Genomic Metrics Applied to Rhizobiales (Hyphomicrobiales): Species Reclassification, Identification of Unauthentic Genomes and False Type Strains.

Camila Gazolla Volpiano,Emanuel Maltempi De Souza,Fernando Hayashi Sant’Anna,William B Whitman,Jackson Freitas Brilhante De São José,Luciano Kayser Vargas,Bruno Brito Lisboa,Adriana Ambrosini,Anelise Beneduzi,Luciane Maria Pereira Passaglia

doi:10.3389/fmicb.2021.614957

Abstract

Taxonomic decisions within the order Rhizobiales have relied heavily on the interpretations of highly conserved 16S rRNA sequences and DNA–DNA hybridizations (DDH). Currently, bacterial species are defined as including strains that present 95–96% of average nucleotide identity (ANI) and 70% of digital DDH (dDDH). Thus, ANI values from 520 genome sequences of type strains from species of Rhizobiales order were computed. From the resulting 270,400 comparisons, a ≥95% cut-off was used to extract high identity genome clusters through enumerating maximal cliques. Coupling this graph-based approach with dDDH from clusters of interest, it was found that: (i) there are synonymy between Aminobacter lissarensis and Aminobacter carboxidus, Aurantimonas manganoxydans and Aurantimonas coralicida, “Bartonella mastomydis,” and Bartonella elizabethae, Chelativorans oligotrophicus, and Chelativorans multitrophicus, Rhizobium azibense, and Rhizobium gallicum, Rhizobium fabae, and Rhizobium pisi, and Rhodoplanes piscinae and Rhodoplanes serenus; (ii) Chelatobacter heintzii is not a synonym of Aminobacter aminovorans; (iii) “Bartonella vinsonii” subsp. arupensis and “B. vinsonii” subsp. berkhoffii represent members of different species; (iv) the genome accessions GCF_003024615.1 (“Mesorhizobium loti LMG 6125T”), GCF_003024595.1 (“Mesorhizobium plurifarium LMG 11892T”), GCF_003096615.1 (“Methylobacterium organophilum DSM 760T”), and GCF_000373025.1 (“R. gallicum R-602 spT”) are not from the genuine type strains used for the respective species descriptions; and v) “Xanthobacter autotrophicus” Py2 and “Aminobacter aminovorans” KCTC 2477T represent cases of misuse of the term “type strain”. Aminobacter heintzii comb. nov. and the reclassification of Aminobacter ciceronei as A. heintzii is also proposed. To facilitate the downstream analysis of large ANI matrices, we introduce here ProKlust (“Prokaryotic Clusters”), an R package that uses a graph-based approach to obtain, filter, and visualize clusters on identity/similarity matrices, with settable cut-off points and the possibility of multiple matrices entries.

Highlights

The classification of Rhizobiales species is complex and has undergone many changes over the years. Frank (1889) described Rhizobium, the type genus of the order, to accommodate different symbiotic nitrogen-fixing bacteria based only on their selective interaction with legume plants
After the removal of sequences belonging to recently described genera that were absent in the classifier, a total of 17 genomes remained assigned to suspicious copies of the 16S rRNA (Table 2)
We found a total of four and two 16S rRNA gene copies in the A. manganoxydans SI85-9A1T and A. coralicida DSM 14790T genomes, respectively, which shared a high identity with the reference sequences (AJ786361 and AJ786360)

Summary

Introduction

The classification of Rhizobiales species is complex and has undergone many changes over the years. Frank (1889) described Rhizobium, the type genus of the order, to accommodate different symbiotic nitrogen-fixing bacteria based only on their selective interaction with legume plants. The minimal standards for species descriptions have usually incorporated DNA–DNA hybridization (DDH) and the 16S rRNA sequence analyses, as well as morphological, physiological, and biochemical features (Graham et al, 1991). In the pre-genomic era, the DDH was considered the gold standard for prokaryotic species circumscriptions. The DDH measures the fraction of DNA that hybridizes under optimal conditions, and a threshold of at least 70% between two strains was widely recognized as the species boundary (Tindall et al, 2010). In the first formal species definition based on the DDH proposed by Wayne et al (1987), the change in the melting temperature ( Tm) was recommended as a second measure of genetic relatedness to be considered with DDH, and a Tm value of 5◦C or less was established as the cut-off for prokaryotic species. Because DDH is technically easier to measure, Tm is usually not determined in most species’ descriptions (Li et al, 2015)

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Conclusion