[Show abstract][Hide abstract] ABSTRACT: A marine, magnetotactic bacterium, designated strain MMS-1(T), was isolated from mud and water from a salt marsh in Woods Hole, Massachusetts, USA, after enrichment in defined oxygen-concentration/redox-gradient medium. Strain MMS-1(T) is an obligate microaerophile capable of chemoorganoheterotrophic and chemolithoautotrophic growth. Optimal growth occurred at pH 7.0 and 24-26 °C. Chemolithoautotrophic growth occurred with thiosulfate as the electron donor and autotrophic carbon fixation was via the Calvin-Benson-Bassham cycle. The G+C content of the DNA of strain MMS-1(T) was 47.2 mol%. Cells were Gram-negative and morphologically variable, with shapes that ranged from that of a lima bean to fully helical. Cells were motile by means of a single flagellum at each end of the cell (amphitrichous). Regardless of whether grown in liquid or semi-solid cultures, strain MMS-1(T) displayed only polar magnetotaxis and possessed a single chain of magnetosomes containing elongated octahedral crystals of magnetite, positioned along the long axis of the cell. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MMS-1(T) belongs to the family Rhodospirillaceae within the Alphaproteobacteria, and is distantly related to species of the genus Magnetospirillum. Strain MMS-1(T) is therefore considered to represent a novel species of a new genus, for which the name Magnetospira thiophila gen. nov., sp. nov. is proposed. The type strain of Magnetospira thiophila is MMS-1(T) ( = ATCC BAA-1438(T) = JCM 17960(T)).
Full-text · Article · Dec 2011 · International Journal of Systematic and Evolutionary Microbiology
[Show abstract][Hide abstract] ABSTRACT: Scanning tunnelling microscopy (STM) images of Pt/Ir- and Pt/Ir/C-coated cell plugs of Methanospirillum hungatei showed paracrystalline structures with P6 symmetry and an 18-nm lattice constant, in agreement with electron microscopy studies. The three-dimensional STM images unambiguously distinguished the two morphologically different proteinaceous plug assemblies and led to an improved understanding of the natural internal organization of whole plugs. Tip convolution effects and the grain size of the metal coating complicated interpretation of finer structures. We discuss possible imaging mechanisms to explain observations in which part of the film was removed but the remaining part of the structure was still imaged reproducibly.
No preview · Article · Aug 2011 · Journal of Microscopy
[Show abstract][Hide abstract] ABSTRACT: The hexagonal S-layer from the unicellular cyanobacterium Synechococcus strain GL24 can act as a template for sulfate and carbonate mineral formation by providing regularly spaced, chemically identical nucleation sites. This novel ability is a reflection of the S-layer's chemistry in addition to its structure. Few, if any, other cyanobacterial S-layers have been subjected to detailed chemical analysis and no other reports of the involvement of an S-layer in mineral formation can be found. We have investigated the surface charge and chemical characteristics of the Synechococcus strain GL24 S-layer in an attempt to unravel the mechanism(s) of mineral formation on this proteinaceous surface layer. The constituent proteins of the S-layer show up as two bands (Mr = 109 000 and 104 000) on SDS-PAGE gels. When assembled, the S-layer confers a net neutral charge on the cell surface with negatively charged sites (which can bind cations) exposed in the large holes of the array. Treatment of whole cells with a wide range of chemical agents failed to disrupt intralayer bonding and binding of the S-layer to the underlying outer membrane, indicating that this is a remarkably resilient S-layer in comparison to those found on other bacteria.Key words: S-layer, cyanobacteria, chemistry, carbonate minerals.
Preview · Article · Feb 2011 · Canadian Journal of Microbiology
[Show abstract][Hide abstract] ABSTRACT: F420-reducing hydrogenase was isolated from spheroplast lysates of Methanospirillum hungatei by sedimentation, followed by sucrose gradient centrifugations and gel filtration. These procedures resulted in a preparation free of methyl reductase and cytoplasmic membranes. The hydrogenase was a brown protein with an absorption spectrum characteristic for a nonheme iron protein. In electron micrographs it was a coin-shaped, multisubunit protein complex of 15.9 nm diameter with a central depression on one surface. On phenyl Sepharose chromatography the hydrogenase exhibited hydrophobic properties. The holoenzyme was about 720 kilodaltons (kDa), composed of 50.7 and 30.7 kDa subunits in a ratio of 1:3. Each enzyme particle contained 6 or 7 Ni2+ atoms. H2-dependent reduction of F420 activity was readily, but transiently, reactivated by anaerobic conditions following exposure of the enzyme to air. Mg2+ or Ca2+ were stimulatory, but added FAD was not required. Antibody raised against the purified hydrogenase of strain GP1 gave a negative reaction with extracts of nine other methanogens and a reaction of identity with strain JF1 and Methanosarcina barkeri MS. Direct comparisons with the hyrogenase from Methanobacterium thermoautotrophicum ΔH revealed striking differences in subunit composition and in the acidity of the holoenzyme.
No preview · Article · Feb 2011 · Canadian Journal of Microbiology
[Show abstract][Hide abstract] ABSTRACT: Purified cell walls of Bacillus subtilis were reacted with various metals in dilute solutions and a simulated Hamilton Harbour (Lake Ontario) water. Atomic absorption spectrophotometry revealed that iron was sorbed to the walls preferentially and caused the walls to adhere together to form macroscopic floes, which were easily sedimented by gravity. Electron microscopy and energy dispersive X-ray analysis of the floes showed that the bacterial walls were embedded in an iron matrix presumed to be iron oxyhydroxide. The sorption of iron to the walls was also found to enhance the uptake of other metals such as chromium and aluminum. This sorption suggests that bacteria may play a role in the initial formation of metal rich sediments. It may also have important implications for the general transport of metals to the sediments from the water column in fresh water environments like Hamilton Harbour by all types of particulate biological debris.Key words: metal sorption to bacterial walls.
No preview · Article · Feb 2011 · Canadian Journal of Microbiology
[Show abstract][Hide abstract] ABSTRACT: Methanospirillum hungatei GP1 consists of chains of rod-shaped cells separated from one another by "cell spacers" comprised of two spacer plugs sandwiching a loose, amorphous material. The chain is encased within a highly ordered sheath to form a cylindrical, multicelled filament about 5 to 10 cells long under our growth conditions. Cells within the filament divide by septation in a manner similar to gram-positive eubacteria; the plasma membrane and wall grow inward to partition the cell in two. Yet, unlike gram-positive eubacteria, the wall is flexible, since cells round up when extruded from the sheath; the shape-maintaining structures are the sheath and spacer plugs. After septation and daughter cell separation, the cell spacer grows between the new cells. Initially, the growth of a spacer plug is detected by electron microscopy as the addition of electron-dense layers, exhibiting an 18.0-nm periodicity, at the surface of one new cell pole. Usually three layers develop at this pole before plug assembly is initiated at the opposite pole. As assembly proceeds, the two newly formed plugs separate from each other to form the loose, amorphous central zone of the spacer. Presumably, cell and cell spacer elongation requires sheath extension, since filament growth is observed. The amorphous cell spacer zone continues to expand as the spacer grows larger until lesions appear in the sheath near the zone's midpoint. Usually, the largest spacer zones are found towards each filament's centre and the lesions split the chain in two. Consequently, M. hungatei requires two separate events for filament division: cell replication which is a septation process and filament splitting which is a "cell spacer" breakage.
No preview · Article · Feb 2011 · Canadian Journal of Microbiology
[Show abstract][Hide abstract] ABSTRACT: Adherent epilithic microorganisms recovered from rocks submerged 10 to 20 cm in two different rivers were examined by electron microscopy and enumerated after dispersion in M-9 salts by viable plate counts. Bacterial cells concentrated in microcolonies were often observed attached to the surface of algae, cyanobacteria, and organic detritus. This structured communal mode of growth was common among epilithic microbial communities of different rock types. However, counts of heterotrophic bacteria from limestone (106 to 107 cfu/cm2) were 10- to 100-fold greater than corresponding values from granite, gabbro, rhyolite, basalt, and quartz. Cyanobacteria and algae were an order of magnitude less abundant compared with their bacterial counterparts. These variations in population densities of epilithic microorganisms present on different rocks were inversely related to mineral substrate hardness.Key words: epilithic microorganisms, mineral hardness.
Full-text · Article · Feb 2011 · Canadian Journal of Microbiology
[Show abstract][Hide abstract] ABSTRACT: On Ellesmere Island, in the Canadian Arctic, dark-colored biofilms proliferate on moist surfaces, including exposed granodiorite outcrops. Transmission electron microscopy of these biofilms indicates that complex epilithic microbial communities developed, consisting of cyanobacteria and fungi symbiotically associated in a lichen, along with a consortium of free-living algae and gram-negative bacteria. The epilithic cyanobacteria and bacteria were shown to extensively precipitate phosphatic minerals, ranging from relatively large polyphosphate granules (approximately 250 nm in diameter) within their cytoplasmic membranes to smaller iron phosphate grains (generally less than 50 nm in diameter) associated with the periplasmic space and encompassing capsule. Complete encrustation of some bacterial cells by the iron phosphates was observed. Energy-dispersive X-ray spectroscopy suggested that these grains are compositionally similar to the mineral strengite (FePO4∙2H2O). This study clearly indicates that the Arctic supports a thriving microbial community that influences the biogeochemical cycling of PO4 in an environment of low nutrient availability. Nutritional requirements by the microorganisms were actively maintained through a relatively closed recycling mechanism, which restricted the immediate loss of phosphorus from the biofilm.
Full-text · Article · Feb 2011 · Canadian Journal of Earth Sciences
[Show abstract][Hide abstract] ABSTRACT: All biomass contains a significant quantity of metallic constituents, and mineralization in living and dead biodebris may contribute to element transport from the hydrosphere into sediments. The anionic cell walls of bacteria are remarkable in their ability to fix metals and provide sites for nucleation and growth of minerals. Results presented show the types of cell wall polymers that are responsible for metal binding in walls of Gram-positive and Gram-negative bacteria.
Preview · Article · Feb 2011 · Canadian Journal of Earth Sciences
[Show abstract][Hide abstract] ABSTRACT: Environmental growth conditions and cell physiology have the potential to influence bacterial surface-metal interactions in both planktonic and biofilm systems. Here, Pseudomonas aeruginosa was studied to determine the influence of these factors (pH, redox potential, and active respiration) on surface electrostatics and metal immobilization. Acid-base titrations revealed a decrease in ionizable ligands at pKa 5 (putative carboxyls) in cells grown below pH 6.2 and in cells grown anaerobically relative to cells grown under oxic and circumneutral pH conditions. This observation correlates with Western immunoblotting assays that revealed a reduction in carboxylated B-band lipopolysaccharide in these cells. Furthermore, spectrophotometric analysis revealed a decrease in zinc, copper, and iron immobilization in these cells, suggesting that lipopolysaccharide modification in response to environmental stimuli influences metal binding. The effect of active versus inactive metabolism on metal adsorption was also examined using respiration inhibitors carbonyl cyanide m-chlorophenylhydrazone and sodium azide. Cells treated with these compounds bound more zinc, copper, and iron than untreated controls, suggesting proton extrusion through respiration competes with metal cations for reactive groups on the cell surface. Accumulation of gold did not show the same trend, and transmission electron microscopy studies confirmed it was not a surface-mediated process. These results suggest that variations in growth environment and cell physiology influence metal accumulation by bacterial cell surfaces and may help to explain discontinuous accumulation of metal observed throughout microbial communities.
No preview · Article · Jul 2010 · Canadian Journal of Microbiology
[Show abstract][Hide abstract] ABSTRACT: Geomicrobiology is a relatively new field that aims to establish how microorganisms interact with the surrounding inorganic environment. Most of what is known so far has been derived from laboratory experiments using free-swimming (or planktonic) bacteria, however, throughout nature, nutrient limitations and other environmental challenges encourage bacteria to live in a complex and dynamic surface-associated community known as a biofilm. These assemblages exhibit micron-scale variations in environmental chemistry (such as pH and redox potential), bacterial cell physiology, and are exemplified by a viscous extracellular matrix that also shows significant heterogeneity over very small scales. The inherent complexity of these systems leaves us with very little knowledge about how microbes drive geochemical cycles in the natural environment in which they are found. Fortunately, recent developments in instrumentation in the laboratory (particularly in microscopy and other analytical techniques) have generated a greater understanding of how microbial biofilms influence the geochemistry of their surroundings. This chapter discusses several properties of bacteria that make them efficient mediators of metal ion chemistry, the range of metalmicrobe interactions found in natural environments, and a number of physico-chemical differences between the planktonic and biofilm modes of growth. To conclude, the chapter outlines the recent developments in the field that have allowed us to gain a greater appreciation of the added controls biofilm communities exert on the fate and speciation of metal ions in the natural environment.
[Show abstract][Hide abstract] ABSTRACT: A thermophilic bacterium, designated strain CR11(T), was isolated from a filamentous sample collected from a terrestrial hot spring on the south-western foothills of the Rincón volcano in Costa Rica. The Gram-negative cells are approximately 2.4-3.9 microm long and 0.5-0.6 microm wide and are motile rods with polar flagella. Strain CR11(T) grows between 65 and 85 degrees C (optimum 75 degrees C, doubling time 4.5 h) and between pH 4.8 and 7.8 (optimum pH 5.9-6.5). The isolate grows chemolithotrophically with S(0), S(2)O(2)(3)(-) or H(2) as the electron donor and with O(2) (up to 16 %, v/v) as the sole electron acceptor. The isolate can grow on mannose, glucose, maltose, succinate, peptone, Casamino acids, starch, citrate and yeast extract in the presence of oxygen (4 %) and S(0). Growth occurs only at NaCl concentrations below 0.4 % (w/v). The G+C content of strain CR11(T) is 40.3 mol%. Phylogenetic analysis of the 16S rRNA gene sequence places the strain as a close relative of Thermocrinis ruber OC 1/4(T) (95.7 % sequence similarity). Based on phylogenetic and physiological characteristics, we propose the name Thermocrinis minervae sp. nov., with CR11(T) (=DSM 19557(T) =ATCC BAA-1533(T)) as the type strain.
Full-text · Article · Sep 2009 · International Journal of Systematic and Evolutionary Microbiology
[Show abstract][Hide abstract] ABSTRACT: Bacterial biofilms are responsible for the majority of all microbial infections and have profound impact on industrial and geochemical processes. While many studies documented phenotypic differentiation and gene regulation of biofilms, the importance of their structural and mechanical properties is poorly understood. Here we investigate how changes in lipopolysaccharide (LPS) core capping in Pseudomonas aeruginosa affect biofilm structure through modification of adhesive, cohesive, and viscoelastic properties at an early stage of biofilm development. Microbead force spectroscopy and atomic force microscopy were used to characterize P. aeruginosa biofilm interactions with either glass substrata or bacterial lawns. Using isogenic migA, wapR, and rmlC mutants with defined LPS characteristics, we observed significant changes in cell mechanical properties among these strains compared to wild-type strain PAO1. Specifically, truncation of core oligosaccharides enhanced both adhesive and cohesive forces by up to 10-fold, whereas changes in instantaneous elasticity were correlated with the presence of O antigen. Using confocal laser scanning microscopy to quantify biofilm structural changes with respect to differences in LPS core capping, we observed that textural parameters varied with adhesion or the inverse of cohesion, while areal and volumetric parameters were linked to adhesion, cohesion, or the balance between them. In conclusion, this report demonstrated for the first time that changes in LPS expression resulted in quantifiable cellular mechanical changes that were correlated with structural changes in bacterial biofilms. Thus, the interplay between architectural and functional properties may be an important contributor to bacterial community survival.
Full-text · Article · Sep 2009 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: Lipopolysaccharide (LPS) monolayers deposited on planar, hydrophobic substrates were used as a defined model of outer membranes of Pseudomonas aeruginosa strain dps 89. To investigate the influence of ions on the (out-of-plane) monolayer structure, we measured specular X-ray reflectivity at high energy (22 keV) to ensure transmission through water. Electron density profiles were reconstructed from the reflectivity curves, and they indicate that the presence of Ca(2+) ions induces a significant change in the conformation of the charged polysaccharide head groups (O-side chains). Monte Carlo simulations based on a minimal computer model of LPS molecules allow for the modelling of 100 or more molecules over 10(-3) s and theoretically explained the tendency found by experiments.
Full-text · Article · Aug 2009 · Journal of The Royal Society Interface
[Show abstract][Hide abstract] ABSTRACT: The biofilm matrix contributes to the chemistry, structure, and function of biofilms. Biofilm-derived membrane vesicles (MVs)
and DNA, both matrix components, demonstrated concentration-, pH-, and cation-dependent interactions. Furthermore, MV-DNA
association influenced MV surface properties. This bears consequences for the reactivity and availability for interaction
of matrix polymers and other constituents.
Preview · Article · Jun 2009 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: Bacterial biofilms are the most prevalent mode of bacterial growth in nature. Adhesive and viscoelastic properties of bacteria play important roles at different stages of biofilm development. Following irreversible attachment of bacterial cells onto a surface, a biofilm can grow in which its matrix viscoelasticity helps to maintain structural integrity, determine stress resistance, and control ease of dispersion. In this study, a novel application of force spectroscopy was developed to characterize the surface adhesion and viscoelasticity of bacterial cells in biofilms. By performing microbead force spectroscopy with a closed-loop atomic force microscope, we accurately quantified these properties over a defined contact area. Using the model gram-negative bacterium Pseudomonas aeruginosa, we observed that the adhesive and viscoelastic properties of an isogenic lipopolysaccharide mutant wapR biofilm were significantly different from those measured for the wild-type strain PAO1 biofilm. Moreover, biofilm maturation in either strain also led to prominent changes in adhesion and viscoelasticity. To minimize variability in force measurements resulting from experimental parameter changes, we developed standardized conditions for microbead force spectroscopy to enable meaningful comparison of data obtained in different experiments. Force plots measured under standard conditions showed that the adhesive pressures of PAO1 and wapR early biofilms were 34 +/- 15 Pa and 332 +/- 47 Pa, respectively, whereas those of PAO1 and wapR mature biofilms were 19 +/- 7 Pa and 80 +/- 22 Pa, respectively. Fitting of creep data to a Voigt Standard Linear Solid viscoelasticity model revealed that the instantaneous and delayed elastic moduli in P. aeruginosa were drastically reduced by lipopolysaccharide deficiency and biofilm maturation, whereas viscosity was decreased only for biofilm maturation. In conclusion, we have introduced a direct biophysical method for simultaneously quantifying adhesion and viscoelasticity in bacterial biofilms under native conditions. This method could prove valuable for elucidating the contribution of genetic backgrounds, growth conditions, and environmental stresses to microbial community physiology.
Full-text · Article · May 2009 · Biophysical Journal
[Show abstract][Hide abstract] ABSTRACT: There have been considerable strides made in the characterization of the dispensability of teichoic acid biosynthesis genes
in recent years. A notable omission thus far has been an early gene in teichoic acid synthesis encoding the N-acetylmannosamine transferase (tagA in Bacillus subtilis; tarA in Staphylococcus aureus), which adds N-acetylmannosamine to complete the synthesis of undecaprenol pyrophosphate-linked disaccharide. Here, we show that the N-acetylmannosamine transferases are dispensable for growth in vitro, making this biosynthetic enzyme the last dispensable
gene in the pathway, suggesting that tagA (or tarA) encodes the first committed step in wall teichoic acid synthesis.
Preview · Article · May 2009 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: Slow-growing bacteria and biofilms are notoriously tolerant to antibiotics. Oritavancin is a lipoglycopeptide with multiple
mechanisms of action that contribute to its bactericidal action against exponentially growing gram-positive pathogens, including
the inhibition of cell wall synthesis and perturbation of membrane barrier function. We sought to determine whether oritavancin
could eradicate cells known to be tolerant to many antimicrobial agents, that is, stationary-phase and biofilm cultures of
Staphylococcus aureus in vitro. Oritavancin exhibited concentration-dependent bactericidal activity against stationary-phase inocula of methicillin-susceptible
S. aureus (MSSA) ATCC 29213, methicillin-resistant S. aureus (MRSA) ATCC 33591, and vancomycin-resistant S. aureus (VRSA) VRS5 inoculated into nutrient-depleted cation-adjusted Mueller-Hinton broth. As has been described for exponential-phase
cells, oritavancin induced membrane depolarization, increased membrane permeability, and caused ultrastructural defects including
a loss of nascent septal cross walls in stationary-phase MSSA. Furthermore, oritavancin sterilized biofilms of MSSA, MRSA,
and VRSA at minimal biofilm eradication concentrations (MBECs) of between 0.5 and 8 μg/ml. Importantly, MBECs for oritavancin
were within 1 doubling dilution of their respective planktonic broth MICs, highlighting the potency of oritavancin against
biofilms. These results demonstrate a significant activity of oritavancin against S. aureus in phases of growth that exhibit tolerance to other antimicrobial agents.
Preview · Article · Mar 2009 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Spirochaeta aurantia is a free-living saprophytic spirochete that grows easily in simple laboratory media, and thus can be used as a model for the investigation of surface carbohydrate structures in spirochetae, which are normally not available in sufficient amounts. Freeze-substitution electron microscopy indicated the presence of a capsule-like material projecting from the surface of S. aurantia. Extraction of cells gave two major glycolipids, the one with a higher molecular mass glycolipid was designated large glycolipid A (LGLA). LGLA contained small amount of branched and unsaturated O-linked fatty acids, L: -rhamnose, L: -fucose, D: -xylose, D: -mannose, D: -glucosamine, D: -glycero-D: -gluco-heptose (DDglcHep), D: -glycero-D: -manno-heptose (DDHep), and a novel branched tetradeoxydecose monosaccharide, which we proposed to call aurantose (Aur). The carbohydrate structure of LGLA was extremely complex and consisted of the repeating units built of 11 monosaccharides, arrangement of nine of them was determined as: - [- 3 - beta - DDglcHep - 3 - beta - D - GlcNAc - 2 - beta - D - Man - ] - which wasdeduced from the NMR and chemical data on the LGLA and its fragments, obtained by various degradations. Tentative position of two remaining sugars is proposed. LGLA was negative for gelation of Limulus amebocyte lysate, did not contain lipid A, and was unable to activate any known Toll-like receptors.
Full-text · Article · Mar 2009 · Glycoconjugate Journal