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PubMed2005

Analysis of 16S-23S intergenic spacer regions of the rRNA operons in Edwardsiella ictaluri and Edwardsiella tarda isolates from fish.

AIMS: To analyse interspecies and intraspecies differences based on the 16S-23S rRNA intergenic spacer region (ISR) sequences of the fish pathogens Edwardsiella ictaluri and Edwardsiella tarda. METHODS AND RESULTS: The 16S-23S rRNA spacer regions of 19 Edw. ictaluri and four Edw. tarda isolates from four geographical regions were amplified by PCR with primers complementary to conserved sequences within the flanking 16S-23S rRNA coding sequences. Two products were generated from all isolates, without interspecies or intraspecific size polymorphisms. Sequence analysis of the amplified fragments revealed a smaller ISR of 350 bp, which contained a gene for tRNA(Glu), and a larger ISR of 441 bp, which contained genes for tRNA(Ile) and tRNA(Ala). The sequences of the smaller ISR of different Edw. ictaluri isolates were essentially identical to each other. Partial sequences of larger ISR from several Edw. ictaluri isolates also revealed no differences from the one complete Edw. ictaluri large ISR sequence obtained. The sequences of the smaller ISR of Edw. tarda were 97% identical to the Edw. ictaluri smaller ISR and the larger ISR were 96-98% identical to the Edw. ictaluri larger ISR sequence. The Edw. tarda isolates displayed limited ISR sequence heterogeneity, with > or =97% sequence identity among isolates for both small and large ISR. CONCLUSIONS: There is a high degree of size and sequence similarity of 16S-23S ISR both among isolates within Edw. ictaluri and Edw. tarda species and between the two species. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results confirm a close genetic relationship between Edw. ictaluri and Edw. tarda and the relative homogeneity of Edw. ictaluri isolates compared with Edw. tarda isolates. Because no differences were found in ISR sequences among Edw. ictaluri isolates, sequence analysis of the ISR will not be useful to distinguish isolates of Edw. ictaluri. However, we identified restriction sites that differ between ISR sequences of Edw. ictaluri and Edw. tarda, which will be useful in distinguishing the two species.

Animals↗
PubMed1996

Genetic diversity of rRNA operons of unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid of neonates suffering from meningitis.

The genetic diversity of a collection of 54 unrelated Streptococcus agalactiae strains isolated from the cerebrospinal fluid of neonates and of 60 unrelated carrier strains was evaluated by investigating the restriction fragment length polymorphism of the rRNA gene region. Three restriction enzymes were selected for use: PstI, HindIII, and CfoI. Clustering analysis revealed two phylogenetic groups of strains with 40% divergence. Group I contained two clusters, A and B, and group II contained three clusters, C, D, and E. Strains of serotype Ia were mostly distributed in cluster A, and strains of serotype Ib were mostly distributed in cluster E. Serotype III isolates did not cluster. Nevertheless, 37 of 39 isolates belonging to cluster B were serotype III. With HindIII, two rRNA gene banding patterns characterized 38 of the 39 strains of cluster B, which represents a high-virulence group. In addition, two rRNA gene banding patterns with each enzyme and/or a pair of CfoI fragments of 905 and 990 bp identified 81% of the invasive strains. On account of the genetic homogeneity of the cerebrospinal fluid strains, ribotyping is a powerful typing method for investigation of nosocomial or epidemic invasive infections only when all three enzymes are used or when PstI and HindIII or PstI and CfoI are combined with serotyping (index of discrimination, > 0.95).

Bacterial Typing Techniques↗
PubMed1991

Restriction fragment length polymorphisms in rRNA operons for subtyping Shigella sonnei.

Shigella sonnei is the most frequent cause of shigellosis in the United States. Epidemiologic studies of this organism have been hampered by the lack of adequate typing procedures. Ribosomal DNA analysis (ribotyping), a method which analyzes restriction fragment length polymorphisms in the chromosomal genes that encode rRNA, has recently been shown to be useful for microbial species identification and subtyping. To determine whether ribotyping could be used to distinguish between S. sonnei isolates, we conducted Southern hybridization studies on isolates from 16 different geographic locations and from four recent outbreaks. S. sonnei genomic DNA fragments generated following digestion with SalI hybridized with Escherichia coli 16S and 23S rRNAs to produce six distinct patterns; strains with patterns 1, 2, and 3 were each further subdivided into two additional patterns by using PvuII, SmaI, and SstI, respectively. Epidemiologically related strains had identical patterns. Ribotyping appears to be a useful tool for epidemiologic studies of shigellosis caused by S. sonnei.

Bacterial Typing Techniques↗
PubMed2006

RFLP analysis of the rRNA operon of a Brazilian collection of bradyrhizobial strains from 33 legume species.

Genetic diversity in tropical rhizobial species is still poorly known. With the aim of increasing this knowledge, three ribosomal regions of 119 strains belonging to the official Brazilian culture collection of rhizobia and classified as Bradyrhizobium based on morphological and physiological characteristics in vitro were analysed by RFLP-PCR. The strains were isolated from 33 legume species, representing nine tribes and all three subfamilies; they all form very effective N2-fixing nodules and 43 of them are recommended for use in Brazilian commercial inoculants as the most effective for their hosts. For the 16S rRNA gene, type and reference strains of Bradyrhizobium japonicum fell into two major clusters, joined at a level of similarity of 50 %, which included 52 strains, 90 % of which were isolated from soybean. Two other clusters, joined at a similarity of 53 %, included reference strains of Bradyrhizobium elkanii, but not USDA 76T; furthermore, two other major clusters were identified and all strains were clustered at a final level of similarity of only 28 %. For the intergenic spacer (IGS) between genes coding for the 16S and 23S rRNA, strains were clustered at a final level of similarity of 27 %. Reference strains of B. japonicum fell into a major group with 51 strains, 84 % isolated from soybean, with a similarity of 59 %, while strains of B. elkanii fell into another major group, with a similarity of 55 %, clustering 44 strains, 59 % of which were isolated from hosts other than soybean. New clusters were also observed for the IGS region. The largest number of differences was detected in the analysis of the 23S rRNA gene, and 16 groups and isolated strains were joined at a very low level of similarity (16 %). In a combined analysis with the three ribosomal regions, the majority of strains isolated from soybean clustered with a similarity of 54 % with type and reference strains of B. japonicum, while most strains isolated from Brazilian indigenous legume species grouped with B. elkanii at a level of similarity of 46 %. All strains were clustered at a very low level of similarity (27 %), and at least two new clusters were clearly defined. These new clusters might be related to intraspecific differences or to novel subspecies, or even to novel species; indeed, strains from one of these clusters show higher 16S rRNA gene sequence similarity to members of the genus Burkholderia. The results obtained in this study emphasize the high level of diversity of symbiotic diazotrophic bacteria in the tropics that still remains to be determined.

Bradyrhizobium↗
PubMed1990

FIS-dependent trans-activation of tRNA and rRNA operons of Escherichia coli.

Two mechanisms controlling stable RNA synthesis have been described: growth rate-dependent control and stringent response. Although the mechanism underlying growth rate-dependent control is still a matter of dispute, this control is commonly assumed to operate through repression of transcription initiation of stable RNA operons. The same is true for the stringent response. Here we show that the cell utilizes an additional control system operating through activation of the thrU(tufB) operon. This operon, the tyrT and the rrnB operon share a common trans-activating protein that binds to cis-acting DNA regions upstream of the promoters of the two tRNA operons and of the P1 promoter of the rrnB operon. Conceivably, more stable RNA operons may be regulated by trans-activation. Both in vivo and in vitro experiments show that the Escherichia coli protein FIS (Factor for Inversion Stimulation) is involved in the trans-activation. This protein is known to stimulate the inversion of various DNA segments by binding to cis-acting recombinational enhancers and functions as a host factor for the bacteriophages Mu and Lambda.

Bacteriophage mu↗
PubMed1980

Fusion of the promoter region of rRNA operon rrnB to lac Z gene.

A Lambda phage was constructed in which the structural gene for beta galactosidase is fused to a DNA segment carrying the ribosomal promoter rrnB of E. coli. In this hybrid operon beta galactosidase synthesis in vitro is repressed by ppGpp. Repression of beta galactosidase synthesis by cAMP is reported.

Bacteriophage lambda↗
PubMed1981

The rRNA operon from Zea mays chloroplasts: nucleotide sequence of 23S rDNA and its homology with E.coli 23S rDNA.

The nucleotide sequence of 23S rDNA from Zea mays chloroplasts has been determined. Alignment with 23S rDNA from E.coli reveals 71 percent homology when maize 4.5S rDNA is included as an equivalent of the 3' end of E.coli 23S rDNA. Among the conserved sequences are sites for base modification. Chloramphenicol sensitivity and ribosomal subunit interaction. A proposal for the base pairs formed between 16S and 23S rRNAs during the 30S/50S subunit interaction is presented. The alignment of maize 23S rDNA with that of E.coli reveals three small insertion sequences of 25, 65 and 78 base pairs, whereas maize 16S rDNA shows only deletions when compared with the E.coli species.

Base Sequence↗
PubMed1998

Physical and genetic chromosomal maps of Streptococcus agalactiae, serotypes II and III; rRNA operon organization.

A detailed analysis of two Streptococcus agalactiae (group B streptococcus, GBS) strains was performed by pulsed field gel electrophoresis (PFGE). Digestion of the chromosomal DNA with SmaI and SgrAI endonucleases, followed by separation and analysis of fragments by PFGE was carried out. Physical chromosomal maps of serotype II/(alpha + beta) and III/alpha strains of S. agalactiae were constructed. The GBS genome size was estimated to be 2200 kb. Sixteen GBS genes were used as probes and were located on the restriction maps of both strains by DNA-DNA hybridization. Six copies of ribosomal operons were found in the genome of the analyzed strains. Significant differences in the restriction patterns of chromosomal DNA and DNA-DNA hybridization between the two analyzed strains were detected so that DNA restriction patterns may be used to trace outbreaks of disease. The overall GBS chromosomal organization as determined is fairly conserved.

Base Sequence↗
PubMed1999

A small and compact genome in the marine cyanobacterium Prochlorococcus marinus CCMP 1375: lack of an intron in the gene for tRNA(Leu)(UAA) and a single copy of the rRNA operon.

Data obtained by pulsed field gel electrophoresis revealed for Prochlorococcus marinus CCMP 1375 a genome size of 1.81+/-0.04 Mbp. This value is significantly smaller than for all other cyanobacteria investigated so far. The absence of an intron in the gene for tRNA(Leu)(UAA), which otherwise is widespread among cyanobacteria, and the additional finding that the ribosomal operon exists as a single copy suggest that the deletion of non-essential sequences played a major role in the evolution of P. marinus. A small genome may have been advantageous in the adaptation to very oligotrophic marine conditions.

Base Sequence↗
PubMed1997

Assessment of genetic diversity among strains of Pseudomonas syringae by PCR-restriction fragment length polymorphism analysis of rRNA operons with special emphasis on P. syringae pv. tomato.

Phylogenetic relationships among 77 bacterial strains belonging to Pseudomonas syringae and Pseudomonas viridiflava species were assessed by analysis of the PCR-restriction fragment length polymorphism (RFLP) patterns of three DNA fragments corresponding to rrs and rrl genes and the internal transcribed spacer, ITS1. No difference among all strains in rrs and rrl genes was observed with 14 restriction enzymes, which confirms the close relationships existing between these two species. The nucleotidic sequence of the internal transcripted spacer (ITS1) between rrs and rrl for the P. syringae pv. syringae strain CFBP1392 was determined. Restriction maps of the PCR-amplified ITS1 region were prepared and compared for all 77 strains. Seventeen RFLP patterns, forming three main clusters, were distinguished. One contained all strains of P. syringae pv. tomato and of other pathovars which had been previously described as closely related by either pathogenicity studies or biochemical analyses. This cluster was equally far from P. viridiflava and from other P. syringae pathovars. These other pathovars of P. syringae formed a less coherent taxon.

DNA, Ribosomal↗
PubMed1993

Two tRNA gene clusters associated with rRNA operons rrnD and rrnE in Bacillus subtilis.

Sequence analysis of cloned rescued DNA fragments from a Bacillus subtilis strain with an inserted recombinant plasmid in ribosomal operon rrnE revealed the presence of two tRNA genes for Met and Asp at the 3' end of the operon. Probing chromosomal DNA from a strain carrying a plasmid inserted in rrnD with a fragment containing the genetically unassigned cluster of 16 tRNA genes revealed that the cluster is located immediately following the rrnD operon. Our findings show that all 10 rrn operons in B. subtilis are associated with tRNA gene clusters.

Amino Acid Sequence↗
PubMed1996

Comparison of the small 16S to 23S intergenic spacer region (ISR) of the rRNA operons of some Escherichia coli strains of the ECOR collection and E. coli K-12.

Several 16S to 23S spacers of 354 bp have been sequenced from six Escherichia coli strains belonging to the ECOR collection. Four phylogenetically informative variable sites were identified. The results of their comparison confirm the existence of two major phylogenetic branches in this species, as previously reported. Remarkable intercistronic heterogeneity was found in strain ECOR35 and its closest relatives, in which at least one of the operons has suffered a major mutagenic event or has an independent phylogenetic origin.

Base Sequence↗