Atypical 16S rRNA Gene Copies in Ochrobactrum intermedium Strains Reveal a Large Genomic Rearrangement by Recombination between rrn Copies

Abstract

Ochrobactrum intermedium is an opportunistic human pathogen belonging to the alpha 2 subgroup of proteobacteria. The 16S rDNA sequences of nine O. intermedium isolates from a collection of clinical and environmental isolates exhibited a 46-bp insertion at position 187, which was present in only one sequence among the 82 complete or partial 16S rDNA sequences of Ochrobactrum spp. available in data banks. Reverse transcription-PCR experiments showed that the 46-bp insertion remained in the 16S rRNA. The inserted sequence folded into a stem-loop structure, which took place in and prolonged helix H184 of the 16S rRNA molecule. Helix H184 has been described as conserved in length among eubacteria, suggesting the idiosyncratic character of the 46-bp insertion. Pulsed-field gel electrophoresis experiments showed that seven of the clinical isolates carrying the 46-bp insertion belonged to the same clone. Insertion and rrn copy numbers were determined by hybridization and I-CeuI digestion. In the set of clonal isolates, the loss of two insertion copies revealed the deletion of a large genomic fragment of 150 kb, which included one rrn copy; deletion occurred during the in vivo evolution of the clone. Determination of the rrn skeleton suggested that the large genomic rearrangement occurred during events involving homologous recombination between rrn copies. The loss of insertion copies suggested a phenomenon of concerted evolution among heterogeneous rrn copies.The genus Ochrobactrum belongs to the alpha 2 subgroup of Proteobacteria and is the closest relative to the genus Brucella. Ochrobactrum anthropi was considered the sole and type species of the genus Ochrobactrum (15) until 1998, when Ochrobactrum intermedium was proposed as a new species of this genus (50). Factors discriminating between O. anthropi and O. intermedium were their low DNA-DNA hybridization, their different 16S ribosomal DNA (rDNA) sequences, the different Western blot profiles of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell proteins, and the resistance of O. intermedium to colistin and polymyxin B (50). In a recent study, Lebuhn et al. (21) performed a polyphasic analysis of a large collection of Ochrobactrum sp. strains isolated from the wheat rhizoplane. They described two new species, Ochrobactrum grignonense and Ochrobactrum tritici and proposed the separation of the Brucella-Ochrobactrum group into five clades with O.

Paenibacillus maceransPossesses Two Types of 16S rDNA Copies in a Genome with a Length Difference of Twelve Base Pairs

“…the same helix 10) and Ochrobactrum intermedium exhibits a polymorphism with a length difference of 42 nucleotides for helix H9, a near neighbor of H10. 3) Based on the E. coli 16S rRNA secondary structure model, a polymorphism accompanied with a long length difference was found for helix H10 in the present study, in contrast with previously detected 16S rRNA polymorphisms. In the Database of Ribosomal CrossLinks, 11) there are no known interactions with rRNA or ribosomal proteins listed for helix H10.…”

“…1) Although mutations such as transition or transversion are introduced by misincorporation or misrepair by DNA polymerase during DNA replication, 2) certain cases of intra-genomic heterogeneity of 16S rDNA cannot be fully explained by these phenomena. For example, Ochrobactrum intermedium, 3) Streptomyces spp., 2) Thermobispora bispora, 4) and Thermomonospora chromogena 5) show heterogeneity, differing from those introduced by misincorporation or misrepair.…”

Molecular and phenotypic features for identification of the opportunistic pathogens Ochrobactrum spp.

“…Intact genomic DNAs were prepared in agarose plugs for enzymic digestion as previously described (Teyssier et al, 2003). DNAs were digested with 40 U HindIII or EcoRI (New England Biolabs), and then electrophoresed for 3 h at 80 V in a 0 .…”

“…Bacteria were grown on tryptic soy agar (TSA) for 24 h at 37 8C. One colony was suspended in 50 ìl sterile water, and the DNA was liberated by a boiling-freezing method (Teyssier et al, 2003). The 16S rRNA gene was selectively amplified from this crude lysate by PCR using universal primers 27f and 1492r, as previously described (Teyssier et al, 2003).…”

Gluconobacter as Well as Asaia Species, Newly Emerging Opportunistic Human Pathogens among Acetic Acid Bacteria

“…16S rRNA gene amplification was performed as described previously (31). The housekeeping gene dnaK was amplified using primers dnaK-01-F and dnaK-02-R, as described by Cleenwerck et al (4).…”