Application of Deoxyribozymes on Studies of 23S rRNA

Harjot Mann; El-moussa, Ahmad; Jiang, Jun; Chow, Christine S.
Wayne State University, Detroit, MI 48202

Helix 69 (H69) serves important roles in almost all stages of translation. Protruding from the 23S ribosomal RNA (rRNA), H69 forms the intersubunit bridge B2a with helix 44 (h44) of the 16S rRNA and interacts with tRNAs and other translation factors. The sequence and secondary structure of H69 are highly conserved. The post-transcriptional modifications of H69 also show conservation, and the pseudouridylations in H69 confer a growth advantage of the wild-type over the mutants without these modifications. Therefore, H69 is a potential target for antibacterial drugs and obtaining H69 with natural modifications from 23S rRNA will be beneficial for model studies. A deoxyribozyme was designed and employed in current study to cleave the 5' terminus of H69 within the context of 23S rRNA. It is revealed in a polyacrylamide gel electrophoresis (PAGE) that the deoxyribozyme binds to transcribed H69 oligonucleotides. Results from an agarose gel electrophoresis confirm that a reaction occurs on the 23S rRNA when both the deoxyribozymes and a reaction buffer are added. The 5' terminus of H69 is possibly cleaved by the deoxyribozyme, as suggested by a reverse transcription on the 23S rRNA treated with deoxyribozymes followed by a PAGE. Future directions include optimizing the 5' terminus cleavage reaction, designing a deoxyribozyme to cleave the 3' H69 terminus, and developing deoxyribozymes for the ligation of probes to H69.

top

Folate-Functionalized Antibiotic Encapsulated Nanoparticles Improve Chlamydial Drug Delivery

Christopher M McPharlin1, Lin, Tayson1, Kirtane, Ameya2, Hali, Mirabela1, Gerard Herve C1, Hudson, Alan P1, Panyam, Jayanth2, Whittum-Hudson, Judith A2, 1Wayne State University, Detroit, MI 48201 and 2University of Minnesota, Minneapolis, MN 55455

Chlamydia trachomatis is a sexually transmitted obligate intracellular bacterial pathogen with a biphasic developmental cycle. A Chlamydial infection may deviate from its normal developmental cycle and enter a reversible state called persistence in which the bacteria are refractory to antibiotics. Persistence is associated with serious sequelae including reactive arthritis in both men and women and pelvic inflammatory disease in women. To have a therapeutic effect, antibiotics must traverse three cell membrane, the Chlamydial inclusion membrane, and the bacterial membrane, leading to modest drug concentrations. Previous studies in our labs have shown that Chlamydia-infected cells up-regulate folic acid receptors; this offers a well-established targeting strategy. Azithromycin (AZ) and Rifampicin (Rif) have been suggested for combination therapy for treating persistent Chlamydiae in reactive arthritis patients. We used folate-functionalized, biodegradable poly(d-l-lactide-co-glycolide) (PLGA) nanoparticles (FA-NP-Combo) for targeted combination antibiotic delivery to acutely and persistently infected cells in vitro. Non-targeted antibiotic-loaded nanoparticles (NP-Combo) and free AZ and RIF in combination (Free-Combo) were the controls. The methods used included fluorescent antibody staining of Chlamydial inclusions and RT-qPCR of Chlamydial primary transcripts of the 16S rRNA gene.  FA-NP-Combo significantly reduced acute and persistent Chlamydial inclusion size and numbers by two to three fold when comparing serial dilutions of FA-NP-Combo and Free-combo at 8, 16, and 32 ng/mL (AZ) with 4.4, 8.8, and 17.2 ng/mL (Rif). Transcripts of the Chlamydial 16S rRNA gene decreased greater than tenfold at 8 and 16 ng/mL in acute infected cells, and two to three fold in persistently infected cells. These results demonstrate an increased drug efficacy and a decrease in Chlamydial infectious load when combination antibiotics were delivered in NP. Folate-targeted nanoparticles have a potential for improving current Chlamydial treatments and establishing new treatments for Chlamydia-associated chronic sequelae.

top

Microbial 16S rRNA Gene Clone Libraries from Hydrocarbon Contaminated Sediments  with Elevated Magnetic Susceptibility Measurements

Anja Williams1, Carol Beaver1, Silvia Rossbach1, Estella Atewana2, Gamal Abdel Aal2, Farag Mewafy2, and Lee Slater3

1Biological Sciences, Western Michigan University, Kalamazoo, USA
2 Boone Pickens School of Geology, Oklahoma State University, USA
3Earth and Environmental Sciences, Rutgers University-Newark, USA

Contaminated soil from oil spills is a common and widespread environmental problem. It has been shown that microbial degradation of hydrocarbons can effectively reduce contamination in subsurface soil. However, techniques that can be used to observe the presence and progression of biodegradation remain to be evaluated. Magnetic susceptibility (MS) is one geophysical technique that was applied to a subsurface petroleum-contaminated site in Bemidji, Minnesota. Spikes in MS measurements were found in zones around the water table. Some microorganisms involved in bioremediation couple hydrocarbon degradation to iron reduction, which may lead to the production of magnetite. We sought to analyze the microbial communities present at different depths of the petroleum-contaminated site through the construction of clone libraries. Several 16S rRNA clone libraries of Bacteria and Archaea from different depths of a contaminated soil core were constructed. Representative DNA sequences for each clone were analyzed using BLAST and the Ribosomal and Silva sequence databases, and clones were grouped by phyla. In the vadose zone, bacteria similar to the iron-reducing Albidiferax and Thermincola formed the majority populations of the bacterial community. Below the water table, bacteria similar to the synthrophic Smithella and the methanogenic archaeon Methanoregula dominated the microbial community. Desulfosporosinus, a possible iron reducer, was also prominent beneath the water table. The geophysical technique of measuring magnetic susceptibilities may be a useful tool for indicating microbial biodegradation activities.

top

Characterization of Winter Bacterial Communities in the Fluid of the Purple Pitcher Plant (Sarracenia purpurea)

Reid J. Tornquist and Margaret M. Hanes, Eastern Michigan University, Ypsilanti, MI 48197

Carnivorous pitcher plants trap insects in cone-shaped leaves and digest them to gain vital nutrients. For digestion to occur, plants in the genus Sarracenia require mutualistic microorganisms living in their leaves. Few studies have investigated the microbial communities in this genus and even fewer have examined how these communities change over time. This study specifically examines the bacterial composition in the most widely distributed species, Sarracenia purpurea, in the winter. The leaves of this plant species live for several years and it is unknown whether microbes overwinter in pitcher fluid or if community structure must be reestablished each spring. This study aims to characterize the phyllosphere in one population of Sarracenia collected between the months of November and March over the last two years. DNA from the fluid of 53 pitchers was extracted and amplified using ARISA-PCR. ARISA peaks were visualized and identified using ADAPT, then blasted against NCIB and SEED databases.   Results indicate a large and diverse winter community of microbes. Bacteria from 26 phyla were present throughout all winter samples. The number of unique genera identified per sample ranged from 27-60 and the number of unique species per sample ranged from 59-186. To further explore the role of microbes throughout the winter, samples collected in the winter of 2013-2014 will be loaded onto 96-well Biolog Eco plates containing various carbon substrates. Preliminary results collected in summer 2013 will be compared to winter samples collected later this year in order to discern patterns of potential functional diversity of the fluid’s microbial inhabitants throughout the year.

top  

Reciprocal Preferences for Mucus O-linked Glycan Foraging by
Symbiotic Human Gut Bacteria

Nicholas A. Pudlo1, Elizabeth A. Cameron1, J. Bruce German2, David A. Mills2
and Eric C. Martens1

1University of Michigan Medical School, Ann Arbor, MI 48109
2University of California, Davis, Davis, CA 95616

The human gut symbiont Bacteroides thetaiotaomicron (Bt) can metabolize over a dozen different complex carbohydrates (glycans), including the O-linked glycans that are attached to host mucins and contain energy-rich sugars. We conducted a variety of experiments to investigate how Bt responds when O-linked glycans are simultaneously presented with any one of the other usable polysaccharides. As evidenced by diauxic growth profiles, and corresponding growth phase-dependent regulation of genes involved in glycan metabolism, expression of genes involved in O-linked glycan degradation is repressed by the presence of all competing polysaccharides. Furthermore, Bt displays these same growth and expression trends when grown on human milk oligosaccharides (HMOs), which are structurally similar to O-linked glycans. In several cases tested, repression was mediated by monosaccharides, including D-galactose, which is also present in O-linked glycans. We identified one O-linked glycan-responsive gene cluster that is not subject to strong repression. Molecular genetic experiments in which the regulatory regions of this locus were exchanged with a system that exhibits strong repression suggest that glycan catabolite repression occurs independently of the promoters involved and may instead occur at the level of substrate sensing by outer membrane receptors. Finally, we identified another human gut symbiont, Bacteroides massiliensis, (Bm) which is much more specialized on mucin O-glycan foraging. Compared to Bt, this species exhibits reciprocal prioritization for O-linked glycans, revealing that the observed preferences are not merely intrinsic to the biochemistry of the glycans involved but may have been wired differently in each bacterium as it has evolved to occupy its own distinct niche.

top

Inositol Transport and Catabolism in Sinorhizobium meliloti

Ee Leng Choong, Mary Thwaites, Petra Kohler, Silvia Rossbach, Biological Sciences Western Michigan University, Kalamazoo, USA

Atmospheric nitrogen is in a form that is unavailable to plants. Nitrogen fixing bacteria can be found either free-living or in association with plants. Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that infects the plant and forms specialized organs known as root nodules.  In the nodule, the bacteria will fix the atmospheric nitrogen for use by the host plant, and in return, the plants supply the bacteria with carbon sources. Specialized carbon sources such as inositols may play a role in determining bacterial composition of the rhizosphere. The focus of this study is to characterize essential genes that involved in the inositol transport and catabolism in S. meliloti.
The S. meliloti ibpA gene was found to be the first gene in an operon that encodes other proteins of an ABC transporter responsible for the transport of inositol isomers. A mutant with an insertion in the ibpA gene was tested for the ability to use inositol as sole carbon source. The ibpA mutant was not able to grow for the first three days, but after five days, it reached an optical density similar to the wild type strain. It was concluded that the ibpA-iatA-iatP operon encodes a major inositol transporter, but that there may be also a second, minor inositol transporter present in S. meliloti. A candidate gene was identified based on homology, and mutants with insertions in this gene were constructed and analyzed for their phenotype.
The inositol (iol) genes are required for bacterial growth with inositol; S. meliloti mutant strains with Tn5 insertion in each individual iol gene were characterized with growth studies offering myo-inositol as the sole carbon source. The regulation of the pathway was investigated using NAD(H)-dependent myo-inositol dehydrogenase assays as well as β-glucuronidase assays. The iolA mutant strain showed that iolA is constitutively expressed, but the expression of the gene increased in the presence of inositol. Probably due to inducer accumulation, the myo-inositol dehydrogenase activity increased by two-fold. By providing the iolA gene in trans, the iolA gene expression and myo-inositol dehydrogenase activity remained the same level as the wild type. The finding that the iolA gene is constitutively expressed indicates that its gene product is not only part of the central inositol catabolic pathway; but also plays a role in other pathways, for example in the degradation of valine. Plant inoculation studies will show whether inositol transport and catabolism of S. meliloti is involved in establishing and maintaining the nitrogen fixing symbiosis between rhizobia and legume plants.

top

Laser-Based Rapid Identification of Bacterial Pathogens

Josh S.Sharp1 and Haavig, David L.2, 1Northern Michigan University, Marquette, MI 49855, 2Micro Identification Technologies, San Clemente, CA 92673 

With an aging population and rising healthcare costs, pathogen identification methods that are fast, accurate, and cost effective are needed.  Micro Identification Technologies (MIT) has developed a novel technology that analyzes the different laser light scattering patterns that specific species of bacteria exhibit to rapidly (and cheaply) identify bacteria and other pathogens. My laboratory at Northern Michigan University will be collaborating with MIT to expand their technology to identify and differentiate Staphylococcus aureus, and Methicillin Resistant Staphylococcus aureus (MRSA).  Our goals are to determine the laser light scattering patterns for known strains of S. aureus and MRSA, and then utilize that data to identify these pathogens in clinical samples.  S. aureus and MRSA represent a continuing healthcare threat that result in roughly 500,000 hospitalizations per year in the United States (and even more doctor’s office visits).  Moreover, MRSA in particular can be difficult to treat because of its resistance to many current antibiotic therapies.  Being able to quickly identify if a patient has a S. aureus infection, and whether or not that S. aureus is MRSA would be extremely useful in dictating the proper course of treatment for that patient, and ultimately increase the likelihood of a successful patient outcome.  Here we describe the basics of the laser light scattering technology, and how it can be utilized to identify specific bacteria species.

top

Gene regulation by a Novel Two-Component System in Pseudomonas entomophila

Kristin M Jacob1, Purdy, Alexandra2, Sharp, Josh S1, 1 Northern Michigan University, Marquette MI 49855, 2Amherst College, Aherst MA 01002

Two-component systems are common regulatory pathways seen in bacteria.  Gene expression in these systems are controlled through a series of auto-phosphorylation events between two proteins, the sensor kinase and response regulator.  Activation can be caused by biological or environmental stimuli, resulting in altered gene expression. A conserved two-component system was recently discovered in entomopathogenic bacteria, including our model organism Pseudomonas entomophila. It has been shown that if either part of this novel two-component system is deleted, the pathogen’s ability to kill Drosophila melanogaster is significantly reduced. The goal in this study is to determine what genes are being regulated by this novel two-component system and how they play a role in virulence. Two major virulence factors of P. entomophila, hemolysins and the AprA protease, were tested for regulation by this system.  It was found that these known virulence factors remained unaffected in P. entomophila strains that contain mutations in this novel two-component system. Future goals will be to determine which genes are specifically regulated by this system utilizing global transcriptome analysis. Information acquired from this study will be beneficial in increasing our understanding of gene regulation by two-component systems.

top

Antibiotic-Producing Streptomyces griseus and Biodiesel Glycerol Byproduct in Undergraduate Research

James F. Graves, Kyle L. Carrizales, and Ali A. Zeitoun, University of Detroit Mercy, Detroit, MI 48221

Actinomycete isolation agar (AIA) and glycerol yeast extract agar (GYEA) are used for recovery and growth of Actinomycetes and contain glycerol. This work examined growth and antimicrobial activity of Streptomyces griseus on media prepared with biodiesel glycerol byproduct made by transesterification or catalyst column. On AIA, S. griseus and Pseudomonas aeruginosa would grow, but not Staphylococcus epidermidis, Streptococcus faecalis, Escherichia coli, Klebsiella pneumoniae and Serratia marcescens. On minimal agar, containing only essential nutrients, with glucose, glycerol or glycerol byproducts as carbon sources, S. griseus, E. coli, K. pneumoniae, P. aeruginosa and S. marcescens exhibited growth, but not S. epidermidis and S. faecalis. All the bacteria grew on yeast extract agar (YEA), without or with addition of glucose, glycerol, or glycerol byproducts. Antibiograms made by the disk diffusion technique indicated that E. coli, K. pneumoniae, P. aeruginosa and S. marcescens were sensitive to streptomycin, but S. epidermidis and S. faecalis showed resistance. S. griseus inhibition of antibiotic sensitive E. coli growth was detected by a cross-streak inoculation test which included additional streak inoculations of E. coli on subsequent days. S. griseus did not produce enough antibiotic to inhibit sensitive bacteria inoculated on the same day. Inhibition of E. coli growth was observed on minimal agar containing glycerol, glycerol byproduct made by catalyst column, and other added carbon sources tested by 7, 9 and 14 days respectively. Inhibition of growth of E. coli occurred on YEA with all added carbon sources tested by three days incubation of S. griseus. Distance of growth inhibition from S. griseus increased with incubation period. Byproduct from catalyst column produced the most rapid increase in distance of inhibition. Factors associated with production of antimicrobial may be complex. These findings suggested that biodiesel glycerol byproduct can be used in media where a product is synthesized.

top

Microbial Communities Influence Magnetic Susceptibility in
Hydrocarbon-Contaminated Sediments

Carol L. Beaver1, Anja Williams1, Silvia Rossbach1, Estella Atewana2, Gamal Abdel Aal2, Farag Mewafy2, and Lee Slater3, 1Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan, USA , 2School of Geology, Oklahoma State University, USA, 3Earth and Environmental Sciences, Rutgers University-Newark, USA

Oil spills become a long-lasting problem when large quantities of hydrocarbons escape into the environment. Some of the contamination may be eradicated by physical means, but in many cases, the oil percolates through the vadose zone down to the water table, making it inaccessible to removal efforts. Once the spill becomes impossible to clean up, the only solution for its elimination is biodegradation.  In the past, hydrocarbon bioremediation was monitored by geochemical and microbiological testing.  However, geophysical methods such as magnetic susceptibility (MS) measurements may also be used to assess hydrocarbon bioremediation. For our experiment, one core contaminated with hydrocarbons and one uncontaminated core were acquired from a field site in Bemidji, Minnesota. First, MS was measured with a Bartington MS probe from each of the boreholes at the contaminated site. Then molecular microbiological methods such as denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene amplicon sequencing with the Illumina Miseq platform were used to assess the microbial diversity in the two cores. The results showed that MS was higher in the contaminated sediments, particularly in the smear zone above the water table and in the oil plume at the water table. Further, there were differences in community structure along the contaminated core, in contrast to the uncontaminated core, which showed little variance. The composition of microbial communities in the contaminated core changed from iron-reducing bacteria in the smear zone above the water table to syntrophic bacteria and methanogenic archaea in the oil plume, indicating that the smear zone was in an area of iron reduction and that the oil plume was undergoing methanogenesis. In conclusion, since high MS values were coincident with active hydrocarbon-degrading microbiological communities, this geophysical method may be a suitable tool to assess hydrocarbon bioremediation.

top

Investigating scyllo-Inositol Catabolism in the Soil Bacterium Sinorhizobium meliloti

Olivia Walser, Petra Kohler, Silvia Rossbach, Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008-5410

scyllo-Inositol catabolism is part of the larger inositol catabolism pathway in Sinorhizobum meliltoi, the nitrogen-fixing endosymbiont of alfafa. Previous work in our laboratory shows that iolY encodes a scyllo-inositol dehydrogenase, and also suggests the presence of a second scyllo-inositol dehydrogenase. We complemented the iolY mutant using triparental mating and ran a growth study in order to verify the phenotype of this mutant. Additionally, we screened a second potential candidate for involvement in the inositol catabolism pathway using transcriptional analysis. We were able to verify the phenotype of the iolY-1 mutant, but we are still in the process of identifying a second scyllo-inositol dehydrogenase.

top

Effect of decreasing enrichment time for ANSR assay for Salmonella detection

Madeline Lipp and Matthew Flood, Michigan State University, East Lansing, MI

EPA Method 1682 is the culture-based method currently used to detect Salmonella in water. This method is time consuming due to its multiple stages of incubation, and is non-specific for pathogenic species. Use of a Nicking Enzyme Amplification Reaction (NEAR)1 would provide a more efficient process for determining presence and concentration of Salmonella. The current protocol for ANSR™ (Neogen, Lansing, MI) requires a 24 hour incubation before amplification. It is possible that this incubation time could be shortened while maintaining equivalent recovery. To test this, reagent water samples were spiked with a known concentration of Salmonella  typhimurium. Spiked samples were assayed seven times over a 24 hour period using ANSR™. Results indicate that detection of Salmonella peaked after 8 hours of incubation and matched results from the 24 hour incubation from this point forward. Recovery remained constant at 82% for hours 8-24. This data indicates that incubation time for the ANSR™ assay could be decreased by 16 hours, improving the efficiency of the assay. Future assays should be performed as the same timed series from environmental samples in order to provide more evidence for this finding, and to investigate any matrix-specific efficiency problems.

top

Using Microscopy to Visualize Cyanobacteria of the Maxton Plains Alvar

Travis Bean and Gregory M. Colores, Biology Department, Central Michigan University, Mount Pleasant MI, 48859

Grassland alvars are a rare type of prairie composed of areas of little to no soil upon a mostly flat bed of limestone or dolomite. Nearly all North American alvars are found within the Great Lakes region. Many unique species have adapted to live in this harsh environment that is highly impacted by sporadic rainfall and large fluctuations in temperature. The development of soil upon the alvar is paramount in the transition from bare substrate to a system that can support vascular plant growth. Cyanobacteria are some of the earliest colonizers of bare substrate and are structurally and functionally important in soil development. Large filamentous cyanobacteria form matrices capable of trapping soil particles that lead to initial soil development; cyanobacteria are also important as primary producers that fix nitrogen, making it available for successional species. Alvar material was collected from different sites on Drummond Island, Michigan. Both lithic and soil cyanobacteria were cultivated from these samples using a liquid cyanobacterial growth medium, BG-11. Results of scanning electron and light microscopy show the diversity of both communities and the possible roles that cyanobacteria play in stabilizing the delicate soil of the alvar.

top