Description of Maribellus sediminis sp. nov., a marine nitrogen-fixing bacterium isolated from sediment of cordgrass and mangrove.

Two marine bacterial strains designated Y2-1-60T and GM1-28 were isolated from sediments of cordgrass and mangrove along the Luoyang estuary in Quanzhou Bay, China, respectively. Both strains were Gram-staining-negative, straight rod-shaped, non-flagellum, facultatively anaerobic, nitrogen-fixing, and did not contain carotenoid pigment. Catalase activities were found to be weak positive and oxidase activities negative. The 16S rRNA gene sequences of the two strains were identical and had maximum similarity of 98.0% with Maribellus luteus XSD2T, and of <94.5% with other species. ANI value (96.9%) and DDH estimate (71.5%) between the two strains supported that they belonged to the same species. ANI value and DDH estimate between the two strains and M. luteus XSD2T was 74.3% and 19.4%, respectively, indicating that they represent a novel species. Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis indicated that strains Y2-1-60T and GM1-28 formed a monophyletic branch within the genus Maribellus. The respiratory quinone was menaquinone MK-7. The major fatty acid (>10%) consisted of iso-C15:0, and iso-C17:0 3-OH. The polar lipids consisted of phosphatidylethanolamine and several unidentified lipids. The genomic G+C contents were 41.9-42.0mol%. Gene annotation revealed that strains Y2-1-60T and GM1-28 contained a set of nif gene cluster (nifHDKENB) responsible for nitrogen fixation. Based on the above characteristics, strains Y2-1-60T and GM1-28 represent a novel species within the genus Maribellus. Thus, Maribellus sediminis sp. nov. is proposed with type strain Y2-1-60T (=MCCC 1K04285T=KCTC 72884T), isolated from cordgrass sediment and strain GM1-28 (=MCCC 1K04384=KCTC 72880), isolated from mangrove sediment.

Candidatus Abditibacter, a novel genus within the Cryomorphaceae, thriving in the North Sea.

Coastal phytoplankton blooms are frequently followed by successive blooms of heterotrophic bacterial clades. The class Flavobacteriia within the Bacteroidetes has been shown to play an important role in the degradation of high molecular weight substrates that become available in the later stages of such blooms. One of the flavobacterial clades repeatedly observed over the course of several years during phytoplankton blooms off the coast of Helgoland, North Sea, is Vis6. This genus-level clade belongs to the family Cryomorphaceae and has been resistant to cultivation to date. Based on metagenome assembled genomes, comparative 16S rRNA gene sequence analyses and fluorescence in situ hybridization, we here propose a novel candidate genus Abditibacter, comprising three novel species Candidatus Abditibacter vernus, Candidatus Abditibacter forsetii and Candidatus Abditibacter autumni. While the small genomes of the three novel photoheterotrophic species encode highly similar gene repertoires, including genes for degradation of proteins and algal storage polysaccharides such as laminarin, two of them - Ca. A. vernus and Ca. A. forsetii - seem to have a preference for spring blooms, while Ca. A. autumni almost exclusively occurs in late summer and autumn.

Arthrospiribacter ruber gen. nov., sp. nov., a novel bacterium isolated from Arthrospira cultures.

Polyphasic analysis of ten isolates of the red-pigmented bacteria isolated from ten Arthrospira cultures originating from different parts of the world is described. The 16S rRNA analysis showed <95 % identity with the known bacteria on public databases, therefore, additional analyses of fatty acids profiles, MALDI-TOF/MS, genome sequencing of the chosen isolate and following phylogenomic analyses were performed. Gram-stain-negative, strictly aerobic rods were positive for catalase, negative for oxidase, proteolytic and urease activity. Major fatty acids were 15 : 0 iso, 17:0 iso 3 OH and 17:1 iso w9c/16:0 10-methyl. The whole phylogenomic analyses revealed that the genomic sequence of newly isolated strain DPMB0001 was most closely related to members of Cyclobacteriaceae family and clearly indicated distinctiveness of newly isolated bacteria. The average nucleotide identity and in silico DNA-DNA hybridisation values were calculated between representative of the novel strains DPMB0001 and its phylogenetically closest species, Indibacter alkaliphilus CCUG57479 (LW1)T (ANI 69.2 % is DDH 17.2 %) and Mariniradius saccharolyticus AK6T (ANI 80.02 % isDDH 26.1 %), and were significantly below the established cut-off <94 % (ANI) and <70 % (isDDH) for species and genus delineation. The obtained results showed that the analysed isolates represent novel genus and species, for which names Arthrospiribacter gen nov. and Arthrospiribacter ruber sp. nov. (type strain DPMB0001=LMG 31078=PCM 3008) is proposed.

Pontibacter beigongshangensis sp. nov., Isolated from the Mash of Wine.

A Gram-negative, aerobic, oval-shaped, and light red pigmented bacterium, designated T6-1T, was isolated from the mash of wine collected from a wine-making laboratory simulated fermenter located in Beijing, China. The optimal growth of T6-1T occurred at 30 °C, pH 7.0 with 1% NaCl. The sole respiratory quinone was menaquinone-7 (MK-7). The principal cellular fatty acids (>5%) were iso-C15:0, iso-C17:0 3OH, C16:1 ω5c, and iso-C17:0. The major polar lipids were PE (phosphatidylethanolamine), PL (unidentified phospholipid), and L1-2 (unidentified lipids). 16S rRNA phylogenetic analysis indicated that strain T6-1T belonged to the genus Pontibacter. The 16S rRNA gene sequence of strain T6-1T was most similar to Pontibacter amylolyticus 9-2T (95.92%). The genomic DNA G+C content of strain T6-1 was 50.34 mol%. The digital DNA-DNA relatedness and average nucleotide identity value between T6-1T and 9-2T was 20.20% and 74.18%, respectively. Polyphasic taxonomy analysis indicated that strain T6-1T represents a novel species of the genus Pontibacter, for which the name Pontibacter beigongshangensis sp. nov. is proposed, with the type strain T6-1T (= CGMCC 1.17104T = KCTC 72413T).

Impact of anaerobic digestion and centrifugation/decanting processes in bacterial communities fractions.

Sewage sludge can be treated by anaerobic processes that frequently are followed by physical separation processes. In this work, a high-throughput sequencing technology, based on variation in the bacterial 16S rRNA gene, has been used to characterise the bacterial populations present in samples taken from different points of an industrial anaerobic digestion process fed with sewage sludge. Relative abundances of phyla and classes throughout the biological process and the subsequent separation steps were determined. Results revealed that the Bacteroidetes, Firmicutes and Proteobacteria phyla were the most representative. However, significant changes in relative abundance were detected along treatments, showing the influence of operational parameters on the distribution of microorganisms throughout the process. After anaerobic digestion, phylum Firmicutes doubled its relative abundance, which seems to indicate that the anaerobic conditions and the nutrients favoured its growth, in contrast to other phyla that almost disappeared. After centrifugation, Proteobacteria went preferentially to the solid phase, in contrast to Firmicutes which was the dominant phylum in the liquid phase. After decanting the liquid phase during 45 h, an important growth of Proteobacteria, Spirochaetes and Tenericutes was detected. At class level, only significantly changes were observed for Proteobacteria classes being α-proteobacteria dominant in the digestate, while γ-proteobacteria was the majority since this point to the final steps. To know the changes on the kind and abundance of microbial populations throughout the anaerobic and separation processes is very important to understand how the facilities design and operation conditions can influence over the efficiencies of next biological treatments.

Chitinophaga salinisoli sp. nov., isolated from saline soil.

A novel aerobic bacterium, designated strain LAM9153T, was isolated from a saline soil sample collected from Lingxian County, Shandong Province, China. Cells of strain LAM9153T were observed to be Gram-stain negative, non-motile, non-spore-forming and rod-shaped. The new isolate grew optimally at 30-35 °C, pH 7.0 and 0.5% of NaCl concentration (w/v). According to the phylogenetic analysis based on the 16S rRNA gene sequence, strain LAM9153T shares high similarity with Chitinophaga terrae Gsoil 238T (96.9%) and Chitinophaga niabensis JS 13-10T (95.9%), forming a subcluster with C. terrae Gsoil 238T, Chitinophaga cymbidii R156-2T, C. niabensis JS 13-10T and Chitinophaga soli Gsoil 219T in the phylogenetic tree. The major cellular fatty acids (> 10%) were identified as iso-C15:0, iso-C17:0 3-OH and summed features 3 (C16:1 ω6c and/or C16:1 ω7c). The predominant respiratory quinone was identified as menaquinone MK-7. The polar lipids consisted of phosphatidylethanolamine, aminophospholipid, three unidentified aminolipids and five unidentified lipids. The genomic DNA G+C content was determined to be 53.2 ± 1.6 mol%. On the basis of phylogenetic, chemotaxonomic and phenotypic data, strain LAM9153T is concluded to represent a novel species of the genus Chitinophaga, for which the name Chitinophaga salinisoli sp. nov. is proposed. The type strain is LAM9153T (= ACCC 19960T = JCM 30847T).

Comparative Analysis of Cellulophaga algicola and Flavobacterium johnsoniae Gliding Motility.

UNLABELLED: Gliding motility is common in members of the phylum Bacteroidetes, including Flavobacterium johnsoniae and Cellulophaga algicola. F. johnsoniae gliding has been extensively studied and involves rapid movement of the cell surface adhesin SprB. Genetic analysis of C. algicola allowed a comparative analysis of gliding. Sixty-three HimarEm1-induced mutants that formed nonspreading colonies were characterized. Each had an insertion in an ortholog of an F. johnsoniae motility gene, highlighting similarities between the motility systems. Differences were also observed. C. algicola lacks orthologs of the F. johnsoniae motility genes gldA, gldF, and gldG that are thought to encode the components of an ATP-binding cassette (ABC) transporter. In addition, mutations in any of 12 F. johnsoniae gld genes result in complete loss of motility, whereas all C. algicola gld mutants retained slight residual motility. This may indicate that C. algicola has multiple motility systems, that the motility proteins exhibit partial redundancy of function, or that essential components of the motility machinery of both C. algicola and F. johnsoniae remain to be discovered. IMPORTANCE: The development of genetic tools for C. algicola and comparative analysis of F. johnsoniae and C. algicola motility mutants identified similarities and differences between their gliding motility machineries. Gliding motility is common in the phylum Bacteroidetes Proteins that are important for gliding in both C. algicola and F. johnsoniae are potential core components of the Bacteroidetes gliding motility machinery.

Hymenobacter sedentarius sp. nov., isolated from a soil.

A novel Gram-negative and red-pinkish bacterium designated DG5B(T) was isolated from a dry soil. Cells were rods that were catalase- and oxidase-positive, and non-motile. The strain was found to grow at temperatures from 10 to 30°C (optimum 25°C) and pH 6.0-8.0, (optimum pH 7) on R2A broth. 16S rRNA gene sequence (1,452 bp) analysis of this strain identified it as a member of the genus Hymenobacter that belongs to the class Cytophagia. The highest gene sequence similarities were with Hymenobacter arizonensis OR362-8(T) (98.3%), Hymenobacter humi DG31A(T) (97.6%), and Hymenobacter glaciei VUG-A130(T) (96.6%). Strain DG5B(T) exhibited <70% DNA-DNA relatedness with H. arizonensis (34.7 ± 7.0%; reciprocally, 29.7 ± 1.2%) and H. humi (39.4 ± 4.3%; reciprocally, 39.5 ± 3.3%) as a different genomic species, and its genomic DNA G+C content was 59.8%. Strain DG5B(T) had the following chemotaxonomic characteristics: the major fatty acids are iso-C15:0, anteiso-C15:0, C16:1 ω5c, and summed feature 3 (C16:1 ω7c / C16:1 ω6c); polar lipid profile contained phosphatidylethanolamine (PE), unknown aminophospholipid (APL), unknown glycolipids (GL), unknown phospholipids (PL), and unknown polar lipids (L); the major quinone is MK-7. The absorbance peak of pigment is at 481.0 nm. Strain DG5B(T) showed low-level resistance to gamma-ray irradiation. Phenotypic, chemotaxonomic, and genotypic properties indicated that isolate DG5B(T) represents a novel species within the genus Hymenobacter for which the name Hymenobacter sedentarius sp. nov. is proposed. The type strain is DG5B(T) (=KCTC 32524(T) =JCM 19636(T)).

Previously Uncultured Marine Bacteria Linked to Novel Alkaloid Production.

Low-nutrient media and long incubation times facilitated the cultivation of 20 taxonomically diverse Gram-negative marine bacteria within the phyla Bacteroidetes and Proteobacteria. These strains comprise as many as three new families and include members of clades that had only been observed using culture-independent techniques. Chemical studies of the type strains representing two new families within the order Cytophagales led to the isolation of nine new alkaloid secondary metabolites that can be grouped into four distinct structure classes, including azepinones, aziridines, quinolones, and pyrazinones. Several of these compounds possess antibacterial properties and appear, on structural grounds, to be produced by amino acid-based biosynthetic pathways. Our results demonstrate that relatively simple cultivation techniques can lead to the isolation of new bacterial taxa that are capable of the production of alkaloid secondary metabolites with antibacterial activities. These findings support continued investment in cultivation techniques as a method for natural product discovery.

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism.

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a 'selfish' model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

Localization and morphological variation of three bacteriome-inhabiting symbionts within a planthopper of the genus Oliarus (Hemiptera: Cixiidae).

Many planthoppers of the family Cixiidae (Hemiptera: Fulgoroidea) host three bacteriome-inhabiting bacteria: a gammaproteobacterium: 'Ca.Purcelliella pentastirinorum', a betaproteobacterium: 'Ca. Vidania fulgoroidea', and a member of the bacteroidetes: 'Ca.Sulcia muelleri'. Through light microscopy observations, DGGE PCR and FISH analysis, we examined the morphology and localization of these three endosymbionts within the abdomens of females of the planthopper Oliarus filicicola. Our results indicate a complex distribution and variation in bacterial morphologies. 'Ca. Sulcia muelleri' singularly colonize one pair of bacteriomes and have cells of irregular shape with an average diameter of approximately 4-5 µm. 'Ca.Purcelliella pentastirinorum' bacteria are roughly globular and have an average diameter of approximately 1.5-2 µm in a pair of bacteriomes located near the posterior end of the abdomen, which are surrounded by giant and highly degenerated cells of 'Ca.Vidania fulgoroidea'. In addition, 'Ca.Vidania fulgoroidea' colonizes the 'rectal organ' (sensu Buchner) and the bacterial cells appear as a small, roughly globular with an average diameter of 3 µm; whereas, 'Ca.Purcelliella pentastirinorum' infects an additional two bacteriomes and the bacterial cells appear tightly packed and highly degenerated. All three endosymbionts colocalize in the forming eggs inside the host's ovaries. Based on the abdominal distribution of bacteriomes and bacterial morphologies, we suggest that 'Ca.Vidania fulgoroidea' and 'Ca.Purcelliella pentastirinorum' correspond to the symbionts described by Buchner as the 'x-' and the 'c + d symbiont' respectively.

Abundance and single-cell activity of heterotrophic bacterial groups in the western Arctic Ocean in summer and winter.

Environmental conditions in the western Arctic Ocean range from constant light and nutrient depletion in summer to complete darkness and sea ice cover in winter. This seasonal environmental variation is likely to have an effect on the use of dissolved organic matter (DOM) by heterotrophic bacteria in surface water. However, this effect is not well studied and we know little about the activity of specific bacterial clades in the surface oceans. The use of DOM by three bacterial subgroups in both winter and summer was examined by microautoradiography combined with fluorescence in situ hybridization. We found selective use of substrates by these groups, although the abundances of Ant4D3 (Antarctic Gammaproteobacteria), Polaribacter (Bacteroidetes), and SAR11 (Alphaproteobacteria) were not different between summer and winter in the Beaufort and Chukchi Seas. The number of cells taking up glucose within all three bacterial groups decreased significantly from summer to winter, while the percentage of cells using leucine did not show a clear pattern between seasons. The uptake of the amino acid mix increased substantially from summer to winter by the Ant4D3 group, although such a large increase in uptake was not seen for the other two groups. Use of glucose by bacteria, but not use of leucine or the amino acid mix, related strongly to inorganic nutrients, chlorophyll a, and other environmental factors. Our results suggest a switch in use of dissolved organic substrates from summer to winter and that the three phylogenetic subgroups examined fill different niches in DOM use in the two seasons.

The chance identification of unusual-appearing bacteria in bronchoalveolar lavage fluid.

BACKGROUND: A well-known indication for the cytologic examination of bronchoalveolar lavage (BAL) fluid is the identification of infectious organisms. However, an important distinction must be made as to whether the organisms seen represent a true opportunistic lower respiratory tract infection or a non-pathologic contamination. CASE: We describe herein the case of a 13-month-old male infant who presented with persistent chest congestion and tracheobronchitis and who underwent BAL as part of his clinical work-up. On cytological examination of the BAL fluid, the Romanowsky-stained cytospin slides contained numerous squamous epithelial cells with some showing rare striated rod-like structures on their surfaces. The peculiar structures also had rounded ends and were very large when compared to adjacent known bacterial cocci. CONCLUSION: We have determined that the striated rod-like structures in the infant's BAL fluid were indeed bacteria, Simonsiella sp. Simonsiella has reportedly been found in up to 32% of oral swabs in normal humans and it is considered a commensal and non-pathogenic organism. The characteristically large size, the association with normal oral-derived squamous cells and the striated appearance is diagnostic and will hopefully eliminate any possibility of confusion with a truly pathogenic organism.

A hybrid two-component system of Tannerella forsythia affects autoaggregation and post-translational modification of surface proteins.

Tannerella forsythia is a Gram-negative oral anaerobe closely associated with both periodontal and periapical diseases. The ORF TF0022 of strain ATCC 43037 encodes a hybrid two-component system consisting of an N-terminal histidine kinase and a C-terminal response regulator. Disruption of the TF0022 locus enhanced autoaggregation of the broth-cultured cells. Comparative proteome analyses revealed that two S-layer proteins in the TF0022 mutant exhibited decreased apparent masses by denaturing gel electrophoresis, suggesting a deficiency in post-translational modification. Furthermore, the mutant decreased the production of a glycosyltransferase encoded by TF1061 that is located in a putative glycosylation-related gene cluster. Quantitative real-time PCR revealed reduced transcription of TF1061 and the associated genes in the TF0022 mutant. These results indicate that TF0022 upregulates the expression of the glycosylation-related genes and suggest modulation of the autoaggregation of T. forsythia cells by a possible post-translational modification of cell-surface components.

A protein secretion system linked to bacteroidete gliding motility and pathogenesis.

Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum.

Phylogenetic classification of heterotrophic bacteria associated with filamentous marine cyanobacteria in culture.

Fifty-one heterotrophic bacterial strains were isolated from the marine cyanobacterial cultures of heterocystous Nodularia harveyana strain Bo53 and non-heterocystous Oscillatoria brevis strain Bo10. Fluorescence in situ hybridisation and fingerprinting methods were used for a preliminary taxonomical classification of 44 of the 51 isolates. The strains obtained from Bo53 were mostly Alphaproteobacteria (10/24), followed by Bacteroidetes (7/24), and Gammaproteobacteria (3/24). The affiliation of the isolates originating from Bo10 was dominated by Alphaproteobacteria (8/20) and Bacteroidetes (7/20), followed by Gammaproteobacteria (3/20). The 16S rRNA genes of four selected isolates were sequenced. A red-coloured bacterium from Bo53 grouped with the alphaproteobacterial genus Porphyrobacter, while the other three strains, obtained from Bo10, belonged to the alphaproteobacterial genera Roseobacter (pink) and Rhodobacter (colourless), and to the genus Muricauda (yellow) of Bacteroidetes. The findings indicated that the aerobic anoxygenic phototroph Porphyrobacter and its relatives only occurred in Bo10 culture, whereas members of the Roseobacter clade and the Bacteroidetes bacterium Muricauda sp. seemed to be more ubiquitous.

Characterizing a model human gut microbiota composed of members of its two dominant bacterial phyla.

The adult human distal gut microbial community is typically dominated by 2 bacterial phyla (divisions), the Firmicutes and the Bacteroidetes. Little is known about the factors that govern the interactions between their members. Here, we examine the niches of representatives of both phyla in vivo. Finished genome sequences were generated from Eubacterium rectale and E. eligens, which belong to Clostridium Cluster XIVa, one of the most common gut Firmicute clades. Comparison of these and 25 other gut Firmicutes and Bacteroidetes indicated that the Firmicutes possess smaller genomes and a disproportionately smaller number of glycan-degrading enzymes. Germ-free mice were then colonized with E. rectale and/or a prominent human gut Bacteroidetes, Bacteroides thetaiotaomicron, followed by whole-genome transcriptional profiling, high-resolution proteomic analysis, and biochemical assays of microbial-microbial and microbial-host interactions. B. thetaiotaomicron adapts to E. rectale by up-regulating expression of a variety of polysaccharide utilization loci encoding numerous glycoside hydrolases, and by signaling the host to produce mucosal glycans that it, but not E. rectale, can access. E. rectale adapts to B. thetaiotaomicron by decreasing production of its glycan-degrading enzymes, increasing expression of selected amino acid and sugar transporters, and facilitating glycolysis by reducing levels of NADH, in part via generation of butyrate from acetate, which in turn is used by the gut epithelium. This simplified model of the human gut microbiota illustrates niche specialization and functional redundancy within members of its major bacterial phyla, and the importance of host glycans as a nutrient foundation that ensures ecosystem stability.

Adherence and invasion of Bacteroidales isolated from the human intestinal tract.

Members of the genera Bacteroides and Parabacteroides are important constituents of both human and animal intestinal microbiota, and are significant facultative pathogens. In this study, the ability of Bacteroides spp. and Parabacteroides distasonis isolated from both diarrhoeal and normal stools (n = 114) to adhere to and invade HEp-2 cells was evaluated. The presence of putative virulence factors such as capsule and fimbriae was also investigated. Adherence to HEp-2 cells was observed in 75.4% of the strains, which displayed non-localized clusters. Invasion was observed in 37.5% and 26% of the strains isolated from diarrhoeal and non-diarrhoeal stools, respectively. All strains displayed a capsule, whereas none of them showed fimbriae-like structures. This is the first report of the ability of Bacteroides spp. and P. distasonis to adhere to and invade cultured HEp-2 epithelial cells.

Mucilaginibacter paludis gen. nov., sp. nov. and Mucilaginibacter gracilis sp. nov., pectin-, xylan- and laminarin-degrading members of the family Sphingobacteriaceae from acidic Sphagnum peat bog.

Two facultatively aerobic, heterotrophic bacteria capable of degrading pectin, xylan, laminarin and some other polysaccharides were obtained from the acidic Sphagnum peat bog Bakchar, in western Siberia, Russia, and were designated strains TPT18(T) and TPT56(T). Cells of these isolates are Gram-negative, non-motile, long rods that are covered by large capsules. On ageing, they transform into spherical L-forms. Strains TPT18(T) and TPT56(T) are acido- and psychrotolerant organisms capable of growth at pH 4.2-8.2 (with an optimum at pH 6.0-6.5) and at 2-33 degrees C (with an optimum at 20 degrees C). The major fatty acids are iso-C(15 : 0), anteiso-C(15 : 0), iso-C(17 : 0) 3-OH and summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c); the quinones are MK-7 and MK-6. Comparative 16S rRNA gene sequence analysis revealed that the novel strains share 97 % sequence similarity and belong to the family Sphingobacteriaceae; however, they are related only distantly to members of the genera Pedobacter (91.8-93.3 % similarity) and Sphingobacterium (89.6-91.2 % similarity). The DNA G+C content of strains TPT18(T) and TPT56(T) is 42.4 and 46.1 mol%, respectively. The low DNA-DNA hybridization value (42 %) and a number of phenotypic differences between strains TPT18(T) and TPT56(T) indicated that they represent two separate species. Since the two isolates are clearly distinct from all currently described members of the family Sphingobacteriaceae, we propose a novel genus, Mucilaginibacter gen. nov., containing two novel species, Mucilaginibacter gracilis sp. nov. and Mucilaginibacter paludis sp. nov. The type strains of Mucilaginibacter gracilis and Mucilaginibacter paludis are respectively TPT18(T) (=ATCC BAA-1391(T) =VKM B-2447(T)) and TPT56(T) (=ATCC BAA-1394(T) =VKM B-2446(T)).

'Candidatus Aquirestis calciphila' and 'Candidatus Haliscomenobacter calcifugiens', filamentous, planktonic bacteria inhabiting natural lakes.

Filamentous bacteria frequently occurring in the pelagic zone of natural freshwater lakes and ponds were previously identified as being related to Haliscomenobacter hydrossis based upon their 16S rRNA gene sequences. These bacteria exhibit a specific morphology characterized by the formation of straight, stick-like filaments of variable length (5 to >100 microm) and quite stable, but narrow, width (0.25 to 0.35 microm). Bacteria with these morphological characteristics form a monophyletic but broad phylogenetic group with a maximal divergence of 16S rRNA gene sequences of 12.0 %. This monophyletic group consists of at least three monophyletic subclusters. H. hydrossis is affiliated to one of these subclusters and represents the sole recognized species affiliated to the broad monophyletic group. 'Candidatus Haliscomenobacter calcifugiens' and 'Candidatus Aquirestis calciphila' are uncultured representatives of the other two subclusters and have 16S rRNA gene sequence dissimilarities of 5.4 % and 8.2 %, respectively, with the type strain of H. hydrossis. 'Candidatus H. calcifugiens' and 'Candidatus A. calciphila' have a 16S rRNA gene sequence dissimilarity of 8.5 %. These large ribosomal divergences justify the classification of these environmentally important bacteria as a novel species and a new genus, respectively. Intensive attempts to cultivate these filamentous bacteria have resulted in the establishment of mixed cultures, however, attempts to establish pure cultures have failed.