The Effects and Genetic Mechanisms of Bacterial Species Interactions on Biofilm Formation.

Bacteria can increase their survival in stressed environments by forming sessile biofilms on surfaces. Natural ecosystems are usually occupied by multiple species, which may interact with and therefore affect biofilm formation of an incoming species. This dissertation research explores the effects of species interactions and investigates genetic mechanisms of species interactions between an environmental strain Stenotrophomonas maltophilia and a water quality indicator species Escherichia coli on biofilm formation of E. coli. It was found that E. coli biofilm development was promoted in dynamic flow systems, but inhibited in static batch plates in mixed species culture compared with pure culture conditions. The opposite effects of co-culture on E. coli biofilm formation suggested that species interactions may have different impacts under different culture conditions. To enable the mechanistic study of species interactions, a separation method was developed to allow transcriptome analysis of mixed species communities. Transcriptomic responses of E. coli to S. maltophilia were analyzed to investigate genetic mechanisms of inhibited E. coli biofilm formation in static co-culture. Eighty-nine and 108 genes exhibited genetic responses of E. coli to S. maltophilia co-cultured in biofilm and suspensions, respectively. Several genes were involved with inhibited biofilm formation of E. coli in static co-culture. One highly up-regulated gene, fliA, was selected for a mechanistic study. It was found that the production of a major monomer of curli, CsgA, as well as cell aggregation were greatly repressed in E. coli with fliA overexpression. Knocking out fliA partially restored the inhibitive effect of co-culture on E. coli biofilm growth. Therefore, it was concluded that inhibited E. coli biofilm formation by interactions with S. maltophilia partially was caused by the induction of gene fliA to suppress curli production. Overall, this dissertation examined the effects of species interactions on biofilm formation of E. coli, highlighted the impact of environmental conditions on the effect, and revealed partial understanding of species interactions at a genetic level. This fundamental study contributes to understanding of biofilm formation in real environments with mixed species, and serves as a starting point towards the development of bacteriotherapy for pathogen control using indigenous species for environmental health.Ph.D.Environmental Health SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/78983/1/joandai_1.pd

Separation of the bacterial species, Escherichia coli, from mixed-species microbial communities for transcriptome analysis

Abstract Background The study of bacterial species interactions in a mixed-species community can be facilitated by transcriptome analysis of one species in the community using cDNA microarray technology. However, current applications of microarrays are mostly limited to single species studies. The purpose of this study is to develop a method to separate one species, Escherichia coli as an example, from mixed-species communities for transcriptome analysis. Results E. coli cells were separated from a dual-species (E. coli and Stenotrophomonas maltophilia) community using immuno-magnetic separation (IMS). High recovery rates of E. coli were achieved. The purity of E. coli cells was as high as 95.0% separated from suspended mixtures consisting of 1.1 - 71.3% E. coli, and as high as 96.0% separated from biofilms with 8.1% E. coli cells. Biofilms were pre-dispersed into single-cell suspensions. The reagent RNAlater (Ambion, Austin, TX) was used during biofilm dispersion and IMS to preserve the transcriptome of E. coli. A microarray study and quantitative PCR confirmed that very few E. coli genes (only about eight out of 4,289 ORFs) exhibited a significant change in expression during dispersion and separation, indicating that transcriptional profiles of E. coli were well preserved. Conclusions A method based on immuno-magnetic separation (IMS) and application of RNAlater was developed to separate a bacterial species, E. coli as an example, from mixed-species communities while preserving its transcriptome. The method combined with cDNA microarray analysis should be very useful to study species interactions in mixed-species communities.http://deepblue.lib.umich.edu/bitstream/2027.42/112838/1/12866_2011_Article_1346.pd

Bayesian Network Expansion Identifies New ROS and Biofilm Regulators

Signaling and regulatory pathways that guide gene expression have only been partially defined for most organisms. However, given the increasing number of microarray measurements, it may be possible to reconstruct such pathways and uncover missing connections directly from experimental data. Using a compendium of microarray gene expression data obtained from Escherichia coli, we constructed a series of Bayesian network models for the reactive oxygen species (ROS) pathway as defined by EcoCyc. A consensus Bayesian network model was generated using those networks sharing the top recovered score. This microarray-based network only partially agreed with the known ROS pathway curated from the literature and databases. A top network was then expanded to predict genes that could enhance the Bayesian network model using an algorithm we termed ‘BN+1’. This expansion procedure predicted many stress-related genes (e.g., dusB and uspE), and their possible interactions with other ROS pathway genes. A term enrichment method discovered that biofilm-associated microarray data usually contained high expression levels of both uspE and gadX. The predicted involvement of gene uspE in the ROS pathway and interactions between uspE and gadX were confirmed experimentally using E. coli reporter strains. Genes gadX and uspE showed a feedback relationship in regulating each other's expression. Both genes were verified to regulate biofilm formation through gene knockout experiments. These data suggest that the BN+1 expansion method can faithfully uncover hidden or unknown genes for a selected pathway with significant biological roles. The presently reported BN+1 expansion method is a generalized approach applicable to the characterization and expansion of other biological pathways and living systems

Shotgun Metagenomics Reveals Taxonomic and Functional Shifts in Hot Water Microbiome Due to Temperature Setting and Stagnation

Hot water premise plumbing has emerged as a critical nexus of energy, water, and public health. The composition of hot water microbiomes is of special interest given daily human exposure to resident flora, especially opportunistic pathogens (OPs), which rely on complex microbial ecological interactions for their proliferation. Here, we applied shotgun metagenomic sequencing to characterize taxonomic and functional shifts in microbiomes as a function of water heater temperature setting, stagnation in distal pipes, and associated shifts in water chemistry. A cross-section of samples from controlled, replicated, pilot-scale hot water plumbing rigs representing different temperature settings (39, 42, and 51°C), stagnation periods (8 h vs. 7 days), and time-points, were analyzed. Temperature setting exhibited an overarching impact on taxonomic and functional gene composition. Further, distinct taxa were selectively enriched by specific temperature settings (e.g., Legionella at 39°C vs. Deinococcus at 51°C), while relative abundances of genes encoding corresponding cellular functions were highly consistent with expectations based on the taxa driving these shifts. Stagnation in distal taps diminished taxonomic and functional differences induced by heating the cold influent water to hot water in recirculating line. In distal taps relative to recirculating hot water, reads annotated as being involved in metabolism and growth decreased, while annotations corresponding to stress response (e.g., virulence disease and defense, and specifically antibiotic resistance) increased. Reads corresponding to OPs were readily identified by metagenomic analysis, with L. pneumophila reads in particular correlating remarkably well with gene copy numbers measured by quantitative polymerase chain reaction. Positive correlations between L. pneumophila reads and those of known protozoan hosts were also identified. Elevated proportions of genes encoding metal resistance and hydrogen metabolism were noted, which was consistent with elevated corrosion-induced metal concentrations and hydrogen generation. This study provided new insights into real-world factors influencing taxonomic and functional compositions of hot water microbiomes. Here metagenomics is demonstrated as an effective tool for screening for potential presence, and even quantities, of pathogens, while also providing diagnostic capabilities for assessing functional responses of microbiomes to various operational conditions. These findings can aid in informing future monitoring and intentional control of hot water microbiomes

Multiphasic analysis of the temporal development of the distal gut microbiota in patients following ileal pouch anal anastomosis

Abstract Background The indigenous gut microbiota are thought to play a crucial role in the development and maintenance of the abnormal inflammatory responses that are the hallmark of inflammatory bowel disease. Direct tests of the role of the gut microbiome in these disorders are typically limited by the fact that sampling of the microbiota generally occurs once disease has become manifest. This limitation could potentially be circumvented by studying patients who undergo total proctocolectomy with ileal pouch anal anastomosis (IPAA) for the definitive treatment of ulcerative colitis. A subset of patients who undergo IPAA develops an inflammatory condition known as pouchitis, which is thought to mirror the pathogenesis of ulcerative colitis. Following the development of the microbiome of the pouch would allow characterization of the microbial community that predates the development of overt disease. Results We monitored the development of the pouch microbiota in four patients who underwent IPAA. Mucosal and luminal samples were obtained prior to takedown of the diverting ileostomy and compared to samples obtained 2, 4 and 8 weeks after intestinal continuity had been restored. Through the combined analysis of 16S rRNA-encoding gene amplicons, targeted 16S amplification and microbial cultivation, we observed major changes in structure and function of the pouch microbiota following ileostomy. There is a relative increase in anaerobic microorganisms with the capacity for fermentation of complex carbohydrates, which corresponds to the physical stasis of intestinal contents in the ileal pouch. Compared to the microbiome structure encountered in the colonic mucosa of healthy individuals, the pouch microbial community in three of the four individuals was quite distinct. In the fourth patient, a community that was much like that seen in a healthy colon was established, and this patient also had the most benign clinical course of the four patients, without the development of pouchitis 2 years after IPAA. Conclusions The microbiota that inhabit the ileal-anal pouch of patients who undergo IPAA for treatment of ulcerative colitis demonstrate significant structural and functional changes related to the restoration of fecal flow. Our preliminary results suggest once the pouch has assumed the physiologic role previously played by the intact colon, the precise structure and function of the pouch microbiome, relative to a normal colonic microbiota, will determine if there is establishment of a stable, healthy mucosal environment or the reinitiation of the pathogenic cascade that results in intestinal inflammation.http://deepblue.lib.umich.edu/bitstream/2027.42/112442/1/40168_2012_Article_10.pd

Interactive effects of temperature, organic carbon, and pipe material on microbiota composition and Legionella pneumophila in hot water plumbing systems

Abstract Background Several biotic and abiotic factors have been reported to influence the proliferation of microbes, including Legionella pneumophila, in hot water premise plumbing systems, but their combined effects have not been systematically evaluated. Here, we utilize simulated household water heaters to examine the effects of stepwise increases in temperature (32–53 °C), pipe material (copper vs. cross-linked polyethylene (PEX)), and influent assimilable organic carbon (0–700 μg/L) on opportunistic pathogen gene copy numbers and the microbiota composition, as determined by quantitative polymerase chain reaction and 16S rRNA gene amplicon sequencing. Results Temperature had an overarching influence on both the microbiota composition and L. pneumophila numbers. L. pneumophila peaked at 41 °C in the presence of PEX (1.58 × 105 gene copies/mL). At 53 °C, L. pneumophila was not detected. Several operational taxonomic units (OTUs) persisted across all conditions, accounting for 50% of the microbiota composition from 32 to 49 °C and 20% at 53 °C. Pipe material most strongly influenced microbiota composition at lower temperatures, driven by five to six OTUs enriched with each material. Copper pipes supported less L. pneumophila than PEX pipes (mean 2.5 log10 lower) at temperatures ≤ 41 °C, but showed no difference in total bacterial numbers. Differences between pipe materials diminished with elevated temperature, probably resulting from decreased release of copper ions. At temperatures ≤ 45 °C, influent assimilable organic carbon correlated well with total bacterial numbers, but not with L. pneumophila numbers. At 53 °C, PEX pipes leached organic carbon, reducing the importance of dosed organic carbon. L. pneumophila numbers correlated with a Legionella OTU and a Methylophilus OTU identified by amplicon sequencing. Conclusions Temperature was the most effective factor for the control of L. pneumophila, while microbiota composition shifted with each stepwise temperature increase. While copper pipe may also help shape the microbiota composition and limit L. pneumophila proliferation, its benefits might be constrained at higher temperatures. Influent assimilable organic carbon affected total bacterial numbers, but had minimal influence on opportunistic pathogen gene numbers or microbiota composition. These findings provide guidance among multiple control measures for the growth of opportunistic pathogens in hot water plumbing and insight into the mediating role of microbial ecological factors

miR-196a-2 Promotes Malignant Progression of Thyroid Carcinoma by Targeting NRXN1

Thyroid cancer (TC) is the most common endocrine malignant disease with a rising morbidity year by year. Accumulating studies have shown that microRNAs (miRNAs) play a regulatory role in the progression of various tumors, but the molecular regulatory mechanism of miR-196a-2 in TC is still unknown. qRT-PCR was employed to measure the expression of miR-196a-2 and NRXN1 mRNA in TC cells, while western blot was used to detect the protein expression of NRXN1. CCK-8, colony formation and flow cytometry assays were used to measure cell proliferation and apoptosis of TC cells. Dual-luciferase reporter gene assay was used to predict and verify the targeted binding relationship between miR-196a-2 and NRXN1. Our study results manifested that miR-196a-2 was dramatically overexpressed in cells of TC, while NRXN1 was lowly expressed. miR-196a-2 could promote cell proliferation and inhibit cell apoptosis of TC. Additionally, miR-196a-2 could also target and inhibit the expression of NRXN1. Silencing NRXN1 could reverse the inhibitory effect of miR-196a-2 downregulation on cell proliferation of TC, as well as the promoting effect on cell apoptosis. In a conclusion, we found that miR-196a-2 could promote cell proliferation and inhibit cell apoptosis of TC by targeting NRXN1. Therefore, miR-196a-2/NRXN1 is potential to be a molecular therapeutic target for TC

miR-301b-3p Regulates Breast Cancer Cell Proliferation, Migration, and Invasion by Targeting NR3C2

Objectives. Breast cancer is the most common malignant tumor among females, and miRNAs have been reported to play an important regulatory role in breast cancer progression. This study aimed to explore the function and underlying molecular mechanism of miR-301b-3p in breast cancer. Methods. Differential analysis and survival analysis were performed based on the data accessed from the TCGA-BRCA dataset for identification of the target miRNA. Bioinformatics analysis was conducted to predict the downstream target gene of the miRNA. Real-time quantitative PCR was carried out to detect the expression of miR-301b-3p and nuclear receptor subfamily 3 group C member 2 (NR3C2). Western blot was used to assess the protein expression of NR3C2. Cell counting kit-8 assay was performed to evaluate the proliferation of breast cancer cells. Transwell assay was conducted to determine the migratory and invasive abilities of breast cancer cells. Dual-luciferase reporter assay was employed to verify the targeting relationship between miR-301b-3p and NR3C2. Results. miR-301b-3p was elevated in breast cancer cell lines and promoted cell proliferation, migration, and invasion in terms of its biological function in breast cancer. NR3C2 was validated as a direct target of miR-301b-3p via bioinformatics analysis and dual-luciferase reporter assay, and NR3C2 was downregulated in breast cancer cell lines. The rescue experiment indicated that NR3C2 was involved in the mechanism by which miR-301b-3p regulated the malignant phenotype of breast cancer cells. Conclusion. The present study revealed for the first time that miR-301b-3p could foster breast cancer cell proliferation, migration, and invasion by targeting NR3C2, unveiling that miR-301b-3p is a novel carcinogen in breast cancer