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At least 991 records · Page 55Linked to original sources
PubMed1984

Transcription of ribosomal component genes and lac in a relA+/relA pair of Escherichia coli strains.

To determine the stringent response, a repression of gene activity during amino acid starvation assumed to be mediated by the effector necleotide guanosine tetraphosphate (ppGpp), of metabolically regulated constitutive genes, we measured the transcription of ribosomal protein genes, the constitutive lac operon, and stable RNA genes in a variety of growth media and after amino acid starvation in a relA+/relA pair of Escherichia coli B/r strains. For rRNA and tRNA (stable RNA) it has previously been shown that the distinction between stringent control and growth rate control is unfounded, as the function describing the stable RNA gene activities at different concentrations of guanosine tetraphosphate is independent of growth conditions (exponential growth or amino acid starvation) and of the relA allele present. Here, the results indicated that the stringent responses of ribosomal protein genes and lac differ from their metabolic control during exponential growth in different media. This can be explained by polarity and RNA polymerase sink effects during amino acid starvation which are irrelevant for stable RNA genes but which are superimposed on mRNA gene activities.

Amino Acids↗
PubMed1996

Genomic diversity and differentiation among phytoplasma strains in 16S rRNA groups I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas).

Conserved gene sequences, including 16S rRNA and ribosomal protein gene sequences, were used to evaluate genetic variations in phytoplasma strains belonging to 16S rRNA groups I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas). We used PCR to amplify the sequences of the 16S ribosomal DNA and a segment of the ribosomal protein gene operon (encoding the 3' region of rps19, all of rp122, and rps3) from diverse phytoplasma group I and III strains. Additional chromosomal gene sequences of group I strains were also amplified. The PCR products amplified from members of each group of phytoplasmas were compared by performing restriction fragment length polymorphism (RFLP) analyses. On the basis of the RFLP patterns observed and similarity coefficients derived from combined RFLP analyses, the phytoplasma strains belonging to groups I and III were placed in distinct 16S rRNA, ribosomal protein, and 16S rRNA-ribosomal protein subgroups. Analyses of two or more conserved gene sequences revealed that members of the two groups were more diverse than previously thought. Subgroup differentiation on the basis of our combined analyses of 16S rRNA and ribosomal protein gene sequences seemed to adequately reflect the levels of chromosomal homology determined by DNA-DNA hybridization assays. On the basis of unique RFLP profiles, we identified new, previously unclassified group I phytoplasma strains, including the organisms that are associated with Ipomoea obscura witches'-broom [subgroup 16SrI-F(rr-rp)], maize bushy stunt [subgroup 16SrI-I(rr-rp)], and Mexican periwinkle virescence [subgroup 16SrI-J(rr-rp)], and new, previously unclassified group III phytoplasma strains, including the organism that is associated with pecan bunch [subgroup 16SrIII-H(rr-rp)]. On the basis of the results of our analyses of 16S rRNA and ribosomal protein conserved gene sequences, we recognized 9 group I subgroups and eight group III subgroups. We propose that phytoplasma strains belonging to each group I and III subgroup should be distinguished taxonomically at a level equivalent to the subspecies level.

Base Sequence↗
PubMed2004

Inter-genomic displacement via lateral gene transfer of bacterial trp operons in an overall context of vertical genealogy.

BACKGROUND: The growing conviction that lateral gene transfer plays a significant role in prokaryote genealogy opens up a need for comprehensive evaluations of gene-enzyme systems on a case-by-case basis. Genes of tryptophan biosynthesis are frequently organized as whole-pathway operons, an attribute that is expected to facilitate multi-gene transfer in a single step. We have asked whether events of lateral gene transfer are sufficient to have obscured our ability to track the vertical genealogy that underpins tryptophan biosynthesis. RESULTS: In 47 complete-genome Bacteria, the genes encoding the seven catalytic domains that participate in primary tryptophan biosynthesis were distinguished from any paralogs or xenologs engaged in other specialized functions. A reliable list of orthologs with carefully ascertained functional roles has thus been assembled and should be valuable as an annotation resource. The protein domains associated with primary tryptophan biosynthesis were then concatenated, yielding single amino-acid sequence strings that represent the entire tryptophan pathway. Lateral gene transfer of several whole-pathway trp operons was demonstrated by use of phylogenetic analysis. Lateral gene transfer of partial-pathway trp operons was also shown, with newly recruited genes functioning either in primary biosynthesis (rarely) or specialized metabolism (more frequently). CONCLUSIONS: (i) Concatenated tryptophan protein trees are congruent with 16S rRNA subtrees provided that the genomes represented are of sufficiently close phylogenetic spacing. There are currently seven tryptophan congruency groups in the Bacteria. Recognition of a succession of others can be expected in the near future, but ultimately these should coalesce to a single grouping that parallels the 16S rRNA tree (except for cases of lateral gene transfer). (ii) The vertical trace of evolution for tryptophan biosynthesis can be deduced. The daunting complexities engendered by paralogy, xenology, and idiosyncrasies of nomenclature at this point in time have necessitated an expert-assisted manual effort to achieve a correct analysis. Once recognized and sorted out, paralogy and xenology can be viewed as features that enrich evolutionary histories.

Bacteria↗
PubMed1984

Effects of site-directed mutations in the central domain of 16 S ribosomal RNA upon ribosomal protein binding, RNA processing and 30 S subunit assembly.

Using a multicopy plasmid encoding the Escherichia coli rrnB ribosomal RNA operon and the techniques of in vitro site-directed mutagenesis, we have introduced several small alterations into the central domain of 16 S rRNA, which encompasses nucleotides 560 to 890. Four of the rRNAs studied contained deletions and one contained an insertion. The altered small ribosomal subunit rRNAs were used to investigate relationships among 16 S rRNA processing, protein-16 S rRNA interactions and assembly of the 30 S ribosomal subunit. Analysis of plasmid-coded transcripts from maxicells revealed that products from wild-type 16 S rRNA genes were fully processed and assembled into mature 30 S subunits. Under the same conditions, the processing and assembly of transcripts derived from the mutant plasmids were severely impaired. In some instances, the mutations completely blocked both processes, while in other cases rRNA maturation and ribosome assembly were retarded, but not eliminated completely. In all cases, the mutations led to the accumulation of the 17 S precursor to 16 S rRNA. The mutant 17 S rRNAs were purified and incubated with various combinations of E. coli ribosomal proteins S6, S8, S15 and S18, which are known to bind to the central domain of 16 S rRNA. Ribonuclease digestion of the resulting protein-17 S rRNA complexes and fractionation of the products permitted detection of three distinct protein-RNA fragment complexes which contained S8, S8 + S15, or S6 + S8 + S15 + S18. Whereas wild-type 17 S rRNA was able to form all three of these complexes, deletion of nucleotides 693 to 721 or 822 to 874 abolished the interaction of S6 and S18, and removal of nucleotides 659 to 718 prevented the binding of S6, S15 and S18. In contrast, elimination of residue 614, or the presence of a 16-base insertion between nucleotides 614 and 615, had no significant effect on the binding of any of the four proteins tested. Together, our results demonstrate that 16 S rRNA maturation and 30 S subunit assembly are tightly coupled, and show that, in at least some cases, defects in these processes can be correlated with the inability of particular ribosomal proteins to associate with altered rRNA molecules. Moreover, we have confirmed the essentiality of certain rRNA sequences for the formation and/or stabilization of these protein-rRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Centrifugation, Density Gradient↗
PubMed2004

Intragenomic heterogeneity of the 16S rRNA-23S rRNA internal transcribed spacer among Pseudomonas syringae and Pseudomonas fluorescens strains.

The 16S-23S rRNA internal transcribed spacer regions (ITS1) from 14 strains of Pseudomonas syringae and P. fluorescens were sequenced. ITS1 exhibited significant sequence variability among different operons within a single genome. From 1 to 4 types of ITS1 were found in individual genomes of the P. syringae and P. fluorescens strains. A total of eight ITS1 types were identified among strains studied. The ITS1 nucleotide sequences consisted of conserved blocks including, among others, a stem-forming region of box B, tRNAIle and tRNAAla genes and several variable blocks. The differences in the variable regions were mostly due to insertions and/or deletions of nucleotide blocks. The intragenomic heterogeneity of ITS1 was brought about by different combinations of variable blocks, which possibly have resulted from recombination and horizontal transfer.

Base Sequence↗
PubMed2006

Nitrogen fixation at 92 degrees C by a hydrothermal vent archaeon.

A methanogenic archaeon isolated from deep-sea hydrothermal vent fluid was found to reduce N(2) to NH(3) at up to 92 degrees C, which is 28 degrees C higher than the current upper temperature limit of biological nitrogen fixation. The 16S ribosomal RNA gene of the hyperthermophilic nitrogen fixer, designated FS406-22, was 99% similar to that of non-nitrogen fixing Methanocaldococcus jannaschii DSM 2661. At its optimal growth temperature of 90 degrees C, FS406-22 incorporated (15)N(2) and expressed nifH messenger RNA. This increase in the temperature limit of nitrogen fixation could reveal a broader range of conditions for life in the subseafloor biosphere and other nitrogen-limited ecosystems than previously estimated.

Amino Acid Sequence↗
PubMed1985

Transcription of herpes simplex virus tk sequences under the control of wild-type and mutant human RNA polymerase I promoters.

We studied RNA polymerase I transcription in cells transfected with a plasmid, prHuTK, containing the herpes simplex virus tk gene fused to a human rRNA promoter. Primer extension analysis of tk RNA isolated from COS cells transfected with prHuTK reveals that transcription from the RNA polymerase I promoter is highly efficient and initiates at the same position used for the synthesis of endogenous rRNA in HeLa cells. The RNA products derived from prHuTK are distinguishable from normal RNA polymerase II transcripts of tk in that they are not polyadenylated, are extremely unstable, and are found predominantly in the nucleus. Moreover, the transcription observed is resistant to 300 micrograms of alpha-amanitin per ml. These results strongly suggest that prHuTK transcription is under the control of the human rRNA promoter and RNA polymerase I. To further characterize the activity of the human rDNA promoter in vivo, a series of 5' and 3' deletion mutants was tested in this transfection assay. The deletion analysis indicates that a core region of ca. 40 base pairs overlapping the initiation site is critical for transcription. In addition, a region between nucleotides -234 and -131 upstream from the core sequence serves to modulate the efficiency of transcription. Insertion into prHuTK of additional ribosomal nontranscribed spacer DNA or the simian virus 40 enhancer element has no apparent effect on the promoter activity. Surprisingly, RNA polymerase II transcripts synthesized at low levels from two start sites within the core control element of the wild-type RNA polymerase I promoter are activated upon deletion of upstream RNA polymerase I promoter sequences. However, these RNA polymerase II transcripts are not expressed from the endogenous rRNA promoter.

Amanitins↗
PubMed1998

Distribution of the pressure-regulated operons in deep-sea bacteria.

DNA regions corresponding to portions of two different pressure-regulated operons previously identified in two deep-sea barophilic bacteria were separately PCR amplified from a variety of deep-sea microorganisms and sequenced. With the two sets of primers employed, amplification was particularly successful from the more barophilic bacteria examined. 16S rRNA sequence analysis revealed that these bacteria are all phylogenetically related and belong in a sub-branch of the genus Shewanella containing only the deep-sea Shewanella barophilic bacteria. We define this sub-branch as the 'Shewanella barophile branch' containing at least two different species. Our results suggest that the DNA sequences of the pressure-regulated operons can be regarded as marker sequences to identify the Shewanella barophilic strains.

Amino Acid Sequence↗
PubMed1998

16S rRNA mutation associated with tetracycline resistance in a gram-positive bacterium.

A genetic basis for tetracycline resistance in cutaneous propionibacteria was suggested by comparing the nucleotide sequences of the 16S rRNA genes from 16 susceptible and 21 resistant clinical isolates and 6 laboratory-selected tetracycline-resistant mutants of a susceptible strain. Fifteen clinical isolates resistant to tetracycline were found to have cytosine instead of guanine at a position cognate with Escherichia coli 16S rRNA base 1058 in a region important for peptide chain termination and translational accuracy known as helix 34. Cytosine at base 1058 was not detected in the laboratory mutants or the tetracycline-susceptible strains. The apparent mutation was recreated by site-directed mutagenesis in the cloned E. coli ribosomal operon, rrnB, encoded by pKK3535.E. coli strains carrying the mutant plasmid were more resistant to tetracycline than those carrying the wild-type plasmid both in MIC determinations and when grown in tetracycline-containing liquid medium. These data are consistent with a role for the single 16S rRNA base mutation in clinical tetracycline resistance in cutaneous propionibacteria.

DNA, Bacterial↗
PubMed2005

Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression.

Certain nascent peptide sequences, when within the ribosomal exit tunnel, can inhibit translation termination and/or peptide elongation. The 24 residue leader peptidyl-tRNA of the tna operon of E. coli, TnaC-tRNA(Pro), in the presence of excess tryptophan, resists cleavage at the tnaC stop codon. TnaC residue Trp12 is crucial for this inhibition. The approximate location of Trp12 in the exit tunnel was determined by crosslinking Lys11 of TnaC-tRNA(Pro) to nucleotide A750 of 23S rRNA. Methylation of nucleotide A788 of 23S rRNA was reduced by the presence of Trp12 in TnaC-tRNA(Pro), implying A788 displacement. Inserting an adenylate at position 751, or introducing the change U2609C in 23S rRNA or the change K90H or K90W in ribosomal protein L22, virtually eliminated tryptophan induction. These modified and mutated regions are mostly located near the putative site occupied by Trp12 of TnaC-tRNA(Pro). These findings identify features of the ribosomal exit tunnel essential for tna operon induction.

Biotinylation↗
PubMed2001

Phylogenetic analyses of Synechococcus strains (cyanobacteria) using sequences of 16S rDNA and part of the phycocyanin operon reveal multiple evolutionary lines and reflect phycobilin content.

The genus Synechococcus (cyanobacteria), while containing morphologically similar isolates, is polyphyletic and organisms presently classified as such require reclassification into several independent genera. Studies based on analysis of 16S rRNA gene sequences have shown that members of the genus Synechococcus are affiliated to three of seven deeply branching cyanobacterial lineages. In addition, some strains do not appear to be associated with any of these lineages and may represent novel clades. In this report, a cyanobacterial phylogeny based on 16S rDNA sequences, including 14 newly sequenced Synechococcus isolates, is presented. One newly sequenced Synechococcus strain (PCC 7902) did not have any close relatives amongst cyanobacterial isolates currently contained in 16S rDNA sequence databases and was only loosely affiliated to a cyanobacterial lineage in which no other Synechococcus strains were found. Three hot-spring Synechococcus isolates, including two that were newly sequenced in this study (PCC 6716 and PCC 6717), formed an additional cyanobacterial lineage. These results indicated that Synechococcus species are affiliated to five of eight deeply branching cyanobacterial lineages. Part of the phycocyanin (PC) gene sequence (cpc), including the intergenic spacer (IGS) between cpcB and cpcA and the corresponding flanking regions (cpcBA-IGS), was used to investigate relationships between closely related Synechococcus isolates. Previously described PCR primers did not amplify this region from the majority of strains under investigation, so a new set of primers was designed that allowed amplification and sequencing of the cpcBA-IGS and flanking regions from 38 Synechococcus species. Phylogenetic analysis of this region was largely consistent with that obtained from 16S rDNA sequence analysis and revealed a relationship between the primary PC DNA sequence and the phycobilin content of cells.

Base Sequence↗
PubMed1985

Evidence for functional interaction between domains II and V of 23S ribosomal RNA from an erythromycin-resistant mutant.

A mutation affording low levels of erythromycin resistance has been obtained by in vitro hydroxylamine mutagenesis of a cloned ribosomal RNA operon from Escherichia coli. The site of the mutational event responsible for antibiotic resistance was localized to the gene region encoding domain II of 23S rRNA by replacement of restriction fragments in the wild-type plasmid by corresponding fragments from the mutant plasmid. DNA sequencing showed that positions 1219-1230 of the 23S rRNA gene are deleted in the mutant. Since all previously characterized rRNA mutations conferring resistance to erythromycin show changes exclusively in domain V, our present findings provide direct evidence for functional interaction between domains II and V of 23S rRNA.

Ampicillin↗
PubMed2003

Differences in clinical manifestations and hematological and serological responses after experimental infection with genetic variants of Anaplasma phagocytophilum in sheep.

Five-month-old lambs were experimentally infected with two 16S rRNA genetic variants of Anaplasma phagocytophilum, variants 1 (GenBank accession no. M73220) and 2 (GenBank accession no. AF336220). Additional sequencing of the groESL heat shock operon gene indicated that these variants differ in three nucleotides at positions 782, 824, and 890. The variants were obtained by blood sampling of A. phagocytophilum-infected lambs from one sheep flock in Norway and were stored at -70 degrees C with 10% dimethyl sulfoxide as a cryoprotectant before being inoculated intravenously into susceptible lambs. The infectious blood contained, per ml, approximately 0.5 x 10(6) neutrophils infected with either of the variants. Six weeks after the primary inoculation, the lambs were challenged with the same infectious dose of the heterologous variant. The results of the study indicate a marked difference in clinical manifestation, neutropenia, antibody response, and cross-protection after experimental infection with the two variants of A. phagocytophilum.

Anaplasma phagocytophilum↗
PubMedDate not supplied

[Molecular structure of the locus for sucrose utilization by Lactobacillus plantarum: comparison with Pediococcus pentosaceus].

Structure of the sucrose utilization locus in a Lactobacillus plantarum type strain was studied using PCR and Southern hybridization. Restriction map analysis revealed its high similarity to the sequenced sucrose utilization locus of Pediococcus pentosaceus pSRQ1. The L. plantarum locus proved containing oppositely oriented scrA and the scrBRagl operon, but not agaS. The L. plantarum sucrase gene (scrB) was partly sequenced. A higher (98.6%) homology was revealed between scrB than between the 16S rRNA genes of L. plantarum and P. pentosaceus, suggesting horizontal transfer of the sucrose utilization locus between the genera of lactic acid bacteria. Amino acid sequence analysis showed that the ScrB proteins of the two species belong to a subfamily of glycosyl hydrolase family GH32 which includes various beta-fructosidases.

Amino Acid Sequence↗
PubMed1981

Nucleotide sequence of an Escherichia coli tRNA (Leu 1) operon and identification of the transcription promoter signal.

Fourteen different DNA fragments containing Escherichia coli tRNA genes have been cloned using the vector pBR322. We report the methods of cloning, the identification of specific tRNA genes, and the presence or absence of rRNA genes on these cloned DNA fragments. In particular, one chimeric plasmid contains a 17.0 kilobase pair EcoRI fragment bearing tRNA(Leu 1) sequences. Using nucleotide sequence analysis we have identified a cluster of three tandem tRNA(Leu 1) genes separated by intergenic spacers of 27 and 34 base pairs, respectively. The nucleotide sequence upstream of the first gene contains a transcription promoter site. A G-C rich sequence (5'-CGCCTCC-3') found between the Pribnow box and initiation site is very similar to the corresponding sequence found in other genes which are under stringent control.

Base Sequence↗
PubMed1983

Functional interrelationship between two tandem E. coli ribosomal RNA promoters.

The Escherichia coli chromosome carries seven cistrons encoding ribosomal RNA sequences. In all cases studied, in vitro and in vivo, it has been established that transcription is initiated from two tandem promoters. The expression of the rRNA cistrons is regulated in response to growth rate as well as to aminoacyl tRNA availability. In the present study, a plasmid (pPS1) carrying the promoter region of the rrnA cistron fused to the terminator region of rrnB has been used for in vitro transcription experiments. The presence of the terminators (T1 and T2) together with the fact that supercoiled DNA is found to be a highly efficient template, provide an ideal in vitro system in which to study the functional interrelationship between the two tandem promoters of E. coli rRNA cistrons. The results suggest that the rate of rRNA synthesis in E. coli cells growing in various conditions, as reflected by the availability of RNA polymerase, is primarily dependent on the properties of the two tandem rRNA promoters.

Escherichia coli↗
PubMed1984

Species-specific rDNA transcription is due to promoter-specific binding factors.

RNA polymerase I transcription factors were purified from HeLa and mouse L cell extracts by phosphocellulose chromatography. Three fractions from each species were found to be required for transcription. One of these fractions, virtually devoid of RNA polymerase I activity, was found to form a stable preinitiation complex with small DNA fragments containing promoter sequences from the homologous but not the heterologous species. These species-specific DNA-binding factors can explain nucleolar dominance in vivo in mouse-human hybrid somatic cells and species specificity in cell-free, RNA polymerase I-dependent transcription systems. The evolution of species-specific transcriptional control signals may be the natural outcome of a special relationship that exists between the RNA polymerase I transcription machinery and the multigene family coding for rRNA.

Animals↗