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Real-time imaging of fluorescent flagellar filaments.

Bacteria swim by rotating flagellar filaments that are several micrometers long, but only about 20 nm in diameter. The filaments can exist in different polymorphic forms, having distinct values of curvature and twist. Rotation rates are on the order of 100 Hz. In the past, the motion of individual filaments has been visualized by dark-field or differential-interference-contrast microscopy, methods hampered by intense scattering from the cell body or shallow depth of field, respectively. We have found a simple procedure for fluorescently labeling cells and filaments that allows recording their motion in real time with an inexpensive video camera and an ordinary fluorescence microscope with mercury-arc or strobed laser illumination. We report our initial findings with cells of Escherichia coli. Tumbles (events that enable swimming cells to alter course) are remarkably varied. Not every filament on a cell needs to change its direction of rotation: different filaments can change directions at different times, and a tumble can result from the change in direction of only one. Polymorphic transformations tend to occur in the sequence normal, semicoiled, curly 1, with changes in the direction of movement of the cell body correlated with transformations to the semicoiled form.

Escherichia coli↗

Molecular Identification and Localization of Filamentous Symbiotic Bacteria Associated with the Hydrothermal Vent Annelid Alvinella pompejana.

Alvinella pompejana is a polychaetous annelid that inhabits high-temperature environments associated with active deep-sea hydrothermal vents along the East Pacific Rise. A unique and diverse epibiotic microflora with a prominent filamentous morphotype is found associated with the worm's dorsal integument. A previous study established the taxonomic positions of two epsilon proteobacterial phylotypes, 13B and 5A, which dominated a clone library of 16S rRNA genes amplified by PCR from the epibiotic microbial community of an A. pompejana specimen. In the present study deoxyoligonucleotide PCR primers specific for phylotypes 13B and 5A were used to demonstrate that these phylotypes are regular features of the bacterial community associated with A. pompejana. Assaying of other surfaces around colonies of A. pompejana revealed that phylotypes 13B and 5A are not restricted to A. pompejana. Phylotype 13B occurs on the exterior surfaces of other invertebrate genera and rock surfaces, and phylotype 5A occurs on a congener, Alvinella caudata. The 13B and 5A phylotypes were identified and localized on A. pompejana by in situ hybridization, demonstrating that these two phylotypes are, in fact, the prominent filamentous bacteria on the dorsal integument of A. pompejana. These findings indicate that the filamentous bacterial symbionts of A. pompejana are epsilon Proteobacteria which do not have an obligate requirement for A. pompejana.

Journal Article↗

Bacterial filamentation of Yersinia pestis by beta-lactam antibiotics in experimentally infected mice.

OBJECTIVE: To identify alternatives to streptomycin for treating pneumonic plague, we evaluated beta-lactam antibiotics to treat experimental pneumonic plague in mice. METHODS: Mice were exposed to a lethal inhaled dose of Yersinia pestis and treated with beta-lactam antibiotics administered every 6 hours, starting 42 hours postexposure. RESULTS: The mice died or were euthanized in extremis 3 days postexposure. We observed marked bacterial filamentation of Y pestis in the tissues of mice treated with ceftazidime (10/10 mice), aztreonam (9/10 mice), or ampicillin (1/10 mice), but not in the tissues of mice treated with cefotetan, cefazolin, ceftriaxone, or saline. There was no evidence of septation of the filamentous bacteria by light or electron microscopy. The filamentous bacteria were confirmed as Y pestis based on their reactivity with rabbit anti-Y pestis F1 serum. CONCLUSIONS: Marked bacterial filamentation of Y pestis can be produced in vivo by certain beta-lactam antibiotics. This antibiotic-induced morphologic change is important because filamentous bacteria in clinical samples could possibly be confused with filamentous actinomycotic organisms.

Animals↗

Numbers and locations of native bacteria on field-grown wheat roots quantified by fluorescence in situ hybridization (FISH).

Native bacteria, Pseudomonas and filamentous bacteria were quantified and localized on wheat roots grown in the field using fluorescence in situ hybridization (FISH). Seminal roots were sampled through the season from unploughed soil in a conservation farming system. Such soils are spatially heterogeneous, and many roots grow slowly through hard soil with cracks and pores containing dead roots remnant from previous crops. Root and rhizosphere morphology, and contact with soil particles were preserved, and autofluorescence was avoided by observing sections in the far-red with Cy5 and Cy5.5 fluorochromes. Spatial analyses showed that bacteria were embedded in a stable matrix (biofilm) within 11 microm of the root surface (range 2-30 microm) and were clustered on 40% of roots. Half the clusters co-located with axial grooves between epidermal cells, soil particles, cap cells or root hairs; the other half were not associated with visible features. Across all wheat roots, although variable, bacteria averaged 15.4 x 10(5) cells per mm(3) rhizosphere, and of these, Pseudomonas and filaments comprised 10% and 4%, respectively, with minor effects of sample time, and no effect of plant age. Root caps were most heavily colonized by bacteria along roots, and elongation zones least heavily colonized. Pseudomonas varied little with root development and were 17% of bacteria on the elongation zone. Filamentous bacteria were not found on the elongation zone. The most significant factor to rhizosphere populations along a wheat root, however, was contact with dead root remnants, where Pseudomonas were reduced but filaments increased to 57% of bacteria (P < 0.001). This corresponded with analyses of root remnants showing they were heavily colonized by bacteria, with 48% filaments (P < 0.001) and 1.4%Pseudomonas (P = 0.014). Efforts to manage rhizosphere bacteria for sustainable agricultural systems should continue to focus on root cap and mucilage chemistry, and remnant roots as sources of beneficial bacteria.

Agriculture↗

In situ detection of cell surface hydrophobicity of probe-defined bacteria in activated sludge.

The surface hydrophobicity of different types of bacteria in activated sludge were investigated under in situ conditions by following the adhesion of fluorescent microspheres with defined surface properties to bacterial surfaces (the MAC-method). This technique was combined with identification of the bacteria with fluorescence in situ hybridization with rRNA-targeted oligonucleotides (FISH) and could thus be used for characterization of surface properties of probe-defined bacteria directly in a complex system without prior enrichment or isolation. This MAC-FISH technique could be used for single bacteria as well as filamentous bacteria. In the investigated activated sludge from an industrial wastewater treatment plant, two types of filamentous bacteria dominated. One morphotype consistently attracted only very few hydrophobic microspheres, indicating that the thin sheath of exopolymers around the cells had a hydrophilic surface. Use of a hierarchical set of gene probes revealed that these filaments were sulphide oxidising Thiothrix spp. The other predominating filamentous morphotype had a thick, very hydrophobic exopolymeric sheath. This filamentous bacterium was found to belong to the alpha-Proteobacteria. The relevance of the significant differences in surface hydrophobicity for the two morphotypes in respect to substrate uptake and floc formation is discussed.

Bacteria↗

Filamentous sulfur bacteria of activated sludge: characterization of Thiothrix, Beggiatoa, and Eikelboom type 021N strains.

Seventeen strains of filamentous sulfur bacteria were isolated in axenic culture from activated sludge mixed liquor samples and sulfide-gradient enrichment cultures. Isolation procedures involved plating a concentrated inoculum of washed filaments onto media containing sulfide or thiosulfate. The isolates were identified as Thiothrix spp., Beggiatoa spp., and an organism of uncertain taxonomic status, designated type 021N. All bacteria were gram negative, reduced nitrate, and formed long, multicellular trichomes with internal reserves of sulfur, volutin, and sudanophilic material. Thiothrix spp. formed rosettes and gonidia, and four of six strains were ensheathed. Type 021N organisms utilized glucose, lacked a sheath, and differed from Thiothrix spp. in several aspects of cellular and cultural morphology. Beggiatoa spp. lacked catalase and oxidase, and filaments were motile. Biochemical and physiological characterization of the isolates revealed important distinguishing features between the three groups of bacteria. Strain differences were most evident among the Thiothrix cultures. A comparison of the filamentous sulfur bacteria with freshwater strains of Leucothrix was made also.

Bacteroidetes↗

Quantification of cell-specific substrate uptake by probe-defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization.

A technique based on quantitative microautoradiography (QMAR) and fluorescence in situ hybridization (FISH) was developed and evaluated in order to determine the quantitative uptake of specific substrates in probe-defined filamentous bacteria directly in a complex system. The technique, QMAR-FISH, has a resolution of a single cell and is based on an improved fixation protocol and the use of an internal standard of bacteria with known specific radioactivity. The method was used to study the in situ ecophysiology of the filamentous bacteria 'Candidatus Meganema perideroedes' and Thiothrix sp. directly in an activated sludge system. The cellular uptake rate of tritium-labelled substrates revealed an average cell-specific uptake rate of 4.1 yen 10-15 mol of acetate cell-1 h-1 and 3.1 yen 10-15 mol of acetate cell-1 h-1 for the two filamentous species respectively. The two filamentous species had very similar activity in all cells along each filament. Surprisingly, the filaments within both probe-defined populations had threefold variation in activity between the different filaments, demonstrating a large variation in activity level within a single population in a complex system. The substrate affinity (Ks) for uptake of acetate of the cells within the two filamentous bacteria was determined by incubation with variable concentrations of labelled acetate. The Ks values of the 'Candidatus Meganema perideroedes' and the Thiothrix filamentous bacteria were determined to be 1.8 micro M and 2.4 micro M acetate respectively.

Acetates↗

Effect of feeding pattern and storage on the sludge settleability under aerobic conditions.

The selection of filamentous bacteria is often assumed to be associated with specific microbial properties such as growth rate, substrate uptake rate, substrate affinity and potential for substrate storage. In this study we aimed to verify some of these factors. Sequencing batch reactor (SBR) systems were used to scale-down aerobic activated sludge systems with an aerobic selector. Adding acetate in different aerobic feeding periods allowed us to simulate a variable relative size of aerobic selector with different bulk liquid substrate concentrations. The experiments showed that as expected, the aerobic fill time ratio (FTR(ox)) and the corresponding feast period, which can be assumed similar to contact time in an aerobic selector, had a strong effect on the sludge settleability. Promoting a strong substrate gradient in the SBR (FTR(ox)<5.4%) resulted in good sludge settleability (SVI<120mLg(-1)). Whenever acetate was added in a limiting rate (FTR(ox)>6.2%), a condition in which the acetate concentration in the reactor was always very low, the sludge settleability decreased (SVI>150mLg(-1)). Sludge settleability could be improved by changing the feeding strategy to a pulse feed. The maximum specific acetate uptake rate and poly beta-hydroxybutyrate (PHB) production rate of bad settling sludge, including bulking sludge, was similar to well-settling sludge, which is not in accordance with the general assumptions that well settling sludge have a higher maximal substrate uptake rate and better storage capacities. An alternative hypothesis for the development of filamentous structures in biological flocs has been formulated. It is hypothesized that bulking sludge originates from the presence of substrate gradients in sludge aggregates. Whereas at low bulk liquid substrate concentration filamentous bacteria give easier access to the substrate at the outside of the flocs and thereby proliferate, at high bulk liquid substrate concentration there is no substrate advantage for filamentous organisms and smooth bacterial structures predominate. In this hypothesis there is no need for an intrinsic difference in kinetic parameters between floc and filamentous bacteria. Where presence of filamentous bacteria is related to process conditions, the presence of a specific filament is likely due to presence of a specific limiting substrate.

Bacteria, Aerobic↗

Morphological and compositional changes in a planktonic bacterial community in response to enhanced protozoan grazing.

We analyzed changes in bacterioplankton morphology and composition during enhanced protozoan grazing by image analysis and fluorescent in situ hybridization with group-specific rRNA-targeted oligonucleotide probes. Enclosure experiments were conducted in a small, fishless freshwater pond which was dominated by the cladoceran Daphnia magna. The removal of metazooplankton enhanced protozoan grazing pressure and triggered a microbial succession from fast-growing small bacteria to larger grazing-resistant morphotypes. These were mainly different types of filamentous bacteria which correlated in biomass with the population development of heterotrophic nanoflagellates (HNF). Small bacterial rods and cocci, which showed increased proportion after removal of Daphnia and doubling times of 6 to 11 h, belonged nearly exclusively to the beta subdivision of the class Proteobacteria and the Cytophaga-Flavobacterium cluster. The majority of this newly produced bacterial biomass was rapidly consumed by HNF. In contrast, the proportion of bacteria belonging to the gamma and alpha subdivisions of the Proteobacteria increased throughout the experiment. The alpha subdivision consisted mainly of rods that were 3 to 6 microm in length, which probably exceeded the size range of bacteria edible by protozoa. Initially, these organisms accounted for less than 1% of total bacteria, but after 72 h they became the predominant group of the bacterial assemblage. Other types of grazing-resistant, filamentous bacteria were also found within the beta subdivision of Proteobacteria and the Cytophaga-Flavobacterium cluster. We conclude that the predation regimen is a major structuring force for the bacterial community composition in this system. Protozoan grazing resulted in shifts of the morphological as well as the taxonomic composition of the bacterial assemblage. Grazing-resistant filamentous bacteria can develop within different phylogenetic groups of bacteria, and formerly underepresented taxa might become a dominant group when protozoan predation is the major selective pressure.

Animals↗

The effect of depth on the accrual of marine biofilms on glass substrata deployed in the Clyde Sea, Scotland.

Recent concerns about fouling problems caused by biofilms affecting optical oceanographic instruments have highlighted the need for a better understanding of their nature and extent in the marine environment. Glass slides were deployed in April and August for periods of up to 3 weeks at 5, 10, 20, 40, 80 and 160 m in the water column of Loch Fyne, Clyde Sea, W Scotland. Biofilms were enumerated using epifluorescence and bright field microscopy. During April the biofilm community varied significantly with depth, although this effect was attributable solely to changes in the diatom community. Diatom numbers peaked between 10 and 20 m. During August the biofilm community also showed a significant depth effect, although in this case there were significant effects for diatoms, rods, and filamentous bacteria. Cell numbers for diatoms, filamentous bacteria, and rod shaped bacteria peaked at 5 m. There was a significant linear relationship between the number of diatoms and bacteria on the slides deployed in August. No such relationship was found for the April data. The results indicate that optical performance may be significantly degraded after a few weeks and highlights the need for provision of suitable strategies to protect such surfaces from biofilm accumulation.

Bacteria↗

Observations of fouling biofilm formation.

Fouling biofilm development was monitored in a completely mixed tubular recycle reactor. A unique sampling system allowed direct (brightfield, epifluorescence, and scanning electron photomicroscopy) and indirect (increased fluid frictional resistance) observations of biofilms. Low fluid velocity (138.5 cm/s) experiments had shorter induction times and biofilm matrixes which included firmly adherent filamentous bacteria. High fluid velocity (265.4 cm/s) experiments had longer induction times with firmly adherent filamentous bacteria present only after the accumulation of extracellular materials. In both cases the fluid frictional resistance increased after filamentous bacteria became a permanent part of the biofilm.

Adhesiveness↗

[Phylogeny of anoxygenic filamentous phototrophic bacteria of the family Oscillochloridaceae as inferred from comparative analyses of the rrs, cbbL, and nifH genes].

Phylogeny of anoxygenic filamentous phototrophic bacteria (AFPB) of the family Oscillochloridaceae (Oscillochloris trichoides DG6T and the recently isolated strains Oscillochloris sp. R and C6) was studied based on comparative analyses of the genes coding for 16S rRNA (rrs), ribulose- 1,5-bisphosphate carboxylase/oxygenase (cbbL), and nitrogenase (nifH). The sequences of the genes studied proved to be identical in the three strains, which is in agreement with data obtained earlier that showed lack of differentiating phenotypic distinctions between these strains; therefore, it is proposed that the new strains should be identified as representatives of the species O. trichoides. Using an earlier designed system of oligonucleotide primers and a specially designed additional primer, fragments of the cbbL genes of the "red-like" form I RuBPC were amplified and sequenced for all of the O. trichoides strains. Analysis of the cbbL genes suggested a separate position of the bacteria studied in the phylogenetic tree, where O. trichoides strains formed an independent branch, which, apart from this species, also included the only studied species of gram-positive facultatively chemoautotrophic bacteria, Sulfobacillus acidophilus. In the phylogenetic tree inferred from the analysis of nifH genes, the bacteria under study also formed a new separate branch, deviating near the root, which indicated lack of relatedness between them and other phototrophic bacteria. The data obtained support the conclusion that AFPB has an ancient origin and their identification as one of the main evolutionary lineages of eubacteria, which was made based on the analysis of ribosomal genes.

Bacteria, Anaerobic↗

Diversity and distribution in hypersaline microbial mats of bacteria related to Chloroflexus spp.

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of "green nonsulfur bacteria." PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.

Chlorobi↗

Microbial composition and structure of a rotating biological contactor biofilm treating ammonium-rich wastewater without organic carbon.

High nitrogen losses were observed in a rotating biological contactor (RBC) treating ammonium-rich (up to 500 mg NH4(+)-N/L) but organic-carbon-poor leachate from a hazardous waste landfill in Kölliken, Switzerland. The composition and spatial structure of the microbial community in the biofilm on the RBC was analyzed with specific attention for the presence of aerobic ammonium and nitrite oxidizing bacteria and anaerobic ammonium oxidizers. Anaerobic ammonium oxidation (anammox) involves the oxidation of ammonium with nitrite to N2. First the diversity of the biofilm community was determined from sequencing cloned PCR-amplified 16S rDNA fragments. This revealed the presence of a number of very unusual 16S rDNA sequences, but very few sequences related to known ammonium or nitrite oxidizing bacteria. From analysis of biofilm samples by fluorescence in situ hybridization with known phylogenetic probes and by dot-blot hybridization of the same probes to total RNA purified from biofilm samples, the main groups of microorganisms constituting the biofilm were found to be ammonium-oxidizing bacteria from the Nitrosomonas europaea/eutropha group, anaerobic ammonium-oxidizing bacteria of the "Candidatus Kuenenia stuttgartiensis" type, filamentous bacteria from the phylum Bacteroidetes, and nitrite-oxidizing bacteria from the genus Nitrospira. Aerobic and anaerobic ammonium-oxidizing bacteria were present in similar amounts of around 20 to 30% of the biomass, whereas members of the CFB phylum were present at around 7%. Nitrite oxidizing bacteria were only present in relatively low amounts (less than 5% determined with fluorescence in situ hybridization). Data from 16S rRNA dot-blot and in situ hybridization were not in all cases congruent. FISH analysis of thin-sliced and fixed biofilm samples clearly showed that the aerobic nitrifiers were located at the top of the biofilm in an extremely high density and in alternating clusters. Anammox bacteria were exclusively present in the lower half of the biofilm, whereas CFB-type filamentous bacteria were present throughout the biofilm. The structure and composition of these biofilms correlated very nicely with the proposed physiological functional separations in ammonium conversion.

Bacteria, Aerobic↗

Structural proteins involved in emergence of microbial aerial hyphae.

Filamentous fungi and filamentous bacteria (i.e., the streptomycetes) belong to different kingdoms that diverged early in evolution. Yet, they adopted similar lifestyles. After a submerged feeding mycelium has been established, hyphae grow into the air and form aerial structures from which (a)sexual spores can develop. These spores are dispersed and can give rise to a new mycelium. Some of the key processes involved in the formation of aerial hyphae by these microbes appear to be very similar. In both cases molecules that lower the surface tension are secreted into the aqueous environment, thereby enabling hyphae to grow into the air. Aerial hyphae are then covered with a hydrophobic film. In fungi, this film is characterized by a mosaic of parallel rodlets, while similar rodlets have also been observed on aerial structures of filamentous bacteria. Although the erection of aerial hyphae in both filamentous fungi and filamentous bacteria is dependent upon (poly)peptides that are structurally unrelated, they can, at least partially, functionally substitute for each other.

Bacterial Proteins↗

Operational strategies for a small wastewater treatment plant using OUR, microscopic analysis, and toxicity test.

This study was focused on determining the optimal operational conditions of a small domestic wastewater treatment plant (SWP) with a flow rate of 1000 m3 d(-1) using oxygen uptake rate (OUR) data, microscopic analyses, and effluent toxicity testing with Daphnia magna in parallel to wastewater characterization. Chemical analyses of nine samples, taken from different points of the treatment plant over a nine months period, presented strong domestic wastewater characteristics. The plant performance for meeting discharge standards was not sufficient enough for TP parameter. Aeration capacity and sludge recycle ratio were adjusted to improve the performance of the system and to prevent any operational problems, such as bulking. Although the inflow was 100% toxic, effluent toxicity following treatment was decreased to 5%. Filamentous bacteria were in "excessive" amount. The dominant filamentous bacteria species were determined as Nostocoioda limicola II. Reevaluation of the discharge limits with respect to nutrient removal, geographic location, and receiving water quality subject to treatment effluent discharges.

Animals↗

Clinical, pathological, and epizootiological features of long-segmented filamentous organisms (bacteria, LSFOs) in the small intestines of chickens, turkeys, and quails.

Long-segmented filamentous organisms (LSFOs) are easily recognized gram-positive bacteria that infect several species of animals. The present study describes the epizootiological, clinical, and pathological characteristics of LSFOs in chicks, turkey poults, and quails in Georgia and California. LSFOs are most likely to be seen in young poultry that have gastrointestinal illnesses during winter. Concomitant infections with other bacteria and protozoans are common. Although inflammation and displacement of microvilli are characteristics of LSFO infections in these birds, LSFOs are not necessarily pathogens. They may be normal intestinal flora or commensal organisms that overgrow when certain unknown gastrointestinal conditions are correct or certain unknown events occur.

Animals↗

Monitoring granule formation in anaerobic upflow bioreactors using oligonucleotide hybridization probes.

The process of granule formation in upflow anaerobic sludge blanket (UASB) reactors was studied using oligonucleotide hybridization probes. Two laboratory-scale UASB reactors were inoculated with sieved primary anaerobic digester sludge from a municipal wastewater treatment plant and operated similarly except that reactor G was fed glucose, while reactor GP was fed glucose and propionate. Size measurements of cell aggregates and quantification of different populations of methanogens with membrane hybridization targeting the small-subunit ribosomal RNA demonstrated that the increase in aggregate size was associated with an increase in the abundance of Methanosaeta concilii in both reactors. In addition, fluorescence in situ hybridization showed that the major cell components of small aggregates collected during the early stages of reactor startup were M. concilii cells. These results indicate that M. concilii filaments act as nuclei for granular development. The increase in aggregate size was greater in reactor GP than in reactor G during the early stages of startup, suggesting that the presence of propionate-oxidizing syntrophic consortia assisted the formation of granules. The mature granules formed in both reactors exhibited a layered structure with M. concilii dominant in the core, syntrophic consortia adjacent to the core, and filamentous bacteria in the surface layer. The excess of filamentous bacteria caused delay of granulation, which was corrected by increasing shear through an increase of the recycling rate.

Bioreactors↗