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PubMed2004

Infection with a proposed new subspecies of Babesia canis, Babesia canis subsp. presentii, in domestic cats.

Parasitemia with a large Babesia species was identified in two domestic cats from Israel. One cat, also coinfected with feline immunodeficiency virus and "Candidatus Mycoplasma haemominutum," had profound icterus and anemia which resolved after therapy, whereas a second cat was an asymptomatic carrier. Amplification and sequencing of the 18S rRNA gene, followed by phylogenetic analyses, indicated that infection was caused by Babesia canis. However, the sequences of the internal transcribed and 5.8S rRNA regions of the ribosomal operon used for subspeciation of B. canis were markedly different from the recognized subspecies of B. canis, which include B. canis vogeli, B. canis canis, and B. canis rossi. Based on phylogenetic comparisons of the 18S rRNA gene, 5.8S, and internal transcribed spacer sequences of the isolates from the cats and on the smaller sizes of the merozoite and trophozoite stages of this parasite, which distinguish it from the subspecies of B. canis present in dogs, we propose to identify the novel feline genotype of B. canis described in the present study as a new subspecies, B. canis subsp. presentii.

Animals↗

PubMed2001

Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro.

To determine the role of ppGpp in both negative and positive regulation of transcription initiation during exponential growth in Escherichia coli, we examined transcription in vivo and in vitro from the growth-rate-dependent rRNA promoter rrnB P1 and from the inversely growth-rate-dependent amino acid biosynthesis/transport promoters PargI, PhisG, PlysC, PpheA, PthrABC, and PlivJ. rrnB P1 promoter activity was slightly higher at all growth-rates in strains unable to synthesize ppGpp (deltarelAdeltaspoT) than in wild-type strains. Consistent with this observation and with the large decrease in rRNA transcription during the stringent response (when ppGpp levels are much higher), ppGpp inhibited transcription from rrnB P1 in vitro. In contrast, amino acid promoter activity was considerably lower in deltarelAdeltaspoT strains than in wild-type strains, but ppGpp had no effect on amino acid promoter activity in vitro. Detailed kinetic analysis in vitro indicated that open complexes at amino acid promoters formed much more slowly and were much longer-lived than rrnB P1 open complexes. ppGpp did not increase the rates of association with, or escape from, amino acid promoters in vitro, consistent with its failure to stimulate transcription directly. In contrast, ppGpp decreased the half-lives of open complexes at all promoters, whether the half-life was seconds (rrnB P1) or hours (amino acid promoters). The results described here and in the accompanying paper indicate that ppGpp directly inhibits transcription, but only from promoters like rrnB P1 that make short-lived open complexes. The results indicate that stimulation of amino acid promoters occurs indirectly. The accompanying paper evaluates potential models for positive control of amino acid promoters by ppGpp that might explain the requirement of ppGpp for amino acid prototrophy.

Amino Acids↗

PubMed1995

UGA suppression by a mutant RNA of the large ribosomal subunit.

A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 168 rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations. In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA (UGA211) of E. coli. The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination. The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon. Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA. Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L11, or 16S rRNA. Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon.

Base Sequence↗

PubMed2000

Gene conversion drives within genic sequences: concerted evolution of ribosomal RNA genes in bacteria and archaea.

Multiple copies of a given ribosomal RNA gene family undergo concerted evolution such that sequences of all gene copies are virtually identical within a species although they diverge normally between species. In eukaryotes, gene conversion and unequal crossing over are the proposed mechanisms for concerted evolution of tandemly repeated sequences, whereas dispersed genes are homogenized by gene conversion. However, the homogenization mechanisms for multiple-copy, normally dispersed, prokaryotic rRNA genes are not well understood. Here we compared the sequences of multiple paralogous rRNA genes within a genome in 12 prokaryotic organisms that have multiple copies of the rRNA genes. Within a genome, putative sequence conversion tracts were found throughout the entire length of each individual rRNA genes and their immediate flanks. Individual conversion events convert only a short sequence tract, and the conversion partners can be any paralogous genes within the genome. Interestingly, the genic sequences undergo much slower divergence than their flanking sequences. Moreover, genomic context and operon organization do not affect rRNA gene homogenization. Thus, gene conversion underlies concerted evolution of bacterial rRNA genes, which normally occurs within genic sequences, and homogenization of flanking regions may result from co-conversion with the genic sequence.

Archaea↗

PubMed1985

Feedback regulation of rRNA and tRNA synthesis and accumulation of free ribosomes after conditional expression of rRNA genes.

We have constructed a conditional rRNA gene expression system by fusing a plasmid-encoded rrnB operon to the lambda PL promoter/operator. It was thereby possible to study the events that lead to the regulation of chromosomal rRNA and tRNA synthesis after overproduction of rRNA. rRNA induction resulted in a 2-fold increase in 30S and 50S free ribosomal subunits, whereas the polysome fraction was unaffected. Overproduction of rRNA and "free" ribosomes produced a large repression of rRNA and tRNA synthesis from chromosomal genes and a smaller increase in the concentration of guanosine tetraphosphate. These results lend support to the ribosome feedback regulation model: rRNA and tRNA operons are negatively regulated, either directly or through some intermediate, by free, nontranslating ribosomes.

Bacteriophage lambda↗

PubMed1993

Characterization of intergenic spacers in two rrn operons of Enterococcus hirae ATCC 9790.

Two DNA restriction enzyme fragments coding for the 3' termini of 16S rRNA, the 5' termini of 23S rRNA, and the intergenic spaces between them in Enterococcus hirae ATCC 9790 were cloned and sequenced. The intergenic space of one of these genes contains a tRNA(Ala) sequence, whereas the other does not. Nevertheless, the intergenic spaces contain several regions that exhibit high levels of sequence homology and are capable of forming structures with similar base pairs. An analysis of Southern blots of chromosomal DNA cut with one and two restriction enzymes indicated that E. hirae has a total of six rrn operons.

Base Sequence↗

PubMed1988

Mutations within the decoding site of Escherichia coli 16S rRNA: growth rate impairment, lethality and intragenic suppression.

Several C----U transitions and small deletions were introduced into the conserved region centered on base C1400 in Escherichia coli 16S rRNA by in vitro mutagenesis. The mutations were placed within rrnB operons on multicopy plasmids under the transcriptional regulation of either the normal rrnB P1P2 promoters or the temperature-inducible PL promoter from bacteriophage lambda and introduced into E. coli hosts. When expressed from the P1P2 promoters, several of the mutant 16S rRNAs impaired cell growth while others, including one in which U replaced C at position 1400 within the ribosomal decoding site, had little or no effect on cell doubling time. However, C----U transitions at positions 1395 and 1407, as well as the deletion of C1400, appeared to render their hosts inviable. Cells in which these mutations were expressed from the lambdaPL promoter died within four generations after induction. Unexpectedly, the lethal phenotype was suppressed intragenically by replacement of G1505 with A, C or U. Suppression may alleviate a functional defect in 30S subunits containing the U1395, U1407 or deltaC1400 mutations.

Base Sequence↗

PubMed2000

A Brevibacterium lactofermentum 16S rRNA gene used as target site for homologous recombination.

Genes for rRNA are highly conserved and present in multiple copies in most prokaryotic organisms increasing the number of theoretical sites for homologous recombination. They might be targets for integration events between unrelated microorganisms providing that an efficient genetic transfer is present. We have used a plasmid containing a portion of the 16S rRNA gene from the rrnD operon of Brevibacterium lactofermentum to transform the same strain resulting in non-essential inactivation of various rrn operons. Integration of the transforming DNA occurs in all cases. The system may be used to test possible gene transfer at least among closely related strains and is of great interest for integration of foreign DNA and for mapping.

Brevibacterium↗

PubMed1988

Visualization and quantitative analysis of complex formation between E. coli RNA polymerase and an rRNA promoter in vitro.

We have established conditions that stabilize the interaction between RNA polymerase and the rrnB P1 promoter in vitro. The requirements for quantitative complex formation are unusual for E. coli promoters: (1) The inclusion of a competitor is required to allow visualization of a specific footprint. (2) Low salt concentrations are necessary since complex formation is salt sensitive. (3) The addition of the initiating nucleotides ATP and CTP, resulting in a low rate of dinucleotide production, is required in order to prevent dissociation of the complexes. The complex has been examined using DNAase I footprinting and filter binding assays. It is characterized by a region protected from DNAase I cleavage that extends slightly upstream of the region protected by RNA polymerase in most E. coli promoters. We find that only one mole of active RNA polymerase is required per mole of promoter DNA in order to detect filter-bound complexes. Under the conditions measured, the rate of association of RNA polymerase with rrnB P1 is as rapid as, or more rapid than, that reported for any other E. coli or bacteriophage promoter.

Binding, Competitive↗

PubMed2003

Ribosomal RNA and ribosomal proteins in corynebacteria.

Ribosomal RNAs (rRNAs) (16S, 23S, 5S) encoded by the rrn operons and ribosomal proteins play a very important role in the formation of ribosomes and in the control of translation. Five copies of the rrn operon were reported by hybridization studies in Brevibacterium (Corynebacterium) lactofermentum but the genome sequence of Corynebacterium glutamicum provided evidence for six rrn copies. All six copies of the C. glutamicum 16S rRNA have a size of 1523 bp and each of the six copies of the 5S contain 120 bp whereas size differences are found between the six copies of the 23S rRNA. The anti-Shine-Dalgarno sequence at the 3'-end of the 16S rRNA was 5'-CCUCCUUUC-3'. Each rrn operon is transcribed as a large precursor rRNA (pre-rRNA) that is processed by RNaseIII and other RNases at specific cleavage boxes that have been identified in the C. glutamicum pre-rRNA. A secondary structure of the C. glutamicum 16S rRNA is proposed. The 16S rRNA sequence has been used as a molecular evolution clock allowing the deduction of a phylogenetic tree of all Corynebacterium species. In C. glutamicum, there are 11 ribosomal protein gene clusters encoding 42 ribosomal proteins. The organization of some of the ribosomal protein gene cluster is identical to that of Escherichia coli whereas in other clusters the organization of the genes is rather different. Some specific ribosomal protein genes are located in a different cluster in C. glutamicum when compared with E. coli, indicating that the control of expression of these genes is different in E. coli and C. glutamicum.

Corynebacterium↗

PubMed1988

Effects of induction of rRNA overproduction on ribosomal protein synthesis and ribosome subunit assembly in Escherichia coli.

Overproduction of rRNA was artificially induced in Escherichia coli cells to test whether the synthesis of ribosomal protein (r-protein) is normally repressed by feedback regulation. When rRNA was overproduced more than twofold from a hybrid plasmid carrying the rrnB operon fused to the lambda pL promoter (pL-rrnB), synthesis of individual r-proteins increased by an average of about 60%. This demonstrates that the synthesis of r-proteins is repressed under normal conditions. The increase of r-protein production, however, for unknown reasons, was not as great as the increase in rRNA synthesis and resulted in an imbalance between the amounts of rRNA and r-protein synthesis. Therefore, only a small (less than 20%) increase in the synthesis of complete 30S and 50S ribosome subunits was detected, and a considerable fraction of the excess rRNA was degraded. Lack of complete cooperativity in the assembly of ribosome subunits in vivo is discussed as a possible explanation for the absence of a large stimulation of ribosome synthesis observed under these conditions. In addition to the induction of intact rRNA overproduction from the pL-rrnB operon, the effects of unbalanced overproduction of each of the two large rRNAs, 16S rRNA and 23S rRNA, on r-protein synthesis were examined using pL-rrnB derivatives carrying a large deletion in either the 23S rRNA gene or the 16S rRNA gene. Operon-specific derepression after 23S or 16S rRNA overproduction correlated with the overproduction of rRNA containing the target site for the operon-specific repressor r-protein. These results are discussed to explain the apparent coupling of the assembly of one ribosomal subunit with that of the other which was observed in earlier studies on conditionally lethal mutants with defects in ribosome assembly.

Coliphages↗

PubMed2003

A functional plasmid-borne rrn operon in soil isolates belonging to the genus Paracoccus.

Plasmid analysis of isolates from a small Paracoccus population revealed that all 15 representatives carried at least one endogenous plasmid of 23 or 15 kb in size, in addition to further plasmids of different sizes. It was shown by restriction analysis and hybridization that the 23 and 15 kb plasmids from the different isolates were identical or very similar to each other. By partial sequencing of pOL18/23, one of the 23 kb plasmids, a complete rrn operon with the structural genes for 16S, 23S and 5S rRNA, two genes for tRNA(Ile) and tRNA(Ala) within the spacer between the 16S and 23S rRNA genes, and a final tRNA(fMet) at the end of the operon were discovered. Expression of a green fluorescent protein gene (gfp) after insertion of a DNA fragment from the region upstream of the rRNA genes into a promoter-probe vector demonstrated that the rrn promoter region is functional. The rrn operon encoded by plasmid pOL18/23 is the first complete rrn operon sequenced from a strain of the genus Paracoccus, and only the second example of an rrn operon on a small plasmid.

Base Sequence↗

PubMed1995

Refinement of the high-resolution physical and genetic map of Rhodobacter capsulatus and genome surveys using blots of the cosmid encyclopedia.

Cosmids from a library containing Rhodobacter capsulatus DNA fragments were previously ordered in two contigs: one corresponding to the chromosome and one to a 134 kb plasmid. This map contained 40 regions connected only by colony hybridization. To confirm the linkage and correct the map, the actual sizes of the overlaps were determined by blot-hybridization with Rhodobacter chromosomal DNA and by mapping of additional cosmids. Several revisions of the earlier map include single cosmid shifts and inversions. One additional gap in a cosmid contig was also found, raising the possibility that the chromosome is not a contiguous circle. About 2500 additional EcoRI,BamHI and HindIII restriction sites were added to the 560 EcoRV sites previously mapped onto the Rhodobacter chromosome, increasing the resolution of the physical map to the size of individual genes. Twenty-five new markers were located on the genetic map. The 48 markers now mapped represent nearly 300 genes and ORFs cloned from different species of Rhodobacter. The orientation of transcription of the four rrn operons was established using 16S rRNA- and 23S rRNA-specific probes and digestion with the rare-cutting enzyme, CeuI. Gel blots of 192 cosmids of the miniset of R.capsulatus digested with EcoRV were prepared. Such a hybridization template represents the whole genome cut into 560 DNA fragments varying in size from 0.4 to 25 kb. This template was used for high-resolution mapping of single genes, analysis of total genomic DNAs from related Rhodobacter strains and differentially expressed RNAs.

Chromosome Mapping↗

PubMed2004

Nature of the genome of the saprophytic spirochete Spirochaeta aurantia and its ribosomal RNA operons.

Using restriction endonucleases DraI, AseI, and I-CeuI in conjunction with pulsed-field gel electrophoresis, we have shown that Spirochaeta aurantia M1 possesses a circular 3.98-Mb genome. This is the second largest spirochete chromosome yet analyzed. The observation that the latter enzyme cuts in 3 places suggests the presence of 3 copies of the large subunit (23S) rRNA gene (rrl), which was confirmed by Southern hybridizations. The complete sequence of 2 of the ribosomal RNA operons was determined, revealing that their structure resembled that of the typical member of the bacterial superkingdom: rrs (16S; 1561 bp), tRNA, rrl (23S; 2972 bp), and rrf (5S; 110 bp). The S. aurantia rrs-rrl intergenic regions, as with Treponema denticola, contain genes specifying a 73-nt tRNA(Ala) (anticodon TGC) and a 77-nt tRNA(Ile) (anticodon GAT).

Base Sequence↗

PubMed2002

Isolation and characterization of two European strains of Ehrlichia phagocytophila of equine origin.

We report the isolation and partial genetic characterization of two equine strains of granulocytic Ehrlichia of the genogroup Ehrlichia phagocytophila. Frozen whole-blood samples from two Swedish horses with laboratory-verified granulocytic ehrlichiosis were inoculated into HL-60 cell cultures. Granulocytic Ehrlichia was isolated and propagated from both horses. DNA extracts from the respective strains were amplified by PCR using primers directed towards the 16S rRNA gene, the groESL heat shock operon gene, and the ank gene. The amplified gene fragments were sequenced and compared to known sequences in the GenBank database. With respect to the 16S rRNA gene, the groESL gene, and the ank gene, the DNA sequences of the two equine Ehrlichia isolates were identical to sequences found in isolates from clinical cases of granulocytic ehrlichiosis in humans and domestic animals in Sweden. However, compared to amplified DNA from an American Ehrlichia strain of the E. phagocytophila genogroup, differences were found in the groESL gene and ank gene sequences.

Animals↗

PubMed2002

Mycoplasma gallisepticum rpoA gene cluster.

Two conservative gene clusters, the S10 ribosomal protein region and one (of the two) set of rRNA genes, were split in a genome crossover rearrangement event in Mycoplasma gallisepticum. As a result of the rearrangement the major part of the S10 ribosomal protein cluster is located upstream of genes for 23S-5S rRNA (rrn23-5), but the genes infA-rpl36-rps13-rpoA-rpl17 are located immediately downstream of the isolated gene for 16S rRNA (rrn16). A new ribosomal protein cluster infA-rpl36-rps13-rpoA-rpl17-rps16-trmD-rpl19 was formed. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that this ribosomal protein cluster is an operon.

Chromosome Mapping↗

PubMedDate not supplied

Bacterial molecular epidemiology based on a nonradioactive probe complementary to ribosomal RNA.

The use of plasmid pKK3535 (a pBR322-derived plasmid containing a ribosomal RNA operon) is described for determining rRNA gene restriction patterns. The plasmid allowed the use of both radioactive as well as nonradioactive procedures and provided features that are necessary for the inclusion of internal and/or external standards that facilitate comparison of strains. The patterns generated are identical to those obtained by hybridization with 32P-labelled rRNA.

Bacteria↗

PubMed2004

Reconstructing evolutionary relationships from functional data: a consistent classification of organisms based on translation inhibition response.

The last two decades have witnessed an unsurpassed effort aimed at reconstructing the history of life from the genetic information contained in extant organisms. The availability of many sequenced genomes has allowed the reconstruction of phylogenies from gene families and its comparison with traditional single-gene trees. However, the appearance of major discrepancies between both approaches questions whether horizontal gene transfer (HGT) has played a prominent role in shaping the topology of the Tree of Life. Recent attempts at solving this controversy and reaching a consensus tree combine molecular data with additional phylogenetic markers. Translation is a universal cellular function that involves a meaningful, highly conserved set of genes: both rRNA and r-protein operons have an undisputed phylogenetic value and rarely undergo HGT. Ribosomal function reflects the concerted expression of that genetic network and consequently yields information about the evolutionary paths followed by the organisms. Here we report on tree reconstruction using a measure of the performance of the ribosome: antibiotic sensitivity of protein synthesis. A large database has been used where 33 ribosomal systems belonging to the three major cellular lineages were probed against 38 protein synthesis inhibitors. Different definitions of distance between pairs of organisms have been explored, and the classical algorithm of bootstrap evaluation has been adapted to quantify the reliability of the reconstructions obtained. Our analysis returns a consistent phylogeny, where archaea are systematically affiliated to eukarya, in agreement with recent reconstructions which used information-processing systems. The integration of the information derived from relevant functional markers into current phylogenetic reconstructions might facilitate achieving a consensus Tree of Life.

Archaea↗