Microtiter plate tests for segregation of bioluminescent bacteria

It has been recently shown that bioluminescence imaging can be usefully applied to provide new insights into bacterial self‐organization. In this work we employ bioluminescence imaging to record images of nutrient rich liquid cultures of the lux‐gene reporter Escherichia coli in microtiter plate wells. The images show that patterns of inhomogenous bioluminescence form along the three‐phase contact lines. The paper analyzes the dependencies of the average number of luminous aggregates (clouds) on various environmental factors. In particular, our results show that optimal (neutral) pH and high aeration rates determine the highest mean number of clouds, and that spatiotemporal patterns do not form in the pH buffered suspensions. In addition, a sigmoidal (switch‐like) dependence of the number of aggregates on the rate of aeration was observed. The obtained bioluminescence imaging data was interpreted by employing the Keller–Segel–Fisher (KSF) model of chemotaxis and logistic growth, adapted to systems of metabolically flexible (two‐state) bacteria. The modified KSF model successfully simulated the observed switch‐like responses. The results of the microtiter plate tests and their simulations indicate that the segregation of bacteria with different activities proceeds in the three‐phase contact line region. Copyright © 2015 John Wiley & Sons, Ltd.     

Robust Min‐system oscillation in the presence of internal photosynthetic membranes in cyanobacteria

The oscillatory Min system of Escherichia coli defines the cell division plane by regulating the site of FtsZ‐ring formation and represents one of the best‐understood examples of emergent protein self‐organization in nature. The oscillatory patterns of the Min‐system proteins MinC, MinD and MinE (MinCDE) are strongly dependent on the geometry of membranes they bind. Complex internal membranes within cyanobacteria could disrupt this self‐organization by sterically occluding or sequestering MinCDE from the plasma membrane. Here, it was shown that the Min system in the cyanobacterium Synechococcus elongatus PCC 7942 oscillates from pole‐to‐pole despite the potential spatial constraints imposed by their extensive thylakoid network. Moreover, reaction‐diffusion simulations predict robust oscillations in modeled cyanobacterial cells provided that thylakoid network permeability is maintained to facilitate diffusion, and suggest that Min proteins require preferential affinity for the plasma membrane over thylakoids to correctly position the FtsZ ring. Interestingly, in addition to oscillating, MinC exhibits a midcell localization dependent on MinD and the DivIVA‐like protein Cdv3, indicating that two distinct pools of MinC are coordinated in S. elongatus. Our results provide the first direct evidence for Min oscillation outside of E. coli and have broader implications for Min‐system function in bacteria and organelles with internal membrane systems.     

Multistability and dynamic transitions of intracellular Min protein patterns

Cells owe their internal organization to self‐organized protein patterns, which originate and adapt to growth and external stimuli via a process that is as complex as it is little understood. Here, we study the emergence, stability, and state transitions of multistable Min protein oscillation patterns in live Escherichia coli bacteria during growth up to defined large dimensions. De novo formation of patterns from homogenous starting conditions is observed and studied both experimentally and in simulations. A new theoretical approach is developed for probing pattern stability under perturbations. Quantitative experiments and simulations show that, once established, Min oscillations tolerate a large degree of intracellular heterogeneity, allowing distinctly different patterns to persist in different cells with the same geometry. Min patterns maintain their axes for hours in experiments, despite imperfections, expansion, and changes in cell shape during continuous cell growth. Transitions between multistable Min patterns are found to be rare events induced by strong intracellular perturbations. The instances of multistability studied here are the combined outcome of boundary growth and strongly nonlinear kinetics, which are characteristic of the reaction–diffusion patterns that pervade biology at many scales.     

Tetrathionate Disproportionation by Thiomonas intermedia K12

Thiomonas intermedia K12, a moderately acidophilic bacterium, which oxidises sulphur compounds, – exhibited the capability to use tetrathionate under oxic and anoxic conditions. Whereas under oxic conditions, the reduced sulphur tetrathionate compound was oxidised, under anoxic conditions, the organism disproportionated the compound. In both cases, trithionate and sulphate were produced but in different amounts. The results of the tetrathionate degradation experiments under oxic conditions pointed towards a cyclic degradation process with a transient formation of trithionate and sulphate as the final products, similar to the mechanism described for acidophilic sulphur compound oxidising bacteria. The results of the tetrathionate degradation experiments under anoxic conditions hinted to a partial reduction of tetrathionate to thiosulphate and a fractional oxidation to trithionate and sulphate. 4 M tetrathionate were converted to 6 M thiosulphate, 1 M trithionate, 1 M sulphate, and 8 M protons. The ΔG₀' of this reaction was found to be –16.1 kJ per mol tetrathionate degraded. Additionally, Thiomonas intermedia K12 grew under anoxic conditions with tetrathionate as the sole energy source. The cell numbers increased from 10⁵ as the start value to 10⁷/mL at the end. Organic compounds, excluding traces of yeast extract, did not enhance growth. Therefore, it is proposed that tetrathionate disproportionation is a novel lithotrophic metabolism, which allowed Thiomonas intermedia K12 to survive changing conditions of oxygen supply in sulphur‐compound‐rich environments and even to grow during this reaction. The extensive sulphur compound analysis was carried out by ion‐pair chromatography.     

Immobilization of bioluminescent Escherichia coli cells using natural and artificial fibers treated with polyethyleneimine

Biosensors based on whole-cell bioluminescence have the potential to become a cost-effective alternative to conventional detection methods upon validation of target selectivity and sensitivity. However, quantitative analysis of bioluminescence is greatly hindered due to lack of control over the total number of cells in a suspending culture. In this study, the effect of surface properties of genetically engineered luminous E. coli cells and fibrous matrices on the immobilization capacity and effectiveness under various environmental conditions were characterized. Four different fibers, including cotton, polyester, viscose rayon, and silk, were investigated. Although cell adhesion was observed on untreated viscose and cotton fibers, viscose fiber pretreated with 0.667% polyethyleneimine (PEI) was found capable of immobilizing the most viable E. coli DPD2234 cells, followed by viscose treated with 0.33% and 1% PEI. The cells immobilized on PEI-treated viscose remained viable and yielded 20% or more bioluminescence signals immediately upon contact with the inducer up to 72 h without feeding nutrients to the cells, suggesting that viscose treated with 0.667% PEI could provide a stable immobilization mechanism for bioluminescent E. coli cells with long sensing period, quick response time, and good signal reproducibility.     

The cytochrome bd oxidase of Escherichia coli prevents respiratory inhibition by endogenous and exogenous hydrogen sulfide

When sulfur compounds are scarce or difficult to process, Escherichia coli adapts by inducing the high‐level expression of sulfur‐compound importers. If cystine then becomes available, the cystine is rapidly overimported and reduced, leading to a burgeoning pool of intracellular cysteine. Most of the excess cysteine is exported, but some is adventitiously degraded, with the consequent release of sulfide. Sulfide is a potent ligand of copper and heme moieties, raising the prospect that it interferes with enzymes. We observed that when cystine was provided and sulfide levels rose, E. coli became strictly dependent upon cytochrome bd oxidase for continued respiration. Inspection revealed that low‐micromolar levels of sulfide inhibited the proton‐pumping cytochrome bo oxidase that is regarded as the primary respiratory oxidase. In the absence of the back‐up cytochrome bd oxidase, growth failed. Exogenous sulfide elicited the same effect. The potency of sulfide was enhanced when oxygen concentrations were low. Natural oxic‐anoxic interfaces are often sulfidic, including the intestinal environment where E. coli dwells. We propose that the sulfide resistance of the cytochrome bd oxidase is a key trait that permits respiration in such habitats.     

Characterization of biogenic iron oxides collected by the newly designed liquid culture method using diffusion chambers

We designed a new culture method for neutrophilic iron‐oxidizing bacteria using liquid medium (i) to study the formation and mineralogical characteristics of biogenic iron oxides (BIOS) and (ii) to apply BIOS to various scientific and engineering applications. An iron‐oxidizing bacterium, Mariprofundus ferrooxydans PV‐1^T (ATCC, BAA–1020), was cultured using a set of diffusion chambers to prepare a broad anoxic–oxic interface, upon which BIOS formation is typically observed in natural environments. Iron oxide precipitates were generated in parallel with bacterial growth. A scanning electron microscopy analysis indicated that the morphological features of the iron oxide precipitates in the medium (in vitro BIOS) were similar to those of BIOS collected from natural deep‐sea hydrothermal environments in the Northwest Eifuku Seamount field in the northern Mariana Arc (in situ BIOS). Further chemical speciation of both the in vitro and in situ BIOS was examined with X‐ray absorption fine structure (XAFS). A bulk XAFS analysis showed that the minerals in both BIOS were mainly ferrihydrite and oligomeric stages of amorphous iron oxyhydroxides with edge‐sharing octahedral linkages. The amount of in vitro BIOS produced with the diffusion‐chamber method was greater than those produced previously with other culture methods, such as gel‐stabilized gradient and batch liquid culture methods. The larger yields of BIOS produced with the new culture method will allow us to clarify in the future the mineralization mechanisms during bacterial growth and to examine the physicochemical properties of BIOS, such as their adsorption to and coprecipitation with various elements and substances.     

Variation of the folding and dynamics of the Escherichia coli chromosome with growth conditions

We examine whether the Escherichia coli chromosome is folded into a self‐adherent nucleoprotein complex, or alternately is a confined but otherwise unconstrained self‐avoiding polymer. We address this through in vivo visualization, using an inducible GFP fusion to the nucleoid‐associated protein Fis to non‐specifically decorate the entire chromosome. For a range of different growth conditions, the chromosome is a compact structure that does not fill the volume of the cell, and which moves from the new pole to the cell centre. During rapid growth, chromosome segregation occurs well before cell division, with daughter chromosomes coupled by a thin inter‐daughter filament before complete segregation, whereas during slow growth chromosomes stay adjacent until cell division occurs. Image correlation analysis indicates that sub‐nucleoid structure is stable on a 1 min timescale, comparable to the timescale for redistribution time measured for GFP–Fis after photobleaching. Optical deconvolution and writhe calculation analysis indicate that the nucleoid has a large‐scale coiled organization rather than being an amorphous mass. Our observations are consistent with the chromosome having a self‐adherent filament organization.     

Escherichia coli K‐12 (pEGFPluxABCDEamp): a tool for analysis of bacterial killing by antibacterial agents and human complement activities on a real‐time basis

Photorhabdus luminescens luxCDABE genes were integrated into E. coli K‐12 using a high copy number plasmid containing modified luxABCDE genes under the control of the powerful Lac promoter. This strain emitted 10 times higher bioluminescence (BL) than P. luminescens. BL production under different growth conditions was studied. In both bacterial strains, the increase in BL signal correlated with the increase in optical density (OD) in a rich growth medium. However, at the logarithmic growth phase, the BL signal was roughly constant. By contrast, in minimal growth media, there was no substantial growth and the BL/cell was approximately five times higher than in the rich medium. The dynamic measurement range of BL was 10²–10⁷ colony‐forming units (CFU) in E. coli and 10³–10⁷ CFU in P. luminescens. Because the decrease in the BL signal correlated with the decrease in CFU and OD, i.e. the number of bacterial cells killed, it proved to be very suitable for assessing the antibacterial effects of different antimicrobial agents. Unlike with plate counting, the kinetics of killing can be monitored on a real‐time basis using BL measurements. Complement activities in different samples can be estimated using only one serum dilution. The transformed E. coli strain appeared to be superior to P. luminescens in these applications because E. coli was complement sensitive, the detection limit of E. coli was one order lower and the BL‐producing system of P. luminescens appeared to be quite unstable. Copyright © 2012 John Wiley & Sons, Ltd.     

Effects of a transient oxic period on mineralization of organic matter to CH4 and CO2 in anoxic peat incubations

Rates of organic matter mineralization in peatlands, and hence production of the greenhouse gases CH₄ and CO₂, are highly dependent on the distribution of oxygen in the peat. Using laboratory incubations of peat, we investigated the sensitivity of the anoxic production of CH₄ and CO₂ to a transient oxic period of a few weeks’ duration. Production rates during 3 successive anoxic periods were compared with rates in samples incubated in the presence of oxygen during the second period. In surface peat (5–10‐cm depth), with an initially high level of CH₄ production, oxic conditions during period 2 did not result in a lower potential CH₄ production rate during period 3, although production was delayed ～1 week. In permanently anoxic, deep peat (50–55‐cm depth) with a comparatively low initial production of CH₄, oxic conditions during period 2 resulted in zero production of CH₄ during period 3. Thus, the methanogens in surface peal—but not in deep peat—remained viable after several weeks of oxic conditions. In contrast to CH₄ production, the oxic period had a negligible effect on anoxic CO₂ production during period 3, in surface as well as deep peat. In both surface and deep peat, CO₂ production was several times higher under oxic than under anoxic conditions. However, for the first 2 weeks of oxic conditions, CO₂ production in the deep peat was very low. Still, deep peat obviously contained facultative microorganisms that, after a relatively short period, were able to maintain a considerably higher rate of organic matter mineralization under oxic than under anoxic conditions.     

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

Aims: Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol‐acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen‐restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. Methods and Results: Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1·4‐fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro‐oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6·5 ± 0·3 g l^?1 ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l^?1 ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl‐coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two‐stage bioreactor cultures were conducted in a minimal medium containing 100 μg l^?1 calcium d ‐pantothenate to evaluate oxic acetyl‐CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15·4 ± 0·9 g l^?1 with a volumetric productivity of 0·34 ± 0·02 g l^?1 h^?1. Conclusions: Escherichia coli responded to adhE over‐expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl‐CoA played a key role in micro‐oxic ethanol synthesis and growth. Significance and Impact of the Study: Insight into the micro‐oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis.     

Reduced inorganic sulfur oxidation supports autotrophic and mixotrophic growth of Magnetospirillum strain J10 and Magnetospirillum gryphiswaldense

Magnetotactic bacteria are present at the oxic–anoxic transition zone where opposing gradients of oxygen and reduced sulfur and iron exist. Growth of non‐magnetotactic lithoautotrophic Magnetospirillum strain J10 and its close relative magnetotactic Magnetospirillum gryphiswaldense was characterized in microaerobic continuous culture. Both strains were able to grow in mixotrophic (acetate + sulfide) and autotrophic (sulfide or thiosulfate) conditions. Autotrophically growing cells completely converted sulfide or thiosulfate to sulfate and produced 7.5 g dry weight per mol substrate at a maximum observed growth rate of 0.09 h^?1 for strain J10 and 0.07 h^?1 for M. gryphiswaldense. The respiratory activity for acetate was repressed in autotrophic and also in mixotrophic cultures, suggesting acetate was used as C‐source in the latter. We have estimated the proportions of substrate used for assimilatory processes and evaluated the biomass yields per mol dissimilated substrate. The yield for lithoheterotrophic growth using acetate as the C‐source was approximately twice the autotrophic growth yield and very similar to the heterotrophic yield, showing the importance of reduced sulfur compounds for growth. In the draft genome sequence of M. gryphiswaldense homologues of genes encoding a partial sulfur‐oxidizing (Sox) enzyme system and reverse dissimilatory sulfite reductase (Dsr) were identified, which may be involved in the oxidation of sulfide and thiosulfate. Magnetospirillum gryphiswaldense is the first freshwater magnetotactic species for which autotrophic growth is shown.     

Spatial and seasonal variation in nutrient excretion by benthic invertebrates in a eutrophic reservoir

1. Nitrogen (N) and phosphorus (P) fluxes via excretion by benthic invertebrates were quantified in a eutrophic reservoir (Acton Lake, Ohio, U.S.A.). We quantified variation in nutrient fluxes seasonally (June until November 1997), spatially (three sites) and among taxa (chironomids, tubificid oligochaetes and Chaoborus). 2. The three taxa differed in spatial distribution and contribution to nutrient fluxes. Tubificids were the most abundant taxon at two oxic sites (1.5 and 4 m depth), and were exceedingly rare at an anoxic, hypolimnetic site (8 m). Chironomids were abundant only at the shallowest oxic site. Chaoborus was the only abundant taxon at the anoxic site. Total benthic invertebrate biomass was greatest at the shallowest site and lowest at the anoxic, hypolimnetic site. 3. Mass‐specific excretion rate [μmol NH₄–N or soluble reactive P (SRP) excreted mg dry mass^–1 h^–1] varied among experiments and was influenced by temperature. Differences among taxa were not significant. Thus, nutrient flux through benthic invertebrates was affected more by total invertebrate biomass and temperature than by species composition. 4. Fluxes of N and P via benthic invertebrate excretion (μmol NH₄–N or SRP m^–2 day^–1) were greatest at the oxic sites, where fluxes were dominated by the excretion of tubificids and chironomids. The N and P fluxes at the anoxic site were much lower, and were dominated by excretion by Chaoborus. The ratio of N and P excreted by the benthic invertebrate assemblage varied seasonally and was lowest at the anoxic site. 5. Comparison with other measured inputs shows that excretion by benthic invertebrates could be an important source of nutrients, especially of P. However, the relative importance of nutrient excretion by the benthos varies greatly spatially and temporally.     

Impact of osmotic stress on protein diffusion in Lactococcus lactis

We measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Gram‐positive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12‐transmembrane helix membrane proteins are similar to those in Escherichia coli. The translational diffusion of GFP in L. lactis drops by two orders of magnitude when the medium osmolality is increased by ～ 1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L. lactis are similar to those reported in E. coli, the decrease in translational diffusion upon osmotic challenge in L. lactis is smaller than in E. coli. An even more striking difference is that in L. lactis the GFP diffusion coefficient drops much more rapidly with volume than in E. coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram‐positive and Gram‐negative bacteria.     

Microbial processes of the carbon and sulfur cycles in an ice‐covered,iron‐rich meromictic lake Svetloe (Arkhangelsk region,Russia)

Biogeochemical, isotope geochemical and microbiological investigation of Lake Svetloe (White Sea basin), a meromictic freshwater was carried out in April 2014, when ice thickness was ～0.5 m, and the ice‐covered water column contained oxygen to 23 m depth. Below, the anoxic water column contained ferrous iron (up to 240 μμM), manganese (60 μM), sulfide (up to 2 μM) and dissolved methane (960 μM). The highest abundance of microbial cells revealed by epifluorescence microscopy was found in the chemocline (redox zone) at 23–24.5 m. Oxygenic photosynthesis exhibited two peaks: the major one (0.43 μmol C L^?1 day^?1) below the ice and the minor one in the chemocline zone, where cyanobacteria related to Synechococcus rubescens were detected. The maximum of anoxygenic photosynthesis (0.69 μmol C L^?1 day^?1) at the oxic/anoxic interface, for which green sulfur bacteria Chlorobium phaeoclathratiforme were probably responsible, exceeded the value for oxygenic photosynthesis. Bacterial sulfate reduction peaked (1.5 μmol S L^?1 day^?1) below the chemocline zone. The rates of methane oxidation were as high as 1.8 μmol CH₄ L^?1 day^?1 at the oxi/anoxic interface and much lower in the oxic zone. Small phycoerythrin‐containing Synechococcus‐related cyanobacteria were probably involved in accumulation of metal oxides in the redox zone.     

High-resolution 2D and 3D cryo-TEM reveals structural adaptations of two stalk-forming bacteria to an Fe-oxidizing lifestyle

Aerobic neutrophilic Fe‐oxidizing bacteria (FeOB) thrive where oxic and iron‐rich anoxic waters meet. Here, iron microbial mats are commonly developed by stalk‐forming Fe‐oxidizers adapted to these iron‐rich gradient environments, somehow avoiding iron encrustation. Few details are known about FeOB physiology; thus, the bases of these adaptations, notably the mechanisms of interactions with iron, are poorly understood. We examined two stalked FeOB: the marine Zetaproteobacterium Mariprofundus ferrooxydans and a terrestrial Betaproteobacterium Gallionella‐like organism. We used cryo‐transmission electron microscopy and cryo‐electron tomography to provide unprecedented ultrastructural data on intact cell‐mineral systems. Both FeOB localize iron mineral formation at stalk extrusion sites, while avoiding surface and periplasmic mineralization. The M. ferrooxydans cell surface is densely covered in fibrils while the terrestrial FeOB surface is smooth, suggesting a difference in surface chemistry. Only the terrestrial FeOB exhibited a putative chemotaxis apparatus, which may be due to differences in chemotaxis mechanisms. Both FeOB have a single flagellum, which alone is insufficient to account for cell motion during iron oxidation, suggesting that stalk extrusion is a mechanism for motility. Our results delineate the physical framework of iron transformations and characterize possible structural adaptations to the iron‐oxidizing lifestyle. This study shows ultrastructural similarities and differences between two distinct FeOB, setting the stage for further (e.g. genomic) comparisons that will help us understand functional differences and evolutionary history.     

Effect of concentrating and exposing the bioluminescent bacteria to the non‐luminescent allo‐bacterial extracellular products on their luminescence

Bioluminescence is a biochemical process occurring in many organisms. Bacterial bioluminescence has been investigated extensively that lead to many applications of such knowledge. Quorum sensing in the bioluminescent bacteria is a chemical signal process to recognize the strength of its own population to start luminescence in harmony. There is a mechanism in these bacteria to also recognize inter‐species strength. When there is a higher number of these bacteria, the possibility and frequency of cell–cell physical contact will be high. In this study, the physical proximity was artificially enhanced between cells and the effect on luminescence in the concentrated cells in the normal culture medium and in the presence of other non‐bacterial cell‐free supernatants was investigated. The role of such physical contact in the quorum sensing in the bioluminescence is not known. Increase in the luminescence of V. fischeri when concentrated shows that the presence of physical proximity facilitates the quorum sensing for their bioluminescence. Copyright © 2009 John Wiley & Sons, Ltd.     

The Leucine Incorporation Method Estimates Bacterial Growth Equally Well in Both Oxic and Anoxic Lake Waters

Bacterial biomass production is often estimated from incorporation of radioactively labeled leucine into protein, in both oxic and anoxic waters and sediments. However, the validity of the method in anoxic environments has so far not been tested. We compared the leucine incorporation of bacterial assemblages growing in oxic and anoxic waters from three lakes differing in nutrient and humic contents. The method was modified to avoid O₂ contamination by performing the incubation in syringes. Isotope saturation levels in oxic and anoxic waters were determined, and leucine incorporation rates were compared to microscopically observed bacterial growth. Finally, we evaluated the effects of O₂ contamination during incubation with leucine, as well as the potential effects of a headspace in the incubation vessel. Isotope saturation occurred at a leucine concentration of above about 50 nM in both oxic and anoxic waters from all three lakes. Leucine incorporation rates were linearly correlated to observed growth, and there was no significant difference between oxic and anoxic conditions. O₂ contamination of anoxic water during 1-h incubations with leucine had no detectable impact on the incorporation rate, while a headspace in the incubation vessel caused leucine incorporation to increase in both anoxic and O₂-contaminated samples. The results indicate that the leucine incorporation method relates equally to bacterial growth rates under oxic and anoxic conditions and that incubation should be performed without a headspace.