Natural communities of novel archaea and bacteria with a string-of-pearls-like morphology: molecular analysis of the bacterial partners

A recently discovered bacterial/archaeal association, growing in a string-of-pearls-like structure, thrives in the cold (approximately 10 degrees C) sulfidic marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany. It forms characteristic, macroscopically visible globules, the pearls, containing microcolonies of novel euryarchaeota, which are surrounded by mainly filamentous bacteria (C. Rudolph, G. Wanner, and R. Huber, Appl. Environ. Microbiol. 67:2336-2344, 2001). Single pearls in series are connected by white threads. Here we report the first detailed molecular investigations of the taxonomic affiliation of the bacteria contributing to the strings of pearls. Phylogenetic analysis showed the dominance of a single phylotype (clone sipK4) within single pearls most closely related to Thiothrix unzii. The presence of Thiothrix sipK4 as a major constituent of single pearls and of the pearl-connecting white threads was verified by fluorescence in situ hybridization.     

Natural Communities of Novel Archaea and Bacteria Growing in Cold Sulfurous Springs with a String-of-Pearls-Like Morphology

We report the identification of novel archaea living in close association with bacteria in the cold (approximately 10°C) sulfurous marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany. These microorganisms form a characteristic, macroscopically visible structure, morphologically comparable to a string of pearls. Tiny, whitish globules (the pearls; diameter, about 0.5 to 3.0 mm) are connected to each other by thin, white-colored threads. Fluorescent in situ hybridization (FISH) studies have revealed that the outer part of the pearls is mainly composed of bacteria, with a filamentous bacterium predominating. Internally, archaeal cocci are the predominant microorganisms, with up to 10⁷ cells estimated to be present in a single pearl. The archaea appear to be embedded in a polymer of unknown chemical composition. According to FISH and 16S rRNA gene sequence analysis, the archaea are affiliated with the euryarchaeal kingdom. The new euryarchaeal sequence represents a deep phylogenetic branch within the 16S rRNA tree and does not show extensive similarity to any cultivated archaea or to 16S rRNA gene sequences from environmental samples.     

Natural communities of novel archaea and bacteria growing in cold sulfurous springs with a string-of-pearls-like morphology

We report the identification of novel archaea living in close association with bacteria in the cold (approximately 10 degrees C) sulfurous marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany. These microorganisms form a characteristic, macroscopically visible structure, morphologically comparable to a string of pearls. Tiny, whitish globules (the pearls; diameter, about 0.5 to 3.0 mm) are connected to each other by thin, white-colored threads. Fluorescent in situ hybridization (FISH) studies have revealed that the outer part of the pearls is mainly composed of bacteria, with a filamentous bacterium predominating. Internally, archaeal cocci are the predominant microorganisms, with up to 10(7) cells estimated to be present in a single pearl. The archaea appear to be embedded in a polymer of unknown chemical composition. According to FISH and 16S rRNA gene sequence analysis, the archaea are affiliated with the euryarchaeal kingdom. The new euryarchaeal sequence represents a deep phylogenetic branch within the 16S rRNA tree and does not show extensive similarity to any cultivated archaea or to 16S rRNA gene sequences from environmental samples.     

The pea (Pisum sativum L.) genes sym33 and sym40 control infection thread formation and root nodule function

Two novel non-allelic mutants that were unable to fix nitrogen (Fix^?) were obtained after EMS (ethyl methyl sulfonate) mutagenesis of pea (Pisum sativum L.). Both mutants, SGEFix^?–1 and SGEFix^?–2, form two types of nodules: SGEFix^?–1 forms numerous white and some pink nodules, while mutant SGEFix^?–2 forms white nodules with a dark pit at the distal end and also some pinkish nodules. Both mutations are monogenic and recessive. In both lines the manifestation of the mutant phenotype is associated with the root genotype. White nodules of SGEFix^?–1 are characterised by hypertrophied infection threads and infection droplets, mass endocytosis of bacteria, abnormal morphological differentiation of bacteroids, and premature degradation of nodule symbiotic structures. The structure of the pink nodules of SGEFix^?–1 does not differ from that of the parental line, SGE. White nodules of SGEFix^?–2 are characterised by “locked” infection threads surrounded with abnormally thick plant cell walls. In these nodules there is no endocytosis of bacteria into host-cell cytoplasm. The pinkish nodules of SGEFix^?–2 are characterised by virtually undifferentiated bacteroids and premature degradation of nodule tissues. Thus, the novel pea symbiotic genes, sym40 and sym33, identified after complementation analysis in SGEFix^?–1 and SGEFix^?–2 lines, respectively, control early nodule developmental stages connected with infection thread formation and function.     

Scanning Electron Microscopy of Rhizobium trifolii Infection Sites on Root Hairs of White Clover

White clover root hairs which were inoculated with Rhizobium trifolii 4S (infectious strain) contained infection threads which were observed by light microscopy and scanning electron microscopy. Three morphological types of root hairs retaining infection threads were recognized. The bacteria were strongly attached between the surfaces of two plant cell walls as follows: between surfaces of a root hair tip curled back on itself, between a protuberance from a root hair and its cell surface, or between two root hair tips clinging together. An anatomical analysis documented the attachment site of the infection thread sheath from the inside of the root hair cell.     

Seasonal Population Changes and Characterization of Ice-Nucleating Bacteria in Farm Fields of Central Alberta

During the summer of 1983 in central Alberta, changes in the bacterial population inhabiting the leaves of field beans (Phaseolus vulgaris L.) and canola (Brassica napus L. Altex) were studied to determine if ice-nucleating bacteria were present on these plants. Three colony types (white, yellow, and peach-colored) were found on field beans and canola leaves. Approximately 25% of the isolates from the white colony group, which dominated the population, were ice-nucleating bacteria. No ice-nucleating bacteria were present on canola leaves. Out of a total of 76 ice-nucleating bacteria isolated, 5 representative cultures were characterized in detail and identified as Pseudomonas fluorescens. The fatty acid composition of these cultures was essentially identical to that of typical P. fluorescens cultures and was altered by varying the growth temperature from 10 to 30°C.     

Geochemical Evidence of the Seasonality,Affinity and Pigmenation of Solenopora jurassica

Solenopora jurassica is a fossil calcareous alga that functioned as an important reef-building organism during the Palaeozoic. It is of significant palaeobiological interest due to its distinctive but poorly understood pink and white banding. Though widely accepted as an alga there is still debate over its taxonomic affinity, with recent work arguing that it should be reclassified as a chaetetid sponge. The banding is thought to be seasonal, but there is no conclusive evidence for this. Other recent work has, however demonstrated the presence of a unique organic boron-containing pink/red pigment in the pink bands of S. jurassica. We present new geochemical evidence concerning the seasonality and pigmentation of S. jurassica. Seasonal growth cycles are demonstrated by X-ray radiography, which shows differences in calcite density, and by varying δ¹³C composition of the bands. Temperature variation in the bands is difficult to constrain accurately due to conflicting patterns arising from Mg/Ca molar ratios and δ¹⁸O data. Fluctuating chlorine levels indicate increased salinity in the white bands, when combined with the isotope data this suggests more suggestive of marine conditions during formation of the white band and a greater freshwater component (lower chlorinity) during pink band precipitation (δ¹⁸O). Increased photosynthesis is inferred within the pink bands in comparison to the white, based on δ¹³C. Pyrolysis Gas Chromatography Mass Spectrometry (Py-GCMS) and Fourier Transform Infrared Spectroscopy (FTIR) show the presence of tetramethyl pyrrole, protein moieties and carboxylic acid groups, suggestive of the presence of the red algal pigment phycoerythrin. This is consistent with the pink colour of S. jurassica. As phycoerythrin is only known to occur in algae and cyanobacteria, and no biomarker evidence of bacteria or sponges was detected we conclude S. jurassica is most likely an alga. Pigment analysis may be a reliable classification method for fossil algae.     

Comparison of Two Pearl Sacs Formed in the Same Recipient Oyster with Different Genetic Background Involved in Yellow Pigmentation in <Emphasis Type="Italic">Pinctada fucata</Emphasis>

Color is one of the most important factors determining the commercial value of pearls. Pinctada fucata is a well-known pearl oyster producing high-quality Akoya pearls. Phenotypic variation in amount of yellow pigmentation produces white and yellowish pearls. It has been reported that polymorphism of yellow pigmentation of Akoya pearls is genetically regulated, but the responsible gene(s) has remained unknown. Here, we prepared pearl sac pairs formed in the same recipient oyster but coming from donor oysters that differ in their color. These two pearl sacs produced pearls with different yellowness even in the same recipient oyster. Yellow tone of produced pearls was consistent with shell nacre color of donor oysters from which mantle grafts were prepared, indicating that donor oysters strongly contribute to the yellow coloration of Akoya pearls. We also conducted comparative RNA-seq analysis and retrieved several candidate genes involved in the pearl coloration. Whole gene expression patterns of pair sacs were not grouped by pearl color they produced, but grouped by recipient oysters in which they were grown, suggesting that the number of genes involved in the yellow coloration is quite small, and that recipient oyster affects gene expression of the majority of genes in the pearl sac.     

Fine structure of the chorionic projections of the egg of Rhithrogena kimminsi Thomas (Ephemeroptera : Heptageniidae) and their role in egg adhesion

Scanning (SEM) and transmission electron microscopical (TEM) observations of the eggs of Rhithrogena kimminsi (Ephemeroptera : Heptageniidae), a species of the alpestris group, revealed 2 kinds of chorionic projections, both characterized by knob-terminated coiled threads (KCTs). The former are concentrated at one egg pole, and arise directly from the shell surface. The latter are scattered on the egg chorion and are supported by a basal excrescence, giving them a peglike feature. At TEM level, KCTs, arising directly from the chorion, appear to be composed of fibers that are enveloped by filaments. The fibers are tightly twisted together and joined at their apicals, which end in a terminal knob. KCTs, supported by peglike projections, show a similar organization, but each thread derives from a single coiled fiber. The different numbers of fibers form wider threads at the egg polar region and thinner ones on the peglike projections. The involvement of both kinds of KCTs in egg adhesion is documented through the discharge of their threads.     

Nodules Initiated by Rhizobium meliloti Exopolysaccharide Mutants Lack a Discrete, Persistent Nodule Meristem

Infection of alfalfa with Rhizobium meliloti exo mutants deficient in exopolysaccharide results in abnormal root nodules that are devoid of bacteria and fail to fix nitrogen. Here we report further characterization of these abnormal nodules. Tightly curled root hairs or shepherd's crooks were found after inoculation with Rm 1021-derived exo mutants, but curling was delayed compared with wild-type Rm 1021. Infection threads were initiated in curled root hairs by mutants as well as by wild-type R. meliloti, but the exo mutant-induced threads aborted within the peripheral cells of the developing nodule. Also, nodules elicited by Rm 1021-derived exo mutants were more likely to develop on secondary roots than on the primary root. In contrast with wild-type R. meliloti-induced nodules, the exo mutant-induced nodules lacked a well defined apical meristem, presumably due to the abortion of the infection threads. The relationship of these findings to the physiology of nodule development is discussed.     

Inactivation of Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 μg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.     

Distinct glycoconjugate cell surface structures make the pelagic diatom Thalassiosira rotula an attractive habitat for bacteria

Interactions between marine diatoms and bacteria have been studied for decades. However, the visualization of physical interactions between these diatoms and their colonizers is still limited. To enhance our understanding of these specific interactions, a new Thalassiosira rotula isolate from the North Sea (strain 8673) was characterized by scanning electron microscopy and confocal laser scanning microscopy (CLSM) after staining with fluorescently labeled lectins targeting specific glycoconjugates. To investigate defined interactions of this strain with bacteria the new strain was made axenic and co-cultivated with a natural bacterial community and in two- or three-partner consortia with different bacteria of the Roseobacter group, Gammaproteobacteria and Bacteroidetes. The CLSM analysis of the consortia identified six out of 78 different lectins as very suitable to characterize glycoconjugates of T. rotula. The resulting images show that fucose-containing threads were the dominant glycoconjugates secreted by the T. rotula cells but chitin and to a lesser extent other glycoconjugates were also identified. Bacteria attached predominantly to the fucose glycoconjugates. The colonizing bacteria showed various attachment patterns such as adhering to the diatom threads in aggregates only or attaching to both the surfaces and the threads of the diatom. Interestingly the colonization patterns of single bacteria differed strikingly from those of bacterial co-cultures, indicating that interactions between two bacterial species impacted the colonization of the diatom. Our observations help to better understand physical interactions and specific colonization patterns of distinct bacterial mono- and co-cultures with an abundant diatom of costal seas.     

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g. by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-Pérot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a ''real time'' observation of bacteria attachment.The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 10⁴ cells/ml) in less than an hour.     

Binding of Complement Inhibitor C4b-binding Protein to a Highly Virulent Streptococcus pyogenes M1 Strain Is Mediated by Protein H and Enhances Adhesion to and Invasion of Endothelial Cells

Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of ¹²⁵I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92–109, which specifically bound C4BP. Biacore was used to determine K_D = 6 × 10⁻⁷ m between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.     

The Temperature-Sensitive brush Mutant of the Legume Lotus japonicus Reveals a Link between Root Development and Nodule Infection by Rhizobia

The brush mutant of Lotus japonicus exhibits a temperature-dependent impairment in nodule, root, and shoot development. At 26°C, brush formed fewer nodules, most of which were not colonized by rhizobia bacteria. Primary root growth was retarded and the anatomy of the brush root apical meristem revealed distorted cellular organization and reduced cell expansion. Reciprocal grafting of brush with wild-type plants indicated that this genotype only affected the root and that the shoot phenotype was a secondary effect. The root and nodulation phenotype cosegregated as a single Mendelian trait and the BRUSH gene could be mapped to the short arm of chromosome 2. At 18°C, the brush root anatomy was rescued and similar to the wild type, and primary root length, number of infection threads, and nodule formation were partially rescued. Superficially, the brush root phenotype resembled the ethylene-related thick short root syndrome. However, treatment with ethylene inhibitor did not recover the observed phenotypes, although brush primary roots were slightly longer. The defects of brush in root architecture and infection thread development, together with intact nodule architecture and complete absence of symptoms from shoots, suggest that BRUSH affects cellular differentiation in a tissue-dependent way.     

Tissue Turnover Rates and Isotopic Trophic Discrimination Factors in the Endothermic Teleost,Pacific Bluefin Tuna (Thunnus orientalis)

Stable isotope analysis (SIA) of highly migratory marine pelagic animals can improve understanding of their migratory patterns and trophic ecology. However, accurate interpretation of isotopic analyses relies on knowledge of isotope turnover rates and tissue-diet isotope discrimination factors. Laboratory-derived turnover rates and discrimination factors have been difficult to obtain due to the challenges of maintaining these species in captivity. We conducted a study to determine tissue- (white muscle and liver) and isotope- (nitrogen and carbon) specific turnover rates and trophic discrimination factors (TDFs) using archived tissues from captive Pacific bluefin tuna (PBFT), Thunnus orientalis, 1–2914 days after a diet shift in captivity. Half-life values for ¹⁵N turnover in white muscle and liver were 167 and 86 days, and for ¹³C were 255 and 162 days, respectively. TDFs for white muscle and liver were 1.9 and 1.1‰ for δ ¹⁵N and 1.8 and 1.2‰ for δ ¹³C, respectively. Our results demonstrate that turnover of ¹⁵N and ¹³C in bluefin tuna tissues is well described by a single compartment first-order kinetics model. We report variability in turnover rates between tissue types and their isotope dynamics, and hypothesize that metabolic processes play a large role in turnover of nitrogen and carbon in PBFT white muscle and liver tissues. ¹⁵N in white muscle tissue showed the most predictable change with diet over time, suggesting that white muscle δ ¹⁵N data may provide the most reliable inferences for diet and migration studies using stable isotopes in wild fish. These results allow more accurate interpretation of field data and dramatically improve our ability to use stable isotope data from wild tunas to better understand their migration patterns and trophic ecology.     

Cytofluorometric detection of wine lactic acid bacteria: application of malolactic fermentation to the monitoring

In this study we report for the first time a rapid, efficient and cost-effective method for the enumeration of lactic acid bacteria (LAB) in wine. Indeed, up to now, detection of LAB in wine, especially red wine, was not possible. Wines contain debris that cannot be separated from bacteria using flow cytometry (FCM). Furthermore, the dyes tested in previous reports did not allow an efficient staining of bacteria. Using FCM and a combination of BOX/PI dyes, we were able to count bacteria in wines. The study was performed in wine inoculated with Oenococcus oeni (10⁶ CFU ml^?1) stained with either FDA or BOX/PI and analyzed by FCM during the malolactic fermentation (MLF). The analysis show a strong correlation between the numbers of BOX/PI-stained cells determined by FCM and the cell numbers determined by plate counts (red wine: R ² ≥ 0.97, white wine R ² ≥ 0.965). On the other hand, we found that the enumeration of O. oeni labeled with FDA was only possible in white wine (R ² ≥ 0.97). Viable yeast and LAB populations can be rapidly discriminated and quantified in simultaneous malolactic-alcoholic wine fermentations using BOX/PI and scatter parameters in a one single measurement. This rapid procedure is therefore a suitable method for monitoring O. oeni populations during winemaking, offers a detection limit of <10⁴ CFU ml^?1 and can be considered a useful method for investigating the dynamics of microbial growth in wine and applied for microbiological quality control in wineries.     

DNA Fingerprinting of Pearls to Determine Their Origins

We report the first successful extraction of oyster DNA from a pearl and use it to identify the source oyster species for the three major pearl-producing oyster species Pinctada margaritifera, P. maxima and P. radiata. Both mitochondrial and nuclear gene fragments could be PCR-amplified and sequenced. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in the internal transcribed spacer (ITS) region was developed and used to identify 18 pearls of unknown origin. A micro-drilling technique was developed to obtain small amounts of DNA while maintaining the commercial value of the pearls. This DNA fingerprinting method could be used to document the source of historic pearls and will provide more transparency for traders and consumers within the pearl industry.     

Effects of dominant microalgae species and bacterial quantity on shrimp production in the final culture season

The dominant microalgal species, quantity of heterotrophic bacteria and Vibrio in the intestines and gills of Litopenaeus vannamei (Pacific white shrimp), positive detection rate of white spot syndrome virus (WSSV), and water quality indices were investigated at the final culture stage (88th day in culture season). Correlation of microalgal community, bacteria quantity, and shrimp production were analyzed by statistical analysis methods. Every 10 days, probiotics were used in groups A, B, and C, consisting of Bacillus, photosynthetic bacteria (PSB), and equal parts Bacillus and PSB, respectively. The results showed that production (25597.33?±?928.46 kg ha^?1) and survival rate (77.06?±?9.00 %) was the highest in group C, but positive detection rate of WSSV was the lowest. The microalgal community of group C was significantly dominated by Chlorella pyrenoidosa, with an average density and dominance of (289.52?±?142.10)?×?10⁷ cells L^?1 and 0.878?±?0.161, respectively. The correlation analysis indicated a significant negative correlation between Cyanophyta dominance and shrimp production (P?<?0.05), while the relationship between production and Vibrio quantity was not significantly correlated (P?>?0.05). Accordingly, microalgal dominant species should be controlled as a key factor in the shrimp culture season; in particular, the dominance of Cyanophyta should be restricted to a low level. Meanwhile, the combined use of Bacillus and PSB probiotics was considered an effective solution to optimize microalgal communities and controlling the cell density of Cyanophyta.