The elite strain INPA03-11B approved as a cowpea inoculant in Brazil represents a new Bradyrhizobium species and it has high adaptability to stressful soil conditions.

The strain INPA03-11BT, isolated in the 1980s from nodules of Centrosema sp. collected in Manaus, Amazonas, Brazil, was approved by the Brazilian Ministry of Agriculture as a cowpea inoculant in 2004. Since then, several studies have been conducted regarding its phenotypic, genetic, and symbiotic characteristics under axenic and field conditions. Phenotypic features demonstrate its high adaptability to stressful soil conditions, such as tolerance to acidity, high temperatures, and 13 antibiotics, and, especially, its high symbiotic efficiency with cowpea and soybean, proven in the field. The nodC and nifH phylogenies placed the INPA strain in the same clade as the species B. macuxiense BR 10303T which was also isolated from the Amazon region. The sequencing of the 16S rRNA ribosomal gene and housekeeping genes, as well as BOX-PCR profiles, showed its potential as a new species, which was confirmed by a similarity percentage of 94.7% and 92.6% in Average Nucleotide Identity with the closest phylogenetically related species Bradyrhizobium tropiciagri CNPSo1112T and B. viridifuturi SEMIA690T, respectively. dDDH values between INPA03-11BT and both CNPSo 1112T and SEMIA690T were respectively 58.5% and 48.1%, which are much lower than the limit for species boundary (70%). Therefore, we propose the name Bradyrhizobium amazonense for INPA03-11BT (= BR3301 = SEMIA6463).

Bacillus mexicanus sp. nov., a biological control bacterium isolated from the common bean (Phaseolus vulgaris L.) crop in Sinaloa, Mexico.

Strain FSQ1T was isolated from the rhizosphere of the common bean (Phaseolus vulgaris L.) crop sampled in a commercial field located in the Gabriel Leyva Solano community, which belongs to the Guasave municipality (state of Sinaloa, Mexico). Based on its full-length 16S rRNA gene sequence, strain FSQ1T was assigned to the genus Bacillus (100 % similarity). This taxonomic affiliation was supported by its morphological and metabolic traits. Strain FSQ1T was a Gram-stain-positive bacterium with the following characteristics: rod-shaped cells, strictly aerobic, spore forming, catalase positive, reduced nitrate to nitrite, hydrolysed starch and casein, grew in the presence of lysozyme and 2 % NaCl, utilized citrate, grew at pH 6.0-8.0, produced acid from glucose, was unable to produce indoles from tryptophan, and presented biological control against Sclerotinia sclerotiorum. The whole-genome phylogenetic results showed that strain FSQ1T formed an individual clade in comparison with highly related Bacillus species. In addition, the maximum values for average nucleotide identity and from Genome-to-Genome Distance Calculator analysis were 91.57 and 44.20 %, respectively, with Bacillus spizizenii TU-B-10T. Analysis of its fatty acid content showed the ability of strain FSQ1T to produce fatty acids that are not present in closely related Bacillus species, such as C18 : 0 and C20 : 0. Thus, these results provide strong evidence that strain FSQ1T represents a novel species of the genus Bacillus, for which the name Bacillus mexicanus sp. nov. is proposed. The type strain is FSQ1T (CM-CNRG TB51T=LBPCV FSQ1T).

Using average nucleotide identity (ANI) to evaluate microsporidia species boundaries based on their genetic relatedness.

Microsporidia are obligatory intracellular parasites related to fungi and since their discovery their classification and origin has been controversial due to their unique morphology. Early taxonomic studies of microsporidia were based on ultrastructural spore features, characteristics of their life cycle and transmission modes. However, taxonomy and phylogeny based solely on these characteristics can be misleading. SSU rRNA is a traditional marker used in taxonomical classifications, but the power of SSU rRNA to resolve phylogenetic relationships between microsporidia is considered weak at the species level, as it may not show enough variation to distinguish closely related species. Overall genome relatedness indices (OGRI), such as average nucleotide identity (ANI), allows fast and easy-to-implement comparative measurements between genomes to assess species boundaries in prokaryotes, with a 95% cutoff value for grouping genomes of the same species. Due to the increasing availability of complete genomes, metrics of genome relatedness have been applied for eukaryotic microbes taxonomy such as microsporidia. However, the distribution of ANI values and cutoff values for species delimitation have not yet been fully tested in microsporidia. In this study we examined the distribution of ANI values for 65 publicly available microsporidian genomes and tested whether the 95% cutoff value is a good estimation for circumscribing species based on their genetic relatedness.

A new microsporidian parasite, Ordospora pajunii sp. nov (Ordosporidae), of Daphnia longispina highlights the value of genomic data for delineating species boundaries.

Speciation is a complex and continuous process that makes the delineation of species boundaries a challenging task in particular in species with little morphological differentiation, such as parasites. In this case, the use of genomic data is often necessary, such as for the intracellular Microsporidian parasites. Here, we characterize the genome of a gut parasite of the cladoceran Daphnia longispina (isolate FI-F-10), which we propose as a new species within the genus Ordospora: Ordospora pajunii sp. nov (Ordosporidae). FI-F-10 closest relative, Ordospora colligata is only found in D. magna. Both microsporidian species share several morphological features. Although it is not possible to estimate divergence times for Microsporidia due to the lack of fossil records and accelerated evolutionary rates, we base our proposal on the phylogenomic and genomic distances between both microsporidian lineages. The phylogenomic reconstruction shows that FI-F-10 forms an early diverging branch basal to the cluster that contains all known O. colligata strains. Whole-genome comparisons show that FI-F-10 presents a greater divergence at the sequence level than observed among O. colligata strains, and its genomic average nucleotide identity (ANI) values against O. colligata are beyond the intraspecific range previously established for yeast and prokaryotes. Our data confirm that the ANI metrics are useful for fine genetic divergence calibration across Microsporidia taxa. In combination with phylogenetic and ecological data, genome-based metrics provide a powerful approach to delimitate species boundaries.

Unexpected genomic, biosynthetic and species diversity of Streptomyces bacteria from bats in Arizona and New Mexico, USA.

BACKGROUND: Antibiotic-producing Streptomyces bacteria are ubiquitous in nature, yet most studies of its diversity have focused on free-living strains inhabiting diverse soil environments and those in symbiotic relationship with invertebrates. RESULTS: We studied the draft genomes of 73 Streptomyces isolates sampled from the skin (wing and tail membranes) and fur surfaces of bats collected in Arizona and New Mexico. We uncovered large genomic variation and biosynthetic potential, even among closely related strains. The isolates, which were initially identified as three distinct species based on sequence variation in the 16S rRNA locus, could be distinguished as 41 different species based on genome-wide average nucleotide identity. Of the 32 biosynthetic gene cluster (BGC) classes detected, non-ribosomal peptide synthetases, siderophores, and terpenes were present in all genomes. On average, Streptomyces genomes carried 14 distinct classes of BGCs (range = 9-20). Results also revealed large inter- and intra-species variation in gene content (single nucleotide polymorphisms, accessory genes and singletons) and BGCs, further contributing to the overall genetic diversity present in bat-associated Streptomyces. Finally, we show that genome-wide recombination has partly contributed to the large genomic variation among strains of the same species. CONCLUSIONS: Our study provides an initial genomic assessment of bat-associated Streptomyces that will be critical to prioritizing those strains with the greatest ability to produce novel antibiotics. It also highlights the need to recognize within-species variation as an important factor in genetic manipulation studies, diversity estimates and drug discovery efforts in Streptomyces.

Paraburkholderia youngii sp. nov. and 'Paraburkholderia atlantica' - Brazilian and Mexican Mimosa-associated rhizobia that were previously known as Paraburkholderia tuberum sv. mimosae.

Previous studies have recognized South and Central/Latin American mimosoid legumes in the genera Mimosa, Piptadenia and Calliandra as hosts for various nodulating Paraburkholderia species. Several of these species have been validly named in the last two decades, e.g., P. nodosa, P. phymatum, P. diazotrophica, P. piptadeniae, P. ribeironis, P. sabiae and P. mimosarum. There are still, however, a number of diverse Paraburkholderia strains associated with these legumes that have an unclear taxonomic status. In this study, we focus on 30 of these strains which originate from the root nodules of Brazilian and Mexican Mimosa species. They were initially identified as P. tuberum and subsequently placed into a symbiovar (sv. mimosae) based on their host preferences. A polyphasic approach for the delineation of these strains was used, consisting of genealogical concordance analysis (using atpD, gyrB, acnA, pab and 16S rRNA gene sequences), together with comparisons of Average Nucleotide Identity (ANI), DNA G+C content ratios and phenotypic characteristics with those of the type strains of validly named Paraburkholderia species. Accordingly, these 30 strains were delineated into two distinct groups, of which one is conspecific with 'P. atlantica' CNPSo 3155T and the other new to Science. We propose the name Paraburkholderia youngii sp. nov. with type strain JPY169T (= LMG 31411T; SARCC751T) for this novel species.

The assessment of leading traits in the taxonomy of the Bacillus cereus group.

Bacillus cereus sensu lato strains (B. cereus group) are widely distributed in nature and have received interest for decades due to their importance in insect pest management, food production and their positive and negative repercussions in human health. Consideration of practical uses such as virulence, physiology, morphology, or ill-defined features have been applied to describe and classify species of the group. However, current comparative studies have exposed inconsistencies between evolutionary relatedness and biological significance among genomospecies of the B. cereus group. Here, the combined analyses of core-based phylogeny and all versus all Average Nucleotide Identity values based on 2116 strains were conducted to update the genomospecies circumscriptions within B. cereus group. These analyses suggested the existence of 57 genomospecies, 37 of which are novel, thus indicating that the taxonomic identities of more than 39% of the analyzed strains should be revised or updated. In addition, we found that whole-genome in silico analyses were suitable to differentiate genomospecies such as B. anthracis, B. cereus and B. thuringiensis. The prevalence of toxin and virulence factors coding genes in each of the genomospecies of the B. cereus group was also examined, using phylogeny-aware methods at wide-genome scale. Remarkably, Cry and emetic toxins, commonly assumed to be associated with B. thuringiensis and emetic B. paranthracis, respectively, did not show a positive correlation with those genomospecies. On the other hand, anthrax-like toxin and capsule-biosynthesis coding genes were positively correlated with B. anthracis genomospecies, despite not being present in all strains, and with presumably non-pathogenic genomospecies. Hence, despite these features have been so far considered relevant for industrial or medical classification of related species of the B. cereus group, they were inappropriate for their circumscription. In this study, genomospecies of the group were accurately affiliated and representative strains defined, generating a rational framework that will allow comparative analysis in epidemiological or ecological studies. Based on this classification the role of specific markers such as Type VII secretion system, cytolysin, bacillolysin, and siderophores such as petrobactin were pointed out for further analysis.

Draft genome sequence of Bacillus paralicheniformis TRQ65, a biological control agent and plant growth-promoting bacterium isolated from wheat (Triticum turgidum subsp. durum) rhizosphere in the Yaqui Valley, Mexico.

The strain denominated TRQ65 was isolated from wheat (Triticum turgidum subsp. durum) commercial fields in the Yaqui Valley, Mexico. Here, we report its draft genome sequence, which presented ~ 4.5 million bp and 45.5% G + C content. Based on the cutoff values on species delimitation established for average nucleotide identity (> 95 to 96%), genome-to-genome distance calculator (> 70%), and the reference sequence alignment-based phylogeny builder method, TRQ65 was strongly affiliated to Bacillus paralicheniformis. The rapid annotation using subsystem technology server revealed that TRQ65 contains genes related to osmotic, and oxidative stress response, as well as auxin biosynthesis (plant growth promotion traits). In addition, antiSMASH and BAGEL revealed the presence of genes involved in lipopeptides and antibiotic biosynthesis. The function of those annotated genes was validated at a metabolic level, observing that strain TRQ65 was able to tolerate saline (91.0%), and water (155.0%) stress conditions, besides producing 28.8 ± 0.9 µg/mL indoles. In addition, strain TRQ65 showed growth inhibition (1.6 ± 0.4 cm inhibition zone) against the causal agent of wheat spot blotch, Bipolaris sorokiniana. Finally, plant-microbe interactions assays confirm the ability of strain TRQ65 to regulate wheat growth, showing a significant increment in shoot height (26%), root length (40%), shoot dry weight (48%), stem diameter (55%), and biovolume index (246%). These findings provide insights for future agricultural studies of this strain.

Genomic metrics made easy: what to do and where to go in the new era of bacterial taxonomy.

With the advent of high-throughput DNA sequencing technologies, traditional methodologies for taxonomic classification of bacteria as DNA-DNA hybridization and 16S rRNA identity analyses are being challenged by the development of a fast-growing number of genomic metrics. The large amount of portable and digitized genome sequences available in public repositories constitutes an invaluable data for bacterial classification. Consequently, several genomic metrics and tools were developed to aid the interpretation of these massive data. Genomic metrics are based on the assumption that higher genome similarities would reflect closer phylogenetic relationships. Different metrics would vary in their methodology of analysis, resolution power, limitations and easiness of use. The aim of this review is to highlight the differences among available genome-based methods and tools to provide a guide for in silico bacterial identification and classification.

Classification of the inoculant strain of cowpea UFLA03-84 and of other strains from soils of the Amazon region as Bradyrhizobium viridifuturi (symbiovar tropici).

Cowpea (Vigna unguiculata L.) is a legume species that considerably benefits from inoculation with nitrogen fixing bacteria of the genus Bradyrhizobium. One of the strains recommended for inoculation in cowpea in Brazil is UFLA03-84 (Bradyrhizobium sp.). The aim of our study was to define the taxonomic position of the UFLA03-84 strain and of two other strains of Bradyrhizobium (UFLA03-144 and INPA237B), all belonging to the same phylogenetic group and isolated from soils of the Brazilian Amazon. Multilocus sequence analysis (MLSA) of the housekeeping genes atpD, gyrB, recA, and rpoB grouped (with similarity higher than 99%) the three strains with Bradyrhizobium viridifuturi SEMIA 690T. The analyses of average nucleotide identity and digital DNA-DNA hybridization supported classification of the group as Bradyrhizobium viridifuturi. The three strains exhibited similar behavior in relation to the most of the phenotypic characteristics evaluated. However, some characteristics exhibited variation, indicating phenotypic diversity within the species. Phylogenetic analysis of the nodC and nifH genes showed that the three strains are members of the same symbiovar (tropici) that contains type strains of Bradyrhizobium species coming from tropical soils (SEMIA 690TB. viridifuturi, CNPSo 1112TB. tropiciagri, CNPSo 2833TB. embrapense, and B. brasilense UFLA03-321T).

Identification of Acinetobacter seifertii isolated from Bolivian hospitals.

Acinetobacter seifertii is a recently described species that belongs to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. It has been recovered from clinical samples and is sometimes associated with antimicrobial resistance determinants. We present here the case of three A. seifertii clinical isolates which were initially identified as Acinetobacter sp. by phenotypic methods but no identification at the species level was achieved using semi-automated identification methods. The isolates were further analysed by whole genome sequencing and identified as A. seifertii. Due to the fact that A. seifertii has been isolated from serious infections such as respiratory tract and bloodstream infections, we emphasize the importance of correctly identifying isolates of the genus Acinetobacter at the species level to gain a deeper knowledge of their prevalence and clinical impact.

Reclassification of Paenibacillus riograndensis as a Genomovar of Paenibacillus sonchi: Genome-Based Metrics Improve Bacterial Taxonomic Classification.

Species from the genus Paenibacillus are widely studied due to their biotechnological relevance. Dozens of novel species descriptions of this genus were published in the last couple of years, but few utilized genomic data as classification criteria. Here, we demonstrate the importance of using genome-based metrics and phylogenetic analyses to identify and classify Paenibacillus strains. For this purpose, Paenibacillus riograndensis SBR5T, Paenibacillus sonchi X19-5T, and their close relatives were compared through phenotypic, genotypic, and genomic approaches. With respect to P. sonchi X19-5T, P. riograndensis SBR5T, Paenibacillus sp. CAR114, and Paenibacillus sp. CAS34 presented ANI (average nucleotide identity) values ranging from 95.61 to 96.32%, gANI (whole-genome average nucleotide identity) values ranging from 96.78 to 97.31%, and dDDH (digital DNA-DNA hybridization) values ranging from 68.2 to 73.2%. Phylogenetic analyses of 16S rRNA, gyrB, recA, recN, and rpoB genes and concatenated proteins supported the monophyletic origin of these Paenibacillus strains. Therefore, we propose to assign Paenibacillus sp. CAR114 and Paenibacillus sp. CAS34 to P. sonchi species, and reclassify P. riograndensis SBR5T as a later heterotypic synonym of P. sonchi (type strain X19-5T), with the creation of three novel genomovars, P. sonchi genomovar Sonchi (type strain X19-5T), P. sonchi genomovar Riograndensis (type strain SBR5T), P. sonchi genomovar Oryzarum (type strain CAS34T = DSM 102041T; = BR10511T).

New Insights into the Diversity of the Genus Faecalibacterium.

Faecalibacterium prausnitzii is a commensal bacterium, ubiquitous in the gastrointestinal tracts of animals and humans. This species is a functionally important member of the microbiota and studies suggest it has an impact on the physiology and health of the host. F. prausnitzii is the only identified species in the genus Faecalibacterium, but a recent study clustered strains of this species in two different phylogroups. Here, we propose the existence of distinct species in this genus through the use of comparative genomics. Briefly, we performed analyses of 16S rRNA gene phylogeny, phylogenomics, whole genome Multi-Locus Sequence Typing (wgMLST), Average Nucleotide Identity (ANI), gene synteny, and pangenome to better elucidate the phylogenetic relationships among strains of Faecalibacterium. For this, we used 12 newly sequenced, assembled, and curated genomes of F. prausnitzii, which were isolated from feces of healthy volunteers from France and Australia, and combined these with published data from 5 strains downloaded from public databases. The phylogenetic analysis of the 16S rRNA sequences, together with the wgMLST profiles and a phylogenomic tree based on comparisons of genome similarity, all supported the clustering of Faecalibacterium strains in different genospecies. Additionally, the global analysis of gene synteny among all strains showed a highly fragmented profile, whereas the intra-cluster analyses revealed larger and more conserved collinear blocks. Finally, ANI analysis substantiated the presence of three distinct clusters-A, B, and C-composed of five, four, and four strains, respectively. The pangenome analysis of each cluster corroborated the classification of these clusters into three distinct species, each containing less variability than that found within the global pangenome of all strains. Here, we propose that comparison of pangenome subsets and their associated α values may be used as an alternative approach, together with ANI, in the in silico classification of new species. Altogether, our results provide evidence not only for the reconsideration of the phylogenetic and genomic relatedness among strains currently assigned to F. prausnitzii, but also the need for lineage (strain-based) differentiation of this taxon to better define how specific members might be associated with positive or negative host interactions.

Bradyrhizobium sacchari sp. nov., a legume nodulating bacterium isolated from sugarcane roots.

Members of the genus Bradyrhizobium are well-known as nitrogen-fixing microsymbionts of a wide variety of leguminous species, but they have also been found in different environments, notably as endophytes in non-legumes such as sugarcane. This study presents a detailed polyphasic characterization of four Bradyrhizobium strains (type strain BR 10280T), previously isolated from roots of sugarcane in Brazil. 16S rRNA sequence analysis, multilocus sequence analysis (MLSA) and analysis of the 16S-23S rRNA internal transcribed spacer showed that these strains form a novel clade close to, but different from B. huanghuaihaiense strain CCBAU 23303T. Average nucleotide identity (ANI) analyses confirmed that BR 10280T represents a novel species. Phylogenetic analysis based on nodC gene sequences also placed the strains close to CCBAU 23303T, but different from this latter strain, the sugarcane strains did not nodulate soybean, although they effectively nodulated Vigna unguiculata, Cajanus cajan and Macroptilium atropurpureum. Physiological traits are in agreement with the placement of the strains in the genus Bradyrhizobium as a novel species for which the name Bradyrhizobium sacchari sp. nov. is proposed.