Charge-directed targeting of antimicrobial protein-nanoparticle conjugates

Use of antimicrobial enzymes covalently attached to nanoparticles is of great interest as an antibiotic-free approach to treat microbial infections. Intrinsic properties of nanoparticles can also be used to add functionality to their conjugates with biomolecules. Here, we show in a model system that nanoparticle charge can be used to enhance delivery and increase bactericidal activity of an antimicrobial enzyme, lysozyme. Hen egg lysozyme was covalently attached to two types of polystyrene latex nanoparticles: positively charged, containing aliphatic amine surface groups, and negatively charged, containing sulfate and chloromethyl surface groups. In the case of bacterial lysis assay with a Gram-positive bacteria Micrococcus lysodeikticus, activity of lysozyme conjugated to positively charged nanoparticles was approximately twice as large as that of free lysozyme, while lysozyme conjugated to negatively charged nanoparticles showed little detectable activity. At the same time, when assayed using a low-molecular weight oligosaccharide substrate, lysozyme attached to both positively and negatively charged nanoparticles showed slightly lower activity than free enzyme. A possible explanation of these results is that lysozyme attached to negatively charged nanoparticles cannot be effectively targeted to the bacteria because of the electrostatic Coulombic repulsion from the negatively charged bacterial cell walls, whereas lysozyme conjugated to positively charged nanoparticles was targeted better than free enzyme due to stronger electrostatic attraction to bacteria. Zeta potential measurements confirmed the validity of this hypothesis. Thus, nanoparticle charge is an important factor that can be used to control targeting and activity of protein-nanoparticle conjugates.     

Biochemistry and physiology of aerobic carbon monoxide-utilizing bacteria

Abstract The use of CO as a growth substrate by aerobic CO-oxidizing (carboxydotrophic) bacteria requires some features not obvious in other bacteria. These are the presence of the enzyme CO dehydrogenase, a branched respiratory chain with an alternative CO-insensitive terminal oxidase (cytochrome b ₆₅₃) and formation of reduced pyridine nucleotides by a pmf-driven reversed electron transfer. Immunocytochemical localization studies revealed that CO dehydrogenase is attached to the inner aspect of the cytoplasmic membrane of Pseudomonas carboxydovorans . The enzyme is a molybdo iron-sulfur flavoprotein containing bactopterin as the organic portion of the molybdenum cofactor. Recent findings suggest that this novel pterin is universal to eubacterial molybdenum enzymes, whereas molybdopterin is universal to eukaryotic molybdoenzymes.     

Attached and Free Bacteria in an Estuary with Abundant Suspended Solids

Concentrations and glucose mineralization potentials ( V _max) of attached and free bacteria in waters from the Humber estuary were determined. The waters had high concentrations of suspended solids. Most bacteria were attached to suspended solids and V _max of attached bacteria was greater than that of free bacteria. These observations suggest that breakdown of organic matter is carried out mainly by attached bacteria. Examination of samples from other estuaries which contained less suspended solids than the Humber suggested that in these estuaries free bacteria might be more abundant than attached bacteria.     

Diel and Seasonal Variations in Abundance,Activity, and Community Structure of Particle-Attached and Free-Living Bacteria in NW Mediterranean Sea

Diel and seasonal variations in abundance, activity, and structure of particle-attached vs free-living bacterial communities were investigated in offshore NW Mediterranean Sea (0–1000 m). Attached bacteria were always less abundant and less diverse but generally more active than free-living bacteria. The most important finding of this study was that the activity of attached bacteria showed pronounced diel variations in the upper mixed water column with higher activities at night. Under mesotrophic conditions, the contribution of attached bacteria to total bacterial activity increased from less than 10% at day time to 83% at night time. At high chlorophyll a concentration, the highest cell-specific activities and contribution to total bacterial activity were due to free-living bacteria at day and to attached bacteria at night. Under summer oligotrophic conditions, free-living bacteria dominated and contributed to the most important part of the bacterial activity at both day and night, whereas attached bacteria were much less abundant but presented the highest cell-specific activities. These diel and seasonal variations in activities were concomitant to changes in bacterial community structure, mainly in the upper layer. The number of attached ribotypes was fairly constant suggesting that particles are colonized by a relatively limited number of ubiquitous ribotypes. Most of these ribotypes were also free-living ribotypes suggesting that attached bacteria probably originate from colonization of newly formed particles by free-living bacteria in the upper layer. These results reinforce the biogeochemical role of attached bacteria in the cycling of particulate organic carbon in the NW Mediterranean Sea and the importance of diel variability in these processes.     

Comparison of cell-specific activity between free-living and attached bacteria using isolates and natural assemblages

Marine snow aggregates are microbial hotspots that support high bacterial abundance and activities. We conducted laboratory experiments to compare cell-specific bacterial protein production (BPP) and protease activity between free-living and attached bacteria. Natural bacterial assemblages attached to model aggregates (agar spheres) had threefold higher BPP and two orders of magnitude higher protease activity than their free-living counterpart. These observations could be explained by preferential colonization of the agar spheres by bacteria with inherently higher metabolic activity and/or individual bacteria increasing their metabolism upon attachment to surfaces. In subsequent experiments, we used four strains of marine snow bacteria isolates to test the hypothesis that bacteria could up- and down-regulate their metabolism while on and off an aggregate. The protease activity of attached bacteria was 10-20 times higher than that of free-living bacteria, indicating that the individual strains could increase their protease activity within a short time (2 h) upon attachment to surfaces. Agar spheres with embedded diatom cells were colonized faster than plain agar spheres and the attached bacteria were clustered around the agar-embedded diatom cells, indicating a chemosensing response. Increased protease activity and BPP allow attached bacteria to quickly exploit aggregate resources upon attachment, which may accelerate remineralization of marine snow and reduce the downward carbon fluxes.     

Amino Acid Assimilation and Electron Transport System Activity in Attached and Free-Living Marine Bacteria

Amino acid assimilation and electron transport system activity of a marine Pseudomonas sp. was evaluated to determine whether the activity of bacteria attached to solid surfaces differed from that of free-living bacteria or bacteria which had been attached but subsequently desorbed from the substratum (detached bacteria). Bacteria were allowed to attach to glass and to a range of plastic surfaces (Thermanox, polyvinylidene fluoride, polyethylene, polytetrafluoroethylene). Microautoradiography and staining with a tetrazolium salt to demonstrate electron transport system activity were used to compare the activity of these organisms with that of free-living or detached cells. The water-wettability of the surfaces was evaluated by measuring the advancing contact angle (θ_A) of water on each surface, to determine whether there was a relationship between activity and substratum hydrophilicity. There was an increase in the proportion of leucine-assimilating attached bacteria and in the proportion of attached cells demonstrating electron transport system activity with an increase in substratum θ_A, but the relationship between activity of attached and free-living cells depended on the substratum. Activity appeared to promote firm attachment, and detached bacteria assimilated fewer amino acids than did attached cells. There was no general effect of surfaces on attached bacterial activity, and attached cells may be more, or less, active than free-living cells, depending on the amino acid, its concentration, and substratum properties.     

Bacterial production and growth rate estimation from [h]thymidine incorporation for attached and free-living bacteria in aquatic systems

Production and specific growth rates of attached and free-living bacteria were estimated in an oligotrophic marine system, La Salvaje Beach, Vizcaya, Spain, and in a freshwater system having a higher nutrient concentration, Butron River, Vizcaya, Spain. Production was calculated from [methyl-H]thymidine incorporation by estimating specific conversion factors (cells or micrograms of C produced per mole of thymidine incorporated) for attached and free-living bacteria, respectively, in each system. Conversion factors were not statistically different between attached and free-living bacteria: 6.812 x 10 and 8.678 x 10 mug of C mol for free-living and attached bacteria in the freshwater system, and 1.276 x 10 and 1.354 x 10 mug of C mol for free-living and attached bacteria in the marine system. Therefore, use of a unique conversion factor for the mixed bacterial population is well founded. However, conversion factors were higher in the freshwater system than in the marine system. This could be due to the different trophic conditions of the two systems. Free-living bacteria contributed the most to production in the two systems (85% in the marine system and 67% in the freshwater system) because of their greater contribution to total biomass. Specific growth rates calculated from production data and biomass data were similar for attached and free-living bacteria.     

A microautoradiographic study of the activity of attached and free-living bacteria

Microautoradiography, combined with epifluorescent microscopy, was used to evaluate the uptake of tritiated amino acids by a marine Pseudomonas sp. A comparison was made between the activity of bacteria free-living in the medium and bacteria which were attached to glass, polyethylene or polystyrene substrata. The proportion of active bacteria was lower for free-living cells (53–82%) and those attached to polystyrene (53–76%) than for those attached to glass (77–99%) or polyethylene (73–96%). For bacteria attached to glass, assimilated labelled substrate was retained within the cell over 3 h, whereas with polyethylene, labelled material was released from the cells and adsorbed on the surrounding substratum. Hence the physiological activity of attached bacteria depended on the chemical composition of the substratum.     

Sphinganase, a new endoglycanase that cleaves specific members of the gellan family of polysaccharides.

A sporeforming gram-positive aerobic bacterium was isolated from soil and shown to secrete an endoglycanase that cleaves the tetrasaccharide backbone structure of specific members within the gellan family of related bacterial exopolysaccharides. We refer to these polysaccharides as sphingans. The structures of the sphingans differ by the type and position of side groups that are attached to the backbone. The new enzyme named sphinganase degrades welan, gellan, deacylated gellan, and polysaccharides S-88, S-7, and S-198. However, the enzyme does not attack rhamsan or polysaccharide NW11. Methods for growing the bacteria, isolating the enzyme, and assaying sphinganase activity are presented, and uses for the enzyme are proposed.     

The invasion of HeLa cells by Salmonella typhimurium: reversible and irreversible bacterial attachment and the role of bacterial motility

The interactions which brought about the invasion of HeLa cells by Salmonella typhimurium consisted of a sequence of three phases. Initially, the motility of the bacteria facilitated their contact with the HeLa cells whereupon the bacteria became attached in a reversible manner (i.e. the bacteria could be removed readily by washing the HeLa cell monolayers with Hanks' Balanced Salt solution). The binding forces responsible for reversible attachment were probably the weak long-range forces of the secondary minimum level of attractive interactions between the bacterium and the HeLa cell. Reversible attachment was a necessary interlude before the bacteria became irreversibly attached to the surfaces of the HeLa cells (i.e. the bacteria were no longer removed by the washing procedure that removed the reversibly attached salmonellae). Irreversible attachment was prevented in solutions of low ionic strength; the forces responsible were probably those of the primary minimum generated between the HeLa cell and a bacterial adhesion which was capable of acting over only short distances between the reversibly attached bacterium and the HeLa cell (i.e. probably less than 15 nm). Only irreversibly attached bacteria proceeded to the third phase and were internalized by the HeLa cells.     

Grazing of attached bacteria by heterotrophic microflagellates

Four species of heterotrophic microflagellates were examined for their ability to graze attached and unattached bacteria. The species tested displayed pronounced differences in their ability to graze the bacteriumPseudomonas halodurans attached to chitin particles. Two species of microflagellates (Monas andCryptobia sp.) efficiently grazed unattached bacteria but showed little or no ability to graze attached or aggregated cells. In contrast,Rhynchomonas nasuta andBodo sp. showed marked preferences for attached and aggregated bacteria and a limited ability to graze unattached cells. The density of attached bacteria was reduced by an order of magnitude due to grazing byBodo andR. nasuta, even though the density of unattached bacteria was 5–90× the density of attached cells. The maximum densities attained by microflagellates in the cultures were related to the density of unattached bacteria forMonas andCryptobia but not forBodo andR. nasuta. Growth of the latter two species appeared to be related to the density of attached or aggregated bacteria. Based on the results of these experiments, it is concluded that the pelagic existence of microflagellates that graze attached bacteria may be strongly linked to the distribution of suspended particles and their associated bacteria. In addition, the removal of attached bacteria by microflagellates can significantly affect the density of bacteria attached to particles in the plankton. This activity may have important implications for the controversy concerning the relative importance of attached and free-living bacteria in the plankton.     

Attached and Free-Floating Bacteria in a Diverse Selection of Water Bodies

The contribution of attached and free-floating bacteria to the bacterial numbers and heterotrophic uptake in 44 diverse aquatic environments was studied. A factor analysis reduced the variability of the raw data base to three major factors explaining 53.6% of total variance. These factors were (i) salinity, (ii) heterotrophic uptake, and (iii) particulate load. A cluster analysis categorized the 44 habitats into five distinct environmental types based on these three factors. There was no significant pattern in the distribution of attached versus free-floating bacteria when assessed by epifluorescent microscopy. However the contribution of attached bacteria to the uptake of an amino acids mix was reduced in marine waters. Heavy particulate loads resulted in an increased percentage uptake of amino acids and glucose from the attached bacteria. Uptake response was found to be substrate specific especially in oliogotrophic freshwater. Amino acid uptake was more associated with the attached fraction, whereas glucose uptake was mediated more by the free-floating fraction.     

Bacterial Colonization and Ectoenzymatic Activity in Phytoplankton-Derived Model Particles: Cleavage of Peptides and Uptake of Amino Acids

Abstract Phytoplankton-derived model particles were created in laboratory from a mixture of autoclaved diatom cultures. These particles were colonized by a marine bacterial community and incubated in rolling tanks in order to examine the relationship between aminopeptidase activity and leucine uptake. Bacteria inhabiting particles and ambient water were characterized for abundance, biovolume, aminopeptidase activity, leucine uptake, and growth rate. Particles were a less favorable habitat than ambient water for bacterial growth since growth rates of particle-attached bacteria were similar or even lower than those of free-living bacteria. During the first ∼100 h of the particle decomposition process, there were not statistically significant differences in the aminopeptidase activity:leucine uptake ratio between attached and free-living bacteria. From ∼100 h to ∼200 h, this ratio was higher for attached bacteria than for free-living bacteria. This indicates an uncoupling of aminopeptidase activity and leucine uptake. During this period, attached and free-living bacteria showed similar hydrolytic activities on a cell-specific basis. In the free-living bacterial community, variations in aminopeptidase activity per cell were associated with variations in leucine uptake per cell and growth rates. However, in the attached bacterial community, when leucine uptake and growth rates decreased, aminopeptidase activity remained constant. Thus, after ∼100 h, particle-attached bacteria were not taking advantage of their high aminopeptidase activity; consequently the hydrolysed amino acids were released into the ambient water, supporting the growth of free-living bacteria. These results demonstrate that over the particle decomposition process, the relationship between hydrolysis and uptake of the protein fraction shows different patterns of variation for attached and free-living bacterial communities. However, in our experiments, this uncoupling was not based on a hyperproduction of enzymes by attached bacteria, but on lower uptake rates when compared to the free-living bacteria. Received: 4 February 1997; Accepted: 9 May 1997     

Improved Method of Enumeration of Attached Bacteria for Study of Fluctuation in the Abundance of Attached and Free-Living Bacteria in Response to Diel Variation in Seawater Turbidity

Sample preparation for enumerating attached bacteria in turbid seawater by epifluorescence microscopy was improved by treating samples with a surfactant (Tween 80) followed by sonication. With optimal treatment with Tween 80 (final concentration, 10 ppm [10 μg/ml]) and sonication, as many as 10 times more attached bacteria were enumerated from turbid seawater relative to the number enumerated from an untreated control. Dispersion of bacteria by sonication alone resulted in the enumeration of only 42 to 72% of the attached bacteria. By this technique, fluctuations in the number of attached and free-living bacteria were determined in water from Aransas Pass, Tex., where surface sediments are resuspended on a regular basis by tidal currents. The abundance of attached bacteria increased in proportion to the seawater turbidity that resulted from sediment resuspension. The variation in abundance of free-living bacteria was not directly related to seawater turbidity. However, the magnitude of fluctuation in the abundance of free-living bacteria was related to the extent of turbidity variation during diurnal tides.     

Seasonal Variation in Population Density and Heterotrophic Activity of Attached and Free-Living Bacteria in Coastal Waters

The abundance and heterotrophic activity of attached and free-living bacteria were examined seasonally in coastal water. Heterotrophic activity was determined by the uptake of [¹⁴C]glucose. The density of attached bacteria was always minor, not showing a seasonal variation, whereas the free-living bacteria were more numerous and showed a marked seasonal variation, their density being higher under warmer conditions. The contribution of the attached bacteria to the total assimilation of [¹⁴C]glucose (from 10 to 38%) was lower than that of the free-living bacteria, neither of them showing a seasonal variation. On a cellular basis, attached bacteria were more active, since they assimilated more [¹⁴C]glucose and showed, under warmer conditions, a higher cellular volume (0.102 versus 0.047 μm³). We consider that the factors responsible for these observations were the amount and quality of the particulate material, the different availability of organic matter for the two types of bacteria, and in a fundamental way, the variation in water temperature.     

Methodology for Estimating Numbers of Free-Living and Attached Bacteria in Estuarine Water

A fundamental problem in estuarine microbiology studies is the accurate determination of the density in the water column of both free-living bacteria and those attached to suspended particulate matter. When a water sample is filtered and the filter is viewed by epifluorescence microscopy, counts can be made of the numbers of bacteria which are seen on the filter background (free-living) and those which appear to lie on sediment particles (both free-living and attached). With only the additional knowledge of the proportion of the filter area covered by particles (a quantity that is straightforwardly determined by stereological point counting), results from geometric probability were used to determine the expected number of bacteria which are hidden by particles and hence to provide an estimation scheme for the true densities of free-living and attached bacteria. Variance equations based on a Taylor series are given, and a partial check of the method is attempted with controlled mixtures of bacteria and sediment. An alternative procedure is also proposed, in which the natural attached/free-living ratio is altered by an intervention experiment, allowing an estimation which is less model dependent but more labor intensive. Both methods are applied to a series of samples from the Tamar estuary, United Kingdom, taken in April 1985. A notable conclusion is that there are always more free-living than attached bacteria in the water column throughout the estuary.     

Attached and free-living bacteria: Production and polymer hydrolysis during a diatom bloom

Abundance, production and extracellular enzymatic activity of free-living and attached bacteria were measured during the development and collapse of a spring bloom in a eutrophic lake. Free-living bacteria accounted for most of the total bacterial production during the first part of the bloom. Their production had a significant positive correlation to chlorophyll (P < .01) and polysaccharide concentration (P < .02) and to potential -glucosidase and aminopeptidase activity (P < .05), suggesting that algal release of dissolved polymeric compounds provided an important carbon source for bacterial production. As the bloom collapsed, we observed a change in the activity and structure of the microbial community. The mean contribution of attached bacteria to total bacterial production increased from 12% during the first part of the bloom to 26% at the end. Also, the extracellular enzymatic activity of attached bacteria increased as the bloom collapsed and constituted up to 75% of the total hydrolytic activity. An estimated disparity between hydrolytic activity and the corresponding carbon demand of attached bacteria suggested a net release of dissolved organic compounds from organic particles via polymer hydrolysis by attached bacteria. Correspondence to: M. Middelboe     

Zooplankton and aggregates as refuge for aquatic bacteria: protection from UV,heat and ozone stresses used for water treatment

Aggregates and zooplankton may provide refuge for aquatic bacteria against external hazards. The ability of attached bacteria to survive and recover from stressors commonly used for water treatment was tested in the laboratory. Without zooplankton or aggregates, both UV and ozone significantly reduced abundance of free‐living bacteria in both freshwater and marine medium. The presence of zooplankton carcasses and aggregates, however, allowed some of the attached bacteria to survive and recover quickly within 3 days. Heat exposure was the least effective as both free‐living and attached bacteria were able to recover quickly. Selective survival of bacterial phylotypes led to large changes in bacterial community composition after stress exposures, and some of the bacteria that recovered belonged to groups with known pathogens. This study demonstrates that zooplankton and aggregates protected various aquatic bacteria from external stressors, and organic remains generated from zooplankton and aggregates after stress exposure even enabled the surviving bacteria to quickly regrow and subsequently be released into the surrounding water. Hence, water disinfection treatments that overlooked the potential persistence of bacteria associated with organisms and aggregates may not be effective in preventing the spread of undesirable bacteria.     

Investigation of energy gene expressions and community structures of free and attached acidophilic bacteria in chalcopyrite bioleaching

In order to better understand the bioleaching mechanism, expression of genes involved in energy conservation and community structure of free and attached acidophilic bacteria in chalcopyrite bioleaching were investigated. Using quantitative real-time PCR, we studied the expression of genes involved in energy conservation in free and attached Acidithiobacillus ferrooxidans during bioleaching of chalcopyrite. Sulfur oxidation genes of attached A. ferrooxidans were up-regulated while ferrous iron oxidation genes were down-regulated compared with free A. ferrooxidans in the solution. The up-regulation may be induced by elemental sulfur on the mineral surface. This conclusion was supported by the results of HPLC analysis. Sulfur-oxidizing Acidithiobacillus thiooxidans and ferrous-oxidizing Leptospirillum ferrooxidans were the members of the mixed culture in chalcopyrite bioleaching. Study of the community structure of free and attached bacteria showed that A. thiooxidans dominated the attached bacteria while L. ferrooxidans dominated the free bacteria. With respect to available energy sources during bioleaching of chalcopyrite, sulfur-oxidizers tend to be on the mineral surfaces whereas ferrous iron-oxidizers tend to be suspended in the aqueous phase. Taken together, these results indicate that the main role of attached acidophilic bacteria was to oxidize elemental sulfur and dissolution of chalcopyrite involved chiefly an indirect bioleaching mechanism.     

Coupling the microbial food web with fish: are bacteria attached to cyanobacteria an important food source for underyearling roach?

1. After observing that juvenile roach fed intensively on cyanobacteria and that cyanobacteria were densely colonized by heterotrophic bacteria, we tested whether the bacteria are used by underyearling roach and the extent to which they contribute to the energy requirements of the fish.
2. We radiolabelled attached bacteria in a natural cyanobacterial suspension, fed the fish with these particles, and estimated their assimilation by roach. Biomass of attached bacteria on cyanobacteria increased with the proportion of the cyanobacterium Microcystis in total cyanobacteria. Biomass-specific thymidine incorporation of attached bacteria was higher than that of free bacteria.
3. In feeding experiments, we detected assimilation of bacterial biomass into muscle tissue of underyearling roach. Fish consumed Microcystis to a lesser extent compared with Aphanizomenon but assimilation of attached bacteria was higher when roach fed on Microcystis because of the higher biomass of epibacteria on this cyanobacterium. However, biomass of attached bacteria was too low to be an important food source for underyearling roach.
4. We conclude that assimilation of epibacteria from cyanobacteria cannot explain the success of roach in eutrophic lakes.