rpoB gene as a novel molecular marker to infer phylogeny in Planctomycetales

Abstract

The 16S rRNA gene has been used in the last decades as a gold standard for determining the phylogenetic position of bacteria and their taxonomy. It is a well conserved gene, with some variations, present in all bacteria and allows the reconstruction of genealogies of microorganisms. Nevertheless, this gene has its limitations when inferring phylogenetic relationships between closely related isolates. To overcome this problem, DNA-DNA hybridization appeared as a solution to clarify interspecies relationships when the sequence similarity of the 16S rRNA gene is above 97 %. However, this technique is time consuming, expensive and laborious and so, researchers developed other molecular markers such as sequencing of housekeeping or functional genes for accurate determination of bacterial phylogeny. One of these genes that have been used successfully, particularly in clinical microbiology, codes for the beta subunit of the RNA polymerase (rpoB). The rpoB gene is sufficiently conserved to be used as a molecular clock, it is present in all bacteria and it is a mono-copy gene. In this study, rpoB gene sequencing was applied to the phylum Planctomycetes. Based on the genomes of 19 planctomycetes it was possible to determine the correlation between the rpoB gene sequence and the phylogenetic position of the organisms at a 95-96 % sequence similarity threshold for a novel species. A 1200-bp fragment of the rpoB gene was amplified from several new planctomycetal isolates and their intra and inter-species relationships to other members of this group were determined based on a 96.3 % species border and 98.2 % for intraspecies resolution.

Genome biology of a novel lineage of planctomycetes widespread in anoxic aquatic environments

“…In addition, RNA polymerase beta subunit (RpoB) protein sequences were analysed, because several MAGs lacked 16S rRNA genes, but encoded almost complete RpoB sequences, which could be used as an alternative phylogenetic marker for the reconstruction of evolutionary relationships (Mollet et al, 1997;Bondoso et al, 2013). The placement of the analysed RpoB protein sequences within the Phycisphaerae class is illustrated in Supporting Information Fig.…”

Geographic distribution at subspecies resolution level: closely related Rhodopirellula species in European coastal sediments

“…Geographic distribution of Rhodopirellula species M Žure et al (Bondoso et al, 2013;Žure et al, 2015). In this case, acsA gene resulted as a good candidate to delimitate the amplification to three species with a 16S rRNA gene identity of 499.5%.…”

“…However, an adequate gene marker is necessary to circumvent the low resolution of the 16S rRNA gene and to resolve fine-scale biogeographic structures in environmental studies (Ramette and Tiedje, 2007). So far, rpoB coding for the beta subunit of the RNA polymerase (Bondoso et al, 2013) and carB coding for the large subunit of carbamoylphosphate synthetase (Žure et al, 2015) have been used for the genus Rhodopirellula. These amplicons were designed to cover the whole genus.…”

Description of Polystyrenella longa gen. nov., sp. nov., isolated from polystyrene particles incubated in the Baltic Sea

“…The average nucleotide identity (ANI) was calculated using OrthoANI (Lee et al 2016), the average amino acid identity (AAI) was computed with the aai.rb script from the enveomics collection (Rodriguez-R and Konstantinidis 2016) and the percentage of conserved proteins (POCP) was determined as previously described (Qin et al 2014). The rpoB nucleotide sequences (encoding the b-subunit of the DNA-dependent RNA polymerase) were taken from the genome annotations and the sequence identities were determined as described before (Bondoso et al 2013). Upon extracting only those parts of the sequences that would have been sequenced with the described primer set, the alignment and matrix calculation was done with Clustal Omega (Sievers et al 2011).…”

“…Other phylogenetic markers, such as rpoB similarity (Bondoso et al 2013), AAI (Konstantinidis and Tiedje 2005) and POCP (Qin et al 2014) provide Table 1 continued additional means to determine if strain Pla110 T is indeed part of a new genus (Fig. 3).…”