Dissection of the genus Actinobaculum: Reclassification of Actinobaculum schaalii Lawson et al. 1997 and Actinobaculum urinale Hall et al. 2003 as Actinotignum schaalii gen. nov., comb. nov. and Actinotignum urinale comb. nov., description of Actinotignum sanguinis sp. nov. and emended descriptions of the genus Actinobaculum and Actinobaculum suis; and re-examination of the culture deposited as Actinobaculum massiliense CCUG 47753T ( = DSM 19118T), revealing that it does not represent a strain of this species.

The remarkable host specificity of the species of the genus Actinobaculum led us to recharacterize these species by a polyphasic approach. A comparative chemotaxonomic study including analysis of whole-cell sugars, amino acid composition of the peptidoglycan, fatty acid methyl esters, respiratory quinones and polar lipids revealed significant differences that, in combination with molecular data, support a dissection of the genus Actinobaculum. The proposals of this study include the reclassification of Actinobaculum schaalii and Actinobaculum urinale as Actinotignum schaalii gen. nov., comb. nov. (type strain DSM 15541(T) = CCUG 27420(T)) and Actinotignum urinale comb. nov. (type strain DSM 15805(T) = CCUG 46093(T)), respectively. Emended descriptions of the genus Actinobaculum and Actinomyces suis are also provided. The results of 16S rRNA gene sequence analysis and DNA-DNA hybridization also indicated that the type strain of Actinobaculum massiliense deposited as CCUG 47753(T) ( = DSM 19118(T)) should in fact be considered a member of the species Actinobaculum schaalii. In addition, comparative 16S rRNA gene sequencing and DNA-DNA relatedness studies of four strains recovered from clinical materials demonstrated that three of the isolates belonged to Actinotignum schaalii; the remaining strain represents a novel species, for which the name Actinotignum sanguinis sp. nov. is proposed. The type strain is IMMIB L-2199(T) ( = DSM 26039(T) = CCUG 64068(T)).

The status of the species Actinobaculum massiliense (Greub and Raoult 2006). Request for an Opinion.

A recent study on members of the genus Actinobaculum revealed that cultures of the species Actinobaculum massiliense CCUG 47753(T) ( = DSM 19118(T)) currently being distributed do not conform to the properties of the type strain of A. massiliense CIP 107404(T) given by Greub & Raoult [Greub, G. & Raoult, D. (2002). J Clin Microbiol 40, 3938-3941]. The original strain, CIP 107404(T) is no longer available from the Biological Resource Center of Institut Pasteur, Paris. Based on data currently available, the organism currently deposited as CCUG 47753(T) and DSM 19118(T) is a member of the species Actinobaculum schaalii. Clearly, the organism deposited as CCUG 47753(T) and DSM 19118(T) as the type strain of the species Actinobaculum massiliense does not have the properties given by Greub & Raoult. Based on the absence of an authentic type strain, the Judicial Commission is requested to examine the status of the name Actinobaculum massiliense Greub and Raoult 2006 and to issue an Opinion.

Reclassification of Koreibacter algae as a later heterotypic synonym of Paraoerskovia marina and emended descriptions of the genus Paraoerskovia Khan et al. 2009 and of Paraoerskovia marina Khan et al. 2009.

16S rRNA gene sequences deposited for the type strains of Paraoerskovia marina (CTT-37(T); GenBank accession no. AB445007) and Koreibacter algae (DSW-2(T); FM995611) show a similarity of 100 %. Consequently, the type strains were subjected to a polyphasic recharacterization under direct comparison in order to clarify their taxonomic position. PvuII RiboPrint patterns and quantitative ratios of cellular fatty acids revealed strain-specific differences between P. marina DSM 21750(T) ( = CTT-37(T)) and K. algae DSM 22126(T) ( = DSW-2(T)). The percentage of DNA-DNA binding of 94 % indicated that the two type strains belong to the same genomospecies. Agreement in the peptidoglycan structure and polar lipid pattern, highly similar fatty acid profiles and MALDI-TOF mass spectra, the ability to produce acid from the same carbon sources, corresponding enzymic activities and DNA G+C contents of 70.8 ± 0.3 mol%, in addition to the consistent characteristics reported in the original descriptions, support the view that the two strains should be affiliated to the same species. According to Rules 38 and 42 of the Bacteriological Code, Koreibacter algae should be reclassified as later heterotypic synonym of Paraoerskovia marina, and the descriptions of the genus Paraoerskovia Khan et al. 2009 and of Paraoerskovia marina Khan et al. 2009 are emended accordingly.

Psychrobacillus gen. nov. and proposal for reclassification of Bacillus insolitus Larkin & Stokes, 1967, B. psychrotolerans Abd-El Rahman et al., 2002 and B. psychrodurans Abd-El Rahman et al., 2002 as Psychrobacillus insolitus comb. nov., Psychrobacillus psychrotolerans comb. nov. and Psychrobacillus psychrodurans comb. nov.

The taxonomic status of three Bacillus species, Bacillus insolitus, B. psychrodurans and B. psychrotolerans was reexamined using a polyphasic approach. In our analysis, these three Bacillus species formed a cluster separate from other members of Bacillus rRNA group 2 [5] and from Bacillus sensu stricto. These three species shared high 16S rRNA gene sequence similarities between them (97.8-99.7%) and showed closest sequence similarity (95.3-96.3%) to Paenisporosarcina quisquiliarum gen. nov., sp. nov. [18]. Sequence similarities with other related genera ranged between 90.9% and 94.5%. Phylogenetic coherence of the three species was supported by phenotypic characteristics, such as growth at low temperatures, negative oxidation and assimilation of many carbohydrates, MK8 as the major isoprenoid quinine and broadly similar polar lipid profiles. All three species had a similar peptidoglycan type of the variation A4ß and similar genomic G+C contents (35.7-36.6 mol% [1]). Genomic relatedness among them was shown to be less than 70% and justified their separate species status [1]. These three species could be differentiated from each other and from related taxa on the basis of phenotypic, including chemotaxonomic, characteristics and ribotype patterns. On the basis of our analysis, we propose a new genus Psychrobacillus gen. nov. and to transfer B. insolitus, B. psychrodurans and B. psychrotolerans to the new genus as Psychrobacillus insolitus comb. nov. (type species of the genus; type strain W16B(T)=DSM 5(T)), P. psychrodurans comb. nov. (type strain 68E3(T)=DSM 11713(T)) and P. psychrotolerans comb. nov. (type strain 3H1(T)=DSM 11706(T)).

Arthrobacter phenanthrenivorans sp. nov., to accommodate the phenanthrene-degrading bacterium Arthrobacter sp. strain Sphe3.

A novel phenanthrene-degrading bacterium, designated strain Sphe3(T), was isolated from a creosote-contaminated soil in Greece. Cells were non-motile, Gram-positive, aerobic, and rod- to coccus-shaped. The strain was isolated on the basis of formation of a clear zone on agar plates sprayed with phenanthrene. Optimal growth occurred at 30 degrees C. The G+C content of the DNA was 65.7 mol%. The polar lipid pattern of strain Sphe3(T) consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The dominant fatty acids were iso-C(15 : 0), anteiso-C(15 : 0), iso-C(16 : 0), C(16 : 0) and anteiso-C(17 : 0), representing >86 % of the total fatty acids. The predominant isoprenoid quinone of strain Sphe3(T) was menaquinone-8 (MK-8). Based on 16S rRNA gene sequence analysis, strain Sphe3(T) showed 99 and 98.9 % similarity to the type strains of Arthrobacter oxydans and Arthrobacter polychromogenes, respectively. Strain Sphe3(T) showed 91 % similarity to homologues of A. oxydans and A. polychromogenes based on recA gene sequence analysis. Based on 16S rRNA and recA gene sequence analysis and DNA-DNA hybridization analysis, as well as physiological and chemotaxonomic characteristics, it is concluded that strain Sphe3(T) represents a novel species of the genus Arthrobacter, for which the name Arthrobacter phenanthrenivorans sp. nov. is proposed. The type strain is Sphe3(T) (=DSM 18606(T) =LMG 23796(T)).

Bacillus aeolius DSM 15084T (=CIP 107628T) is a strain of Bacillus licheniformis.

Based on phenotypic properties, 16S rRNA gene sequence and MALDI-TOF analysis, strains DSM 15084T and CIP 107628T, deposited as the type strain of Bacillus aeolius, do not represent the original type strain, strain 4-1T. It is therefore proposed that the Judicial Commission of the International Committee on Systematics of Prokaryotes places the name Bacillus aeolius on the list of rejected specific and subspecific epithets in names of species and subspecies of bacteria if a suitable replacement for the type strain or a neotype cannot be found within 2 years of publication of this Request.

Culture-independent analysis of bacterial species from an anaerobic mat from Lake Fryxell, Antarctica: prokaryotic diversity revisited.

Using a lake sediment mat sample from Lake Fryxell, Antarctica, different DNA extraction and purification methods were compared by denaturing gradient gel electrophoresis (DGGE). Based on the analyses of cloned 16S rRNA gene sequences a high degree of as yet uncultured prokaryotes have been reported in this sample. Although the vast majority of these as yet uncultured organisms seem to be classified as representatives of Firmicutes, Proteobacteria and Bacteriodetes, many of these taxa should be regarded novel species as judged from the distance of their gene sequences to those of their nearest cultured phylogenetic neighbours. The physiological properties of cultured strains from Lake Fryxell and of those of described species that are phylogenetically affiliated to the as yet uncultured species from this environment, suggest the presence of a well developed food web of primary producers, anaerobic degraders and fermenters, and aerobes. The few novel species described from this sample add to the increasing number of species characterized from various Antarctic habitats. Determination of the phylogenetic relatedness of the mat clone sequences of Clostridia with recent entries into public databases revealed that many of the putative species are closely related to other putative species detected in a broad range of environments, ranging from rumen and gut, anaerobic and polluted soil to sediment and groundwater samples.

Paenibacillus agarexedens sp. nov., nom. rev., and Paenibacillus agaridevorans sp. nov.

Twenty-two agarolytic, aerobic, spore-forming strains were characterized taxonomically by DNA-DNA reassociation experiments, riboprint analyses, 16S rDNA sequencing and phenetic similarity analyses. Based on riboprint analyses, the strains formed eight ribogroups, six of which contained 2-6 strains and two encompassed single strains. Within the multi-strain ribogroups, similarities ranged from 91-99%. Phylogenetic analyses of representatives of the eight groups by 16S rDNA sequence analysis showed that the strains were affiliated to the genus Paenibacillus, but relatedness to described Paenibacillus species was only moderate (<97.8% sequence similarity). Published DNA-DNA similarity values for most of the agarolytic strains, supplemented with new data, supported the distinctiveness of the eight ribogroups. Intragroup DNA-DNA similarity values ranged from 80 to 104%, while intergroup DNA-DNA similarities were <35%. Based on genomic distinctiveness and supported by the presence of distinguishing phenotypic properties, multi-strain groups 1 and 2 are proposed as novel species, Paenibacillus agarexedens sp. nov., nom. rev. (type strain, DSM 1327T=CIP 107437T) and Paenibacillus agaridevorans sp. nov. (type strain, DSM 1355T=CIP 107436T).

Salana multivorans gen. nov., sp. nov., a novel actinobacterium isolated from an anaerobic bioreactor and capable of selenate reduction.

Three facultatively anaerobic, Gram-positive bacteria, strains Se-3111T, Se-13111 and Se-1311A, were isolated from an anaerobic, dechlorinating bioreactor culture enriched from sediment of the River Saale in Germany. All strains were isolated from the dechlorinating mixed culture through their ability to reduce selenate anaerobically to elemental selenium. All three strains shared identical 16S rDNA sequences and phylogenetic analysis revealed that strain Se-3111T forms a novel taxon within the suborder Micrococcineae of the class Actinobacteria, related most closely to Beutenbergia cavernae. On the basis of genotypic, chemotaxonomic and physiological characteristics, it is proposed that the novel strains Se-3111T, Se-13111 and Se-1311A be classified in a new genus as Salana multivorans gen. nov., sp. nov. The type strain of the novel species is Se-3111T (= DSM 13521T = NRRL B-24118T).

Streptocidins A-D, novel cyclic decapeptide antibiotics produced by Streptomyces sp. Tü 6071. I. Taxonomy, fermentation, isolation and biological activities.

Four novel cyclic homodecapetide antibiotics, streptocidins A-D were detected in the mycelium extract of Streptomyces sp. Tü 6071 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening. The peptides which were closely related in structure to the tyrocidines and gramicidins of Bacillus brevis show antibiotic activities against Gram-positive bacteria.

Ureibacillus gen. nov., a new genus to accommodate Bacillus thermosphaericus (Andersson et al. 1995), emendation of Ureibacillus thermosphaericus and description of Ureibacillus terrenus sp. nov.

A polyphasic taxonomic study was performed on the type strain of Bacillus thermosphaericus DSM 10633T and three related soil isolates. On the basis of phenotypic characteristics, chemotaxonomic profiles and phylogenetic data a new genus, Ureibacillus gen. nov., is proposed for the strains in the Bacillus thermosphaericus cluster. Strains of this cluster fall into two DNA-DNA similarity groups: while one group contains the type strain of Ureibacillus thermosphaericus comb. nov. and a single soil isolate, the other contains two soil isolates. The two groups differed in the composition of isoprenoid quinones and some phenotypic properties. These data support the description of a novel species of Ureibacillus for which the name Ureibacillus terrenus is proposed. The type strain of this new species is TH9AT (= DSM 12654T = LMG 19470T).

Reclassification of bioindicator strains Bacillus subtilis DSM 675 and Bacillus subtilis DSM 2277 as Bacillus atrophaeus.

On the basis of high DNA-DNA reassociation values and confirmatory automated RiboPrint analysis, two aerobic spore-forming strains hitherto allocated to Bacillus subtilis and used as bioindicators (DSM 675, hot-air sterilization control; DSM 2277, ethylene oxide sterilization control) are reclassified as Bacillus atrophaeus.

Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in the genetic diversity of bacterial populations.

A mesocosm experiment was performed to study the influence of nutrients on activity and diversity of bacterial assemblages from the Mediterranean Sea. Changes in the diversity of the predominant bacterial populations were monitored by DGGE fingerprinting of PCR products derived from 16S rRNA encoding genes. Fluctuations in the diversity of the most active populations was inferred by performing the DGGE fingerprinting on the basis of the cellular rRNA after reverse transcription and PCR amplification. DNA-derived DGGE patterns obtained from duplicate control and nutrient-enriched mesocosms showed differences in the development of the bacterial communities between control and nutrient-enriched experimental mesocosms. Multidimensional scaling analysis of the DNA-derived DGGE fingerprints indicated that duplicate treatments were reproducible. DNA- and RNA-derived DGGE fingerprints of bacterial assemblages changed over time, showing that the composition of the bacterial assemblages, as well as the most active bacterial populations changed during different phases of the incubation. Sequences of predominant DGGE bands in RNA-derived patterns were similar to 16S rRNA gene sequences of members of the alpha-, gamma- and delta-Proteobacteria and of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB). Bands corresponding to Ruegeria-like bacteria and members of the CFB became especially dominant during the course of incubation, suggesting that these populations were important contributors to bacterial production and activity in the post-grazing phase of the experiment.

Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in abundances, activity and composition.

Quantitative and qualitative changes in bacterial communities from the Mediterranean Sea were compared in duplicate batch mesocosms with or without addition of inorganic nutrients. Methods including traditional microbial ecology techniques, molecular biology and flow cytometry were combined to determine abundances, production, cell size, activity, culturability and taxonomic diversity of bacterial cells. Addition of nutrients and confinement resulted in an increase of bacterial densities which were rapidly controlled by protozoan grazing. Changes in bacterial activity and morphology were observed during the growth phase of bacteria and under grazing pressure. The proportion of medium-size and culturable cells increased during the growth phase. These cells were preferentially consumed by grazers resulting in a strong limitation of bacterial production. As a consequence of the grazing pressure, large cells were produced and contributed to the remaining bacterial productivity after grazing. Grazing had an effect on the taxonomic composition of bacterial communities by preferentially eliminating gamma-Proteobacteria, alpha-Proteobacteria were preserved. It seems that some species from the genera Ruegeria and Cytophaga may have developed defence strategies to escape predation.

Microbial diversity of cultivatable bacteria associated with the North Sea bryozoan Flustra foliacea.

The microbial diversity of cultivatable bacteria associated with the bryozoan species Flustra foliacea from the North Sea was investigated by a molecular approach. Amplified ribosomal RNA restriction analyses (ARDRA) and 16S rDNA partial sequence analysis revealed differences in the composition of cultivatable bacteria populations from single bryozoan colonies collected from two different sampling sites in the North Sea as well from one site taken at different points in time. Whereas gamma-Proteobacteria identified as Shewanella frigidimarina, Pseudoalteromonas ssp. and Psycbrobacter ssp. were predominant on samples of Flustra I (taken near the island of Helgoland), most bacteria isolated from Flustra II, originating from the Steingrund, could be affiliated to Gram-positive taxa. Survey of the bryozoan samples from the latter site in February 2000 led to the detection of a phylogenetically mixed bacterial population, consisting of gamma-, and alpha-Proteobacteria and Gram-positive bacteria with low and high GC-content (Flustra III). As these bacteria are among the most widely isolated organisms from the marine environment, it may be concluded that the bryozoan Flustra foliacea accepts colonization of surfaces by bacteria which are common inhabitants of the marine environment and which may have been transferred into this environment from terrestrial sites.

Bacillus thermodenitrificans sp. nov., nom. rev.

A polyphasic study was performed on 10 soil isolates of thermophilic denitrifying Bacillus strains from different geographical areas. The presence of two main characteristic bands following amplification of the internal transcribed spacer (ITS) region of rrn operons suggests a close relatedness to 'Bacillus thermodenitrificans'. The isolates cluster around two strains of 'B. thermodenitrificans' in riboprint and fatty acid analyses, though differences occur at the strain level. Subsequent DNA-DNA reassociation studies including the 10 isolates, 'B. thermodenitrificans' DSM 465T and DSM 466, and Bacillus stearothermophilus ATCC 12980T and Bacillus thermoleovorans ATCC 43513T revealed such a high level of genomic relatedness between the isolates and the DSM strains (> 73% similarity) that they must be considered strains of the same taxon. The degree of DNA-DNA similarity between the 12 strains of 'B. thermodenitrificans' and the type strains of the other two phylogenetically neighbouring Bacillus species was significantly lower (21-43% similarity). Based upon phylogenetic, chemotaxonomic and phenotypic evidence, the designation of B. thermodenitrificans sp. nov., nom. rev. is proposed. The type strain of B. thermodenitrificans is DSM 465T.

Cultivatable microbial biodiversity: gnawing at the Gordian knot.

Rapid and inexpensive sorting of bacterial isolates may be achieved using Fourier transform infrared spectroscopy (FT-IR), a method that has hitherto been applied to identification and classification. The comprehensive characterization of environmental samples requires the isolation of large numbers of isolates using different growth media and growth conditions. In such cases, sorting the isolates is critical before isolates are subjected to more detailed studies. Using FT-IR, isolates are grown under standardized conditions, and 100 strains can be tested within less than 8 h. Chemotaxonomic and molecular characterization of members of clusters emerging from FT-IR analysis either at a level of spectral distance values below 20-30 (analysis of region 600-800 cm(-1), average linkage algorithm) or at spectral heterogeneity values below 75 (regions 1,200-900, 3,000-2,798 and 901-698, scaling to first region, Ward's algorithm) reveals great similarities in fatty acids and 16S rDNA sequences. As judged from riboprinting analyses and fatty acid analyses, FT-IR analysis is able to unravel intraspecific subclustering. The example used in this study of 100 isolates from a mat system, Lake Fryxell, Dry Valleys, Antarctica, selected from a larger number of isolates, picked mainly on the basis of colony pigmentation and form, reveals the utility of the method for identifying the number of putative species quickly. The method described is able to select strains rapidly that represent clusters at the specific and intraspecific level for subsequent characterization.

Sulfitobacter mediterraneus sp. nov., a new sulfite-oxidizing member of the alpha-Proteobacteria.

Analysis of PCR products of 16S rDNA of 680 isolates from Mediterranean Sea mesocosm experiments with taxon-specific 16S rDNA oligonucleotides revealed that 262 isolates belonged to the alpha subclass of the class Proteobacteria. Partial 16S rDNA sequence analysis of selected isolates and oligonucleotide probing with a Sulfitobacter-specific 16S rDNA probe affiliated 33 strains to the genus Sulfitobacter. Analysis of the HaeIII digest pattern of 16S rDNA revealed the presence of two groups; while 30 strains showed a pattern identical with that obtained for Sulfitobacter pontiacus DSM 10014T, a second group of three strains had a unique pattern that was different from that of the type strain. Five isolates of group 1 and one isolates of group 2, strain CH-B427T, were selected for detailed taxonomic analysis. All six isolates closely resembled the type strain Sulfitobacter pontiacus DSM 10014T in physiological reactions. However, strain CH-B427T differed quantitatively in the composition of fatty acids from Sulfitobacter pontiacus DSM 10014T and showed only 98.2% 16S rDNA sequence similarity with strain DSM 10014T. DNA-DNA reassociation value obtained for strains DSM 10014T and CH-B427T revealed 46% similarity. Based on the results of DNA-DNA reassociation and discrete differences in the nucleotide composition of 16S rDNA, a new species of the genus Sulfitobacter is proposed, designated Sulfitobacter mediterraneus sp. nov., the type strain being strain CH-B427T (= DSM 12244T).

Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR.

Primer systems for PCR amplification of different replicon-specific DNA regions were designed on the basis of published sequences for plasmids belonging to the incompatibility (Inc) groups IncP, IncN, IncW, and IncQ. The specificities of these primer systems for the respective Inc groups were tested with a collection of reference plasmids belonging to 21 different Inc groups. Almost all primer systems were found to be highly specific for the reference plasmid for which they were designed. In addition, the primers were tested with plasmids which had previously been grouped by traditional incompatibility testing to the IncN, IncW, IncP, or IncQ group. All IncQ plasmids gave PCR products with the IncQ primer systems tested. However, PCR products were obtained for only some of the IncN, IncP, and IncW group plasmids. Dot blot and Southern blot analyses of the plasmids revealed that PCR-negative plasmids also failed to hybridize with probes derived from the reference plasmids. The results indicated that plasmids assigned to the same Inc group by traditional methods might be partially or completely different from their respective reference plasmids at the DNA level. With a few exceptions, all plasmids related to the reference plasmid at the DNA level also reacted with the primer systems tested. PCR amplification of total DNA extracted directly from different soil and manure slurry samples revealed the prevalence of IncQ- and IncP-specific sequences in several of these samples. In contrast, IncN- and IncW-specific sequences were detected mainly in DNA obtained from manure slurries.