New genus-specific primers for PCR identification of Rubrobacter strains

Abstract

A set of oligonucleotide primers, Rubro223f and Rubro454r, were found to amplify a 267 nucleotide sequence of 16S rRNA genes of Rubrobacter type strains. The primers distinguished members of this genus from other deeply-rooted actinobacterial lineages corresponding to the genera Conexibacter, Gaiella, Parviterribacter, Patulibacter, Solirubrobacter and Thermoleophilum of the class Thermoleophilia. Amplification of DNA bands of about 267 nucleotides were generated from environmental DNA extracted from soil samples taken from two locations in the Atacama Desert. Sequencing of a DNA library prepared from the bands showed that all of the clones fell within the evolutionary radiation occupied by the genus Rubrobacter. Most of the clones were assigned to two lineages that were well separated from phyletic lines composed of Rubrobacter type strains. It can be concluded that primers Rubro223f and Rubro454r are specific for the genus Rubrobacter and can be used to detect the presence and abundance of members of this genus in the Atacama Desert and other biomes.

Keywords: Actinobacteria; Atacama desert; Genus-specific primers; Rubrobacter; Taxonomy.

Fig. 1

Conserved nucleotide regions of 16S rRNA genes of Rubrobacter type strains used to design the specific primers Rubro223f and Rubro454r. The bar represents the 16S rRNA gene sequence of Escherichia coli; black boxes indicate conserved regions and the grey ones variable regions (V) with corresponding numbers (Brosius et al. ; Yarza et al. 2014). Arrows above the bar represent the position of the primers within the 16S rRNA gene sequence. Nucleotide alignment for primers Rubro223f and Rubro454r and for primer Rubro749r (Holmes et al. 2000) are highlighted in orange boxes and nucleotides in white represent those unique to the genus Rubrobacter and hence absent in the type strains of species classified in the genera Conexibacter, Gaiella, Parviterribacter, Patulibacter, Solirubrobacter and Thermoleophilum. (Color figure online)

Fig. 2

Verification of the specificity of primers Rubro223f and Rubro454r in PCR runs using genomic DNA extracted from Rubrobacter type strains and corresponding strains of the closely-related genera. Electrophoresis in 2% agarose gels shows PCR amplification of a region of 267 nt that was only found in the Rubrobacter strains

Fig. 3

Maximum-likelihood phylogenetic tree generated using the GTR + CAT model and rooted by midpoint-rooting showing relationships between the 267 nt sequences amplified with primers Rubro223f and Rubro454r, using community DNA extracted from Salar de Tara (ST1) and Quebrada Nacimiento (QN) soils and the corresponding full 16S rRNA gene sequences of the type strains of representatives of the genera Conexibacter, Gaiella, Parviterribacter, Patulibacter, Solirubrobacter and Thermoleophilum. The branches of the tree are scaled in terms of the expected number of substitutions per site and the numbers above the branches are bootstrap support values greater than 60% for the ML (left) and MP (right) analyses