Intracellular Amyloid-ß Detection from Human Brain Sections Using a Microfluidic Immunoassay in Tandem with MALDI-MS.

Alzheimer's disease (AD) currently affects more than 30 million people worldwide. The lack of understanding of AD's physiopathology limits the development of therapeutic and diagnostic tools. Soluble amyloid-ß peptide (Aß) oligomers that appear as intermediates along the Aß aggregation into plaques are considered among the main AD neurotoxic species. Although a wealth of data are available about Aß from in vitro and animal models, there is little known about intracellular Aß in human brain cells, mainly due to the lack of technology to assess the intracellular protein content. The elucidation of the Aß species in specific brain cell subpopulations can provide insight into the role of Aß in AD and the neurotoxic mechanism involved. Here, we report a microfluidic immunoassay for in situ mass spectrometry analysis of intracellular Aß species from archived human brain tissue. This approach comprises the selective laser dissection of individual pyramidal cell bodies from tissues, their transfer to the microfluidic platform for sample processing on-chip, and mass spectrometric characterization. As a proof-of-principle, we demonstrate the detection of intracellular Aß species from as few as 20 human brain cells.

MIMAS: microfluidic platform in tandem with MALDI mass spectrometry for protein quantification from small cell ensembles.

Understanding cell-to-cell variation at the molecular level provides relevant information about biological phenomena and is critical for clinical and biological research. Proteins carry important information not available from single-cell genomics and transcriptomics studies; however, due to the minute amount of proteins in single cells and the complexity of the proteome, quantitative protein analysis at the single-cell level remains challenging. Here, we report an integrated microfluidic platform in tandem with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the detection and quantification of targeted proteins from small cell ensembles (> 10 cells). All necessary steps for the assay are integrated on-chip including cell lysis, protein immunocapture, tryptic digestion, and co-crystallization with the matrix solution for MALDI-MS analysis. We demonstrate that our approach is suitable for protein quantification by assessing the apoptotic protein Bcl-2 released from MCF-7 breast cancer cells, ranging from 26 to 223 cells lysed on-chip (8.75 nL wells). A limit of detection (LOD) of 11.22 nM was determined, equivalent to 5.91 × 107 protein molecules per well. Additionally, the microfluidic platform design was further improved, establishing the successful quantification of Bcl-2 protein from MCF-7 cell ensembles ranging from 8 to 19 cells in 4 nL wells. The LOD in the smaller well designs for Bcl-2 resulted in 14.85 nM, equivalent to 3.57 × 107 protein molecules per well. This work shows the capability of our approach to quantitatively assess proteins from cell lysate on the MIMAS platform for the first time. These results demonstrate our approach constitutes a promising tool for quantitative targeted protein analysis from small cell ensembles down to single cells, with the capability for multiplexing through parallelization and automation.

Characterization of microbial mixtures by mass spectrometry.

MS applications in microbiology have increased significantly in the past 10 years, due in part to the proliferation of regulator-approved commercial MALDI MS platforms for rapid identification of clinical infections. In parallel, with the expansion of MS technologies in the "omics" fields, novel MS-based research efforts to characterize organismal as well as environmental microbiomes have emerged. Successful characterization of microorganisms found in complex mixtures of other organisms remains a major challenge for researchers and clinicians alike. Here, we review recent MS advances toward addressing that challenge. These include sample preparation methods and protocols, and established, for example, MALDI, as well as newer, for example, atmospheric pressure ionization (API) techniques. MALDI mass spectra of intact cells contain predominantly information on the highly expressed house-keeping proteins used as biomarkers. The API methods are applicable for small biomolecule analysis, for example, phospholipids and lipopeptides, and facilitate species differentiation. MS hardware and techniques, for example, tandem MS, including diverse ion source/mass analyzer combinations are discussed. Relevant examples for microbial mixture characterization utilizing these combinations are provided. Chemometrics and bioinformatics methods and algorithms, including those applied to large scale MS data acquisition in microbial metaproteomics and MS imaging of biofilms, are highlighted. Select MS applications for polymicrobial culture analysis in environmental and clinical microbiology are reviewed as well.

MALDI TOF MS profiling of bacteria at the strain level: a review.

Since the advent of the use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) as a tool for microbial characterization, efforts to increase the taxonomic resolution of the approach have been made. The rapidity and efficacy of the approach have suggested applications in counter-bioterrorism, prevention of food contamination, and monitoring the spread of antibiotic-resistant bacteria. Strain-level resolution has been reported with diverse bacteria, using library-based and bioinformatics-enabled approaches. Three types of characterization at the strain level have been reported: strain categorization, strain differentiation, and strain identification. Efforts to enhance the library-based approach have involved sample pre-treatment and data reduction strategies. Bioinformatics approaches have leveraged the ever-increasing amount of publicly available genomic and proteomic data to attain strain-level characterization. Bioinformatics-enabled strategies have facilitated strain characterization via intact biomarker identification, bottom-up, and top-down approaches. Rigorous quantitative and advanced statistical analyses have fostered success at the strain level with both approaches. Library-based approaches can be limited by effects of sample preparation and culture conditions on reproducibility, whereas bioinformatics-enabled approaches are typically limited to bacteria, for which genetic and/or proteomic data are available. Biological molecules other than proteins produced in strain-specific manners, including lipids and lipopeptides, might represent other avenues by which strain-level resolution might be attained. Immunological and lectin-based chemistries have shown promise to enhance sensitivity and specificity. Whereas the limits of the taxonomic resolution of MALDI TOF MS profiling of bacteria appears bacterium-specific, recent data suggest that these limits might not yet have been reached.

Automating data acquisition affects mass spectrum quality and reproducibility during bacterial profiling using an intact cell sample preparation method with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) has emerged as a promising tool to rapidly profile bacteria at the genus and species level and, more recently, at the sub-species (strain) level. Recently, it has been proposed that the approach can be enhanced with regard to reproducibility and throughput by automating spectrum acquisition; however, effects of automating spectrum acquisition on spectrum quality and reproducibility have not been investigated. Using an intact cell-based sample preparation method, we directly compared the quality and reproducibility of spectra acquired in a fully automated fashion to those acquired manually by two operators with different levels of experience. While automation tended to increase base peak resolution, other measures of spectrum quality, including signal-to-noise (S:N) ratio, data richness, and reproducibility were reduced. Negative effects of automation on the performance of this approach to bacterial profiling may be particularly important during profiling of closely related strains of bacteria that yield very similar spectra.

Identification of putative transcriptomic biomarkers in irritable bowel syndrome (IBS): Differential gene expression and regulation of TPH1 and SERT by vitamin D.

Irritable bowel syndrome (IBS) is one of the most common gastrointestinal disorders and affects approximately 4% of the global population. The diagnosis of IBS can be made based on symptoms using the validated Rome criteria and ruling out commonly occurring organic diseases. Although biomarkers exist for "IBS mimickers" such as celiac disease and inflammatory bowel disease (IBD), no such test exists for IBS. DNA microarrays of colonic tissue have been used to identify disease-associated variants in other gastrointestinal (GI) disorders. In this study, our objective was to identify biomarkers and unique gene expression patterns that may define the pathological state of IBS. Mucosal tissue samples were collected from the sigmoid colon of 29 participants (11 IBS and 18 healthy controls). DNA microarray analysis was used to assess gene expression profiling. Extraction and purification of RNA were then performed and used to synthesize cDNA. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was employed to identify differentially expressed genes in patients diagnosed with IBS compared to healthy, non-IBS patient-derived cDNA. Additional testing probed vitamin D-mediated regulation of select genes associated with serotonergic metabolism. DNA microarray analyses led to the identification of 858 differentially expressed genes that may characterize the IBS pathological state. After screening a series of genes using a combination of gene ontological analysis and RT-qPCR, this spectrum of potential IBS biomarkers was narrowed to 23 genes, some of which are regulated by vitamin D. Seven putative IBS biomarkers, including genes involved in serotonin metabolism, were identified. This work further supports the hypothesis that IBS pathophysiology is evident within the human transcriptome and that vitamin D modulates differential expression of genes in IBS patients. This suggests that IBS pathophysiology may also involve vitamin D deficiency and/or an irregularity in serotonin metabolism.

Characterization of environmental isolates of Enterococcus spp. by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Currently available bacterial source-tracking tools are often technically demanding, time consuming, and have limited accuracy in grouping isolates according to their respective sources. There is a need for the development of bacterial source-tracking tools that would allow for more rapid and accurate grouping of isolates by source. We examined the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the characterization of environmental isolates of Enterococcus. Our main objectives were to develop sample preparation protocols for obtaining reproducible MALDI-TOF mass spectra from Enterococcus isolates and to evaluate methods of data analysis to maximize repeatability of the method and its ability to group isolates according to their respective sources. Our data showed that treatment of 21 Enterococcus isolates from seven unique sources with lysozyme for 20 h, followed by calculation of similarity coefficients using the Pearson product-moment correlation coefficient, facilitated a repeatability level of 91% as well as grouping by source for isolates obtained from several sources including human waste. Our data suggest that MALDI-TOF-MS-based fingerprinting of environmental isolates of Enterococcus has potential as a rapid and accurate bacterial source tracking (BST) tool, but requires further development, specifically regarding the time requirements needed for pre-treatment of isolates with lysozyme.

MALDI-TOF MS Affords Discrimination of Deinococcus aquaticus Isolates Obtained From Diverse Biofilm Habitats.

Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectroscopy (MALDI-TOF MS) has been used routinely over the past decade in clinical microbiology laboratories to rapidly characterize diverse microorganisms of medical importance both at the genus and species levels. Currently, there is keen interest in applying MALDI-TOF MS at taxonomic levels beyond species and to characterize environmental isolates. We constructed a model system consisting of 19 isolates of Deinococcus aquaticus obtained from biofilm communities indigenous to diverse substrates (concrete, leaf tissue, metal, and wood) in the Fox River - Lake Winnebago system of Wisconsin to: (1) develop rapid sample preparation methods that produce high quality, reproducible MALDI-TOF spectra and (2) compare the performance of MALDI-TOF MS-based profiling to common DNA-based approaches including 16S rRNA sequencing and genomic diversity by BOX-A1R fingerprinting. Our results suggest that MALDI-TOF MS can be used to rapidly and reproducibly characterize environmental isolates of D. aquaticus at the subpopulation level. MALDI-TOF MS provided higher taxonomic resolution than either 16S rRNA gene sequence analysis or BOX-A1R fingerprinting. Spectra contained features that appeared to permit characterization of isolates into two co-occurring subpopulations. However, reliable strain-level performance required rigorous and systematic standardization of culture conditions and sample preparation. Our work suggests that MALDI-TOF MS offers promise as a rapid, reproducible, and high-resolution approach to characterize environmental isolates of members of the genus Deinococcus. Future work will focus upon application of methods described here to additional members of this ecologically diverse and ubiquitous genus.

Gene Expression Profiling and Assessment of Vitamin D and Serotonin Pathway Variations in Patients With Irritable Bowel Syndrome.

BACKGROUND/AIMS: Irritable bowel syndrome (IBS) is a multifaceted disorder that afflicts millions of individuals worldwide. IBS is currently diagnosed based on the presence/duration of symptoms and systematic exclusion of other conditions. A more direct manner to identify IBS is needed to reduce healthcare costs and the time required for accurate diagnosis. The overarching objective of this work is to identify gene expression-based biological signatures and biomarkers of IBS. METHODS: Gene transcripts from 24 tissue biopsy samples were hybridized to microarrays for gene expression profiling. A combination of multiple statistical analyses was utilized to narrow the raw microarray data to the top 200 differentially expressed genes between IBS versus control subjects. In addition, quantitative polymerase chain reaction was employed for validation of the DNA microarray data. Gene ontology/pathway enrichment analysis was performed to investigate gene expression patterns in biochemical pathways. Finally, since vitamin D has been shown to modulate serotonin production in some models, the relationship between serum vitamin D and IBS was investigated via 25-hydroxyvitamin D (25[OH]D) chemiluminescence immunoassay. RESULTS: A total of 858 genetic features were identified with differential expression levels between IBS and asymptomatic populations. Gene ontology enrichment analysis revealed the serotonergic pathway as most prevalent among the differentially expressed genes. Further analysis via real-time polymerase chain reaction suggested that IBS patient-derived RNA exhibited lower levels of tryptophan hydroxylase-1 expression, the enzyme that catalyzes the rate-limiting step in serotonin biosynthesis. Finally, mean values for 25(OH)D were lower in IBS patients relative to non-IBS controls. CONCLUSIONS: Values for serum 25(OH)D concentrations exhibited a trend towards lower vitamin D levels within the IBS cohort. In addition, the expression of select IBS genetic biomarkers, including tryptophan hydroxylase 1, was modulated by vitamin D. Strikingly, the direction of gene regulation elicited by vitamin D in colonic cells is "opposite" to the gene expression profile observed in IBS patients, suggesting that vitamin D may help "reverse" the pathological direction of biomarker gene expression in IBS. Thus, our results intimate that IBS pathogenesis and pathophysiology may involve dysregulated serotonin production and/or vitamin D insufficiency.

Discrimination and characterization of environmental strains of Escherichia coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

A rapid and reliable bacterial source tracking (BST) method is essential to counter risks to human health posed by fecal contamination of surface waters. Genetic fingerprinting methods, such as repetitive sequence based-PCR (rep-PCR), have shown promise as BST tools but are time-consuming and labor-intensive. In this work, we investigate the ability of MALDI-TOF-MS to characterize and discriminate between closely related environmental strains of Escherichia coli and to classify them according to their respective sources. We compared the performance of a rapid MALDI-TOF-MS-based method to a commonly used rep-PCR-based method that employs the BOX-A1R primer. Among the criteria evaluated were repeatability and the ability of each method to group E. coli isolates according to their respective sources. Our data suggest that the MALDI-TOF-MS-based approach has a lower repeatability level compared to rep-PCR but offers an improved ability to correctly assign E. coli isolates to specific source groups. In addition, we have identified five biomarkers that appear conserved among avian species. We conclude that MALDI-TOF-MS may represent a promising, novel and rapid approach to addressing the problem of fecal contamination of surface waters and warrants further investigation.

Lead pollution in a large, prairie-pothole lake (Rush Lake, WI, USA): effects on abundance and community structure of indigenous sediment bacteria.

Rush Lake (WI, USA), the largest prairie-pothole lake east of the Mississippi River, has been contaminated with lead pollution as a result of over 140 years of waterfowl hunting. We examined: (1) the extent of lead pollution in Rush Lake sediments and (2) whether lead pollution in Rush Lake is affecting the abundance and community structure of indigenous sediment bacteria. Sediment lead concentrations did not exceed 59 mg Pb kg(-1) dry sediment. No relationship was observed between sediment lead concentration and the abundance of aerobic (P=0.498) or anaerobic (P=0.416) heterotrophic bacteria. Similarly, lead did not appear to affect bacterial community structure when considering both culturable and nonculturable community members. In contrast, the culturable fraction of sediment bacteria in samples containing 59 mg Pb kg(-1) exhibited a unique community structure. While factors other than lead content likely play roles in determining bacterial community structure in the sediments of Rush Lake, these data suggest that the culturable fraction of sediment bacterial communities is affected by elevated lead levels.

A Designed Experiments Approach to Optimizing MALDI-TOF MS Spectrum Processing Parameters Enhances Detection of Antibiotic Resistance in Campylobacter jejuni.

MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the collection consisting of 31 strains resistant to beta-lactams and 141 strains sensitive to beta-lactams. Applying optimal preprocessing parameters, beta-lactam resistance detection was increased by 34%. These results suggest that spectrum processing parameters, which are rarely optimized or adjusted, affect the performance of MALDI-TOF MS-based detection of antibiotic resistance and can be fine-tuned to enhance screening performance.

Medium composition affects the degree and pattern of cadmium inhibition of naphthalene biodegradation.

Metals have been reported to inhibit organic pollutant biodegradation; however, widely varying degrees and patterns of inhibition have been reported. To investigate the roles of medium composition and metal bioavailability on these different degrees and patterns of inhibition, we assessed the impact of cadmium on naphthalene biodegradation by a newly isolated strain of Comamonas testosteroni in three chemically-defined minimal salts media (MSM): Tris-buffered MSM, PIPES-buffered MSM, and Bushnell-Haas medium. Cadmium (total concentrations of 100 and 500 microM) inhibited biodegradation in each medium. Degrees of inhibition were different in each medium. Cadmium was most inhibitory in PIPES-buffered MSM and least inhibitory in Bushnell-Haas. For example, in Bushnell-Haas medium, 100 microM cadmium reduced the cell yield more than 4-fold compared to controls not containing cadmium. The same concentration of cadmium completely inhibited growth in PIPES-buffered MSM. No difference in inhibition was observed in any medium when cadmium was added 24 h before inoculation rather than when added within one minute of inoculation. Two patterns of inhibition were observed. Inhibition occurred in a dose dependent pattern in Tris- and PIPES-buffered MSM and in a non-dose dependent pattern in Bushnell-Haas. Specifically, in Bushnell-Haas, 100 microM total cadmium extended the lag phase by 23+/-8.66 h, whereas 500 microM did not extend the lag phase. Soluble, ionic cadmium (Cd2+) concentrations were measured and modeled in each medium to assess cadmium bioavailability. In media containing 500 microM total cadmium, bioavailability was highest in Tris- and PIPES-buffered MSM and lowest in Bushnell-Haas. In Bushnell-Haas, cadmium bioavailability was initially higher in the 500 microM treatments (196+/-21.2 microM) than in the 100 microM treatments (78.2+/-2.04 microM); however, after 12 h, bioavailability was higher in the 100 microM treatments (56.4+/-24.8 micro) than the 500 microM treatments (13.3+/-1.2 microM). These data suggest that the type of medium determines the degrees and patterns by which metals inhibit biodegradation and emphasize the importance of coupling metal toxicity and bioavailability data.

Biomarker- and similarity coefficient-based approaches to bacterial mixture characterization using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

MALDI-TOF MS profiling has been shown to be a rapid and reliable method to characterize pure cultures of bacteria. Currently, there is keen interest in using this technique to identify bacteria in mixtures. Promising results have been reported with two- or three-isolate model systems using biomarker-based approaches. In this work, we applied MALDI-TOF MS-based methods to a more complex model mixture containing six bacteria. We employed: 1) a biomarker-based approach that has previously been shown to be useful in identification of individual bacteria in pure cultures and simple mixtures and 2) a similarity coefficient-based approach that is routinely and nearly exclusively applied to identification of individual bacteria in pure cultures. Both strategies were developed and evaluated using blind-coded mixtures. With regard to the biomarker-based approach, results showed that most peaks in mixture spectra could be assigned to those found in spectra of each component bacterium; however, peaks shared by two isolates as well as peaks that could not be assigned to any individual component isolate were observed. For two-isolate blind-coded samples, bacteria were correctly identified using both similarity coefficient- and biomarker-based strategies, while for blind-coded samples containing more than two isolates, bacteria were more effectively identified using a biomarker-based strategy.

Use of MALDI-TOF mass spectrometry and a custom database to characterize bacteria indigenous to a unique cave environment (Kartchner Caverns, AZ, USA).

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Impact of metals on the biodegradation of organic pollutants.

Forty percent of hazardous waste sites in the United States are co-contaminated with organic and metal pollutants. Data from both aerobic and anaerobic systems demonstrate that biodegradation of the organic component can be reduced by metal toxicity. Metal bioavailability, determined primarily by medium composition/soil type and pH, governs the extent to which metals affect biodegradation. Failure to consider bioavailability rather than total metal likely accounts for much of the enormous variability among reports of inhibitory concentrations of metals. Metals appear to affect organic biodegradation through impacting both the physiology and ecology of organic degrading microorganisms. Recent approaches to increasing organic biodegradation in the presence of metals involve reduction of metal bioavailability and include the use of metal-resistant bacteria, treatment additives, and clay minerals. The addition of divalent cations and adjustment of pH are additional strategies currently under investigation.

A designed experiments approach to optimization of automated data acquisition during characterization of bacteria with MALDI-TOF mass spectrometry.

MALDI-TOF MS has been shown capable of rapidly and accurately characterizing bacteria. Highly reproducible spectra are required to ensure reliable characterization. Prior work has shown that spectra acquired manually can have higher reproducibility than those acquired automatically. For this reason, the objective of this study was to optimize automated data acquisition to yield spectra with reproducibility comparable to those acquired manually. Fractional factorial design was used to design experiments for robust optimization of settings, in which values of five parameters (peak selection mass range, signal to noise ratio (S:N), base peak intensity, minimum resolution and number of shots summed) commonly used to facilitate automated data acquisition were varied. Pseudomonas aeruginosa was used as a model bacterium in the designed experiments, and spectra were acquired using an intact cell sample preparation method. Optimum automated data acquisition settings (i.e., those settings yielding the highest reproducibility of replicate mass spectra) were obtained based on statistical analysis of spectra of P. aeruginosa. Finally, spectrum quality and reproducibility obtained from non-optimized and optimized automated data acquisition settings were compared for P. aeruginosa, as well as for two other bacteria, Klebsiella pneumoniae and Serratia marcescens. Results indicated that reproducibility increased from 90% to 97% (p-value[Formula: see text]0.002) for P. aeruginosa when more shots were summed and, interestingly, decreased from 95% to 92% (p-value [Formula: see text] 0.013) with increased threshold minimum resolution. With regard to spectrum quality, highly reproducible spectra were more likely to have high spectrum quality as measured by several quality metrics, except for base peak resolution. Interaction plots suggest that, in cases of low threshold minimum resolution, high reproducibility can be achieved with fewer shots. Optimization yielded more reproducible spectra than non-optimized settings for all three bacteria.

Multi-scale temporal and spatial variation in genotypic composition of Cladophora-borne Escherichia coli populations in Lake Michigan.

High concentrations of Escherichia coli in mats of Cladophora in the Great Lakes have raised concern over the continued use of this bacterium as an indicator of microbial water quality. Determining the impacts of these environmentally abundant E. coli, however, necessitates a better understanding of their ecology. In this study, the population structure of 4285 Cladophora-borne E. coli isolates, obtained over multiple three day periods from Lake Michigan Cladophora mats in 2007-2009, was examined by using DNA fingerprint analyses. In contrast to previous studies that have been done using isolates from attached Cladophora obtained over large time scales and distances, the extensive sampling done here on free-floating mats over successive days at multiple sites provided a large dataset that allowed for a detailed examination of changes in population structure over a wide range of spatial and temporal scales. While Cladophora-borne E. coli populations were highly diverse and consisted of many unique isolates, multiple clonal groups were also present and accounted for approximately 33% of all isolates examined. Patterns in population structure were also evident. At the broadest scales, E. coli populations showed some temporal clustering when examined by year, but did not show good spatial distinction among sites. E. coli population structure also showed significant patterns at much finer temporal scales. Populations were distinct on an individual mat basis at a given site, and on individual days within a single mat. Results of these studies indicate that Cladophora-borne E. coli populations consist of a mixture of stable, and possibly naturalized, strains that persist during the life of the mat, and more unique, transient strains that can change over rapid time scales. It is clear that further study of microbial processes at fine spatial and temporal scales is needed, and that caution must be taken when interpolating short term microbial dynamics from results obtained from weekly or monthly samples.

Carboxymethyl-beta-cyclodextrin mitigates toxicity of cadmium, cobalt, and copper during naphthalene biodegradation.

Hazardous waste sites are commonly contaminated with both organic and metal pollutants. Many metal pollutants have been shown to inhibit organic pollutant biodegradation. We investigated the ability of a modified, polydentate cyclodextrin (carboxymethyl-beta-cyclodextrin, CMCD) to reduce the toxicity of 33.4 microM cadmium, cobalt or copper during naphthalene degradation by a Burkholderia sp. in 120 h aerobic, batch studies. The highest investigated concentration of CMCD, 3340 microM, reduced cadmium, cobalt, and copper toxicity. With each metal, the length of the lag phase was reduced (by as much as 108 h with cobalt or copper), the cell yield was increased (by as much as a factor of 16 with cobalt), and the growth rate was increased (by as much as a factor of 31 with cobalt). The degrader was unable to use CMCD as the sole source of carbon and energy. Our data suggest that the ability of CMCD to complex metals plays an important role in its ability to mitigate metal toxicity and that CMCD has the potential to enhance biodegradation in organic and metal co-contaminated environments.

Environmental pH affects transcriptional responses to cadmium toxicity in Escherichia coli K-12 (MG1655).

It has been widely reported that pH mediates cadmium toxicity to bacteria. We used a tripartite approach to investigate mechanisms by which pH affects cadmium toxicity that included analyses of: (1) growth kinetics, (2) global gene expression, and (3) cadmium speciation. Cadmium extended the lag phase at pH 7, but not at pH 5. DNA microarray analysis revealed that stress response genes including hdeA, otsA, and yjbJ were more highly expressed at pH 5 than at pH 7 after only 5 min of exposure to cadmium, suggesting that acidic pH more rapidly induced genes that confer cadmium resistance. In addition, genes involved in transport and many hypothetical genes were more highly expressed at pH 5 than at pH 7 in the presence of cadmium. Concentrations of two cadmium species, including one previously implicated in the mechanism by which pH mediates cadmium toxicity (CdOH+), increased with pH. Our data demonstrate that transcriptional responses of Escherichia coli to cadmium are substantially affected by pH and suggest that several stress response, transport, and hypothetical genes play roles in the mechanism by which pH mediates cadmium toxicity.