Stenotrophomonas riyadhensis sp. nov., isolated from a hospital floor swab.

During the analysis of a collection of Pseudomonas strains linked to an outbreak in an intensive care unit at King Faisal Specialist Hospital and Research Center in 2019, one isolate (CFS3442T) was identified phenotypically as Pseudomonas aeruginosa. However, whole-genome sequencing revealed its true identity as a member of the genus Stenotrophomonas, distinct from both P. aeruginosa and Stenotrophomonas maltophilia. The isolate demonstrated: (i) a significant phylogenetic distance from P. aeruginosa; (ii) considerable genomic differences from several S. maltophilia reference strains and other Stenotrophomonas species; and (iii) unique phenotypic characteristics. Based on the combined geno- and phenotypic data, we propose that this isolate represents a novel species within the genus Stenotrophomonas, for which the name Stenotrophomonas riyadhensis sp. nov. is proposed. The type strain is CFS3442T (=NCTC 14921T=LMG 33162T).

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).

High prevalence of colistin heteroresistance in specific species and lineages of Enterobacter cloacae complex derived from human clinical specimens.

BACKGROUND: Colistin (CST) is a last-line drug for multidrug-resistant Gram-negative bacterial infections. CST-heteroresistant Enterobacter cloacae complex (ECC) has been isolated. However, integrated analysis of epidemiology and resistance mechanisms based on the complete ECC species identification has not been performed. METHODS: Clinical isolates identified as "E. cloacae complex" by MALDI-TOF MS Biotyper Compass in a university hospital in Japan were analyzed. Minimum inhibitory concentrations of CST were determined by the broth microdilution method. The population analysis profiling (PAP) was performed for detecting the heteroresistant phenotype. The heat shock protein 60 (hsp60) cluster was determined from its partial nucleotide sequence. From the data of whole-genome sequencing, average nucleotide identity (ANI) for determining ECC species, multilocus sequence type, core genome single-nucleotide-polymorphism-based phylogenetic analysis were performed. phoPQ-, eptA-, and arnT-deleted mutants were established to evaluate the mechanism underlying colistin heteroresistance. The arnT mRNA expression levels were determined by reverse transcription quantitative PCR. RESULTS: Thirty-eight CST-resistant isolates, all of which exhibited the heteroresistant phenotype by PAP, were found from 138 ECC clinical isolates (27.5%). The prevalence of CST-resistant isolates did not significantly differ among the origin of specimens (29.0%, 27.8%, and 20.2% for respiratory, urine, and blood specimens, respectively). hsp60 clusters, core genome phylogeny, and ANI revealed that the CST-heteroresistant isolates were found in all or most of Enterobacter roggenkampii (hsp60 cluster IV), Enterobacter kobei (cluster II), Enterobacter chuandaensis (clusters III and IX), and Enterobacter cloacae subspecies (clusters XI and XII). No heteroresistant isolates were found in Enterobacter hormaechei subspecies (clusters VIII, VI, and III) and Enterobacter ludwigii (cluster V). CST-induced mRNA upregulation of arnT, which encodes 4-amino-4-deoxy-L-arabinose transferase, was observed in the CST-heteroresistant isolates, and it is mediated by phoPQ pathway. Isolates possessing mcr-9 and mcr-10 (3.6% and 5.6% of total ECC isolates, respectively) exhibited similar CST susceptibility and PAP compared with mcr-negative isolates. CONCLUSIONS: Significant prevalence (approximately 28%) of CST heteroresistance is observed in ECC clinical isolates, and they are accumulated in specific species and lineages. Heteroresistance is occurred by upregulation of arnT mRNA induced by CST. Acquisition of mcr genes contributes less to CST resistance in ECC.

First Report of Bacterial Leaf Streak of Barley (Hordeum vulgare) Caused by Xanthomonas translucens pv. translucens in British Columbia, Canada.

Bacterial leaf streak (BLS) of barley is caused by the Gram-negative bacterial pathogen Xanthomonas translucens (Sapkota et al. 2020). In 2021, we observed multiple hill plots with BLS symptomatic plants in a barley stripe rust nursery in Vancouver, BC, Canada. We collected 29 leaf samples showing typical BLS symptoms (e.g. necrotic lesions; Fig. S1) and stored at 4 oC until bacterial isolation. Samples were surface-sterilized in 10% NaOCl for 20 sec and rinsed twice. About 1 cm2 of leaf tissue containing BLS characteristic lesions was macerated in 200 µL sterile H2O on a petri dish, incubated for 15 min, and 10 µl of the homogenates was streaked onto Wilbrink's - Boric Acid - Cephalexin (WBC) agar medium. Plates were incubated at 28-30 oC for 48 hrs. Four single colonies were obtained: BC10-1-2a (USask BC10-2a), BC10-1-2b (USask BC10-2b), UBC026 and UBC028. Colonies were grown in WBC broth and gDNA was extracted using E.Z.N.A. Bacterial DNA Kit (Omega Bio-Tek) or DNeasy Plant Pro Kit® (Qiagen) following manufacturer protocols. Genus-level identification was achieved using 16S rRNA sequencing with 27F/1492R primers (Lane 1991) of UBC026 (1,399 bp; NCBI # OP327375) and UBC028 (1,415 bp; NCBI #OP327376). Complete 16S rRNA sequences (1,533bp) of BC10-2a and BC10-2b (1,533 bp) were extracted from the draft whole-genome sequences (WGS) generated in this study. The 16S rRNA sequence homology values of 99.0-100% were recorded between the 4 strains. BLAST analyses of the 16S rRNA sequences to GenBank entries exhibited 99.5-100% similarity values (100% coverage) with the pathotype strains of Xtt DSM 18974T (LT604072) and X. translucens pv. undulosa (Xtu) CFBP 2055 (CP074361). Whole genomes of BC10-2a (JANUQY01) and BC10-2b (JANUQZ01) were sequenced (150-bp; reads 33.1 million; mean coverage 2125x) using NovaSeq Illumina, assembled (Unicycler v0.4.8; Wick et al. 2017) and analyzed to identify the strains to the species-level (Tambong et al. 2021). WGS of strains USask BC10-2a and USask BC10-2b exhibited genome-based DNA-DNA hybridization (dDDH; Meier-Kolthoff et al. 2013) and BLAST-based average nucleotide identity (ANIb; Richter et al. 2015) of 100%. The two strains also showed dDDH and ANIb of 90.4% (species-leel cut-off of 70%) and 98.780% and 98.80% (cut-off of 96%), respectively, with Xtt DSM 18974T (LT604072). In contrast, the WGS of BC10-2a and BC10-2b exhibited only 78.2% dDDH homology values with Xtu CFBP 2055T, suggesting that the strains are genetically more similar to Xtt. The assignment of these strains to Xtt is corroborated by phylogenomic analysis (Fig. S2; Meier-Kolthoff and Göker 2019) that showed the two strains clustering together (100% bootstrap) with the type strain DSM 18974T. These data suggest that these strains are taxonomically members of Xtt. Identification was also confirmed to the genus-level by LAMP assay using published X. translucens primers (Langlois et al. 2017). Pathovar-level identification was confirmed using a cbsA and S8.pep multiplex PCR diagnostic assay (Roman-Reyna et al. 2022). Koch's postulates were verified by greenhouse inoculation via leaf infiltration of UBC026 and UBC028 on 21-day old barley plants (line HB522) using an inoculum of 108 CFU ml-1 followed by re-isolation of the bacteria on WBC. The inoculated plants showed typical BLS symptoms similar to those observed in the field (Fig. S1). Water-inoculated plants had no symptoms. To our knowledge, this is the first published report of BLS of barley in British Columbia.

Sphingopyxis lutea sp. nov., a novel moderately halotolerant bacterium isolated from pebbles.

A Gram-stain-negative, aerobic, motile and rod-shaped bacterium was isolated from pebbles collected from the coast near Taejongdae Park, Busan, South Korea. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that strain DHUNG17T belonged to the family Sphingomonadaceae, and it showed the highest sequence similarity found with Sphingopyxis panaciterrulae DCY34T (98.4%) and Sphingopyxis granuli TFAT (98.4%). The strain grew at 10-45 °C, at pH 5.0-9.0 and with 0-12% (w/v) NaCl. Chemotaxonomic data revealed that strain DHUNG17T possessed ubiquinone Q-10 as the predominant respiratory lipoquinone. The predominant fatty acids were C16: 0, C18: 0, summed feature 4 (C16: 1 ω7c and/or C15: 0 iso 2-OH) and summed feature 8 (C19: 1 ω6c and/or unknown 18.864). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and a sphingoglycolipid and spermidine were detected as the major polyamines. Strain DHUNG17T was able to produce carotenoid-type pigment. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values with its closest neighbors were 79.3-81.7% and 22.0-24.4%, respectively. The genome of strain DHUNG17T is 3,129,415 bp long with a DNA G + C content of 64.7% and encodes 2,951 predicted proteins, 3 rRNAs and 47 tRNAs. Gene related to transportation of polycyclic aromatic hydrocarbons (PAHs) was found in the genome of strain DHUNG17T. According to the genotypic, phylogenetic and chemotaxonomic data, strain DHUNG17T represents a novel species within the genus Sphingopyxis, for which the name Sphingopyxis lutea sp. nov. is proposed. The type strain is DHUNG17T (= KACC 21746T = NBRC 114643T).

Genetic diversity of Lactobacillus delbrueckii isolated from raw milk in Hokkaido, Japan.

Lactic acid bacteria (LAB) play important roles in acid production and flavor formation in fermented dairy products. Lactic acid bacteria strains with distinct characteristics confer unique features to products. Diverse LAB have been identified in raw milk and traditional fermented milk prepared from raw milk. However, little is known about LAB in raw milk in Japan. To preserve diverse LAB as potential starters or probiotics for future use, we have isolated and identified various kinds of LAB from raw milk produced in Japan. In this study, we focused on Lactobacillus delbrueckii, one of the most important species in the dairy industry. We identified L. delbrueckii subspecies isolated from raw milk in Hokkaido, Japan, by analyzing intraspecific diversity using 4 distinct methods, hsp60 cluster analysis, multilocus sequence analysis, core-genome analysis, and whole-genome analysis based on average nucleotide identity. The subspecies distribution and a new dominant subset of L. delbrueckii from raw milk in Japan were revealed. The discovery of new strains with different genotypes is important for understanding the geographic distribution and characteristics of the bacteria and further their use as a microbial resource with the potential to express unconventional flavors and functionalities. The strains identified in this study may have practical applications in the development of fermented dairy products.

Description of Devosia faecipullorum sp. nov., harboring antibiotic- and toxic compound-resistace genes, isolated from poultry manure.

A polyphasic taxonomic approach was used to characterize a Gram-stain-negative bacterium, designated strain CC-YST696T, harbouring antibiotic- and toxic compound-resistace genes, isolated from poultry manure in Taiwan. Cells of CC-YST696T were short rods, motile with polar flagella, catalase- and oxidase-positive. Optimal growth occurred at 30 °Ð¡, pH 9 and with 1 % NaCl. The results of phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by CC-YST696T associated with Devosia chinhatensis (97.9 % sequence identity), Devosia riboflavina (97.3 %) and Devosia indica (97.2 %), and with lower sequence similarity values to other species. Average nucleotide identity (ANI) values were 72.8-80.0 % (n=17) compared within the type strains of species of of the genus Devosia. CC-YST696T contained C16:0, C18:0, C18:1ω7c 11-methyl and C18:1ω6c/ C18:1ω7c as the predominant fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids, three unidentified glycolipids, two unidentified phospholipids and three unidentified lipids. The DNA G+C content was 62.2 mol% and the predominant quinone was ubiquinone Q-10. On the basis of its distinct phylogenetic, phenotypic and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence and ANI analyses, strain CC-YST696T is proposed to represent a novel species of the genus Devosia, for which the name Devosia faecipullorum sp. nov. (type strain CC-YST696T=BCRC 81284T=JCM 34167T) is proposed.

Aeromicrobium terrae sp. nov., isolated from a maize field.

A polyphasic taxonomic approach was used to characterize a Gram-stain-positive bacterium, designated strain CC-CFT486T, isolated from soil sampled in a maize field in Taiwan. Cells of strain CC-CFT486T were short rods, motile with polar flagella, catalase-positive and oxidase-positive. Optimal growth occurred at 30 °Ð¡, pH 8 and 1 % NaCl. Phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by strain CC-CFT486T associated with Aeromicrobium panacisoli (97.0 % sequence identity), Aeromicrobium lacus (97.0 %), Aeromicrobium erythreum (96.8 %) and Aeromicrobium alkaliterrae (96.8 %), and lower sequence similarity values to other species. Average nucleotide identity (ANI) values were 70.6-77.8 % (n=11) compared within the type strains of the genus Aeromicrobium. Strain CC-CFT486T contained C16 : 0, C17 : 0, C17 : 1 ω8c and C18 : 1 ω9c as the predominant fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, two unidentified aminophospholipids and three unknown phospholipids. The cell wall peptidoglycan of strains CC-CFT486T contained ll-diaminopimelic acid (ll-DAP) and the major polyamine was spermidine. The DNA G+C content was 70.6 mol% and the predominant quinone was menaquinone 9 (MK-9). Based on its distinct phylogenetic, phenotypic and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence and ANI analyses, strain CC-CFT486T is proposed to represent a novel Aeromicrobium species, for which the name Aeromicrobium terrae sp. nov. (type strain CC-CFT486T=BCRC 81217T=JCM 33499T).

Taxonomic note on the family Pseudonocardiaceae based on phylogenomic analysis and descriptions of Allosaccharopolyspora gen. nov. and Halosaccharopolyspora gen. nov.

The taxonomic positions of members within the family Pseudonocardiaceae were assessed based on phylogenomic trees reconstructed using core-proteome and genome blast distance phylogeny approaches. The closely clustered genome sequences from the type strains of validly published names within the family Pseudonocardiaceae were analysed using overall genome-related indices based on average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values. The family Pseudonocardiaceae consists of the type genus Pseudonocardia, as well as the genera Actinoalloteichus, Actinocrispum, Actinokineospora, Actinomycetospora, Actinophytocola, Actinopolyspora, Actinorectispora, Actinosynnema, Allokutzneria, Allosaccharopolyspora gen. nov., Amycolatopsis, Bounagaea, Crossiella, Gandjariella, Goodfellowiella, Haloactinomyces, Haloechinothrix, Halopolyspora, Halosaccharopolyspora gen. nov., Herbihabitans, Kibdelosporangium, Kutzneria, Labedaea, Lentzea, Longimycelium, Prauserella, Saccharomonospora, Saccharopolyspora, Saccharothrix, Salinifilum, Sciscionella, Streptoalloteichus, Tamaricihabitans, Thermocrispum, Thermotunica and Umezawaea. The G+C contents of the Pseudonocardiaceae genomes ranged from 66.2 to 74.6 mol% and genome sizes ranged from 3.69 to 12.28 Mbp. Based on the results of phylogenomic analysis, the names Allosaccharopolyspora coralli comb. nov., Halosaccharopolyspora lacisalsi comb. nov. and Actinoalloteichus caeruleus comb. nov. are proposed. This study revealed that Actinokineospora mzabensis is a heterotypic synonym of Actinokineospora spheciospongiae, Lentzea deserti is a heterotypic synonym of Lentzea atacamensis, Prauserella endophytica is a heterotypic synonym of Prauserella coralliicola, and Prauserella flava and Prauserella sediminis are heterotypic synonyms of Prauserella salsuginis. This study addresses the nomenclature conundrums of Actinoalloteichus cyanogriseus and Streptomyces caeruleus as well as Micropolyspora internatus and Saccharomonospora viridis.

Cerasibacillus terrae sp. nov., isolated from maize field, and emended description of Cerasibacillus quisquiliarum Nakamura et al. 2004.

A polyphasic taxonomic approach was used to characterize a Gram-stain-positive bacterium, designated strain CC-CFT480T, isolated from soil sampled in a maize field in Taiwan, ROC. Cells of strain CC-CFT480T were rod-shaped, motile with polar flagella, catalase-positive and oxidase-positive. Optimal growth occurred at 30 °Ð¡, pH 8 and 3 % NaCl. Phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by strain CC-CFT480T associated with Cerasibacillus quisquiliarum (97.2 % sequence identity), Virgibacillus soli (95.7 %), Virgibacillus carmonensis (95.4 %) and Virgibacillus byunsanensis (95.2 %), and lower sequence similarity values to other species. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain CC-CFT480T and C. quisquiliarum were 74.2 and 20.1 %, respectively. Strain CC-CFT480T contained iso-C15:0, C16:1 ω7c alcohol and iso-C17:1 ω10c as the predominant fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown aminophospholipids, one uncharacterized aminophospholipid and two unknown phospholipids. The major polyamine was spermidine. The DNA G+C content was 34.8 mol% and the predominant quinone was menaquinone 7 (MK-7). Based on its distinct phylogenetic, phenotypic and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence, ANI and dDDH analyses, strain CC-CFT480T is proposed to represent a novel Cerasibacillus species, for which the name Cerasibacillus terrae sp. nov. (type strain CC-CFT480T=BCRC 81216T=JCM 33498T).

Description of Azotobacter chroococcum subsp. isscasi subsp. nov. isolated from paddy soil and establishment of Azotobacter chroococcum subsp. chroococcum subsp. nov.

Three aerobic, asymbiotic, N2-fixing bacterial strains, designated P205T, P204 and P207, were isolated from a paddy soil in Yanting County, China. Based on 16S rRNA gene sequences, the three strains were closely related to Azotobacter chroococcum IAM 12666T (=ATCC 9043T) (99.00-99.79 % similarities). Strain P205T formed an individual branch distinct from the other two newly isolated strains and other related type strains in phylogenetic analyses based on 16S rRNA gene and 92 core genes. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values based on genome sequences of strain P205T and A. chroococcum ATCC 9043T, P204, P207 were near or slightly higher than the thresholds for species circumscription (95-96, 95-96 and 70 %, respectively), and the dDDH values were significantly lower than the threshold for delineating subspecies (79-80 %), which strongly supported that strain P205T belonged to A. chroococcum but was a novel subspecies distinct from the type strain of A. chroococcum. This finding was further corroborated by distinct phenotypic characteristics such as growth in Luria-Bertani (LB) medium, carbon source utilization and chemical sensitivity to vancomycin. Therefore, strain P205T represents a novel subspecies of Azotobacter chroococcum, for which the name Azotobacter chroococcum subsp. isscasi subsp. nov. is proposed with the type strain P205T (=KCTC 72233T=CGMCC 1.16846T=CCTCC AB 2019080T). The subspecies Azotobacter chroococcum subsp. chroococcum subsp. nov. is created automatically with the type strain ATCC 9043T (=DSM 2286T=JCM 20725T=JCM 21503T=LMG 8756T=NBRC 102613T=NCAIM B.01391T=NRRL B-14346T=VKM B-1616T).

Genome-based classification of Acidihalobacter prosperus F5 (=DSM 105917=JCM 32255) as Acidihalobacter yilgarnensis sp. nov.

The genus Acidihalobacter has three validated species, Acidihalobacter ferrooxydans, Acidihalobacter prosperus and Acidihalobacter aeolinanus, all of which were isolated from Vulcano island, Italy. They are obligately chemolithotrophic, aerobic, acidophilic and halophilic in nature and use either ferrous iron or reduced sulphur as electron donors. Recently, a novel strain was isolated from an acidic, saline drain in the Yilgarn region of Western Australia. Strain F5T has an absolute requirement for sodium chloride (>5 mM) and is osmophilic, growing in elevated concentrations (>1 M) of magnesium sulphate. A defining feature of its physiology is its ability to catalyse the oxidative dissolution of the most abundant copper mineral, chalcopyrite, suggesting a potential role in biomining. Originally categorized as a strain of A. prosperus, 16S rRNA gene phylogeny and multiprotein phylogenies derived from clusters of orthologous proteins (COGS) of ribosomal protein families and universal protein families unambiguously demonstrate that strain F5T forms a well-supported separate branch as a sister clade to A. prosperus and is clearly distinguishable from A. ferrooxydans DSM 14175T and A. aeolinanus DSM14174T. Results of comparisons between strain F5T and the other Acidihalobacter species, using genome-based average nucleotide identity, average amino acid identity, correlation indices of tetra-nucleotide signatures (Tetra) and genome-to-genome distance (digital DNA-DNA hybridization), support the contention that strain F5T represents a novel species of the genus Acidihalobacter. It is proposed that strain F5T should be formally reclassified as Acidihalobacter yilgarnenesis F5T (=DSM 105917T=JCM 32255T).

Delineating yeast species with genome average nucleotide identity: a calibration of ANI with haplontic, heterothallic Metschnikowia species.

We determined pairwise average nucleotide identity (ANI) values for the genomes of 71 strains assigned to 36 Metschnikowia species, 28 of which were represented by multiple isolates selected to represent the range of genetic diversity of the species, and most of which were defined on the basis of reproductive isolation. Similar to what has been proposed for prokaryote species delineation, an ANI value of 95% emerged as a good guideline for the delineation of yeast species, although some overlap exists, whereby members of a reproductive community could have slightly lower values (e.g., 94.3% for M. kamakouana), and representatives of distinct sister species could give slightly higher values (e.g., 95.2% for the sister species M. drakensbergensis and M. proteae). Unlike what is observed in prokaryotes, a sizeable gap between intraspecific and interspecific ANI values was not encountered. Given the ease with which yeast draft genomes can now be obtained, ANI values are poised to become the new standard upon which yeast species may be delineated on genetic distance. As borderline cases exist, however, the delineation of yeast species will continue to require careful evaluation of all available data. We also explore the often-neglected distinction between phylogenetic relatedness and sequence identity through the analysis of a tree constructed from ANI' (100 - ANI) values.

Bacillus rugosus sp. nov. producer of a diketopiperazine antimicrobial, isolated from marine sponge Spongia officinalis L.

A novel Gram-positive and endospore-forming bacterium assigned as strain SPB7T which is also a new source of a cyclic diketopiperazine (3S,6S)-3,6-diisobutylpiperazine-2,5-dione is described. A polyphasic (biochemical, phenotypic and genotypic) approach was used to clarify the taxonomic affiliation of this strain. The partial and complete 16S rRNA gene sequences revealed that strain SPB7T is a member of the Bacillus genus [showing high similarity (> 98.70%) with Bacillus spizizenii NRRL B-23049T, Bacillus tequilensis KCTC 13622T, Bacillus inaquosorum KCTC 13429T and Bacillus cabrialesii TE3T]. The maximum values for average nucleotide identity (ANI) and in silico DNA-DNA hybridization (GGDC, Formula 2) of strain SPB7T was obtained for twenty-five strains of Bacillus spizizenii (ANI 95.01-95.48% and GGDC 62.70-60.00%). The whole-genome phylogenetic relationship showed that SPB7T formed an individual and separated clade with the Bacillus spizizenii group. Principal cellular fatty acids identified in strain SPB7T were anteiso C15:0, anteiso C17:0, iso C15:0, iso C17:0, C16:0, C10:0 3OH and iso C17:1 ϖ10c. Polar lipid profile showed presence of diphosphotidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown phospholipids and five unknown lipids. Cells were rod shaped, catalase, oxidase-positive and motile. Growth occurred at 20-45 °C (optimal 35 °C), at pH 6.0-10.0 (optimal pH 8) and 0-10% (w/v) NaCl (optimal 2%). The phenotypic, biochemical, and genotypic traits of strain SPB7T strongly supported its taxonomic affiliation as a novel species of the Bacillus genus, for which the name Bacillus rugosus sp. nov. is proposed. The type strain is SPB7T (= NRRL B-65559T, = CICC 24827T, = MCC 4185T).

Whole genome sequences reveal the presence of 11 heterotypic synonyms in the genus Sphingobium and emended descriptions of Sphingobium indicum, Sphingobium fuliginis, Sphingobium xenophagum and Sphingobium cupriresistens.

Phylogenetic analysis based on 16S rRNA gene sequences of the genus Sphingobium showed the presence of four distinguishable clusters, in each of which the species shared almost the same evolutionary distance. They were Sphingobium indicum, Sphinogbium lucknowense, Sphinogbium chinhatense, Sphinogbium francense and Sphinogbium japonicum in cluster I, Sphinogbium barthaii and Sphinogbium fuliginis in cluster II, Sphinogbium hydrophobicum and Sphinogbium xenophagum in cluster III and Sphinogbium czechense and Sphinogbium cupriresistens in cluster IV. The 16S rRNA gene sequence similarities between the species in each cluster were all higher than 98 %. Genome-based average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) relatedness values between the species of each cluster were all higher than the threshold values of 95-96 % ANI and 70 % dDDH for species discrimination, respectively, suggesting that each cluster represents only one species of the genus Sphingobium. Due to priority of publication, S. lucknowense, S. chinhatense, S. francense and S. japonicum should be taken as later heterotypic synonyms of S. indicum, S. barthaii as a later heterotypic synonym of S. fuliginis, S. hydrophobicum as a later heterotypic synonym of S. xenophagum and S. czechense as a later heterotypic synonym of S. cupriresistens. Correspondingly, the descriptions of S. indicum, S. fuliginis, S. xenophagum and S. cupriresistens are also emended based on this study.

Halorubrum amylolyticum sp. nov., a novel halophilic archaeon isolated from a salt mine.

A pleomorphic and non-motile halophilic archaeon forming light-red pigmented colonies, strain ZC67T, was isolated from the Yuanyongjing Salt Mine, Yunnan, China. Based on similarity search and phylogenetic analysis of the 16S rRNA gene sequence, strain ZC67T belongs to the genus Halorubrum and is closely related to the species of Halorubrum (Hrr.) saccharovorum JCM 8865T, Hrr. persicum C49T, Hrr. halophilum B8T, Hrr. lipolyticum 9-3T, Hrr. salsamenti Y69T and Hrr. depositum Y78T with 16S rRNA gene sequence similarities of 99.0%, 98.7%, 98.5%, 98.4%, 98.1% and 97.7%, respectively. The values of average nucleotide identity (ANI) and average amino-acid identity (AAI) between strain ZC67T and its close relatives were less than 90.5% and 89.3%, respectively. In silico DNA-DNA hybridization (DDH) analysis showed that DNA-DNA relatedness between strain ZC67T and its relatives is less than 45%. Values of ANI, AAI and in silico DDH were clearly below the thresholds used for the delineation of a new species. The major polar lipids of strain ZC67T were similar to other neutrophilic members in the genus Halorubrum containing phosphatidylglycerol, phosphatidylglycerolphosphate methyl ester, phosphatidylglycerol sulfate and sulfated mannosyl-glucosyl-glycerol diether-1. The DNA G+C content was determined to be 66.3 mol% (based on the draft genome). Combined with other diagnostic characteristics, e.g. phenotypic and chemotaxonomic differences, strain ZC67T is concluded to represent a novel species in the genus Halorubrum, for which the name Halorubrum amylolyticum sp. nov. is proposed. The type strain is ZC67T (=CGMCC 1.15718T = JCM 31850T).

Polynucleobacter aenigmaticus sp. nov. isolated from the permanently anoxic monimolimnion of a temperate meromictic lake.

The bacterial strain MWH-K35W1T was isolated from a permanently anoxic water layer of a meromictic lake located in the Austrian Salzkammergut area. The basically chemo-organoheterotrophic strain was isolated and maintained under aerobic conditions. Phylogenetic analyses of the 16S rRNA gene and the glutamine synthetase gene (glnA) of the strain suggested an affiliation to the genus Polynucleobacter and the cryptic species complex PnecC. Strain MWH-K35W1T shares with the type strains of the six free-living species of the genus Polynucleobacter affiliated with this species complex 16S rRNA gene sequence similarities of 99.6-99.9 %, while the type material of the obligate endosymbiont Polynucleobacternecessarius, which is also affiliated with this species complex, shares a gene sequence similarity of 99.1 %. Genome sequencing resulted in a genome size of 2.14 Mbp and a DNA G+C content of 45.98 mol%. Major fatty acids were C16 : 1ω7c, C18 : 1ω7c and C16 : 0. This strain is the first strain of the genus Polynucleobacter found to encode a proteorhodopsin-like protein but, in contrast to some other strains affiliated to this genus, it does not encode a putative anoxygenic photosynthesis system. Multilocus sequence analysis based on partial sequences of eight housekeeping genes, as well as average nucleotide identity (ANI) analyses, did not suggest that strain MWH-K35W1T belongs to a previously described species. We propose the name Polynucleobacter aenigmaticus for a novel species with strain MWH-K35W1T (=DSM 24006T=LMG 29706T) as the type strain.

Genome-wide comparison and taxonomic relatedness of multiple Xylella fastidiosa strains reveal the occurrence of three subspecies and a new Xylella species.

A total of 21 Xylella fastidiosa strains were assessed by comparing their genomes to infer their taxonomic relationships. The whole-genome-based average nucleotide identity and tetranucleotide frequency correlation coefficient analyses were performed. In addition, a consensus tree based on comparisons of 956 core gene families, and a genome-wide phylogenetic tree and a Neighbor-net network were constructed with 820,088 nucleotides (i.e., approximately 30-33 % of the entire X. fastidiosa genome). All approaches revealed the occurrence of three well-demarcated genetic clusters that represent X. fastidiosa subspecies fastidiosa, multiplex and pauca, with the latter appeared to diverge. We suggest that the proposed but never formally described subspecies 'sandyi' and 'morus' are instead members of the subspecies fastidiosa. These analyses support the view that the Xylella strain isolated from Pyrus pyrifolia in Taiwan is likely to be a new species. A widely used multilocus sequence typing analysis yielded conflicting results.

On the road to genomically defining bacterial intra-species units.

Over almost three decades, average nucleotide identity (ANI) analysis has been instrumental in operationally defining species in bacteria. However, barely any attention has been paid to soundly defining intra-species units employing ANI analyses until recently. Notably, some very recent publications are good steps forward in that direction. The level of granularity provided by these intra-species units will be relevant to understanding the eco-evolutionary dynamics and transmission of bacterial lineages and mobile genetic elements, antibiotic resistance, and virulence genes. These intra-species units will undoubtedly advance the genomic epidemiology of many bacterial pathogens. In the coming years, we anticipate that many studies will implement ANI-based definitions of different intra-species units, such as strains or sequence types, for many different bacterial species.

Sequence-discrete species for prokaryotes and other microbes: A historical perspective and pending issues.

Whether prokaryotes, and other microorganisms, form distinct clusters that can be recognized as species remains an issue of paramount theoretical as well as practical consequence in identifying, regulating, and communicating about these organisms. In the past decade, comparisons of thousands of genomes of isolates and hundreds of metagenomes have shown that prokaryotic diversity may be predominantly organized in such sequence-discrete clusters, albeit organisms of intermediate relatedness between the identified clusters are also frequently found. Accumulating evidence suggests, however, that the latter "intermediate" organisms show enough ecological and/or functional distinctiveness to be considered different species. Notably, the area of discontinuity between clusters often-but not always-appears to be around 85%-95% genome-average nucleotide identity, consistently among different taxa. More recent studies have revealed remarkably similar diversity patterns for viruses and microbial eukaryotes as well. This high consistency across taxa implies a specific mechanistic process that underlies the maintenance of the clusters. The underlying mechanism may be a substantial reduction in the efficiency of homologous recombination, which mediates (successful) horizontal gene transfer, around 95% nucleotide identity. Deviations from the 95% threshold (e.g., species showing lower intraspecies diversity) may be caused by ecological differentiation that imposes barriers to otherwise frequent gene transfer. While this hypothesis that clusters are driven by ecological differentiation coupled to recombination frequency (i.e., higher recombination within vs. between groups) is appealing, the supporting evidence remains anecdotal. The data needed to rigorously test the hypothesis toward advancing the species concept are also outlined.