Oriented adhesion of Escherichia coli to polystyrene particles

The adhesion of nonflagellated Escherichia coli strain K-12 to polystyrene (PS) latex spheres or glass capillaries has been observed by using several techniques. Attention was focused on the orientation of the rod-shaped bacteria as they adhered to the surfaces in 100 mM phosphate-buffered saline. Data show that PS particles adhered to the ends of the bacteria more than 90% of the time. Moreover, the PS particles adhered to one end only, never to both. Similarly, for experiments with bacteria adhering to glass, the bacteria adhered on their ends. In order to determine whether the end of a bacterium had a different charge density from that of the middle, rotational electrophoresis experiments were used. These experiments indicated no measurable charge nonuniformity. In order to examine how strongly adhered the bacteria were to the PS particles, differential electrophoresis was used. Almost always, bacteria were found to be irreversibly adhered to the PS spheres. The cause of the oriented adhesion is not likely due to surface lipopolysaccharides (LPS), since the three strains of K-12 that were used, each having a different length of LPS, showed similar behavior. The results are discussed in terms of bacterial cell polarity. The data indicate that nanodomains on the bacterial ends are important for adhesion and that the time scale for irreversible adhesion is short.     

Oriented Adhesion of Escherichia coli to Polystyrene Particles

The adhesion of nonflagellated Escherichia coli strain K-12 to polystyrene (PS) latex spheres or glass capillaries has been observed by using several techniques. Attention was focused on the orientation of the rod-shaped bacteria as they adhered to the surfaces in 100 mM phosphate-buffered saline. Data show that PS particles adhered to the ends of the bacteria more than 90% of the time. Moreover, the PS particles adhered to one end only, never to both. Similarly, for experiments with bacteria adhering to glass, the bacteria adhered on their ends. In order to determine whether the end of a bacterium had a different charge density from that of the middle, rotational electrophoresis experiments were used. These experiments indicated no measurable charge nonuniformity. In order to examine how strongly adhered the bacteria were to the PS particles, differential electrophoresis was used. Almost always, bacteria were found to be irreversibly adhered to the PS spheres. The cause of the oriented adhesion is not likely due to surface lipopolysaccharides (LPS), since the three strains of K-12 that were used, each having a different length of LPS, showed similar behavior. The results are discussed in terms of bacterial cell polarity. The data indicate that nanodomains on the bacterial ends are important for adhesion and that the time scale for irreversible adhesion is short.     

Effective and robust partial nitrification to nitrite by real-time aeration duration control in an SBR treating domestic wastewater

Achieving sustainable partial nitrification to nitrite has been proven difficult in treating low strength nitrogenous wastewater. Real-time aeration duration control was used to achieve efficient partial nitrification to nitrite in a sequencing batch reactor (SBR) to treat low strength domestic wastewater. Above 90% nitrite accumulation ratio was maintained for long-term operation at normal condition, or even lower water temperature in winter. Partial nitrification established by controlling aeration duration showed good performance and robustness even though encountering long-term extended aeration and starvation period. Process control enhanced the successful accumulation of ammonia oxidizing bacteria (AOB) and washout of nitrite oxidizing bacteria (NOB). Scanning electron microscope observations indicated that the microbial morphology showed a shift towards small rod-shaped clusters. Fluorescence in situ hybridization (FISH) results demonstrated AOB were the dominant nitrifying bacteria, up to 8.3 ± 1.1% of the total bacteria; on the contrary, the density of NOB decreased to be negligible after 135 days operation since adopting process control.     

Control of cell morphogenesis in bacteria: two distinct ways to make a rod-shaped cell

Cell shape in most eubacteria is maintained by a tough external peptidoglycan cell wall. Recently, cell shape determining proteins of the MreB family were shown to form helical, actin-like cables in the cell. We used a fluorescent derivative of the antibiotic vancomycin as a probe for nascent peptidoglycan synthesis in unfixed cells of various Gram-positive bacteria. In the rod-shaped bacterium B. subtilis, synthesis of the cylindrical part of the cell wall occurs in a helical pattern governed by an MreB homolog, Mbl. However, a few rod-shaped bacteria have no MreB system. Here, a rod-like shape can be achieved by a completely different mechanism based on use of polar growth zones derived from the division machinery. These results provide insights into the diverse molecular strategies used by bacteria to control their cellular morphology, as well as suggesting ways in which these strategies may impact on growth rates and cell envelope structure.     

The Ultrastructure of Chlorobium tepidum Chlorosomes Revealed by Electron Microscopy

Chlorosomes are the light harvesting structures of green photosynthetic bacteria. Each chlorosome from green sulfur bacteria houses hundreds of thousands of bacteriochlorophyll molecules in addition to smaller amounts of chlorobiumquinone and carotenoids. In electron microscopy studies, chlorosomes exhibit different appearances depending on the fixation method used. Fixation with osmium tetroxide results in electron-transparent chlorosomes. Fixation with potassium permanganate results in clearly delineated electron-dense chlorosomes. This fixation method features an electron-transparent area in the interior of the chlorosome. In addition to electron density patterns that can be considered compositions of rod-shaped elements, chlorosomes exhibit a striation pattern that is oriented parallel to the longitudinal axis. Treatment with osmium tetroxide followed by potassium permanganate treatment results in a more diffused density distribution that outlines connecting elements between the chlorosome and the cytoplasmic membrane, and connecting elements between the cytoplasmic membrane and the outer membrane, which act as a diffusion barrier for electron density.     

Percentage of gelatinolytic bacteria among heterotrophic bacteria as indicator of water quality

The relationship between the physiological group of gelatinolytic bacteria and the abundance of heterotrophic bacteria in freshwater ecosystems was described, based on analysis of 1082 different freshwater samples collected in Croatia. Percentages of gelatinolytic bacteria among the population of heterotrophic bacteria showed a significant negative correlation with the abundance of heterotrophic bacteria. The relation between the physiological group (gelatinolytic bacteria) and heterotrophic bacteria can be considered to be an indicator of the pollution degree of freshwaters. A high relative content of gelatinolytic bacteria (> 76%) always indicates the colony-forming units of heterotrophic bacteria < 1000/mL, which corresponds to the high water quality; gelatinolytic bacteria < 11% indicate polluted waters. Isolated strains of aerobically grown gelatinolytic bacteria were Gram-negative rod-shaped or Gram-positive endospore-forming rod-shaped cells.     

Interconnected Cavernous Structure of Bacterial Fruiting Bodies

The formation of spore-filled fruiting bodies by myxobacteria is a fascinating case of multicellular self-organization by bacteria. The organization of Myxococcus xanthus into fruiting bodies has long been studied not only as an important example of collective motion of bacteria, but also as a simplified model for developmental morphogenesis. Sporulation within the nascent fruiting body requires signaling between moving cells in order that the rod-shaped self-propelled cells differentiate into spores at the appropriate time. Probing the three-dimensional structure of myxobacteria fruiting bodies has previously presented a challenge due to limitations of different imaging methods. A new technique using Infrared Optical Coherence Tomography (OCT) revealed previously unknown details of the internal structure of M. xanthus fruiting bodies consisting of interconnected pockets of relative high and low spore density regions. To make sense of the experimentally observed structure, modeling and computer simulations were used to test a hypothesized mechanism that could produce high-density pockets of spores. The mechanism consists of self-propelled cells aligning with each other and signaling by end-to-end contact to coordinate the process of differentiation resulting in a pattern of clusters observed in the experiment. The integration of novel OCT experimental techniques with computational simulations can provide new insight into the mechanisms that can give rise to the pattern formation seen in other biological systems such as dictyostelids, social amoeba known to form multicellular aggregates observed as slugs under starvation conditions.     

PCR detection of uncultured rumen bacteria

16S rRNA sequences of ruminal uncultured bacterial clones from public databases were phylogenetically examined. The sequences were found to form two unique clusters not affiliated with any known bacterial species: cluster of unidentified sequences of free floating rumen fluid uncultured bacteria (FUB) and cluster of unidentified sequences of bacteria associated with rumen epithelium (AUB). A set of PCR primers targeting 16S rRNA of ruminal free uncultured bacteria and rumen epithelium adhering uncultured bacteria was designed based on these sequences. FUB primers were used for relative quantification of uncultured bacteria in ovine rumen samples. The effort to increase the population size of FUB group has been successful in sulfate reducing broth and culture media supplied with cellulose.     

Selective binding and lateral clustering of α5β1 and αvβ3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

ABSTRACT

Coordination of the specific functions of α5β1 and αvβ3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvβ3 and α5β1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5β1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvβ3 ligand at 30 and 60 nm spacings. Analysis of αvβ3 and α5β1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5β1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvβ3 ligand. αvβ3 integrin clusters are more pronounced on αvβ3 ligand, though they can also be detected in cells adhering to α5β1 ligand. Furthermore, α5β1 integrin clusters are present in cells adhering to α5β1 ligand, and often colocalize with αvβ3 clusters. Taken together, these findings indicate that the activation of αvβ3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions.     

Analysis of bacterial detachment from substratum surfaces by the passage of air-liquid interfaces

A theoretical analysis of the detachment of bacteria adhering to substratum surfaces upon the passage of an air-liquid interface is given, together with experimental results for bacterial detachment in the absence and presence of a conditioning film on different substratum surfaces. Bacteria (Streptococcus sobrinus HG1025, Streptococcus oralis J22, Actinomyces naeslundii T14V-J1, Bacteroides fragilis 793E, and Pseudomonas aeruginosa 974K) were first allowed to adhere to hydrophilic glass and hydrophobic dimethyldichlorosilane (DDS)-coated glass in a parallel-plate flow chamber until a density of 4 x 10(6) cells cm(-2) was reached. For S. sobrinus HG1025, S. oralis J22, and A. naeslundii T14V-J1, the conditioning film consisted of adsorbed salivary components, while for B. fragilis 793E and P. aeruginosa 974K, the film consisted of adsorbed human plasma components. Subsequently, air bubbles were passed through the flow chamber and the bacterial detachment percentages were measured. For some experimental conditions, like with P. aeruginosa 974K adhering to DDS-coated glass and an air bubble moving at high velocity (i.e., 13.6 mm s(-1)), no bacteria detached upon passage of an air-liquid interface, while for others, detachment percentages between 80 and 90% were observed. The detachment percentage increased when the velocity of the passing air bubble decreased, regardless of the bacterial strain and substratum surface hydrophobicity involved. However, the variation in percentages of detachment by a passing air bubble depended greatly upon the strain and substratum surface involved. At low air bubble velocities the hydrophobicity of the substratum had no influence on the detachment, but at high air bubble velocities all bacterial strains were more efficiently detached from hydrophilic glass substrata. Furthermore, the presence of a conditioning film could either inhibit or stimulate detachment. The shape of the bacterial cell played a major role in detachment at high air bubble velocities, and spherical strains (i.e., streptococci) detached more efficiently than rod-shaped organisms. The present results demonstrate that methodologies to study bacterial adhesion which include contact with a moving air-liquid interface (i.e., rinsing and dipping) yield detachment of an unpredictable number of adhering microorganisms. Hence, results of studies based on such methodologies should be referred as "bacterial retention" rather than "bacterial adhesion".     

Electric field induced desorption of bacteria from a conditioning film covered substratum.

Desorption of three oral bacterial strains from a salivary conditioning film on an indium tin oxide electrode during application of a positive (bacterial adhesion to the anode) or a negative electric current was studied in a parallel plate flow chamber. Bacterial adhesion was from a flowing suspension of high ionic strength, after which the bacterial suspension was replaced by a low ionic strength solution without bacteria and currents ranging from -800 to +800 microA were applied. Streptococcus oralis J22 desorbed during application of a positive and negative electric current with a desorption probability that increased with increasing electric current. Two actinomyces strains, however, could not be stimulated to desorb by the electric currents applied. The desorption forces acting on adhering bacteria are electroosmotic in origin and working parallel to the electrode surface in case of a positive current, whereas they are electrophoretic and electrostatic in origin and working perpendicular to the surface in case of a negative current. By comparison of the effect of positive and negative electric currents, it can be concluded that parallel forces are more effective in stimulating bacterial desorption than perpendicular forces. The results of this study point to a new pathway of cleaning industrial and biomedical surfaces without the use of detergents or biocides.     

Random amplification of polymorphic DNA (RAPD) typing of carnobacteria isolated from hindgut chamber and large intestine of Atlantic cod (Gadus morhua l.)

Autochthonous and allochthonous bacteria were isolated from hindgut chamber and large intestine of fed and starved Atlantic cod (Gadus morhua L.). All bacterial strains isolated from hindgut chamber belong to carnobacteria. However, only 10.2% of the bacteria strains from the large intestine belong to carnobacteria. Random amplification of polymorphic DNA (RAPD) analysis using three selective primers, was performed to further identify the carnobacteria strains. Nine of these were isolated from hindgut chamber contents, ten associated with epithelial cells of the hindgut chamber, and six isolated from the large intestines of fed and starved fish. The 25 isolates segregated into eight clusters. The major cluster comprised nine strains isolated from the hindgut chamber of both fed and starved fish showing low similarity with the reference strains. The other strains isolated from the hindgut were located in clusters showing high similarity with Carnobacterium gallinarum or Carnobacterium piscicola. Strains isolated from large intestine appeared more divergent and were located in five different clusters. Autochthonous (indigenous) bacteria were clearly demonstrated in the hindgut chamber as transmission electron microscopy revealed rod-shaped bacteria between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in the hindgut chamber.     

Collective Motion of Spherical Bacteria

A large variety of motile bacterial species exhibit collective motions while inhabiting liquids or colonizing surfaces. These collective motions are often characterized by coherent dynamic clusters, where hundreds of cells move in correlated whirls and jets. Previously, all species that were known to form such motion had a rod-shaped structure, which enhances the order through steric and hydrodynamic interactions. Here we show that the spherical motile bacteria Serratia marcescens exhibit robust collective dynamics and correlated coherent motion while grown in suspensions. As cells migrate to the upper surface of a drop, they form a monolayer, and move collectively in whirls and jets. At all concentrations, the distribution of the bacterial speed was approximately Rayleigh with an average that depends on concentration in a non-monotonic way. Other dynamical parameters such as vorticity and correlation functions are also analyzed and compared to rod-shaped bacteria from the same strain. Our results demonstrate that self-propelled spherical objects do form complex ordered collective motion. This opens a door for a new perspective on the role of cell aspect ratio and alignment of cells with regards to collective motion in nature.     

A quantitative method to study co-adhesion of microorganisms in a parallel plate flow chamber: basic principles of the analysis

Intermicrobial aggregation is described as one of the factors contributing to dental plaque formation. Intermicrobial aggregation is usually measured by mixing potential partners suspended in a liquid phase (‘coaggregation’). However, even if aggregation in the liquid phase occurs, adhesion of microorganisms to partners already adhering to a substratum surface may also occur (‘co-adhesion’). Coaggregation assays have been performed in order to measure coaggregation and to model co-adhesion, although it is not yet clear which the two prevails in vivo. Apart from being semi-quantitative (scores run from 0 to 4) it is questionable whether coaggregation assays really mimic co-adhesion. This study was designed to develop a method to quantitative assess co-adhesion of microbial pairs in order to gain a better understanding of the mechanisms governing co-adhesion. Co-adhesion of coaggregating and non-coaggregating partners (S. oralis, S. sanguis and A. naeslundii) to glass has been studied in a parallel plate flow chamber using real time image analysis. The spatial arrangements of adhering bacteria were analyzed by radial pair distribution functions, revealing the relative density of adhering bacteria around adhering bacteria from the same (g₂₂(r)) or a partner strain (g₂₁(r)). Pair distribution functions g₂₁(r) of coaggregating pairs clearly reveal a preference of coaggregating streptococci (S. oralis J22 and S. sanguis PK2951) to adhere around the actimomyces (A. naeslundii PK213 or T14V-J1), which were used to coat the bare glass substratum. Besides, the distribution function g₂₁(r) showed differences in co-adhesion patterns for strains with the same coaggregation score. From the results presented in this paper it can be concluded that with a parallel plate flow chamber, co-adhesion can be quantified on a continuous scale under well controlled conditions, more closely resembling those occurring in vivo.     

Adherence of clinical isolates ofPseudomonas aeruginosa to hamster trachael epithelium in vitro

The adherence to hamster tracheal epithelium, of mucoid and nonmucoid clinical isolates ofPseudomonas aeruginosa from cystic fibrosis patients, was studied using tracheal organ cultures. Tracheal cultures were infected with 10⁷ colony-forming units per ml of either mucoid or nonmucoid clinical isolates ofP aeruginosa. The tracheal explants were rinsed at various time intervals to remove nonadherent bacteria, fixed, and prepared for transmission-and scanning-electron microscopy. Mucoid isolates were seen adhering to the ciliated epithelium as early as 4 h after initiation of infection, whereas nonmucoid isolates were only observed adhering at 6 to 8 h after infection. Mucoid organisms were found as clusters of bacteria embedded in an extensive extracellular matrix. The nonmucoid isolates were generally found as single organisms with no evidence of an extracellular matrix. These results suggest that the prevalence of mucoid isolates ofP. aeruginosa in cystic fibrosis may be due to adherent properties of the mucoid organism.     

Influence of adhesion to activated carbon particles on the viability of waterborne pathogenic bacteria under flow

In rural areas around the world, people often rely on water filtration plants using activated carbon particles for safe water supply. Depending on the carbon surface, adhering microorganisms die or grow to form a biofilm. Assays to assess the efficacy of activated carbons in bacterial removal do not allow direct observation of bacterial adhesion and the determination of viability. Here we propose to use a parallel plate flow chamber with carbon particles attached to the bottom plate to study bacterial adhesion to individual carbon particles and determine the viability of adhering bacteria. Observation and enumeration is done after live/dead staining in a confocal laser scanning microscope. Escherichiae coli adhered in higher numbers than Raoultella terrigena, except to a coconut-based carbon, which showed low bacterial adhesion compared to other wood-based carbon types. After adhesion, 83-96% of the bacteria adhering to an acidic carbon were dead, while on a basic carbon 54-56% were dead. A positively charged, basic carbon yielded 76-78% bacteria dead, while on a negatively charged coconut-based carbon only 32-37% were killed upon adhesion. The possibility to determine both adhesion as well as the viability of adhering bacteria upon adhesion to carbon particles is most relevant, because if bacteria adhere but remain viable, this still puts the water treatment system at risk, as live bacteria can grow and form a biofilm that can then be shedded to cause contamination.     

Direct observations of cooperative effects in oral streptococcal adhesion to glass by analysis of the spatial arrangement of adhering bacteria

The spatial arrangement of two strains of oral bacteria adhering on glass was studied in order to investigate cooperative effects in their adhesion mechanisms. Streptococcus salivarius HB was a strain which possessed several classes of fibrillar surface appendages, whereas on the cell surface of S. mutants NS no surface appendages could be identified. The bacteria were deposited from a flowing suspension with various buffer concentrations on the bottom glass plate of a parallel plate flow cell and were observed directly with a video camera mounted on a phase contrast microscope. The positions of all adhering bacteria were determined by means of automated real time image analysis and subsequently employed for calculating radial and angular pair distribution functions. Pair distribution functions indicate the average relative number density of bacteria around one deposited bacterium as a function of the radial distance or the angular orientation relative to the flow direction. From the calculated pair distribution functions of both bacterial strains it was concluded that cooperative effects contributed to the adhesion of S. salivarius HB, but not to adhesion of S. mutants NS. It was suggested that these cooperative effects originate from the surface appendages of S. salivarius HB.     

Long-term effect of dissolved oxygen on partial nitrification performance and microbial community structure

In this study, the performance of partial nitrification via nitrite and microbial community structure were investigated and compared in two sequencing batch reactors (SBR) with different dissolved oxygen (DO) levels. Both reactors achieved stable partial nitrification with nitrite accumulation ratio of above 95% by using real-time aeration duration control. Compared with high DO (above 3 mg/l on average) SBR, simultaneous nitrification and denitrification (SND) via nitrite was carried out in low DO (0.4–0.8 mg/l) SBR. The average efficiencies of SND in high DO and low DO reactor were 7.7% and 44.9%, and the specific SND rates were 0.20 and 0.83 mg N/(mg MLSS h), respectively. Low DO did not produce sludge with poorer settling properties but attained lower turbidities of the effluent than high DO. Fluorescence in situ hybridization (FISH) analysis in both the reactors showed that ammonia-oxidizing bacteria (AOB) were the dominant nitrifying bacteria and nitrite-oxidizing bacteria (NOB) did not be recovered in spite of exposing nitrifying sludge to high DO. The morphology of the sludge from both two reactors according to scanning electron microscope indicated that small rod-shaped and spherical clusters were dominant, although filamentous bacteria and few long rod-shaped coexisted in the low DO reactor. By selecting properly DO level and adopting process control method is not only of benefit to the achievement of novel biological nitrogen removal technology, but also favorable to sludge population optimization.     

Mechanisms for maintaining cell shape in rod-shaped Gram-negative bacteria

For the rod-shaped Gram-negative bacterium Escherichia coli, changes in cell shape have critical consequences for motility, immune system evasion, proliferation and adhesion. For most bacteria, the peptidoglycan cell wall is both necessary and sufficient to determine cell shape. However, how the synthesis machinery assembles a peptidoglycan network with a robustly maintained micron-scale shape has remained elusive. To explore shape maintenance, we have quantified the robustness of cell shape in three Gram-negative bacteria in different genetic backgrounds and in the presence of an antibiotic that inhibits division. Building on previous modelling suggesting a prominent role for mechanical forces in shape regulation, we introduce a biophysical model for the growth dynamics of rod-shaped cells to investigate the roles of spatial regulation of peptidoglycan synthesis, glycan-strand biochemistry and mechanical stretching during insertion. Our studies reveal that rod-shape maintenance requires insertion to be insensitive to fluctuations in cell-wall density and stress, and even a simple helical pattern of insertion is sufficient for over sixfold elongation without significant loss in shape. In addition, we demonstrate that both the length and pre-stretching of newly inserted strands regulate cell width. In sum, we show that simple physical rules can allow bacteria to achieve robust, shape-preserving cell-wall growth.     

Comparison of Electrode Reduction Activities of Geobacter sulfurreducens and an Enriched Consortium in an Air-Cathode Microbial Fuel Cell

An electricity-generating bacterium, Geobacter sulfurreducens PCA, was inoculated into a single-chamber, air-cathode microbial fuel cell (MFC) in order to determine the maximum electron transfer rate from bacteria to the anode. To create anodic reaction-limiting conditions, where electron transfer from bacteria to the anode is the rate-limiting step, anodes with electrogenic biofilms were reduced in size and tests were conducted using anodes of six different sizes. The smallest anode (7 cm², or 1.5 times larger than the cathode) achieved an anodic reaction-limiting condition as a result of a limited mass of bacteria on the electrode. Under these conditions, the limiting current density reached a maximum of 1,530 mA/m², and power density reached a maximum of 461 mW/m². Per-biomass efficiency of the electron transfer rate was constant at 32 fmol cell⁻¹ day⁻¹ (178 μmol g of protein⁻¹ min⁻¹), a rate comparable to that with solid iron as the electron acceptor but lower than rates achieved with fumarate or soluble iron. In comparison, an enriched electricity-generating consortium reached 374 μmol g of protein⁻¹ min⁻¹ under the same conditions, suggesting that the consortium had a much greater capacity for electrode reduction. These results demonstrate that per-biomass electrode reduction rates (calculated by current density and biomass density on the anode) can be used to help make better comparisons of electrogenic activity in MFCs.