced if the same DNA fragment is transcribed in a topA mutant. The formation of such topoisomers most likely reflect the presence of extensive R-loops since it is sensitive to the intracellular level of RNase H activity. Finally, the formation of R-looped plasmid DNAs in an in vitro transcription system using phage RNA polymerases is also detected when the 567-base pair HindIII fragment is transcribed on a negatively supercoiled DNA template.
Subject(s)
DNA Topoisomerases, Type I/metabolism , Nucleic Acid Conformation , Transcription, Genetic , rRNA Operon , DNA, Superhelical/metabolism , Deoxyribonuclease HindIII/metabolism , Escherichia coli , Plasmids/metabolism , Ribonuclease H/metabolismABSTRACT
We have recently found that stable R-loop formation occurs in vivo and in vitro when a portion of the Escherichia coli rrnB operon is transcribed preferentially in its physiological orientation. Our results also suggested that the formation of such structures was more frequent in topA mutants and was sensitive to the template DNA supercoiling level. In the present report we investigated in greater detail the involvement of DNA topoisomerases in this process. By using an in vitro transcription system with phage RNA polymerases, we found that hypernegative supercoiling of plasmid DNAs in the presence of DNA gyrase is totally abolished by RNase H, suggesting that extensive R-looping occurs during transcription in the presence of DNA gyrase. When RNase A is present, significant hypernegative supercoiling occurs only when the 567-base pair rrnB HindIII fragment is transcribed in its physiological orientation. This result suggests that more stable R-loops are being produced in this orientation. Our results also suggest that DNA gyrase can participate in the process of R-loop elongation. The strong transcription-induced relaxing activity of E. coli DNA topoisomerase I is shown to efficiently counteract the effect of DNA gyrase and thus inhibit extensive R-looping. In addition, we found that an R-looped plasmid DNA is a better substrate for relaxation by E. coli DNA topoisomerase I as compared with a non-R-looped substrate.
Subject(s)
DNA Topoisomerases, Type I/metabolism , rRNA Operon/genetics , DNA Topoisomerases, Type II/metabolism , DNA, Recombinant/chemistry , DNA, Recombinant/genetics , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , DNA-Directed RNA Polymerases/metabolism , Deoxyribonuclease HindIII , Escherichia coli/enzymology , Escherichia coli/genetics , Nucleic Acid Conformation , Plasmids , Substrate Specificity , Transcription, Genetic , Viral ProteinsABSTRACT
Previous biochemical studies have suggested a role for bacterial DNA topoisomerase (TOPO) I in the suppression of R-loop formation during transcription. In this report, we present several pieces of genetic evidence to support a model in which R-loop formation is dynamically regulated during transcription by activities of multiple DNA TOPOs and RNase H. In addition, our results suggest that events leading to the serious growth problems in the absence of DNA TOPO I are linked to R-loop formation. We show that the overexpression of RNase H, an enzyme that degrades the RNA moiety of an R loop, can partially compensate for the absence of DNA TOPO I. We also note that a defect in DNA gyrase can correct several phenotypes associated with a mutation in the rnhA gene, which encodes the major RNase H activity. In addition, we found that a combination of topA and rnhA mutations is lethal.
Subject(s)
DNA Topoisomerases, Type I/deficiency , Escherichia coli/growth & development , Nucleic Acid Conformation , Ribonuclease H/biosynthesis , Transcription, Genetic , Cold Temperature , DNA, Bacterial/metabolism , Escherichia coli/genetics , Genetic Complementation Test , Nucleic Acid Heteroduplexes/metabolism , Phenotype , RNA, Bacterial/metabolism , Ribonuclease H/geneticsABSTRACT
Wild-type genes which, when overexpressed, are capable of restoring the growth deficiency of the division mutant ftsZ84 of Escherichia coli on L medium containing no added NaCl have been isolated. One of these genes is rcsB, a positive regulator of colanic acid biosynthesis. A direct relationship between rcsB expression and FtsZ activity was observed, suggesting that RcsB specifically increases transcription of ftsZ, thus accounting for the restoration of colony formation by ftsZ84 mutant cells. Analysis of the 5' upstream sequence of rcsB revealed, in addition to the sigma 54 promoter sequence previously reported, a presumptive sigma 70 promoter and LexA-binding site plus an upstream sequence that is found to be essential for the expression of rcsB on a plasmid. The absence of the sigma 54 factor does not have a negative effect on the transcription of rcsB. The RcsB protein is an activator of its own synthesis, particularly in the presence of NaCl. Evidence which suggests that RcsB can be phosphorylated by a presumably modified EnvZ or PhoM sensor protein leading to a suppression of the growth deficiency of ftsZ84 mutant cells and to an increase in colanic acid production was obtained. We also demonstrated that the level of colanic acid is reduced when the cells carry a multicopy rcsC plasmid, suggesting that the RcsC sensor has phosphatase activity.
Subject(s)
Bacterial Proteins/genetics , Cytoskeletal Proteins , DNA-Binding Proteins , DNA-Directed RNA Polymerases , Escherichia coli Proteins , Escherichia coli/genetics , Gene Expression Regulation, Bacterial/genetics , Polysaccharides/biosynthesis , Serine Endopeptidases , Transcription Factors , Amino Acid Sequence , Bacterial Proteins/chemistry , Base Sequence , Escherichia coli/growth & development , Escherichia coli/metabolism , Genetic Complementation Test , Molecular Sequence Data , Phosphorylation , Plasmids/genetics , Polysaccharides, Bacterial/biosynthesis , Promoter Regions, Genetic , RNA Polymerase Sigma 54 , Recombinant Fusion Proteins/genetics , Sigma Factor/geneticsABSTRACT
Mutant strains of Escherichia coli were screened for the ability to grow on L agar plates containing 3.4 or 4.6 mM sodium azide. Most mutants had mutations located in the leucine region, presumably at the azi locus. Two of these mutants were found to have a mutation in the secA gene, but expression of the resistance phenotype also required the presence of upstream gene X. While a plasmid carrying the X-secA mutant gene pair was able to confer azide resistance to a sensitive host, a similar plasmid harboring the wild-type secA allele rendered a resistant strain sensitive to azide, indicating codominance of the two alleles. That azide inhibits SecA is consistent with the fact that SecA has ATPase activity, an activity that is often prone to inhibition by azide.
Subject(s)
Adenosine Triphosphatases/genetics , Azides/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Microbial/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Genes, Bacterial/drug effects , Membrane Transport Proteins , Mutation , DNA, Bacterial/genetics , Escherichia coli/drug effects , Phenotype , R Factors , Restriction Mapping , SEC Translocation Channels , SecA Proteins , Sodium Azide , Suppression, GeneticABSTRACT
The Fts proteins play an important role in the control of cell division in Escherichia coli. These proteins, which possibly form a functional complex, are encoded by genes that form an operon. In this study, we examined the properties of the temperature-sensitive mutation ftsZ84 harbored by low- or high-copy-number plasmids. Cells of strain AB1157, which had the ftsZ84 mutation, did not form colonies on salt-free L agar at 30 degrees C. When a low-copy-number plasmid containing the ftsZ84 mutation was present in these mutant cells, colony formation was restored on this medium at 30 degrees C, suggesting that FtsZ84 is probably less active than the wild-type protein and is therefore limiting in its capacity to trigger cell divisions. On the other hand, when the ftsZ84 mutation was harbored by the high-copy-number plasmid pBR325, colony formation was prevented on salt-free L agar plates whether the recipients were ftsZ84 mutant or parental cells, suggesting that, at high levels, FtsZ84 acts as a division inhibitor. The fact that colony formation was also prevented at 42 degrees C indicates that the FtsZ84 protein is not inactivated at the nonpermissive temperature. The possibility that FtsZ84 is a more efficient division inhibitor than the wild-type FtsZ is discussed. Evidence is also presented showing that a gene adjacent to mutT codes for a product that, under certain conditions, suppresses the ftsZ84 mutation.
Subject(s)
Bacterial Proteins/genetics , Escherichia coli/growth & development , Suppression, Genetic , Cloning, Molecular , DNA Restriction Enzymes , Escherichia coli/genetics , Genes, Bacterial , Genetic Complementation Test , Mutation , Phenotype , Plasmids , TemperatureABSTRACT
The effects of streptomycin and neomycin on the phenotypic suppression of frameshift mutations in the lacZ gene of Escherichia coli and on the efficiency of suppression of amber mutations in T4 phage by the informational supE tRNA nonsense suppressor were compared. Neomycin stimulated much more efficiently than streptomycin the phenotypic suppression of frameshift mutations. Because neomycin favors mismatches of the central codon base whereas streptomycin favors mismatches of the first codon base, this result suggests that mismatching of the central codon base pair and shifting of the reading frame are two correlated phenomena. In contrast, both streptomycin and neomycin stimulated about equally the efficiency of the tRNA nonsense suppressor, an effect probably related to their interference with the proofreading control in tRNA selection.
Subject(s)
Escherichia coli/genetics , Genes, Bacterial/drug effects , Genes/drug effects , Mutation , Neomycin/pharmacology , Streptomycin/pharmacology , T-Phages/genetics , Escherichia coli/drug effects , Phenotype , Species Specificity , Suppression, Genetic , T-Phages/drug effects , beta-Galactosidase/geneticsABSTRACT
This review surveys the different experimental approaches which describe the binding of tRNA to mRNA-programmed ribosomes and the control of tRNA selection. This selection is best described by the two-step model proposed by Hopfield and demonstrated by Thompson and his collaborators. The model involves a first control at the initial reversible binding of tRNA to the ribosome and a second control, the proofreading control, which promotes rejection of the incorrect tRNA from a high-energy intermediate during the transition from the initial to the final binding state. Streptomycin, neomycin, and ribosomal fidelity mutations appear to affect both control steps. Their effect can be related to the location of the mutated ribosomal proteins and to the conformational changes induced in the ribosome by the misreading agents. An alteration of the first control probably results from a distortion of the codon-anticodon interaction, while an alteration of the second control may be caused by a change in the association between ribosomal subunits.
Subject(s)
Bacterial Proteins/biosynthesis , Escherichia coli/metabolism , Neomycin/pharmacology , Protein Biosynthesis/drug effects , Ribosomal Proteins/genetics , Streptomycin/pharmacology , Anticodon/metabolism , Bacterial Proteins/genetics , Codon/metabolism , Escherichia coli/genetics , Kinetics , Models, Biological , Mutation , Peptide Chain Elongation, Translational/drug effects , RNA, Bacterial/metabolism , RNA, Transfer/metabolism , Ribosomes/metabolismABSTRACT
Neomycin-resistant mutants of Escherichia coli K12 were selected on a minimal succinate medium containing neomycin. In an in vitro poly(U)-dependent protein synthesizing assay, the ribosomes from the mutant strains showed lower levels of misreading than wild-type ribosomes in the presence of neomycin or other error-promoting aminoglycoside antibiotics or ethanol. The mutation was shown to affect a component of the 30S subunit and to facilitate the dissociation of ribosomes into subunits. It is suggested that an impairment in the subunit interaction may be responsible for the observed restriction of the stimulation of misreading.