- Volume 74, Issue 9, 2024

An erratum of this article has been published full details can be found at https://doi.org/10.1099/ijsem.0.006541

Two-novel filamentous actinobacteria designated strains 2-2T and 2-15T were isolated from soil of a coal mining site in Mongolia, and their taxonomic positions were determined using a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that each of the strains formed a distinct clade within the genus Amycolatopsis. The 16S rRNA gene sequence similarity analysis showed that both strains were mostly related to Amycolatopsis rhabdoformis NCIMB 14900T with 99.0 and 99.4% sequence similarity, respectively. The genome-based comparison indicated that strain 2-2T shared the highest digital DNA–DNA hybridization value of 35.6% and average nucleotide identity value of 86.9% with Amycolatopsis pretoriensis DSM 44654T, and strain 2-15T shared the corresponding values of 36.5 and 87.9% with A. rhabdoformis NCIMB 14900T, all of which being well below the thresholds for species delineation. The chemotaxonomic properties of both strains were typical of the genus Amycolatopsis. In silico prediction of chemotaxonomic markers was also carried out, and the results were consistent with the chemotaxonomic profiles of the genus. Genome mining for secondary metabolite production in strains 2-2T and 2-15T revealed the presence of 29 and 24 biosynthetic gene clusters involved in the production of polyketide synthase, non-ribosomal peptide synthetase, ribosomally synthesized and post-translationally modified peptides, lanthipeptide, terpenes, siderophore, and a number of other unknown type compounds. Both strains showed broad antifungal activity against several filamentous fungi and also antibacterial activity against methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii. The phenotypic, biochemical, and chemotaxonomic properties indicated that both strains could be clearly distinguished from other species of Amycolatopsis, and thus the names Amycolatopsis nalaikhensis sp. nov. (type strain, 2-2T=KCTC 29695T=JCM 30462T) and Amycolatopsis carbonis (type strain, 2-15T=KCTC 39525T=JCM 30563T) are proposed accordingly.

Six bacterial strains, Mut1T, Mut2, Alt1, Alt2, Alt3T, and Alt4, were isolated from soil samples collected in parks in Gothenburg, Sweden, based on their ability to utilize the insoluble polysaccharides α−1,3-glucan (mutan; Mut strains) or the mixed-linkage α−1,3/α−1,6-glucan (alternan; Alt strains). Analysis of 16S rRNA gene sequences identified all strains as members of the genus Streptomyces. The genomes of the strains were sequenced and subsequent phylogenetic analyses identified Mut2 as a strain of Streptomyces laculatispora and Alt1, Alt2 and Alt4 as strains of Streptomyces poriferorum, while Mut1T and Alt3T were most closely related to the type strains Streptomyces drozdowiczii NBRC 101007T and Streptomyces atroolivaceus NRRL ISP-5137T, respectively. Comprehensive genomic and biochemical characterizations were conducted, highlighting typical features of Streptomyces, such as large genomes (8.0–9.6 Mb) with high G+C content (70.5–72.0%). All six strains also encode a wide repertoire of putative carbohydrate-active enzymes, indicating a capability to utilize various complex polysaccharides as carbon sources such as starch, mutan, and cellulose, which was confirmed experimentally. Based on phylogenetic and phenotypic characterization, our study suggests that strains Mut1T and Alt3T represent novel species in the genus Streptomyces for which the names Streptomyces castrisilvae sp. nov. and Streptomyces glycanivorans sp. nov. are proposed, with strains Mut1T (=DSM 117248T=CCUG 77596T) and Alt3T (=DSM 117252T=CCUG 77600T) representing the respective type strains.

Two novel strains, YIM 133132T and YIM 133296, were isolated from lichen samples collected from Yunnan Province, Southwest PR China. YIM 133132T and YIM 133296 are aerobic, Gram-staining-positive, non-motile actinomycetes. They are also catalase-positive and oxidase-negative, and YIM 133132T formed flat yellowish colonies that were relatively dry on YIM38 agar medium. Flat yellowish colonies of YIM 133296 were also observed on YIM38 agar medium. YIM 133132T grew at 25–35 °C (optimum 25–30 °C), pH 6.0–9.0 (optimum pH 7.0) and in the presence of 0–8% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains YIM 133132T and YIM 133296 represented members of the genus Luteipulveratus and exhibited high sequence similarity (96.93%) with Luteipulveratus halotolerans C296001T. The genomic DNA G+C content of both strains was 71.8%. The DNA–DNA hybridisation (dDDH) values between YIM 133132T and YIM 133296 were 85.1%, and the DNA–DNA hybridisation value between YIM 133132T and YIM 133296 and L. halotolerans C296001T was 23.4%. On the basis of the draft genome sequences, the average nucleotide identity (ANI) between strains YIM 133132T and YIM 133296 and L. halotolerans C296001T was 80.8%. The major menaquinones that were identified were MK-8(H4), MK-9 and MK-8(H2). The polar lipids were diphosphatidylglycerol and phosphatidylinositol. On the basis of the morphological, physiological, biochemical, genomic, phylogenetic and chemotaxonomic characteristics, strains YIM 133132T and YIM 133296 can be clearly distinguished from L. halotolerans C296001T, and the two strains represent a novel species for which the name L. flavus sp. nov. is proposed. The type strain is YIM 133132T (CGMCC= 1.61357T and KCTC= 49824T).

Two obligately acidophilic, mesophilic and aerobic soil ammonia-oxidising archaea were isolated from a pH 4.5 arable sandy loam (UK) and pH 4.7 acidic sulphate paddy soil (PR China) and designated strains Nd1T and Nd2T, respectively. The strains shared more than 99 % 16S rRNA gene sequence identity and their genomes were both less than 2 Mb in length, sharing 79 % average nucleotide identity, 81 % average amino acid identity and a DNA G+C content of approximately 37 mol%. Both strains were chemolithotrophs that fixed carbon dioxide and gained energy by oxidising ammonia to nitrite, with no evidence of mixotrophic growth. Neither strain was capable of using urea as a source of ammonia. Both strains were non-motile in culture, although Nd1T does possess genes encoding flagella components and therefore may be motile under certain conditions. Cells of Nd1T were small angular rods 0.5–1 µm in length and grew at pH 4.2–5.6 and at 20–30 °C. Cells of Nd1T were small angular rods 0.5–1 µm in length and grew at pH 4.0–6.1 and at 20–42 °C. Nd1T and Nd2T are distinct with respect to genomic and physiological features and are assigned as the type strains for the species Nitrosotalea devaniterrae sp. nov. (type strain, Nd1T=NCIMB 15248T=DSM 110862T) and Nitrosotalea sinensis sp. nov. (type strain, Nd2T=NCIMB 15249T=DSM 110863T), respectively, within the genus Nitrosotalea gen. nov. The family Nitrosotaleaceae fam. nov. and order Nitrosotaleales ord. nov. are also proposed officially.

The haloarchaeal genera Halomicroarcula and Haloarcula, belonging to the family Haloarculaceae, order Halobacteriales, class Halobacteria, within the phylum Methanobacteriota, have previously exhibited significant phylogenetic and taxonomic overlaps. This issue was recently resolved by merging the two genera into a single genus, Haloarcula. However, Halomicroarcula saliterrae and Halomicroarcula onubensis were described almost simultaneously with the proposal to unify the genera Haloarcula and Halomicroarcula. Their names were validly published under the International Code of Nomenclature of Prokaryotes (ICNP) according to Validation List no. 217, alongside six Haloarcula species and the transfer of the existing Halomicroarcula species into the genus Haloarcula. Therefore a phylogenetic, phylogenomic, and comparative genomic analysis was carried out to clarify the taxonomic status of these two haloarchaeal species, Halomicroarcula saliterrae and Halomicroarcula onubensis, with lower priority than the six new species of the genus Haloarcula. Phylogenetic studies of 16S rRNA and rpoB′ gene sequences, along with phylogenomic reconstructions using single-copy core-orthologous proteins, indicated that the two species clustered with the members of the genus Haloarcula. The overall genome relatedness indexes (OGRIs), comparative analyses of phenotypic features, and polar lipid profiles further supported their taxonomic reassignment as two separate species within the genus Haloarcula. Consequently, we propose the reclassification of Halomicroarcula saliterrae Straková et al. 2024 and Halomicroarcula onubensis Straková et al. 2024 into the genus Haloarcula, as Haloarcula saliterrae comb. nov. and Haloarcula onubensis comb. nov., respectively, in accordance with the ICNP.

Three Gram-stain-negative, aerobic, non-motile, chemoheterotrophic, short-rod-shaped bacteria, designated CDY1-MB1T, CDY2-MB3, and BDY3-MB2, were isolated from three marine sediment samples collected in the eastern Pacific Ocean. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains were related to the genus Aequorivita and close to the type strain of Aequorivita vitellina F4716T (with similarities of 98.0–98.1%). Strain CDY1-MB1T can grow at 15–37 °C (optimum 30 °C) and in media with pH 6–9 (optimum, pH 7), and tolerate up to 10% (w/v) NaCl. The predominant cellular fatty acids of strain CDY1-MB1T were iso-C15 : 0 (20.7%) and iso-C17 : 0 3-OH (12.8%); the sole respiratory quinone was menaquinone 6; the major polar lipids were phosphatidylethanolamine, two unidentified aminolipids and two unidentified polar lipids. The digital DNA–DNA hybridization/average nucleotide identity values between strains CDY1-MB1T, CDY2-MB3, and BDY3-MB2 and A. vitellina F4716T were 24.7%/81.6–81.7%, thereby indicating that strain CDY1-MB1T should represent a novel species of the genus Aequorivita. The genomic DNA G+C contents were 37.6 % in all three strains. Genomic analysis showed the presence of genes related to nitrogen and sulphur cycling, as well as metal reduction. The genetic traits of these strains indicate their possible roles in nutrient cycling and detoxification processes, potentially shaping the deep-sea ecosystem’s health and resilience. Based upon the consensus of phenotypic and genotypic analyses, strain CDY1-MB1T should be classified as a novel species of the genus Aequorivita, for which the name Aequorivita flava sp. nov. is proposed. The type strain is CDY1-MB1T (=MCCC 1A16935T=KCTC 102223T).

The 16S rRNA gene of Thermobacterium salinum TK19130T had the highest sequence similarity to that of Luteirhabdus pelagi A3-108T (99.7%). Phylogeny of 16S rRNA gene and whole genome sequences indicated that T. salinum TK19130T and L. pelagi A3-108T are closely related, and represented an independent clade. Whole genome comparisons showed that T. salinum TK19130T and L. pelagi A3-108T shared average amino acid identity of 95.3%, indicating they could be merged into the same genus. The digital DNA–DNA hybridization and average nucleotide identity values between T. salinum TK19130T and L. pelagi A3-108T were 52.5 and 93.3%, respectively. These values were below the recommended threshold values of prokaryotic species delineation. Thus, based on the principle of priority, we proposed the transfer of Thermobacterium salinum Chen et al. 2023 to the genus Luteirhabdus Ren et al. 2022 as Luteirhabdus salina comb. nov.

In this article, the author addresses the issue of nomenclatural illegitimacy of Paenimyroides aquimaris (García-López et al. 2020) Zhang et al. 2023. This name was formed without re-establishment of the earlier legitimate epithet marinum and should be considered to be illegitimate according to Rule 41a. As required by Rule 54, the author proposes Paenimyroides marinum (Song et al. 2013) as a new combination to replace the illegitimate name Paenimyroides aquimaris.

A Gram-stain-negative, yellow-pigmented, and strictly aerobic bacterium, designated as strain MSW5T, was isolated from seawater of the Yellow Sea in South Korea. The cells were non-motile rods exhibiting oxidase- and catalase-positive activities. Growth was observed at 15–25 °C (optimum, 25 °C) and pH 5.0–9.0 (optimum, pH 7.0–8.0) and in the presence of 1.0–5.0% (w/v) NaCl (optimum, 2.0%). Menaquinone-6 was the sole respiratory quinone, and iso-C15 : 0, summed feature 3 (C16 : 1  ω7c and/or C16 : 1  ω6c), iso-C15 : 0 3-OH, and C15 : 1  ω6c were the major cellular fatty acids. Major polar lipids included phosphatidylethanolamine, two unidentified aminolipids, and three unidentified lipids. Phylogenetic analyses based on 16S rRNA gene sequences and 92 concatenated core protein sequences revealed that strain MSW5T formed a distinct lineage within the genus Polaribacter. The genome of strain MSW5T was 3582 kb in size with a 29.1 mol% G+C content. Strain MSW5T exhibited the highest similarity to Polaribacter atrinae WP25T, with a 97.9% 16S rRNA gene sequence similarity. However, the average nucleotide identity and digital DNA–DNA hybridization values were 79.4 and 23.3%, respectively, indicating that strain MSW5T represents a novel species. Based on its phenotypic, chemotaxonomic, and phylogenetic characteristics, strain MSW5T is proposed to represent a novel species, with the name Polaribacter ponticola sp. nov. The type strain is MSW5T (=KACC 22340T=NBRC 116025T). In addition, whole genome sequence comparisons and phenotypic features suggested that Polaribacter sejongensis and Polaribacter undariae belong to the same species, with P. undariae proposed as a later heterotypic synonym of P. sejongensis. An emended description of Polaribacter sejongensis is also proposed.

The reclassification of Butyrivibrio crossotus Moore et al. 1976 (Approved Lists 1980) as Eshraghiella crossota gen. nov., comb. nov. is proposed within the family Lachnospiraceae. This reclassification is based on differences revealed through the analysis of 16S rRNA, groEL, recA, and rpoB genes, as well as genome sequences, distinguishing it from other Butyrivibrio species. Comparative analysis showed that B. crossotus exhibited digital DNA–DNA hybridization (dDDH) values of 19.40–27.20% and average nucleotide identities based on blast (ANIb) values of 67.06–67.64% with other Butyrivibrio species. These values are significantly below the species delineation thresholds (dDDH, 70%; ANIb, 95–96%), justifying the proposed reclassification. Additionally, the results of the average amino acid identity (AAI) analysis indicated that this species shares 59.22–60.17% AAI with the other species of the genus Butyrivibrio, which is below the AAI threshold (65%) for a genus boundary. In addition, biochemical and morphological characteristics also support the proposal that this species is different from other species of the genus Butyrivibrio. The type strain is ATCC 29175T (DSM 2876T=T9-40AT).

Seven novel lactic acid bacterial strains (BF125T, BF186, TKL145, YK3, YK6, YK10 and NSK) were isolated from the fresh faeces of Japanese black beef cattle and weanling piglets, spent mushroom substrates, or steeping water of a corn starch production plant. These strains are rod-shaped, Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, cytochrome oxidase-negative, facultatively anaerobic, and homofermentative. Strain BF125T did not produce any gas from glucose; both d- and l-lactate were produced as end-products of glucose (D/L, 40 : 60). Growth occurred at 30–45 °C (optimum, 37 °C), pH 5.0–8.0 (optimum, pH 6.0), and with NaCl concentration of 1.0–3.0% (w/v). The G+C content of genomic DNA of strain BF125T was 37.8 mol% (whole-genome analysis). The major fatty acids were C16 : 0, C18 : 1 ω9c, C19 cyclopropane 9, 10, and summed feature 10. The 16S rRNA gene in strain BF125T showed high similarity to that of the type strain of Lactobacillus amylovorus (99.93%), and the other isolates were also identified as L. amylovorus based on these similarities. A phylogenetic tree based on the core genomes of L. amylovorus strains (n=54), including the seven isolates, showed that they could be divided into two clusters. Strains YK3, YK6, YK10, and NSK were in the first cluster, along with the type strain DSM 20531T, while the second cluster included isolates BF125T, BF186, TKL145, and other strains isolated from various animal origins. Phenotypic differences in fermentability were observed for lactose, salicin, and gentiobiose between these two groups. The intergroup digital DNA–DNA hybridization values (72.9–78.6%) and intergroup average nucleotide identity values (95.64–96.92%) were comparable to values calculated using datasets of other valid subspecies of the genus (ex-) Lactobacillus. In light of the physiological, genotypic, and phylogenetic evidence, we propose a novel subspecies of L. amylovorus, named Lactobacillus amylovorus subsp. animalis subsp. nov. (type strain BF125T=MAFF 212522T=DSM 115528T). Our findings also led to the automatic creation of Lactobacillus amylovorus subsp. amylovorus subsp. nov. and an emended description of the species L. amylovorus.

One bacterial strain, designated as C22-A2T, was isolated from Lake LungmuCo in Tibet. Cells of strain C22-A2T were long rod-shaped, Gram-stain-negative, non-spore-forming, with positive catalase and oxidase activity. Optimal growth occurred at 20–25 °C, pH 8.0 and with 3.0–7.0% (w/v) NaCl. Phylogenetic analysis of 16S rRNA gene and whole genome sequences revealed that strain C22-A2T belonged to the genus Virgibacillus, showing the highest 16S rRNA gene similarity to Virgibacillus halodenitrificans DSM 10037T (97.6%). The average nucleotide identity values between strain C22-A2T and the type strains of related species in the genus Virgibacillus were less than 74.4% and the digital DNA–DNA hybridization values were less than 20.2%, both below the species delineation thresholds of 95 and 70% respectively. The genome analysis revealed that strain C22-A2T harboured genes responsible for osmotic and oxidative stress, enabling it to adapt to its surrounding environment. In terms of biochemical and physiological characteristics, strain C22-A2T shared similar characteristics with the genus Virgibacillus, including the predominant cellular fatty acid anteiso-C15 : 0, the major respiratory quinone MK-7, as well as the polar lipids phosphatidylglycerol and diphosphatidylglycerol. Based on the comprehensive analysis of phylogenetic, phylogenomic, morphological, physiological and biochemical characteristics, strain C22-A2T is proposed to represent a novel species of the genus Virgibacillus, named as Virgibacillus tibetensis sp. nov. (=CGMCC 1.19202T=KCTC 43426T).

The family Anoxybacillaceae was recently proposed encompassing the genera Anoxybacillus, Geobacillus, Parageobacillus, Saccharococcus and Thermolongibacillus. Of these genera, Anoxybacillus contains >50% of the Anoxybacillaceae species. However, Anoxybacillus species form multiple unrelated clades in phylogenetic trees and their evolutionary relationships are unclear. To clarify the evolutionary relationships of Anoxybacillus and other Anoxybacillaceae species, detailed phylogenomic and comparative analyses were conducted on 38 Anoxybacillaceae species with available genomes. In a phylogenomic tree based on 1148 core proteins, all Anoxybacillus, Geobacillus, Parageobacillus, Saccharococcus and Thermolongibacillus species, excepting Anoxybacillus sediminis, formed a strongly supported clade representing the family Anoxybacillaceae. Five conserved signature indels (CSIs) reported here are also uniquely found in these species, providing robust means for the demarcation of family Anoxybacillaceae in molecular terms. In our phylogenomic tree and in the Genomic Taxonomy Database, Anoxybacillus species formed four distinct clades designated as Anoxybacillus sensu stricto (containing the type species A. pushchinoensis), Anoxybacillus_A, Anoxybacillus_B and Anoxybacillus_C. Our analyses have identified 17 novel CSIs which offer means to reliably distinguish species from these clades based upon multiple uniquely shared molecular characteristics. Additionally, we have identified three and seven CSIs specific for the genera Geobacillus and Brevibacillus, respectively. All seven Brevibacillus-specific CSIs are also shared by Anoxybacillus sediminis, which branches reliably with this genus. Based on the strong phylogenetic and molecular evidence presented here, we are proposing that the genus Anoxybacillus should be restricted to only the species from Anoxybacillus sensu stricto clade, whereas the species from Anoxybacillus_A, Anoxybacillus_B, and Anoxybacillus_C clades should be transferred into three novel genera Anoxybacteroides gen. nov., Paranoxybacillus gen. nov. and Thermaerobacillus gen. nov., respectively. Additionally, we are also proposing the transfer of Anoxybacillus sediminis to the genus Brevibacillus. The proposed changes, which reliably depict the evolutionary relationships among Anoxybacillaceae species, should be helpful in the studies of these organisms.

A Gram-stain-negative, strictly aerobic, motile, flagellated, rod-shaped, halotolerant, and poly-β-hydroxyalkanoate-producing bacterium, designated DP4N28-3T, was isolated from offshore sediment surrounding hard coral in the Dapeng peninsula (Guangdong, PR China). Growth occurred at 15–35 °C (optimal at 30 °C), pH 6.0–9.5 (optimal at 6.0–7.0), and 0.0–30.0 % NaCl concentration (w/v, optimal at 0.0–2.0 %), showing halotolerance. Phylogeny based on 16S rRNA gene sequences, five housekeeping genes, and genome sequences identified Pseudohoeflea suaedae DSM 23348T (98.1 %, 16S rRNA gene sequence similarity) as the most related species to strain DP4N28-3T. Average nucleotide identity, digital DNA–DNA hybridization, and average amino acid identity values between strain DP4N28-3T and P. suaedae DSM 23348T were all below the threshold of species demarcation. Major phenotypic differences were the flagella type and the limited sources of single carbon utilization by strain DP4N28-3T, which only included acetic acid, acetoacetic acid, d-glucuronic acid, and glucuronamide. Strain DP4N28-3T harboured the class I poly-β-hydroxyalkanoate synthase gene (phaC) and produced poly-β-hydroxybutyrate. The fatty acids were summed feature 8 (C18 : 1  ω6c and/or C18 : 1  ω7c, 49.4 %) and C16 : 0 (13.4 %). The major cellular polar lipids consisted of phosphatidylcholine, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, and sulfoquinovosyl diacylglycerol. The respiratory quinone was Q-10. The results of the phylogenetic, genomic, phenotypic, and chemotaxonomic analysis indicated that the isolated strain represents the type strain of a novel species. Based on these results, strain DP4N28-3T (=MCCC 1K05639T=KCTC 82803T) is proposed as the type strain of the novel species Pseudohoeflea coraliihabitans sp. nov.

Two bacterial strains, Y60-23T and HN-65T, were isolated from marine sediment samples collected from Xiaoshi Island, Weihai, and Dongzhai Harbour, Haikou, PR China, respectively. Based on the 16S rRNA gene sequences, strain Y60-23T exhibited 96.0% similarity to its most related type strain Hyphobacterium vulgare KCTC 52487T, while strain HN-65T exhibited 97.3% similarity to its most related type strain Hyphobacterium indicum 2ED5T. The 16S rRNA gene sequence similarity between the two strains was 95.8%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains Y60-23T and HN-65T belonged to the genus Hyphobacterium. Cells of strains Y60-23T and HN-65T were rod-shaped, Gram-stain-negative, aerobic, non-motile, prosthecate and multiplied by binary fission. The major cellular fatty acids (>10.0%) of strain Y60-23T were C18 : 1  ω7c and C17 : 0, while those of strain HN-65T were iso-C17 : 1  ω9c, iso-C17 : 0 and C18 : 1  ω7c. The major respiratory quinone in both strains was ubiquinone-10 (Q-10) and the major polar lipids were monoglycosyl diglyceride, sulfoquinovosyl diacylglycerol and glucuronopyranosyl diglyceride. The genomic DNA G+C contents of strains Y60-23T and HN-65T were 63.9 and 60.7 mol%, respectively. The average nucleotide identity value between the two strains was 72.1% and the DNA–DNA hybridization value was 18.4%, clearly distinguishing them from each other. According to the results of the phenotypic, chemotaxonomic, phylogenetic and genomic analyses, the two strains represented two novel species within the genus Hyphobacterium, for which the names Hyphobacterium marinum sp. nov. and Hyphobacterium lacteum sp. nov. were proposed with the type strains Y60-23T (=MCCC 1H01433T=KCTC 8172T) and HN-65T (=MCCC 1H01434T=KCTC 8169T), respectively.

Three bacterial strains, 1AS14IT, 1AS12I and 6AS6, isolated from root nodules of Acacia saligna, were characterized using a polyphasic approach. Phylogenetic analysis based on rrs sequences placed all three strains within the Rhizobium leguminosarum complex. Further phylogeny, based on 1 756 bp sequences of four concatenated housekeeping genes (recA, atpD, glnII and gyrB), revealed their distinction from known rhizobia species of the R. leguminosarum complex (Rlc), forming a distinct clade. The closest related species, identified as Rhizobium laguerreae, with a sequence identity of 96.4% based on concatenated recA-atpD-glnII-gyrB sequences. The type strain, 1AS14IT, showed average nucleotide identity (ANI) values of 94.9, 94.3 and 94.1% and DNA–DNA hybridization values of 56.1, 57.4 and 60.0% with the type strains of closest known species: R. laguerreae, Rhizobium acaciae and ‘Rhizobium indicum’, respectively. Phylogenomic analyses using 81 up-to-date bacteria core genes and the Type (Strain) Genome Server pipeline further supported the uniqueness of strains 1AS14IT, 1AS12I and 6AS6. The relatedness of the novel strains to NCBI unclassified Rhizobium sp. (396 genomes) and metagenome-derived genomes showed ANI values from 76.7 to 94.8% with a species-level cut-off of 96%, suggesting that strains 1AS14I, 1AS12I and 6AS6 are a distinct lineage. Additionally, differentiation of strains 1AS14IT, 1AS12I and 6AS6 from their closest phylogenetic neighbours was achieved using phenotypic, physiological and fatty acid content analyses. Based on the genomic, phenotypic and biochemical data, we propose the establishment of a novel rhizobial species, Rhizobium aouanii sp. nov., with strain 1AS14IT designated as the type strain (=DSM 113914T=LMG 33206T). This study contributes to the understanding of microbial diversity in nitrogen-fixing symbioses, specifically within Acacia saligna ecosystems in Tunisia.