The number of rRNA genes in Escherichia coli (original) (raw)
Related papers
Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools
Annales de l'Institut Pasteur / Microbiologie, 1986
Deoxyribonucleic acid from 41 different bacterial species (including Gramnegative and Gram-positive species) were cleaved by different restriction endonucleases, electrophoresed in agarose and transferred to nylon filters. The DNA fragments carrying rRNA genes (rDNA) were localized by hybridization with a 32p-labelled Escherichia coli 16 + 23S rRNA probe. A pattern of hybridized fragments was obtained for each DNA tested. Within a bacterial species (defined as a DNA hybridization group), one or several rDNA restriction patterns were observed. When DNA hybridization data were available, strains showing identical patterns were highly related with insignificant divergence. In a species, different patterns corresponded to significant divergence, as evaluated by thermal stability studies of DNA/DNA hybrids. Sets of rDNA restriction fragment sizes might constitute useful data for inclusion in species and type strain descriptions. Such data might later prove useful in identification of bacteria when biochemical characteristics are poor or atypical.
An assessment of 10 tetrameric restriction enzymes (TREs) was conducted by using a computer-simulated restriction fragment length polymorphism (RFLP) analysis for over 100 proximally and distally related bacterial small-subunit (SSU) rRNA gene sequences. Screening SSU rDNA clone libraries with TREs has become an effective strategy because of logistic simplicity, commercial availability, and economy. However, the rationale for selecting the type and number of TREs has not been systematically evaluated. Our objective was to identify the optimal combination of TREs for RFLP screening of cloned SSU rRNA genes from undefined bacterial clone libraries. After computer-simulated TRE digestion, the resultant fragments were categorized on the basis of the frequency of different restriction fragment size classes. Three groups of distribution patterns for the TREs were determined and further examined via graphical exploratory data analysis. The RFLP size-frequency distribution data for each group of enzymes were then used to infer phylogenetic relationships via the neighbor-joining method. The resulting bootstrap values and the correct placement of node bifurcations were used as additional criteria to evaluate the efficacy of the selected TREs. These RFLP data were compared with known phylogenetic relationships based on SSU rRNA sequence analysis as defined by the Ribosomal Database Project. A heuristic approach testing random combinations of TREs showed that three or more TRE combinations detected >99% of the operational taxonomic units (OTUs) within the model data set. OTUs that remained undetected after three TRE treatments had a median sequence similarity of 96.1%. Of the 10 restriction enzymes examined, HhaI, RsaI, and BstUI (group 3) were the most efficacious at detecting and differentiating bacterial SSU rRNA genes on the basis of their ability to correctly classify OTUs. Group 3 TREs are therefore recommended for screening in studies using bacterial SSU rRNA genes as descriptors of in situ microbial diversity.
Computer identification of Escherichia coli rRNA gene restriction patterns
Research in Microbiology, 1998
A total of 191 strains of Escherichia coil comprising 164 serovar reference strains and 28 clinical strains were characterized by rRNA gene restriction patterns (ribotypes) generated after cleavage of total DNA with Mlul, Clan or Hindlll restriction endonucleases and hybridization of fragments with acetylaminofluorene-labelled 16+23S rRNA. A wide diversity of ribotypes was observed with endonucleases MIul (104 patterns}, Clal (90 patterns) and Hindlll (98 patterns). When Miul was used, 85% of patterns (11 to 15 fragments) shared five fragments 17.09, 3.94, 3.06, 2.23 and 1.76 kb in size. When these fragments were used as internal standards, the percent errors in fragment length determination was half of that obtained with an external standard. Two fragment size databases of Mlul and Clal ribotypes were built. Automatic identification was obtained after setting the percent fragment size variation tolerance (error) at 5%.
An analysis of the ribosomal ribonucleic acids of Escherichia coli by hybridization techniques
The Biochemical journal, 1969
From analyses of the hybridization of Escherichia coli rRNA (ribosomal RNA) to homologous denatured DNA, the following conclusions were drawn. (1) When a fixed amount of DNA was hybridized with increasing amounts of RNA, only 0.35+/-0.02% of E. coli DNA was capable of binding (16s+23s) rRNA. Although preparations of 16s and 23s rRNA were virtually free from cross-contamination, the hybridization curves for purified 16s or 23s rRNA were almost identical with that of the parent specimen containing 1 weight unit of 16s rRNA mixed with 2 weight units of 23s rRNA. The 16s and 23s rRNA also competed effectively for the same specific DNA sites. It appears that these RNA species each possess all hybridizing species typical of the parent (16s+23s) rRNA specimen, though probably in different relative amounts. (2) By using hybridization-efficiency analysis of DNA-RNA hybridization curves (Avery & Midgley, 1969) it was found that (a) 0.45% of the DNA would hybridize total rRNA and (b) when so l...
Detection of specific sequences among DNA fragments separated by gel electrophoresis
Journal of molecular biology, 1975
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Esoherichia coli and Xenopus lacvis, and from several mammals.
Intraspecific diversity of the 23S rRNA gene and the spacer region downstream in Escherichia coli
Journal of bacteriology, 1999
The molecular microevolution of the 23S rRNA gene (rrl) plus the spacer downstream has been studied by sequencing of different operons from some representative strains of the Escherichia coli ECOR collection. The rrl gene was fully sequenced in six strains showing a total of 67 polymorphic sites, a level of variation per nucleotide similar to that found for the 16S rRNA gene (rrs) in a previous study. The size of the gene was highly conserved (2902 to 2905 nucleotides). Most polymorphic sites were clustered in five secondary-structure helices. Those regions in a large number of operons were sequenced, and several variations were found. Sequences of the same helix from two different strains were often widely divergent, and no intermediate forms existed. Intercistronic variability was detected, although it seemed to be lower than for the rrs gene. The presence of two characteristic sequences was determined by PCR analysis throughout all of the strains of the ECOR collection, and some ...
Cloning of an E. coli ribosomal RNA gene and its promoter region from
Gene, 1978
The DNA of the specialized transducing phage ~rifd18, which carries a bacterial rRNA transcription unit, was digested with restriction enzymes EcoRI and/or BamHI. Attempts were made to clone fragments containing the presumed rRNA promoter region or the entire rRNA gene in RSF2124 or pBR313 plasmid vectors with the following results:
Applied Microbiology and Biotechnology, 2008
Real-time polymerase chain reaction (PCR)-based methodology for the determination of rRNA gene (rrn) copy number was introduced and demonstrated. Both absolute and relative quantifications were tested with Escherichia coli. The separate detection of rRNA gene and chromosomal DNA was achieved using two primer sets, specific for 16S rRNA gene and for D-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. As dxs is a single-copy gene of E. coli chromosomal DNA, the rrn copy number can be determined as the copy ratio of rrn to dxs. This methodology was successfully applied to determine the rrn copy number in E. coli cells. The results from absolute and relative quantifications were identical and highly reproducible with coefficient of variation (CV) values of 1.8–4.6%. The estimated rrn copy numbers also corresponded to the previously reported value in E. coli (i.e., 7), indicating that the results were reliable. The methodology introduced in this study is faster and cost-effective without safety problems compared to the traditionally used Southern blot analysis. The fundamentals in our methodology would be applicable to any microorganism, as long as having the sequence information of the rRNA gene and another chromosomal gene with a known copy number.