Abstract
Sphingomonads comprise a physiologically versatile group many of which appear to be adapted to oligotrophic environments, but several also had features in their genomes indicative of host associations. In this study, the extent variability of the 16S–23S rDNA intergenic spacer (ITS) sequences of 14 ATCC reference sphingomonad strains and 23 isolates recovered from drinking water was investigated through PCR amplification and sequencing. Sequencing analysis of the 16S–23S rRNA gene ITS region revealed that the ITS sizes for all studied isolates varied between 415 and 849 bp, while their G+C content was 42.2–57.9 mol%. Five distinct ITS types were identified: ITSnone (without tRNA genes), ITSAla(TGC), ITSAla(TGC)+Ile(GAT), ITSIle(GAT)+Ala(TGC), and ITS Ile(GAT)+Pseudo. All of the identified tRNAAla(TGC) molecules consisted of 73 bases, and all of the tRNAIle(GAT) molecules consisted of 74 bases. We also detected striking variability in the size of the ITS region among the various examined isolates. Highest variability was detected within the ITS-2. The importance of this study is that this is the first comparison of the 16S–23S rDNA ITS sequence similarities and tRNA genes from sphingomonads. Collectively the data obtained in this study revealed the heterogeneity and extent of variability within the ITS region compared to the 16S rRNA gene within closely related isolates. Sequence and length polymorphisms within the ITS region along with the ITS types (tRNA-containing or lacking and the type of tRNA) and ITS-2 size and sequence similarities allowed us to overcome the limitation we previously encountered in resolving closely related isolates based on the 16S rRNA gene sequence.
Highlights
Sphingomonads are Gram-negative, chemoheterotrophic, non-spore forming, straight rods, strictly aerobic, and characterized by an outer membrane containing glycosphingolipids as cell envelope components, but lacking lipopolysaccharide (Yabuuchi et al, 1990; White et al, 1996)
The intergenic spacer (ITS) region has been increasingly used to differentiate bacterial species or strains which cannot be differentiated using the 16S rRNA gene (Man et al, 2010). This is the first comparison of the 16S–23S rDNA ITS sequence similarities and tRNA genes from sphingomonads, where we analyzed the phylogenetic relationships based on ITS sequencing for a number of chosen sphingomonad ATCC reference strains along with representative sphingomonads recovered from drinking water in Lebanon (Tokajian et al, 2008)
16S rRNA gene sequencing has been widely used for typing bacterial isolates, it was shown previously that this region does not provide enough information to discern between closely related bacterial strains at the sub-generic level, especially for diverging species (Fox et al, 1992)
Summary
Sphingomonads are Gram-negative, chemoheterotrophic, non-spore forming, straight rods, strictly aerobic, and characterized by an outer membrane containing glycosphingolipids as cell envelope components, but lacking lipopolysaccharide (Yabuuchi et al, 1990; White et al, 1996). Sphingomonads are able to survive the chlorination of tap water and have the ability to co-aggregate and form biofilms. Large numbers of phenotypically and phylogenetically similar strains belonging to this group have been isolated. As a result Takeuchi et al (2001) examined the complete 16S rRNA gene sequences, fatty acid profiles and polyamine patterns of several strains of the genus Sphingomonas and related genera. Based on the phylogenetic analyses of the 16S rRNA gene sequences and on some chemotaxonomic and phenotypic differences, the genus Sphingomonas was divided into four clusters and three new genera were proposed. Today sphingomonads encompass eight genera: Novosphingobium, Sphingobium, Sphingomonas, Sphingopyxis, Sphingosinicella, Sphingomicrobium, Sphingorhabdus, and Parasphingopyxis (Stolz, 2013)
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