Ultrastructure of Thiothrix spp. and “Type 021N” Bacteria

The ultrastructural features of two groups of filamentous sulfur bacteria, Thiothrix spp. and an unnamed organism designated “type 021N,” were examined by transmission electron microscopy. Negative staining of whole cells and filaments with uranyl acetate revealed the presence of tufts of fimbriae located at the ends of individual gonidia of Thiothrix sp. strain A1 and “type 021N” strain N7. Holdfast material present at the center of mature rosettes was observed in thin sections stained with ruthenium red. A clearly defined sheath enveloped the trichomes of two of three Thiothrix strains but was absent from “type 021N” filaments. The outer cell wall appeared more complex in “type 021N” strains than in Thiothrix isolates. Bulbs or clusters of irregularly shaped cells, often present in filaments of “type 021N” bacteria, appeared to result from crosswalls which formed at angles oblique to the filament axis. The multicellular nature of these sulfur bacteria was apparent in that only the cytoplasmic membrane and peptidoglycan layer of the cell wall were involved in the septation process. Sulfur inclusions which developed in the presence of sodium thiosulfate were enclosed by a single-layered envelope and located within invaginations of the cytoplasmic membrane.     

Symbiotic Relationship of Thiothrix spp. with an Echinoderm

Immunoassay procedures were used to investigate the symbiotic relationship of Thiothrix spp. in the intestinal cecum of the spatangoid species Echinocardium cordatum. Thiothrix spp. were identified in nodule samples from E. cordatum digestive tubes based on microscopic examination, enzyme-linked immunosorbent assay, and indirect immunofluorescence. Thiothrix spp. protein made up as much as 84% of the total protein content of the nodules. This is the first identification of Thiothrix spp. internally symbiotic with marine invertebrates.     

Molecular phylogeny and taxonomy of colorless, filamentous sulfur bacteria of the genus Thiothrix

Comprehensive investigation combining molecular genetic techniques and comparative studies of morphological and physiological properties made it possible to resolve the disputed issue of the taxonomic status of the groups ??T. nivea?? and ??Eikelboom type 021N?? of the genus Thiothrix. The phylogenetic trees constructed on the basis of 16S rRNA and gyrB gene sequences demonstrated that members of the genus Thiothrix formed a cluster within the order Thiotrichales. According to the ??ribosomal?? tree, the cluster of the genus Thiothrix was divided into two main groups, I and II, corresponding to the groups ??T. nivea?? and ??Eikelboom type 021N??. The levels of similarity between the 16S rRNA gene sequences of Thiothrix species reached 88.9?C100%. On the contrary, in the ??gyrase?? tree, these species were not divided into ??T. nivea?? and ??Eikelboom type 021N?? groups. The levels of similarity between the amino acid sequences of the gyrB gene fragments of Thiothrix species varied from 74.5 to 99.2%. Importantly, members of the groups ??T. nivea?? and ??Eikelboom type 021N?? formed very similar 16S rRNA secondary structures in the variable region V3, where a 30-nucleotide deletion characteristic of all Thiothrix species was detected. Phenotypic analysis of the studied bacteria revealed some morphological and physiological properties shared by the groups ??T. nivea?? and ??Eikelboom type 021N??. The data obtained indicate that members of the groups ??T. nivea?? and ??Eikelboom type 021N?? are phenotypically and genetically heterogeneous species within the single monophyletic genus Thiothrix..     

Sorption of Heavy Metals to the Filamentous Bacterium Thiothrix Strain A1

A study was undertaken to determine the ability of the filamentous bacterium Thiothrix strain A1 to sorb heavy metals from solution. Cells of Thiothrix strain A1 were harvested, washed, and suspended in solutions of metals. After an equilibration period, biomass was separated from solution and the metal content in acid-digested cells and/or filtrates was determined by atomic absorption spectrophotometry. Sorption of nickel and zinc was very rapid; most of the sorbed metal was bound in less than 10 min. The sorption data for copper fit the Freundlich isotherm, and nickel and zinc data fit biphasic Freundlich isotherms. Sorption of both nickel and zinc was dependent on cell age. Cells harvested 24 h after inoculation sorbed approximately one-half of the amount of metal per gram cell protein than did cells harvested after 48, 72, or 96 h. Calcium and magnesium effectively competed with zinc for binding sites, whereas potassium had only a slight effect on the capacity of cells to sorb zinc. The primary mechanism of metal sorption apparently was ion exchange, because 66 to 75% of nickel or zinc could be desorbed by placing metal-laden cells in a solution of 5 mM CaCl₂. A competition experiment with nickel and zinc indicated that both metals occupied the same sorption sites. The strong chelating agents EDTA and NTA effectively prevented metal uptake, but lactate enhanced the uptake of nickel. Thiothrix strain A1 grown in nickel-containing medium had a relatively low uptake of nickel compared with uptake by resting cells suspended in a simple buffer solution.     

Novel Epibiotic Thiothrix Bacterium on a Marine Amphipod

Comparative analysis of the 16S rRNA gene and fluorescent in situ hybridization (FISH) was used to identify epibiotic filamentous bacteria living on the marine amphipod crustacean Urothoe poseidonis. The epibionts belong to the gamma proteobacteria and represent a novel marine phylotype within the genus Thiothrix. FISH and denaturing gradient gel electrophoresis revealed that the Thiothrix filaments are present on the majority of the amphipods examined.     

Biofouling of Groundwater Systems by Thiothrix spp.

Thiothrix spp., sulfide-oxidizing filamentous bacteria, were found to be a principal bacterial component of aquatic biofilms causing biofouling in selected municipal water storage tanks, private wells, and drip irrigation systems in Florida. Treatments of up to 200 ppm chlorine in the affected systems could not prevent return of the biofouling problem. The water originated from the upper Floridan aquifer and associated surficial aquifers in central and north Florida. Samples were examined where visible biofilms had a white, filamentous appearance, indicative of Thiothrix spp. The detection of Thiothrix spp. was confirmed by enzyme-liked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and microbiological procedures. It was estimated through immunocytochemical procedures that Thiothrix spp. comprised 18% of the biofilm in the municipal water storage tanks. These observations confirm that specific biological and chemical interactions may induce physical changes leading to significant biofouling. Received: 6 November 1996 / Accepted: 14 March 1997     

Remediation of Thiothrix spp. associated bulking problems by raw wastewater feeding: A full-scale experience

An industrial wastewater treatment plant (WWTP) in Australia has long suffered from bulking problems associated with the proliferation of Thiothrix spp. The WWTP consists of a covered anaerobic lagoon (CAL) followed by a sequencing batch reactor (SBR). The CAL functions as both an anaerobic digester and surge lagoon for the irregular flow of wastewater generated from the production of seasonal products. Chemical analysis of the raw influent showed it was composed of a mixture of organic acids, phenols and alcohols. The CAL effluent was characterised by high acetic acid and phenolic concentrations. An attempt was made to manipulate the SBR microbial community to improve settling by direct feeding small volumes of raw influent into the SBR. After raw feeding, the plant ceased bulking as the settled sludge volume reduced from 930 to 200 mL L^?1. 16S rRNA gene profiling and biovolumes of SBR samples revealed major changes in the microbial community. The Thiothrix spp. population decreased from 36.8% to 0.2%, and Zoogloea spp. dominated all samples after raw feeding. Therefore, direct feeding is proposed as a control method for industrial plants with surge/anaerobic lagoons in order to manage the bulking problems caused by Thiothrix spp. in downstream SBRs.     

Capacity for nitrate respiration as a new aspect of metabolism of the filamentous sulfur bacteria of the genus Thiothrix

Capacity of Thiothrix species (T. lacustris strains AS and BL^T, T. caldifontis G1^T, T. unzii A1^T, and T. eikelboomii AR3^T) for anaerobic respiration in the presence of nitrate was discovered. The dynamics of nitrate reduction to nitrite was studied and the coupling of this process to thiosulfate oxidation was shown. The investigated Thiothrix representatives performed anaerobic thiosulfate-dependent reduction of nitrate only to nitrite. The presence of the narG gene, encoding the α-subunit of respiratory nitrate reductase NarGHI, was revealed in the cells. The induction of this gene expression was shown for the T. lacustris strain AS under anaerobic conditions of growth. The activity of several enzymes involved in the conversion of reduced sulfur compounds was determined.     

Isolation ofThiothrix in pure culture and observation of a filamentous epiphyte onThiothrix

Thiothrix was isolated from two sites, one in Arizona and the other in Florida, although the former strain was subsequently lost. This is the first report of the isolation ofThiothrix in pure culture. The cells in aThiothrix bloom found in a stock tank had filamentous “appendages”, which proved to consist of a segmented, trichomatous, epiphytic bacterium.     

HCO3− Fixation by Naturally Occurring Tufts and Pure Cultures of Thiothrix nivea

Naturally occurring tufts of the mixotroph Thiothrix nivea blanketed the East Everglades (Dade County, Fla.) Chekika artesian well and runoff areas. The rate of HCO₃⁻ fixation by these Thiothrix tufts was determined to be 14.0 ± 5.4 nmol of HCO₃⁻ per min per mg of dry weight, which reflected a growth rate of 5.0%/h. The addition of 10 mM glucose, ribose, acetate, or pyruvate or 0.05% Casamino Acids (Difco Laboratories, Detroit, Mich.) did not appear to alter the HCO₃⁻ fixation rate. Whereas 1 mM acetate or 10 mM lactate, ethanol, glycerol, α-ketoglutarate, succinate, fumarate, or citrate slightly stimulated HCO₃⁻ fixation, 5 to 10 mM malate inhibited HCO₃⁻ fixation by 90%. Pure Thiothrix cultures isolated from Chekika fixed HCO₃⁻ at rates as high as 29.9 ± 2.8 nmol of HCO₃⁻ per min per mg of dry weight in the presence of growth medium. Malate did not have a suppressive effect but rather slightly stimulated in vivo HCO₃⁻ fixation.     

Chemolithoutotrophic growth of Thiothrix ramosa

Thiothrix has been shown for the first time to be able to grow chemolithoautotrophically with thiosulphate or carbon disulphide as sole energy substrate. Thiosulphate served as the growth-limiting substrate for Thiothrix ramosa in chemostat culture. Maximum growth yield (Y_max) from yields at growth rates between 0.029–0.075 h^-1 was 4.0 g protein/mol thiosulphate oxidized. The key enzyme of the Calvin cycle, ribulose 1,5-bisphosphate carboxylase, was present in these cells, as were rhodanese, adenylyl sulphate (APS) reductase and sulphur-oxidizing enzyme. Thiosulphate-grown cells oxidized thiosulphate, sulphide, tetrathionate and carbon disulphide. Oxidation kinetics for sulphide, thiosulphate and tetrathionate were biphasic: oxygen consumption during the fast first phase of oxidation indicated oxidation of sulphide, and the sulphane moieties of thiosulphate and tetrathionate, to elemental sulphur, before further oxidation to sulphate. Kinetic constants for these four substrates were determined. T. ramosa also grew mixotrophically in batch culture on lactate with a number of organic sulphur compounds: carbon disulphide, methanethiol and diethyl sulphide. Substituted thiophenes were also used as sole substrates. The metabolic versatility of T. ramosa is thus much greater than previously realised.     

Dominance of Epiphytic Filamentous Thiothrix spp. on an Aquatic Macrophyte in a Hydrothermal Vent Flume in Sedge Bay, Yellowstone Lake, Wyoming

Sublacustrine hydrothermal vents, geysers, and fumaroles impart regions of Yellowstone Lake with distinctive chemical compositions that generate unique freshwater habitats and support diverse microbial life. Some microbial communities within Sedge Bay manifest themselves as accumulations of white-colored films on the surfaces of aquatic macrophytes located within the hydrothermal flow of vents. It was hypothesized that the white films were the product of microbial growth, particularly sulfur-oxidizing bacteria. An investigation of the relevant biological compounds in the vent waters was conducted. Microscopy, non-culture molecular techniques, and phylogenetic analysis were used to assay the bacterial diversity associated with the films. Microscopic analysis of the white films revealed the presence of long filaments (>200 μm) that contained sulfur granules. Filaments with these characteristics were not detected on the normal macrophyte samples. Nucleic acids were extracted from the surface of macrophyte coated with the white film (SB1, SB2) and from the surface of an uncoated macrophyte (SC). 16S ribosomal (rRNA) genes were amplified with the polymerase chain reaction (PCR) and cloned. Amplified ribosomal DNA restriction analysis (ARDRA) was used to examine 100 clones from each library and identify unique phylotypes. S_Chao1 and the Shannon Index, mathematical measures of richness and heterogeneity, were employed to assess the ARDRA pattern diversity of each sample. The SC community contained 50 unique phylotypes, predominantly cyanobacteria and proteobacteria, and was the most heterogeneous. SB1 and SB2 communities were less heterogeneous and dominated by Thiothrix. Dilution to extinction PCR conducted with specific primers indicated that the relative abundance of Thiothrix 16S rRNA gene copies in all three samples were similar. However, reduced sulfur compounds from the vent resulted in a more narrow habitat that supported the sulfur-oxidizing Thiothrix in the white film to the exclusion of cyanobacteria and other proteobacteria found on the normal macrophyte. The majority of 16S rRNA gene sequences obtained in this study displayed similarities ≤98% to any known sequence in public data bases which suggests an abundance of new bacterial species in Sedge Bay.     

Biodiversity and monitoring of colorless filamentous bacteitrtn sulfide aquatic systems of North Caucasus region

Bacterial mats in sulfide aquatic systems of North Caucasus are basically composed by the species of genera Thiothrix and Sphaerotilus. Additionally, several non-filamentous sulfur-oxidizing bacteria were isolated from the mats and several minor 16S rRNA phylotypes were found in clone libraries from these mats. The minor components were affiliated with Proteobacteria, Chlorobia, Cyanobacteria and Firmicutes. Even in an individual mat population heterogeneity of Thiothrix spp. was revealed by analysis of 16S rRNA gene and RAPD-PCR. Five Thiothrix isolates were described as new species Thiothrix caldifontis sp. nov. and Thiothrix lacustris sp. nov. In the Thiothrix-Sphaerotilus type of bacterial mat the proportion of dominant organisms might be influenced by sulfide concentration in the spring water. The higher sulfide concentration (more than 10 mg/1) in the spring water is more favorable for the development of bacterial mats with dominant Thiothrix organisms than for Thiothrix-Sphaerotilus type of sulfur mat.     

Purification of two pressure-regulated c-type cytochromes from a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F

From a deep-sea barophilic bacterium, Shewanella sp. strain DB-172F, a membrane-bound cytochrome c-551 and a cytoplasmic cytochrome c-552 were purified. The cytochrome c-551 contained 44.2 nmol of heme c mg protein⁻¹ and cytochrome c-552 contained 31.3 nmol of heme c mg protein⁻¹. The CO difference spectrum of cytochrome c-551 showed a peak at 413.7 nm and troughs at 423.2, 522 and 552 nm which indicated that this cytochrome combined with CO. Cytochrome c-551 was found to consist of two subunits with molecular masses of 29.1 kDa and 14.7 kDa, respectively, and each subunit contained one heme c molecule. Cytochrome c-552 also consisted of two subunits with molecular masses of 16.9 kDa and 14.7 kDa, respectively, and only one of these subunits contained heme c. Cytochrome c-551 was constitutively synthesized when the cells were grown at pressures of either 0.1 MPa or 60 MPa, whereas cytochrome c-552 was synthesized only at 0.1 MPa. These results together with the results of analysis of membrane-associated catalytic activities suggest that the respiratory system of DB-172F is regulated by pressure and may be intimately related to the baroadaptability mechanism of this deep-sea bacterium.     

Croatian mayflies (Insecta,Ephemeroptera): species diversity and distribution patterns

Knowledge of the mayfly biodiversity in the Balkan Peninsula is still far from complete. Compared to the neighbouring countries, the mayfly fauna in Croatia is very poorly known. Situated at the crossroads of central and Mediterranean Europe and the Balkan Peninsula, Croatia is divided into two ecoregions: Dinaric western Balkan and Pannonian lowland. Mayflies were sampled between 2003 and 2013 at 171 sites, and a total of 66 species was recorded. Combined with the literature data, the Croatian mayfly fauna reached a total of 79 taxa. Of these, 29 species were recorded for the first time in Croatia while 15 species were not previously recorded in Dinaric western Balkan ecoregion. Based on the mayfly assemblage, sampling sites were first structured by ecoregion and then by habitat type. In comparison with the surrounding countries, the Croatian mayfly fauna is the most similar to the Hungarian and Bosnian fauna. Some morphologically interesting taxa such as Baetis cf. nubecularis Eaton, 1898 and Rhithrogena from the diaphana group were recorded. Ephemera cf. parnassiana Demoulin, 1958, the species previously recorded only from Greece, was also recorded.     

Repeatedly Evolved Host-Specific Ectosymbioses between Sulfur-Oxidizing Bacteria and Amphipods Living in a Cave Ecosystem

Ectosymbioses between invertebrates and sulfur-oxidizing bacteria are widespread in sulfidic marine environments and have evolved independently in several invertebrate phyla. The first example from a freshwater habitat, involving Niphargus ictus amphipods and filamentous Thiothrix ectosymbionts, was recently reported from the sulfide-rich Frasassi caves in Italy. Subsequently, two new Niphargus species, N. frasassianus and N. montanarius, were discovered within Frasassi and found to co-occur with N. ictus. Using a variety of microscopic and molecular techniques, we found that all three Frasassi-dwelling Niphargus species harbor Thiothrix ectosymbionts, which belong to three distinct phylogenetic clades (named T1, T2, and T3). T1 and T3 Thiothrix dominate the N. frasassianus ectosymbiont community, whereas T2 and T3 are prevalent on N. ictus and N. montanarius. Relative distribution patterns of the three ectosymbionts are host species-specific and consistent over different sampling locations and collection years. Free-living counterparts of T1–T3 are rare or absent in Frasassi cave microbial mats, suggesting that ectosymbiont transmission among Niphargus occurs primarily through inter- or intraspecific inoculations. Phylogenetic analyses indicate that the Niphargus-Thiothrix association has evolved independently at least two times. While ectosymbioses with T1 and T2 may have been established within Frasassi, T3 ectosymbionts seem to have been introduced to the cave system by Niphargus.     

Identification of bacterial chemoattractants for oyster (Crassostrea virginica) hemocytes

The cytoplasmic and cell wall components of the Gram-positive bacterium Bacillus megaterium and the cytoplasmic and cell envelope components of the Gram-negative bacterium Escherichia coli were assayed for chemotactic activity for the hemocytes of Crassostrea virginica. The cellular components were separated by differential centrifugation and gel filtration was used to determine the approximate molecular weights of the chemoattractant molecules. Active fractions were assayed for glycoproteins and lipoproteins. As a result, it is known that hemocytes are chemotactically attracted to proteins of approximately 10,000 daltons which are associated with the cell wall of B. megaterium and the cell envelope of E. coli.     

Evaluation of the Redox Dye 5-Cyano-2,3-Tolyl-Tetrazolium Chloride for Activity Studies by Simultaneous Use of Microautoradiography and Fluorescence In Situ Hybridization

Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only 65% of the active Thiothrix cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells.     

Phylogenetic in situ/ex situ analysis of a sulfur mat microbial community from a thermal sulfide spring in the north Caucasus

A phylogenetic in situ/ex situ analysis of a sulfur mat formed by colorless filamentous sulfur bacteria in a thermal sulfide spring (northern spur of the main Caucasian ridge) was carried out. Nine phylotypes were revealed in the mat. Thiothrix sp. and Sphaerotilus sp. were the dominant phylotypes (66.3% and 26.3%, respectively). The 16S rRNA gene nucleotide sequence of Sphaerotilus sp. phylotype from the clone library was identical to the sequences of the seven Sphaerotilus strains isolated from the same source. A very high degree of similarity of Sphaerotilus strains revealed by ERIC-PCR fingerprints indicated little or no population diversity of this species in the mat. Thiothrix phylotype from the clone library and two Thiothrix strains isolated from the same mat sample differed in one to three nucleotides of 16S rRNA genes; this is an indication of this organism’s population variability in the mat. 16S rRNA genes of the strains and clones of Thiothrix sp. exhibited the highest similarity (ca. 99%) with Thiothrix unzii; the strains and clones of Sphaerotilus had 99% similarity with the type species Sphaerotilus natans (the only species of this genus) and therefore can be assigned to this species. The minor seven components belong to the phylotypes from the Proteobacteria (3%), as well as the Chlorobia, Cyanobacteria, Clostridia, and Bacteroidetes phylogenetic groups, each of them constituting not more than 1%. Intracellular accumulation of elemental sulfur by Sphaerotilus similar to other filamentous sulfur bacteria was demonstrated for the first time (both in the population of the sulfur spring and in cultures with sulfide). Although mass growth of Sphaerotilus and Thiothrix is typical of bacterial populations of anthropogenic ecosystems (the activated sludge of treatment facilities), stable communities of these bacteria have not been previously found in the sulfur mats or “threads” of natural sulfide springs.     

Involvement of Two Plasmids in Fenitrothion Degradation by Burkholderia sp. Strain NF100

A bacterium capable of utilizing fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) as a sole carbon source was isolated from fenitrothion-treated soil. This bacterium was characterized taxonomically as being a member of the genus Burkholderia and was designated strain NF100. NF100 first hydrolyzed an organophosphate bond of fenitrothion, forming 3-methyl-4-nitrophenol, which was further metabolized to methylhydroquinone. The ability to degrade fenitrothion was found to be encoded on two plasmids, pNF1 and pNF2.