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At least 721 records · Page 40Linked to original sources
PubMed1995

Intraspecific variation in small-subunit rRNA sequences in GenBank: why single sequences may not adequately represent prokaryotic taxa.

Small-subunit rRNA (SSU rRNA) sequencing is a powerful tool to detect, identify, and classify prokaryotic organisms, and there is currently an explosion of SSU rRNA sequencing in the microbiology community. We report unexpectedly high levels of intraspecific variation (within and between strains) of prokaryote SSU rRNA sequences deposited in GenBank. A total of 82% of the prokaryote species with two published SSU rRNA sequences had more variable positions than a 0.1% random sequencing error would predict, and 48% of these sequence pairs had more variable positions than predicted by a 1.0% random sequencing error. Other sources of sequence variability must account for some of this intraspecific variation. Given these results, phylogenetic studies and biodiversity estimates obtained by using prokaryotic SSU rRNA sequences cannot proceed under the assumption that rRNA sequences of single operons from single isolates adequately represent their taxa. Sequencing SSU rRNA molecules from multiple operons and multiple isolates is highly recommended to obtain meaningful phylogenetic hypotheses, as is careful attention to accurate strain identification.

Bacteria↗
PubMed1992

The 16S rRNA gene of Streptomyces lividans TK64 contains internal promoters.

A 632-bp Sau3AI fragment of Streptomyces lividans TK64 genome was found to confer promoter activity in Streptomyces and Escherichia coli. This fragment showed almost identical sequence (97.8%) to the S. coelicolor 16S rRNA segment encompassing from nucleotide 706 to 1337 region. The transcription start points of this fragment were identified by the primer extension method. Analysis of the nucleotide sequence upstream the transcription start points revealed two putative E. coli-like promoters resided within this fragment. The occurrence of internal promoters active in Streptomyces and E. coli was also confirmed in the 16S rRNA gene of rrnE operon from TK64.

Base Sequence↗
PubMed1992

Detection of intraspecific DNA polymorphism in Streptococcus salivarius subsp. thermophilus by a homologous rDNA probe.

Three ribosomal probes from Streptococcus salivarius subsp. thermophilus were cloned. Sequence data demonstrate that their juxtaposition corresponds to an entire operon. They were used in order to study ribosomal operon number and organization. rRNA genes were shown to be clustered in the order 5'-16S-23S-5S-3' and the number of rrn loci to vary within the subspecies. The smallest of the 3 probes was used for strain characterization. Substantial variability in hybridization patterns was observed among strains, resulting not only from, restriction fragment length polymorphism (RFLP) but also from the variability of ribosomal operon number.

Autoradiography↗
PubMed1999

5S rRNA gene deletions cause an unexpectedly high fitness loss in Escherichia coli.

In Escherichia coli, ribosomal RNAs (16S, 23S and 5S) are co-transcribed in a highly regulated manner from seven genomically dispersed operons. Previous studies on the cellular effects of altered levels of two of these rRNAs (16S and 23S) have been useful in better understanding the regulation of rRNA expression. Furthering these studies, we have investigated the effect of 5S rRNA deficiencies on cell fitness through the sequential deletion of 5S rRNA genes. Our findings indicate that the loss of 5S rDNA from multiple genes decreases cell fitness more rapidly than loss of a similar number of 16S and 23S rRNA genes. These results suggest that the cell's innate ability to up-regulate rRNA operons does not compensate for 5S rRNA deficiencies, as was previously shown for 16S and 23S rRNAs. A plasmid-borne 5S rRNA gene is able to compensate for the deleted 5S rRNA genes.

Escherichia coli↗
PubMed1993

Replacement of the L11 binding region within E.coli 23S ribosomal RNA with its homologue from yeast: in vivo and in vitro analysis of hybrid ribosomes altered in the GTPase centre.

Replacement of the protein L11 binding domain within Escherichia coli 23S ribosomal RNA (rRNA) by the equivalent region from yeast 26S rRNA appeared to have no effect on the growth rate of E.coli cells harbouring a plasmid carrying the mutated rrnB operon. The hybrid rRNA was correctly processed and assembled into ribosomes, which accumulated normally in polyribosomes. Of the total ribosomal population, < 25% contained wild-type, chromosomally encoded rRNA; the remainder were mutant. The hybrid ribosomes supported GTP hydrolysis dependent upon E.coli elongation factor G, although at a somewhat reduced rate compared with wild-type particles, and were sensitive to the antibiotic, thiostrepton, a potent inhibitor of ribosomal GTPase activity that binds to 23S rRNA within the L11 binding domain. That thiostrepton could indeed bind to the mutant ribosomes, although at a reduced level relative to that seen with wild-type ribosomes, was confirmed in a non-equilibrium assay. The rationale for the ability of the hybrid ribosomes to bind the antibiotic, given that yeast ribosomes do not, was provided when yeast rRNA was shown by equilibrium dialysis to bind thiostrepton only 10-fold less tightly than did E.coli rRNA. The extreme conservation of secondary, but not primary, structure in this region between E.coli and yeast rRNAs allows the hybrid ribosomes to function competently in protein synthesis and also preserves the interaction with thiostrepton.

Base Sequence↗
PubMed1990

Functional importance of the Escherichia coli ribosomal RNA leader box A sequence for post-transcriptional events.

To shed more light on the controversial findings concerning the functional participation of the highly conserved nut-like leader box A sequence element in ribosomal RNA transcription antitermination we have carried out a mutational study. We have substituted the box A and combined this mutation with several deletions comprising the rRNA leader elements box B, box C and the tL region. The mutations are located within the genuine rrnB operon cloned on multicopy plasmids. We determined the effects of the mutations on cell growth, rRNA accumulation and ribosomal subunit stoichiometry. Cells transformed with the mutated plasmids were affected in their growth rate, and showed a surprising deficiency of the promoter-proximal 16S compared to the 23S RNA, indicative of a post-transcriptional degradation event. Accordingly, we could demonstrate a reduced amount of free 30S relative to 50S ribosomal subunits in exponentially growing cells. Similar stoichiometric aberrations in the ribosome pool were detected in conditionally Nus factor-defective strains. The results show that the leader box A sequence within rRNA operons has important post-transcriptional functions for 16S RNA stability and ribosomal subunit stoichiometry. A model is proposed, describing the biogenesis and quality control of ribosomes based on rRNA leader and Nus-factor interactions. It is compatible with the previously observed effects of box A in antitermination.

Base Sequence↗
PubMed2000

Characterization and comparative study of the rrn operons of alkaliphilic Bacillus halodurans C-125.

The ribosomal RNA operons (rrn) of alkaliphilic Bacillus halodurans C-125 were characterized and compared with those of B. subtilis. We isolated clones containing rrn operons from a lambda phage library of the C-125 chromosome, and the complete nucleotide sequence of each was determined. Eight rrn operons were identified by PFGE analysis of the C-125 chromosome digested with I-CeuI. The transcriptional orientation of the rrn operons mapped on the chromosome by Southern hybridization analysis was the same as the direction of replication of the chromosome. These operons were designated as rrnA-H, starting from the oriC locus in clockwise rotation. Sequence and structural analyses of these operons suggested that six of the rrn operons in the C-125 chromosome, rrnA, rrnB, rrnC-rrnD, rrnE, and rrnH, correspond to rrnO, rrnA, rrnJ-rrnW, rrnI, and rrnD in B. subtilis, whereas the other rrn operons (rrnF and rrnG) were specifically observed in C-125. The rrn loci were positioned from 0 degrees to 90 degrees on the physical map, with the oriC locus assigned the position zero degrees. Two ORFs annotated as tnpA and ykfC, whose gene products are likely to act as transposases, were found downstream of these six operons. Comparative analysis of the 16S-23S and 23S-5S ITS (internally transcribed sequence) regions of B. halodurans C-125 and those of B. subtilis revealed that the ITS regions in C-125 were much longer than those in B. subtilis. There was no substantial difference in the length of potential promoter sequences in B. halodurans and B. subtilis.

Alkalies↗
PubMed1984

Sequence and evolution of the regions between thr rrn operons in the chloroplast genome of Euglena gracilis bacillaris.

The rRNA genes are arranged in three sequential operons preceded by a fourth partial operon. Part or all of a 1462 nucleotide sequence extending from within the 3'-end of the 23S rRNA gene, across the 5S rRNA gene and a presumptive transcription terminator, to within the first structural gene (for 16S rRNA) of the rrn operon was determined for each region between operons. Homologies of the 3'-end of the 23S rRNA gene with the 4.5S rRNA genes of higher plant chloroplasts, and of the 5S rRNA gene with other 5S rRNA genes were examined. The region preceding the 16S rRNA gene, which is expected to contain sites for initiation and regulation of rrn transcription, includes a 305 base-pair sequence with substantial homology with structural genes elsewhere in the chloroplast genome. The homologies suggest that this portion of the leader evolved from copies of parts of the structural genes which had been inserted before the 16S rRNA genes. Thus the chloroplast rrn leader may provide a unique opportunity to study how a regulatory sequence evolved from well-defined structural genes.

Base Sequence↗
PubMed1999

Signalling by the fungus Pythium ultimum represses expression of two ribosomal RNA operons with key roles in the rhizosphere ecology of Pseudomonas fluorescens F113.

Pseudomonas fluorescens F113 produces antifungal metabolites that protect the roots of sugarbeet from the fungus Pythium ultimum. The phytopathogen, in turn, has the ability to downregulate the expression of genes fundamental to the rhizosphere competence of the bacterial strain. This paper describes the characterization of two of these genes, which were isolated by screening a mini-Tn5::lacZ mutant bank for differential expression of beta-galactosidase in the presence of P. ultimum. In order to identify the genes affected in reporter mutants SF3 and SF5, the transposons and flanking regions were cloned. Sequence analysis of the regions flanking the transposons in SF3 revealed that mini-Tn5::lacZ had inserted into a tRNA(Ile) gene, which maps within a ribosomal RNA (rrn) operon. In SF5, the transposon inserted between the promoter of a second rrn operon and a gene encoding a 16S rRNA. Southern blot analysis demonstrated that there are five rrn operons in P. fluorescens F113 and that the transposons in SF3 and SF5 had inserted into two different operons. Further characterization of these mutants suggests that their reduced rhizosphere competence is not the result of reduced viability in the short term but may be accounted for partly by reduced growth rates under conditions that support rapid growth. Analysis of lacZ expression in the reporter mutants indicate that the marked rrn operons are regulated differently, suggesting different physiological roles.

Base Sequence↗
PubMed1987

Deletions in the tL structure upstream to the rRNA genes in the E. coli rrnB operon cause transcription polarity.

A number of deletions have been constructed within the leader region of the rrnB operon from E. coli. The deletions remove a potential transcription terminator structure downstream from an antitermination recognition sequence (Box A), which precedes the structural gene for the 16S RNA. Cells harbouring plasmids, where the terminator structure was deleted, partially or totally, showed a reduction in growth rate under minimal growth conditions. Measurement of the ribosomal RNA synthesis rates of such cells determined by pulselabeling and hybridisation to appropriate DNA probes, showed that the amount of the more distally located 23S RNA was reduced compared to the promoter-proximal 16S RNA. This polarity in transcription, resulting in a non-stoichiometric synthesis of the ribosomal RNAs, is most likely the result of a defective antitermination. The reduction in the amount of 23S RNA in such cells is compensated for by an increase in the overall ribosomal RNA synthesis, in concordance with the ribosomal RNA feedback regulation model. The accumulation of transcripts of the tRNAGlu2 gene, coded in the spacer region between the 16S and 23S RNA genes, in cells with an altered rRNA stoichiometry supports this interpretation.

Base Sequence↗
PubMed2003

Understanding bias in microbial community analysis techniques due to rrn operon copy number heterogeneity.

Molecular tools based on rRNA (rrn) genes are valuable techniques for the study of microbial communities. However, the presence of operon copy number heterogeneity represents a source of systematic error in community analysis. To understand the types and magnitude of such bias, four commonly used rrn-based techniques were used to perform an in silico analysis of a hypothetical community comprised organisms from the Comprehensive Microbial Resource database. Community profiles were generated, and diversity indices were calculated for length heterogeneity PCR, automated ribosomal integenic spacer analysis, denaturing gradient gel electrophoresis, and terminal RFLP (using RsaI, MspI, and HhaI). The results demonstrate that all techniques present a quantitative bias toward organisms with higher copy numbers. In addition, techniques may underestimate diversity by grouping similar ribotypes or overestimate diversity by allowing multiple signals for one organism. The results of this study suggest that caution should be used when interpreting rrn-based community analysis techniques.

DNA, Bacterial↗
PubMed1985

Increased expression of ribosomal genes during inhibition of ribosome assembly in Escherichia coli.

We have examined a prediction made from the ribosome feedback regulation model of ribosomal RNA and transfer RNA synthesis in Escherichia coli. This model proposes that non-translating "free" ribosomes act directly or indirectly as negative feedback inhibitors to regulate the transcription of rRNA and tRNA operons. One prediction of this model is that preferential inhibition of the assembly of ribosomes (without inhibiting macromolecular synthesis) should lead to a deficiency of free ribosomes which should, in turn, cause a stimulation of rRNA (and tRNA) synthesis. We have examined this prediction in vivo by causing the preferential inhibition of synthesis of certain ribosomal proteins by an overproduction of the translational repressor ribosomal protein S4. In agreement with our model, we have observed a preferential stimulation of ribosomal protein messenger RNA and rRNA synthesis under these conditions. These results suggest that ribosomes in the cellular pool, rather than incomplete ribosomal particles or free rRNA, are monitored by cells to regulate the rate of rRNA synthesis, and give further support to this proposed model.

Bacterial Proteins↗
PubMed2001

Molecular organization of the ribosomal RNA transcription unit and the phylogenetic study of Zymomonas mobilis ZM4.

Previously we reported that Zymomonas mobilis ZM4 contains three ribosomal transcription units (rrnA to C operons) which are clustered around the 50 min region, PacI fragments 13 and 6, on the physical map of Z. mobilis ZM4 [Kang, H. L. and Kang, H. S. (1998) Gene 206, 223-228]. The physical map reveals that the rrnA gene set is located on the 76 kb PacI fragment 13. The complete nucleotide sequence of the rrnA gene set has been determined. The total number of nucleotides of the rrnA gene set is about 6,250 bp. The structural genes of the 16S, 23S and 5S rRNA code for the 1,478 nt, 2,786 nt and 121 nt RNA chains, respectively. The length of the spacer regions between the 16S and 23S rRNA genes, and between the 23S and 5S rRNA genes, are 606 bp and 101 bp, respectively. Two tRNA genes, Ile-tRNA and Ala-tRNA, are found between the 16S and 23S rRNA genes and a fMet-tRNA gene is identified downstream of the 5S rRNA gene. Thus, the molecular organization of this rrnA gene set is the order of 5'-16S rRNA-tRNAIle-tRNAAla-23S rRNA-5S rRNA-tRNAfMet-3'. The secondary structure models of 16S, 23S and 5S rRNA are proposed. The phylogenetic tree, based on the 16S rRNA sequence, was constructed by neighbor-joining and maximum-parsimony methods. Zymomonas belongs to a group in which Aqorbacterium, Rhodobacter and Sphingomonas are included.

Base Sequence↗
PubMed2005

Localization, mobility and fidelity of retrotransposed Group II introns in rRNA genes.

We previously showed that the group II Lactococcus lactis Ll.LtrB intron could retrotranspose into ectopic locations on the genome of its native host. Two integration events, which had been mapped to unique sequences, were localized in the present study to separate copies of the six L.lactis 23S rRNA genes, within operon B or D. Although further movement within the bacterial chromosome was undetectable, the retrotransposed introns were able to re-integrate into their original homing site provided on a plasmid. This finding indicates not only that retrotransposed group II introns retain mobility properties, but also that movement occurs back into sequence that is heterologous to the sequence of the chromosomal location. Sequence analysis of the retrotransposed introns and the secondary mobility events back to the homing site showed that the introns retain sequence integrity. These results are illuminating, since the reverse transcriptase (RT) of the intron-encoded protein, LtrA, has no known proofreading function, yet the mobility events have a low error rate. Enzymatic digests were used to monitor sequence changes from the wild-type intron. The results indicate that retromobility events have approximately 10(-5) misincorporations per nucleotide inserted. In contrast to the high RT error rates for retroviruses that must escape host defenses, the infrequent mutations of group II introns would ensure intron spread through retention of sequences essential for mobility.

Base Sequence↗
PubMed2000

Comparison of the nucleotide sequences of 16S rRNA, 444 Ep-ank, and groESL heat shock operon genes in naturally occurring Ehrlichia equi and human granulocytic ehrlichiosis agent isolates from Northern California.

We examined 11 naturally occurring isolates of Ehrlichia equi in horses and two human granulocytic ehrlichiosis agent isolates in California for sequence diversity in three genes. Ehrlichia equi isolates were from Sierra (n = 6), Mendocino (n = 3), Sonoma (n = 1), and Marin (n = 1) counties, and human granulocytic ehrlichiosis (HGE) agent isolates were obtained from Humboldt county. PCR with specific primers for 16S rRNA, 444 Ep-ank and groESL heat shock operon genes successfully produced amplicons for all 13 clinical samples. The 444 Ep-ank gene of the HGE agent and E. equi isolates from northern California is different from the eastern U.S. isolates BDS and USG3. The translated amino acid sequence of the groESL heat shock operon gene fragment is identical among E. equi, the HGE agent, and E. phagocytophila, with the exception of the northern Californian equine CASOLJ isolate. Microheterogeneity was observed in the 16S rRNA gene sequences of HGE agent and E. equi isolates from northern California. These results suggest that E. equi and the HGE agent found in California are similar or identical but may differ from the isolates of equine and human origin found in the eastern United States.

Animals↗
PubMed1996

Functional Escherichia coli 23S rRNAs containing processed and unprocessed intervening sequences from Salmonella typhimurium.

We have introduced the intervening sequence (IVS) from 23S rRNA of the rrnD operon of Salmonella typhimurium into the equivalent position of Escherichia coli 23S rRNA. Salmonella typhimurium 23S rRNA is fragmented due to the RNase III-dependent removal of the approximately 100 nt stem-loop structure that comprises the IVS. In this study, we have found that insertion of the S. typhimurium IVS into E. coli 23S rRNA causes fragmentation of the RNA but does not affect ribosome function. Cells expressing the fragmented 23S rRNA exhibited wild-type growth rates. Fragmented RNA was found in the actively translating polysome pool and did not alter the sedimentation profile of ribosomal subunits, 70S ribosomes or polysomes. Finally, hybrid 23S rRNA carrying the A2058G mutation conferred high level erythromycin resistance indistinguishable from that of intact 23S rRNA carrying this mutation. These observations indicate that the presence of this IVS and its removal are phenotypically silent. As observed in an RNase III-deficient strain, processing of the IVS was not required for the production of functional ribosomes.

Base Sequence↗
PubMed1992

Inactivation of the fis gene leads to reduced growth rate.

The FIS protein (factor for inversion stimulation) is known to activate the transcription of rRNA and tRNA operons in Escherichia coli. In this report the growth behaviour of a fis bacterium was studied under steady-state conditions and in competition experiments with a wild-type bacterium in a chemostat. The growth rate of the fis bacterium was reduced as compared to that of an isogenic wild-type and this reduction increased linearly with increasing growth rate. A similar result was obtained in competition experiments in a chemostat, with the selection factor increasing linearly with the growth rate.

Carrier Proteins↗
PubMed1998

Inter- and intra-phage type differentiation of Salmonella enterica subsp. enterica serovar enteritidis isolates using molecular typing methods.

Seventy-six Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) isolates which represented 14 phage types and RDNC isolates were investigated by several independent molecular methods. Eleven different plasmid profiles were detected. Plasmid-encoded virulence genes of the spv gene cluster were shown by hybridization with a spvB/C gene probe to be located on plasmids of either 55 kbp or 95 kbp. Ribotyping as performed with gene probes that recognized either the entire rrn operon or 16S rRNA genes allowed no differentiation between the S. Enteritidis isolates. Three different hybridization patterns could be observed by IS200-typing. Macrorestriction analysis of XbaI-, SpeI- and NotI-digested whole cell DNA proved to be the most discriminatory method as confirmed by the calculation of discriminatory indices. Based on the data obtained from plasmid analysis and macrorestriction analyses with three suitable restriction endonucleases, the 76 S. Enteritidis isolates were assigned to 26 different genomic groups. The observation that several genomic groups included isolates of different phage types has suggested that a combination of suitable molecular methods and phage typing is most useful for tracing S. Enteritidis isolates in epidemiological investigations.

Animals↗