Community-like genome in single cells of the sulfur bacterium Achromatium oxaliferum.

Polyploid bacteria are common, but the genetic and functional diversity resulting from polyploidy is unknown. Here we use single-cell genomics, metagenomics, single-cell amplicon sequencing, and fluorescence in situ hybridization, to show that individual cells of Achromatium oxaliferum, the world's biggest known freshwater bacterium, harbor genetic diversity typical of whole bacterial communities. The cells contain tens of transposable elements, which likely cause the unprecedented diversity that we observe in the sequence and synteny of genes. Given the high within-cell diversity of the usually conserved 16S ribosomal RNA gene, we suggest that gene conversion occurs in multiple, separated genomic hotspots. The ribosomal RNA distribution inside the cells hints to spatially differential gene expression. We also suggest that intracellular gene transfer may lead to extensive gene reshuffling and increased diversity.The cells of Achromatium bacteria are remarkably large and contain multiple chromosome copies. Here, Ionescu et al. show that chromosome copies within individual cells display high diversity, similar to that of bacterial communities, and contain tens of transposable elements.

Traqueobronquitis por Elizabethkingia meningoseptica en una paciente portadora de traqueostomía / Tracheobronchitis by Elizabethkingia meningoseptica in a patient with tracheostomy

No disponible

Potential of the essential oil from Pimenta pseudocaryophyllus as an antimicrobial agent.

This study evaluated the effectiveness of the essential oil of Pimenta pseudocaryophyllus in inhibiting the growth of the main bacteria responsible for bad perspiration odor (Staphylococcus epidermidis, Proteus hauseri, Micrococcus yunnanensis and Corynebacterium xerosis). The chemical profile of the essential oil was evaluated by high-resolution gas chromatography (HR-GC) and four constituents were identified, eugenol being the major component (88.6%). The antimicrobial activity was evaluated by means of the turbidimetric method, using the microdilution assay. The minimum inhibitory concentration (MIC) values of the essential oil ranged from 500 to 1,000 µg mL⁻¹. Scanning electron microscope (SEM) observations confirmed the physical damage and morphological alteration of the test bacteria treated with the essential oil, reference drugs and eugenol. The findings of the study demonstrated that this essential oil can be used in the formulation of personal care products.

Bacterial biofilm diversity in contact lens-related disease: emerging role of Achromobacter, Stenotrophomonas, and Delftia.

PURPOSE: Multi-species biofilms associated with contact lens cases and lenses can predispose individuals to contact lens-related inflammatory complications. Our study used culture-independent methods to assess the relationship between the severity of contact lens-related disease and bacteria residing in biofilms of contact lens cases and lenses. METHODS: Contact lens cases and lenses from 28 patients referred to the West Virginia University Eye Institute and diagnosed as having mild keratitis, keratitis with focal infiltrates, or corneal ulcers were processed and evaluated for bacterial composition based on 16S ribosomal RNA gene sequencing. Cases and lenses from nine asymptomatic contact lens wearers were processed in a manner similar to controls. Relationships between disease severity, bacterial types, and bacterial diversity were evaluated statistically. RESULTS: Disease severity and presenting visual acuity correlated with an increase in the diversity of bacterial types isolated from contact lens cases. A significant difference also was observed in the number of bacterial types associated with the three clinical groups. Achromobacter, Stenotrophomonas, and Delftia were prevalent in all disease groups, and Achromobacter and Stenotrophomonas were present in one asymptomatic control. Scanning electron microscopy revealed that Achromobacter and Stenotrophomonas formed a biofilm on the surface of contact lenses. CONCLUSIONS: Culture-independent methods identified an association between disease severity and bacterial diversity in biofilms isolated from cases and lenses of patients with contact lens-related corneal disease. Achromobacter, Stenotrophomonas, and Delftia were predominant bacteria identified in our study, drawing attention to their emerging role in contact lens-related disease.

Optimization of hot water treatment for removing microbial colonies on fresh blueberry surface.

UNLABELLED: Blueberries for the frozen market are washed but this process sometimes is not effective or further contaminates the berries. This study was designed to optimize conditions for hot water treatment (temperature, time, and antimicrobial concentration) to remove biofilm and decrease microbial load on blueberries. Scanning electron microscopy (SEM) image showed a well-developed microbial biofilm on blueberries dipped in room temperature water. The biofilm consisted of yeast and bacterial cells attached to the berry surface in the form of microcolonies, which produced exopolymer substances between or upon the cells. Berry exposure to 75 and 90 °C showed little to no microorganisms on the blueberry surface; however, the sensory quality (wax/bloom) of berries at those temperatures was unacceptable. Response surface plots showed that increasing temperature was a significant factor on reduction of aerobic plate counts (APCs) and yeast/mold counts (YMCs) while adding Boxyl® did not have significant effect on APC. Overlaid contour plots showed that treatments of 65 to 70 °C for 10 to 15 s showed maximum reductions of 1.5 and 2.0 log CFU/g on APCs and YMCs, respectively; with acceptable level of bloom/wax score on fresh blueberries. This study showed that SEM, response surface, and overlaid contour plots proved successful in arriving at optima to reduce microbial counts while maintaining bloom/wax on the surface of the blueberries. PRACTICAL APPLICATION: Since chemical sanitizing treatments such as chlorine showed ineffectiveness to reduce microorganisms loaded on berry surface (Beuchat and others 2001, Sapers 2001), hot water treatment on fresh blueberries could maximize microbial reduction with acceptable quality of fresh blueberries.

Bryobacter aggregatus gen. nov., sp. nov., a peat-inhabiting, aerobic chemo-organotroph from subdivision 3 of the Acidobacteria.

Bryobacter aggregatus gen. nov., sp. nov. is proposed to accommodate three strains of slowly growing, chemo-organotrophic bacteria isolated from acidic Sphagnum peat bogs. These bacteria were strictly aerobic, Gram-negative, colourless, non-motile coccoids or short rods that multiplied by normal cell division and formed irregularly shaped cell aggregates. Strains MPL3(T), MPL1011 and MOB76 were acidotolerant, mesophilic organisms capable of growth at pH 4.5-7.2 and between 4 and 33 degrees C (optimum growth at pH 5.5-6.5 and 22-28 degrees C). The preferred growth substrates were sugars, some heteropolysaccharides and galacturonic and glucuronic acids, which are released during decomposition of Sphagnum moss. The major fatty acids were iso-C(15 : 0), C(16 : 0) and summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c); the major quinones were MK-9 and MK-10. The DNA G+C content was 55.5-56.5 mol%. Strains MPL3(T), MPL1011 and MOB76 possessed nearly identical 16S rRNA gene sequences and belonged to the phylum Acidobacteria. They represent the first taxonomically characterized members of acidobacterial subdivision 3 and display only 81.7-86.7 % 16S rRNA gene sequence similarity to other members of the Acidobacteria with validly published names. Therefore, strains MPL3(T), MPL1011 and MOB76 are classified as representatives of a novel species in a new genus, for which the name Bryobacter aggregatus gen. nov., sp. nov. is proposed; strain MPL3(T) (=ATCC BAA-1390(T) =DSM 18758(T)) is the type strain of Bryobacter aggregatus.

Sulfurihydrogenibium azorense, sp. nov., a thermophilic hydrogen-oxidizing microaerophile from terrestrial hot springs in the Azores.

Five hydrogen-oxidizing, thermophilic, strictly chemolithoautotrophic, microaerophilic strains, with similar (99-100%) 16S rRNA gene sequences were isolated from terrestrial hot springs at Furnas, São Miguel Island, Azores, Portugal. The strain, designated Az-Fu1T, was characterized. The motile, 0.9-2.0 microm rods were Gram-negative and non-sporulating. The temperature growth range was from 50 to 73 degrees C (optimum at 68 degrees C). The strains grew fastest in 0.1% (w/v) NaCl and at pH 6, although growth was observed from pH 5.5 to 7.0. Az-Fu1T can use elemental sulfur, sulfite, thiosulfate, ferrous iron or hydrogen as electron donors, and oxygen (0.2-9.0%, v/v) as electron acceptor. Az-Fu1T is also able to grow anaerobically, with elemental sulfur, arsenate and ferric iron as electron acceptors. The Az-Fu1T G+C content was 33.6 mol%. Maximum-likelihood analysis of the 16S rRNA phylogeny placed the isolate in a distinct lineage within the Aquificales, closely related to Sulfurihydrogenibium subterraneum (2.0% distant). The 16S rRNA gene of Az-Fu1T is 7.7% different from that of Persephonella marina and 6.8% different from Hydrogenothermus marinus. Based on the phenotypic and phylogenetic characteristics presented here, it is proposed that Az-Fu1T belongs to the recently described genus Sulfurihydrogenibium. It is further proposed that Az-Fu1T represents a new species, Sulfurihydrogenibium azorense.

Bacteriologic characterization of 36 strains of Roseomonas species and proposal of Roseomonas mucosa sp nov and Roseomonas gilardii subsp rosea subsp nov.

We used a polyphasic approach (sequencing analysis of the 16S ribosomal RNA gene and phenotypic analyses) to characterize 36 strains of Roseomonas species isolated from blood. Five strains, represented by strain MDA5176 (M.D. Anderson Cancer Center), were identified as Roseomonas gilardii. One strain belonged to Roseomonas genomospecies 4. The 22 strains represented by strain MDA5527 showed significant differences genotypically and phenotypically with R gilardii and other Roseomonas species and represented a new Roseomonas species; Roseomonas mucosa sp nov was proposed to denote its prominent mucoid, almost runny colonies. Eight strains, represented by strain MDA5605, had minor differences with R gilardii and displayed obvious pink to red colonies; Roseomonas gilardii subsp rosea subsp nov was proposed. For subspecies differentiation, R gilardii was proposed to be R gilardii subsp gilardii subsp nov. Unique patterns of biochemical reactions were established for these Roseomonas species, which may assist routine identification of these organisms. All 36 strains and 2 American Type Culture Collection strains were susceptible to amikacin and ciprofloxacin but resistant to cefepime and ceftazidime. They also were frequently susceptible to imipenem and ticarcillin-clavulanate but far less susceptible to ceftriaxone, trimethoprim-sulfamethoxazole, and ampicillin. R mucosa strains were most resistant, whereas R gilardii subsp gilardii strains were most susceptible.

Methylopila helvetica sp. nov. and Methylobacterium dichloromethanicum sp. nov.--novel aerobic facultatively methylotrophic bacteria utilizing dichloromethane.

Eight strains of Gram-negative, aerobic, asporogenous, neutrophilic, mesophilic, facultatively methylotrophic bacteria are taxonomically described. These icl- serine pathway methylobacteria utilize dichloromethane, methanol and methylamine as well as a variety of polycarbon compounds as the carbon and energy source. The major cellular fatty acids of the non-pigmented strains DM1, DM3, and DM5 to DM9 are C18:1, C16:0, C18:0, Ccy19:0 and that of the pink-pigmented strain DM4 is C18:1. The main quinone of all the strains is Q-10. The non-pigmented strains have similar phenotypic properties and a high level of DNA-DNA relatedness (81-98%) as determined by hybridization. All strains belong to the alpha-subgroup of the alpha-Proteobacteria. 16S rDNA sequence analysis led to the classification of these dichloromethane-utilizers in the genus Methylopila as a new species - Methylopila helvetica sp.nov. with the type strain DM9 (=VKM B-2189). The pink-pigmented strain DM4 belongs to the genus Methylobacterium but differs from the known members of this genus by some phenotypic properties, DNA-DNA relatedness (14-57%) and 16S rDNA sequence. Strain DM4 is named Methylobacterium dichloromethanicum sp. nov. (VKM B-2191 = DSMZ 6343).

Citromicrobium bathyomarinum, a novel aerobic bacterium isolated from deep-sea hydrothermal vent plume waters that contains photosynthetic pigment-protein complexes.

We have taxonomically and phylogenetically characterized a new aerobic bacterial strain (JF-1) that contains photosynthetic pigment-protein complexes and which was recently isolated from black smoker plume waters of the Juan de Fuca Ridge. Strain JF-1 is a gram-negative, yellow-pigmented, motile bacterium that is salt-, pH-, and thermotolerant. These properties are consistent with an oligotrophic adaptation to varied environmental conditions thought to exist around deep-sea hydrothermal vents. The analysis of 16S rDNA sequences revealed that strain JF-1 forms a separate phylogenetic branch between the genus Erythromonas and the Erythromicrobium-Porphyrobacter-Erythrobacter cluster within the alpha subclass of the Proteobacteria. The taxonomic name Citromicrobium bathyomarinum (gen. nov., sp. nov.) is proposed for strain JF-1.

Cell surface analysis techniques: What do cell preparation protocols do to cell surface properties?

Cell surface analysis often requires manipulation of cells prior to examination. The most commonly employed procedures are centrifugation at different speeds, changes of media during washing or final resuspension, desiccation (either air drying for contact angle measurements or freeze-drying for sensitive spectroscopic analysis, such as X-ray photoelectron spectroscopy), and contact with hydrocarbon (hydrophobicity assays). The effects of these procedures on electrophoretic mobility, adhesion to solid substrata, affinity to a number of Sepharose columns, structural integrity, and cell viability were systematically investigated for a range of model organisms, including carbon- and nitrogen-limited Psychrobacter sp. strain SW8 (glycocalyx-bearing cells), Escherichia coli (gram-negative cells without a glycocalyx), and Staphylococcus epidermidis (gram-positive cells without a glycocalyx). All of the cell manipulation procedures severely modified the physicochemical properties of cells, but with each procedure some organisms were more susceptible than others. Considerable disruption of cell surfaces occurred when organisms were placed in contact with a hydrocarbon (hexadecane). The majority of cells became nonculturable after air drying and freeze-drying. Centrifugation at a high speed (15,000 x g) modified many cell surface parameters significantly, although cell viability was considerably affected only in E. coli. The type of washing or resuspension medium had a strong influence on the values of cell surface parameters, particularly when high-salt solutions were compared with low-salt buffers. The values for parameters obtained with different methods that allegedly measure similar cell surface properties did not correlate for most cells. These results demonstrate that the methods used to prepare cells for cell surface analysis need to be critically investigated for each microorganism so that the final results obtained reflect the nature of the in situ microbial cell surface as closely as possible. There is an urgent need for new, reliable, nondestructive, minimally manipulative cell surface analysis techniques that can be used in situ.

Morphological and biochemical properties of a Sphaerotilus sp. Isolated from paper mill slimes.

Four strains of filamentous bacteria were isolated from slimes collected in different paper mill factories. Morphological and physiological characterization of the isolates indicated an affiliation with the genus Sphaerotilus. However, while the physiological properties of the isolates were almost identical, pronounced physiological differences between the isolates and Sphaerotilus natans DSM 6575(T), DSM 565, and DSM 566 with respect to their ability to metabolize complex polysaccharides, sugars, polyalcohols, or organic acids as carbon sources were detected. In contrast to the analyzed culture collection strains of S. natans, all paper mill isolates were able to grow at elevated temperatures of up to 40 degrees C. Comparative sequence analysis of nearly complete 16S ribosomal DNA (rDNA) sequences from the four new isolates demonstrated that the retrieved sequences were highly similar to each other (99.6 to 99.8% similarity) and to previously published partial 16S rDNA sequences of S. natans DSM 6575(T) and ATCC 15291. Polyphasic characterization of the isolated Sphaerotilus strains revealed interesting adaptations of the strains to the environmental paper mill conditions with regard to temperature tolerance and utilization of cellulose and starch.

Xanthobacter tagetidis sp. nov., an organism associated with Tagetes species and able to grow on substituted thiophenes.

Members of the marigold genus of flowering plants (the genus Tagetes), which synthesize and accumulate thiophene compounds in their roots, were investigated as potential sources of bacteria able to degrade substituted thiophenes. Batch and continuous enrichment cultures inoculated with compost from root balls of Tagetes patula and Tagetes erecta reproducibly produced the same predominant type of bacterium when they were supplied with thiophene-2-carboxylate (T2C) or thiophene-2-acetate (T2A) as a carbon and energy substrate. This organism was a yellow-pigmented, neutrophilic, mesophilic, gram-negative, pleomorphic, rodshaped bacterium, which we classify as a new species of the genus Xanthobacter, Xanthobacter tagetidis; strain TagT2C (= DSM 11105) is the type strain. Strain TagT2CT (T = type strain) grew on simple thiophenes, such as T2C, thiophene-3-carboxylate, and T2A, on analogs of these compounds (pyrrole-2-carboxylate and furan-2-carboxylate), and on the condensed thiophene dibenzothiophene. X. tagetidis was facultatively autotrophic, fixing carbon dioxide by means of ribulose bisphosphate carboxylase, and was able to grow on hydrogen, thiosulfate, or sulfide as an energy substrate. It also grew on a wide range of other heterotrophic, chemolithotrophic, and methylotrophic substrates. Its growth on T2C was optimal at 28 to 31 degrees C and pH 7.6 to 7.8, and the maximum growth rate in batch culture was 0.22 h-1. The DNA base composition of X.tagetidis is 68 mol% G + C. A 16S ribosomal DNA sequence analysis of strain TagT2CT showed that this organism represents a distinct lineage within the Aquabacter-Azorhizobium-Xanthobacter cluster of the alpha-2 subclass of the Pro-teobacteria. Discrimination of X. tagetidis from the other genera in this group and from other Xanthobacter species is discussed.

Metal selectivity of in situ microcolonies in biofilms of the Elbe river.

The ultrastructure of natural complex biofilm communities of the Elbe river grown in situ on microscopic glass coverslips was studied by using transmission electron microscopy and energy-dispersive x-ray (EDX) analysis. Characteristic microcolonies which measured between 3.3 and 9.3 microm in diameter were frequently observed. They had an outer envelope and harbored 6 to 30 cells. The cells formed short rods measuring 1.09 +/- 0.28 microm (n = 10) in length and 0.55 + 0.07 microm (n = 21) in width. They were surrounded by a thick layer of electron-transparent, nonosmicated matter, 120 to 300 nm thick. Individual cells exhibited a unique ultrastructural trait, namely, a concentric membrane stack which completely surrounded the cytoplasm. It consisted of three membrane doublets, which showed an overall thickness of 57 to 66 nm. The center-to-center spacing between two membrane doublets was 22.2 +/- 1.0 nm (n = 12). The bacterial cell wall seemed to be of the gram-negative type. The fact that upon shrinkage hexagonal clefts appeared proved the cells to be tightly packed, and septum formation by binary fissions was observed. All of these morphological details indicate that the cells within these microcolonies were actively growing and did not represent spore-like states. EDX analysis showed that only the electron-dense surface deposit of the microcolonies contained Mn and Fe in significant amounts, while these two elements were absent from the intercellular space and the cytoplasm of the microorganisms. In contrast, aluminum ions were able to penetrate the outer envelope of the microcolonies and were detected in the intercellular space. They were, however, completely absent from the microbial cytoplasm, indicating a filter cascade with respect to aluminum. From the ultrastructural data together with the deposition of iron and manganese on the microcolony surface, it appears that these organisms may belong to the genus Siderocapsa or Nitrosomonas. They do not precisely match any of the described species and may therefore represent a new species.

Analysis of the PHA granule-associated proteins GA20 and GA11 in Methylobacterium extorquens and Methylobacterium rhodesianum.

Electrophoretic analysis of the proteins bound to poly(3-hydroxybutyric acid), PHB-, granules in Methylobacterium extorquens, M. rhodesianum as well as the PHB-leaky mutants Mu 1 and Mu 11, which were isolated from the latter, resulted in two dominant low-molecular weight proteins, which were referred to as GA11 and GA20. After purification of these proteins antibodies against the GA11 and GA20 protein of M. extorquens were obtained. Both proteins bound to the surface of PHB granules as revealed by immunoelectron microscopy of whole cells of M. extorquens and M. rhodesianum. With cells of the PHB-leaky mutants Mu 1 and Mu 11 no specific labeling was observed. The N-terminal amino acid sequences of the GA11 and the GA20 protein were determined. We found significant homologies between the sequences of the investigated strains. The use of oligonucleotide probes based on the N-terminal sequences of the GA20 protein from M. rhodesianum to identify the corresponding structural genes in various genomic libraries failed.

Multiparametric analysis of waterline contamination in dental units.

Microbial contamination of dental unit waterlines is thought to be the result of biofilm formation within the small-bore tubing used for these conduits. Systematic sampling of 121 dental units located at the dental school of Université de Montréal showed that none of the waterlines was spared from bacterial contamination. Multilevel statistical analyses showed significant differences between samples taken at the beginning of the day and samples taken after a 2-min purge. Differences were also found between water from the turbine and the air/water syringe. Random variation occurred mainly between measurements (80%) and to a lesser extent between dental units (20%). In other analyses, it was observed to take less than 5 days before initial bacterial counts reached a plateau of 2 x 10(5) CFU/ml in newly installed waterlines. Sphyngomonas paucimobilis, Acinetobacter calcoaceticus, Methylobacterium mesophilicum, and Pseudomonas aeruginosa were the predominant isolates. P. aeruginosa showed a nonrandom distribution in dental unit waterlines, since 89.5% of the all the isolates were located in only three of the nine clinics tested. Dental units contaminated by P. aeruginosa showed significantly higher total bacterial counts than the others. By comparison, P. aeruginosa was never isolated in tap water remote from or near the contaminated dental unit waterlines. In conclusion, dental unit waterlines should be considered an aquatic ecosystem in which opportunistic pathogens successfully colonize synthetic surfaces, increasing the concentration of the pathogens in water to potentially dangerous levels. The clinical significance of these findings in relation to routine dental procedures is discussed.

The phylogenetic position and ultrastructure of the uncultured bacterium Achromatium oxaliferum.

Achromatium oxaliferum is a large, morphologically conspicuous, sediment-dwelling bacterium. Nothing is known concerning its phylogeny and it has eluded all attempts at laboratory cultivation. The limited physiological description of A. oxaliferum has been based on morphological features of the bacterium such as the presence of intracellular sulphur inclusions. A. oxaliferum cells were purified from a wetland region close to Rydal Water (Cumbria, UK). Scanning and transmission electron microscopy revealed that a number of morphologically distinct A. oxaliferum cell-types, based on cell surface features and the size and abundance of calcite and sulphur inclusions within the cells, were present in a single sample of purified cells. PCR was used to amplify almost full-length 16S rRNA gene sequences from DNA extracted from A. oxaliferum cells directly purified from sediments. The PCR products were cloned and partial sequences (approx. 400 bp) were determined for seven of the clones. Three different sequence clusters were recovered from the clone libraries. A near full-length (1489 bp) 16S rRNA gene sequence was determined for a representative clone of the most dominant sequence-type (52% of the sequences). Comparative sequence analysis showed A. oxaliferum to form a deep branching lineage within the gamma-subdivision of the Proteobacteria. A. oxaliferum was related most closely to the Chromatium assemblage that includes sulphur-oxidizing symbiotic bacteria, purple sulphur bacteria, and sulphur- and iron-oxidizing thiobacilli. Phylogenetic inferences made using distance, parsimony and maximum likelihood methods all placed A. oxaliferum with this group of bacteria. Bootstrap support for a relationship with any particular lineage within the assemblage was weak. The seven clone sequences recovered from the A. oxaliferum cells however formed a monophyletic group well supported by bootstrap analysis (85-100% support depending on the analysis done). It was concluded that A. oxaliferum was related to organisms of the Chromatium assemblage but constituted a novel lineage within this group of bacteria. A. oxaliferum cells were confirmed as the source of the 16S rRNA sequence obtained, by the use of a fluorescently-labelled 16S rRNA-targeted oligonucleotide specific for the A. oxaliferum rRNA sequence.

Sensitivity of ribosomes of the hyperthermophilic bacterium Aquifex pyrophilus to aminoglycoside antibiotics.

A poly(U)-programmed cell-free system from the hyperthermophilic bacterium Aquifex pyrophilus has been developed, and the susceptibility of Aquifex ribosomes to the miscoding-inducing and inhibitory actions of all known classes of aminoglycoside antibiotics has been assayed at temperatures (75 to 80 degrees C) close to the physiological optimum for cell growth. Unlike Thermotoga maritima ribosomes, which are systematically refractory to all known classes of aminoglycoside compounds (P. Londei, S. Altamura, R. Huber, K. O. Stetter, and P. Cammarano, J. offteriol. 170-4353-4360, 1988), Aquifex ribosomes are susceptible to all of the aminoglycosides tested (disubstituted 2-deoxystreptamines, monosubstituted 2-deoxystreptamines, sand streptidine compounds). The significance of this result in light of the Aquifex and Thermotoga placements in phylogenetic trees of molecular sequences is discussed.

Phylogeny of the Sphaerotilus-Leptothrix group inferred from morphological comparisons, genomic fingerprinting, and 16S ribosomal DNA sequence analyses.

Phase-contrast light microscopy revealed that only one of eight cultivated strains belonging to the Sphaerotilus-Leptothrix group of sheathed bacteria actually produced a sheath in standard growth media. Two Sphaerotilus natans strains produced branched cells, but other morphological characteristics that were used to identify these bacteria were consistent with previously published descriptions. Genomic fingerprints, which were obtained by performing PCR amplification with primers corresponding to enterobacterial repetitive intergenic consensus sequences, were useful for distinguishing between the genera Sphaerotilus and Leptothrix, as well as among individual strains. The complete 16S ribosomal DNA (rDNA) sequences of two strains of "Leptothrix discophora" (strains SP-6 and SS-1) were determined. In addition, partial sequences (approximately 300 nucleotides) of one strain of Leptothrix cholodnii (strain LMG 7171), an unidentified Leptothrix strain (strain NC-1), and four strains of Sphaerotilus natans (strains ATCC 13338T [T = type strain], ATCC 15291, ATCC 29329, and ATCC 29330) were determined. We found that two of the S. natans strains (ATCC 15291 and ATCC 13338T), which differed in morphology and in their genomic fingerprints, had identical sequences in the 300-nucleotide region sequenced. Both parsimony and distance matrix methods were used to infer the evolutionary relationships of the eight strains in a comparison of the 16S rDNA sequences of these organisms with 16S rDNA sequences obtained from ribosomal sequence databases. All of the strains clustered in the Rubrivivax subdivision of the beta subclass of the Proteobacteria, which confirmed previously published conclusions concerning selected individual strains. Additional analyses revealed that all of the S. natans strains clustered in one closely related group, while the Leptothrix strains clustered in two separate lineages that were approximately equidistant from the S. natans cluster. This finding suggests that the tentative species "L. discophora" needs to be more clearly defined and compared with other species belonging to the genus Leptothrix.