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.     

Microbial Community Composition and Denitrifying Enzyme Activities in Salt Marsh Sediments

Denitrifying microbial communities and denitrification in salt marsh sediments may be affected by many factors, including environmental conditions, nutrient availability, and levels of pollutants. The objective of this study was to examine how microbial community composition and denitrification enzyme activities (DEA) at a California salt marsh with high nutrient loading vary with such factors. Sediments were sampled from three elevations, each with different inundation and vegetation patterns, across 12 stations representing various salinity and nutrient conditions. Analyses included determination of cell abundance, total and denitrifier community compositions (by terminal restriction fragment length polymorphism), DEA, nutrients, and eluted metals. Total bacterial (16S rRNA) and denitrifier (nirS) community compositions and DEA were analyzed for their relationships to environmental variables and metal concentrations via multivariate direct gradient and regression analyses, respectively. Community composition and DEA were highly variable within the dynamic salt marsh system, but each was strongly affected by elevation (i.e., degree of inundation) and carbon content as well as by selected metals. Carbon content was highly related to elevation, and the relationships between DEA and carbon content were found to be elevation specific when evaluated across the entire marsh. There were also lateral gradients in the marsh, as evidenced by an even stronger association between community composition and elevation for a marsh subsystem. Lastly, though correlated with similar environmental factors and selected metals, denitrifier community composition and function appeared uncoupled in the marsh.     

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.     

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.     

Bacterial Community Composition and Extracellular Enzyme Activity in Temperate Streambed Sediment during Drying and Rewetting

Droughts are among the most important disturbance events for stream ecosystems; they not only affect stream hydrology but also the stream biota. Although desiccation of streams is common in Mediterranean regions, phases of dryness in headwaters have been observed more often and for longer periods in extended temperate regions, including Central Europe, reflecting global climate change and enhanced water withdrawal. The effects of desiccation and rewetting on the bacterial community composition and extracellular enzyme activity, a key process in the carbon flow of streams and rivers, were investigated in a typical Central European stream, the Breitenbach (Hesse, Germany). Wet streambed sediment is an important habitat in streams. It was sampled and exposed in the laboratory to different drying scenarios (fast, intermediate, slow) for 13 weeks, followed by rewetting of the sediment from the fast drying scenario via a sediment core perfusion technique for 2 weeks. Bacterial community structure was analyzed using CARD-FISH and TGGE, and extracellular enzyme activity was assessed using fluorogenic model substrates. During desiccation the bacterial community composition shifted toward composition in soil, exhibiting increasing proportions of Actinobacteria and Alphaproteobacteria and decreasing proportions of Bacteroidetes and Betaproteobacteria. Simultaneously the activities of extracellular enzymes decreased, most pronounced with aminopeptidases and less pronounced with enzymes involved in the degradation of polymeric carbohydrates. After rewetting, the general ecosystem functioning, with respect to extracellular enzyme activity, recovered after 10 to 14 days. However, the bacterial community composition had not yet achieved its original composition as in unaffected sediments within this time. Thus, whether the bacterial community eventually recovers completely after these events remains unknown. Perhaps this community undergoes permanent changes, especially after harsh desiccation, followed by loss of the specialized functions of specific groups of bacteria.     

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.     

Relationship between the Intracellular Integrity and the Morphology of the Capsular Envelope in Attached and Free-Living Marine Bacteria

The integrity of the intracellular structures and the presence and dimension of the capsular envelope were investigated in marine snow-associated and marine free-living bacteria by transmission electron microscopy and special fixation techniques. Three categories depending on the presence of internal structures were differentiated. In marine snow, 51% of the marine snow-associated bacterial community was considered intact, 26% had a partly degraded internal structure, and 23% were empty with only the cell wall remaining. For the free-living bacterial community, 34% were intact cells, 42% exhibited damage, and 24% of the cells were lacking any internal structure. We also investigated the morphology and the extent of the bacterial capsular envelope. More than 95% of all intact marine snow-associated bacteria were surrounded by a capsule while (apprx=)55% of empty marine snow-associated bacteria had no capsule. For free-living bacteria, (apprx=)65% of the intact cells had a capsule while (apprx=)80% of the empty free-living bacteria lacked a capsule. Thus there is a clear trend from intact cells which are commonly surrounded by a capsular envelope to empty bacteria for which only the cell wall is remaining. Since bacterioplankton represent the largest living surface in the ocean, it is concluded that the release of intracellular material from bacteria into the environment as well as the release of extracellular capsular material might fuel the dissolved organic matter pool of the ocean.     

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer''s particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil type and temperature can influence enzyme kinetics.     

Effects of Soil Organic Matter Properties and Microbial Community Composition on Enzyme Activities in Cryoturbated Arctic Soils

Enzyme-mediated decomposition of soil organic matter (SOM) is controlled, amongst other factors, by organic matter properties and by the microbial decomposer community present. Since microbial community composition and SOM properties are often interrelated and both change with soil depth, the drivers of enzymatic decomposition are hard to dissect. We investigated soils from three regions in the Siberian Arctic, where carbon rich topsoil material has been incorporated into the subsoil (cryoturbation). We took advantage of this subduction to test if SOM properties shape microbial community composition, and to identify controls of both on enzyme activities. We found that microbial community composition (estimated by phospholipid fatty acid analysis), was similar in cryoturbated material and in surrounding subsoil, although carbon and nitrogen contents were similar in cryoturbated material and topsoils. This suggests that the microbial community in cryoturbated material was not well adapted to SOM properties. We also measured three potential enzyme activities (cellobiohydrolase, leucine-amino-peptidase and phenoloxidase) and used structural equation models (SEMs) to identify direct and indirect drivers of the three enzyme activities. The models included microbial community composition, carbon and nitrogen contents, clay content, water content, and pH. Models for regular horizons, excluding cryoturbated material, showed that all enzyme activities were mainly controlled by carbon or nitrogen. Microbial community composition had no effect. In contrast, models for cryoturbated material showed that enzyme activities were also related to microbial community composition. The additional control of microbial community composition could have restrained enzyme activities and furthermore decomposition in general. The functional decoupling of SOM properties and microbial community composition might thus be one of the reasons for low decomposition rates and the persistence of 400 Gt carbon stored in cryoturbated material.     

Bacterial Production and Growth Rate Estimation from [3H]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-³H]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 × 10¹¹ and 8.678 × 10¹¹ μg of C mol⁻¹ for free-living and attached bacteria in the freshwater system, and 1.276 × 10¹¹ and 1.354 × 10¹¹ μg of C mol⁻¹ 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.     

Diversity of Free-Living and Attached Bacteria in Offshore Western Mediterranean Waters as Depicted by Analysis of Genes Encoding 16S rRNA

In a previous study (S. G. Acinas, F. Rodríguez-Valera, and C. Pedrós-Alió, FEMS Microbiol. Ecol. 24:27–40, 1997), community fingerprinting by 16S rDNA restriction analysis applied to Mediterranean offshore waters showed that the free-living pelagic bacterial community was very different from the bacterial cells aggregated or attached to particles of more than about 8 μm. Here we have studied both assemblages at three depths (5, 50, and 400 m) by cloning and sequencing the 16S rDNA obtained from the same samples, and we have also studied the samples by scanning electron microscopy to detect morphology patterns. As expected, the sequences retrieved from the assemblages were very different. The subsample of attached bacteria contained very little diversity, with close relatives of a well-known species of marine bacteria, Alteromonas macleodii, representing the vast majority of the clones at every depth. On the other hand, the free-living assemblage was highly diverse and varied with depth. At 400 m, close relatives of cultivated γ Proteobacteria predominated, but as shown by other authors, near the surface most clones were related to phylotypes described only by sequence, in which the α Proteobacteria of the SAR11 cluster predominated. The new technique of rDNA internal spacer analysis has been utilized, confirming these results. Clones representative of the A. macleodii cluster have been completely sequenced, producing a picture that fits well with the idea that they could represent a genus with at least two species and with a characteristic depth distribution.     

胞外和胞内菌胞外囊泡的功能及应用

细胞外囊泡(Extracellular Vesicles,EVs)是从细胞膜上脱落或者分泌的双层膜结构的囊泡状小体.真核生物、细菌、古细菌和支原体等具有细胞结构的生物均能够释放EVs.细菌分泌的EVs含有DNA、RNA及蛋白质等多种成分,其在细菌毒力保持、免疫逃逸、细菌间物质运输、宿主细胞免疫调节、宿主转录基因调节、耐...     

Extracellular Enzyme Activities and Carbon Chemistry as Drivers of Tropical Plant Litter Decomposition

Litter quality parameters such as nitrogen and lignin content correlate with decomposition rates at coarse scales, but fine‐scale mechanisms driving litter decomposition have proven more difficult to generalize. One potentially important driver of decomposition is the activity of extracellular enzymes that catalyze the degradation of complex compounds present in litter. To address the importance of this mechanism, we collected 15 Hawaiian plant litter types and decomposed them in fertilized and control plots for up to two years. We measured litter nutrient content and carbon chemistry prior to decomposition, as well as extracellular enzyme activities, mass loss, and litter nutrient content over time. We found that water‐soluble carbon content, cellobiohydrolase activities, and polyphenol oxidase activities were significantly correlated with mass loss. Enzyme activities and decomposition rate constants both varied significantly by litter type, and fertilization increased mass loss rates in five litter types. Some litter types that decayed faster under fertilization also showed time‐dependent increases in carbon‐degrading enzyme activities, but others decayed faster independent of enzyme changes. These results suggest that extracellular enzyme activities partially determine litter decomposition rates, but high soluble carbon content may circumvent the requirement for enzyme‐catalyzed decomposition.     

Effect of the Medium Composition and Cultivation Conditions on Sporulation in Chemolithotrophic Bacteria

The possibility of regulating endospore formation by changing cultivation conditions was for the first time shown in acidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans type strain 1269 and the thermotolerant strain K1 formerly described as S. thermosulfidooxidans subsp. thermotolerans. Suppression of sporulation occurred when these strains were cultured in Manning's liquid medium with yeast extract. This medium was optimized by gradually reducing the concentrations of ferrous iron salts (the source of energy), phosphorous, nitrogen, and yeast extract and simultaneously increasing the concentrations of calcium, magnesium, and manganese (the elements important for sporogenesis) to attain higher yields of endospores by strains 1269 and K1. As a result, a new medium A was proposed, in which, under aeration, the life cycle of the strains studied culminated in sporulation at a level of 45 and 60%, respectively, of the total cell number. In a series of additional tests, the growth temperature and medium pH were adjusted to obtain the maximum yield of endospores. The optimal ranges found were 40–50°C and pH 1.8–2.2 for strain 1269 and 35–40°C and pH 2.5–2.7 for strain K1. An even higher yield of endospores, amounting to 55 and 75% for strains 1269 and K1, respectively, was obtained when the above growth conditions were combined (growth on medium A at optimal temperatures and pH under static conditions). Our results suggest a new approach to optimizing sporulation by acidophilic chemolithotrophs, which consists in limiting the energy and nutrient sources and using temperature and pH values within the tolerance bounds of these cultures but outside their growth optimum ranges.     

Nitrogen Metabolizing Enzyme Activities In the Free-Living Soil Amoebae Acanthamoeba Castellanii, Acanthamoeba Polyphaga and Hartmannella Vermiformis

ABSTRACT. Free-living soil amoebae consume a wide variety of food, including algae, yeast, small protozoa and especially bacteria, which they digest to fulfil their nutritional requirements. Amoebae play an active role in the nitrogen mineralization in soils due to their nitrogen metabolizing capacities. However, little is known about nitrogen metabolizing enzyme activities in these free-living soil amoebae. In this study a number of key enzymes involved in the nitrogen metabolism of the axeaically cultivated free-living soil amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga and two different strains of Hartmannella vermiformis were determined. the specific enzyme activities for exponential growth phase ceils were calculated and it appeared that the species tested possessed urate oxidase, glutamine synthetase, NADH-dependent glutamate dehydrogenase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase activity. Glutamate synthase activity could not be detected in any of these species. the levels of specific activities varied depending on the enzymes tested. For all species the highest activities were detected for the transaminase reactions yielding glutamate, and for glutamate dehydrogenase. A general conclusion is that the pathway of nitrogen assimilation in free-living soil amoebae is similar to the one observed for other eukaryotes. Differences in specific activities were detected between the species.     

Comparison of Proteolytic Activities Produced by Entomopathogenic Photorhabdus Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes

Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (~74, ~55, ~54, and ~37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokházi, G. Kóczán, F. Hudecz, L. Gráf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.     

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.     

Effect of Modified Atmospheres on the Growth and Extracellular Enzyme Activities of Psychrotrophs in Raw Milk

The conservation of food products within a controlled atmosphere is efficient in packaging. To extend the cold storage of raw milk, the effects of five gas atmospheres enriched with carbon dioxide and nitrogen were investigated. Treated and control milk were stored at 7 °C for 10 days and analyzed for microbial counts, pH, proteolysis and lipolysis. The addition of CO₂, N₂, or their mixture had a significant inhibitory effect on psychrotrophic growth. The generation times of these microorganisms were significantly longer in treated milk, particularly for yeasts where they amounted to 16.63 h. The maximum inhibition was observed when a gas mixture of 50 % CO₂ and 50 % N₂ was used. As a result, psychrotrophic growth was affected to 98 % whereas this inhibition did not exceed 78 % when CO₂ and 41 % N₂ were applied. Milk treatment under the conditions of 50 % CO₂ and 50 % N₂ gave significantly lower counts for all groups of psychrotrophs being more efficient against Enterobacteriaceae with 99.5 % of inhibition. Storage of raw milk under the tested atmospheres had a different effect on extracellular enzyme productions. Significant decreases in protease and lipase activities were observed during the storage at 7 °C. These enzyme activities were not detectable with pure CO₂ and a 50 % CO₂ and 50 % N₂ mixture. N₂ has shown to be the less efficient treatment against lipases (65 %) and proteases (95 %). With regard to growth, the course of the pH and the protease and lipase activities, the tested gas mixture of 50 % CO₂ and 50 % N₂ was more suitable for extending the shelf life of raw milk.