The role of recombination and mutation in 16S-23S rDNA spacer rearrangements

The intragenomic heterogeneity of the bacterial intergenic (16S-23S rDNA) spacer region (ISR) was analysed from the following species in which sequences for the complete rRNA operon (rrn) set have been determined (rrn number): Enterococcus faecalis (6) and E. faecium (6), Bacillus subtilis (10), Staphylococcus aureus (9), Vibrio cholerae (4), Haemophilus influenzae (6) and Escherichia coli (7). It was found that some spacer sequence blocks were highly conserved between operons of a genome, whereas the presence of others was variable. When these variations were analysed using the program PLATO and partial likelihood phylogenies determined by DNAml for each operon set, three regions showed significant (Z>3.3) spatial variation [Region I was 78-184 nt long (2.14.4) possibly due to recombination or selection. Within Region I, there was sequence block variation in all operon sets [some operons contained tRNA genes (tRNAala, tRNAile or tRNAglu), whereas others had sequence blocks such as VS2 (S. aureus) or rsl (E. coli)]. Q Analysis of the ISR sequence from E. faecalis and E. faecium showed that there was more interspecies than intraspecies variation (both in DNA sequence and in the presence or absence of blocks). Dot matrix analysis of the sequence blocks in the nine rrn ISRs from S. aureus showed that there was significant homology between VS2 and VS5/VS6. Furthermore, repeat motifs with only A or T were present in higher copy numbers in VS5/VS6 than in VS2. Since these sequence blocks (VS2 and VS5-VS6) are related, intragenic evolution resulting in AT expansion may have occurred between these two regions. A model is proposed that postulates a role for recombination and AT-expansion in intra-genomic ISR variations. This process may represent a general mechanism of concerted evolution for bacterial ISR rearrangements.     

16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium-oxidizing bacteria: implications for phylogeny and in situ detection

Recently, anaerobic ammonium-oxidizing bacteria (AAOB) were identified by comparative 16S rDNA sequence analysis as a novel, deep-branching lineage within the Planctomycetales . This lineage consists currently of only two, not yet culturable bacteria which have been provisionally described as Candidatus 'Brocadia anammoxidans' and Candidatus 'Kuenenia stuttgartiensis'. In this study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2 000 bases of the 23S rDNA, was polymerase chain reaction (PCR) amplified, cloned and sequenced from both AAOB. The retrieved 16S rDNA sequences of both species contain an insertion at helix 9 with a previously overlooked pronounced secondary structure (new subhelices 9a and 9b). This insertion, which is absent in all other known prokaryotes, is detectable by fluorescence in situ hybridization (FISH) and thus present in the mature 16S rRNA. In contrast with the genera Pirellula , Planctomyces and Gemmata that possess unlinked 16S and 23S rRNA genes, both AAOB have the respective genes linked together by an ISR of approximately 450 bp in length. Phylogenetic analysis of the obtained 23S rRNA-genes confirmed the deep branching of the AAOB within the Planctomycetales and allowed the design of additional specific FISH probes. Remarkably, the ISR of the AAOB also could be successfully detected by FISH via simultaneous application of four monolabelled oligonucleotide probes. Quantitative FISH experiments with cells of Candidatus 'Brocadia anammoxidans' that were inhibited by exposure to oxygen for different time periods demonstrated that the concentration of transcribed ISR reflected the activity of the cells more accurately than the 16S or 23S rRNA concentration. Thus the developed ISR probes might become useful tools for in situ monitoring of the activity of AAOB in their natural environment.     

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.     

PCR amplification of species specific sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region for identification of Streptococcus phocae

Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.     

Characterization of thermophilic spore-forming bacteria from a geothermal spring in Lithuania based on 16S rDNA and 16S-23S rDNA intergenic spacers analyses

Forty-two strains of gram-positive, aerobic, heterotrophic, obligately thermophilic, spore-forming bacteria were isolated from a geothermal site near the Baltic Sea in Lithuania. All of the strains were able to hydrolyze collagen and/or casein. Since characteristics of proteolytic activity are correlated with taxonomic positions of bacteria, the strains were grouped on the basis of molecular biological analyses. On the basis of RFLP patterns of 16S rDNA and 16S-23S rDNA ITS-PCR analysis, the strains were subdivided into nine groups.     

Restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer of Aeromonas spp

We analyzed restriction fragment length polymorphism (RFLP) of 16S-23S rDNA intergenic spacer region (ISR) of Aeromonas species. A total of 69 isolates belonging to 18 DNA hybridization groups (HG; equivalent of genomic species) were used in this study. ISRs were amplified by PCR and the products were digested with four restriction endonucleases: Hin6I, Csp6I, TaqI, and TasI. The RFLP patterns obtained after digesting by particular enzymes revealed ISR polymorphism of isolates allocated to individual genomic species. The combined Hin6I, Csp6I, TaqI, and TasI restriction profiles were examined by Dice coefficient (SD) and unweighted pair group method of clustering (UPGMA). The isolates were allocated into 15 groups, three strains were unclustered. The strains belonging to the following genomic species: A. hydrophila, A. bestiarum, A. salmonicida, A. caviae, A. media, A. schubertii, A. allosaccharophila, A. popoffii, and A. culicicola formed distinct clusters. Strains belonging to HG 6, HG 7, HG 11, and HG 16 revealed similar combined RFLP patterns and constituted one group. Similarly, the strains of A. jandaei (HG 9) and the type strain of A. trota were allocated into one cluster. Two isolates of HG 14 formed distinct cluster. We noticed a genetic diversity among A. veronii isolates, the strains were clustered in two groups. Our study showed that combined ISR-RFLP analysis may be used for identification of some species of Aeromonas.     

Cloning and sequencing of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC)

AIMS: To clone and sequence the 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC). METHODS AND RESULTS: The primer sets for 16S rDNA and 16S-23S rDNA ISR amplified almost the full length of 16S rDNA and 16S-23S rDNA ISR. About 1500 bp for 16S rDNA and about 720 bp for 16S-23S rDNA ISR of the rrn operon of four strains of UPTC were identified after molecular cloning and sequencing. CONCLUSIONS: The four strains and CCUG18267 of UPTC showed approximately 99% sequence homology of 16S rDNA to each other, 96-97% to Camp. coli, 97-98% to Camp. jejuni and 97-98% to Camp. lari. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the nucleotide sequence of 16S-23S rDNA ISR of UPTC has been analysed. The sequence of ISR was almost identical among the four strains of UPTC. It is interesting that the UPTC intercistronic tRNAs demonstrated an order of tRNA of 5'-16S-tRNAAla-tRNAIle-23S-3' in the organisms.     

Typing of clinical and environmental Aeromonas veronii strains based on the 16S-23S rDNA spacers

Genetic relationships of Aeromonas veronii strains isolated from human and environmental sources were investigated by restriction fragment length polymorphism (RFLP) of the polymerase chain reaction-amplified intergenic spacer region (ISR) flanked by the 16S and 23S rRNA genes. When using endonucleases AluI, HinfI and CfoI the 16S-23S rDNA-RFLP patterns showed considerable overall similarity, although most strains yielded specific profiles. Several intra-specific lines of descent comprised clinical strains linked to isolates from environmental sources. Strains having identical patterns may be individuals derived from highly similar, if not the same, microorganism. Results suggest that the ISR sequence-based method can be used to demonstrate colonization of a public water supply with a particular microorganism. In addition it could be very useful for tracing recurrent episodes of diarrhea and Aeromonas infection outbreaks.     

Genetic diversity of Acacia tortilis ssp. raddiana rhizobia in Tunisia assessed by 16S and 16S-23S rDNA genes analysis

AIMS: In order to understand the genetic diversity of Acacia tortilis ssp. raddiana-rhizobia in Tunisia, isolates from nine geographical locations were obtained and analysed. METHODS AND RESULTS: Characterization using restriction fragment length polymorphism analysis (RFLP) of PCR-amplified 16S rRNA gene and the intergenic spacer (IGS) between the 16S and 23S rRNA genes was undertaken. Symbiotic efficiency of the strains was also estimated. Analysis of the 16S rRNA by PCR-RFLP showed that the isolates were phylogenetically related to Ensifer ssp., Rhizobium tropicii-IIA, and Rhizobium tumefaciens species. Analysis of 16S-23S spacer by PCR-RFLP showed a high diversity of these rhizobia and revealed eleven additional groups, which indicates that these strains are genetically very diverse. Full 16S rRNA gene-sequencing showed that the majority of strains form a new subdivion inside the genera Ensifer, with Ensifer meliloti being its nearest neighbour. Nodulation test performed on the plant host demonstrated differences in the infectivity among the strains. CONCLUSION: Rhizobial populations that nodulate specifically and efficiently Acacia tortilis ssp. raddiana in representative soils of Tunisia is dominated by E. meliloti-like genomospecies. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the first clear characterization and symbiotic efficiency data of rhizobia strains nodulating A. tortilis in Tunisia.     

Identification of Thai isolates assigned to the genus Gluconobacter based on 16S-23S rDNA ITS restriction analysis

Forty-four Thai isolates phenotypically assigned to the genus Gluconobacter were examined for 16S-23S rDNA ITS restriction analysis by MboII and SduI (=Bsp1286I) digestions. The Thai isolates tested were divided into seven groups: Group I for fourteen isolates, Group IX for one isolate, Group X for two isolates, Group V-2 for four isolates, Group XI for three isolates, Group IV for one isolate, and Group III for nineteen isolates. There were no isolates of either Group II or Group V-1 that were identified as G. cerinus. The isolates of Group III, Group IV, and Group XI were subjected to an additional 16S-23S rDNA ITS restriction analysis by AvaII, TaqI, BsoBI, and BstNI digestions. The isolates of Group III were divided into three groups and two subgroups: Group III-2 for five isolates, Group III-6 for two isolates, and Group III-4, which was divided into two subgroups, Subgroup III-4a for four isolates and Subgroup III-4b for eight isolates. The fourteen isolates of Group I were identified as G. oxydans, and the two isolates of Group X were temporarily identified as G. oxydans. The five isolates of Group III-2 and the one isolate of Group IV were identified as G. frateurii. The remaining twenty-two isolates of Group V-2, Group III-4, Group III-6, Group IX, and Group XI were not identified but are candidates for several new species.     

16S to 23S rDNA spacer fragment length polymorphism of Aeromonas sp

We analyzed polymorphism of the PCR-amplified 16S-23S rDNA spacer of Aeromonas species. A total of 69 isolates representing 18 DNA hybridization groups were used in this study. The analysis of PCR products of 16S-23S rDNA spacers revealed patterns consisting of two to eight DNA fragments. The fragment sizes ranged from 730 to 1050 bp. DNA patterns revealed a considerable genetic diversity between species and within a species. When a procedure to eliminate heteroduplex formation was performed, the number of bands was reduced to 2-5. Nevertheless the homoduplex ISR (intergenic spacer region) patterns obtained were not useful for species distinguishing.     

Inter- and intraspecies variations of the 16S-23S rDNA intergenic spacer region of various streptococcal species

The 16S-23S rDNA intergenic spacer regions (ISR) of different streptococcal species and subspecies were amplified with primers derived from the highly conserved flanking regions of the 16S rRNA and 23S rRNA genes. The single sized amplicons showed a uniform pattern for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. uberis, S. parauberis, S. pyogenes and S. equi subsp. equi, respectively. The amplicons of S. equi subsp. zooepidemicus, S. porcinus and S. suis appeared with 3, 5 and 3 different sizes, respectively. ISR of selected strains of each species or subspecies investigated were sequenced and multiple aligned. This allowed a separation of ISR into regions, with 7 regions for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. pyogenes and S. suis, 8 regions for S. uberis and S. parauberis and mostly 9 regions for S. equi subsp. equi, S. equi subsp. zooepidemicus and S. porcinus. Region 4, encoding the transfer RNA for alanine (tRNA(Ala)), was present and identical for all isolates investigated. The size and sequence of ISR appears to be a unique marker for streptococci of various species and subspecies and could be used for bacterial identification. In addition the size and sequence variations of ISR of S. equi subsp. zooepidemicus, S. porcinus and S. suis allows a molecular typing of isolates of these species possibly useful in epidemiological aspects.     

Long-Term Stability of Mercury-Reducing Microbial Biofilm Communities Analyzed by 16S-23S rDNA Interspacer Region Polymorphism

The composition of mercury-reducing communities in two bioreactors retaining Hg(II) from chloralkali electrolysis wastewater for 485 days was analyzed based on effluent community DNA. Packed bed bioreactors with lava chips as carrier of the biofilm were inoculated with nine Hg(II)-resistant isolates that belonged to the alpha and gamma subdivisions of the proteobacteria. A rapid DNA-fingerprinting method was applied, using the intergenic spacer region (ISR) of the 16S-23S rDNA for analysis of the community composition. This allowed discrimination of the inoculum strains down to subspecies level. A merA specific PCR permitted the discrimination of the community's merA genes. During the 485 days of operation, the bioreactors were exposed to various physical stresses (mixing, gas bubbles, temperature increase up to 41°C, increased flow velocity) and repeated high mercury inflow concentrations, resulting in reduced bioreactor performance and decreased culturable cell numbers in the reactor effluent. Nevertheless, the composition of the microbial community remained rather stable throughout the investigated time period. Of the inoculum strains, two could be detected throughout, whereas three were sometimes present with varying periods of nondetection. Two inoculum strains were only detected within the first month. Two strains of gamma-proteobacteria that were able to reduce ionic mercury invaded the bioreactor community. They did not outcompete established strains and had no negative effect on the Hg(II)-retention activity of the bioreactors. The community comprised diverse merA genes. The abundance of merA genes matched the abundance of their respective strains as confirmed by ISR community analysis. The continuously high selection pressure for mercury resistance maintained a stable and highly active mercury-reducing microbial community within the bioreactors.     

tRNA genes were found in Piscirickettsia salmonis 16S-23S rDNA spacer region (ITS)

Piscirickettsia salmonis is the etiological agent of Salmonid Rickettsial Septicemia, a disease affecting salmon aquaculture industry. We analyzed the 16S-23S rDNA spacer region (internal transcribed spacer, ITS) of Chilean P. salmonis isolates LF-89 and EM-90. Two main ITS amplification products were obtained by PCR using L1 and G1 primers, differing from that described where only one ITS region was found. By Southern blot, it was established that these two amplification products contained sequences related to P. salmonis ITS. Sequence analysis confirmed that P. salmonis had two ITS regions: ITS A and ITS B. In both isolates, the smaller (ITS B) corresponded to ITS sequences previously described for each one, and the larger (ITS A) were almost the same as their respective ITS B sequences interrupted by an insert which contained two tRNAs genes: tRNA-Ile and tRNA-Ala.     

Differentiation of group VIII Spiroplasma strains with sequences of the 16S-23S rDNA intergenic spacer region

Spiroplasma species (Mollicutes: Spiroplasmataceae) are associated with a wide variety of insects, and serology has classified this genus into 34 groups, 3 with subgroups. The 16S rRNA gene has been used for phylogenetic analysis of spiroplasmas, but this approach is uninformative for group VIII because the serologically distinct subgroups generally have similarity coefficients >0.990. Therefore, we investigated the utility of the 16S-23S rRNA spacer region as a means to differentiate closely related subgroups or strains. We generated intergenic sequences and detailed serological profiles for 8 group VIII Spiroplasma strains. Sequence analyses using Maximum Parsimony, Neighbor Joining, and Maximum Likelihood placed the strains into 2 clades. One clade consisted of strains BARC 2649 and GSU5367. The other clade was divided into clusters containing representatives of the 3 designated group VIII subgroups (EA-1, DF-1, and TAAS-1) and 3 previously unclassified strains. The stability of the positions of the strains in various analytical models and the ability to provide robust support for groupings tentatively supported by serology indicates that the 16S-23S intergenic rDNA sequence will prove useful in intragroup analysis of group VIII spiroplasmas.     

Re-identification of Gluconobacter strains based on restriction analysis of 16S-23S rDNA internal transcribed spacer regions

Thirty Gluconobacter strains maintained at Culture Collection NBRC were re-identified at the species level on the basis of restriction analysis of 16S-23S rDNA internal transcribed spacer (ITS) regions by digestion with two restriction endonucleases MboII and Bsp1286I. The strains examined were divided into seven groups, designated as Group I and Group III-VIII, by the combination of the restriction patterns obtained with the two restriction endonucleases. Group I included seven strains, which gave "G. oxydans patterns" with the two restriction endonucleases and were re-identified as G. oxydans. Group III included 12 strains, which gave "G. frateurii patterns" and were re-identified as G. frateurii. Group IV included six strains, which gave "G. cerinus pattern" with MboII and "G. frateurii pattern" with Bsp1286I and were re-identified as G. frateurii. Group V included one strain (NBRC 3274), which gave respectively "G. frateurii pattern" and "G. cerinus pattern" and was re-identified as G. cerinus. Group VI included one strain (NBRC 3990), which gave respectively "G. oxydans pattern" and an unidentified restriction pattern and was re-identified temporarily as G. oxydans. Group VII included two strains (NBRC 3250 and NBRC 3273), which gave respectively an unidentified restriction pattern and "G. oxydans pattern." Group VIII included one strain (NBRC 3266), which gave unidentified restriction patterns. The three strains of Group VII and Group VIII were suggested to constitute new taxa by sequencing of 16S-23S rDNA ITS regions.     

Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains

AIMS: To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. METHODS AND RESULTS: The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. CONCLUSIONS: Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. SIGNIFICANCE AND IMPACT OF THE STUDY: A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.