Measurement of protein concentration in bacteria and small organelles under a light transmission microscope.

Protein concentration (PC) is an essential characteristic of cells and organelles; it determines the extent of macromolecular crowding effects and serves as a sensitive indicator of cellular health. A simple and direct way to quantify PC is provided by brightfield-based transport-of-intensity equation (TIE) imaging combined with volume measurements. However, since TIE is based on geometric optics, its applicability to micrometer-sized particles is not clear. Here, we show that TIE can be used on particles with sizes comparable to the wavelength. At the same time, we introduce a new ImageJ plugin that allows TIE image processing without resorting to advanced mathematical programs. To convert TIE data to PC, knowledge of particle volumes is essential. The volumes of bacteria or other isolated particles can be measured by displacement of an external absorbing dye ("transmission-through-dye" or TTD microscopy), and for spherical intracellular particles, volumes can be estimated from their diameters. We illustrate the use of TIE on Escherichia coli, mammalian nucleoli, and nucleolar fibrillar centers. The method is easy to use and achieves high spatial resolution.

Bacterial communities of tyre monofill sites: growth on tyre shreds and leachate.

AIMS: To investigate bacterial communities of tyre monofill sites, colonization of tyre material by bacteria and the effect of tyre leachate on bacteria. METHODS AND RESULTS: Culturable bacteria were isolated from buried tyre shreds and identified using fatty acid methyl ester analysis. Isolates belonged to taxonomic groups such as Bacilli, Actinobacteria, Clostridia, Flavobacteria, beta and gamma-proteobacteria. For tyre material colonization experiments, Bacillus megatarium, Bacillus cereus, Hydrogenophaga flava, Janthinobacterium lividum, Cellulosimicrobium cellulans, Arthrobacter globiformis (isolated from tyre shreds or leachate at the study site); Escherichia coli and Acidithiobacillus ferrooxidans were used. Beakers containing tyre shreds and artificial rain water were inoculated with a given bacterial culture, incubated at room temperature and sampled at regular intervals. 4',6-diamidino-2-phenylindole (DAPI) staining followed by epifluorescent microscopy was used to enumerate bacteria in samples. Of the bacteria tested, B. megatarium, J. lividum, E. coli, C. cellulans and A. globiformis exhibited the most extensive colonization of the tyre shreds. However, the extent of colonization varied among bacteria. Response to tyre leachate was also examined using B. cereus and J. lividum. Both bacteria increased in abundance due to the addition of leachate. CONCLUSIONS: Bacteria associated with buried tyre shreds were identified and found to include typical soil and freshwater organisms. The majority of indigenous isolates grew on tyre material (or leachate) suggesting that they play an active role in the ecology of these sites and that their potential role in tyre degradation should be explored. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on bacterial communities of tyre-waste disposal sites, explores the interaction between tyre material and bacteria and identifies bacteria that could be involved in or employed for recycling tyre-waste.

Abundance of three bacterial populations in selected streams.

The population sizes of three bacterial species, Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida, were examined in water and sediment from nine streams in different parts of the United States using fluorescent in situ hybridization (FISH). Population sizes were determined from three sites (upstream, midstream, and downstream) in each stream to compare differences in the occurrence and distribution of the species within each stream and among streams. Physical and chemical variables measured reflected differences in environmental conditions among the streams. In the water, B. cepacia numbers were highest in the agricultural, Iowa stream. P. putida numbers were highest in the southern coastal plain streams, Black Creek (GA) and Meyers Branch (SC). Compared to the other two species, the abundance of A. calcoaceticus was similar in all the streams. In sediment, the greatest abundance of all three species was found in the Iowa stream, while the lowest was in Hugh White Creek (NC). Detrended correspondence analysis (DCA) explained 95.8% and 83.9% of the total variation in bacterial numbers in water and sediment of the streams, respectively. In sediments and water, B. cepacia numbers were related to nitrate concentrations. A. calcoaceticus in water clustered with several environmental variables (i.e., SRP, pH, and conductivity) but benthic populations were less well correlated with these variables. This study reveals the potential influence of various environmental conditions on different bacterial populations in stream communities.

Intraspecific differences in bacterial responses to modelled reduced gravity.

AIMS: Bacteria are important residents of water systems, including those of space stations which feature specific environmental conditions, such as lowered effects of gravity. The purpose of this study was to compare responses with modelled reduced gravity of space station, water system bacterial isolates with other isolates of the same species. METHODS AND RESULTS: Bacterial isolates, Stenotrophomonas paucimobilis and Acinetobacter radioresistens, originally recovered from the water supply aboard the International Space Station (ISS) were grown in nutrient broth under modelled reduced gravity. Their growth was compared with type strains S. paucimobilis ATCC 10829 and A. radioresistens ATCC 49000. Acinetobacter radioresistens ATCC 49000 and the two ISS isolates showed similar growth profiles under modelled reduced gravity compared with normal gravity, whereas S. paucimobilis ATCC 10829 was negatively affected by modelled reduced gravity. CONCLUSIONS: These results suggest that microgravity might have selected for bacteria that were able to thrive under this unusual condition. These responses, coupled with impacts of other features (such as radiation resistance and ability to persist under very oligotrophic conditions), may contribute to the success of these water system bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Water quality is a significant factor in many environments including the ISS. Efforts to remove microbial contaminants are likely to be complicated by the features of these bacteria which allow them to persist under the extreme conditions of the systems.

Response of biofilm bacteria to dissolved organic matter from decomposing maple leaves.

Stream bacteria play an important role in the utilization of dissolved organic matter (DOM) leached from leaves, and in transfer of this DOM to other trophic levels. Leaf leachate is a mixture of labile, recalcitrant, and inhibitory compounds, and bacterial communities vary in their ability to utilize leachate. The purpose of this study was to determine the effects of DOM from sugar maple leaves on bacterial populations in biofilms on decomposing leaf surfaces. Populations of Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida were enumerated on decomposing maple leaves in a northeast Ohio stream using fluorescence in situ hybridization. Additionally, artificial substrata consisting of PVC-end caps filled with agar supplemented with leaf leachate and covered with cellulose filters were used to determine bacterial response to leachate from leaves at different stages of decomposition. Population sizes of bacterial species exhibited different responses. Leachate did not affect A. calcoaceticus. B. cepacia was tolerant of phenolic compounds released from leaves and the population size increased when DOM concentrations were greatest. In contrast, P. putida was inhibited by phenolic components of leachate when total DOM concentrations were greatest. Differences in response of the bacterial species to components of leaf leachate indicate the complexity of microbial population dynamics and interactions with DOM. Differences among species in response to DOM have the potential to influence transport and retention of organic matter in stream ecosystems.

Temporal changes in the bacterioplankton of a Northeast Ohio (USA) River.

To examine temporal changes in a bacterial community, water samples were collected monthly for one year from five sites along a major use-reuse river, the Cuyahoga River, in northeastern Ohio (USA). Fluorescent in situ hybridization (FISH) was used to enumerate population sizes of two species of common bacteria, Pseudomonas putida and Acinetobacter calcoaceticus; FISH was also performed with a Domain Bacteria specific probe. In addition, the total bacteria (based on DAPI staining), colony forming units (on modified Nutrient agar) and coliforms were enumerated and supporting physical/chemical data were collected. Each variable examined exhibited a different seasonal pattern. Temporal changes in total number of bacteria and population size of P. putida were correlated with turbidity and precipitation suggesting that allochthonous sources and scouring of the benthos may be major contributors to these portions of the community. In contrast, the number of cells hybridizing the Domain Bacteria and A. calcoaceticus probes were correlated with temperature. Thus, different aspects of the bacterial community are potentially controlled by different factors and the role of allochthonous and autochthonous sources may vary among species.

Use of green fluorescent protein to monitor survival of genetically engineered bacteria in aquatic environments.

Many methods for detecting model genetically engineered microorganisms (GEMs) in experimental ecosystems rely on cultivation of introduced cells. In this study, survival of Escherichia coli was monitored with the green fluorescent protein (GFP) gene. This approach allowed enumeration of GEMs by both plating and microscopy. Use of the GFP-marked GEMs revealed that E. coli persisted in stream water at higher densities as determined microscopically than as determined by CFU enumeration. The GFP gene did not negatively impact the fitness of the host strain.

Comparison of Fatty Acid Methyl Ester Analysis with the Use of API 20E and NFT Strips for Identification of Aquatic Bacteria.

Aquatic bacteria grown on MacConkey agar and modified nutrient agar were identified by using API 20E and NFT strips and fatty acid methyl ester (FAME) analysis. Identifications agreed at the species level 35.7% of the time when API 20E strips and FAME analysis were used and in 4.3% of the cases when API NFT strips and FAME analysis were used. These techniques require further development before extended use in ecological studies.

Identification of aquatic Burkholderia (Pseudomonas) cepacia by hybridization with species-specific rRNA gene probes.

Burkholderia (Pseudomonas) cepacia is a common environmental bacterium which can be pathogenic for plants and humans. In this study, four strategies were used to identify aquatic isolates: API test strips, hybridization with species-specific DNA probes for the 16S and 23S rRNA genes, fatty acid methyl ester (FAME) profiles, and growth on selective medium (TB-T agar [C. Hagedorn, W. D. Gould, T. R. Bardinelli, and D. R. Gustarson, Appl. Environ. Microbiol. 53:2265-2268, 1987]). Only 59% of the isolates identified as B. cepacia with the API test strips were confirmed as B. cepacia by using fatty acid profiles. The 23S rRNA probe generated a few false-positive results but dramatically underestimated the number of B. cepacia isolates (i.e., 40% of the colonies that did not hybridize to the probe were B. cepacia, as determined by FAME). The 16S rRNA probe generated more false-positive results than the 23S rRNA probe but was effective in identifying the majority of the B. cepacia isolates. The selective medium was only partially successful in recovering B. cepacia. Use of the B. cepacia-specific 16S rRNA probe was the most efficient and accurate way of identifying B. cepacia.

Comparison of methods of DNA extraction from stream sediments.

In Upper Three Runs Creek (Aiken, S.C.) and many other environments, less than 1% of bacteria visible microscopically can be cultured. Exploitation of molecular biology techniques has led to development of new methods, such as extraction of nucleic acids from soils or sediments, to study the dominant, nonculturable bacteria. The purpose of this study was to compare three published methods of DNA extraction that fall into two general categories: those in which cells are lysed in sediments (the Ogram and Tsai and methods [A. Ogram, G. S. Sayler, and T. Barkay, J. Microbiol. Methods 7:57-66, 1987; Y. L. Tsai and B. H. Olson, Appl. Environ. Microbiol. 57:1070-1074, 1991]) and those in which cells are removed from sediments prior to lysis (the Jacobsen method [C. S. Jacobsen and O. S. Rasmussen; Appl. Environ. Microbiol. 58:2458-2462, 1992]). DNA yield varied with extraction method; the Ogram method had a significantly higher yield than the other methods. However, DNA extracted via the Ogram method was badly sheared and contained a smaller proportion of eubacterial DNA. The Tsai method was less time consuming than the other methods, but DNA samples were of lower purity. If DNA purity is of paramount concern (as would be the case if PCR was to be performed) and quantity is not important, the Jacobsen method is recommended because of the low concentration of contaminants. If DNA is to be used directly in DNA-DNA hybridizations, the Ogram method is recommended since it gives maximal yields.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of Tn5-like sequences in kanamycin-resistant stream bacteria and environmental DNA.

Resistance to kanamycin and neomycin in the bacterial assemblage of a coastal plain stream was detected by growth of colonies on media containing antibiotics. Three of 184 kanamycin-resistant colonies hybridized with a probe containing the nptII gene from transposon Tn5; the nptII gene encodes the enzyme neomycin phosphotransferase and conveys resistance to kanamycin and neomycin. In one of these isolates, the homologous gene was cloned and shown to confer resistance to a kanamycin-sensitive Escherichia coli strain. Since enumeration of bacteria by acridine orange direct counts revealed that less than 0.2% of the bacteria present were cultivated, direct examination of environmental DNA was used to assess abundance of sequences that hybridize to the nptII gene. To examine the resistance potential of bacteria that were not cultured, total DNA was extracted from environmental samples and hybridized with specific probes. The relative amount of eubacterial DNA in each sample was determined by using a eubacterial specific rDNA probe. Then, the abundance of sequences that hybridize to the eubacterial neomycin phosphotransferase gene was determined by hybridization and expressed relative to the total eubacterial DNA in the assemblage. Relative gene abundance was significantly different among assemblages from different habitats (leaves, midchannel sediments, and bank sediments) but did not differ among stream sites.

Information spiraling: Movement of bacteria and their genes in streams.

Bacteria in transport in streams are largely derived from other parts of the ecosystem. Here we review factors that influence transport of bacteria and their movement between habitats (such as sediment, water column, rocks, wood, and leaves) and consider the role of these movements in ecosystem processes. Bacteria enter the water column by sloughing, scouring, as a consequence of changes in morphology or hydrophobicity, or dislodgment by invertebrates and fish or other aquatic vertebrates. Transported cells (which may be planktonic or particle-associated) that colonize surfaces may establish new gene pools through cell division (vertical transfer) or genetic exchange (lateral transfer). Genetic information is also transported in streams as free or protected DNA or in bacteriophages. Movement of these vectors causes genetic information to spiral along a stream in a manner analogous to that of nutrients and organic carbon. Spiraling refers to the pattern of transport, uptake or attachment, and release of a molecule or cell. The flow of water in streams causes this cycle of attachment and release to be displaced downstream resulting in a spiral rather than a closed, stationary loop.