Comparison of Planktonic and Biofilm Cultures of Pseudomonas fluorescens DSM 8341 Cells Grown on Fluoroacetate

Comparisons between the physiological properties of Pseudomonas fluorescens biofilm cells grown in a tubular biofilm reactor and planktonic cells grown in a chemostat were performed. Fluoroacetate was the sole carbon source for all experiments. The performance of cells was assessed using cell cycle kinetics and by determining specific fluoroacetate utilization rates. Cell cycle kinetics were studied by flow cytometry in conjunction with the fluorescent stain propidium iodide. Determination of the DNA content of planktonic and biofilm cultures showed little difference between the two modes of growth. Cultures with comparable specific glycolate utilization rates had similar percentages of cells in the B phase of the cell cycle, indicating similar growth rates. Specific fluoroacetate utilization rates showed the performance of planktonic cells to be superior to that of biofilm cells, with more fluoroacetate utilized per cell at similar specific fluoroacetate loading rates. A consequence of this decreased biofilm performance was the accumulation of glycolate in the effluent of biofilm cultures. This accumulation of glycolate was not observed in the effluent of planktonic cultures. Spatial stratification of oxygen within the biofilm was identified as a possible explanation for the overflow metabolism of glycolate and the decreased performance of the biofilm cells.There is a general consensus that the adhesion of microbes to a surface influences bacterial metabolism; however, the experimental results are often contradictory (34). Some studies have compared the physiological status of biofilm and planktonic cells by determining their growth rates, with some investigators (2, 9) reporting increased biofilm growth rates in comparison to planktonic growth rates while others (1) have reported the opposite. Other researchers have compared the influence of adhesion on biofilm metabolic activity to planktonic activity, and as with growth rate, conflicting observations have been reported (16, 31, 11). The objective of the present study was to characterize planktonic cells grown in a chemostat and biofilm cells grown in a tubular biofilm reactor (TBR) and compare their performance with respect to the degradation of the model xenobiotic compound fluoroacetate.Halogenated compounds are extensively used in many applications (refrigeration, lubricants, pharmaceuticals, insecticides, and herbicides) and can be considered significant environmental contaminants. The biodegradation of many chlorinated compounds has been widely reported (5, 26, 28). However, in spite of the increased use of organofluorine compounds in the past 60 years, there is limited information on their degradation (17). Currently, a large fraction of wastewater streams containing fluorinated compounds are incinerated (12). Improved biological waste treatment processes require a deeper understanding of microbial degradation of fluorinated compounds.Some previous studies have focused on the biodegradation of fluorinated aromatic compounds using biofilm reactors (3, 10); however, there have been no studies on the degradation of fluorinated aliphatic compounds in biofilm reactors. Thus, sodium fluoroacetate was chosen as the model xenobiotic to study the efficiency of aliphatic organofluorine degradation in biofilms. It was the first naturally occurring fluorinated compound to be isolated, obtained from the South African shrub Dichapetalum cymosum (23). Fluoroacetate is highly toxic to mammals and has found extensive use as a vertebrate pesticide, particularly in Australia and New Zealand. A number of studies have focused on the isolation and identification of microbial soil isolates with the ability to degrade fluoroacetate (14, 33, 35), and other studies have focused on the mechanism of defluorination (13, 15, 21). However, there has been no research on the degradation of fluoroacetate by biofilm cultures. Biofilm systems appear ideal for the degradation of xenobiotics because of the many reported advantages they have over planktonic cultures. Most microorganisms that have the ability to degrade xenobiotic compounds have comparatively slow growth rates, and biofilm reactors allow the enrichment of these microorganisms independent of hydraulic retention times (36). It has been shown in numerous studies that biofilms are less susceptible than suspended bacteria to changes in environmental conditions such as temperature and pH and the presence of metabolic products and toxic substances (8, 25, 27, 36). The high cell concentrations that can be achieved in biofilm systems in combination with high volumetric flow rates could potentially result in high volumetric productivities without the risk of cell washout.The species Pseudomonas fluorescens has been extensively studied; it commonly exists as a biofilm in natural environments and is ubiquitous in industrial environments (6, 29, 30). The specific strain used here, P. fluorescens DSM 8341, was previously isolated from a soil sample in Western Australia, and in a study with 23 other microbial soil isolates, it was shown to be the most efficient degrader of fluoroacetate when fluoroacetate was the sole carbon source (37). The effect of the environmental factors, pH and temperature, on the biodefluorination of fluoroacetate by P. fluorescens was also determined (38); however, at present there are no reported planktonic growth kinetics established for this strain, nor has it previously been grown as a biofilm. In this context a TBR was employed to investigate the degradation of fluoroacetate by a P. fluorescens biofilm, in conjunction with chemostat studies that were conducted to determine the efficiency of planktonic degradation of the substrate. Specific utilization rates, flow cytometry, and fluorescent microscopy were employed to compare the performance and physiological status of biofilm and planktonic cells grown with fluoroacetate as the sole organic substrate.     

Application of Proteomic Marker Ensembles to Subcellular Organelle Identification

Compartmentalization of biological processes and the associated cellular components is crucial for cell function. Typically, the location of a component is revealed through a co-localization and/or co-purification with an organelle marker. Therefore, the identification of reliable markers is critical for a thorough understanding of cellular function and dysfunction. We fractionated macrophage-like RAW264.7 cells, both in the resting and endotoxin-activated states, into six fractions representing the major organelles/compartments: nuclei, mitochondria, cytoplasm, endoplasmic reticulum, and plasma membrane as well as an additional dense microsomal fraction. The identity of the first five of these fractions was confirmed via the distribution of conventional enzymatic markers. Through a quantitative liquid chromatography/mass spectrometry-based proteomics analysis of the fractions, we identified 50-member ensembles of marker proteins (“marker ensembles”) specific for each of the corresponding organelles/compartments. Our analysis attributed 206 of the 250 marker proteins (∼82%) to organelles that are consistent with the location annotations in the public domain (obtained using DAVID 2008, EntrezGene, Swiss-Prot, and references therein). Moreover, we were able to correct locations for a subset of the remaining proteins, thus proving the superior power of analysis using multiple organelles as compared with an analysis using one specific organelle. The marker ensembles were used to calculate the organelle composition of the six above mentioned subcellular fractions. Knowledge of the precise composition of these fractions can be used to calculate the levels of metabolites in the pure organelles. As a proof of principle, we applied these calculations to known mitochondria-specific lipids (cardiolipins and ubiquinones) and demonstrated their exclusive mitochondrial location. We speculate that the organelle-specific protein ensembles may be used to systematically redefine originally morphologically defined organelles as biochemical entities.One of the basic concepts of cell biology is compartmentalization of the cellular processes within subcellular structures, termed organelles. Organelles were originally identified in the 19th century as the morphological entities that are still reflected in their names (e.g. “nucleus” from the Latin “little nut,” “mitochondria” from the Greek “thread” + “grain,” or “reticulum” from the Latin “little net”). Later, the progress of biochemistry made it possible to assign to the various organelles their specific biological functions. Thus, detailed information about the location of biochemical reactions became crucial for the understanding of their roles in cell function or dysfunction. Current technology allows the location of a cell component (a protein or a metabolite) to be linked directly to a morphologically defined organelle (or even a suborganellar compartment) by using electron microscopy. However, more typically, the location of a component is determined on the basis of its co-localization with a known marker for the organelle or subcellular compartment. This co-localization can be either visualized microscopically (imaging approach) to preserve some degree of morphological information or determined through co-purification of the component and the marker in a subcellular fractionation (biochemical approach).For both the imaging and the biochemical approaches, optimal organelle markers are of the utmost importance. Conventional markers include proteins, DNA (for nucleus), and even physical/chemical parameters (electric potential for mitochondria and acidic pH for lysosomes). Protein markers are assayed using either an interaction with specific antibodies or their enzymatic activities. Unfortunately, the former is typically non-quantitative, whereas the latter, although semiquantitative, is subject to interference from multiple parameters of the environment as well as substrate and product sharing with non-marker proteins. For a biochemical approach, tightness of the anchoring of a marker to the corresponding organelle is also an issue. Moreover, an inherent problem is that most proteins are located in several organelles/compartments, which may result in false localization conclusions.Our goal was to identify specific, reliable, and universal protein markers for major subcellular organelles/compartments. The following principles were chosen as the basis for our approach. First, the search had to be conducted without a preconceived notion of the nature of the markers (e.g. we did not expect to necessarily confirm conventional markers as optimal). Second, the search had to be conducted in all major organelles/compartments simultaneously. Third, the aim was to identify relatively large panels (ensembles) of markers as opposed to the best single marker. The last two principles allowed us to address the problem of multiple locations of potential marker proteins. Some of them can be eliminated as markers; for others, the impact of multiple locations on further analysis can be negated by averaging of the data for large numbers of proteins (derivation of marker ensembles).To meet these goals, we performed a complete “quantitative” proteomics analysis of all major subcellular fractions in a single cell type. Numerous reports have focused on the proteomes of specific organelles or interrelated sets of organelles in various cell types (for reviews, see Refs. 1 and 2). However, a need for an integral systematic study in a single cell type has been evident for some time (2), and the present study is the first step aimed at addressing this need.The marker ensembles that we identified from the proteome data were used to quantify the composition of the subcellular fractions. It is becoming appreciated that a physical association of various organelles makes it next to impossible to completely separate the organelles and obtain pure fractions acceptable for detailed proteomics analysis (e.g. see Ref. 3). Therefore, correlative approaches such as protein correlation profiling (1, 3, 4) and localization of organelle proteins by isotope tagging (5, 6) have been suggested to address this problem. These approaches allowed the assignment of protein locations based on co-localization with known markers in a density gradient (1, 4–6) or in multiple fractions (7). We took this approach a step further and derived a quantitative composition of the fractions based on the distribution of the marker ensembles. Furthermore, this enabled us to calculate levels of various components (lipids and proteins) in pure organelles from experimental data obtained with less than pure fractions.The choice of a particular cell type for this study was somewhat arbitrary, and the resulting marker ensembles were optimal for the cell type for which they were generated; of course, they may have to be adjusted to be adapted for other cell types. We chose macrophage cells partly because this study was an integral part of a larger subcellular lipidomics/proteomics study under the auspices of the Lipid Metabolites and Pathways Strategy (LIPID MAPS Consortium). The macrophage plays a central role in inflammation and innate and adaptive immunity. The macrophage detects and attacks pathogens and orchestrates a host response by sending signals to other cells and tissues; in this process, the macrophage itself transits from a resting to an activated state. These two states differ vastly in function, morphology, and underlying protein expression profiles, and therefore, we aimed to identify marker ensembles that would be invariant with regard to the activation process.In the present study, the activation paradigm was treatment with Kdo₂1-lipid A. This defined, nearly homogeneous reagent is a form of lipopolysaccharide endotoxin that has all the essential biological properties of lipopolysaccharide (8).     

Environmental whole-genome amplification to access microbial populations in contaminated sediments

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using phi29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. The reported SSU rRNA sequences and library clone end sequences are listed with their respective GenBank accession numbers, DQ 404590 to DQ 404652, DQ 404654 to DQ 404938, and DX 385314 to DX 389173.     

Partial structures of ketoconazole as modulators of the large conductance calcium-activated potassium channel (BK(Ca))

A series of partial structures of ketoconazole has been synthesized and tested for activity on the large conductance calcium-activated potassium channel (BK) in bovine smooth muscle cells. This has provided openers and blockers of the channel. The results suggest that the phenyl and phenoxy moieties are important for interaction with BK, whereas the imidazole group is unimportant. The properties of the phenoxy moiety seem to determine whether the compounds act to open or block the channel.     

Rapid depletion of dissolved oxygen in 96‐well microtiter plate Staphylococcus epidermidis biofilm assays promotes biofilm development and is influenced by inoculum cell concentration

Biofilm‐related research using 96‐well microtiter plates involves static incubation of plates indiscriminate of environmental conditions, making oxygen availability an important variable which has not been considered to date. By directly measuring dissolved oxygen concentration over time we report here that dissolved oxygen is rapidly consumed in Staphylococcus epidermidis biofilm cultures grown in 96‐well plates irrespective of the oxygen concentration in the gaseous environment in which the plates are incubated. These data indicate that depletion of dissolved oxygen during growth of bacterial biofilm cultures in 96‐well plates may significantly influence biofilm production. Furthermore higher inoculum cell concentrations are associated with more rapid consumption of dissolved oxygen and higher levels of S. epidermidis biofilm production. Our data reveal that oxygen depletion during bacterial growth in 96‐well plates may significantly influence biofilm production and should be considered in the interpretation of experimental data using this biofilm model. Biotechnol. Bioeng. 2009;103: 1042–1047. © 2009 Wiley Periodicals, Inc.     

Comparison of longitudinal change in sST2 vs BNP to predict major adverse cardiovascular events in asymptomatic patients in the community

Biomarker-based preventative and monitoring strategies are increasingly used for risk stratification in cardiovascular (CV) disease. The aim of this study was to investigate the utility of longitudinal change in B-type natriuretic peptide (BNP) and sST2 concentrations for predicting incident major adverse CV events (MACE) (heart failure, myocardial infarction, arrhythmia, stroke/transient ischaemic attack and CV death) in asymptomatic community-based patients with risk factors but without prevalent MACE at enrolment. The study population consisted of 282 patients selected from the longitudinal STOP-HF study of asymptomatic patients with risk factors for development of MACE. Fifty-two of these patients developed a MACE. The study was run in two phases comprising of an initial investigative cohort (n = 195), and a subsequent 2:1 (No MACE: MACE) propensity matched verification cohort (n = 87). BNP and sST2 were quantified in all patients at two time points a median of 2.5 years apart. Results highlighted that longitudinal change in sST2 was a statistically significant predictor of incident MACE, (AUC 0.60). A one-unit increment in sST2 change from baseline to follow up corresponded to approximately 7.99% increase in the rate of one or more incident MACE, independent of the baseline or follow-up concentration. In contrast, longitudinal change value of BNP was not associated with MACE. In conclusion, longitudinal change in sST2 but not BNP was associated with incident MACE in asymptomatic, initially event-free patients in the community. Further work is required to evaluate the clinical utility of change in sST2 in risk prediction and event monitoring in this setting.     

Hypomethylation of Serum Blood Clot DNA,but Not Plasma EDTA-Blood Cell Pellet DNA,from Vitamin B12-Deficient Subjects

Vitamin B12, a co-factor in methyl-group transfer, is important in maintaining DNA (deoxycytidine) methylation. Using two independent assays we examined the effect of vitamin B12-deficiency (plasma vitamin B12<148 pmol/L) on DNA methylation in women of childbearing age. Coagulated blood clot DNA from vitamin B12-deficient women had significantly (p<0.001) lower percentage deoxycytidine methylation (3.23±0.66%; n = 248) and greater [3 H]methyl-acceptance (42,859±9,699 cpm; n = 17) than DNA from B12-replete women (4.44±0.18%; n = 128 and 26,049±2,814 cpm; n = 11) [correlation between assays: r = –0.8538; p<0.001; n = 28]. In contrast, uncoagulated EDTA-blood cell pellet DNA from vitamin B12-deficient and B12-replete women exhibited similar percentage methylation (4.45±0.15%; n = 77 vs. 4.47±0.15%; n = 47) and [3 H]methyl-acceptance (27,378±4,094 cpm; n = 17 vs. 26,610±2,292 cpm; n = 11). Therefore, in simultaneously collected paired blood samples, vitamin B12-deficiency was associated with decreased DNA methylation only in coagulated samples. These findings highlight the importance of sample collection methods in epigenetic studies, and the potential impact biological processes can have on DNA methylation during collection.