Veronia nyctiphanis gen. nov., sp. nov., Isolated from the Stomach of the Euphausiid Nyctiphanes simplex (Hansen, 1911) in the Gulf of California, and Reclassification of Enterovibrio pacificus as Veronia pacifica comb. nov.

The bacterial strain 42Xb2 T was isolated from a female adult krill Nyctiphanes simplex infected with the apostome parasitoid ciliate Pseudocollinia brintoni in January 2007 in the Gulf of California. The strain has the morphological, phenotypic, and molecular characteristics of the bacteria of the family Vibrionaceae. The 16S rRNA gene sequence has a similarity of 97.7% with Enterovibrio pacificus SW014 T and 96.1% similarity with Enterovibrio norvegicus LMG 19839 T. A phylogenomic and a multilocus sequence analyses placed this strain close to the genera Enterovibrio, Grimontia, and Salinivibrio, but clearly forming a separate branch from these bacterial genera. Genomic analyses presented further support this result. A novel genus Veronia gen. nov. and a species Veronia nyctiphanis sp. nov. is here described with CAIM 600 T (= DSM 24592 T = CECT 7578 T) as the type strain. Morphological, physiological, and genetic evidence presented here support the unification of Enterovibrio pacificus and Veronia nyctiphanis in the new genus Veronia. Enterovibrio pacificus is reclassified as Veronia pacifica. V. pacifica is assigned as the type species of the new genus Veronia.Genome Sequencing Data The GenBank/EMBL/DDBJ accession numbers for the genome sequence of Veronia nyctiphanis CAIM 600 T is PEIB01 and of Enterovibrio pacificus CAIM 1920 T is LYBM01. The 16S rRNA gene sequence of V. nyctiphanis CAIM 600 T is JX129353.

Synchronous termination of replication of the two chromosomes is an evolutionary selected feature in Vibrionaceae.

Vibrio cholerae, the causative agent of the cholera disease, is commonly used as a model organism for the study of bacteria with multipartite genomes. Its two chromosomes of different sizes initiate their DNA replication at distinct time points in the cell cycle and terminate in synchrony. In this study, the time-delayed start of Chr2 was verified in a synchronized cell population. This replication pattern suggests two possible regulation mechanisms for other Vibrio species with different sized secondary chromosomes: Either all Chr2 start DNA replication with a fixed delay after Chr1 initiation, or the timepoint at which Chr2 initiates varies such that termination of chromosomal replication occurs in synchrony. We investigated these two models and revealed that the two chromosomes of various Vibrionaceae species terminate in synchrony while Chr2-initiation timing relative to Chr1 is variable. Moreover, the sequence and function of the Chr2-triggering crtS site recently discovered in V. cholerae were found to be conserved, explaining the observed timing mechanism. Our results suggest that it is beneficial for bacterial cells with multiple chromosomes to synchronize their replication termination, potentially to optimize chromosome related processes as dimer resolution or segregation.

Serratia vespertilionis (García-Fraile et al. 2015) is a later heterotypic synonym of Serratia ficaria (Grimont et al. 1981).

A previous 16S rRNA gene sequence comparison had demonstrated that the type strains of Serratia vespertilionis and Serratia ficaria shared 99.5 % sequence similarity. Despite the 56.2 % homology by DNA-DNA hybridization previously found between these strains, the results of an in silico whole-genome sequence comparison and a new DNA-DNA hybridization study have clearly demonstrated that the genomes of the type strain of S. vespertilionis deposited in different Culture Collections (52T=CECT 8595T=DSM 28727T) and the type strain of S. ficaria (culture DSM 4569T), cannot support such a species differentiation. Tests for substrate utilization redone on the deposited cultures of these strains has also shown very few differences between the type strains of both species. Based on these results, and since the name S. ficaria was validly published earlier, S. vespertilionis should be considered as a later heterotypic synonym of S. ficaria, in application of the priority rule. The type strain of the species S. ficaria is strain 4024T=DSM 4569T=NCTC 12148T=ATCC 33105T=CIP 79.23T=ICPB 4050T.

Polynucleobacter paneuropaeus sp. nov., characterized by six strains isolated from freshwater lakes located along a 3000 km north-south cross-section across Europe.

Six Polynucleobacter (Burkholderiaceae, Betaproteobacteria) strains isolated from different freshwater lakes located across Europe were taxonomically investigated. Phylogenetic analyses based on 16S rRNA gene sequences assigns all six strains to the cryptic species complex PnecC within the genus Polynucleobacter. Analyses of partial glutamine synthetase (glnA) genes suggests that all six strains belong to the species-like taxon designated F15 in previous papers. Comparative genome analyses reveal that the six strains form a genomically coherent group characterized by whole-genome average nucleotide identity (gANI) values of >98 % but separated by gANI values of <88 % from the type strains and representatives of the 16 previously described Polynucleobacter species. In phylogenetic analyses based on nucleotide sequences of 319 orthologous genes, the six strains represent a monophyletic cluster that is clearly separated from all other described species. Genome sizes of the six strains range from 1.61 to 1.83 Mbp, which is smaller than genome sizes of the majority of type strains representing previously described Polynucleobacter species. By contrast, the G+C content of the DNA of the strains is well in the range of 44.8-46.6 mol% previously found for other type strains of species affiliated with the subgroup PnecC. Variation among the six strains representing the new species is evident in a number of traits. These include gene content differences, for instance regarding a gene cluster encoding anoxygenic photosynthesis, as well as phenotypic traits. We propose to name the new species represented by the six strains Polynucleobacter paneuropaeus sp. nov. and designate strain MG-25-Pas1-D2T (=DSM 103454T =CIP 111323T) as the type strain.

Polynucleobacter hirudinilacicola sp. nov. and Polynucleobacter campilacus sp. nov., both isolated from freshwater systems.

Strains MWH-EgelM1-30-B4T and MWH-Feld-100T were isolated from the water columns of two freshwater systems. Both strains represent delicate bacteria not easy to work with in laboratory experiments. Phylogenetic analyses of the 16S rRNA genes suggested that both strains were affiliated with the genus Polynucleobacter. Both strains share 16S rRNA gene sequence similarities of >99 % with eight free-living Polynucleobacter type strains, all affiliated with the cryptic species complex PnecC. The full-length 16S rRNA gene sequences of the two strains differ only in two and three positions, respectively, from the sequence of the closest related Polynucleobacter type strain. Genome sequencing of both strains revealed relatively small genome sizes of 2.0 Mbp and G+C contents of 45 mol%. Phylogenetic analyses based on nucleotide sequences of 319 shared protein-encoding genes consistently placed the two strains in taxon PnecC but did not suggest an affiliation with one of the previously described species. Pairwise analyses of whole genome average nucleotide identities (gANI) with representatives of all previously described Polynucleobacter species resulted in both cases throughout in values <80 %. Pairwise comparison of the genomes of the two new strains resulted in gANI values of 83.3 %. All gANI analyses clearly suggested that strains MWH-EgelM1-30-B4T and MWH-Feld-100T represent two novel Polynucleobacter species. We propose for these novel species the names Polynucleobacter hirudinilacicola sp. nov. and Polynucleobacter campilacus sp. nov. and strains MWH-EgelM1-30-B4T (=DSM 23911T=LMG 30144T) and MWH-Feld-100T (=DSM 24007T=LMG 29705T) as the type strains, respectively.

Polynucleobacter meluiroseus sp. nov., a bacterium isolated from a lake located in the mountains of the Mediterranean island of Corsica.

Strain AP-Melu-1000-B4 was isolated from a lake located in the mountains of the Mediterranean island of Corsica (France). Phenotypic, chemotaxonomic and genomic traits were investigated. Phylogenetic analyses based on 16S rRNA gene sequencing referred the strain to the cryptic species complex PnecC within the genus Polynucleobacter. The strain encoded genes for biosynthesis of proteorhodopsin and retinal. When pelleted by centrifugation the strain showed an intense rose colouring. Major fatty acids were C16 : 1ω7c, C16 : 0, C18 : 1ω7c and summed feature 2 (C16 : 1 isoI and C14 : 0-3OH). The sequence of the 16S rRNA gene contained an indel which was not present in any previously described Polynucleobacter species. Genome sequencing revealed a genome size of 1.89 Mbp and a G+C content of 46.6 mol%. In order to resolve the phylogenetic position of the new strain within subcluster PnecC, its phylogeny was reconstructed from sequences of 319 shared genes. To represent all currently described Polynucleobacter species by whole genome sequences, three type strains were additionally sequenced. Our phylogenetic analysis revealed that strain AP-Melu-100-B4 occupied a basal position compared with previously described PnecC strains. Pairwise determined whole genome average nucleotide identity (gANI) values suggested that strain AP-Melu-1000-B4 represents a new species, for which we propose the name Polynucleobacter meluiroseus sp. nov. with the type strain AP-Melu-1000-B4T (=DSM 103591T=CIP 111329T).

Polynucleobacter sphagniphilus sp. nov. a planktonic freshwater bacterium isolated from an acidic and humic freshwater habitat.

Strain MWH-Weng1-1T, isolated from an acidic freshwater habitat located in the Wenger Moor, Austria, was characterized by investigating its phenotypic, chemotaxonomic and genomic traits. Phylogenetic analyses based on 16S rRNA gene sequencing placed the strain in the cryptic species complex PnecC within the genus Polynucleobacter. The strain had a genome of 2.04 Mbp with a G+C content of 45.6 mol%. The major fatty acids of the strain were C16 : 1ω7c, C16 : 0 and C18 : 1ω7c. In order to resolve the systematic position of the strain within the species complex PnecC, concatenated partial sequences of eight housekeeping genes were used for phylogenetic analyses. The obtained trees did not place strain MWH-Weng1-1T close to any of the six previously described species within this cryptic species complex. Pairwise whole genome average nucleotide identity comparisons with genome sequences of strains representing the six previously described species of the subcluster resulted throughout in values <78 %, which clearly suggested that strain MWH-Weng1-1T (DSM 24018T=CIP 111099T) represents a novel species. We propose the name Polynucleobacter sphagniphilus sp. nov. and strain MWH-Weng1-1T as the type strain for this new species.

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

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

Polynucleobacter wuianus sp. nov., a free-living freshwater bacterium affiliated with the cryptic species complex PnecC.

Strain QLW-P1FAT50C-4T, isolated from a shallow, acidic freshwater pond located in the Austrian Alps at an altitude of 1300 m, was characterized by investigation of phenotypic, chemotaxonomic and genomic traits. As shown previously, phylogenetic analyses based on 16S rRNA gene sequences placed the strain in the cryptic species complex PnecC within the genus Polynucleobacter. The major fatty acids of the strain were C16 : 1ω7c and C18 : 1ω7c. The strain has a genome of 2.23 Mbp with a DNA G+C content of 44.9 mol%. The strain encodes a seemingly complete gene cluster for anoxygenic photosynthesis but lacks typical genes for CO2 assimilation. In order to resolve the phylogenetic position of the strain within the species complex PnecC, concatenated partial sequences of eight housekeeping genes were analysed. The phylogenetic reconstruction obtained did not place strain QLW-P1FAT50C-4T close to any of the five previously described species within subcluster PnecC. Pairwise average nucleotide identity (ANI) comparisons of whole-genome sequences suggested that strain QLW-P1FAT50C-4T (=DSM 24008T=CIP 111100T) represents a novel species, for which we propose the name Polynucleobacter wuianus sp. nov.

Silvanigrella aquatica gen. nov., sp. nov., isolated from a freshwater lake, description of Silvanigrellaceae fam. nov. and Silvanigrellales ord. nov., reclassification of the order Bdellovibrionales in the class Oligoflexia, reclassification of the families Bacteriovoracaceae and Halobacteriovoraceae in the new order Bacteriovoracales ord. nov., and reclassification of the family Pseudobacteriovoracaceae in the order Oligoflexales.

The unusual chemo-organoheterotrophic proteobacterial strain MWH-Nonnen-W8redT was isolated from a lake located in the Black Forest (Schwarzwald), Germany, by using the filtration-acclimatization method. Phylogenetic analyses based on the 16S rRNA gene sequence of the strain could not provide clear hints on classification of the strain in one of the current classes of the phylum Proteobacteria. Whole-genome sequencing resulted in a genome size of 3.5 Mbp and revealed a quite low DNA G+C content of 32.6 mol%. In-depth phylogenetic analyses based on alignments of 74 protein sequences of a phylogenetically broad range of taxa suggested assignment of the strain to a new order of the class Oligoflexia. These analyses also suggested that the order Bdellovibrionales should be transferred from the class Deltaproteobacteria to the class Oligoflexia, that this order should be split into two orders, and that the family Pseudobacteriovoracaceae should be transferred from the order Bdellovibrionales to the order Oligoflexales. We propose to establish for strain MWH-Nonnen-W8redT (=DSM 23856T=CCUG 58639T) the novel species and genus Silvanigrella aquatica gen. nov., sp. nov. to be placed in the new family Silvanigrellaceae fam. nov. of the new order Silvanigrellales ord. nov.

Reclassification of four Polynucleobacter necessarius strains as representatives of Polynucleobacter asymbioticus comb. nov., Polynucleobacter duraquae sp. nov., Polynucleobacter yangtzensis sp. nov. and Polynucleobacter sinensis sp. nov., and emended description of Polynucleobacter necessarius.

Genome comparisons based on average nucleotide identity (ANI) values of four strains currently classified as Polynucleobacter necessarius subsp. asymbioticus resulted in ANI values of 75.7-78.4 %, suggesting that each of those strains represents a separate species. The species P. necessarius was proposed by Heckmann and Schmidt in 1987 to accommodate obligate endosymbionts of ciliates affiliated with the genus Euplotes. The required revision of this species is, however, hampered by the fact, that this species is based only on a description and lacks a type strain available as pure culture. Furthermore, the ciliate culture Euplotes aediculatus ATCC 30859, on which the description of the species was based, is no longer available. We found another Euplotes aediculatus culture (Ammermann) sharing the same origin with ATCC 30859 and proved the identity of the endosymbionts contained in the two cultures. A multilocus sequence comparison approach was used to estimate if the four strains currently classified as Polynucleobacternecessarius subsp. asymbioticus share ANI values with the endosymbiont in the Ammermann culture above or below the threshold for species demarcation. A significant correlation (R2 0.98, P<0.0001) between multilocus sequence similarity and ANI values of genome-sequenced strains enabled the prediction that it is highly unlikely that these four strains belong to the species P. necessarius. We propose reclassification of strains QLW-P1DMWA-1T (=DSM 18221T=CIP 109841T), MWH-MoK4T (=DSM 21495T=CIP 110977T), MWH-JaK3T (=DSM 21493T=CIP 110976T) and MWH-HuW1T (=DSM 21492T=CIP 110978T) as Polynucleobacter asymbioticus comb. nov., Polynucleobacter duraquae sp. nov., Polynucleobacter yangtzensis sp. nov. and Polynucleobacter sinensis sp. nov., respectively.

Reclassification of Angiococcus disciformis, Cystobacter minus and Cystobacter violaceus as Archangium disciforme comb. nov., Archangium minus comb. nov. and Archangium violaceum comb. nov., unification of the families Archangiaceae and Cystobacteraceae, and emended descriptions of the families Myxococcaceae and Archangiaceae.

The species Archangium gephyra, Angiococcus disciformis, Cystobacter minus and Cystobacter violaceus are currently classified in three different genera of the order Myxococcales. The 16S rRNA gene sequences of the respective type strains show a similarity higher than 98.4 % and form a tight phylogenetic group. A dendrogram calculating the similarity of MALDI-TOF spectra confirmed the close relatedness of the four species that grouped in a monophyletic cluster in the neighbourhood of other species of the genus Cystobacter. The type strains shared similar fatty acid patterns of high complexity with iso-C15 : 0, C16 : 1ω5c and iso-C14 : 0 3-OH as the major components. The vegetative cells of these species are uniformly long needle-shaped rods, and the myxospores are short rods, ovoid or irregularly spherical thus differing from the myxospores of species related to Cystobacter fuscus, the type species of this genus. Some enzymic and hydrolysing reactions of the type strains are described. As a result of the high relatedness and similarity of the four species, it is proposed to place them into one genus, and due to phylogenetic and morphological distinctness, the species should be classified in a genus distinct from the genus Cystobacter as Archangium gephyra (type strain M18T = DSM 2261T = ATCC 25201T = NBRC 100087T), Archangium disciforme comb. nov. (type strain CMU 1T = DSM 52716T = ATCC 33172T), Archangium minus comb. nov. (proposed neotype strain Cb m2 = DSM 14751 = JCM 12627) and Archangium violaceum comb. nov. (type strain Cb vi61T = DSM 14727T = CIP 109131T = JCM 12629T). Since the family ArchangiaceaeJahn 1924 AL has priority over the family CystobacteraceaeMcCurdy 1970 AL, it is proposed to assign the genera Archangium, Anaeromyxobacter, Cystobacter, Hyalangium, Melittangium and Stigmatella to the family Archangiaceae. Emended descriptions of the families Myxococcaceae and Archangiaceae are also provided.

A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea.

Sequencing of bacterial and archaeal genomes has revolutionized our understanding of the many roles played by microorganisms. There are now nearly 1,000 completed bacterial and archaeal genomes available, most of which were chosen for sequencing on the basis of their physiology. As a result, the perspective provided by the currently available genomes is limited by a highly biased phylogenetic distribution. To explore the value added by choosing microbial genomes for sequencing on the basis of their evolutionary relationships, we have sequenced and analysed the genomes of 56 culturable species of Bacteria and Archaea selected to maximize phylogenetic coverage. Analysis of these genomes demonstrated pronounced benefits (compared to an equivalent set of genomes randomly selected from the existing database) in diverse areas including the reconstruction of phylogenetic history, the discovery of new protein families and biological properties, and the prediction of functions for known genes from other organisms. Our results strongly support the need for systematic 'phylogenomic' efforts to compile a phylogeny-driven 'Genomic Encyclopedia of Bacteria and Archaea' in order to derive maximum knowledge from existing microbial genome data as well as from genome sequences to come.

Improving survival and storage stability of bacteria recalcitrant to freeze-drying: a coordinated study by European culture collections.

The objective of this study is to improve the viability after freeze-drying and during storage of delicate or recalcitrant strains safeguarded at biological resource centers. To achieve this objective, a joint experimental strategy was established among the different involved partner collections of the EMbaRC project ( www.embarc.eu ). Five bacterial strains considered as recalcitrant to freeze-drying were subjected to a standardized freeze-drying protocol and to seven agreed protocol variants. Viability of these strains was determined before and after freeze-drying (within 1 week, after 6 and 12 months, and after accelerated storage) for each of the protocols. Furthermore, strains were exchanged between partners to perform experiments with different freeze-dryer-dependent parameters. Of all tested variables, choice of the lyoprotectant had the biggest impact on viability after freeze-drying and during storage. For nearly all tested strains, skim milk as lyoprotectant resulted in lowest viability after freeze-drying and storage. On the other hand, best freeze-drying and storage conditions were strain and device dependent. For Aeromonas salmonicida CECT 894(T), best survival was obtained when horse serum supplemented with trehalose was used as lyoprotectant, while Aliivibrio fischeri LMG 4414(T) should be freeze-dried in skim milk supplemented with marine broth in a 1:1 ratio. Freeze-drying Campylobacter fetus CIP 53.96(T) using skim milk supplemented with trehalose as lyoprotectant resulted in best recovery. Xanthomonas fragariae DSM 3587(T) expressed high viability after freeze-drying and storage for all tested lyoprotectants and could not be considered as recalcitrant. In contrary, Flavobacterium columnare LMG 10406(T) did not survive the freeze-drying process under all tested conditions.

Zunongwangia mangrovi sp. nov., isolated from mangrove (Avicennia marina) rhizosphere, and emended description of the genus Zunongwangia.

A Gram-stain-negative, strictly aerobic, slightly halophilic, non-motile and rod-shaped bacterial strain, designated P2E16(T), was isolated from mangrove (Avicennia marina) rhizosphere, collected at Devipattinam mangroves, Tamil Nadu, India. Strain P2E16(T) grew optimally at pH 7.0-8.0, at 25-28 °C and in the presence of 2-3% (w/v) NaCl. 16S rRNA gene analysis showed that strain P2E16(T) was phylogenetically closely related to the genus Zunongwangia, with Zunongwangia profunda SM-A87(T) as the closest related type strain (98.2% 16S rRNA gene sequence similarity) and less than 93% 16S rRNA gene sequence similarity to all other members of the family Flavobacteriaceae. Strain P2E16(T) contained MK-6 as the major respiratory quinone, phosphatidylethanolamine as the predominant polar lipid and iso-C(15 : 0) (17.8%), iso-C(17 : 0) 3-OH (15.1%), C(15 : 0) (12.8%), iso-C(17 : 1)ω9c (9.8%), iso-C(15 : 1) G (9.0%), and summed feature 3 (comprising C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH; 7.1%) as the major fatty acids. The DNA G+C content was 34.3 mol%. Differential phenotypic properties, together with the phylogenetic distinctiveness and low DNA-DNA relatedness demonstrated that strain P2E16(T) was distinct from the type strain of Zunongwangia profunda. On the basis of these presented data, strain P2E16(T) is considered to represent a novel species of the genus Zunongwangia, for which the name Zunongwangia mangrovi sp. nov. is proposed. The type strain is P2E16(T) ( = DSM 24499(T) = LMG 26237(T) = KCTC 23496(T)). An emended description of the genus Zunongwangia is also provided.

Designation of type strains for seven species of the order Myxococcales and proposal for neotype strains of Cystobacter ferrugineus, Cystobacter minus and Polyangium fumosum.

Ten species of the order Myxococcales with validly published names are devoid of living type strains. Four species of the genus Chondromyces are represented by dead herbarium samples as the type material. For a species of the genus Melittangium and two species of the genus Polyangium, no physical type material was assigned at the time of validation of the names or later on. In accordance with rule 18f of the International Code of Nomenclature of Bacteria the following type strains are designated for these species: strain Cm a14(T) ( = DSM 14605(T) = JCM 12615(T)) as the type strain of Chondromyces apiculatus, strain Cm c5(T) ( = DSM 14714(T) = JCM 12616(T)) as the type strain of Chondromyces crocatus, strain Sy t2(T) ( = DSM 14631(T) = JCM 12617(T)) as the type strain of Chondromyces lanuginosus, strain Cm p51(T) ( = DSM 14607(T) = JCM 12618(T)) as the type strain of Chondromyces pediculatus, strain Me b8(T) ( = DSM 14713(T) = JCM 12633(T)) as the type strain of Melittangium boletus, strain Pl s12(T) ( = DSM 14670(T) = JCM 12637(T)) as the type strain of Polyangium sorediatum and strain Pl sm5(T) ( = DSM 14734(T) = JCM 12638(T)) as the type strain of Polyangium spumosum. Furthermore, the type strains given for three species of the genera Cystobacter and Polyangium had been kept at one university institute and have been lost according to our investigations. In accordance with Rule 18c of the Bacteriological Code, we propose the following neotype strains: strain Cb fe18 ( = DSM 14716  = JCM 12624) as the neotype strain of Cystobacter ferrugineus, strain Cb m2 ( = DSM 14751 = JCM 12627) as the neotype strain of Cystobacter minus and strain Pl fu5 ( = DSM 14668 = JCM 12636) as the neotype strain of Polyangium fumosum. The proposals of the strains are based on the descriptions and strain proposals given in the respective chapters of Bergey's Manual of Systematic Bacteriology (2005).

Azorhizobium oxalatiphilum sp. nov., and emended description of the genus Azorhizobium.

A gram-negative, motile, non-spore-forming rod, designated NS12(T), was isolated from macerated petioles of Rumex sp. after enrichment with oxalate. On the basis of 16S rRNA gene sequence similarity, strain NS12(T) was phylogenetically related to the genera Azorhizobium and Xanthobacter in the class Alphaproteobacteria. Strain NS12(T) was most closely related to Azorhizobium doebereinerae BR 5401(T) and Azorhizobium caulinodans ORS 571(T) (98.3 and 97.3 % 16S rRNA gene sequence similarity, respectively). Membership of the genus Xanthobacter was excluded by phenotypic characterization. The whole-cell fatty acid compositions of the isolate was typical of members of the genus Azorhizobium with C18 : 1ω7c, cyclo-C19 : 0ω8c, 11-methyl-C18 : 1ω7c and C16 : 0 as the main components. The results of DNA-DNA hybridization and physiological tests allowed the genotypic and phenotypic differentiation of strain NS12(T) from the two members of the genus Azorhizobium. Therefore it is concluded that the isolate represents a novel species of the genus Azorhizobium, for which the name Azorhizobium oxalatiphilum sp. nov. is proposed. The type strain is NS12(T) ( = DSM 18749(T) = CCM 7897(T)). The description of the genus Azorhizobium is also emended.

Budvicia diplopodorum sp. nov. and emended description of the genus Budvicia.

A Gram-negative, rod-shaped, weakly motile, non-spore-forming bacterium (D9(T)) was isolated from the gut of Cylindroiulus fulviceps (Diplopoda) on 1/3-strength nutrient agar plates. On the basis of 16S rRNA gene sequence similarity, strain D9(T) was shown to be phylogenetically closely related to the type strain of Budvicia aquatica, the sole species of the genus Budvicia, family Enterobacteriaceae. The similarity of the 16S rRNA gene sequences of strain D9(T) and B. aquatica DSM 5075(T) was 98.4 %. Other strains that showed high pairwise similarities with the isolate belonged to the genus Yersinia: Y. frederiksenii ATCC 33641(T) (96.8 % 16S rRNA gene sequence similarity), Y. massiliensis CCUG 53443(T) (96.8 %), Y. pestis NCTC 5923(T) (96.8 %), Y. pseudotuberculosis ATCC 29833(T) (96.8 %), Y. similis CCUG 52882(T) (96.7 %) and Y. ruckeri ATCC 29473(T) (96.5 %). The similarities of sequences of the housekeeping genes rpoB, hsp60 and gyrB between strain D9(T) and B. aquatica DSM 5075(T) and other members of the Enterobacteriaceae were less than 94 %. Phylogenetic trees based on all four gene sequences unequivocally grouped the isolate with the type strain of B. aquatica and separately from the genus Yersinia. Cells contained the quinones Q-8, Q-7 and MK-8. The major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. The G+C content of the DNA (48.3 mol%) and the whole-cell fatty acid composition of strain D9(T) (C(14 : 0), C(16 : 1)ω7c, C(16 : 0), cyclo-C(17 : 0) and C(18 : 1)ω7c as major components) were typical for members of the Enterobacteriaceae. DNA-DNA hybridization of strain D9(T) with B. aquatica DSM 5075(T) resulted in a relatedness of 30.4 %, indicating that the isolate did not belong to B. aquatica. Physiological tests allowed the phenotypic differentiation of strain D9(T) from B. aquatica DSM 5075(T) as well as from members of the genus Yersinia. From these results, it is concluded that strain D9(T) represents a novel species, for which the name Budvicia diplopodorum sp. nov. is proposed (type strain D9(T) =DSM 21983(T) = CCM 7845(T)). The description of the genus Budvicia is emended.

Polynucleobacter difficilis sp. nov., a planktonic freshwater bacterium affiliated with subcluster B1 of the genus Polynucleobacter.

Strain AM-8B5(T), isolated from Lake Sevan in Armenia, was characterized phenotypically, chemotaxonomically and phylogenetically. This chemo-organoheterotrophic, aerobic, facultatively anaerobic, catalase- and oxidase-positive, non-motile strain grew on NSY medium at NaCl concentrations of 0.0-0.2 % (w/v) and at 4-30 °C. Whole-cell fatty acids were dominated by summed feature 3 (including C(16 : 1)ω7c and iso-C(15 : 0) 2-OH), C(16 : 0) and C(18 : 1)ω7c. C(12 : 0) 2-OH and C(16 : 1) 2-OH were the only hydroxylated fatty acids detected. Phylogenetic analysis as well as phenotypic and chemotaxonomic similarities indicated that the novel isolate was affiliated with the genus Polynucleobacter. 16S rRNA gene similarity values with the four previously described Polynucleobacter species ranged from 96.2 to 98.7 %. DNA-DNA hybridization experiments showed that the isolate did not belong to any of the previously described Polynucleobacter species. The isolate could be distinguished from all previously established Polynucleobacter species based on chemotaxonomic and phenotypic traits. The bacterium possessed a free-living lifestyle and represents a group of bacteria inhabiting the water column of many freshwater lakes. Based on the revealed phylogeny, and chemotaxonomic and phenotypic differences to previously described Polynucleobacter species, it is proposed that the isolate represents a novel species, Polynucleobacter difficilis sp. nov.; the type strain is AM-8B5(T) ( = DSM 22349(T) = CIP 110078(T)).

Vibrio alfacsensis sp. nov., isolated from marine organisms.

Five strains (CAIM 1831(T), CAIM 1832, CAIM 1833, CAIM 1834 and CAIM 1836) were isolated from cultured sole (Solea senegalensis) in two regions of Spain, two strains (CAIM 404 and CAIM 1294) from wild-caught spotted rose snapper (Lutjanus guttatus) in Mexico, and one strain (CAIM 1835) from corals in Brazil. The 16S rRNA gene sequences of the novel isolates showed similarity to Vibrio ponticus (98.2-98.3%, GenBank accession no. AJ630103) and to a lesser degree to Vibrio furnissii (97.2-97.3%, X76336) and to Vibrio fluvialis (96.9-97.1%, X74703). Multilocus sequence analysis clustered these strains closely together and clearly separated them from phylogenetically related species of the genus Vibrio. Genomic fingerprinting by rep-PCR clustered the novel strains according to their geographical origin. Phenotypic analyses showed a large variation among the new strains, but many tests enabled them to be differentiated from other species of the genus Vibrio. The mean ΔT(m) values between the strains analysed here and closely related type strains were above 6.79 °C. The values between the novel isolates were below 2.35 °C, well outside the limit suggested for the delineation of a bacterial species. The phenotypic and genotypic data presented here clearly place these new strains as a coherent group within the genus Vibrio, for which we propose the name Vibrio alfacsensis sp. nov. with CAIM 1831(T) ( = DSM 24595(T) = S277(T)) as the type strain.