Uncovering Genomic Features and Biosynthetic Gene Clusters in Endophytic Bacteria from Roots of the Medicinal Plant Alkanna tinctoria Tausch as a Strategy To Identify Novel Biocontrol Bacteria.

The world's population is increasing at a rate not seen in the past. Agriculture, providing food for this increasing population, is reaching its boundaries of space and natural resources. In addition, changing legislation and increased ecological awareness are forcing agriculture to reduce its environmental impact. This entails the replacement of agrochemicals with nature-based solutions. In this regard, the search for effective biocontrol agents that protect crops from pathogens is in the spotlight. In this study, we have investigated the biocontrol activity of endophytic bacteria isolated from the medicinal plant Alkanna tinctoria Tausch. To do so, an extensive collection of bacterial strains was initially genome sequenced and in silico screened for features related to plant stimulation and biocontrol. Based on this information, a selection of bacteria was tested in vitro for antifungal activity using direct antagonism in a plate assay and in planta with a detached-leaf assay. Bacterial strains were tested individually and in combinations to assess the best-performing treatments. The results revealed that many bacteria could produce metabolites that efficiently inhibit the proliferation of several fungi, especially Fusarium graminearum. Among these, Pseudomonas sp. strain R-71838 showed a strong antifungal effect, in both dual-culture and in planta assays, making it the most promising candidate for biocontrol application. Using microbes from medicinal plants, this study highlights the opportunities of using genomic information to speed up the screening of a taxonomically diverse set of bacteria with biocontrol properties. IMPORTANCE Phytopathogenic fungi are a major threat to global food production. The most common management practice to prevent plant infections involves the intensive use of fungicides. However, with the growing awareness of the ecological and human impacts of chemicals, there is a need for alternative strategies, such as the use of bacterial biocontrol agents. Limitations in the design of bacterial biocontrol included the need for labor-intensive and time-consuming experiments to test a wide diversity of strains and the lack of reproducibility of their activity against pathogens. Here, we show that genomic information is an effective tool to select bacteria of interest quickly. Also, we highlight that the strain Pseudomonas sp. R-71838 produced a reproducible antifungal effect both in vitro and in planta. These findings build a foundation for designing a biocontrol strategy based on Pseudomonas sp. R-71838.

Phylogenomics reveals insights into the functional evolution of the genus Agrobacterium and enables the description of Agrobacterium divergens sp. nov.

The genus Agrobacterium was initially described as mainly phytopathogenic strains. Nowadays, the genus includes phytopathogenic and non-phytopathogenic bacteria that are distinctive among the Rhizobiaceae family. Recently we have isolated two closely related strains, LMG 31531T and LMG 31532, from soil and plant roots, respectively. Both strains differ from previously reported species based on the genomic and phenotypic data. A. arsenijevicii KFB 330T and A. fabacearum LMG 31642T showed the highest 16S rRNA similarity (98.9 %), followed by A. nepotum LMG 26435T (98.7 %). A clear genomic feature that distinguishes LMG 31531T and LMG 31532 from other Agrobacterium species is the absence of a linear chromid. Nevertheless, typical values of the core-proteome Average Amino Acid Identity (cpAAI > 85 %) and 16S rRNA gene sequence similarity (>96 %) when compared to other members of the genus confirm the position of these two strains as part of the Agrobacterium genus. They are therefore described as Agrobacterium divergens sp. nov. Besides, our comparative genomic study and survey for clade-specific markers resulted in the discovery of conserved proteins that provide insights into the functional evolution of this genus.

More than just hitchhikers: a survey of bacterial communities associated with diatoms originating from sea turtles.

Diatoms and bacteria are known for being the first colonizers of submerged surfaces including the skin of marine reptiles. Sea turtle carapace and skin harbor diverse prokaryotic and eukaryotic microbes, including several epizoic diatoms. However, the importance of diatom-bacteria associations is hardly investigated in biofilms associated with animal hosts. This study provides an inventory of diatoms, bacteria and diatom-associated bacteria originating from loggerhead sea turtles using both metabarcoding and culturing approaches. Amplicon sequencing of the carapace and skin samples chloroplast gene rbcL and 16S rRNA gene detected, in total, 634 diatom amplicon sequence variants (ASVs) and 3661 bacterial ASVs, indicating high diversity. Cultures of putative epizoic and non-epizoic diatoms contained 458 bacterial ASVs and their bacterial assemblages reflected those of their host. Diatom strains allowed for enrichment and isolation of bacterial families rarely observed on turtles, such as Marinobacteraceae, Alteromonadaceae and Alcanivoracaceae. When accounting for phylogenetic relationships between bacterial ASVs, we observed that related diatom genera might retain similar microbial taxa in culture, regardless of the turtle's skin or carapace source. These data provide deeper insights into the sea turtle-associated microbial communities, and reveal the potential of epizoic biofilms as a source of novel microbes and possibly important diatom-bacteria associations.

Description and functional testing of four species of the novel phototrophic genus Chioneia gen. nov., isolated from different East Antarctic environments.

Seven Gram-negative, aerobic, non-sporulating, motile strains were isolated from terrestrial (R-67880T, R-67883, R-36501 and R-36677T) and aquatic (R-39604, R-39161T and R-39594T) East Antarctic environments (i.e. soil and aquatic microbial mats), between 2007 and 2014. Analysis of near-complete 16S rRNA gene sequences revealed that the strains potentially form a novel genus in the family Sphingomonadaceae (Alphaproteobacteria). DNA-DNA reassociation and average nucleotide identity values indicated distinction from close neighbors in the family Sphingomonadaceae and showed that the seven isolates form four different species. The main central pathways present in the strains are the glycolysis, tricarboxylic acid cycle and pentose phosphate pathway. The strains can use only a limited number of carbon sources and mainly depend on ammonia and sulfate as a nitrogen and sulfur source, respectively. The novel strains showed the potential of aerobic anoxygenic phototrophy, based on the presence of bacteriochlorophyll a pigments, which was corroborated by the presence of genes for all building blocks for a type 2 photosynthetic reaction center in the annotated genomes. Based on the results of phenotypic, genomic, phylogenetic and chemotaxonomic analyses, the strains could be assigned four new species in the novel genus Chioneia gen. nov. in the family Sphingomonadaceae, for which the names C. frigida sp. nov. (R-67880T, R-67883 and R-36501), C. hiemis sp. nov. (R-36677T), C. brumae sp. nov. (R-39161T and R-39604) and C. algoris sp. nov. (R-39594T) are proposed.

Roseomonas hellenica sp. nov., isolated from roots of wild-growing Alkanna tinctoria.

Two Gram-negative, aerobic, rod-shaped and yellow-orange pigmented bacterial strains (LMG 31523T and LMG 31524) were isolated from roots of wild-growing Alkanna tinctoria plants collected near Thessaloniki, Greece. Analysis of their 16S rRNA gene sequences revealed that they form a separate cluster related to the genus Roseomonas. A comparative whole genome analysis of the two strains and the type strains of related Roseomonas species revealed average nucleotide identity values from 78.84 and 80.32%. The G + C contents of the genomic DNA of strains LMG 31523T and LMG 31524 were 69.69% and 69.74%, respectively. Combined data from phenotypic, phylogenetic and chemotaxonomic studies indicated that the strains LMG 31523T and LMG 31524 represent a novel species of the genus Roseomonas. Genome analysis of the new strains showed a number of genes involved in survival in the rhizosphere environment and in plant colonization and confirmed the endophytic characteristics of LMG 31523T and LMG 31524. Since the strains LMG 31523T and LMG 31524 were isolated from a plant collected in Greece the name Roseomonas hellenica sp. nov. is proposed. The type strain is LMG 31523T (=CECT 30032T).

Efficient Nitrogen-Fixing Bacteria Isolated from Soybean Nodules in the Semi-arid Region of Northeast Brazil are Classified as Bradyrhizobium brasilense (Symbiovar Sojae).

Soybean (Glycine max L.) is an important legume that greatly benefits from inoculation with nitrogen-fixing bacteria. In a previous study, five efficient nitrogen-fixing bacterial strains, isolated from nodules of soybean inoculated with soil from semi-arid region, Northeast Brazil, were identified as a new group within the genus Bradyrhizobium. The taxonomic status of these strains was evaluated in this study. The phylogenetic analysis of the 16S rRNA gene showed the high similarity of the five strains to Bradyrhizobium brasilense UFLA03-321T (100%), B. pachyrhizi PAC48T (100%), B. ripae WR4T (100%), B. elkanii USDA 76T (99.91%), and B. macuxiense BR 10303T (99.91%). However, multilocus sequence analysis of the housekeeping genes atpD, dnaK, gyrB, recA, and rpoB, average nucleotide identity, and digital DNA-DNA hybridization analyses supported the classification of the group as B. brasilense. Some phenotypic characteristics allowed differentiating the five strains and the type strain of B. brasilense from the two neighboring species (B. pachyrhizi PAC48T and B. elkanii USDA 76T). The nodC and nifH genes' analyses showed that these strains belong to symbiovar sojae, together with B. elkanii (USDA 76T) and B. ferriligni (CCBAU 51502T). The present results support the classification of these five strains as Bradyrhizobium brasilense (symbiovar sojae).

Leeuwenhoekiella aestuarii sp. nov., isolated from salt-water sediment and first insights in the genomes of Leeuwenhoekiella species.

Four Gram-negative, aerobic, rod-shaped and yellow-orange pigmented bacteria (R-46770, R-48165T, R-50232 and R-50233) were isolated from intertidal sediment and water of the Westerschelde estuary between 2006 and 2012. Analysis of their 16S rRNA gene sequences revealed that the four strains form a separate cluster between validly described type strains of the genus Leeuwenhoekiella. DNA-DNA reassociation values of two representative strains (i.e. R-48165T and R-50232) of the new group with type strains of Leeuwenhoekiella species ranged from 18.7 to 56.6 %. A comparative genome analysis of the two strains and the type strains confirmed average nucleotide identity values from 75.6 to 94.4 %. The G+C contents of the genomic DNA of strains R-48165T and R-50232 were 37.80 and 37.83 mol%, respectively. The predominant cellular fatty acids of the four novel strains were summed feature 3 (i.e. C16 : 1ω7c and/or iso-C15 : 0 2-OH), iso-C15 : 0, iso-C15 : 1 G and iso-C17 : 0 3-OH. The four new Leeuwenhoekiella-like strains grew with 0.5-12 % (w/v) NaCl, at pH 5.5-9.0 and displayed optimum growth between 20 and 30 °C. Based on the results of phenotypic, genomic, phylogenetic and chemotaxonomic analyses, the four new strains represent a novel species of the genus Leeuwenhoekiella for which the name Leeuwenhoekiella aestuarii sp. nov. is proposed. The type strain is LMG 30908T (=R-48165T=CECT 9775T=DSM 107866T). Genome analysis of type strains of the genus Leeuwenhoekiella revealed a large number of glycosyl hydrolases, peptidases and carboxyl esterases per Mb, whereas the number of transporters per Mb was low compared to other bacteria. This confirmed the environmental role of Leeuwenhoekiella species as (bio)polymer degraders, with a specialization on degrading proteins and high molecular weight compounds. Additionally, the presence of a large number of genes involved in gliding motility and surface adhesion, and large numbers of glycosyl transferases per Mb confirmed the importance of these features for Leeuwenhoekiella species.

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

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

Spirosoma utsteinense sp. nov. isolated from Antarctic ice-free soils from the Utsteinen region, East Antarctica.

Between 2014 and 2016, 16 Gram-stain-negative, aerobic, rod-shaped and yellow-orange pigmented bacteria were isolated from exposed soils from the Utsteinen region, Sør Rondane Mountains, East Antarctica. Analysis of their 16S rRNA gene sequences revealed that the strains form a separate cluster in the genus Spirosoma, with Spirosoma rigui KCTC 12531T as its closest neighbour (97.8 % sequence similarity). Comparative genome analysis of two representative strains (i.e. R-68523T and R-68079) of the new group with the type strains of Spirosoma rigui (its closest neighbour) and Spirosoma linguale (type species of the genus), yielded average nucleotide identity values of 73.9-78.7 %. Digital DNA-DNA reassociation values of the two strains and these type strains ranged from 20.3 to 22.0 %. The predominant cellular fatty acids of the two novel strains were summed feature 3 (i.e. C16 : 1 ω7c and/or iso-C15 2-OH), C16 : 1 ω5c, C16 : 0 and iso-C15 : 0. The new Spirosoma strains grew with 0-0.5 % (w/v) NaCl, at pH 6.5-8.0 and displayed optimum growth between 15 and 25 °C. Based on the results of phenotypic, genomic, phylogenetic and chemotaxonomic analyses, the new strains represent a novel species of the genus Spirosoma for which the name Spirosoma utsteinense sp. nov. is proposed. The type strain is R-68523T (=LMG 31447T=CECT 9925T).

Abditibacterium utsteinense sp. nov., the first cultivated member of candidate phylum FBP, isolated from ice-free Antarctic soil samples.

Most bacterial lineages are known only by molecular sequence data from environmental surveys and represent the uncultivated majority. One of these lineages, candidate phylum FBP, is widespread in extreme environments on Earth, ranging from polar and desert ecosystems to wastewater and contaminated mine sites. Here we report on the characterization of strain LMG 29911T, the first cultivated representative of the FBP lineage. The strain was isolated from a terrestrial surface sample from Utsteinen, Sør Rondane Mountains, East Antarctica and is a Gram-negative, aerobic, oligotrophic chemoheterotrophic bacterium. It displays growth in a very narrow pH range, use of only a limited number of carbon sources, but also a metabolism optimized for survival in low-nutrient habitats. Remarkably, phenotypic and genome analysis indicated an extreme resistance against antibiotics and toxic compounds. We propose the names Abditibacterium utsteinense for this bacterium and Abditibacteriota for the former candidate phylum FBP. Furthermore, inter- and intra-phylum relationships indicate Armatimonadetes, a neighboring lineage to the Abditibacteriota, to be a superphylum.

Bradyrhizobium forestalis sp. nov., an efficient nitrogen-fixing bacterium isolated from nodules of forest legume species in the Amazon.

Three strains of nitrogen-fixing bacteria isolated from nodules of Inga sp. (INPA54BT) and Swartzia sp. (INPA86A and INPA01-91A) in soils under native forest in the Brazilian Amazon were previously identified as belonging to the Bradyrhizobium genus. In this study, these strains were characterized using a polyphasic approach to establish their taxonomic position. The three strains shared more than 99.5% sequence similarity of the 16S rRNA gene with the type strains of five Bradyrhizobium species (B. japonicum USDA 6T, B. liaoningense LMG 18230T, B. ottawaense OO99T, B. subterraneum 58 2-1T and B. yuanmingense LMG 21827T). However, multilocus sequence analysis of two (recA and glnII) or three (atpD, gyrB, and recA) housekeeping genes indicated that these three strains represent a new Bradyrhizobium species, which is closely related to B. subterraneum 58 2-1T and B. yuanmingense LMG 21827T. DNA-DNA hybridization values between INPA54BT and B. subterraneum 58 2-1T and B. yuanmingense LMG 21827T were only 41.5 and 30.9%, respectively. Phenotypic characterization also allowed the differentiation of the novel species from B. subterraneum 58 2-1T and B. yuanmingense LMG 21827T. In the phylogenetic analysis of the nodC and nifH genes, the three strains showed similar sequences that were divergent from those of type strains of all Bradyrhizobium species. We concluded that these strains represent a novel species, for which the name Bradyrhizobium forestalis is proposed, with INPA54BT (= LMG 10044T) as type strain. The G+C content in the DNA of INPA54BT is 63.7 mol%.

Bradyrhizobium brasilense sp. nov., a symbiotic nitrogen-fixing bacterium isolated from Brazilian tropical soils.

Four strains of rhizobia isolated from nodules of Vigna unguiculata (UFLA03-321T, UFLA03-320 and UFLA03-290) and Macroptilium atropurpureum (UFLA04-0212) in Brazilian soils were previously reported as a new group within the genus Bradyrhizobium. To determine their taxonomic position, these strains were characterized in this study using a polyphasic approach. The analysis of the 16S rRNA gene grouped the four strains with Bradyrhizobium pachyrhizi PAC48T. However, the concatenated sequence analysis of the two (recA and glnII) or three (atpD, gyrB and recA) housekeeping genes indicated that these strains represent a novel species of Bradyrhizobium, which is very closely related to B. pachyrhizi PAC48T and B. elkanii USDA 76T. Genomic relatedness analyses between the UFLA03-321T strain and B. elkanii USDA 76T and B. pachyrhizi PAC48T revealed an average nucleotide identity below 96% and values of estimated DNA-DNA hybridization below 70%, confirming that they represent genomically distinct species. Analysis of MALDI-TOF MS (Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) profiles and phenotypic characteristics also allowed differentiation of the novel species from its two neighboring species. In phylogenetic analysis of nodC and nifH genes, UFLA03-321T exhibited maximum similarity with B. tropiciagri CNPSo 1112T. The data suggest that these four UFLA strains represent a novel species, for which the name Bradyrhizobium brasilense sp. nov. is proposed, with UFLA03-321T (=LMG 29353 =CBAS645) as type strain. G + C content in the DNA of UFLA03-321T is 63.9 mol %.

Chelatococcus thermostellatus sp. nov., a new thermophile for bioplastic synthesis: comparative phylogenetic and physiological study.

The poly(3-hydroxybutyrate), PHB, accumulating thermophilic strain MW9(T), isolated from an aerobic organic waste treatment plant, was characterized by detailed physiological and phylogenetic studies. The strain is a Gram-stain-negative, rod shaped, non-spore forming member of Alphaproteobacteria. It shows optimum growth at 50 °C. Based on 16S rRNA gene sequence similarity, the strain together with five very similar isolates, was affiliated to the genus Chelatococcus (Ibrahim et al. in J Appl Microbiol 109:1579-1590, 2010). Rep-PCR genomic fingerprints and partial dnaK gene sequence also revealed that these isolates are very similar, but differ from other Chelatococcus type strains. The major fatty acids were similar to those of other strains of the genus Chelatococcus. DNA-DNA hybridization of strain MW9(T) with Chelatococcus species type strains revealed 11.0-47.7 % relatedness. G+C content of DNA was 67.1 mol%, which is comparable with the other strains of Chelatococcus species. The physiological and phenotypic characteristics of the new strain MW9(T) are sufficient to differentiate it from previously described species in the genus Chelatococcus. Strain MW9(T) is considered to represent a novel species of the genus Chelatococcus, for which the name Chelatococcus thermostellatus is proposed. The type strain is MW9(T) (=LMG 27009(T) = DSM 28244(T)). Compared to known Chelatococcus strains, strain MW9(T) could be a potent candidate for bioplastic production at elevated temperature.

High diversity of Bradyrhizobium strains isolated from several legume species and land uses in Brazilian tropical ecosystems.

The genus Bradyrhizobium stands out among nitrogen-fixing legume-nodulating bacteria because it predominates among the efficient microsymbionts of forest, forage, and green manure legume species, as well as important species of grain legumes, such as soybean, cowpea, and peanut. Therefore, the diversity of Bradyrhizobium strains is a relevant resource from environmental and economic perspectives, and strains isolated from diverse legume species and land uses in Brazilian tropical ecosystems were assessed in this study. To accomplish this, sequences of four housekeeping genes (atpD, dnaK, gyrB, and recA) were individually analysed, with the first three also being considered using multilocus sequence analysis (MLSA). The sensitivity of the strains to different antibiotics, their tolerance to different levels of salinity, and their ability to nodulate soybean plants were also measured. The phylogenetic trees based on each individual gene, and on the concatenated housekeeping genes, revealed several strain clusters separated from any currently described species. The Bradyrhizobium strains studied were generally resistant to antibiotics. All strains were able to grow at salinity levels of up to 0.5% NaCl, whereas only strains UFLA03-142, UFLA03-143, UFLA03-145, and UFLA03-146 grew in the presence of 1% NaCl. Together, the results indicated that some of the strains studied were potential novel species, indicating that the various soils and ecosystems in Brazil may harbour an as yet unknown diversity of rhizobia.

Emended descriptions of Bacillus sporothermodurans and Bacillus oleronius with the inclusion of dairy farm isolates of both species.

Bacillus sporothermodurans is an industrially important micro-organism because of its ability to produce endospores which resist ultra-high temperature (UHT) and industrial sterilization processes. It was described by Pettersson et al. (1996) [Pettersson, B., Lembke, F., Hammer, P., Stackebrandt, E. & Priest, F. G. (1996). Int J Syst Bacteriol 46, 759-764] based on seven genetically homogeneous isolates all from UHT milk. Bacillus oleronius, the closest phylogenetic neighbour of B. sporothermodurans, was described by Kuhnigk et al. (1995) [Kuhnigk, T., Borst, E.-M., Breunig, A., König, H., Collins, M. D., Hutson, R. A. & Kämpfer, P. (1995). Can J Microbiol 41, 699-706] based on a single strain, isolated from the hindgut of the termite Reticulitermes santonensis. A polyphasic study of a heterogeneous collection of B. sporothermodurans and B. oleronius strains isolated from various sources and geographical origins led to an emended description of both species. Additional data presented are the results of fatty acid, quinone and/or cell wall (polar lipid) analyses. DNA-DNA hybridization confirmed 3 subgroups of strains obtained after SDS-PAGE analysis of cellular proteins as B. sporothermodurans. One named B. sporothermodurans strain (R-7489) was reclassified as a Bacillus fordii strain. The phenotypic profiles of both species were rather heterogeneous, sometimes different from the original descriptions and did not differ in a large number of characteristics, although B. oleronius generally gave stronger reactions in its positive tests than did B. sporothermodurans; the variable and weak reactions for both organisms with some substrates blurred the distinction between the two. However, differences in polar lipid, SDS-PAGE and menaquinone profiles clearly allow distinction between the two species.

Taxonomic revision of the genus Geobacillus: emendation of Geobacillus, G. stearothermophilus, G. jurassicus, G. toebii, G. thermodenitrificans and G. thermoglucosidans (nom. corrig., formerly 'thermoglucosidasius'); transfer of Bacillus thermantarcticus to the genus as G. thermantarcticus comb. nov.; proposal of Caldibacillus debilis gen. nov., comb. nov.; transfer of G. tepidamans to Anoxybacillus as A. tepidamans comb. nov.; and proposal of Anoxybacillus caldiproteolyticus sp. nov.

Sixty-two strains of thermophilic aerobic endospore-forming bacteria were subjected to polyphasic taxonomic study including 16S rRNA gene sequence analysis, polar lipid and fatty acid analysis, phenotypic characterization, and DNA-DNA hybridization experiments. Distinct clusters of the species Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus toebii and Geobacillus thermoglucosidasius were formed, allowing their descriptions to be emended, and the distinctiveness of the poorly represented species Geobacillus jurassicus, Geobacillus subterraneus and Geobacillus caldoxylosilyticus was confirmed. It is proposed that the name Geobacillus thermoglucosidasius be corrected to Geobacillus thermoglucosidans nom. corrig. Bacillus thermantarcticus clustered between Geobacillus species on the basis of 16S rRNA gene sequence analysis, and its transfer to the genus Geobacillus as Geobacillus thermantarcticus comb. nov. (type strain LMG 23032(T)=DSM 9572(T)=strain M1(T)=R-35644(T)) is proposed. The above-mentioned species, together with Geobacillus thermoleovorans and Geobacillus thermocatenulatus, form a monophyletic cluster representing the genus Geobacillus. The distinctiveness of 'Geobacillus caldoproteolyticus' was confirmed and it is proposed that it be accommodated, along with Geobacillus tepidamans, in the genus Anoxybacillus as Anoxybacillus caldiproteolyticus sp. nov. (type strain DSM 15730(T)=ATCC BAA-818(T)=LMG 26209(T)=R-35652(T)) and Anoxybacillus tepidamans comb. nov. (type strain LMG 26208(T)=ATCC BAA-942(T)=DSM 16325(T)=R-35643(T)), respectively. The type strain of Geobacillus debilis was not closely related to any members of the genera Anoxybacillus and Geobacillus, and it is proposed that this species be placed in the new genus Caldibacillus as Caldibacillus debilis gen. nov. comb. nov. The type strain of the type species, Caldibacillus debilis, is LMG 23386(T) (=DSM 16016(T)=NCIMB 13995(T)=Tf(T)=R-35653(T)).

Acidovorax caeni sp. nov., a denitrifying species with genetically diverse isolates from activated sludge.

Four Gram-negative, rod-shaped, non-spore-forming, denitrifying isolates were obtained from the activated sludge of an aerobic-anaerobic wastewater treatment plant in Belgium. Analysis of repetitive sequence-based PCR showed that the four isolates were genetically different from each other. Results of 16S rRNA gene sequence analysis and DNA-DNA hybridization experiments indicated that these four isolates were affiliated to the genus Acidovorax and could be differentiated from all recognized species of the genus. Analysis of whole-cell proteins and results of physiological and biochemical tests allowed differentiation of the new isolates from their closest phylogenetic neighbours. These new isolates therefore represent a novel species of the genus Acidovorax, for which the name Acidovorax caeni sp. nov. is proposed. The type strain is R-24608(T) (=LMG 24103(T) =DSM 19327(T)).

Stenotrophomonas terrae sp. nov. and Stenotrophomonas humi sp. nov., two nitrate-reducing bacteria isolated from soil.

Three Gram-negative, rod-shaped, non-spore-forming, nitrate-reducing isolates (R-32746, R-32768(T) and R-32729(T)) were obtained from soil. Analysis of repetitive sequence-based PCR showed that the three isolates represented two different strains. 16S rRNA gene sequence analysis and DNA-DNA hybridization placed them within the genus Stenotrophomonas and revealed that they were genotypically different from each other and from all recognized Stenotrophomonas species. Analysis of the fatty acid composition and physiological and biochemical tests allowed differentiation from their closest phylogenetic neighbours. They are therefore considered to represent two novel species, for which the names Stenotrophomonas terrae sp. nov. and Stenotrophomonas humi sp. nov. are proposed, with strains R-32768(T) (=LMG 23958(T)=DSM 18941(T)) and R-32729(T) (=LMG 23959(T)=DSM 18929(T)), respectively, as the type strains.

Pseudomonas peli sp. nov. and Pseudomonas borbori sp. nov., isolated from a nitrifying inoculum.

Sixteen Gram-negative, rod-shaped, non-spore-forming isolates were obtained from a nitrifying inoculum. Analysis of repetitive sequence-based PCR and SDS-PAGE banding patterns, 16S rRNA gene sequence analysis and DNA-DNA hybridizations showed that the isolates belonged to various groups within the genus Pseudomonas. One group of isolates could be assigned to Pseudomonas migulae and a second to Pseudomonas veronii. Two groups could be differentiated genotypically from each other and from all other currently known Pseudomonas species. Analysis of the fatty acid composition and physiological and biochemical tests allowed differentiation of these groups from their closest phylogenetic neighbours and they therefore represent two novel species within the genus Pseudomonas, for which the names Pseudomonas peli sp. nov. and Pseudomonas borbori sp. nov. are proposed, with strains LMG 23201(T) (=DSM 17833(T)=R-20805(T)) and LMG 23199(T) (=DSM 17834(T)=R-20821(T)), respectively, as the type strains.

Bacillus ruris sp. nov., from dairy farms.

Four novel ellipsoidal spore-forming Bacillus isolates with swollen sporangia, isolated from raw milk and feed concentrate, showed a high level of similarity in SDS-PAGE, fatty acid methyl esters and routine phenotypic tests. However, 16S rRNA gene sequence comparisons showed that this taxon was different from other related Bacillus species, and only a low level of DNA relatedness was found with the closest phylogenetic and phenotypic relative, Bacillus galactosidilyticus. This taxon could be differentiated from B. galactosidilyticus on the basis of morphological differences, stronger acid reactions with a wide range of substrates after 48 h incubation, and qualitative and quantitative differences in fatty acid content. On the basis of these data, a novel species, Bacillus ruris sp. nov., is proposed, with LMG 22866T (=DSM 17057T) as the type strain.