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PubMed1987

Translational regulation of the L11 ribosomal protein operon of Escherichia coli: mutations that define the target site for repression by L1.

The L11 ribosomal protein operon of Escherichia coli contains the genes for L11 and L1 and is feedback regulated by the translational repressor L1. The mRNA target site for this repression is located close to the Shine-Dalgarno sequence for the first cistron, rp1K (L11). By use of a random mutagenesis procedure we have isolated and characterized a series of point mutations in the L11 leader mRNA which eliminate or greatly diminish the regulation by L1. The mutations define a region essential for translational regulation upstream of the L11 Shine-Dalgarno sequence and identify a region of structural homology with the L1 binding site on 23S rRNA. These results are also consistent with the previously proposed model for the secondary structure of the L11 leader mRNA.

Bacterial Proteins↗
PubMed1991

Cloning and partial characterization of genes for ribosomal ribonucleic acid in Lactococcus lactis subsp. lactis.

A cosmid gene library of the genome of Lactococcus lactis subsp. lactis 712 was probed for the presence of 16S rRNA genes, using 32P 5' end-labelled 16S rRNA fragments. Cosmid DNA from positive clones responsible for hybridisation was subcloned into a high copy number vector and a restriction map was constructed. The location of the 16S, 23S and 5S rRNA genes was determined on this map. Transcriptional promoter activity was identified upstream of the 5' end of the 16S rRNA gene. By probing L. lactis 712 chromosomal DNA cut with a range of restriction endonucleases, with a conserved oligonucleotide to the 5' end of the 16S rRNA gene, 6 copies of rRNA genes were identified.

Base Sequence↗
PubMedDate not supplied

Cloning of the promoters of an Escherichia coli rRNA gene. New experimental system to study the regulation of rRNA transcription.

The promoters of the rrnB gene of Escherichia coli have been cloned on a multicopy, pBR322-derived plasmid by deleting most of the structural part of rrnB and fusing the terminators of the gene immediately to the promoters. Several further deletions were constructed to vary the promoter-terminator distance, destroy or damage selectively any of the promoters or terminators, and vary the distance between the two pairs of P1 P2 and P3 P4 promoters. All these transcription signals were shown to function on the plasmids in vitro and in vivo. The truncated in vivo transcription products initiated at the P1 and P2 promoters of the recombinant plasmids were found to be stable, and the accumulated transcripts could be easily distinguished from the chromosome-coded rRNA. This provides a convenient experimental system to study the regulation of rRNA biosynthesis.

Chromosome Deletion↗
PubMedDate not supplied

A component of Drosophila RNA polymerase I promoter lies within the rRNA transcription unit.

Genes transcribed by RNA polymerase II have been characterized as having several control regions upstream of the site at which transcription is initiated, and the 'CAAT' and 'TAATA' regulatory sequences are conserved in content and location in diverse eukaryotes. The transcription of 5S RNA and tRNA by polymerase III is regulated by promoters that are actually within the genes; these are also conserved in diverse eukaryotes. In addition, sequences upstream of tRNA genes can affect RNA synthesis. Recently, it has been determined that DNA sequences influencing the expression of the 18S and 28S ribosomal RNA genes (rDNA) lie upstream of the site of transcription initiation. We have shown that a major component of the promoter of Drosophila rRNA polymerase I activity involves the region -43 to -27, where initiation is at +1. Here we present evidence that another major component of the polymerase I promoter lies within the first four nucleotides of the external transcribed spacer (ETS). Drosophila polymerase I control signals, therefore, lie both upstream of and within the rRNA transcription unit.

Animals↗
PubMed1981

Sequence of a putative promoter region for the rRNA genes of tobacco chloroplast DNA.

The nucleotide sequence of the segment of tobacco chloroplast DNA adjacent to and including the start of the 16S rRNA gene has been determined. The region just preceding this gene was found to contain a tRNAVal gene and promoter-type sequences similar to those which occur in E. coli were found before this tRNA gene. E. coli RNA polymerase can recognize these sequences and in vitro co-transcribes the tRNA and rRNA genes.

Base Sequence↗
PubMed1999

Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms.

The current taxonomic classification of Chlamydia is based on limited phenotypic, morphologic and genetic criteria. This classification does not take into account recent analysis of the ribosomal operon or recently identified obligately intracellular organisms that have a chlamydia-like developmental cycle of replication. Neither does it provide a systematic rationale for identifying new strains. In this study, phylogenetic analyses of the 16S and 23S rRNA genes are presented with corroborating genetic and phenotypic information to show that the order Chlamydiales contains at least four distinct groups at the family level and that within the Chlamydiaceae are two distinct lineages which branch into nine separate clusters. In this report a reclassification of the order Chlamydiales and its current taxa is proposed. This proposal retains currently known strains with > 90% 16S rRNA identity in the family Chlamydiaceae and separates other chlamydia-like organisms that have 80-90% 16S rRNA relatedness to the Chlamydiaceae into new families. Chlamydiae that were previously described as 'Candidatus Parachlamydia acanthamoebae' Amann, Springer, Schönhuber, Ludwig, Schmid, Müller and Michel 1997, become members of Parachlamydiaceae fam. nov., Parachlamydia acanthamoebae gen. nov., sp. now. 'Simkania' strain Z becomes the founding member of Simkaniaceae fam. nov., Simkania negevensis gen. nov., sp. nov. The fourth group, which includes strain WSU 86-1044, was left unnamed. The Chlamydiaceae, which currently has only the genus Chlamydia, is divided into two genera, Chlamydia and Chlamydophila gen. nov. Two new species, Chlamydia muridarum sp. nov. and Chlamydia suis sp. nov., join Chlamydia trachomatis in the emended genus Chlamydia. Chlamydophila gen. nov. assimilates the current species, Chlamydia pecorum, Chlamydia pneumoniae and Chlamydia psittaci, to form Chlamydophila pecorum comb. nov., Chlamydophila pneumoniae comb. nov. and Chlamydophila psittaci comb. nov. Three new Chlamydophila species are derived from Chlamydia psittaci: Chlamydophila abortus gen. nov., sp. nov., Chlamydophila caviae gen. nov., sp. nov. and Chlamydophila felis gen. nov., sp. nov. Emended descriptions for the order Chlamydiales and for the family Chlamydiaceae are provided. These families, genera and species are readily distinguished by analysis of signature sequences in the 16S and 23S ribosomal genes.

Animals↗
PubMed2002

Determination of the nucleotide sequences of heat shock operon groESL and the citrate synthase gene (gltA) of Anaplasma (Ehrlichia) platys for phylogenetic and diagnostic studies.

The 1,670-bp nucleotide sequence of the heat shock operon groESL and the 1,236-bp sequence of the citrate synthase gene (gltA) of Anaplasma (Ehrlichia) platys were determined. The topology of the groEL- and gltA-based phylogenetic tree was similar to that derived from 16S rRNA gene analyses with distances. Both groESL- and gltA-based PCRs specific to A. platys were also developed based upon the alignment data.

Anaplasma↗
PubMed1998

Genomic analysis reveals chromosomal variation in natural populations of the uncultured psychrophilic archaeon Cenarchaeum symbiosum.

Molecular phylogenetic surveys have recently revealed an ecologically widespread crenarchaeal group that inhabits cold and temperate terrestrial and marine environments. To date these organisms have resisted isolation in pure culture, and so their phenotypic and genotypic characteristics remain largely unknown. To characterize these archaea, and to extend methodological approaches for characterizing uncultivated microorganisms, we initiated genomic analyses of the nonthermophilic crenarchaeote Cenarchaeum symbiosum found living in association with a marine sponge, Axinella mexicana. Complex DNA libraries derived from the host-symbiont population yielded several large clones containing the ribosomal operon from C. symbiosum. Unexpectedly, cloning and sequence analysis revealed the presence of two closely related variants that were consistently found in the majority of host individuals analyzed. Homologous regions from the two variants were sequenced and compared in detail. The variants exhibit >99.2% sequence identity in both small- and large-subunit rRNA genes and they contain homologous protein-encoding genes in identical order and orientation over a 28-kbp overlapping region. Our study not only indicates the potential for characterizing uncultivated prokaryotes by genome sequencing but also identifies the primary complication inherent in the approach: the widespread genomic microheterogeneity in naturally occurring prokaryotic populations.

Animals↗
PubMed1999

Use of the 16S--23S ribosomal genes spacer region in studies of prokaryotic diversity.

The description of microbial diversity by molecular culture-independent techniques most often involves the amplification of the 16S rRNA by PCR gene and either analysis of the diversity of amplified molecules (community fingerprinting) that allows the simultaneous study of many samples or the cloning and sequencing of a significant amount of amplification products. The fact that between the 16S and the 23S genes in the ribosomal operon there is a spacer extremely variable in both sequence and length provides an excellent tool to simplify both approaches. The spacer can be amplified almost as easily as the 16S rDNA taking advantage of conserved nucleotide stretches at the 5' end of the 23S gene and the amplicon can contain different amounts of the 16S rDNA choosing primers at the different conserved areas within this gene. Identified by the acronym RISA (rDNA internal spacer analysis), the spacer addition provides a marker of highly variable size allowing standard separation of the amplification products and the sequence of this hypervariable region is useful in the fine discrimination of operational taxonomic units.

Bacteria↗
PubMed1977

Capacity of ribosomal RNA promoters of E. coli to bind RNA polymerase.

The rate of in vitro transcription of the rRNA genes of E. coli is more than 20 fold higher than the averaged transcription rate of other genome segments of the same size. This "preferential transcription" of rRNA genes reflects a high efficiency of their promoters in chain initiation. We show that the high initiation rate at rRNA promoters results from a high rate of RNA polymerase binding to these promoters as measured by the formation of heparin-resistant RNA polymerase-DNA complexes. The results indicate that the preferential binding of RNA polymerase to rRNA promoters is mainly due to their large binding capacity rather than to a high rate constant of polymerase binding to a single binding site. The polymerase binding capcity of rRNA promoters was estimated from the number of rRNA chains initiated by heparin-resistant complexes under conditions of template saturation and from the number of rRNA transcription units participating in the binding reaction. At least 30 RNA polymerase molecules were found to be protected from heparin per rRNA transcription unit. The rest of the genome (99.4%; possibly sufficient to encode 4000 nonribosomal RNA species) protects under these conditions 2000 enzyme molecules. These results suggest that a high multiplicity of RNA polymerase binding may be responsible for the high efficiency of rRNA promoters. The validity of this hypothesis is discussed.

DNA, Bacterial↗
PubMed2001

Mycobacterium immunogenum sp. nov., a novel species related to Mycobacterium abscessus and associated with clinical disease, pseudo-outbreaks and contaminated metalworking fluids: an international cooperative study on mycobacterial taxonomy.

PCR-restriction enzyme pattern analysis of a 439 bp hsp65 gene segment identified 113 unique isolates among non-pigmented rapidly growing mycobacteria (RGM) from clinical and environmental sources that failed to match currently recognized species patterns. This group represented 40% of isolates recovered from bronchoscope contamination pseudo-outbreaks, 0% of disease-associated nosocomial outbreaks and 4% of routine clinical isolates of the Mycobacterium abscessus/Mycobacterium chelonae group submitted to the Mycobacteria/Nocardia laboratory for identification. It is grouped within the Mycobacterium fortuitum complex, with growth in less than 7 d, absence of pigmentation, positive 3-d arylsulfatase reaction and growth on MacConkey agar without crystal violet. It exhibited overlapping biochemical, antimicrobial susceptibility and HPLC characteristics of M. abscessus and M. chelonae. By 16S rRNA gene sequencing, these isolates comprised a homogeneous group with a unique hypervariable region A sequence and differed by 8 and 10 bp, respectively, from M. abscessus and M. chelonae. Surprisingly, this taxon contained two copies of the ribosomal operon, compared with single copies in the two related species. By DNA-DNA hybridization, this new group exhibited <30% homology with recognized RGM species. The name Mycobacterium immunogenum sp. nov. is proposed for this new taxon.

Bacterial Proteins↗
PubMed2002

Genotyping of axenic and non-axenic isolates of the genus Prochlorococcus and the OMF-'Synechococcus' clade by size, sequence analysis or RFLP of the Internal Transcribed Spacer of the ribosomal operon.

PCR amplicons of the Internal Transcribed Spacer (ITS) of the rrn operon of three axenic OMF (oceanic, marine and freshwater) strains of 'Synechococcus' (WH7803, PCC 7001 and PCC 6307, respectively) differ greatly in length from that of the axenic Prochlorococcus marinus subsp. pastoris PCC 9511(T), although these four cyanobacteria cluster relatively closely in phylogenetic trees inferred from 16S rRNA gene sequences. The ITSs of three strains (PCC 9511(T), PCC 6307 and PCC 7001) were sequenced and compared with those available for strains Prochlorococcus MED4 (CCMP 1378) and MIT9313 from genome sequencing projects. In spite of large differences in length, sequence and mean DNA base composition, conserved domains important for transcriptional antitermination and folding of the rRNA transcripts were identified in all ITSs. A new group-specific primer permitted ITS amplification even with non-axenic isolates of Prochlorococcus and one OMF-'Synechococcus' strain. Prochlorococcus isolates of the high-light-adapted clade (HL) differed from representatives of the low-light-adapted clade (LL) by the length of their ITS. Restriction fragment length polymorphism (RFLP) of the ITS amplicons revealed three subclusters among the HL strains. Size, sequence data and RFLP of the ITS amplicons will therefore be valuable markers for the identification of different Prochlorococcus genotypes and for their discrimination from other cyanobacterial relatives with which they often co-exist in oceanic ecosystems.

Base Sequence↗
PubMed2004

Toxic and nontoxic microcystis colonies in natural populations can be differentiated on the basis of rRNA gene internal transcribed spacer diversity.

Assessing and predicting bloom dynamics and toxin production by Microcystis requires analysis of toxic and nontoxic Microcystis genotypes in natural communities. We show that genetic differentiation of Microcystis colonies based on rRNA internal transcribed spacer (ITS) sequences provides an adequate basis for recognition of microcystin producers. Consequently, ecological studies of toxic and nontoxic cyanobacteria are now possible through studies of rRNA ITS genotypic diversity in isolated cultures or colonies and in natural communities. A total of 107 Microcystis colonies were isolated from 15 lakes in Europe and Morocco, the presence of microcystins in each colony was examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE analysis of amplified ITSa and ITSc fragments, yielding supplementary resolution (I. Janse et al., Appl. Environ. Microbiol. 69:6634-6643, 2003), the colonies could be differentiated into 59 classes. Microcystin-producing and non-microcystin-producing colonies ended up in different classes. Sequences from the rRNA ITS of representative strains were congruent with the classification based on DGGE and confirmed the recognition of microcystin producers on the basis of rRNA ITS. The rRNA ITS sequences also confirmed inconsistencies reported for Microcystis identification based on morphology. There was no indication for geographical restriction of strains, since identical sequences originated from geographically distant lakes. About 28% of the analyzed colonies gave rise to multiple bands in DGGE profiles, indicating either aggregation of different colonies, or the occurrence of sequence differences between multiple operons. Cyanobacterial community profiles from two Dutch lakes from which colonies had been isolated showed different relative abundances of genotypes between bloom stages and between the water column and surface scum. Although not all bands in the community profiles could be matched with isolated colonies, the profiles suggest a dominance of nontoxic colonies, mainly later in the season and in scums.

Base Sequence↗
PubMed2000

16S to 23S rRNA spacer fragment length polymorphism of Salmonella enterica at subspecies and serotype levels.

The variation in the lengths of the internal transcribed spacer (ITS) between 16S and 23S rRNA genes of 101 strains representing 58 serotypes of Salmonella enterica (used for Salm. choleraesuis) subsp. enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV) and indica (VI) was used for typing by PCR amplification. Ten fragment lengths were observed by denaturing polyacrylamide gel electrophoresis on an automatic DNA sequencer resulting in 21 unique fragment patterns. Ten out of the 58 serotypes showed specific patterns but 48 serotypes were not fully differentiated. More than one ITS pattern was observed in seven serotypes. Five of the 21 fragment patterns contained representatives of more than one subspecies. Under non-denaturing electrophoresis conditions, serotype specificity was obtained but precise ITS fragment length determination was not possible. DNA sequence comparison between ITSs of individual rrn operons is needed to further interpret ITS diversity within Salm. enterica at serotype and subspecies levels.

Bacterial Typing Techniques↗
PubMed2000

Detection of the Na(+)-translocating NADH-quinone reductase in marine bacteria using a PCR technique.

To examine the distribution of the Na(+)-translocating NADH-quinone reductase (Na(+)-NQR) among marine bacteria, we developed a simple screening method for the detection of this enzyme. By reference to the homologous sequences of the Na(+)-NQR operons from Vibrio alginolyticus and Haemophilus influenzae, a pair of primers was designed for amplification of a part of the sixth ORF (nqr6) of the Na(+)-NQR operon. When PCR was performed using genomic DNA from 13 marine bacteria, a 0.9-kbp fragment corresponding to nqr6 was amplified in 10 strains. Although there were three PCR-negative strains phylogenetically, based on the sequence of the 16S rRNA, these were placed far from the PCR-positive strains. No product was observed in the case of nonmarine bacteria. The nucleotide and predicted amino acid sequences of nqr6 were highly conserved among the PCR-positive marine bacteria. A phylogenetic analysis of marine bacteria, based on nqr6 sequencing, was performed.

Amino Acid Sequence↗
PubMed2000

Phylogenetic analysis of gram-positive bacteria based on grpE, encoded by the dnaK operon.

The dnaK operon in gram-positive bacteria includes grpE, dnaJ and, in some members, hrcA as well. Both DnaK and DnaJ have been utilized for constructing phylogenetic relationships among various organisms. Multiple copies exist for dnaK and dnaJ genes in some bacterial genera, as opposed to a single gene copy for grpE and for hrcA, according to the currently available data. Here, we present a partial protein-based phylogenetic tree for gram-positive bacteria, derived by using the amino acid sequence identity of GrpE; the results are compared with the phylogenetic trees generated from 5S rRNA, 16S rRNA, dnaK and dnaJ sequences. Our results indicate three main groupings: two are within low-G+C DNA gram-positive bacteria comprising Bacillus species and Staphylococcus aureus on the one hand and Streptococcus species/Lactococcus lactis/Enterococcus faecalis/Lactobacillus sakei on the other hand; the Mycobacterium species and Streptomyces coelicolor, belonging to the high-G+C DNA gram-positive bacteria, form the third cluster. This hierarchical arrangement is in close agreement with that obtained with 16S rRNA and DnaK sequences but not DnaJ-based phylogeny.

Amino Acid Sequence↗
PubMed1990

Development of a highly specific diagnostic 23S rDNA oligonucleotide probe for Mycobacterium leprae.

A 21-mer DNA oligonucleotide probe targeting the 23S rRNA of Mycobacterium leprae was developed and its high specificity demonstrated by dot-blot hybridization. Even under relaxed hybridization and washing conditions (20 degrees C below Tm) the probe was highly selective in that positive signals were only detected with M. leprae, about half of the slow-growing and one of the fast-growing reference mycobacteria and Gordona bronchialis. At more stringent washing temperatures (6 degrees C below Tm) only the rRNA of Mycobacterium leprae was detectable.

Base Sequence↗
PubMed1999

Mutant analysis of Prevotella sp. plaA-lacZ fusion protein expression in Escherichia coli: support for an essential role of the stem-loop.

This study investigated the involvement of RNA folding in the synthesis of a fusion protein with beta-galactosidase activity. The coding gap region of the Prevotella loescheii adhesin gene plaA was fused in-frame with the Escherichia coli lacZ gene on plasmid pSK105. N-Terminal sequencing of the expressed plaA-lacZ protein indicated that it resulted from translational initiation at a fortuitous ribosomal-binding site within the plaA sequence at nt 570. Specific mutations were introduced in the stem-loop region that precedes the gap sequence. Analysis of stem-loop mutants, together with the introduction of compensatory mutations that restored activity, supports a requirement for stem-loop formation within the plaA sequence preceding the translational initiation site. A mutation reducing the predicted size of the loop, but preserving the stem structure, inactivated fusion protein synthesis. A suppressor mutation predicted to restore the size of the loop restored efficient fusion protein synthesis. In addition, the sequence preceding the translational start site of the plaA-lacZ fusion has several similarities to sequences that function as translational enhancers in prokaryotes. These include a stem-loop structure, an A-U rich region preceding the initiation codon, and a region of homology to 16S rRNA.

Adhesins, Bacterial↗