ABSTRACT
It has been shown that in Escherichia coli the chaperone DnaK is necessary for the late stages of 50S and 30S ribosomal subunit assembly in vivo. Here we focus on the roles of other HSPs (heat-shock proteins), including the chaperonin GroEL, in addition to DnaK, in ribosome biogenesis at high temperature. GroEL is shown to be required for the very late 45S-->50S step in the biogenesis of the large ribosome subunit, but not for 30S assembly. Interestingly, overproduction of GroES/GroEL can partially compensate for a lack of DnaK/DnaJ at 44 degrees C.
Subject(s)
Chaperonin 60/metabolism , Escherichia coli Proteins , Escherichia coli/physiology , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Ribosomes/physiology , Alleles , Chaperonin 10/genetics , Chaperonin 10/metabolism , Chaperonin 60/genetics , Escherichia coli/genetics , Genotype , HSP70 Heat-Shock Proteins/genetics , Hot Temperature , Ribosomes/genetics , TemperatureABSTRACT
The Escherichia coli chaperone DnaK is vital for many cellular functions, including ribosome biogenesis at high temperature. Thus, the dnaK756-ts (lambdaR) mutant, at the non-permissive temperature, is inhibited at a late stage of ribosome assembly, yielding 21S, 32S and 45S precursor particles. This defect, unlike the lambda resistance and thermosensitivity phenotypes, is not complemented by lysogenisation with a transducing phage lambda dnaK+ bearing the wild-type dnaK gene. However this dominant phenotype becomes recessive when dnaK+ is expressed from a medium-copy-number plasmid. On the other hand, an excess of DnaK causes an unexpected dominant-lethal effect of the dnaK756 allele near non-permissive temperatures. This interplay between the dnaK+ and dnaK756 alleles supports the idea of that DnaK oligomers form in the cell.
Subject(s)
Bacteriophage lambda/genetics , Escherichia coli Proteins , Escherichia coli/physiology , HSP70 Heat-Shock Proteins/genetics , Ribosomes/metabolism , Alleles , Escherichia coli/virology , Gene Expression Regulation, Bacterial , Genes, Dominant , Genes, Lethal , Genetic Complementation Test , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/metabolism , Phenotype , Plasmids , Protein FoldingABSTRACT
We have isolated an Escherichia coli gene which, when overexpressed, is able to complement the permeability defects of a vancomycin-susceptible mutant. This gene, designated sanA, is located at min 47 of the E. coli chromosome and codes for a 20-kDa protein with a highly hydrophobic amino-terminal segment. A strain carrying a null mutation of the sanA gene, transferred to the E. coli chromosome by homologous recombination, is perfectly viable, but after two generations at high temperature (43 degrees C), the barrier function of its envelope towards vancomycin is defective.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli Proteins , Escherichia coli/drug effects , Escherichia coli/genetics , Genes, Bacterial/genetics , Membrane Proteins/genetics , Vancomycin/pharmacology , Amino Acid Sequence , Bacterial Outer Membrane Proteins/analysis , Base Sequence , Cell Membrane Permeability , Cloning, Molecular , Detergents/pharmacology , Drug Resistance, Microbial/genetics , Escherichia coli/growth & development , Hot Temperature , Membrane Proteins/biosynthesis , Membrane Proteins/metabolism , Molecular Sequence Data , Mutation , Restriction Mapping , Sequence Analysis, DNA , Sodium Dodecyl Sulfate/pharmacologyABSTRACT
The prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11-methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C-terminus of the protein and plasmids overproducing the wild-type and the two mutant proteins have been constructed. The wild-type PrmA protein could be crosslinked to its radiolabelled substrate, S-adenosyl-L-methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H-labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro-constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable.
Subject(s)
Escherichia coli/enzymology , Escherichia coli/genetics , Genes, Bacterial , Methyltransferases/genetics , Alleles , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cross-Linking Reagents , DNA, Bacterial/genetics , Methyltransferases/metabolism , Molecular Sequence Data , Mutagenesis , Phenotype , Plasmids/genetics , Point Mutation , Ribosomal Proteins/metabolism , S-Adenosylmethionine/metabolismABSTRACT
Genetic complementation and enzyme assays have shown that the DNA region between panF, which encodes pantothenate permease, and orf1, the first gene of the fis operon, encodes prmA, the genetic determinant for the ribosomal protein L11 methyltransferase. Sequencing of this region identified one long open reading frame that encodes a protein of 31,830 Da and corresponds to the prmA gene. We found, both in vivo and in vitro, that prmA is expressed from promoters located upstream of panF and thus that the panF and prmA genes constitute a bifunctional operon. We located the major 3' end of prmA transcripts 90 nucleotides downstream of the stop codon of prmA in the DNA region upstream of the fis operon, a region implicated in the control of the expression of the fis operon. Although no promoter activity was detected immediately upstream of prmA, S1 mapping detected 5' ends of mRNA in this region, implying that some mRNA processing occurs within the bicistronic panF-prmA mRNA.
Subject(s)
Escherichia coli Proteins , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression , Genes, Bacterial , Membrane Transport Proteins/genetics , Methyltransferases/genetics , Organic Anion Transporters , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial/isolation & purification , DNA, Bacterial/metabolism , Membrane Transport Proteins/biosynthesis , Methylation , Methyltransferases/biosynthesis , Molecular Sequence Data , Operon , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , RNA, Messenger/isolation & purification , Restriction Mapping , Ribosomal Proteins/metabolism , Transcription, GeneticABSTRACT
To determine whether the biogenesis of ribosomes in Escherichia coli is the result of the self-assembly of their different constituents or involves the participation of additional factors, we have studied the influence of a chaperone, the product of the gene dnaK, on ribosome assembly in vivo. Using three thermosensitive (ts) mutants carrying the mutations dnaK756-ts, dnaK25-ts, and dnaK103-ts, we have observed the accumulation at nonpermissive temperature (45 degrees C) of ribosomal particles with different sedimentation constants--namely, 45S, 35S, and 25S along with the normal 30S and 50S ribosomal subunits. This is the result of a defect not in thermostability but in ribosome assembly at the nonpermissive temperature. These abnormal ribosomal particles are rescued if the mutant cells are returned to 30 degrees C. Thus, the product of the dnaK gene is implicated in ribosome biogenesis at high temperature.
Subject(s)
Escherichia coli Proteins , HSP70 Heat-Shock Proteins , Heat-Shock Proteins/physiology , Proteins/physiology , Ribosomes/ultrastructure , Chaperonins , Escherichia coli/ultrastructure , Morphogenesis , TemperatureABSTRACT
Pantothenate permease, the product of the panF gene, catalyzes the sodium-dependent uptake of extracellular pantothenate. The panF gene was isolated from an Escherichia coli genomic DNA library and subcloned into multicopy plasmids. Increased copy number of the panF+ allele resulted in increased rates of pantothenate uptake and a significant increase in the steady-state intracellular pantothenate concentration. Despite the higher levels of pantothenate, the utilization of pantothenate for coenzyme A formation was not elevated, indicating that pantothenate kinase activity is the dominant regulator of coenzyme A biosynthesis. DNA sequencing of the panF gene revealed the presence of a single open reading frame that encoded a hydrophobic protein with a molecular weight of 51,992. Sequence analysis predicts that pantothenate permease is an integral membrane protein possessing 12 hydrophobic membrane-spanning domains connected by short hydrophilic sequences. The predicted topological profile of pantothenate permease is similar to that of other membrane carriers that catalyze cation-dependent symport.
Subject(s)
Escherichia coli Proteins , Escherichia coli/genetics , Genes, Bacterial , Membrane Transport Proteins/genetics , Organic Anion Transporters , Amino Acid Sequence , Base Sequence , Cloning, Molecular/methods , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Escherichia coli/enzymology , Gene Expression , Genotype , Kinetics , Membrane Transport Proteins/metabolism , Molecular Sequence Data , Plasmids , Protein Conformation , Recombinant Proteins/metabolism , Restriction MappingABSTRACT
I describe a general and rapid procedure allowing the isolation of specialized lambda transducing phages from a lambda library by lysogenic complementation of defective mutants of Escherichia coli. As an example, the cloning of the E. coli fabE gene and of two other adjacent genetic determinants is presented. Subcloning and determination of its nucleotide sequence reveals that fabE codes for the biotin carboxyl carrier protein (BCCP), one of the three subunits of acetyl coenzyme A carboxylase.
Subject(s)
Acetyl-CoA Carboxylase/genetics , Bacteriophage lambda , Cloning, Molecular/methods , Escherichia coli/genetics , Ligases/genetics , Amino Acid Sequence , Bacteriophage lambda/genetics , Base Sequence , Chromosomes, Bacterial , Escherichia coli/enzymology , Gene Library , Genes, Bacterial , Genetic Complementation Test , Molecular Sequence Data , Restriction Mapping , Sequence Homology, Nucleic Acid , Transduction, GeneticSubject(s)
DNA Replication/drug effects , Ethionine/pharmacology , Protein Biosynthesis/drug effects , Adenosine/analogs & derivatives , Adenosine/biosynthesis , Animals , Bacteria/metabolism , Cell Differentiation/drug effects , Cell Division/drug effects , Ethionine/analogs & derivatives , Ethionine/biosynthesis , Ethionine/metabolism , Humans , Liver/metabolism , Methionine/metabolism , Methylation , Protein Processing, Post-Translational/drug effects , RNA Processing, Post-Transcriptional/drug effects , RNA, Ribosomal/biosynthesis , Ribosomes/drug effects , Yeasts/metabolismABSTRACT
Analysis of 16-S rRNA synthesized in Escherichia coli D10 (met-) incubated in a medium containing ethionine in place of methionine shows that it lacks most and probably all of the methyl groups present in normal 16-SrRNA but possesses the same 3'-OH, and 5'-phosphate terminal sequences as the latter. 23-S rRNA formed in ethionine-treated cells also contains normal terminal sequences. 5-S rRNAs of normal and ethionine-treated E. coli D10 are identical. These results lead to the conclusion that methylation of ribosomal precursor RNAs is not necessary for their maturation to products with normal chain lengths and does not influence the conformation of 16-S rRNA.
Subject(s)
Escherichia coli/metabolism , Ethionine/metabolism , RNA, Ribosomal/biosynthesis , Ribosomes/metabolism , Kinetics , Methionine/metabolism , Methylation , Molecular Weight , Ribonucleotides/analysisABSTRACT
In the present study, the nature, proportions and distribution of methylated amino acids in ribosomal proteins from Escherichia coli grown in the presence of ethionine and from mutant prm 1 were studied. The undermethylated ribosomes had been labeled by addition in vitro or in vivo of radioactive methyl groups from S-adenosylmethionine or from methionine. The following compounds were identified : N alpha-mono-, di- and trimethylalanines, N epsilon-mono-, di- and trimethyllysines, methylamine and N alpha-trimethylalanyllysine. Except for the latter compound and N-alpha-dimethylalanine, all other derivatives had been previously identified in the literature. It is shown that the dipeptide had been in the past mistaken for N epsilon-monomethyllysine, and arises through incomplete hydrolysis in 24 hrs of the N-terminal peptide bond of protein L11. The results of the present study are discussed in the light of previous work on ribosomal protein methylation by the authors and other workers in the field.