Fragmentation of 23S rRNA in strains of Proteus and Providencia results from intervening sequences in the rrn (rRNA) genes

Intervening sequences (IVSs) were originally identified in the rrl genes for 23S rRNA (rrl genes, for large ribosomal subunit, part of rrn operon encoding rRNA) of Salmonella enterica serovars Typhimurium LT2 and Arizonae. These sequences are transcribed but later removed during RNase III processing of the rRNA, resulting in fragmentation of the 23S species; IVSs are uncommon, but have been reported in at least 10 bacterial genera. Through PCR amplification of IVS-containing regions of the rrl genes we showed that most Proteus and Providencia strains contain IVSs similar to those of serovar Typhimurium in distribution and location in rrl genes. By extraction and Northern blotting of rRNA, we also found that these IVSs result in rRNA fragmentation. We report the first finding of two very different sizes of IVS (113 bp and 183 to 187 bp) in different rrl genes in the same strain, in helix 25 of Proteus and Providencia spp.; IVSs from helix 45 are 113 to 123 bp in size. Analysis of IVS sequence and postulated secondary structure reveals striking similarities of Proteus and Providencia IVSs to those of serovar Typhimurium, with the stems of the smaller IVSs from helix 25 being similar to those of Salmonella helix 25 IVSs and with both the stem and the central loop domain of helix 45 IVSs being similar. Thus, IVSs of related sequences are widely distributed throughout the Enterobacteriaceae, in Salmonella, Yersinia, Proteus, and Providencia spp., but we did not find them in Escherichia coli, Citrobacter, Enterobacter, Klebsiella, or Morganella spp.; the sporadic distribution of IVSs of related sequence indicates that lateral genetic transfer has occurred.     

Sequence diversity of intervening sequences (IVSs) in the 23S ribosomal RNA in Salmonella spp

Intervening sequences (IVSs) occur sporadically in the rrl (ribosomal RNA large) genes for 23S ribosomal RNA (rRNA) at helix-25 (base pair 550) and helix 45 (base pair 1170) in several bacterial genera, including Salmonella, Yersinia, Proteus, and Providencia, representing the Enterobacteriaceae, but are missing from other genera such as Escherichia. These sequences are transcribed, but later excised without re-ligation during RNaseIII processing of the rRNA, resulting in fragmented 23S rRNA. The IVSs from 22 strains of the SARB (Salmonella Reference Collection B) set were amplified by PCR and sequenced.IVSs with 90% or more sequence identity were placed in the same family; Salmonella has three families of IVSs in helix-25 (A, B, and C) and two in helix-45 (M and O). The rRNA secondary structure for the IVSs predicted from the mfold program reveals a primary stem of about 14bp, which is the postulated RNaseIII cleavage site, and a secondary region of stems and loops. The primary stem is considerably well conserved, with a high rate of compensatory mutations (positional covariants), confirming the reality of the secondary structure and indicating that removal of the IVSs exerts a positive selective pressure to retain the secondary structure. The pattern of possession and presence of families of IVSs was diverse and could not be related to the proposed ancestry of the strains as revealed by the multi-locus enzyme electrophoresis pattern of the strains, suggesting that the IVSs are transferred between strains by lateral transfer. Helix-25 IVSs from families A, B, and C of Salmonella and D of Proteus, which share almost identical primary stems, are placed in superfamily I, while the primary stems of other IVSs from Proteus and Providencia are unrelated to superfamily I and are thus placed into superfamily II; this indicates lateral transfer of members of superfamily I between Proteus and Salmonella, but an independent origin of IVSs of superfamily II in Proteus and Providencia.     

Salmonella typhimurium LT2 possesses three distinct 23S rRNA intervening sequences.

The rrl genes for 23S rRNA of Salmonella typhimurium LT2 are known to carry intervening sequences (IVSs) at two sites, helix-25 and helix-45, which are excised by RNase III during rRNA maturation, resulting in rRNA which is fragmented but nevertheless functional. We isolated DNA fragments containing the seven rrl genes from BlnI, I-CeuI, and SpeI genomic digests following pulsed-field gel electrophoresis and used these DNA fragments as templates for PCRs utilizing primers upstream and downstream of helix-25 and helix-45. Variance in amplicon length and cycle sequencing indicated that rrlG and rrlH have IVSs in helix-25 of approximately 110 bp which are only 56% identical. rrnA, rrnB, rrnC, rrnD, rrnE, and rrnH have IVSs of approximately 90 bp in helix-45, and all have the same nucleotide sequence. Twenty-one independent wild-type strains of S. typhimurium from Salmonella Reference Collection A were analyzed for IVSs by using PCRs with genomic DNAs and by denaturing agarose electrophoresis of RNAs. Many strains resemble LT2, but some have no IVSs in helix-25 and others have IVSs in helix-45 in all seven rrl genes. However, the IVSs in individual wild-type lines are relatively stable, for several LT2 isolates separated over many years by many single-colony isolations are indistinguishable from one another, with the exception of line LB5010, which differs by one helix-25 IVS. We postulate that IVSs have entered strain LT2 by three independent lateral-transfer events and that the IVS in helix-45 was dispersed to and maintained in the same sequence in six of the seven rrl genes by the mechanism of gene conversion.     

Intervening Sequences in rrl Genes and Fragmentation of 23S rRNA in Genera of the Family Enterobacteriaceae

Intervening sequences (IVSs) in the rrl genes for 23S rRNA are transcribed but later removed by RNase III without religation during RNA processing, leading to fragmented rRNA. We examined about 240 strains of the family Enterobacteriaceae for presence of IVSs using PCR. No IVSs were detected in strains belonging to Escherichia, Shigella, Enterobacter, Erwinia, Ewingella, Hafnia, Kluyvera, Morganella, Pantoea, or Serratia. Previously unreported IVSs were detected in Klebsiella oxytoca, Citrobacter amalonaticus, and Providencia stuartii; previously reported IVSs are in species of Salmonella, Proteus, Providencia, and Yersinia. The sporadic distribution of IVSs indicates lateral genetic transfer of IVSs.     

Discordant 16S and 23S rRNA gene phylogenies for the genus Helicobacter: implications for phylogenetic inference and systematics

Analysis of 16S rRNA gene sequences has become the primary method for determining prokaryotic phylogeny. Phylogeny is currently the basis for prokaryotic systematics. Therefore, the validity of 16S rRNA gene-based phylogenetic analyses is of fundamental importance for prokaryotic systematics. Discrepancies between 16S rRNA gene analyses and DNA-DNA hybridization and phenotypic analyses have been noted in the genus Helicobacter. To clarify these discrepancies, we sequenced the 23S rRNA genes for 55 helicobacter strains representing 41 taxa (>2,700 bases per sequence). Phylogenetic-tree construction using neighbor-joining, parsimony, and maximum likelihood methods for 23S rRNA gene sequence data yielded stable trees which were consistent with other phenotypic and genotypic methods. The 16S rRNA gene sequence-derived trees were discordant with the 23S rRNA gene trees and other data. Discrepant 16S rRNA gene sequence data for the helicobacters are consistent with the horizontal transfer of 16S rRNA gene fragments and the creation of mosaic molecules with loss of phylogenetic information. These results suggest that taxonomic decisions must be supported by other phylogenetically informative macromolecules, such as the 23S rRNA gene, when 16S rRNA gene-derived phylogeny is discordant with other credible phenotypic and genotypic methods. This study found Wolinella succinogenes to branch with the unsheathed-flagellum cluster of helicobacters by 23S rRNA gene analyses and whole-genome comparisons. This study also found intervening sequences (IVSs) in the 23S rRNA genes of strains of 12 Helicobacter species. IVSs were found in helices 10, 25, and 45, as well as between helices 31' and 27'. Simultaneous insertion of IVSs at three sites was found in H. mesocricetorum.     

Intervening Sequences in 16S rRNA Genes of Campylobacter sp.: Diversity of Nucleotide Sequences and Uniformity of Location

We found and sequenced intervening sequences (IVSs) in the PCR-amplicons of 16S rRNA genes of 3 strains of Campylobacter rectus, 2 strains of C. curvus and 2 strains of C. sputorum. The lengths of the IVSs were 140 to 233 bp. The IVSs of C. rectus were identical and had a sequence homology of 55 to 79% against those of C. curvus and C. helveticus. The IVSs of C. sputorum were 97.9-100% homologous but poorly homologous to the other IVSs. In spite of the diversities of the lengths and the nucleotide sequences, all of the IVSs were located at the same position in the 16S rRNA genes.     

RNase III deficient Salmonella typhimurium LT2 contains intervening sequences (IVSs) in its 23S rRNA

Salmonella typhimurium LT2 contains intervening sequences (IVSs) of 90–110 nt within all its 23S rRNA that are cleaved out by RNase III, resulting in rRNA fragmentation. In order to determine the functionality of 23S rRNA that contains unexcised IVSs, we constructed an S. typhimurium RNase III (rnc) deficient strain by transducing a mini-Tn10 (rnc-14::Tn10) from Escherichia coli K-12. The resulting strain of S. typhimurium was viable, contained IVSs within all of its 23S rRNA, and showed a growth reduction similar to that observed for the RNase III deficient strain of E. coli. These results indicate that ribosomes containing 23S rRNA in which IVSs are not excised are functional in translation, and make it unlikely that RNase III excision of IVSs from strain LT2 23S rRNA is dictated by a selective pressure to uphold the functional integrity of ribosomes.     

Salmonella typhi contains identical intervening sequences in all seven rrl genes.

Salmonella typhi Ty2 rrl genes contain intervening sequences (IVSs) in helix-25 but not in helix-45 on the basis of observed 23S rRNA fragmentation caused by IVS excision. We have confirmed this and shown all seven IVSs to be identical by isolating genomic DNA fragments containing each of the seven rrl genes from S. typhi Ty2 by use of pulsed-field gel electrophoresis; each rrl gene was amplified by PCR in the helix-25 and helix-45 regions and cycle sequenced. Thirty independent wild-type S. typhi strains, tested by genomic PCR and DraI restriction, also have seven rrl genes with helix-25 IVSs and no helix-45 IVSs. We propose that IVS homogeneity in S. typhi occurs because gene conversion drives IVS sequence maintenance and because adaptation to human hosts results in limited clonal diversity.     

Intervening sequences (IVSs) in the 23S ribosomal RNA genes of pathogenic Yersinia enterocolitica strains. The IVSs in Y enterocolitica and Salmonella typhimurium have a common origin

The 23S ribosomal RNA (rRNA) was shown to be in two fragments in pathogenic Yersinia enterocolitica. The cleavage site in the structural gene of the 23S rRNA was occupied by an intervening sequence (IVS) of about 100 nucleotides, analogous to IVSs found in salmonellae (Burgin et al., 1990). Nucleotide sequences of IVSs of several Y. enterocolitica strains revealed that the IVSs of the highly virulent Y. enterocolitica serotypes strains, and the IVS of Salmonella typhimurium were about 90% similar. On the other hand, the IVSs of the highly and the poorly virulent Y. enterocolitica serotypes were only about 60% similar. These results give the impression that at some point during the IVS evolution, the highly virulent Y. enterocolitica and S. typhimurium both received their IVSs at about the same time from the same source, and that the poorly virulent serotypes received their IVSs earlier. We also found that strain LB5010, derived by extended mutagenization of S. typhimurium LT2, had lost the IVSs originally present in LT2, and that this loss had created a new 'hairpin loop' which substituted for the original 'hairpin loop'.     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.     

Population structure of Salmonella investigated by amplified fragment length polymorphism

AIMS: This study was undertaken to investigate the usefulness of amplified fragment length polymorphism (AFLP) in determining the population structure of Salmonella. METHODS AND RESULTS: A total of 89 strains were subjected to AFLP analysis using the enzymes BglII and BspDI, a combination that is novel in Salmonella. Both species S. bongori and S. enterica and all subsp. of S. enterica were represented with emphasis on S. enterica subsp. enterica using a local strain collection and strains from the Salmonella Reference Collection B (SARB). The amplified fragments were used in a band-based cluster analysis. The tree resulting from the subgroup analysis clearly separated all subgroups with high bootstrap values with the species S. bongori being the most distantly related of the subgroups. The tree resulting from the analysis of the SARB collection showed that some serotypes are very clonal whereas others are highly divergent. CONCLUSIONS: AFLP clearly clustered strains representing the subgroups of Salmonella together with high bootstrap values and the serotypes of subspecies enterica were divided into polyphyletic or monophyletic types corresponding well with multilocus enzyme electrophoresis (MLEE) and sequence-based studies of the population structure in Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP with the enzyme combination BglII and BspDI allows discrimination of individual strains and provides evidence for the usefulness of AFLP in studies of population structure in Salmonella.     

Degradation of rRNA in Salmonella strains: a novel mechanism to regulate the concentrations of rRNA and ribosomes.

We have previously shown that the 23S rRNA of Salmonella strains is highly fragmented by specific enzyme cleavages. In this article, we report that 23S rRNA of Salmonella strains is rapidly degraded as the cells enter the stationary phase. More than 90% of the 23S rRNA is degraded when the cells reach the stationary phase. The rate of degradation of 23S rRNA correlated with its degree of fragmentation. This degradation is probably mediated by newly synthesized protein factor(s), since treatment with chloramphenicol or rifampin inhibits the rRNA degradation. We propose that degradation of 23S rRNA is a novel mechanism in the regulation of the bacterial 23S rRNA and ribosome concentration and that this additional regulatory mechanism provides some selective advantage to cells.     

Genetic relationships within the genus Prevotella analyzed by multilocus enzyme electrophoresis and DNA-DNA hybridization

The genetic diversity and relationships within the genus Prevotella were studied by analyzing twenty-five strains by multilocus enzyme electrophoresis (MLEE) at nine metabolic enzyme loci and DNA-DNA hybridization. MLEE revealed a high genetic diversity with 25 electrophoretic types (ETs) for the 25 strains studied, a mean number of alleles per enzyme locus of 6.8 and a mean genetic diversity per locus of 0.786. The index of association described by Maynard Smith et al. (1993) revealed a clonal structure within the genus Prevotella. A dendrogram generated by cluster analysis of a matrix of ETs showed that species like P. bivia, P. buccae, P. oris, P. oralis, P. nigrescens, and P. denticola form clusters that are consistent with DNA homologies. However, strains identified as P. melaninogenica or P. loescheii by DNA-DNA hybridization did not constitute distinct subpopulations in MLEE. MLEE analysis demonstrated its high power in differentiating closely related strains. It provides an alternative to 16S rRNA analysis for the study of phylogenetic relationships within the genus Prevotella, especially for differentiating strains with high DNA homology or high rRNA homology.     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.