Genome-to-genome Distance Research Articles

Comprehensive genome analysis of Pseudomonas sp. SWRIQ11, a new plant growth-promoting bacterium that alleviates salinity stress in olive.

The study presents the genome analysis of a new Pseudomonas sp. (SWRIQ11), which can alleviate salinity stress effects on growth of olive seedlings in greenhouse study. The strain SWRIQ11 can tolerate salinity up to 6%, produce siderophores, indole acetic acid (IAA), aminocyclopropane-1-carboxylate (ACC) deaminase, and has the phosphate-solubilizing capability. The SWRIQ11 genome contained an assembly size of 6,196,390 bp with a GC content of 60.1%. According to derived indices based on whole-genome sequences for species delineation, including tetra nucleotide usage patterns (TETRA), genome-to-genome distance (GGDC), and average nucleotide identity (ANI), Pseudomonas sp. SWRIQ11 can be considered a novel species candidate. The phylogenetic analysis revealed SWRIQ11 clusters with Pseudomonas tehranensis SWRI196 in the same clade. The SWRIQ11 genome was rich in genes related to stress sensing, signaling, and response, chaperones, motility, attachments, colonization, and enzymes for degrading plant-derived carbohydrates. Furthermore, the genes for production of exopolysaccharides, osmoprotectants, phytohormones, and ACC deaminase, ion homeostasis, nutrient acquisition, and antioxidant defenses were identified in the SWRIQ11 genome. The results of genome analysis (identification of more than 825 CDSs related to plant growth-promoting and stress-alleviating traits in the SWRIQ11 genome which is more than 15% of its total CDSs) are in accordance with laboratory and greenhouse experiments assigning the Pseudomonas sp. SWRIQ11 as a halotolerant plant growth-promoting bacterium (PGPB). This research highlights the potential safe application of this new PGPB species in agriculture as a potent biofertilizer.

Streptococcus humanilactis sp.nov., isolated from healthy nursing mother's breast milk.

Two bacterial strains were isolated from the breast milk of two healthy nursing mothers. The isolates were Gram-positive, catalase-negative, coccus-shaped, chain-forming organisms. Analysis of the 16S rRNA gene sequences of strain IMAU99125T shared 99.7 and 99.6% similarity with Streptococcus mitis ATCC 49456T and Streptococcus pseudopneumoniae ATCC BAA-960T, respectively. The nearly complete 16S rRNA gene sequences of IMAU99125T and IMAU99674 strains were very closely related (with only 0.06% difference between them). Sequence analysis of the gyrB and rpoB genes also indicated that IMAU99125T was closely related to S. mitis ATCC 49456T (94.7% and 97.1%, respectively) and S. pseudopneumoniae ATCC BAA-960T (94.4% and 97.1%, respectively). Average nucleotide identity (ANI) values between strain IMAU99125T and S. mitis ATCC 49456T and S. pseudopneumoniae ATCC BAA-960T, were 93.3% and 92.7%, respectively. Genome-to-genome distance (GGD) values between strain IMAU99125T and S. mitis ATCC 99125T and S. pseudopneumoniae ATCC BAA-960T were 53.4% (50.7-56.0) and 50.4% (47.7-53.0), respectively. The major fatty acids of the strain were C16:0 (51.4%). On the basis of the results of phenotypic and phylogenetic analyses, we propose that the two strains be classified as representing a novel species of the genus Streptococcus, namely Streptococcus humanilactis sp.nov. The type strain is IMAU99125T (= GDMCC 1.1876T = KCTC 21157T). The genome of Streptococcus humanilactis sp. nov. is comprised of 2,027,143bp. The DNA G + C content of the strain is 40.0mol%.

Phylogenomic analysis of Pseudomonas nitroreducens strains FY43 and FY47

Proper identification of strain is essential in understanding the ecology of a bacteria species. The classification of Pseudomonas nitroreducens is still being questioned and revised until now. The novel P. nitroreducens strains FY43 and FY47 used in this study have been reported to show a high level of tolerance to glyphosate. In this study, next-generation sequencing (NGS) and whole genome analysis were used to clarify the delineation of the species. Whole genome analysis showed that P. nitroreducens strains FY43 and FY47 shared high homology to five reference genomes of P. nitroreducens: strain B, Aramco J, NBRC 12694, DF05, and TX01. Phylogenomic and phylogenetic analysis (average nucleotide identity based on BLAST (ANIb), genome-to-genome distance (GGDC) analysis) showed that both P. nitroreducens strains FY43 and FY47 are Pseudomonas nitroreducens members. However, strains DF05 and TX01 were not correctly assigned at the species level for all the analyses. The P. nitroreducens strain DF05 and TX01 should be further investigated for their classification as the correct species classification is the prerequisite for future diversity studies.

Blautia liquoris sp. nov., isolated from the mud in a fermentation cellar used for the production of Chinese strong-flavour liquor.

A novel Gram-positive, non-motile, non-flagellated, strictly anaerobic, non-spore-forming and dumbbell-shaped, coccoid- or chain-shaped bacterium, designated strain LZLJ-3T, was isolated from a mud fermentation cellar which has been used for the production of Chinese strong-flavour liquor for over 100 years. Strain LZLJ-3T grew at 20-40 °C (optimum, 37 °C), at pH 6.0-8.0 (optimum, pH 8.0) and with NaCl concentrations up to 1 % (w/v; optimum, 0 %). Phylogenetic trees established based on 16S rRNA gene sequences showed that strain LZLJ-3T belonged to the genus Blautia of the family Lachnospiraceae, with the highest sequence similarity to Blautia stercoris GAM6-1T (91.7 %) and Blautia faecicola KGMB01111T (91.7 %). Comparative genome analysis showed that the orthologous average nucleotide identity (OrthoANI) and genome-to-genome distance (GGD) values between strain LZLJ-3T and B. stercoris GAM6-1T were respectively 69.1 and 22.9 %; the OrthoANI and GGD values between strain LZLJ-3T and B. faecicola KGMB01111T were respectively 70.86 and 36 % . The DNA G+C content of strain LZLJ-3T genome was 42.1 mol%. The predominant celluar fatty acids (>10 %) of strain LZLJ-3T were C16 : 0 FAME (27.9 %), C14 : 0 FAME (17.6 %) and C16 : 0 DMA (13.0 %). Arabinose, glucose and maltose could be utilized by strain LZLJ-3T as sole carbon sources for growth, with weak utilization of raffinose and l-fucose. API ZYM analysis gave positive reactions with α-galactosidase, β-galactosidase, α-glucosidase and β-glucosidase. The major end product of glucose fermentation was acetic acid. Based on the results of phenotypic, genotypic and phylogenetic analyses, strain LZLJ-3T is considered to represent a novel species of Blautia, for which the name Blautia liquoris sp. nov. is proposed. The type strain is LZLJ-3T (=KCTC 25163T=CGMCC 1.5299T=JCM 34225T).

Analyses of publicly available Hungatella hathewayi genomes revealed genetic distances indicating they belong to more than one species

ABSTRACT Hungatella hathewayi has been observed to be a member of the gut microbiome. Unfortunately, little is known about this organism in spite of being associated with human fatalities; it is important to understand virulence mechanisms and epidemiological prospective to cause disease. In this study, a patient with chronic neurologic symptoms presented to the clinic with subsequent isolation of a strain with phenotypic characteristics suggestive of Clostridium difficile. However, whole-genome sequence found the organism to be H. hathewayi. Analysis including publicly available Hungatella genomes found substantial genomic differences as compared to the type strain, indicating this isolate was not C. difficile. We examined the whole-genome of Hungatella species and related genera, using comparative genomics to fully examine species identification and toxin production. Orthogonal phylogenetic using the 16S rRNA gene and entire genome analyses that included genome distance analyses using Genome-to-Genome Distance (GGDC), Average Nucleotide Identity (ANI), and a pan-genome analysis with inclusion of available public genomes determined the speciation to be Hungatella. Two clearly differentiated groups were identified, one including a reference H. hathewayi genome (strain DSM-13,479) and a second group that was determined to be H. effluvii, which included our clinical isolate. Also, some genomes reported as H. hathewayi were found to belong to other genera, including Clostridium and Faecalicatena. We show that the Hungatella species have an open pan-genome reflecting high genomic diversity. This study highlights the importance of correctly assigning taxonomic identification, particularly in disease-associated strains, to better understand virulence and therapeutic options.

Pan-genome analysis of Exiguobacterium reveals species delineation and genomic similarity with Exiguobacterium profundumPHM 11.

The stint of the bacterial species is convoluting, but the new algorithms to calculate genome-to-genome distance (GGD) and DNA-DNA hybridization (DDH) for comparative genome analysis have rejuvenated the exploration of species and sub-species characterization. The present study reports the first whole genome sequence of Exiguobacterium profundum PHM11. PHM11 genome consist of ~ 2.92 Mb comprising 48 contigs, 47.93% G + C content. Functional annotations revealed a total of 3033 protein coding genes and 33 non-protein coding genes. Out of these, only 2316 could be characterized and others reported as hypothetical proteins. The comparative analysis of predicted proteome of PHM11 with five other Exiguobacterium sp. identified 3806 clusters, out of which the PHM11 shared a total of 2723 clusters having 1664 common clusters, 131 singletons and 928 distributed between five species. The pan-genome analysis of 70 different genomic sequences of Exigubacterium strains devoid of a species taxon was done on the basis of GGD and the DDH which identified eight genomes analogous to the PHM11 at species level and may be characterized as E. profundum. The ANI value and phylogenetic tree analysis also support the same. The results regarding pan-genome analysis provide a convincing insight for delineation of these eight strains to species.

Arthrobacter sedimenti sp. nov., isolated from river sediment in Yuantouzhu park, China.

A Gram-stain positive, motile, aerobic and rod-shaped strain (MIC A30T) was isolated from river sediment in Yuantouzhu park, Wuxi City, China. Growth occurred at 20-40°C, at pH 6.0-9.0 and at 0-5.0% NaCl. Strain MIC A30T was moderately related to Arthrobacter liuii CGMCC 1.12778T (97.9%), Arthrobacter pokkaliiT (97.9%) and Arthrobacter globiformis NBRC 12137T (96.7%) by 16S rRNA analysis. The DNA-DNA relatedness values between strain MIC A30T and these reference strains were below 30%. The DNA G+C content was 63.1mol%. Average nucleotide identity (ANI) and genome-to-genome distance (GGD) values between strain MIC A30T and A. liuii CGMCC 1.12778T were 60.34% and 29.39%, respectively. Quinone was identified as MK-9(H2). Major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. Major fatty acids were iso-C15:0, anteiso-C15:0 and anteiso-C17:0. Whole-cell sugars were galactose, mannose and rhamnose. The cell wall peptidoglycan contained A4α peptidoglycan type with lysine as the diagnostic diamino acid. Based on several taxonomic results, strain MIC A30T is identified as a novel species in genus Arthrobacter, whose name is proposed as Arthrobacter sedimenti sp. nov. The type strain is MIC A30T (= KACC 19599T = CGMCC 1.13474T).

Luteimonas cellulosilyticus sp. nov., Cellulose-Degrading Bacterium Isolated from Soil in Changguangxi National Wetland Park, China.

A Gram-negative, motile, aerobic, and rod-shaped strain (MIC 1.5T) was isolated from soil in Changguangxi national wetland park. Growth occurred at 20-45°C, at pH 6.0-8.0, and at 0-4.0% NaCl. Based on 16S rRNA gene sequence analysis, strain MIC 1.5T was related to were identified as Luteimonas dalianensis CGMCC 1.12191T (95.3%), Luteimonas padinae DSM 101536T (94.5%), Luteimonas huabeiensis DSM 26429T (94.1%), and Luteimonas mephitis DSM 12574T (92.5%). The DNA-DNA relatedness values between strain MIC 1.5T , and these strains were well below 31%. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G+C content of strain MIC 1.5T was 66.3mol%. Average nucleotide identity (ANI) and genome-to-genome distance (GGD) values between strain MIC 1.5T and L. dalianensis CGMCC 1.12191T were 65.39% and 29.52%, respectively. The quinone was identified as Q-8. The major fatty acids were iso-C15:0, iso-C15:0 3OH, and iso-C17:0 3OH and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH). Based on the phylogenetic, physiological, and chemotaxonomic results, strain MIC 1.5T represents a novel species of the genus Luteimonas, for which the name Luteimonas cellulosilyticus sp. nov. is proposed. The type strain is MIC 1.5T (= KACC 19469T = CCTCC AB 2017256T).

Streptococcus chosunense sp. nov., Isolated from Human Postoperative Maxillary Cyst.

A novel facultative anaerobic, non-spore forming, non-motile, and Gram-stain-positive coccus, designated strain ChDC B353T, was isolated from human postoperative maxillary cyst. The 16S ribosomal RNA gene (16S rDNA) sequence of the strain was most closely related to those of Streptococcus pseudopneumoniae ATCC BAA-960T (99.4%), Streptococcus mitis NCTC 12261T (99.3%), and Streptococcus pneumoniae NCTC 7465T (99.2%). The major fatty acids of the strain were C16:0 (43.2%) and C18:1 ω6c/C18:1 ω7c (20.2%). The genome of strain ChDC B353T was composed of 1,902,053 bps. The DNA G+C content of the strain was 40.2mol%. Average nucleotide identity (ANI) values between strain ChDC B353T and S. pseudopneumoniae ATCC BAA-960T, S. mitis NCTC 12261T, and S. pneumoniae NCTC 7465T were 91.9%, 93.5%, and 91.3%, respectively. Genome-to-genome distance (GGD) values between strain ChDC B353T and S. pseudopneumoniae ATCC BAA-960T, S. mitis NCTC 12261T, or S. pneumoniae NCTC 7465T were 46.6% (44.0-49.2%), 53.2% (50.5-55.9%), and 46.0% (43.5-48.7%), respectively. The threshold values of ANI and GGD for species discrimination are 95-96% and 70%, respectively. These results reveal that strain ChDC B353T (= KCOM 1699T = JCM 33453T) is a novel species belonging to genus Streptococcus, for which a name of Streptococcus chosunense sp. nov. is proposed.

Brachyspira catarrhinii sp. nov., an anaerobic intestinal spirochaete isolated from vervet monkeys may have been misidentified as Brachyspira aalborgi in previous studies

To date nine species of anaerobic intestinal spirochaetes have been validly assigned to the genus Brachyspira. These include both pathogenic and non-pathogenic species. In the current study a genomic analysis of a novel spirochaete isolate was undertaken to determine whether it is a distinct species that previously has been misidentified as Brachyspira aalborgi. The genome of spirochaete strain Z12 isolated from the faeces of a vervet monkey was sequenced and compared to the genomes of the type strains of the nine assigned Brachyspira species. Genome to Genome Distance (GGD) values and Average Nucleotide Identity (ANI) values were determined. Single nucleotide polymorphisms (SNP) were used to create a phylogenetic tree to assess relatedness. The 16S rRNA gene sequences of the strains were aligned and the similarity amongst the Brachyspira species was recorded. Multilocus sequence typing (MLST) using five loci was conducted on Z12 and results compared with those for other Brachyspira isolates. Assembly of the Z12 sequences revealed a 2,629,108 bp genome with an average G + C content of 31.3%. The GGD, ANI, 16S rRNA gene sequence comparisons and the MLST results all indicated that Z12 represents a distinct species within the genus Brachyspira, with its nearest neighbour being B. aalborgi. Spirochaete strain Z12T was assigned as the type strain of a new species, Brachyspira catarrhinii sp. nov. The diagnostic PCR currently in use to detect B. aalborgi cross-reacts with Z12, but RFLP analysis of PCR product can be used to distinguish the two species. Previous reports of non-human primates being colonised by B. aalborgi based on PCR results may have been incorrect. The development of an improved diagnostic method will allow future studies on the distribution and possible clinical significance of these two anaerobic spirochaete species.

Fusobacterium pseudoperiodonticum sp. nov., Isolated from the Human Oral Cavity.

In the present study, three strains (ChDC F213T, ChDC F251, and ChDC F267) were classified as novel species of genus Fusobacterium based on average nucleotide identity (ANI) and genome-to-genome distance (GGD) analysis and chemotaxonomic characterization. 16S rDNA sequences of strains ChDC F213T, ChDC F251, and ChDC F267 were highly similar to that of F. periodonticum ATCC 33693T (99.6, 99.4, and 99.4%, respectively). ANI and GGD values of the three isolates with F. periodonticum ATCC 33693T ranged from 92.5 to 92.6% and 47.7 to 48.2%, respectively. Considering that threshold of ANI and GGD values for bacterial species discrimination are 95-96% and 70%, respectively, these results indicate that the three isolates represent a novel Fusobacterium species. DNA G + C contents of the three isolates were 28.0mol% each. Cellular fatty acid analysis of these strains revealed that C14:0, C16:0, and C16:1 ω6c/C16:1 ω7c were major fatty acids. Therefore, these three strains are novel species belonging to genus Fusobacterium. Strain ChDC F213T (= KCOM 1259T = KCTC 5677T = JCM 33009T) is the type strain of a novel species of genus Fusobacterium, for which a name of Fusobacterium pseudoperiodonticum sp. nov. is proposed.

Ochrobactrum quorumnocens sp. nov., a quorum quenching bacterium from the potato rhizosphere, and comparative genome analysis with related type strains.

Ochrobactrum spp. are ubiquitous bacteria attracting growing attention as important members of microbiomes of plants and nematodes and as a source of enzymes for biotechnology. Strain Ochrobactrum sp. A44T was isolated from the rhizosphere of a field-grown potato in Gelderland, the Netherlands. The strain can interfere with quorum sensing (QS) of Gram-negative bacteria through inactivation of N-acyl homoserine lactones (AHLs) and protect plant tissue against soft rot pathogens, the virulence of which is governed by QS. Phylogenetic analysis based on 16S rRNA gene alone and concatenation of 16S rRNA gene and MLSA genes (groEL and gyrB) revealed that the closest relatives of A44T are O. grignonense OgA9aT, O. thiophenivorans DSM 7216T, O. pseudogrignonense CCUG 30717T, O. pituitosum CCUG 50899T, and O. rhizosphaerae PR17T. Genomes of all six type strains were sequenced, significantly expanding the possibility of genome-based analyses in Ochrobactrum spp. Average nucleotide identity (ANIb) and genome-to-genome distance (GGDC) values for A44T and the related strains were below the single species thresholds (95% and 70%, respectively), with the highest scores obtained for O. pituitosum CCUG 50899T (87.31%; 35.6%), O. rhizosphaerae PR17T (86.80%; 34.3%), and O. grignonense OgA9aT (86.30%; 33.6%). Distinction of A44T from the related type strains was supported by chemotaxonomic and biochemical analyses. Comparative genomics revealed that the core genome for the newly sequenced strains comprises 2731 genes, constituting 50–66% of each individual genome. Through phenotype-to-genotype study, we found that the non-motile strain O. thiophenivorans DSM 7216T lacks a cluster of genes related to flagella formation. Moreover, we explored the genetic background of distinct urease activity among the strains. Here, we propose to establish a novel species Ochrobactrum quorumnocens, with A44T as the type strain (= LMG 30544T = PCM 2957T).

Description of Alteromonas abrolhosensis sp. nov., isolated from sea water of Abrolhos Bank, Brazil.

Two Gram-negative, motile, aerobic bacteria isolated from waters of the Abrolhos Bank were classified through a whole genome-based taxonomy. Strains PEL67ET and PEL68C shared 99% 16S rRNA and dnaK sequence identity with Alteromonas marina SW-47T and Alteromonas macleodii ATCC 27126T. In silico DNA-DNA Hybridization, i.e. genome-to-genome distance (GGD), average amino acid identity (AAI) and average nucleotide identity (ANI) showed that PEL67ET and PEL68C had identity values between 33-36, 86-88 and 83-84%, and 85-86 and 83%, respectively, towards their close neighbors A. macleodii ATCC 27126T and A. marina SW-47T. The DNA G+C contents of PEL67ET and PEL68C were 44.5%. The phenotypic features that differentiate PEL67ET and PEL68C strains from their close neighbors were assimilation of galactose and activity of phosphatase, and lack of mannitol, maltose, acetate, xylose and glycerol assimilation and lack of lipase, α and β-glucosidase activity. The new species Alteromonas abrolhosensis is proposed. The type strain is PEL67ET (=CBAS 610T=CAIM 1925T).

Capnocytophaga endodontalis sp. nov., Isolated from a Human Refractory Periapical Abscess.

A novel Gram-negative, capnophilic, fusiform bacterium, designated strain ChDC OS43T, was isolated from a human refractory periapical abscess in the left mandibular second molar and was characterized by polyphasic taxonomic analysis. The 16S rRNA gene sequence revealed that the strain belongs to the genus Capnocytophaga, as it showed sequence similarities to Capnocytophaga ochracea ATCC 27872T (96.30%) and C. sputigena ATCC 33612T (96.16%). The prevalent fatty acids of strain ChDC OS43T were isoC15:0 (57.54%), C16:0 (5.93%), C16:0 3OH (5.72%), and C18:1 cis 9 (4.41%). The complete genome of strain ChDC OS43T was 3,412,686bp, and the G+C content was 38.2mol%. The average nucleotide identity (ANI) value between strain ChDC OS43T and C. ochracea ATCC 27872T or C. sputigena ATCC 33612T was >92.01%. The genome-to-genome distance (GGD) value between strain ChDC OS43T and C. ochracea ATCC 27872T or C. sputigena ATCC 33612T was 32.0 and 45.7%, respectively. Based on the results of phenotypic, chemotaxonomic, and phylogenetic analysis, strain ChDC OS43T (= KCOM 1579T = KCTC 5562T = KCCM 42841T = JCM 32133T) should be classified as the type strain of a novel species of genus Capnocytophaga, for which the name Capnocytophaga endodontalis sp. nov. is proposed.

Genome-Based Reclassification of Fusobacterium nucleatum Subspecies at the Species Level.

Fusobacterium nucleatum is classified as four subspecies, subsp. nucleatum, polymorphum, vincentii, and animalis, based on DNA-DNA hybridization (DDH) patterns, phenotypic characteristics, and/or multilocus sequence analysis (MLSA). The gold standards for classification of bacterial species are DDH and 16S ribosomal RNA gene (16S rDNA) sequence homology. The thresholds of DDH and 16S rDNA similarity for delineation of bacterial species have been suggested to be >70 and 98.65%, respectively. Average nucleotide identity (ANI) and genome-to-genome distance (GGD) analysis based on genome sequences were recently introduced as a replacement for DDH to delineate bacterial species with ANI (95-96%) and GGD (70%) threshold values. In a previous study, F. hwasookii was classified as a new species based on MLSA and DDH results. 16S rDNA similarity between F. hwasookii type strain and F. nucleatum subspecies type strains was higher than that between F. nucleatum subspecies type strains. Therefore, it is possible that the four F. nucleatum subspecies can be classified as Fusobacterium species. In this study, we performed ANI and GGD analyses using the genome sequences of 36 F. nucleatum, five F. hwasookii, and one Fusobacterium periodonticum strain to determine whether the four F. nucleatum subspecies could be classified as species using OrthoANI and ANI web-based softwares provided by ChunLab and Kostas lab, respectively, and GGD calculator offered by German Collection of Microorganisms and Cell Cultures. ANI values calculated from OrthoANI and ANI calculators between the type strains of F. nucleatum subspecies ranged from 89.80 to 92.97 and from 90.40 to 91.90%, respectively. GGD values between the type strains of F. nucleatum subspecies ranged from 42.3 to 46.0%. ANI and GGD values among strains belonging to the same F. nucleatum subspecies, subsp. nucleatum, subsp. polymorphum, subsp. vincentii, and subsp. animalis were >96 and >68.2%, respectively. These results strongly suggest that F. nucleatum subsp. nucleatum, subsp. polymorphum, subsp. vincentii, and subsp. animalis should be classified as F. nucleatum, F. polymorphum, F. vincentii, and F. animalis, respectively.

Characterization of Mycobacterium chelonae-Like Strains by Comparative Genomics.

Isolates of the Mycobacterium chelonae-M. abscessus complex are subdivided into four clusters (CHI to CHIV) in the INNO-LiPA® Mycobacterium spp DNA strip assay. A considerable phenotypic variability was observed among isolates of the CHII cluster. In this study, we examined the diversity of 26 CHII cluster isolates by phenotypic analysis, drug susceptibility testing, whole genome sequencing and single-gene analysis. Pairwise genome comparisons were performed using several approaches, including average nucleotide identity (ANI) and genome-to-genome distance (GGD) among others. Based on ANI and GGD the isolates were identified as M. chelonae (14 isolates), M. franklinii (2 isolates) and M. salmoniphium (1 isolate). The remaining 9 isolates were subdivided into three novel putative genomospecies. Phenotypic analyses including drug susceptibility testing, as well as whole genome comparison by TETRA and delta differences, were not helpful in separating the groups revealed by ANI and GGD. The analysis of standard four conserved genomic regions showed that rpoB alone and the concatenated sequences clearly distinguished the taxonomic groups delimited by whole genome analyses. In conclusion, the CHII INNO-LiPa is not a homogeneous cluster; on the contrary, it is composed of closely related different species belonging to the M. chelonae-M. abscessus complex and also several unidentified isolates. The detection of these isolates, putatively novel species, indicates a wider inner variability than the presently known in this complex.

Pseudomonas cerasi sp. nov. (non Griffin, 1911) isolated from diseased tissue of cherry

Eight isolates of Gram-negative fluorescent bacteria (58T, 122, 374, 791, 963, 966, 970a and 1021) were obtained from diseased tissue of cherry trees from different regions of Poland. The symptoms resembled those of bacterial canker. Based on an analysis of 16S rDNA sequences the isolates shared the highest over 99.9% similarity with Pseudomonas ficuserectae JCM 2400T and P. congelans DSM 14939T. Phylogenetic analysis using housekeeping genes gyrB, rpoD and rpoB revealed that they form a separate cluster and confirmed their closest relation to P. syringae NCPPB 281T and P. congelans LMG 21466T. DNA–DNA hybridization between the cherry isolate 58T and the type strains of these two closely related species revealed relatedness values of 58.2% and 41.9%, respectively. This was further supported by Average Nucleotide Identity (ANIb) and Genome-to-Genome Distance (GGDC) between the whole genome sequences of strain LMG 28609T and closely related Pseudomonas species. The major cellular fatty acids are 16:0 and summed feature 3 (16:1 ω7c/15:0 iso 2OH). Phenotypic characteristics differentiated the novel isolates from other closely related species. The G+C content of the genomic DNA of strain 58T was 59%. The diversity was proved by PCR MP and BOX PCR, eliminating the possibility that they constitute a clonal population. Based on the evidence of this polyphasic taxonomic study the eight strains are considered to represent a novel species of the genus Pseudomonas for which the name P. cerasi sp. nov. (non Griffin, 1911) is proposed. The type strain of this species is 58T (=LMG 28609T=CFBP 8305T).

Thalassospira australica sp. nov. isolated from sea water.

Two Gram-negative, non-pigmented, motile bacteria were isolated from a sea water sample collected at St. Kilda Beach, Port Philip Bay, Victoria, Australia. The two strains were found to grow between 4 and 40°C, pH 5-10 and tolerate up to 10% NaCl. A phylogenetic study, based on a 16S rRNA gene sequence analysis indicated that strains NP 3b2(T) and H 94 belong to the genus Thalassospira. The sequence similarity of the 16S rRNA gene between the two new isolates is 99.8% and between these strains and all validly named Thalassospira species was found to be in the range of 95-99.4%. The DNA-DNA relatedness between the two strains was found to be 80.2%, while relatedness with other validly named species of the genus Thalassospira was between 53 and 65%. The average nucleotide identity (ANI) and the in silico genome-to-genome distance (GGD) between the two bacteria and T. profundimaris WP0211(T), T. xiamenensis M-5(T), 'T. permensis' NBRC 106175(T) and T. lucentensis QMT2(T) was 76-82% and 21-25%, respectively. The results of phylogenetic and genomic analysis, together with physiological and biochemical properties, indicated that the two strains represent a new species of the genus Thalassospira. Based on these data, a new species, Thalassospira australica, is proposed with strain NP 3b2(T) (=KMM 6365(T)=JCM 31222(T)) as the type strain.

Description of Endozoicomonas arenosclerae sp. nov. using a genomic taxonomy approach.

The taxonomic position of strains Ab112(T) (CBAS 572(T)) and Ab227_MC (CBAS 573) was evaluated by means of genomic taxonomy. These isolates represent the dominant flora cultured from the healthy marine sponge Arenosclera brasiliensis, endemic to Rio de Janeiro. Strains CBAS 572(T) and CBAS 573 shared >98 % 16S rRNA sequence identity with Endozoicomonas numazuensis and Endozoicomonas montiporae. In silico DNA-DNA Hybridization, i.e. genome-to-genome distance (GGD), amino acid identity (AAI) and average nucleotide identity (ANI) further showed that these strains had <70 %, at maximum 71.1 and 78 % of identity, respectively, to their closest neighbours E. numazuensis and E. montiporae. The DNA G+C content of CBAS 572(T) and CBAS 573 were 47.6 and 47.7 mol%, respectively. Phenotypic and chemotaxonomic features also allowed a separation from the type strains of their phylogenetic neighbours. Useful phenotypic features for discriminating CBAS 572(T) and CBAS 573 from E. numazuensis and E. montiporae species include C8 esterase, N-acetyl-β-glucosaminidase, citric acid, uridine and siderophore. The species Endozoicomonas arenosclerae sp. nov. is proposed to harbour the new isolates. The type strain is CBAS 572(T) (=Ab112(T)).

Phylogenomics and systematics in Pseudomonas

The genus Pseudomonas currently contains 144 species, making it the genus of Gram-negative bacteria that contains the largest number of species. Currently, multilocus sequence analysis (MLSA) is the preferred method for establishing the phylogeny between species and genera. Four partial gene sequences of housekeeping genes (16S rRNA, gyrB, rpoB, and rpoD) were obtained from 112 complete or draft genomes of strains related to the genus Pseudomonas that were available in databases. These genes were analyzed together with the corresponding sequences of 133 Pseudomonas type strains of validly published species to assess their correct phylogenetic assignations. We confirmed that 30% of the sequenced genomes of non-type strains were not correctly assigned at the species level in the accepted taxonomy of the genus and that 20% of the strains were not identified at the species level. Most of these strains had been isolated and classified several years ago, and their taxonomic status has not been updated by modern techniques. MLSA was also compared with indices based on the analysis of whole-genome sequences that have been proposed for species delineation, such as tetranucleotide usage patterns (TETRA), average nucleotide identity (ANIm, based on MUMmer and ANIb, based on BLAST) and genome-to-genome distance (GGDC). TETRA was useful for discriminating Pseudomonas from other genera, whereas ANIb and GGDC clearly separated strains of different species. ANIb showed the strongest correlation with MLSA. The correct species classification is a prerequisite for most diversity and evolutionary studies. This work highlights the necessity for complete genomic sequences of type strains to build a phylogenomic taxonomy and that all new genome sequences submitted to databases should be correctly assigned to species to avoid taxonomic inconsistencies.