Alignment Fraction Research Articles

Description of a new genus of the Pectobacteriaceae family isolated from water in coastal brackish wetlands of the French Camargue region, Prodigiosinella gen. nov., including the new species Prodigiosinella aquatilis sp. nov

The Pectobacteriaceae family comprises plant pathogens able to provoke diverse diseases, including plant maceration due to the production of pectinases disrupting the plant cell wall. To better understand their diversity, a survey of pectinolytic bacteria was performed in brackish lakes of the French region La Camargue near the Mediterranean Sea. The genome of six atypical isolates was sequenced; their size is around 4.8 to 5.0 Mb, including a plasmid of 59 to 61 kb; their G+C values range from 49.1 to 49.3 mol%. Phylogenetic analyses indicated that the novel strains form a new clade of Pectobacteriaceae that branches at the basis of the group encompassing the genera Lonsdalea, Musicola, and Dickeya. Based on phenotypic, genomic and phylogenetic characteristics, we propose the creation of a new genus with the name Prodigiosinella gen. nov. Both the phenotypic and phylogenetic analyses separated the strains into two distinct subgroups, G1 and G2. The type strain LS101T (CFBP 8826T = LMG 32072T) and strain CE70 (CFBP 9054 = LMG 32867) are representative G1 and G2 members, respectively. Three genomic methods were used to analyze DNA–DNA relatedness: digital DNA–DNA hybridization (isDDH), average nucleotide identity (ANI), and genome alignment fraction (AF). They revealed a close relationship between genomes of the two groups, supporting their appurtenance to a same species for which we propose the name Prodigiosinella aquatilis sp. nov. Four strains previously designated as Serratia sp. (ATCC 39006), Brenneria “ulupoensis” (K61) or Erwinia sp. (MK01 and MK09) belong to the new genus Prodigiosinella.

Physiological and Genomic Characterization of a Hyperthermophilic Archaeon Archaeoglobus neptunius sp. nov. Isolated From a Deep-Sea Hydrothermal Vent Warrants the Reclassification of the Genus Archaeoglobus.

Hyperthermophilic archaea of the genus Archaeoglobus are the subject of many fundamental and biotechnological researches. Despite their significance, the class Archaeoglobi is currently represented by only eight species obtained as axenic cultures and taxonomically characterized. Here, we report the isolation and characterization of a new species of Archaeoglobus from a deep-sea hydrothermal vent (Mid-Atlantic Ridge, TAG) for which the name Archaeoglobus neptunius sp. nov. is proposed. The type strain is SE56T (=DSM 110954T = VKM B-3474T). The cells of the novel isolate are motile irregular cocci growing at 50–85°C, pH 5.5–7.5, and NaCl concentrations of 1.5–4.5% (w/v). Strain SE56T grows lithoautotrophically with H2 as an electron donor, sulfite or thiosulfate as an electron acceptor, and CO2/HCO3− as a carbon source. It is also capable of chemoorganotrophic growth by reduction of sulfate, sulfite, or thiosulfate. The genome of the new isolate consists of a 2,115,826 bp chromosome with an overall G + C content of 46.0 mol%. The whole-genome annotation confirms the key metabolic features of the novel isolate demonstrated experimentally. Genome contains a complete set of genes involved in CO2 fixation via reductive acetyl-CoA pathway, gluconeogenesis, hydrogen and fatty acids oxidation, sulfate reduction, and flagellar motility. The phylogenomic reconstruction based on 122 conserved single-copy archaeal proteins supported by average nucleotide identity (ANI), average amino acid identity (AAI), and alignment fraction (AF) values, indicates a polyphyletic origin of the species currently included into the genus Archaeoglobus, warranting its reclassification.

Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species.

Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales. Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0–4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27–59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis, respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis, R. baikonurensis, R. opacus and R. rhodochrous, would be reclassified either as R. erythropolis or R. qingshengii.

Genome analysis reveals that the correct name of type strain Adlercreutzia caecicola DSM 22242T is Parvibacter caecicola Clavel et al. 2013.

The strain Adlercreutzia caecicola DSM 22242T (=CCUG 57646T=NR06T) was taxonomically described in 2013 and named as Parvibacter caecicola Clavel et al. 2013. In 2018, the name of the strain DSM 22242T was changed to Adlercreutzia caecicola (Clavel et al. 2013) Nouioui et al. 2018 due to taxonomic investigations of the closely related genera Adlercreutzia, Asaccharobacter and Enterorhabdus within the phylum Actinobacteria. However, the first whole draft genome of strain DSM 22242T was published by our group in 2019. Therefore, the genome was not available within the study of Nouioui et al. (2018). The results of the polyphasic approach within this study, including phenotypic and biochemical analyses and genome-based taxonomic investigations [genome-wide average nucleotide identity (gANI), alignment fraction (AF), average amino acid identity (AAI), percentage of orthologous conserved proteins (POCP) and genome blast distance phylogeny (GBDP) tree], indicated that the proposed change of the name Parvibacter caecicola to Adlercreutzia caecicola was not correct. Therefore, it is proposed that the correct name of Adlercreutzia caecicola (Clavel et al. 2013) Nouioui et al. 2018 strain DSM 22242T is Parvibacter caecicola Clavel et al. 2013.

Aequoribacter fuscus gen. nov., sp. nov., a new member of the family Halieaceae, isolated from coastal seawater.

A Gram-stain-negative, rod-shaped, obligately aerobic, nonflagellated, and chemoheterotrophic bacterium, designated IMCC3088T, was isolated from coastal seawater of the Yellow Sea. The 16S rRNA gene sequence analysis indicated that this strain belonged to the family Halieaceae which shared the highest sequence similarities with Luminiphilus syltensis NOR5-1BT (94.5%) and Halioglobus pacificus S1-72T (94.5%), followed by 92.3-94.3% sequence similarities with other species within the aforementioned family. Phylogenetic analyses demonstrated that strain IMCC3088T was robustly clustered with Luminiphilus syltensis NOR5-1BT within the family Halieaceae. However, average amino acid identity (AAI), percentages of conserved proteins (POCP), average nucleotide identity (ANI), and alignment fraction (AF) between strain IMCC3088T and Luminiphilus syltensis NOR5-1BT were 54.5%, 47.7%, 68.0%, and 16.5%, respectively, suggesting that they belonged to different genera. Whole-genome sequencing of strain IMCC3088T revealed a 3.1 Mbp genome size with a DNA G + C content of 51.7 mol%. The genome encoded diverse metabolic pathways including sulfur oxidation, phenol degradation, and proteorhodopsin phototrophy. Mono-unsaturated fatty acids were found to be the predominant cellular fatty acid components in the strain. Phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol were the primarily identified polar lipids, and ubiquinone-8 was identified as a major respiratory quinone. The taxonomic data collected herein suggested that strain IMCC3088T represented a novel genus and species of the family Halieaceae, for which the name Aequoribacter fuscus gen. nov., sp. nov. is proposed with the type strain (= KACC 15529T = NBRC 108213T).

Microbial species delineation using whole genome sequences.

Increased sequencing of microbial genomes has revealed that prevailing prokaryotic species assignments can be inconsistent with whole genome information for a significant number of species. The long-standing need for a systematic and scalable species assignment technique can be met by the genome-wide Average Nucleotide Identity (gANI) metric, which is widely acknowledged as a robust measure of genomic relatedness. In this work, we demonstrate that the combination of gANI and the alignment fraction (AF) between two genomes accurately reflects their genomic relatedness. We introduce an efficient implementation of AF,gANI and discuss its successful application to 86.5M genome pairs between 13,151 prokaryotic genomes assigned to 3032 species. Subsequently, by comparing the genome clusters obtained from complete linkage clustering of these pairs to existing taxonomy, we observed that nearly 18% of all prokaryotic species suffer from anomalies in species definition. Our results can be used to explore central questions such as whether microorganisms form a continuum of genetic diversity or distinct species represented by distinct genetic signatures. We propose that this precise and objective AF,gANI-based species definition: the MiSI (Microbial Species Identifier) method, be used to address previous inconsistencies in species classification and as the primary guide for new taxonomic species assignment, supplemented by the traditional polyphasic approach, as required.