Benchmarking short-, long- and hybrid-read assemblers for metagenome sequencing of complex microbial communities.

Metagenome community analyses, driven by the continued development in sequencing technology, is rapidly providing insights in many aspects of microbiology and becoming a cornerstone tool. Illumina, Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) are the leading technologies, each with their own advantages and drawbacks. Illumina provides accurate reads at a low cost, but their length is too short to close bacterial genomes. Long reads overcome this limitation, but these technologies produce reads with lower accuracy (ONT) or with lower throughput (PacBio high-fidelity reads). In a critical first analysis step, reads are assembled to reconstruct genomes or individual genes within the community. However, to date, the performance of existing assemblers has never been challenged with a complex mock metagenome. Here, we evaluate the performance of current assemblers that use short, long or both read types on a complex mock metagenome consisting of 227 bacterial strains with varying degrees of relatedness. We show that many of the current assemblers are not suited to handle such a complex metagenome. In addition, hybrid assemblies do not fulfil their potential. We conclude that ONT reads assembled with CANU and Illumina reads assembled with SPAdes offer the best value for reconstructing genomes and individual genes of complex metagenomes, respectively.

Acetobacter senegalensis isolated from mango fruits: Its polyphasic characterization and adaptation to protect against stressors in the industrial production of vinegar: A review.

It has been more than a decade since Acetobacter senegalensis was isolated, identified and described as a thermotolerant strain of acetic acid bacteria. It was isolated from mango fruits in Senegal and used for industrial vinegar production in developing countries, mainly in sub-Saharan Africa. The strain was tested during several spirit vinegar fermentation processes at relatively high temperatures in accordance with African acclimation. The upstream fermentation process had significant stress factors, which are highlighted in this review so that the fermentation process can be better controlled. Due to its high industrial potential, this strain was extensively investigated by diverse industrial microbiologists worldwide; they concentrated on its microbiological, physiological and genomic features. A research group based in Belgium proposed an important project for the investigation of the whole-genome sequence of A. senegalensis. It would use a 454-pyrosequencing technique to determine and corroborate features that could give this strain significant diverse bio-industrial applications. For instance, its application in cocoa bean fermentation has made it a more suitable acetic acid bacterium for the making of chocolate than Acetobacter pasteurianus. Therefore, in this paper, we present a review that summarizes the current research on A. senegalensis at its microbial and genomic levels and also its specific bio-industrial applications, which can provide economic opportunities for African agribusiness. This review summarizes the physiological and genomic characteristics of Acetobacter senegalensis, a thermotolerant strain isolated from mango fruits and intended to be used in industrial vinegar fermentation processes. It also explores other bio-industrial applications such as cocoa fermentation. Vinegar fermentation is usually performed with mesophilic strains in temperate regions of the world. Developing countries, such as Senegal, import vinegar or make 'fake' vinegar by diluting acetic acid obtained from petrochemicals. The use of a thermotolerant Acetobacter senegalensis strain as a solid functional starter culture, as well as the design of a new adapted bioreactor, has significantly contributed to food security and the creation of small- to medium-sized enterprises that produce mango vinegar in West Africa.

PaSiT: a novel approach based on short-oligonucleotide frequencies for efficient bacterial identification and typing.

MOTIVATION: One of the most widespread methods used in taxonomy studies to distinguish between strains or taxa is the calculation of average nucleotide identity. It requires a computationally expensive alignment step and is therefore not suitable for large-scale comparisons. Short oligonucleotide-based methods do offer a faster alternative but at the expense of accuracy. Here, we aim to address this shortcoming by providing a software that implements a novel method based on short-oligonucleotide frequencies to compute inter-genomic distances. RESULTS: Our tetranucleotide and hexanucleotide implementations, which were optimized based on a taxonomically well-defined set of over 200 newly sequenced bacterial genomes, are as accurate as the short oligonucleotide-based method TETRA and average nucleotide identity, for identifying bacterial species and strains, respectively. Moreover, the lightweight nature of this method makes it applicable for large-scale analyses. AVAILABILITY AND IMPLEMENTATION: The method introduced here was implemented, together with other existing methods, in a dependency-free software written in C, GenDisCal, available as source code from https://github.com/LM-UGent/GenDisCal. The software supports multithreading and has been tested on Windows and Linux (CentOS). In addition, a Java-based graphical user interface that acts as a wrapper for the software is also available. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Gluconacetobacter dulcium sp. nov., a novel Gluconacetobacter species from sugar-rich environments.

A phylogenomic analysis based on 107 single-copy core genes revealed that three strains from sugar-rich environments, i.e. LMG 1728T, LMG 1731 and LMG 22058, represented a single, novel Gluconacetobacter lineage with Gluconacetobacter liquefaciens as nearest validly named neighbour. OrthoANIu and digital DNA-DNA hybridization analyses among these strains and Gluconacetobacter type strains confirmed that the three strains represented a novel Gluconacetobacter species. Biochemical characteristics and MALDI-TOF mass spectra allowed differentiation of this novel species from the type strains of G. liquefaciens and other closely related Gluconacetobacter species. We therefore propose to classify strains LMG 1728T, LMG 1731 and LMG 22058 in the novel species Gluconacetobacter dulcium sp. nov., with LMG 1728T (=CECT 30142T) as the type strain.

Novel acetic acid bacteria from cider fermentations: Acetobacter conturbans sp. nov. and Acetobacter fallax sp. nov.

Strains LMG 1627T, LMG 1636T and LMG 1637 were all isolated from cider fermentations in the 1940s and 1950s. A recent study based on MALDI-TOF MS and dnaK gene sequence analyses suggested they represented novel Acetobacter species. In the present study, we determined the whole-genome sequences of these strains and analysed their phenotypic and chemotaxonomic characteristics. A phylogenomic analysis based on 107 single-copy core genes revealed that they represented a single Acetobacter lineage with Acetobacter aceti, Acetobacter sicerae, Acetobacter musti and Acetobacter oeni, Acetobacter estunensis and with Acetobacter nitrogenifigens as an outgroup to this cluster. OrthoANIu value and dDDH analyses among these and other Acetobacter type strains confirmed that these three strains represented two novel Acetobacter species, which could be differentiated from other closely related type strains of Acetobacter by different phenotypic tests, such as ketogenesis from glycerol. We therefore propose to classify strain LMG 1627T in the novel species Acetobacter conturbans sp. nov., with LMG 1627T (=NCIMB 8945T) as the type strain, and to classify strains LMG 1636T and LMG 1637 in the novel species Acetobacter fallax sp. nov., with LMG 1636T (=NCIMB 8956T) as the type strain.

Pectobacterium parvum sp. nov., having a Salmonella SPI-1-like Type III secretion system and low virulence.

Pectobacterium strains isolated from potato stems in Finland, Poland and the Netherlands were subjected to polyphasic analyses to characterize their genomic and phenotypic features. Phylogenetic analysis based on 382 core proteins showed that the isolates clustered closest to Pectobacterium polaris but could be divided into two clades. Average nucleotide identity (ANI) analysis revealed that the isolates in one of the clades included the P. polaris type strain, whereas the second clade was at the border of the species P. polaris with a 96 % ANI value. In silico genome-to-genome comparisons between the isolates revealed values below 70%, patristic distances based on 1294 core proteins were at the level observed between closely related Pectobacterium species, and the two groups of bacteria differed in genome size, G+C content and results of amplified fragment length polymorphism and Biolog analyses. Comparisons between the genomes revealed that the isolates of the atypical group contained SPI-1-type Type III secretion island and genes coding for proteins known for toxic effects on nematodes or insects, and lacked many genes coding for previously characterized virulence determinants affecting rotting of plant tissue by soft rot bacteria. Furthermore, the atypical isolates could be differentiated from P. polaris by their low virulence, production of antibacterial metabolites and a citrate-negative phenotype. Based on the results of a polyphasic approach including genome-to-genome comparisons, biochemical and virulence assays, presented in this report, we propose delineation of the atypical isolates as a novel species Pectobacterium parvum, for which the isolate s0421T (CFBP 8630T=LMG 30828T) is suggested as a type strain.

Actinomadura roseirufa sp. nov., producer of semduramicin, a polyether ionophore.

The taxonomic position of 'Actinomadura roseorufa' LMG 30035T, a semduramicin-producing mutant of strain ATCC 53666P, which was isolated from a soil sample collected in Yamae Village, Kamamoto, Japan, was clarified in the present study using a polyphasic approach. This Gram-positive, aerobic actinomycete formed a well-developed, extensively branched, non-fragmenting substrate and aerial mycelia which differentiated into single, smooth-appearing spores. Based on analysis of nearly complete 16S rRNA gene sequence, strain LMG 30035T was found to be closely related to the type strains of Actinomadura fibrosa ATCC 49459T (98.88 %) and Actinomadura formosensis JCM 7474T (98.82 %) (pairwise similarity values in parentheses). Digital DNA-DNA hybridisation experiments revealed unambiguously that strain LMG 30035T represents a novel Actinomadura species (OrthoANIu values less than 83.1 %; dDDH values less than 27.2 % with type strains of validly named Actinomadura species). Analysis of the cell wall revealed the presence of meso-diaminopimelic acid in the peptidoglycan. The whole-cell sugars were glucose, madurose, galactose, ribose and rhamnose. The major polar lipids included phosphatidylinositol and diphosphatidylglycerol. The predominant menaquinones were MK-9(H6), MK-9(H8), MK-9(H4) and MK-9(H2). The major fatty acids were C16 : 00, 10-methyl C18 : 0, C18 : 1 ω9c and C18 : 00. The DNA G+C content of its genome was 72.5 mol%. In summary, these characteristics distinguish strain LMG 30035T from validly named species of the genus Actinomadura, and therefore, we propose to classify this strain formally as the novel species Actinomadura roseirufa sp. nov. with LMG 30035T (=CECT 9808T,=ATCC 53664T) as the type strain.

Entomobacter blattae gen. nov., sp. nov., a new member of the Acetobacteraceae isolated from the gut of the cockroach Gromphadorhina portentosa.

A novel bacterium designated G55GPT and pertaining to the family Acetobacteraceae was isolated from the gut of the Madagascar hissing cockroach Gromphadorhina portentosa. The Gram-negative cells were rod-shaped and non-motile. The complete 16S rRNA sequence of the strain G55GPT showed the highest pairwise similarity to Gluconacetobacter johannae CFN-Cf-55T (95.35 %), suggesting it represents a potential new genus of the family Acetobacteraceae. Phylogenetic analysis based on 16S rRNA gene and 106 orthologous housekeeping protein sequences revealed that G55GPT forms a monophyletic clade with the genus Commensalibacter, which thus far has also been isolated exclusively from insects. The G55GPT genome size was 2.70 Mbp, and the G+C content was 45.4 mol%, which is lower than most acetic acid bacteria (51-68 mol%) but comparable to Swingsia samuiensis AH83T (45.1 mol%) and higher than Commensalibacter intestini A911T (36.8 mol%). Overall genome relatedness indices based on gene and protein sequences strongly supported the assignment of G55GPT to a new genus within the family Acetobacteraceae. The percentage of conserved proteins, which is a useful metric for genus differentiation, was below 54 % when comparing G55GPT to type strains of acetic acid bacteria, thus strongly supporting our hypothesis that G55GPT is a member of a yet-undescribed genus. The fatty acid composition of G55GPT differed from that of closely related acetic acid bacteria, particularly given the presence of C19 : 1 ω9c/ω11c and the absence of C14 : 0 and C14 : 0 2-OH fatty acids. Strain G55GPT also differed in terms of metabolic features such as its ability to produce acid from d-mannitol, and its inability to produce acetic acid from ethanol or to oxidize glycerol to dihydroxyacetone. Based on the results of combined genomic, phenotypic and phylogenetic characterizations, isolate G55GPT (=LMG 31394T=DSM 111244T) is considered to represent a new species in a new genus, for which we propose the name Entomobacter blattae gen. nov., sp. nov.

Characterization of novel Gluconobacter species from fruits and fermented food products: Gluconobacter cadivus sp. nov., Gluconobacter vitians sp. nov. and Gluconobacter potus sp. nov.

Strains LMG 1744T, LMG 1745, LMG 31484T, LMG 1764T and R-71646 were isolated from rotting fruits and fermented food products. A phylogenomic analysis based on 107 single-copy core genes revealed that they grouped in a Gluconobacter lineage comprising Gluconobacter oxydans, Gluconobacter roseus, Gluconobacter sphaericus, Gluconobacter kanchanaburiensis, Gluconobacter albidus, Gluconobacter cerevisiae, Gluconobacter kondonii and Gluconobacter aidae. OrthoANIu and digital DNA hybridization analyses demonstrated that these five strains represented three novel Gluconobacter species, which could be differentiated from the type strains of closely related Gluconobacter species by multiple phenotypic characteristics. We therefore propose to classify strains LMG 1744T and LMG 1745 in the novel species Gluconobacter cadivus sp. nov., with LMG 1744T (=CECT 30141T) as the type strain; to classify strain LMG 31484T as the novel species Gluconobacter vitians sp. nov., with LMG 31484T (=CECT 30132T) as the type strain; and to classify strains LMG 1764T and R-71646 in the novel species Gluconobacter potus sp. nov., with LMG 1764T (=CECT 30140T) as the type strain.

Arcobacter haliotis sp. nov., isolated from abalone species Haliotis gigantea.

A Gram-negative, aerobic, polar-flagellated and rod-shaped, sometimes slightly curved bacterium, designated MA5T, was isolated from the gut of an abalone of the species Haliotis gigantea collected in Japan. Phylogenetic analyses based on 16S rRNA, gyrB, hsp60 and rpoB gene sequences placed strain MA5T in the genus Arcobacter in an independent phylogenetic line. Comparison of the 16S rRNA gene sequence of this strain with those of the type strains of the established Arcobacter species revealed A. nitrofigilis (95.1 %) as nearest neighbour. Strain MA5T grew optimally at 25 °C, pH 6.0 to 9.0 and in the presence of 2 to 5 % (w/v) NaCl under both aerobic and microaerobic conditions. The predominant fatty acids found were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1 ω7c), C12 : 0 3-OH and C18 : 1 ω7c. Menaquinone-6 (MK-6) and menaquinone-7 (MK-7) were found as the major respiratory quinones. The major polar lipids detected were phosphatidylethanolamine and phosphatidylglycerol. Strain MA5T could be differentiated phenotypically from the phylogenetic closest Arcobacter species by its ability to grow on 0.05 % safranin and 0.01 % 2,3,5-triphenyl tetrazolium chloride (TTC), but not on 0.5 % NaCl. The obtained DNA G+C content of strain MA5T was 27.9 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic distinctiveness of MA5T, this strain is considered to represent a novel species of the genus Arcobacter, for which the name Arcobacter haliotis sp. nov. is proposed. The type strain is MA5T (=LMG 28652T=JCM 31147T).

Cellulose synthesis by Komagataeibacter rhaeticus strain P 1463 isolated from Kombucha.

Isolate B17 from Kombucha was estimated to be an efficient producer of bacterial cellulose (BC). The isolate was deposited under the number P 1463 and identified as Komagataeibacter rhaeticus by comparing a generated amplified fragment length polymorphism (AFLP™) DNA fingerprint against a reference database. Static cultivation of the K. rhaeticus strain P 1463 in Hestrin and Schramm (HS) medium resulted in 4.40 ± 0.22 g/L BC being produced, corresponding to a BC yield from glucose of 25.30 ± 1.78 %, when the inoculum was made with a modified HS medium containing 10 g/L glucose. Fermentations for 5 days using media containing apple juice with analogous carbon source concentrations resulted in 4.77 ± 0.24 g/L BC being synthesised, corresponding to a yield from the consumed sugars (glucose, fructose and sucrose) of 37.00 ± 2.61 %. The capacity of K. rhaeticus strain P 1463 to synthesise BC was found to be much higher than that of two reference strains for cellulose production, Komagataeibacter xylinus DSM 46604 and Komagataeibacter hansenii DSM 5602T, and was also considerably higher than that of K. hansenii strain B22, isolated from another Kombucha sample. The BC synthesised by K. rhaeticus strain P 1463 after 40 days of cultivation in HS medium with additional glucose supplemented to the cell culture during cultivation was shown to have a degree of polymerization of 3300.0 ± 122.1 glucose units, a tensile strength of 65.50 ± 3.27 MPa and a length at break of 16.50 ± 0.83 km. For the other strains, these properties did not exceed 25.60 ± 1.28 MPa and 15.20 ± 0.76 km.

Paenibacillus aquistagni sp. nov., isolated from an artificial lake accumulating industrial wastewater.

Strain 11T was isolated from water of an artificial lake accumulating industrial wastewater on the outskirts of Celje, Slovenia. Phenotypic characterisation showed strain 11T to be a Gram-stain positive, spore forming bacterium. The 16S rRNA gene sequence identified strain 11T as a member of the genus Paenibacillus, closely related to Paenibacillus alvei (96.2%). Genomic similarity with P. alvei 29T was 73.1% (gANI), 70.2% (ANIb), 86.7% (ANIm) and 21.7 ± 2.3% (GGDC). The DNA G+C content of strain 11T was determined to be 47.5%. The predominant menaquinone of strain 11T was identified as MK-7 and the major fatty acid as anteiso-C15:0. The peptidoglycan was found to contain meso-diaminopimelic acid. In contrast to its close relatives P. alvei DSM 29T, Paenibacillus apiarius DSM 5581T and Paenibacillus profundus NRIC 0885T, strain 11T was found to be able to ferment D-fructose, D-mannose and D-xylose. A draft genome of strain 11T contains a cluster of genes associated with type IV pilin synthesis usually found in clostridia, and only sporadically in other Gram-positive bacteria. Genotypic, chemotaxonomic, physiological and biochemical characteristics of strain 11T presented in this study support the creation of a novel species within the genus Paenibacillus, for which the name Paenibacillus aquistagni sp. nov. is proposed, with strain 11T (=ZIM B1027T =LMG 29561T =CCM 8679T ) as the type strain.

Reclassification of Pseudomonas sp. PB-6250T as Lysobacter firmicutimachus sp. nov.

Strain PB-6250T, isolated from soil in Japan, was first identified in 1992. In contrast to its original taxonomic classification, its 16S rRNA gene sequence showed the highest similarity (99.2 %) to the sequence of Lysobacter enzymogenes DSM 2043T, with Lysobacter antibioticus DSM 2044T being the next most closely related species (98.7 %) with a validly published name. Chemotaxonomic data (fatty acid profile, quinone and polar lipid composition) and the G+C content of strain PB-6250T were compared with those of the closely related type strains L. enzymogenes LMG 8762T, L. antibioticus LMG 8760T, L. capsici DSM 19286T and L. gummosus LMG 8763T; this supported the affiliation of strain PB-6250T to the genus Lysobacter. Phylogenetic analyses, DNA-DNA-hybridization data, biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiation of strain PB-6250T from species of Lysobacter with validly published names. Strain PB-6250T, therefore represents a novel species, for which the name Lysobacter firmicutimachus sp. nov. is proposed. The type strain is PB-6250T (=LMG 28994T=DSM 102073T).

Erwinia gerundensis sp. nov., a cosmopolitan epiphyte originally isolated from pome fruit trees.

A survey to obtain potential antagonists of pome fruit tree diseases yielded two yellow epiphytic bacterial isolates morphologically similar to Pantoea agglomerans, but showing no biocontrol activity. Whole-cell MALDI-TOF mass spectrometry and analysis of 16S rRNA gene and gyrB sequences suggested the possibility of a novel species with a phylogenetic position in either the genus Pantoea or the genus Erwinia. Multi-locus sequence analysis (MLSA) placed the two strains in the genus Erwinia and supported their classification as a novel species. The strains showed general phenotypic characteristics typical of this genus and results of DNA-DNA hybridizations confirmed that they represent a single novel species. Both strains showed a DNA G+C content, as determined by HPLC, of 54.5 mol% and could be discriminated from phylogenetically related species of the genus Erwinia by their ability to utilize potassium gluconate, potassium 2-ketogluconate, maltose, melibiose and raffinose. Whole-genome sequencing of strain EM595T revealed the presence of a chromosomal carotenoid biosynthesis gene cluster similar to those found in species of the genera Cronobacter and Pantoea that explains the pigmentation of the strain, which is atypical for the genus Erwinia. Additional strains belonging to the same species were recovered from different plant hosts in three different continents, revealing the cosmopolitan nature of this epiphyte. The name Erwinia gerundensis sp. nov. is proposed, with EM595T ( = LMG 28990T = CCOS 903T) as the designated type strain.

Formosa haliotis sp. nov., a brown-alga-degrading bacterium isolated from the gut of the abalone Haliotis gigantea.

Four brown-alga-degrading, Gram-stain-negative, aerobic, non-flagellated, gliding and rod-shaped bacteria, designated LMG 28520T, LMG 28521, LMG 28522 and LMG 28523, were isolated from the gut of the abalone Haliotis gigantea obtained in Japan. The four isolates had identical random amplified polymorphic DNA patterns and grew optimally at 25 °C, at pH 6.0-9.0 and in the presence of 1.0-4.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences placed the isolates in the genus Formosa with Formosa algae and Formosa arctica as closest neighbours. LMG 28520T and LMG 28522 showed 100 % DNA-DNA relatedness to each other, 16-17 % towards F. algae LMG 28216T and 17-20 % towards F. arctica LMG 28318T; they could be differentiated phenotypically from these established species. The predominant fatty acids of isolates LMG 28520T and LMG 28522 were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), iso-C15 : 1 G and iso-C15 : 0. Isolate LMG 28520T contained menaquinone-6 (MK-6) as the major respiratory quinone and phosphatidylethanolamine, two unknown aminolipids and an unknown lipid as the major polar lipids. The DNA G+C content was 34.4 mol% for LMG 28520T and 35.5 mol% for LMG 28522. On the basis of their phylogenetic and genetic distinctiveness, and differential phenotypic properties, the four isolates are considered to represent a novel species of the genus Formosa, for which the name Formosa haliotis sp. nov. is proposed. The type strain is LMG 28520T ( = NBRC 111189T).

Bombella intestini gen. nov., sp. nov., an acetic acid bacterium isolated from bumble bee crop.

In the frame of a bumble bee gut microbiota study, acetic acid bacteria (AAB) were isolated using a combination of direct isolation methods and enrichment procedures. MALDI-TOF MS profiling of the isolates and a comparison of these profiles with profiles of established AAB species identified most isolates as Asaia astilbis or as 'Commensalibacter intestini', except for two isolates (R-52486 and LMG 28161(T)) that showed an identical profile. A nearly complete 16S rRNA gene sequence of strain LMG 28161(T) was determined and showed the highest pairwise similarity to Saccharibacter floricola S-877(T) (96.5%), which corresponded with genus level divergence in the family Acetobacteraceae. Isolate LMG 28161(T) was subjected to whole-genome shotgun sequencing; a 16S-23S rRNA internal transcribed spacer (ITS) sequence as well as partial sequences of the housekeeping genes dnaK, groEL and rpoB were extracted for phylogenetic analyses. The obtained data confirmed that this isolate is best classified into a new genus in the family Acetobacteraceae. The DNA G+C content of strain LMG 28161(T) was 54.9 mol%. The fatty acid compositions of isolates R-52486 and LMG 28161(T) were similar to those of established AAB species [with C18:1ω7c (43.1%) as the major component], but the amounts of fatty acids such as C19:0 cyclo ω8c, C14:0 and C14:0 2-OH enabled to differentiate them. The major ubiquinone was Q-10. Both isolates could also be differentiated from the known genera of AAB by means of biochemical characteristics, such as their inability to oxidize ethanol to acetic acid, negligible acid production from melibiose, and notable acid production from d-fructose, sucrose and d-mannitol. In addition, they produced 2-keto-d-gluconate, but not 5-keto-d-gluconate from d-glucose. Therefore, the name Bombella intestini gen nov., sp. nov. is proposed for this new taxon, with LMG 28161(T) ( =DSM 28636(T) =R-52487(T)) as the type strain of the type species.

Characterization of a novel clade of Xanthomonas isolated from rice leaves in Mali and proposal of Xanthomonas maliensis sp. nov.

Four bacterial strains, designated M89, M92, M97(T), and M106, were isolated in a previous study from surface-sterilized leaves of rice (Oryza sativa) or murainagrass (Ischaemum rugosum) at three sites in Mali, Africa. Here they were examined by a polyphasic taxonomic approach and analysis of a whole-genome sequence. Phylogenetic analyses based on 16S rRNA sequence and multilocus sequence analysis of seven genes showed that these four strains formed a distinct lineage representing a novel species within the genus Xanthomonas. This was supported by whole-genome average nucleotide identity values calculated from comparisons of strain M97(T) with established Xanthomonas species. The strains can be differentiated from the known Xanthomonas species on the basis of their fatty acid and carbohydrate utilization profiles. Population growth studies on rice confirmed that these bacteria multiply in rice leaves without causing symptoms. Identification of this novel species can be accomplished by using diagnostic primer sets or by gyrB gene sequence analysis. We propose to classify these rice- and grass-associated bacteria as Xanthomonas maliensis sp. nov. with strain M97(T) = CFBP7942(T) = LMG27592(T) as the type strain.

Thiouracil-Forming Bacteria Identified and Characterized upon Porcine In Vitro Digestion of Brassicaceae Feed.

In recent years, the frequent detection of the banned thyreostat thiouracil (TU) in livestock urine has been related to endogenous TU formation following digestion of glucosinolate-rich Brassicaceae crops. Recently, it was demonstrated that, upon in vitro digestion of Brassicaceae, fecal bacteria induce TU detection in livestock (porcine livestock > bovines). Therefore, the present study was intended to isolate and identify bacteria involved in this intestinal TU formation upon Brassicaceae digestion and to gain more insight into the underlying mechanism in porcine livestock. Twenty porcine fecal inocula (gilts and multiparous sows) were assessed through static in vitro colonic-digestion simulations with rapeseed. After derivatization and extraction of the fecal suspensions, TU was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS(2)). On average, lower TU concentrations were observed in fecal colonic simulations in gilts (8.35 ng g(-1) rapeseed ± 3.42 [mean ± standard deviation]) than in multiparous sows (52.63 ng g(-1) ± 16.17), which correlates with maturation of the gut microbial population with age. Further exploration of the mechanism showed cell-dependent activity of the microbial conversion and sustained TU-forming activity after subjection of the fecal inoculum to moderate heat over a time span of up to 30 min. Finally, nine TU-producing bacterial species were successfully isolated and identified by a combination of biochemical and molecular techniques as Escherichia coli (n = 5), Lactobacillus reuteri (n = 2), Enterococcus faecium (n = 1), and Salmonella enterica subsp. arizonae (n = 1). This report demonstrates that endogenous formation of TU is Brassicaceae induced and occurs under colonic conditions most likely through myrosinase-like enzyme activity expressed by different common intestinal bacterial species.

Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum sp. nov.

The Ralstonia solanacearum species complex has long been recognized as a group of phenotypically diverse strains that can be subdivided into four phylotypes. Using a polyphasic taxonomic approach on an extensive set of strains, this study provides evidence for a taxonomic and nomenclatural revision of members of this complex. Data obtained from phylogenetic analysis of 16S-23S rRNA ITS gene sequences, 16S-23S rRNA intergenic spacer (ITS) region sequences and partial endoglucanase (egl) gene sequences and DNA-DNA hybridizations demonstrate that the R. solanacearum species complex comprises three genospecies. One of these includes the type strain of Ralstonia solanacearum and consists of strains of R. solanacearum phylotype II only. The second genospecies includes the type strain of Ralstonia syzygii and contains only phylotype IV strains. This genospecies is subdivided into three distinct groups, namely R. syzygii, the causal agent of Sumatra disease on clove trees in Indonesia, R. solanacearum phylotype IV strains isolated from different host plants mostly from Indonesia, and strains of the blood disease bacterium (BDB), the causal agent of the banana blood disease, a bacterial wilt disease in Indonesia that affects bananas and plantains. The last genospecies is composed of R. solanacearum strains that belong to phylotypes I and III. As these genospecies are also supported by phenotypic data that allow the differentiation of the three genospecies, the following taxonomic proposals are made: emendation of the descriptions of Ralstonia solanacearum and Ralstonia syzygii and descriptions of Ralstonia syzygii subsp. nov. (type strain R 001(T) = LMG 10661(T) = DSM 7385(T)) for the current R. syzygii strains, Ralstonia syzygii subsp. indonesiensis subsp. nov. (type strain UQRS 464(T) = LMG 27703(T) = DSM 27478(T)) for the current R. solanacearum phylotype IV strains, Ralstonia syzygii subsp. celebesensis subsp. nov. (type strain UQRS 627(T) = LMG 27706(T) = DSM 27477(T)) for the BDB strains and Ralstonia pseudosolanacearum sp. nov. (type strain UQRS 461(T) = LMG 9673(T) = NCPPB 1029(T)) for the strains of R. solanacearum phylotypes I and III.