A new antibiotic kills pathogens without detectable resistance.

Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Most antibiotics were produced by screening soil microorganisms, but this limited resource of cultivable bacteria was overmined by the 1960s. Synthetic approaches to produce antibiotics have been unable to replace this platform. Uncultured bacteria make up approximately 99% of all species in external environments, and are an untapped source of new antibiotics. We developed several methods to grow uncultured organisms by cultivation in situ or by using specific growth factors. Here we report a new antibiotic that we term teixobactin, discovered in a screen of uncultured bacteria. Teixobactin inhibits cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid). We did not obtain any mutants of Staphylococcus aureus or Mycobacterium tuberculosis resistant to teixobactin. The properties of this compound suggest a path towards developing antibiotics that are likely to avoid development of resistance.

Pararhodobacter marinus sp. nov., a bacterium isolated from marine sediment in the East China Sea and emended description of the genus Pararhodobacter.

A Gram-negative, rod-shaped, non-motile and strictly aerobic bacterium, designated ZQ420T, was isolated from marine sediment sampled on Zhoushan Island located in the East China Sea. Strain ZQ420T was able to grow at 10-45 °C, 0-12.0 % (w/v) NaCl and pH 5.5-9.0. Catalase and oxidase activities, nitrate reduction, H2S production, hydrolysis of starch, casein, Tween 20, 40 and 80 were positive. Indole, methyl red, Voges-Proskauer test, hydrolysis of gelatin and Tween 60 were negative. The major cellular fatty acids were C18 : 1 ω7c, C16 : 0 and 11-methyl C18 : 1ω7c. Ubiquinone-10 (Q-10) was the only detected respiratory quinone. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three unidentified phospholipids, three unidentified glycolipids, two unidentified aminolipid and two unidentified lipids. The DNA G+C content was 64.8 mol%. According to 16S rRNA gene sequence similarities, strain ZQ420T shared 97.9, 96.3 and 96.3 % similarities to the following species with validated names Pararhodobacteraggregans D1-19T, Pseudo rhodobacter psychrotolerans PAMC27389T and Pseudo rhodobacter collinsensis 4-T-34T, respectively. While sharing lower sequence similarities (<96.0 %) to other type species. Phylogenetic analyses showed that strain ZQ420T and P. aggregans D1-19T formed an independent cluster in the phylogenetic trees. The average nucleotide identity value between strain ZQ420T and P. aggregans D1-19T was 79.1 %. The in silico DNA-DNA hybridization analysis revealed that strain ZQ420T shared 21.5 % DNA relatedness with P. aggregans D1-19T. On the basis of its phenotypic, chemotaxonomic and genotypic characteristics, strain ZQ420T is considered to represent a novel species in the genus Pararhodobacter, for which the name Pararhodobactermarinus sp. nov. is proposed. The type strain is ZQ420T (=KCTC 62579T=MCCC 1K03530T).

Megasphaera lornae sp. nov., Megasphaera hutchinsoni sp. nov., and Megasphaera vaginalis sp. nov.: novel bacteria isolated from the female genital tract.

Six strictly anaerobic Gram-negative bacteria representing three novel species were isolated from the female reproductive tract. The proposed type strains for each species were designated UPII 199-6T, KA00182T and BV3C16-1T. Phylogenetic analyses based on 16S rRNA gene sequencing indicated that the bacterial isolates were members of the genus Megasphaera. UPII 199-6T and KA00182T had 16S rRNA gene sequence identities of 99.9 % with 16S rRNA clone sequences previously amplified from the human vagina designated as Megasphaera type 1 and Megasphaera type 2, members of the human vaginal microbiota associated with bacterial vaginosis, preterm birth and HIV acquisition. UPII 199-6T exhibited sequence identities ranging from 92.9 to 93.6 % with validly named Megasphaera isolates and KA00182T had 16S rRNA gene sequence identities ranging from 92.6-94.2 %. BV3C16-1T was most closely related to Megasphaera cerevisiae with a 16S rRNA gene sequence identity of 95.4 %. Cells were coccoid or diplococcoid, non-motile and did not form spores. Genital tract isolates metabolized organic acids but were asaccharolytic. The isolates also metabolized amino acids. The DNA G+C content for the genome sequences of UPII 199-6T, KA00182T and BV3C16-1T were 46.4, 38.9 and 49.8 mol%, respectively. Digital DNA-DNA hybridization and average nucleotide identity between the genital tract isolates and other validly named Megasphaera species suggest that each isolate type represents a new species. The major fatty acid methyl esters include the following: C12 : 0, C16 : 0, C16 : 0 dimethyl acetal (DMA) and summed feature 5 (C15 : 0 DMA and/or C14 : 0 3-OH) in UPII 199-6T; C16 : 0 and C16 : 1 cis 9 in KA00182T; C12 : 0; C14 : 0 3-OH; and summed feature 5 in BV3C16-1T. The isolates produced butyrate, isobutyrate, and isovalerate but there were specific differences including production of formate and propionate. Together, these data indicate that UPII 199-6T, KA00182T and BV3C16-1T represent novel species within the genus Megasphaera. We propose the following names: Megasphaera lornae sp. nov. for UPII 199-6T representing the type strain of this species (=DSM 111201T=ATCC TSD-205T), Megasphaera hutchinsoni sp. nov. for KA00182T representing the type strain of this species (=DSM 111202T=ATCC TSD-206T) and Megasphaera vaginalis sp. nov. for BV3C16-1T representing the type strain of this species (=DSM 111203T=ATCC TSD-207T).

Complete Genome Sequence of Saccharospirillum mangrovi HK-33T Sheds Light on the Ecological Role of a Bacterium in Mangrove Sediment Environment.

We present the genome sequence of Saccharospirillum mangrovi HK-33T, isolated from a mangrove sediment sample in Haikou, China. The complete genome of S. mangrovi HK-33T consisted of a single-circular chromosome with the size of 3,686,911 bp as well as an average G + C content of 57.37%, and contained 3,383 protein-coding genes, 4 operons of 16S-23S-5S rRNA genes, and 52 tRNA genes. Genomic annotation indicated that the genome of S. mangrovi HK-33T had many genes related to oligosaccharide and polysaccharide degradation and utilization of polyhydroxyalkanoate. For nitrogen cycle, genes encoding nitrate and nitrite reductase, glutamate dehydrogenase, glutamate synthase, and glutamine synthetase could be found. For phosphorus cycle, genes related to polyphosphate kinases (ppk1 and ppk2), the high-affinity phosphate-specific transport (Pst) system, and the low-affinity inorganic phosphate transporter (pitA) were predicted. For sulfur cycle, cysteine synthase and type III acyl coenzyme A transferase (dddD) coding genes were searched out. This study provides evidence about carbon, nitrogen, phosphorus, and sulfur metabolic patterns of S. mangrovi HK-33T and broadens our understandings about ecological roles of this bacterium in the mangrove sediment environment.

Saccharospirillum mangrovi sp. nov., a bacterium isolated from mangrove sediment.

A Gram-negative, spirilla, non-spore-forming, motile and strictly aerobic bacterium, designated HK-33T, was isolated from a mangrove sediment sample in Haikou city, Hainan Province, China. Strain HK-33T was able to grow at 10-45 °C (optimum 37 °C), 0.5-12.0 % (w/v) NaCl (2.0 %, w/v) and pH 5.5-8.5 (pH 7.0). The major cellular fatty acids were C16 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). Ubiquinone-8 was the predominant respiratory quinone, and Q-9 was present in trace amounts. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids, two unidentified glycolipids, an unidentified aminophosphoglycolipid and two unidentified lipids. The DNA G+C content was 57.3 mol%. According to 16S rRNA gene sequences similarity, strain HK-33T shared 98.6 %, 96.4%, 95.7 and 94.9 % sequence similarities to the species Saccharospirillum correiae CPA1T, Saccharospirillum impatiens EL-105T, Saccharospirillum salsuginis YIM-Y25T and Saccharospirillum aestuarii IMCC 4453T, respectively. Phylogenetic analysis showed that strain HK-33T was clustered with S. correiae CPA1T, S. impatiens EL-105T, S. salsuginis YIM-Y25T and S. aestuarii IMCC 4453T. Results of DNA-DNA hybridization analysis revealed that strain HK-33T shared 36.3±1.7 % DNA relatedness with S. correiae CPA1T. On the basis of its phenotypic, chemotaxonomic and genotypic characteristics, strain HK-33T is considered to represent a novel species in the genus Saccharospirillum, for which the name Saccharospirillummangrovi sp. nov. is proposed. The type strain is HK-33T (=KCTC 62178T=MCCC 1K03440T).

Gallaecimonas mangrovi sp. nov., a novel bacterium isolated from mangrove sediment.

A Gram-stain negative, rod-shaped, non-motile, strictly aerobic bacterium HK-28T was isolated from a mangrove sediment sample in Haikou city, Hainan Province, China. Strain HK-28T was able to grow at 10-45 °C (optimum 25-30 °C), pH 5.0-8.5 (optimum 6.0-7.0) and 0.5-12.0% (w/v) NaCl (optimum 1.0-3.0%, w/v). The major cellular fatty acids were C16:0, Summed Feature 8 (C18:1 ω7c and/or C18:1 ω6c), Summed Feature 3 (C16:1 ω7c and/or C16:1 ω6c), C17:0, C12:0 3-OH and C17:1ω8c. Ubiquinone-8 (Q-8) was the predominant respiratory quinone. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, four unidentified phospholipids, two unidentified glycolipid, an unidentified glycophospholipid, an unidentified aminolipid and an unidentified lipid. The DNA G+C content was 50.2 mol%. Accoroding to 16S rRNA gene sequence similarities, strain HK-28T shared 97.1 and 96.7% sequence similarities to the validly named species Gallaecimonas xiamenensis MCCC 1A01354T and Gallaecimonas pentaromativorans MCCC 1A06435T, respectively, and shared lower sequence similarities (< 92.0%) to all other genera. Phylogenetic analysis showed strain HK-28T was clustered with G. pentaromativorans MCCC 1A06435T and G. xiamenensis MCCC 1A01354T. Strain HK-28T showed low DNA-DNA relatedness with G. xiamenensis MCCC 1A01354T (28.3 ± 1.5%) and G. pentaromativorans MCCC 1A06435T (25.2 ± 2.4%). On the basis of phenotypic, chemotaxonomic and genotypic characteristics, strain HK-28T is considered to represent a novel species in the genus Gallaecimonas, for which the name Gallaecimonas mangrovi sp. nov. is proposed. The type strain is HK-28T (= KCTC 62177T = MCCC 1K03441).

More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

BACKGROUND: Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture. METHODS: Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing. RESULTS: We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with <98% 16S rRNA sequence identity to validly described species, (2) closely related species within a genus, (3) bacteria previously isolated from body sites other than the vagina, and (4) known bacteria formerly isolated from the vagina. Pyrosequencing showed that novel strains Peptoniphilaceae DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV. CONCLUSIONS: We isolated a diverse set of novel and clinically significant anaerobes from the human vagina using conventional approaches with systematic molecular identification. Several previously "uncultivated" bacteria are amenable to conventional cultivation.

Efficient Preparation of Streptochlorin from Marine Streptomyces sp. SYYLWHS-1-4 by Combination of Response Surface Methodology and High-Speed Counter-Current Chromatography.

Since first isolated from the lipophilic extract of Streptomyces sp. SF2583, streptochlorin, has attracted a lot of attention because of its various pharmacological properties, such as antibiotic, antiallergic, antitumor, and anti-inflammatory activities. For the efficient preparation of streptochlorin from a producing strain Streptomyces sp. SYYLWHS-1-4, we developed a combinative method by using response surface methodology (RSM) and high-speed counter-current chromatography (HSCCC). In the fermentation process, we used RSM to optimize the condition for the efficient accumulation of streptochlorin, and the optimal parameters were: yeast extract 1.889 g/L, soluble starch 8.636 g/L, K2HPO4 0.359 g/L, CaCl2 2.5 g/L, MgSO4 0.625 g/L, marine salt 25 g/L, medium volume 50%, initial pH value 7.0, temperature 27.5 °C, which enhanced streptochlorin yield by 17.7-fold. During the purification process, the preparative HSCCC separation was performed using a petroleum ether-ethyl acetate-methanol-water (9:0.8:5:5, v/v/v/v) biphasic solvent system, where 300 mg of crude sample yielded 16.5 mg streptochlorin with over 95% purity as determined by UPLC. Consequently, the combination method provided a feasible strategy for highly effective preparation of streptochlorin, which ensured the supply of large amounts of streptochlorin for in vivo pharmacological assessments or other requirements.

Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov., obligately anaerobic bacteria from the human oral cavity, and emended description of the genus Oribacterium.

Three strictly anaerobic, Gram-positive, non-spore-forming, rod-shaped, motile bacteria, designated strains ACB1(T), ACB7(T) and ACB8, were isolated from human subgingival dental plaque. All strains required yeast extract for growth. Strains ACB1(T) and ACB8 were able to grow on glucose, lactose, maltose, maltodextrin and raffinose; strain ACB7(T) grew weakly on sucrose only. The growth temperature range was 30-42 °C with optimum growth at 37 °C. Major metabolic fermentation end products of strain ACB1(T) were acetate and lactate; the only product of strains ACB7(T) and ACB8 was acetate. Major fatty acids of strain ACB1(T) were C(14 : 0), C(16 : 0), C(16 : 1)ω7c dimethyl aldehyde (DMA) and C(18 : 1)ω7c DMA. Major fatty acids of strain ACB7(T) were C(12 : 0), C(14 : 0), C(16 : 0), C(16 : 1)ω7c and C(16 : 1)ω7c DMA. The hydrolysate of the peptidoglycan contained meso-diaminopimelic acid, indicating peptidoglycan type A1γ. Genomic DNA G+C content varied from 42 to 43.3% between strains. According to 16S rRNA gene sequence phylogeny, strains ACB1(T), ACB8 and ACB7(T) formed two separate branches within the genus Oribacterium, with 98.1-98.6% sequence similarity to the type strain of the type species, Oribacterium sinus. Predicted DNA-DNA hybridization values between strains ACB1(T), ACB8, ACB7(T) and O. sinus F0268 were <70%. Based on distinct genotypic and phenotypic characteristics, strains ACB1(T) and ACB8, and strain ACB7(T) are considered to represent two distinct species of the genus Oribacterium, for which the names Oribacterium parvum sp. nov. and Oribacterium asaccharolyticum sp. nov. are proposed. The type strains are ACB1(T) ( = DSM 24637(T) = HM-481(T) = ATCC BAA-2638(T)) and ACB7(T) ( = DSM 24638(T) = HM-482(T) = ATCC BAA-2639(T)), respectively.

Stomatobaculum longum gen. nov., sp. nov., an obligately anaerobic bacterium from the human oral cavity.

A strictly anaerobic Gram-stain-variable but positive by structure, non-spore-forming bacterium designated Lachnospiraceae bacterium ACC2 strain DSM 24645(T) was isolated from human subgingival dental plaque. Bacterial cells were 4-40 µm long non-motile rods, often swollen and forming curved filaments up to 200 µm. Cells contained intracellular, poorly crystalline, nanometre-sized iron- and sulfur-rich particles. The micro-organism was able to grow on yeast extract, trypticase peptone, milk, some sugars and organic acids. The major metabolic end-products of glucose fermentation were butyrate, lactate, isovalerate and acetate. The growth temperature and pH ranges were 30-42 °C and 4.9-7.5, respectively. Major fatty acids were C14 : 0, C14 : 0 DMA (dimethyl aldehyde), C16 : 0, C16 : 1ω7c DMA. The whole-cell hydrolysate contained meso-diaminopimelic acid, indicating peptidoglycan type A1γ. The DNA G+C content was calculated to be 55.05 mol% from the whole-genome sequence and 55.3 mol% as determined by HPLC. There were no predicted genes responsible for biosynthesis of respiratory lipoquinones, mycolic acids and lipopolysaccharides. Genes associated with synthesis of teichoic and lipoteichoic acids, diaminopimelic acid, polar lipids and polyamines were present. According to the 16S rRNA gene sequence phylogeny, strain DSM 24645(T) formed, together with several uncultured oral clones, a separate branch within the family Lachnospiraceae, with the highest sequence similarity to the type strain of Moryella indoligenes at 94.2 %. Based on distinct phenotypic and genotypic characteristics, we suggest that strain DSM 24645(T) represents a novel species in a new genus, for which the name Stomatobaculum longum gen. nov., sp. nov. is proposed. The type strain of Stomatobaculum longum is DSM 24645(T) ( = HM-480(T); deposited in BEI Resources, an NIH collection managed by the ATCC).

Endophytic bacterial communities in ungerminated and germinated seeds of commercial vegetables.

Chile is a prominent seed exporter globally, but the seed microbiome of vegetables (46% of seeds) and its role in the early stages of plant growth have remained largely unexplored. Here, we employed DNA metabarcoding analysis to investigate the composition and putative functions of endophytic bacterial communities in ungerminated and germinated seeds of the commercial vegetables Apiaceae (parsley and carrot), Asteraceae (lettuce), Brassicaceae (cabbage and broccoli), and Solanaceae (tomato). Bacterial quantification showed 104 to 108 copies of the 16S rRNA gene per gram of ungerminated and germinated seeds. Alpha diversity analysis (e.g., Chao1, Shannon, and Simpson indices) did not indicate significant differences (Kruskal-Wallis test) between ungerminated and germinated seeds, except for Solanaceae. However, beta diversity (PCoA) analysis showed distinctions (Adonis test) between ungerminated and germinated seeds, except Apiaceae. Pseudomonadota and Bacillota were identified as the dominant and specialist taxa in both ungerminated and germinated seed samples. Chemoheterotrophy and fermentation were predicted as the main microbial functional groups in the endophytic bacterial community. Notably, a considerable number of the 143 isolated endophytic strains displayed plant growth-promoting traits (10 to 64%) and biocontrol activity (74% to 82%) against plant pathogens (Xanthomonas and Pseudomonas). This study revealed the high variability in the abundance, diversity, composition, and functionality of endophytic bacteria between ungerminated and germinated seeds in globally commercialized vegetables. Furthermore, potential beneficial endophytic bacteria contained in their seed microbiomes that may contribute to the microbiome of the early stages, development, growth and progeny of vegetables were found.

A targeted liquid cultivation method for previously uncultured non-colony forming microbes.

A large number of microbes are not able to form colonies using agar-plating methods, which is one of the reasons that cultivation based on solid media leaves the majority of microbial diversity in the environment inaccessible. We developed a new Non-Colony-Forming Liquid Cultivation method (NCFLC) that can selectively isolate non-colony-forming microbes that exclusively grow in liquid culture. The NCFLC method involves physically separating cells using dilution-to-extinction (DTE) cultivation and then selecting those that could not grow on a solid medium. The NCFLC was applied to marine samples from a coastal intertidal zone and soil samples from a forest area, and the results were compared with those from the standard direct plating method (SDP). The NCFLC yielded fastidious bacteria from marine samples such as Acidobacteriota, Epsilonproteobacteria, Oligoflexia, and Verrucomicrobiota. Furthermore, 62% of the isolated strains were potential new species, whereas only 10% were novel species from SDP. From soil samples, isolates belonging to Acidobacteriota and Armatimonadota (which are known as rare species among identified isolates) were exclusively isolated by NCFLC. Colony formation capabilities of isolates cultivated by NCFLC were tested using solid agar plates, among which approximately one-third of the isolates were non-colony-forming, approximately half-formed micro-colonies, and only a minority could form ordinary size colonies. This indicates that the majority of the strains cultivated by NCFLC were previously uncultured microbial species unavailable using the SDP method. The NCFCL method described here can serve as a new approach to accessing the hidden microbial dark matter.

Draft genome sequence for Microbacterium laevaniformans strain OR221, a bacterium tolerant to metals, nitrate, and low pH.

Microbacterium laevaniformans strain OR221 was isolated from subsurface sediments obtained from the Field Research Center (FRC) in Oak Ridge, TN. It was characterized as a bacterium tolerant to heavy metals, such as uranium, nickel, cobalt, and cadmium, as well as nitrate and low pH. We present its draft genome sequence.

Genealogical analyses of multiple loci of litostomatean ciliates (Protista, Ciliophora, Litostomatea).

The class Litostomatea is a highly diverse ciliate taxon comprising hundreds of free-living and endocommensal species. However, their traditional morphology-based classification conflicts with 18S rRNA gene phylogenies indicating (1) a deep bifurcation of the Litostomatea into Rhynchostomatia and Haptoria+Trichostomatia, and (2) body polarization and simplification of the oral apparatus as main evolutionary trends in the Litostomatea. To test whether 18S rRNA molecules provide a suitable proxy for litostomatean evolutionary history, we used eighteen new ITS1-5.8S rRNA-ITS2 region sequences from various free-living litostomatean orders. These single- and multiple-locus analyses are in agreement with previous 18S rRNA gene phylogenies, supporting that both 18S rRNA gene and ITS region sequences are effective tools for resolving phylogenetic relationships among the litostomateans. Despite insertions, deletions and mutational saturations in the ITS region, the present study shows that ITS1 and ITS2 molecules can be used to infer phylogenetic relationships not only at species level but also at higher taxonomic ranks when their secondary structure information is utilized to aid alignment.

Class Cariacotrichea, a novel ciliate taxon from the anoxic Cariaco Basin, Venezuela.

The majority of environmental micro-organisms identified with the rRNA approach have never been visualized. Thus, their reliable classification and taxonomic assignment is often difficult or even impossible. In our preliminary 18S rRNA gene sequencing work from the world's largest anoxic marine environment, the Cariaco Basin (Caribbean Sea, Venezuela), we detected a ciliate clade, designated previously as CAR_H [Stoeck, S., Taylor, G. T. & Epstein, S. S. (2003). Appl Environ Microbiol 63, 5656-5663]. Here, we combine the traditional rRNA detection method of fluorescent in situ hybridization (FISH) with scanning electron microscopy (SEM) and confirm the phylogenetic separation of the CAR_H sequences from all other ciliate classes by showing an outstanding morphological feature of this group: a unique, archway-shaped kinety surrounding the oral apparatus and extending to the posterior body end in CAR_H cells. Based on this specific feature and the molecular phylogenies, we propose a novel ciliate class, Cariacotrichea nov. cl.

Phylogeny and classification of the Litostomatea (Protista, Ciliophora), with emphasis on free-living taxa and the 18S rRNA gene.

The class Litostomatea is a highly diverse ciliate taxon comprising hundreds of species ranging from aerobic, free-living predators to anaerobic endocommensals. This is traditionally reflected by classifying the Litostomatea into the subclasses Haptoria and Trichostomatia. The morphological classifications of the Haptoria conflict with the molecular phylogenies, which indicate polyphyly and numerous homoplasies. Thus, we analyzed the genealogy of 53 in-group species with morphological and molecular methods, including 12 new sequences from free-living taxa. The phylogenetic analyses and some strong morphological traits show: (i) body polarization and simplification of the oral apparatus as main evolutionary trends in the Litostomatea and (ii) three distinct lineages (subclasses): the Rhynchostomatia comprising Tracheliida and Dileptida; the Haptoria comprising Lacrymariida, Haptorida, Didiniida, Pleurostomatida and Spathidiida; and the Trichostomatia. The curious Homalozoon cannot be assigned to any of the haptorian orders, but is basal to a clade containing the Didiniida and Pleurostomatida. The internal relationships of the Spathidiida remain obscure because many of them and some "traditional" haptorids form separate branches within the basal polytomy of the order, indicating one or several radiations and convergent evolution. Due to the high divergence in the 18S rRNA gene, the chaeneids and cyclotrichiids are classified incertae sedis.

Isolation and physiology of bacteria from contaminated subsurface sediments.

The majority of environmental microorganisms cannot be grown by traditional techniques. Here we employed, and contrasted with conventional plating, an alternative approach based on cultivation of microorganisms inside diffusion chambers incubated within natural samples, followed by subculturing in petri dishes. Using this approach, we isolated microorganisms from subsurface sediments from the Field Research Center (FRC) in Oak Ridge, TN. The sediments were acidic and highly contaminated with uranium, heavy metals, nitrate, and organic pollutants. Phylogenetic analysis of 16S rRNA gene sequences revealed clear differences between diversity of isolates obtained by the diffusion chamber approach and those obtained by conventional plating. The latter approach led to isolation of members of the Alpha- and Gammaproteobacteria, Actinobacteria, and Verrucomicrobia. Isolates obtained via the diffusion chamber approach represented the Alpha-, Beta-, and Gammaproteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. Notably, one-third of the isolates obtained by the new method were closely related to species known from previous molecular surveys conducted in the FRC area. Since the initial growth of microorganisms inside diffusion chambers occurred in the presence of the environmental stress factors, we expected the isolates we obtained to be tolerant of these factors. We investigated the physiologies of selected isolates and discovered that the majority were indeed capable of growth under low pH and/or high concentrations of heavy metals and nitrate. This indicated that in contrast to conventional isolation, the diffusion chamber-based approach leads to isolation of species that are novel, exhibit tolerance to extant environmental conditions, and match some of the species previously discovered by molecular methods.