Lithotrophic iron-oxidizing bacteria produce organic stalks to control mineral growth: implications for biosignature formation

Neutrophilic Fe-oxidizing bacteria (FeOB) are often identified by their distinctive morphologies, such as the extracellular twisted ribbon-like stalks formed by Gallionella ferruginea or Mariprofundus ferrooxydans. Similar filaments preserved in silica are often identified as FeOB fossils in rocks. Although it is assumed that twisted iron stalks are indicative of FeOB, the stalk''s metabolic role has not been established. To this end, we studied the marine FeOB M. ferrooxydans by light, X-ray and electron microscopy. Using time-lapse light microscopy, we observed cells excreting stalks during growth (averaging 2.2 μm h⁻¹). Scanning transmission X-ray microscopy and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy show that stalks are Fe(III)-rich, whereas cells are low in Fe. Transmission electron microscopy reveals that stalks are composed of several fibrils, which contain few-nanometer-sized iron oxyhydroxide crystals. Lepidocrocite crystals that nucleated on the fibril surface are much larger (∼100 nm), suggesting that mineral growth within fibrils is retarded, relative to sites surrounding fibrils. C and N 1s NEXAFS spectroscopy and fluorescence probing show that stalks primarily contain carboxyl-rich polysaccharides. On the basis of these results, we suggest a physiological model for Fe oxidation in which cells excrete oxidized Fe bound to organic polymers. These organic molecules retard mineral growth, preventing cell encrustation. This model describes an essential role for stalk formation in FeOB growth. We suggest that stalk-like morphologies observed in modern and ancient samples may be correlated confidently with the Fe-oxidizing metabolism as a robust biosignature.     

Differential modes of crosslinking establish spatially distinct regions of peptidoglycan in Caulobacter crescentus

The diversity of cell shapes across the bacterial kingdom reflects evolutionary pressures that have produced physiologically important morphologies. While efforts have been made to understand the regulation of some prototypical cell morphologies such as that of rod‐shaped Escherichia coli, little is known about most cell shapes. For Caulobacter crescentus, polar stalk synthesis is tied to its dimorphic life cycle, and stalk elongation is regulated by phosphate availability. Based on the previous observation that C. crescentus stalks are lysozyme‐resistant, we compared the composition of the peptidoglycan cell wall of stalks and cell bodies and identified key differences in peptidoglycan crosslinking. Cell body peptidoglycan contained primarily DD‐crosslinks between meso‐diaminopimelic acid and D‐alanine residues, whereas stalk peptidoglycan had more LD‐transpeptidation (meso‐diaminopimelic acid‐meso‐diaminopimelic acid), mediated by LdtD. We determined that ldtD is dispensable for stalk elongation; rather, stalk LD‐transpeptidation reflects an aging process associated with low peptidoglycan turnover in the stalk. We also found that lysozyme resistance is a structural consequence of LD‐crosslinking. Despite no obvious selection pressure for LD‐crosslinking or lysozyme resistance in C. crescentus, the correlation between these two properties was maintained in other organisms, suggesting that DAP‐DAP crosslinking may be a general mechanism for regulating bacterial sensitivity to lysozyme.     

Morphology of biogenic iron oxides records microbial physiology and environmental conditions: toward interpreting iron microfossils

Despite the abundance of Fe and its significance in Earth history, there are no established robust biosignatures for Fe(II)‐oxidizing micro‐organisms. This limits our ability to piece together the history of Fe biogeochemical cycling and, in particular, to determine whether Fe(II)‐oxidizers played a role in depositing ancient iron formations. A promising candidate for Fe(II)‐oxidizer biosignatures is the distinctive morphology and texture of extracellular Fe(III)‐oxyhydroxide stalks produced by mat‐forming microaerophilic Fe(II)‐oxidizing micro‐organisms. To establish the stalk morphology as a biosignature, morphologic parameters must be quantified and linked to the microaerophilic Fe(II)‐oxidizing metabolism and environmental conditions. Toward this end, we studied an extant model organism, the marine stalk‐forming Fe(II)‐oxidizing bacterium, Mariprofundus ferrooxydans PV‐1. We grew cultures in flat glass microslide chambers, with FeS substrate, creating opposing oxygen/Fe(II) concentration gradients. We used solid‐state voltammetric microelectrodes to measure chemical gradients in situ while using light microscopy to image microbial growth, motility, and mineral formation. In low‐oxygen (2.7–28 μm ) zones of redox gradients, the bacteria converge into a narrow (100 μm–1 mm) growth band. As cells oxidize Fe(II), they deposit Fe(III)‐oxyhydroxide stalks in this band; the stalks orient directionally, elongating toward higher oxygen concentrations. M. ferrooxydans stalks display a narrow range of widths and uniquely biogenic branching patterns, which result from cell division. Together with filament composition, these features (width, branching, and directional orientation) form a physical record unique to microaerophilic Fe(II)‐oxidizer physiology; therefore, stalk morphology is a biosignature, as well as an indicator of local oxygen concentration at the time of formation. Observations of filamentous Fe(III)‐oxyhydroxide microfossils from a ~170 Ma marine Fe‐Si hydrothermal deposit show that these morphological characteristics can be preserved in the microfossil record. This study demonstrates the potential of morphological biosignatures to reveal microbiology and environmental chemistry associated with geologic iron formation depositional processes.     

Visual system of the stalk-eyed fly,Cyrtodiopsis quinqueguttata (Diopsidae,Diptera): an anatomical investigation of unusual eyes

Diopsid flies have eye stalks up to a centimeter in length, displacing the retina laterally from the rest of the head. This bizarre condition, called hypercephaly, is rare, but has evolved independently among several insect orders and is most common in flies (Diptera). Earlier studies of geometrical optics and behavior have led to various hypotheses about possible adaptive advantages of eye stalks, such as enhanced stereoscopic vision while other hypothesis suggest that eye stalks are an outcome of sexual selection. Here, we focus on how these curious distortions of head/eye morphology are accompanied by changes in the neural organization of the visual system of Cyrtodiopsis quinqueguttata. Histological examinations reveal that the optic lobes, lamina (La), medulla (Me), lobula (Lo), and lobula plate (LP) are contained entirely within the fly's eye bulbs, which are located at the distal ends of the eye stalks. We report that the organization of the peripheral visual system (La and Me) is similar to that of other Diptera (e.g., Musca and Drosophila), but deeper visual areas (Lo and LP) have been more strongly modified. For example, in both the lobula and lobula plate, fewer but larger giant collector neurons are found. The most pronounced difference is the reduction in the number of wide-field vertical cells of the lobula plate, where there are only four relatively large fibers, as opposed to 11 in Musca. The “fewer but larger” neural organization may enhance the conduction velocities of these cells, but may result in a loss of spatial resolution. At the base of the eye bulb, axon bundles collect and form a long optic nerve that extends the length of the eye stalk. We suggest that this organization of the diopsid visual system provides evidence for the costs of possessing long eye stalks. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 449–468, 1998     

THE CELL WALL OF PYROCYSTIS SPP. (DINOCOCCALES)1,2

The cell of Pyrocystis spp. is covered by an outer layer of material resistant to strong acids and bases. Internal to this layer much of the cell wall is composed of cellulose fibrils. The presence of cellulose fibrils was established by staining raw and ultra-violet–peroxide-cleaned cell walls and by combining X-ray diffraction spectroscopy with electron microscope observation. Carbon replicas of freeze-etched preparations and thin sections of P. lunula walls show outer layers, inside them ca. 24 layers of crossed parallel cellulose fibrils (4–5 nm thick, ca. 12 nm wide), then a region of smaller (ca. 6–12 nm diameter) fibrils in a disperse texture, and then the plasma membrane. Cellulose fibrils in the parallel texture are constructed of 3–5 elementary fibrils ca. 3 nm in diameter. Walls of P. fusiformis and P. pseudonctiluca also have cellulose fibrils in a crossed parallel texture similar to those of P. lunula. The Gymnodinium-type swarmer from lunate P. lunula appears to have a cell wall ultrastructure typical of other “naked” dinoflagellates.     

Isolation and ultrastructure of freshwater strains ofPlanctomyces

Four strains of a freshwaterPlanctomyces species—different in a number of respects from those hitherto described—have been isolated and their morphology and ultrastructure examined by transmission electron microscopy. The ovoid or spherical prokaryotic cells have a cell envelope consisting of outer and inner membranes, but apparently lacking a peptidoglycan wall layer. The cell envelopes of these osmotically sensitive organisms are stabilized in the presence of 5 mM MgSO₄ or CaCl₂; in the absence of divalent cations, autolysis is a common occurrence. Reproduction of these motile, stalked bacteria occurs by an asymmetric budding process in nonaxenic enrichment cultures and in pure cultures grown in very dilute (0.005% or less) peptone medium; but in higher concentrations of nutrients, division is more frequently symmetric and the multifibrillar stalks or appendages are seldom detectable. The cell diameters and the proportion of motile, flagellated cells as a stage of the life cycle are variable features, dependent on cultural conditions.     

THE MORPHOLOGY AND DEVELOPMENT OF SOME PROMINENTLY STALKED SOUTHERN AUSTRALIAN HALYMENIACEAE (CRYPTONEMIALES,RHODOPHYTA). II. THE SPONGE-ASSOCIATED GENERA THAMNOCLONIUM KUETZING AND CODIOPHYLLUM GRAY1

Three members of the red algal family Halymeniaceae (Thamnoclonium dichotomum (J. Ag.) J. Ag., Codiophyllum flabelliforme (Sond.) Schmitz, and C. decipiens (J. Ag.) Schmitz) are investigated. All are endemic to southern and southwestern Australia, possess basal stalks of substantial size and firmness, and are consistently associated with specific sponge taxa. In each case, the sponges are bonded by collagen-like fibrils to the host cuticle without modifying the algal tissue at the ultrastructural level. Secondary cortication and prominent growth rings occur in the stalks of all three species, and in each the pit plugs between cells become wider, more convoluted and less electron dense with increasing distance from the surface. Such pit plugs are apparently a unique attribute of the stalked Halymeniaceae. The three species share pit plug, sponge association and stalk morphological features but are not otherwise closely related, as they actually represent three distinct genera.     

Organisms of deep sea hydrothermal vents as a source for studying adaptation and evolution

It has been postulated that life originated in a similar environment to those of deep sea hydrothermal vents. These environments are located along volcanic ridges and are characterized by extreme conditions such as unique physical properties (temperature, pressure), chemical toxicity, and absence of photosynthesis. However, numerous living organisms have been discovered in these hostile environments, including a variety of microorganisms and many animal species which live in intimate and complex symbioses with sulfo-oxidizing and methanotrophic bacteria. Recent proteomic analyses of the endosymbiont ofRiftia pachyptila and genome sequences of some free living and symbiotic bacteria have provided complementary information about the potential metabolic and genomic capacities of these organisms. The evolution of these adaptive strategies is connected with different mechanisms of genetic adaptation including horizontal gene transfer and . various structural and functional mutations. Therefore, the organisms in this environment are good models for studying the evolution of prokaryotes and eukaryotes as well as different aspects of the biology of adaptation. This review describes some current research concerning metabolic and plausible genetic adaptations of organisms in a deep sea environment, usingRiftia pachyptila as model.     

Modeling human neurodegenerative diseases in transgenic systems

Transgenic systems are widely used to study the cellular and molecular basis of human neurodegenerative diseases. A wide variety of model organisms have been utilized, including bacteria (Escherichia coli), plants (Arabidopsis thaliana), nematodes (Caenorhabditis elegans), arthropods (Drosophila melanogaster), fish (zebrafish, Danio rerio), rodents (mouse, Mus musculus and rat, Rattus norvegicus) as well as non-human primates (rhesus monkey, Macaca mulatta). These transgenic systems have enormous value for understanding the pathophysiological basis of these disorders and have, in some cases, been instrumental in the development of therapeutic approaches to treat these conditions. In this review, we discuss the most commonly used model organisms and the methodologies available for the preparation of transgenic organisms. Moreover, we provide selected examples of the use of these technologies for the preparation of transgenic animal models of neurodegenerative diseases, including Alzheimer’s disease (AD), frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD) and Parkinson’s disease (PD) and discuss the application of these technologies to AD as an example of how transgenic modeling has affected the study of human neurodegenerative diseases.     

Phosphorus distribution in perichromatin granules and surrounding nucleoplasm as visualized by electron spectroscopic imaging

Summary— The in situ distribution of phosphorus in perichromatin granules (PCGs), and in the surrounding nucleoplasm was investigated in rat liver cells by means of electron spectroscopic imaging of unstained preparations. A 2–3 nm fibril containing high concentration of phosphorus was found to be the main substructural feature of the PCGs revealed in the maps of phosphorus. This fibril is folded within the PCG with no apparent order. Fibrils of similar diameter and phosphorus content were also found in both the halo surrounding the PCG and dispersed in the nucleoplasm. Some of such fibrils are in continuity with those occurring within PCGs. Sometimes these fibrils are grouped forming a stalk connecting the PCG to chromatin. Some stalked PCGs are U-shaped or kidneyshaped, resembling Balbiani ring granules in the process of formation as observed in Chironomus salivary gland cell nuclei. The external fibrils are interpreted as perichromatin fibrils considered to be precursors of PCGs.     

Cell wall composition and synthesis via Golgi-directed scale formation in the marine eucaryote,Schizochytrium aggregatum,with a note on Thraustochytrium sp.

Summary Cell walls of Schizochytrium aggregatum and Thraustochytrium sp. were mechanically isolated and subjected to chemical analysis. On a dry weight basis the cell walls contain 21–36% carbohydrate and 30–43% protein. The principal sugar (>95%) of the Schizochytrium wall is l-galactose, while the Thraustochytrium cell wall contains l-galactose, d-galactose and xylose with l-galactose predominating. Ultrastructurally the cell walls of both organisms consist of a laminated structure which yields thin, flexible, nearly circular scales (0.5–1.1 in diameter) upon sonic disintegration. Structures presumed to be developing wall scales are found within cisternae of the Golgi apparatus in both organisms. The chemical composition and method of formation of the cell wall in these two protists is distinctly different from that found in the Saprolegniales (Oomycetes), the group with which these organisms have hitherto been aligned.     

Peculiar Epibionts in Euplotidium itoi (Ciliata, Hypotrichida)

ABSTRACT. Euplotidium itoi share with some other species of the same genus a peculiar feature: the presence of a band of particles running along the right and left borders of the cell body and forming a sort of "scarf" at the dorsal anterior end. The ultrastructural analysis, here performed, revealed that these particles (reported in the literature as extrusomes) are always external to the cell and are inserted in matching depressions on the euplotidium cortex. They are present in two different forms: type I, whose ultrastructure recalls that of bacteria, are able to reproduce by binary fission; type II are not able to divide and contain peculiar structures (a granular dome-shaped zone, a complex extrusive apparatus and a network of regularly arranged fibrils) which render them more complicated with respect to the majority of prokaryotic organisms. These observations, together with the finding that these particles contain DNA, indicate that we are dealing with epibionts, that will be referred to as "epixenosomes" (ecto-organisms), rather than extrusomes. Some ideas about the nature of "epixenosomes" and their relationship with the host cell are proposed and discussed.     

Antimicrobial protegrin-1 forms amyloid-like fibrils with rapid kinetics suggesting a functional link

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich β-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-β (Aβ_1–42) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Aβ_1–42 fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Aβ_1–42 fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing β-rich AMPs and amyloids such as Aβ, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and β-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function.     

Origin of diderm (Gram-negative) bacteria: antibiotic selection pressure rather than endosymbiosis likely led to the evolution of bacterial cells with two membranes

The prokaryotic organisms can be divided into two main groups depending upon whether their cell envelopes contain one membrane (monoderms) or two membranes (diderms). It is important to understand how these and other variations that are observed in the cell envelopes of prokaryotic organisms have originated. In 2009, James Lake proposed that cells with two membranes (primarily Gram-negative bacteria) originated from an ancient endosymbiotic event involving an Actinobacteria and a Clostridia (Lake 2009). However, this Perspective argues that this proposal is based on a number of incorrect assumptions and the data presented in support of this model are also of questionable nature. Thus, there is no reliable evidence to support the endosymbiotic origin of double membrane bacteria. In contrast, many observations suggest that antibiotic selection pressure was an important selective force in prokaryotic evolution and that it likely played a central role in the evolution of diderm (Gram-negative) bacteria. Some bacterial phyla, such as Deinococcus-Thermus, which lack lipopolysaccharide (LPS) and yet contain some characteristics of the diderm bacteria, are postulated as evolutionary intermediates (simple diderms) in the transition between the monoderm bacterial taxa and the bacterial groups that have the archetypal LPS-containing outer cell membrane found in Gram-negative bacteria. It is possible to distinguish the two stages in the evolution of diderm-LPS cells (viz. monoderm bacteria → simple diderms lacking LPS → LPS containing archetypal diderm bacteria) by means of conserved inserts in the Hsp70 and Hsp60 proteins. The insert in the Hsp60 protein also distinguishes the traditional Gram-negative diderm bacterial phyla from atypical taxa of diderm bacteria (viz. Negativicutes, Fusobacteria, Synergistetes and Elusimicrobia). The Gram-negative bacterial phyla with an LPS-diderm cell envelope, as defined by the presence of the Hsp60 insert, are indicated to form a monophyletic clade and no loss of the outer membrane from any species from this group seems to have occurred. This argues against the origin of monoderm prokaryotes from diderm bacteria by loss of outer membrane.     

Further observations on the microanatomy of the haptonema inChrysochromulina chiton andPrymnesium parvum

Summary The structure of a bulbous swelling near the haptonema base inChrysochromulina chiton is shown to possess some unique features interpreted as (a) mechanically significant in maintaining stability of the organelle as a whole and (b) functionally significant with respect to control of its movements. Identical structural features have been detected inPrymnesium parvum in a comparable position though this is not marked externally by a swelling. Below this region continuity has been demonstrated in both organisms between the haptonema cavity and a layer of superficial endoplasmic reticulum within the cell. Finally the numerical and geometrical changes affecting the axial tubes or fibres of the haptonema inC. chiton after entry into the subtending cytoplasm have been shown to agree precisely with those already traced in a similar position inPrymnesium parvum. In both organisms the haptonema starts at the extreme base as a close-packed group of 9 tubes or fibres which becomes reduced to 8 and eventually to 7 during growth towards the cell surface; thereafter an arc of 7 and eventually a ring of 7 tubes or fibres constitutes the core of the main free part of the organelle irrespective of its length. At the distal tip in both organisms the haptonema cavity passes over the rounded end of the core without other elaboration.     

Crystallographic aspects of sub-elementary cellulose fibrils occurring in the wall of rose cells culturedin vitro

Summary Quantities of disencrusted sub-elementary cellulose fibrils from the cell wall of rose cells culturedin vitro were prepared. Following an X-ray and electron diffraction analysis, these fibrils gave a cellulose diffraction pattern which presented only two strong equatorial diffraction spacings at 0.409 and 0.572 nm indicating that the fibrils have a crystalline structure resembling that of cellulose IV_I. This observation is best explained in terms of a lateral disorganization of the cellulose chains within the fibrils. This disorganization cannot be eliminated and is connected with the small width of the fibrils which contain from 12 to 25 cellulose chains only. In these fibrils, most of the cellulose chains are superficial and not locked with neighboring chains in a tight hydrogen bond system as in thicker cellulose microfibrils.     

Prosthecomicrobium and Ancalomicrobium: New Prosthecate Freshwater Bacteria

Direct microscopic examination of natural freshwater samples reveals a variety of small microorganisms having elaborate cellular appendages. Several strains have been isolated from crude cultures containing low concentrations of organic nutrients. All of the isolates are procaryotic. They are aerobic chemoorganotrophs that require vitamins for growth. Because they cannot be assigned to any of the existing bacterial genera, two new genera are proposed: Ancalomicrobium for organisms which have several long appendages and which reproduce by budding; Prosthecomicrobium for organisms which have many short appendages tapering toward a blunt tip and which reproduce by binary fission. Gas vacuoles have been found in strains of each genus. The term prostheca is proposed for the rigid appendages of procaryotic cells bounded by the cell wall, and is defined to include the structures on these new bacteria, as well as the stalks of the caulobacters and the hyphae of the hyphomicrobia.     

Preservation of soft tissues in Silurian graptolites from Latvia

The contractile stalks of graptoloid zooids are preserved as organic carbon residues in thecae of the middle Llandovery graptoloid graptolites Rastrites geinitzii and Neolagarograptus? sp. from the Aizpute‐41 core, Latvia. The contractile stalks are surrounded by equant pyrite crystals, resulting in three‐dimensional preservation of the graptolite rhabdosomes, and are associated with sediment of similar composition to, and derived from, the adjacent matrix. Matrix entered the thecae after pyrite crystal growth and filled some of the space left by collapse of the contractile stalks and some intercrystalline cavities; other space is partially infilled by diagenetic minerals. The contractile stalks are parallel‐sided and occupy up to one‐half the metathecal width, which is not inconsistent, assuming post‐mortem shrinkage, with the suggestion that graptoloid zooids filled their thecal tubes in life. The location of the preserved soft tissues, towards the distal ends of the metathecae, is very different from that predicted by decay experiments on the extant pterobranch hemichordate Rhabdopleura; the latter's soft tissues may thus not be a reliable analogue for those of these Silurian graptoloids.     

On the binding of Thioflavin-T to HET-s amyloid fibrils assembled at pH 2

Amyloid fibrils are ordered β-sheet protein or peptide polymers. The benzothiazole dye Thioflavin-T (ThT) shows a strong increase in fluorescence upon binding to amyloid fibrils and has hence become the most commonly used amyloid-specific dye. In spite of this widespread use, the mechanism underlying specific binding and fluorescence enhancement upon interaction with amyloid fibrils remains largely unknown. Recent contradictory reports have proposed radically different modes of binding. We have studied the interaction of ThT with fibrils of the prion forming domain of the fungal HET-s prion protein assembled at pH 2 in order to try to gain some insight into the general mechanism of ThT-binding and fluorescence. We found that ThT does not bind to HET-s(218–289) fibrils as a micelle as previously proposed in the case of insulin fibrils. We have measured binding kinetics, affinity and stoichiometry at pH values above and below the pI of the HET-s(218–289) fibrils and found that binding is dramatically affected by pH and ionic strength. Binding is poor at acidic pH, presumably as a result of repulsive electrostatic interaction between the positively charged ThT molecule and the fibril surface. Finally, we found that ThT acquires chiral properties when it is fibril-bound. These results are discussed in relation to the different ThT-binding modes that have been proposed.