Mechanically resolved imaging of bacteria using expansion microscopy.

Imaging dense and diverse microbial communities has broad applications in basic microbiology and medicine, but remains a grand challenge due to the fact that many species adopt similar morphologies. While prior studies have relied on techniques involving spectral labeling, we have developed an expansion microscopy method (µExM) in which bacterial cells are physically expanded prior to imaging. We find that expansion patterns depend on the structural and mechanical properties of the cell wall, which vary across species and conditions. We use this phenomenon as a quantitative and sensitive phenotypic imaging contrast orthogonal to spectral separation to resolve bacterial cells of different species or in distinct physiological states. Focusing on host-microbe interactions that are difficult to quantify through fluorescence alone, we demonstrate the ability of µExM to distinguish species through an in vitro defined community of human gut commensals and in vivo imaging of a model gut microbiota, and to sensitively detect cell-envelope damage caused by antibiotics or previously unrecognized cell-to-cell phenotypic heterogeneity among pathogenic bacteria as they infect macrophages.

Acetification of rice wine by Acetobacter aceti using loofa sponge in a low-cost reciprocating shaker.

AIMS: To maximize acetification rate (ETA) by adsorption of acetic acid bacteria (AAB) on loofa sponge matrices (LSM). METHODS AND RESULTS: AAB were adsorbed on LSM, and the optimal shaking rate was determined for maximized AAB growth and oxygen availability. Results confirm that the 1 Hz reciprocating shaking rate with 40% working volume (liquid volume 24 l, tank volume 60 l) achieved a high oxygen transfer coefficient (k(L)a). The highest ETA was obtained at 50% (w:v) LSM-AAB:culture medium at 30 ± 2°C (P ≤ 0·05). To test process consistency, nine sequential acetification cycles were run using LSM-AAB and comparing it with no LSM. The highest ETA (1·701-2·401 g l(-1) d(-1)) was with LSM-AAB and was associated with the highest biomass of AAB, confirmed by SEM images. CONCLUSIONS: Results confirm that LSM-AAB works well as an inert substrate for AAB. High oxygenation was maintained by a reciprocating shaker. Both shaking and LSM were important in increasing ETA. SIGNIFICANCE AND IMPACT OF THE STUDY: High cell biomass in LSM-AAB provides good conditions for higher ETAs of quick acetification under adequate oxygen transfer by reciprocating shaker. It is a sustainable process for small-scale vinegar production system requiring minimal set-up cost.

Proteome analysis of Acetobacter pasteurianus during acetic acid fermentation.

Acetic acid bacteria (AAB) are Gram-negative, strictly aerobic microorganisms that show a unique resistance to ethanol (EtOH) and acetic acid (AcH). Members of the Acetobacter and Gluconacetobacter genera are capable of transforming EtOH into AcH via the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes and are used for the industrial production of vinegar. Several mechanisms have been proposed to explain how AAB resist high concentrations of AcH, such as the assimilation of acetate through the tricarboxylic acid (TCA) cycle, the export of acetate by various transporters and modifications of the outer membrane. However, except for a few acetate-specific proteins, little is known about the global proteome responses to AcH. In this study, we used 2D-DIGE to compare the proteome of Acetobacter pasteurianus LMG 1262(T) when growing in glucose or ethanol and in the presence of acetic acid. Interesting protein spots were selected using the ANOVA p-value of 0.05 as threshold and 1.5-fold as the minimal level of differential expression, and a total of 53 proteins were successfully identified. Additionally, the size of AAB was reduced by approximately 30% in length as a consequence of the acidity. A modification in the membrane polysaccharides was also revealed by PATAg specific staining.

Culture medium optimization for acetic acid production by a persimmon vinegar-derived bacterium.

A new acetic acid-producing microorganism, Acetobacter sp. RKY4, was isolated from Korean traditional persimmon vinegar, and we optimized the culture medium for acetic acid production from ethanol using the newly isolated Acetobacter sp. RKY4. The optimized culture medium for acetic acid production using this microorganism was found to be 40 g/L ethanol, 10 g/L glycerol, 10 g/L corn steep liquor, 0.5 g/L MgSO4.7H2O, and 1.0 g/L (NH4)H2PO4. Acetobacter sp. RKY4 produced 47.1 g/L of acetic acid after 48 h of fermentation in a 250 mL Erlenmeyer flask containing 50 mL of the optimized medium.

Structural changes in the cells of some bacteria during population growth: a Fourier transform infrared-attenuated total reflectance study.

Structural changes occurring in the cells of several bacteria during their growth curves have been investigated by Fourier transform infrared (FT-IR) spectroscopy using the sampling technique of attenuated total reflectance (ATR). Spectra reflect all of the components of the cells, including the cell walls, cell membranes, internal structures, and the cytoplasm. The bacteria studied were Bacillus stearothermophilus, Halobacterium salinarum, Halococcus morrhuae, and Acetobacter aceti. All species showed significant spectral changes during their growth curves, indicating structural changes in the cells during increases in cell numbers. The major change for B. stearothermophilus was in the lipid content, which was at a maximum during the exponential phase of the growth curve. For the halophiles H. salinarum and H. morrhuae, the major change was that the concentration of sulfate ion in the cells varied during the growth curve and was at a maximum during the mid-part of the exponential phase of the growth curve. A. aceti cells showed increasing polysaccharide content during the growth curve as well as maximum lipid content during the exponential phase of growth.

Mechanical properties of primary plant cell wall analogues.

Mechanical effects of turgor pressure on cell walls were simulated by deforming cell wall analogues based on Acetobacter xylinus cellulose under equi-biaxial tension. This experimental set-up, with associated modelling, allowed quantitative information to be obtained on cellulose alone and in composites with pectin and/or xyloglucan. Cellulose was the main load-bearing component, pectin and xyloglucan leading to a decrease in modulus when incorporated. The cellulose-only system could be regarded as an essentially linear elastic material with a modulus ranging from 200 to 500 MPa. Pectin incorporation modified extensibility properties of the system by topology/architecture changes of cellulose fibril assemblies, but the cellulose/pectin composites could still be described as a linear elastic material with a modulus ranging from 120 to 250 MPa. The xyloglucan/cellulose composite could not be modelled as a linear elastic material. Introducing xyloglucan into a cellulose network or a cellulose/pectin composite led to very compliant materials characterised by time-dependent creep behaviour. Modulus values obtained for the composite materials were compared with mechanical data found for plant-derived systems. After comparing bi-axial and uni-axial behaviour of the different composites, structural models were proposed to explain the role of each polysaccharide in determining the mechanical properties of these plant primary cell wall analogues.

Comparison of benefit to sugarcane plant growth and 15N2 incorporation following inoculation of sterile plants with Acetobacter diazotrophicus wild-type and Nif- mutants strains.

The ability of the nitrogen-fixing bacterial endophyte Acetobacter diazotrophicus strain PAl5 to enhance the growth of sugarcane SP70-1143 was evaluated in the growth chamber, greenhouse, and field by comparing plants inoculated with wild-type and Nif mutant MAd3A in two independent experiments. The wild-type and Nif mutant strains colonized sugarcane plants equally and persisted in mature plants. In N-deficient conditions, sugarcane plants inoculated with A. diazotrophicus PAl5 generally grew better and had a higher total N content 60 days after planting than did plants inoculated with mutant MAd3A or uninoculated plants. These results indicate that the transfer of fixed N from A. diazotrophicus to sugarcane might be a significant mechanism for plant growth promotion in this association. When N was not limiting, growth enhancement was observed in plants inoculated with either wild-type or Nif- mutants, suggesting the additional effect of a plant growth promoting factor provided by A. diazotrophicus. A 15N2 incorporation experiment demonstrated that A. diazotrophicus wild-type strains actively fixed N2 inside sugarcane plants, whereas the Nif- mutants did not.

Gravity effects on cellulose assembly.

The effect of microgravity on cellulose synthesis using the model system of Acetobacter xylinum was the subject of recent investigations using The National Aeronautics and Space Administration's Reduced Gravity Laboratory, a modified KC-135 aircraft designed to produce 20 sec of microgravity during the top of a parabolic dive. Approximately 40 parabolas were executed per mission, and a period of 2 x g was integral to the pullout phase of each parabola. Cellulose biosynthesis was initiated on agar surfaces, liquid growth medium, and buffered glucose during parabolic flight and terminated with 2.0% sodium azide or 50.0% ethanol. While careful ground and in-flight controls indicated normal, compact ribbons of microbial cellulose, data from five different flights consistently showed that during progression into the parabola regime, the cellulose ribbons became splayed. This observation suggests that some element of the parabola (the 20 sec microgravity phase, the 20 sec 2 x g phase, or a combination of both) was responsible for this effect. Presumably the cellulose I alpha crystalline polymorph normally is produced under strain, and the microgravity/hypergravity combination may relieve this stress to produce splayed ribbons. An in-flight video microscopy analysis of bacterial motions during a parabolic series demonstrated that the bacteria continue to synthesize cellulose during all phases of the parabolic series. Thus, the splaying may be a reflection of a more subtle alteration such as reduction of intermicrofibrillar hydrogen bonding. Long-term microgravity exposures during spaceflight will be necessary to fully understand the cellulose alterations from the short-term microgravity experiments.

Biogenesis of bacterial cellulose.

Cellulose is the most abundant biological polymer on Earth. It is found in wood and cotton, and forms the basic structural foundation of the cell wall of almost all eukaryotic plants. Bacteria are known to secrete cellulose as part of their metabolism of glucose and other sugars. The focus of this review is upon bacterial cellulose synthesis. We emphasize recent literature directed primarily upon Acetobacter xylinum, which has been most widely studied. Our review covers the following topics relating to cellulose synthesis: genetics, biochemistry, ultrastructure, growth conditions, and ecological considerations as they relate to the diversity of microbes capable of synthesizing this abundant, unique polymer--cellulose.

Alteration of in vivo cellulose ribbon assembly by carboxymethylcellulose and other cellulose derivatives.

In vivo cellulose ribbon assembly by the Gram-negative bacterium Acetobacter xylinum can be altered by incubation in carboxymethylcellulose (CMC), a negatively charged water-soluble cellulose derivative, and also by incubation in a variety of neutral, water-soluble cellulose derivatives. In the presence of all of these substituted celluloses, normal fasciation of microfibril bundles to form the typical twisting ribbon is prevented. Alteration of ribbon assembly is most extensive in the presence of CMC, which often induces synthesis of separate, intertwining bundles of microfibrils. Freeze-etch preparations of the bacterial outer membrane suggest that particles that are thought to be associated with cellulose synthesis or extrusion may be specifically organized to mediate synthesis of microfibril bundles. These data support the previous hypothesis that the cellulose ribbon of A. xylinum is formed by a hierarchical, cell-directed, self-assembly process. The relationship of these results to the regulation of cellulose microfibril size and wall extensibility in plant cell walls is discussed.

Calcofluor white ST Alters the in vivo assembly of cellulose microfibrils.

The fluorescent brightener, Calcofluor White ST, prevents the in vivo assembly of crystalline cellulose microfibrils and ribbons by Acetobacter xylinum. In the presence of more than 0.01 percent Calcofluor, Acetobacter continues to synthesize high-molecular-weight beta-1,4 glucans. X-ray crystallography shows that the altered product exhibits no detectable crystallinity in the wet state, but upon drying it changes into crystalline cellulose I. Calcofluor alters cellulose crystallization by hydrogen bonding with glucan chains. Synthesis of this altered product is reversible and can be monitored with fluorescence and electron microscopy. Use of Calcofluor has made it possible to separate the processes of polymerization and crystallization leading to the biogenesis of cellulose microfibrils, and has suggested that crystallization occurs by a cell-directed. self-assembly process in Acetobacter xylinum.

Lipid and fatty acid composition of Gluconobacter oxydans before and after intracytoplasmic membrane formation.

Gluconobacter oxydans differentiates by forming quantities of intracytoplasmic membranes at the end of exponential growth, and this formation occurs concurrently with a 60% increase in cellular lipid. The present study was initiated to determine whether this newly synthesized lipid differed from that extracted before intracytoplasmic membrane synthesis. Undifferentiated exponential-phase cells were found to contain 30% phosphatidylcholine, 27.1% caridolipin, 25% phosphatidylethanolamine, 12.5% phosphatidylglycerol, 0.4% phosphatidic acid, 0.2% phosphatidylserine, and four additional unidentified lipids totaling less than 5%. The only change detected after formation of intracytoplasmic membranes was a slight decrease in phosphatidylethanolamine and a corresponding increase in phosphatidylcholine. An examination of lipid hydrolysates revealed 11 different fatty acids in the lipids from each cell type. Hexadecanoic acid and monounsaturated octadecenoic accounted for more than 75% of the total fatty acids for both cell types. Proportional changes were noted in all fatty acids except octadecenoate. Anteiso-pentadecanoate comprised less than 1% of the fatty acids from undifferentiated cells but more than 13% of the total fatty acids from cells containing intracytoplasmic membranes. These results suggest that anteiso-pentadecanoate formation closely parallels the formation of intracytoplasmic membranes. Increased concentrations of this fatty acid may contribute to the fluidity necessary for plasma membrane convolution during intracytoplasmic membrane development.

Electron microscopic investigation of the hydrogen-oxidizing acetate-forming anaerobic bacterium Acetobacterium woodii.

Acetobacterium woodii is a Gram-positive anaerobic nonsporeforming bacterium able to grow on H2 and CO2 as sole sources of energy. The product of fermentation is acetic acid. Fine structural analysis showed rod-shaped flagellated cells, and coccoid cells without flagella arranged predominantly in pairs and chains. The cell wall was found to be composed of three layers. The cell surface exhibited a periodic array of particles consisting of subunits. The cytoplasmic membrane showed particles either in random distribution or in a hexagonal pattern. Intracytoplasmic membranes were rarely observed, whereas inclusion bodies of varying shapes, predominantly in an uncommon disc-shape, could frequently be observed. Their content was dissolved in ultrathin sections indicating hydrophobic nature.

Nascent stage of cellulose biosynthesis.

Freeze-etching of never-dried pellicles or of incubated suspensions of both Acetobacter xylinum and Acetobacter acetigenum show a nascent form of the cellulose microfibril which has a core surrounded by an amorphous sheath. Drying of the pellicle or suspension reduces the diameter of the sheath and changes the form of the microfibril to the one usually seen. This nascent form of the cellulose microfibril is consistent with previous postulations of an intermediate polymer or polymers in the biosynthesis of cellulose.