Effects of fullerene (C60), multi-wall carbon nanotubes (MWCNT), single wall carbon nanotubes (SWCNT) and hydroxyl and carboxyl modified single wall carbon nanotubes on riverine microbial communities.

Commercial production of nanoparticles (NP) has created a need for research to support regulation of nanotechnology. In the current study, microbial biofilm communities were developed in rotating annular reactors during continuous exposure to 500 µg L(-1) of each nanomaterial and subjected to multimetric analyses. Scanning transmission X-ray spectromicroscopy (STXM) was used to detect and estimate the presence of the carbon nanomaterials in the biofilm communities. Microscopy observations indicated that the communities were visibly different in appearance with changes in abundance of filamentous cyanobacteria in particular. Microscale analyses indicated that fullerene (C60) did not significantly (p < 0.05) impact algal, cyanobacterial or bacterial biomass. In contrast, MWCNT exposure resulted in a significant decline in algal and bacteria biomass. Interestingly, the presence of SWCNT products increased algal biomass, significantly in the case of SWCNT-COOH (p < 0.05) but had no significant impact on cyanobacterial or bacterial biomass. Thymidine incorporation indicated that bacterial production was significantly reduced (p < 0.05) by all nanomaterials with the exception of fullerene. Biolog assessment of carbon utilization revealed few significant effects with the exception of the utilization of carboxylic acids. PCA and ANOSIM analyses of denaturing gradient gel electrophoresis (DGGE) results indicated that the bacterial communities exposed to fullerene were not different from the control, the MWCNT and SWNT-OH differed from the control but not each other, whereas the SWCNT and SWCNT-COOH both differed from all other treatments and were significantly different from the control (p < 0.05). Fluorescent lectin binding analyses also indicated significant (p < 0.05) changes in the nature and quantities of exopolymer consistent with changes in microbial community structure during exposure to all nanomaterials. Enumeration of protozoan grazers showed declines in communities exposed to fullerene or MWCNT but a trend for increases in all SWCNT exposures. Observations indicated that at 500 µg L(-1), carbon nanomaterials significantly alter aspects of microbial community structure and function supporting the need for further evaluation of their effects in aquatic habitats.

Soft X-ray spectromicroscopy for speciation, quantitation and nano-eco-toxicology of nanomaterials.

There is a critical need for methods that provide simultaneous detection, identification, quantitation and visualization of nanomaterials at their interface with biological and environmental systems. The approach should allow speciation as well as elemental analysis. Using the intrinsic X-ray absorption properties, soft X-ray scanning transmission X-ray spectromicroscopy (STXM) allows characterization and imaging of a broad range of nanomaterials, including metals, oxides and organic materials, and at the same time is able to provide detailed mapping of biological components. Thus, STXM offers considerable potential for application to research on nanomaterials in biology and the environment. The potential and limitations of STXM in this context are discussed using a range of examples, focusing on the interaction of nanomaterials with microbial cells, biofilms and extracellular polymers. The studies outlined include speciation and mapping of metal-containing nanomaterials (Ti, Ni, Cu) and carbon-based nanomaterials (multiwalled carbon nanotubes, C60 fullerene). The benefits of X-ray fluorescence detection in soft X-ray STXM are illustrated with a study of low levels of Ni in a natural river biofilm.

Resilience and recovery: the effect of triclosan exposure timing during development, on the structure and function of river biofilm communities.

Triclosan (TCS) is a ubiquitous antibacterial agent found in soaps, scrubs, and consumer products. There is limited information on hazardous effects of TCS in the environment. Here, rotating annular reactors were used to cultivate river biofilm communities exposed to 1.8 µg l(-1) TCS with the timing and duration of exposure and recovery during development varied. Two major treatment regimens were employed: (i) biofilm development for 2, 4 or 6 weeks prior to TCS exposure and (ii) exposure of biofilms to TCS for 2, 4 or 6 weeks followed by recovery. Biofilms not exposed to TCS were used as a reference condition. Communities cultivated without and then exposed to TCS all exhibited reductions in algal biomass and significant (p<0.05) reductions in cyanobacterial biomass. No significant effects were observed on bacterial biomass. CLSM imaging of biofilms at 8 weeks revealed unique endpoints in terms of community architecture. Community composition was altered by any exposure to TCS, as indicated by significant shifts in denaturing gradient gel electrophoresis fingerprints and exopolymer composition relative to the reference. Bacterial, algal and cyanobacterial components initially exposed to TCS were significantly different from those TCS-free at time zero. Pigment analyses suggested that significant changes in composition of algal and cyanobacterial populations occurred with TCS exposure. Bacterial thymidine incorporation rates were reduced by TCS exposure and carbon utilization spectra shifted in terms substrate metabolism. Direct counts of protozoans indicated that TCS was suppressive, whereas micrometazoan populations were, in some instances, stimulated. These results indicate that even a relatively brief exposure of a river biofilm community to relatively low levels of TCS alters both the trajectory and final community structure. Although some evidence of recovery was observed, removal of TCS did not result in a return to the unexposed reference condition.

Biogeochemical activity of microbial biofilms in the water column overlying uranium mine tailings.

AIMS: To describe microbial diversity, biofilm composition and biogeochemical potential within biofilms in the water overlying uranium tailings characterized by high pH, high metal concentration and low permeability. METHODS AND RESULTS: To estimate microbial diversity in biofilms formed in water columns overlying uranium mine tailings, culture-dependent and culture-independent methods were employed. High-throughput sequencing revealed the presence of 11 phyla; however, the majority of the sequences were affiliated with four major lineages (Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes) as confirmed by culture-based methods. Dominant phylotypes were closely related to methylotrophs (Methylobacterium) and bacterial groups able to utilize complex hydrocarbons (Aquabacterium and Dechloromonas). Microbial diversity in biofilms from the 13 m depth was significantly different that in biofilms from 1 to 41 m (P < 0·05). Phylotypes closely related to iron-reducing bacteria were identified at each depth; whereas sulphate-, thio-sulphate-, sulphite- and sulphur-reducing bacteria, at low abundance, were only detected at lower depths. Confocal scanning laser microscopy (CSLM) was used to investigate polymer quantity and composition of the biofilm components, and principal component analysis of the CLSM data revealed that the relative abundance of α-L-fucose and N-acetyl-glucosamine/lipopolysaccharide residues separated tailings-water interface biofilms from those from other depths. Reduced (ferrous) iron was detected within all the biofilm samples examined by scanning X-ray transmission microscopy. CONCLUSIONS: Microbial communities within the water column covering a highly alkaline uranium tailings body form biofilms with microenvironments where iron reduction takes place. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the biogeochemical potential of microbial biofilm communities in the water column covering an alkaline uranium tailings body; specifically, the nature of the bacterial groups detected (Aquabacterium, Dechloromonas) and the presence of reduced iron suggest that complex hydrocarbons are available for bacterial growth and geochemical change, such as iron reduction, can occur even though the system bulk phase is predominantly oxic.

Relationship between water quality parameters and bacterial indicators in a large prairie reservoir: Lake Diefenbaker, Saskatchewan, Canada.

Lake Diefenbaker (LD) is a large reservoir on the South Saskatchewan River used for agricultural irrigation, drinking water, and recreation. Our objectives were to determine the distribution and abundance of bacterial indicators in embayments and the main channel of LD and to relate these to environmental factors. Total coliforms (TCs), fecal coliforms (FCs), and fecal indicator bacteria (i.e., Escherichia coli) were measured concurrently with water quality parameters. Although TCs, FCs, and E. coli were present in LD, they rarely exceeded the TC and FC Canadian Council of Ministers of the Environment (CCME) water quality standards for agricultural use (1000 colony-forming units (CFU) per 100 mL and 100 CFU per 100 mL, respectively). The correlation between the bacterial indicators in the sediments and the water column indicates that higher embayment abundances may be related to sediment loading and (or) resuspension events in these frequently mixed embayments. With higher water temperatures and water levels, as well as higher microbial activity, CCME bacterial limits may be exceeded. The greatest contributor to bacterial indicator abundance was water temperature. We predict that water quality standards will be exceeded more frequently with climate warming.

Glycerol and environmental factors: effects on 1,3-propanediol production and NAD(+) regeneration in Lactobacillus panis PM1.

AIMS: This study was conducted to understand the influences of fermentation factors in NADH recycling and mechanisms of 1,3-propanediol (1,3-PDO) production in Lactobacillus panis PM1. METHODS AND RESULTS: We conducted metabolite analyses, qRT-PCR of the glycerol reductive pathway [glycerol dehydratase (DhaB) and 1,3-PDO dehydrogenase (DhaT)] and DhaT activity assays at different pH, temperature and initial glycerol concentrations. The supplementation of 150 mmol l(-1) glycerol caused a shift in NADH flux from ethanol to 1,3-PDO production, whereas 300 mol l(-1) glycerol negatively affected the regeneration of NAD(+) via 1,3-PDO production. This retardation decreased transcription levels and specific activities of DhaT. The decreased DhaT activity eventually caused the shutdown of 1,3-PDO production. Temperature and pH did not significantly affect the specific activity of DhaT, whereas expression of genes for DhaB and DhaT was activated under acidic conditions. Moreover, fresh glucose addition after its depletion could not restart the glycerol reduction, but increased ethanol production. CONCLUSIONS: Those environmental factors affect 1,3-PDO production in different ways through changing the expression level of enzymes and shifting the NAD(+) regeneration pathways. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings elucidated a key element to optimize 1,3-PDO production by Lact. panis PM1, which potentially improves 1,3-PDO manufacturing efficiencies.

Microbial communities in low permeability, high pH uranium mine tailings: characterization and potential effects.

AIMS: To describe the diversity and metabolic potential of microbial communities in uranium mine tailings characterized by high pH, high metal concentration and low permeability. METHODS AND RESULTS: To assess microbial diversity and their potential to influence the geochemistry of uranium mine tailings using aerobic and anaerobic culture-based methods, in conjunction with next generation sequencing and clone library sequencing targeting two universal bacterial markers (the 16S rRNA and cpn60 genes). Growth assays revealed that 69% of the 59 distinct culturable isolates evaluated were multiple-metal resistant, with 15% exhibiting dual-metal hypertolerance. There was a moderately positive correlation coefficient (R = 0·43, P < 0·05) between multiple-metal resistance of the isolates and their enzyme expression profile. Of the isolates tested, 17 reduced amorphous iron, 22 reduced molybdate and seven oxidized arsenite. Based on next generation sequencing, tailings depth was shown to influence bacterial community composition, with the difference in the microbial diversity of the upper (0-20 m) and middle (20-40 m) tailings zones being highly significant (P < 0·01) from the lower zone (40-60 m) and the difference in diversity of the upper and middle tailings zone being significant (P < 0·05). Phylotypes closely related to well-known sulfate-reducing and iron-reducing bacteria were identified with low abundance, yet relatively high diversity. CONCLUSIONS: The presence of a population of metabolically-diverse, metal-resistant micro-organisms within the tailings environment, along with their demonstrated capacity for transforming metal elements, suggests that these organisms have the potential to influence the long-term geochemistry of the tailings. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first investigation of the diversity and functional potential of micro-organisms present in low permeability, high pH uranium mine tailings.

Transient response of microbial communities in a water well field to application of an impressed current.

Deterioration of water wells due to clogging and corrosion over time is a common problem where solutions may be costly and ineffective. Pilot studies have suggested that impressed current or cathodic protection may be used to reduce microbially-induced declines in water well performance. Two water wells in an alluvial aquifer close to the North Saskatchewan River were selected to study the response of subsurface microbial communities to the application of an impressed current as an anti-fouling technology. The treated well was exposed to an impressed current while the untreated well was used as a reference site. Biofilms grown on in situ coupons under the influence of the impressed current were significantly (p < 0.05) thicker (mean thickness = 67.3 µm) when compared to the biofilms (mean thickness = 19.3 µm) grown outside the electric field. Quantitative PCR analyses showed significantly (p < 0.05) higher numbers of total bacteria, iron- and nitrate-reducers in the electrified zone. Molecular analysis revealed that the predominant bacteria present in biofilms grown under the influence of the impressed current belonged to Rhodobacter spp., Sediminibacterium spp. and Geobacter spp. In addition to favouring the growth of biofilms, direct microscopic and ICP-AES analyses revealed that the impressed current also caused the deposition of iron and manganese on, and in the vicinity of, the well screen. Together, these factors contributed to rapid clogging leading to reduced specific pumping capacities of the treated well. The study revealed that the impressed current system was not effective as an anti-fouling technology but actually promoted both microbial growth and physical clogging in this aquifer.

Spatial variation in microbial community structure, richness, and diversity in an alluvial aquifer.

Relatively little is known regarding the spatial variability of microbial communities in aquifers where well fouling is an issue. In this study 2 water wells were installed in an alluvial aquifer located adjacent to the North Saskatchewan River and an associated piezometer network developed to facilitate the study of microbial community structure, richness, and diversity. Carbon utilization data analysis revealed reduced microbial activity in waters collected close to the wells. Functional PCR and quantitative PCR analysis indicated spatial variability in the potential for iron-, sulphate-, and nitrate-reducing activity at all locations in the aquifer. Denaturing gradient gel electrophoresis analysis of aquifer water samples using principal components analyses indicated that the microbial community composition was spatially variable, and denaturing gradient gel electrophoresis sequence analysis revealed that bacteria belonging to the genera Acidovorax , Rhodobacter , and Sulfuricurvum were common throughout the aquifer. Shannon's richness (H') and Pielou's evenness (J') indices revealed a varied microbial diversity (H' = 1.488-2.274) and an even distribution of microbial communities within the aquifer (J' = 0.811-0.917). Overall, these analyses revealed that the aquifer's microbial community varied spatially in terms of composition, richness, and metabolic activity. Such information may facilitate the diagnosis, prevention, and management of fouling.

Effect of pumping on the spatio-temporal distribution of microbial communities in a water well field.

A water well field adjacent to the North Saskatchewan River (City of North Battleford, Saskatchewan, Canada) with a history of rapid deterioration of both well water quality and yield was selected to study the spatial and temporal distribution of subsurface microbial communities and their response to water pumping. A range of conventional cultural, microscopic and molecular techniques, including confocal laser scanning microscopy (CLSM), Biolog, qPCR and Denaturing Gradient Gel Electrophoresis (DGGE), was used during this study. Redox data and water and sediment chemistry showed that the aquifer was anoxic and harbored substantial amounts of Fe and Mn. CLSM analyses of incubated coupons indicated extensive biofilm growth in the zone immediately surrounding the well and was coincident with reduced water well yield. PCR screening and qPCR analyses showed that the potential for iron- and sulfate-reducing activity increased with proximity to the well. Bacterial communities inhabiting the zone closest to the well showed the greatest changes and differences in metabolic activities and composition as revealed by PCA (Principal Components Analysis) of the Biolog and DGGE data. The sequence analysis of all the samples revealed that Sulfuricurvum spp., Methylobacter spp., Geobacter spp. and Rhodobacter spp. were most commonly detected in this aquifer. Overall the findings demonstrated that the microbial numbers, metabolic activities and the community composition changed in response to water pumping but effects did not extend beyond 1-2m zone from the well.

Soft X-ray spectromicroscopy of nickel sorption in a natural river biofilm.

Scanning transmission X-ray microscopy (STXM) at the C 1s, O 1s, Ni 2p, Ca 2p, Mn 2p, Fe 2p, Mg 1s, Al 1s and Si 1s edges was used to study Ni sorption in a complex natural river biofilm. The 10-week grown river biofilm was exposed to 10 mg L(-1) Ni(2+) (as NiCl(2)) for 24 h. The region of the biofilm examined was dominated by filamentous structures, which were interpreted as the discarded sheaths of filamentous bacteria, as well as a sparse distribution of rod-shaped bacteria. The region also contained discrete particles with spectra similar to those of muscovite, SiO(2) and CaCO(3). The Ni(II) ions were selectively adsorbed by the sheaths of the filamentous bacteria. The sheaths were observed to be metal rich with significant amounts of Ca, Fe and Mn, along with the Ni. In addition, the sheaths had a large silicate content but little organic material. The metal content of the rod-shaped bacterial cells was much lower. The Fe on the sheath was mainly in the Fe(III) oxidation state. Mn was found in II, III and IV oxidation states. The Ni was likely sorbed to Mn-Fe minerals on the sheath. These STXM results have probed nano-scale biogeochemistry associated with bacterial species in a complex, natural biofilm community. They have implications for selective Ni contamination of the food chain and for developing bioremediation strategies.

Whole-cell protein and ITS rDNA profiles as diagnostic tools to discriminate Fusarium avenaceum intraspecific variability and associated virulence.

A total of 91 isolates of Fusarium avenaceum were regrouped into 15 phenotypes and 10 vegetative compatibility groups showing specific one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1-D SDS-PAGE) protein profiles and less-specific internal transcribed spacer rDNA profiles. Each isolate possessed reproducible signature protein bands. Indeed, the unweighted pair group method with arithmetic averages clustering revealed that the protein profile of each group of isolates correlated with fungus virulence. The use of SDS-PAGE offers a simple and sensitive technique for routine differentiation between pathogenic and nonpathogenic isolates within unknown F. avenaceum populations. The discovery has significant implications for risk assessment of cereal yield to ensure food and feed safety. This low-cost approach has the potential to be optimized and extended to a broad spectrum of Fusarium head blight pathogens.

Comparative microscale analysis of the effects of triclosan and triclocarban on the structure and function of river biofilm communities.

The broad spectrum antimicrobials triclosan (TCS) and triclocarban (TCC) are commonly detected in the environment. However, there is very limited understanding of the aquatic ecological implications of these agents. During this study, river biofilms were cultivated using 10 microg l(-1) of TCS or TCC and the equivalent in nutrients (carbon, nitrogen) over a developmental period of 8 weeks. Confocal laser microscopy showed that the biofilm communities developing under the influence of TCS and TCC had community architecture and composition different from either control or nutrient exposed communities. Microscale analyses of biofilm community structure indicated a significant reduction in algal biomass (p<0.05) as a result of exposure to either TCS or TCC. Thymidine incorporation did not detect significant differences between control and treated communities. The use of carbon utilization assays based on growth indicated that, in general, TCS and TCC suppressed utilization. The community was altered from one dominated by autotrophic processes to one dominated by heterotrophic processes. Both TCS and TCC treatments resulted in significant (p<0.05) alterations in the composition of the EPS matrix of the communities, suggesting significant changes in community composition. Denaturing gradient gel electrophoresis and PCA-ANOSIM analyses indicated a significant change occurred in the bacterial community as a consequence of TCS treatments. Enumeration of micrometazoa and protozoa revealed an increase in micrometazoan numbers over control values, whereas no clear impact on protozoa was detected in any treatment. This study indicated significant effects of 10 microg l(-1) TCS and TCC on microbial community composition, algal biomass, architecture and activity.

Microbial distribution and diversity in saturated, high pH, uranium mine tailings, Saskatchewan, Canada.

Microbiological analyses were conducted on core samples collected along a vertical profile (0-66 m below surface) from the tailings management facility (TMF) at the Rabbit Lake uranium mine in northern Saskatchewan, Canada. Bacterial numbers in the core materials were similar to surrounding soils and surface waters, regardless of the seemingly unfavorable pH (mean=9.9) and temperature (approximately 0 degrees C) in the TMF. The greatest number of viable cells (105 CFU/g) was detected at the interface between the tailings and overlying standing water, below which cell counts decreased rapidly with depth. Whole-community metabolic profiles for samples from the different depths grouped into 3 clusters; however, these groups could not be positively correlated with sampling depth, temperature, redox potential, pH, or ore-mill feed. Flow-cell studies demonstrated microbial communities in the tailings surface water could develop biofilms and maintain cell activity at both pH 10 and 7, and altering the pH between these 2 values had little effect on biofilm viability. These results demonstrate the resilience and adaptive nature of naturally occurring microbial communities and signify a potential role of microbial activity in the long-term geochemical evolution of the TMF.

Community-level assessment of the effects of the broad-spectrum antimicrobial chlorhexidine on the outcome of river microbial biofilm development.

Chlorhexidine is a common-use antibacterial agent found in a range of personal-care products. We used rotating annular reactors to cultivate river biofilms under the influence of chlorhexidine or its molar equivalent in nutrients. Studies of the degradation of [(14)C]chlorhexidine demonstrated that no mineralization of the compound occurred. During studies with 100 microg liter(-1) chlorhexidine, significant changes were observed in the protozoan and micrometazoan populations, the algal and cyanobacterial biomass, the bacterial biomass, and carbon utilization. Denaturing gradient gel electrophoresis (DGGE) in combination with statistical analyses showed that the communities developing under control and 100 microg liter(-1) chlorhexidine were significantly different. At 10 microg liter(-1) chlorhexidine, there was significantly increased algal and cyanobacterial biomass while the bacterial biomass was not significantly affected (P < 0.05). No significant effects on protozoan or metazoan grazing were detected at the 10-microg liter(-1) chlorhexidine level. Fluorescent in situ hybridization indicated a significant reduction in the abundance of betaproteobacteria and gammaproteobacteria (P < 0.05). Archaeal cell counts were significantly reduced by both chlorhexidine and nutrient treatments. DGGE and statistical analyses indicated that 10 microg liter(-1) chlorhexidine and molar equivalent nutrient treatments were significantly different from control communities. In contrast to community level observations, toxicological testing with a panel of cyanobacteria, algae, and protozoa indicated no detectable effects at 10, 50, and 100 microg liter(-1) chlorhexidine. Thus, community level assessment indicated a risk of low levels of chlorhexidine in aquatic habitats while conventional approaches did not.

Microbial response to environmental gradients in a ceramic-based diffusion system.

A solid, porous matrix was used to establish steady-state concentration profiles upon which microbial responses to concentration gradients of nutrients or antimicrobial agents could be quantified. This technique relies on the development of spatially defined concentration gradients across a ceramic plate resulting from the diffusion of solutes through the porous ceramic matrix. A two-dimensional, finite-element numerical transport model was used to predict the establishment of concentration profiles, after which concentration profiles of conservative tracers were quantified fluorometrically and chemically at the solid-liquid interface to verify the simulated profiles. Microbial growth responses to nutrient, hypochloride, and antimicrobial concentration gradients were then quantified using epifluorescent or scanning confocal laser microscopy. The observed microbial response verified the establishment and maintenance of stable concentration gradients along the solid-liquid interface. These results indicate the ceramic diffusion system has potential for the isolation of heterogeneous microbial communities as well as for testing the efficacy of antimicrobial agents. In addition, the durability of the solid matrix allowed long-term investigations, making this approach preferable to conventional gel-stabilized systems that are impeded by erosion as well as expansion or shrinkage of the gel.

Planktonic or biofilm growth affects survival, hydrophobicity and protein expression patterns of a pathogenic Campylobacter jejuni strain.

The effect of planktonic or biofilm modes of growth on survival, hydrophobicity and cellular protein expression patterns of a pathogenic Campylobacter jejuni strain were determined. This was achieved by growing the strain in brain heart infusion broth (with 1% yeast extract), or attached to glass beads in the same medium, at 37 degrees C for 48 h under microaerophilic conditions. Cells from the broth or the bead surfaces were stored at different temperatures (4, 10, 25 and 37 degrees C) for 28 days in phosphate buffered saline (PBS) and monitored at appropriate time intervals for culturable numbers and hydrophobicity by standard methods. In addition, cells were inoculated onto the surface of two processed meat products (a bologna and a summer sausage) vacuum packaged and stored at 4 degrees C for 28 days. Numbers of culturable cells were monitored at appropriate time intervals by standard methods. Cells from the broth or the bead surfaces were also examined for protein expression using two-dimensional protein electrophoresis. Results indicated that numbers of culturable cells in phosphate buffered saline decreased from approximately 6 log colony forming units (cfu) g(-1) to undetectable levels within 14-day storage in a temperature dependent manner. Hydrophobicity of broth grown cells decreased from 15% to 0% adherence to xylene over the same time in a temperature independent manner. Cells grown in a biofilm mode initially displayed a <0.3% adherence to xylene which was maintained during storage. Furthermore, cells grown in the biofilm mode decreased in number more rapidly on storage in buffer than their counterparts grown in broth. Numbers of culturable cells on meat decreased from approximately 5 log cfu g(-1) to undetectable levels within 14-day storage in a product dependent manner, with the most rapid decrease observed for the more acidic summer sausage. Cells grown in a biofilm mode decreased in number more rapidly on storage than broth grown cells. The protein expression patterns differed between planktonic and biofilm cells with seven unique and 12 up-regulated protein spots expressed in a growth mode specific manner. A number of the differentially expressed spots were tentatively identified, by comparison to existing literature, as surface- and stress-associated proteins. Despite the elicitation of some putative stress proteins, this study importantly indicates that biofilm cells of C. jejuni are less resistant to stress than their planktonic counterparts and may lack a sophisticated adaptive stress-resistance response. These findings have implication in determining the risks of infection associated with C. jejuni contamination on food.

Efficacy enhancement of trisodium phosphate against spoilage and pathogenic bacteria in model biofilms and on adipose tissue.

A two-step approach for enhancing the efficacy of trisodium phosphate (TSP) was evaluated using meat spoilage and pathogenic bacteria in flow cell biofilms and adipose tissue model systems. The process was based on the plasmolysis of attached bacteria (biofilms) with a hyperosmotic solution (1.5 M NaCl) and the subsequent deplasmolysis of cells with a low-osmotic-strength solution containing different concentrations of TSP (0.1, 0.25, 0.5, 0.625, and 1.0 % [wt/vol]). Escherichia coli, Salmonella Enteritidis, Pseudomonas sp., Listeria monocytogenes, and Brochothrix thermosphacta strains were cultivated for 24 h as pure culture biofilms in glass flow cells with complex media and were then treated with either 0.1, 0.25, 0.5, 0.625, and 1.0% TSP, or the same TSP concentrations delivered in conjunction with plasmolysis-deplasmolysis (PDP). Confocal scanning laser microscopy, a commercial fluorescent viability probe, and image analysis were then used to quantify the relative abundances of living and dead cells remaining after the different treatment regimes. With the exception of L. monocytogenes (which was resistant to TSP concentrations of up to 5%), the PDP process increased the sensitivity of the test strains to TSP. However, when similar experiments were conducted with pork adipose tissue, it became evident that higher TSP concentrations were necessary to produce significant decreases in the number of viable cells and that the PDP process generally failed to enhance TSP efficacy. An exception was L. monocytogenes, which exhibited an increase in sensitivity to TSP when inoculated tissue was pretreated with 1.5 M NaCl. It is thought that factors contributing to the failure of the PDP process to enhance the activity of TSP in meat systems involves the mode of TSP antimicrobial activity, alkaline pH stress, and the chemically complex, buffered nature of meats. It remains to be determined whether the PDP process is suitable for use with other food grade antimicrobial agents or can be used in nonfood biofilm control applications.

Two parameters improve efficiency of mitochondrial uptake of adenylate kinase: decreased folding velocity and increased propensity of N-terminal alpha-helix formation.

The long isoform of eukaryotic adenylate kinase has a dual subcellular location in the cytoplasm and in the mitochondrial intermembrane space. Protein sequences and modifications are identical in both locations. In yeast, the bulk of the major form of adenylate kinase (Aky2p) is in the cytoplasm and, in the steady state, only 5-8% is sorted to the mitochondrial intermembrane space. Since the reasons for exclusion from mitochondrial import are unclear, we have constructed aky2 mutants with elevated mitochondrial uptake efficiency of Aky2p in vivo and in vitro. We have analyzed the effect of the mutations on secondary structure prediction in silico and have tested folding velocity and folding stability. One type of mutants displayed decreased proteolytic stability and retarded renaturation kinetics after chaotropic denaturation implying that deterioration of folding leads to prolonged presentation of target information to mitochondrial import receptors, thereby effecting improved uptake. In a second type of mutants, increased import efficiency was correlated with an increased probability of formation of an alpha-helix with increased amphipathic moment at the N-terminus suggesting that targeting interactions with mitochondrial import receptors had been improved at the level of binding affinity.

Adaptation of bacterial communities to environmental transitions from labile to refractory substrates.

The aim of this work was to assess the adaptation of bacterial communities to environmental transitions from labile to refractory substrates. This involved testing the hypothesis that bacteria self-organize and propagate not only as individual cellular systems, but also as functional sets of interacting organisms. A biofilm community was cultivated in a flow-cell irrigated with tryptic soy broth and subjected to a cyclic series of environmental transitions, from labile to refractory substrates, followed by a period of starvation (30 days). The appearance and disappearance of specific colony morphotypes when the emigrants were plated onto tryptic soy agar was used to monitor the restructuring of the community. Confocal laser microscopy of flow cells showed that these transitions decreased the biofilm thickness and coverage. Substrate shifts also changed the architecture of the biofilm communities. Repeated inoculation of flow-cell communities with a composite inoculum increased the number and diversity of emigrants. Their biofilms were thicker and covered a wider area than those of communities that had been inoculated only at the beginning of the experiment. With repeated inoculation, the time required for the community to restructure and stabilize decreased during most transitions. This suggested that organismal recombination acted as a mechanism of adaptation, enhancing the growth of microbial communities exposed to environmental stresses. Changes in the profiles of emigrants during the adaptation of biofilm communities to environmental transitions showed the appearance and disappearance of discrete sets of organisms. This suggested that the biofilm communities responded to environmental stresses as sets of interacting organisms. Enhanced growth of biofilm communities due to repeated environmental cycling suggested that the functionality of cellular positioning accrued from one cycle to the next and was thus heritable, although it was not necessarily genetically encoded.