Cyclic di-GMP in light-dependent responses in cyanobacteria

Marco Agostoni, Christopher M. Waters and Beronda L. Montgomery, Michigan State University, East Lansing, MI, 48824.

Microorganisms use a variety of metabolites and signaling molecules to respond to external signals, among which are second messengers. These molecules often amplify first messenger signals and elicit biochemical or physiological changes in a cell. The turnover of second messengers is energetically affordable and enables rapid, adaptive phenotypic changes. Levels of the second messenger cyclic dimeric GMP (c-di-GMP) play critical roles in regulating cellular processes such as biofilm formation and motility. C-di-GMP signaling systems have been mainly characterized in pathogenic bacteria; however remain largely unexplored in cyanobacteria. In cyanobacteria, many putative c-di-GMP synthesis or degradation domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. Indeed, such domains are often the second most common output domain in photoreceptors – only outnumbered by a histidine kinase output domain. Cyanobacteria differ from other bacteria regarding the number and type of photoreceptor domains associated with c-di-GMP domains. Bioinformatics analyses of available genomes have established that the presence of c-di-GMP modulating domains reflects environmental characteristics and that these domains are extensively spread in the genomes of cyanobacteria. We are conducting functional explorations into the photoregulation and regulatory roles of c-di-GMP homeostasis in two cyanobacteria, Fremyella diplosiphon and Synechocystis sp. PCC 6803. These two organisms displayed different intracellular c-di-GMP concentrations under blue, green, and red light. We suggest that c-di-GMP could be associated with physiological adaptations attributed to different light quality such as biofilm formation and morphology changes. This research will examine the importance of environmental light fluctuations on the functions of c-di-GMP modulating-domain proteins linked to photoreceptor domains and the intracellular regulation of c-di-GMP levels in cyanobacteria.

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Connecting Function and Ecology: Defining Bacterial Cr(VI) Reduction Pathways In the Environment and Laboratory

Michael W Henson1, Ethan Wogolo1, Nathan Young1, Peter Kourtev2, Jorge Santo Domingo3 and Deric R Learman1. 1Institute for Great Lakes Research, 2Department of Biology, Central Michigan University, Mt. Pleasant, MI 48859, 3National Risk Management Research Laboratory, Environmental Protection Agency, Cincinnati, OH, 45268

Bacteria have been shown to be an active component in the geochemical cycling of chromium (Cr), but the mechanisms governing Cr (VI) reduction or how Cr (VI) affects bacterial community composition is unrefined. Within the environment, chromium mainly persists in two forms: Cr (III) and Cr (VI). Specifically, hexavalent chromium [Cr (VI)] has caused widespread contamination of soil and water in the United States and other industrial nations such as China. This is of particular concern because the toxicity of chromium to soil microorganisms can inhibit growth of natural communities.  Therefore, we sought to combine laboratory and molecular techniques to begin to elucidate key Cr (VI) reduction pathways.  Here we identified 34 environmental isolates using 16S rRNA gene sequences and physiologically characterized them for their growth and Cr (VI) reducing abilities. The 34 isolates were grouped into 12 operational taxonomic units (OTUs) represented by the genera Microbacterium, Cellulomonas, Cellulomicrobactium, and Bacillus. Although several strains were nearly identical at the 16S rRNA gene level, analysis of growth and chromium reduction revealed physiological differences between strains that belong to the same or closely related OTUs.  Furthermore, resistance to chromium was correlated with the strain’s ability to reduce chromium. Isolates that reduce chromium at a high rate (~2mM in 48 hours) were resistant concentration of ≥80 mM of Cr (VI), while isolates that had low to little reduction showed lower resistance (≤10 mM of Cr (VI)).  Future studies will focus on sequencing the genomes of these isolates to develop gene-specific assays and study the expression of chromium reducing genes in environmental samples.

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The Effect of Garlic on Streptococcus mutans Biofilm Formation on Orthodontic Wire in Mono- and Dual-Species Cultures

Lauren Bauer and Jennifer Hess, Department of Biology, Aquinas College, 1607 Robinson Road SE, Grand Rapids, Michigan 49506

It has long been known that garlic has an inhibitory effect on microbial growth; however, it is not quite understood how this antimicrobial agent affects the formation of biofilms in both mono- and dual-species cultures.  In this study, biofilms were grown in both sucrose and non-sucrose media containing varying concentrations of garlic and the relative abundance of growth was qualitatively assessed and recorded.  Expression of genes, SpaP, GtfB, and GbpB, known to be associated with bacterial attachment in sucrose and non-sucrose dependent pathways were observed using RT-PCR and the presence of banding was recorded.  Garlic effectively inhibited bacterial growth of Streptococcus mutans and Streptococcus salivarius in agar diffusion tests.  Biofilm experiments show that garlic may increase biofilm formation in single-species models, but it is unclear if the same is true for dual-species models.  Some gene expression in both models was observed but expression did not follow a noticeable trend.  Replication of this study is needed in order to determine the effect of garlic on microbial biofilm formation in dual-species models.  Effective characterization of the relationships amongst naturally occurring oral flora and the effect of garlic on their adhesion pathways may be important in developing new methods for treating common dental caries and thus reduce the occurrence of associated diseases.

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Prevalence and Characterization of Tetrathionate-Reducing Bacteria Isolated from Environmental Samples

Amber L. Carr and Anne M. Spain, Ferris State University, Big Rapids, MI 49307

Tetrathionate can be used as an electron acceptor for anaerobic growth in many well-characterized bacteria, including Salmonella and sulfate-reducing species.  However, the prevalence of tetrathionate reduction and the bacteria that participate in this process in anaerobic environments remains unknown.  The main focus of this study was to help fill in a small gap of the vast unknown in microbial diversity by isolating bacteria from the Muskegon River (Big Rapids, MI), and identifying those that potentially reduce tetrathionate.  Basic microbiology techniques were used to isolate bacteria on diluted Luria-Bertani (1/10X LB) agar plates under both aerobic and anaerobic conditions.  Nineteen isolates were obtained from Muskegon River sediment and surrounding soil and were further characterized by gram-staining and growth experiments designed to determine aerobic vs. anaerobic growth in 1/10X LB as well to compare anaerobic growth with and without tetrathionate.  Eight isolates were obtained under aerobic conditions.  Of these, most (87.5%) were gram-negative, and 25% and 75% were strict aerobes vs. facultative anaerobes, respectively.  Eleven isolates were obtained under anaerobic conditions, and of these, most were gram-positive (64%), while 64% and 36% were strict anaerobes vs. facultative anaerobes, respectively.  Growth experiments also showed that five of our isolates (26% of the total) had improved growth with tetrathionate compared to without, demonstrating their potential to reduce this compound during anaerobic growth. These findings suggest that tetrathionate-reducing bacteria are more prevalent in sediments and soils than we had thought.  We hope to continue this research by PCR-amplifying and sequencing 16S rRNA and tetrathionate reductase genes from these isolates as well as from isolates obtained by direct plating of sediment from a second site (Zodletone sulfur spring in SW Oklahoma) on solid minimal medium containing only non-fermentable substrates (H2 and acetate) and tetrathionate as available electron donors and acceptor, respectively, for anaerobic growth.

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Dehalogenation of PCE Contaminants by Dehalobacter restrictus, Desulfuromonas michiganensis and/or Dehalococcoides ethenogenes in a Model Aquifer

Bench Scale Analysis of a Copper Based Chemical for Disinfection of Bacteriophages and Indicator Bacteria from Constructed Challenge Water

Zachery Geurin, Rebecca Ives, Kaitlyn Delancey, and Joan Rose, Michigan State University, East Lansing, MI 48824

There are a variety of metal and halogen based disinfectants available for sanitizing water. Our experiment analyzed a copper based disinfectant marketed as being more affordable than chlorine without altering water’s taste or smell.
Disinfectant performance was evaluated using the bacteria R. terrigena and E. coli and the bacteriophage MS2 and P22. Parameters of the challenge water, such as microbial concentration levels, pH, and turbidity, were constructed to be representative of the conditions of National Sanitization Foundation (NSF) Protocol P231 Microbiological Water Purifiers.  After the defined contact time, the disinfectant was neutralized and samples were processed.   Bacteria concentrations were analyzed using APHA Standard Method 9222C (Colilert®). Bacteriophage were assayed using a modified double layer agar procedure based on EPA Method 1602.
After 2 hours of contact time, the 0 ppm copper control resulted in  0% reduction of R. terrigena and E. coli, 7.6% of P22 phage, and  2.6%  of MS2 phage. The 0.92 ppm copper concentration reduced R. terrigena 36.42%, E. coli 84.2%, P22 phage 4.4%, and MS2 phage 91%.  The 1.9 ppm copper concentration resulted in 0% reduction of R. terrigena, E. coli, and MS2 phage and 4.3% P22 phage reduction. At 2.5 ppm copper, R. terrigena reduced 91.5%, E. coli 91.5%,P22 phage 73%, and MS2 phage 85%.  At 4.0 ppm, all organisms showed 0% reduction.
The overall results showed small reductions when using the disinfectant. After 2 hours of contact time, the copper disinfectant was not successful in reducing the microbial concentrations by the 6 log10 (99.9999%) for bacteria and 4 log10 (99.99%) for viruses required to make the legal claim of microbiological water purifier. In previous studies, it has been shown metal based disinfectants may require longer contact time than other commonly used disinfectants (i.e. chlorine) to achieve 4-6 log10 reductions. In addition, a bell shaped curve of efficacy was shown where there may be an optimal concentration of the metal, and above or below that the metal is not as effective.

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A Comparative Analysis of Salmonella Detection Techniques in Surface Waters

Matthew T. Flood and Joan B. Rose, Department of Fisheries & Wildlife, Michigan State University, East Lansing, MI 48824

In recent years, waterborne pathogens have been of growing concern, specifically Salmonella.  As national water treatment infrastructure deteriorates, the risk of new outbreaks continues to rise.  Since 2006, forty-five Salmonella outbreaks have been documented by the CDC with contaminated water being implicated in a large number of the produce related outbreaks.  This study sought to determine if a novel detection technique from Neogen® is comparable to the current culture based EPA 1682 method modified for the detection of Salmonella in surface waters.  The ANSR™ (Amplified Nucleic Single Temperature Reaction) system originally developed for use in the testing of food safety, uses nicking enzyme amplification reaction (NEAR™) technology, and has shown 99.1% inclusivity in testing of >100 serovars of S. enterica and S. bongori.  A total of 9 samples were analyzed for six separate sampling events at four surface water sites (River Raisin, Grand River, Red Cedar River and an East Lansing farm canal).  Environmental samples and laboratory reagent water were also spiked with  S. typhimurium for additional comparison and recovery efficiency analysis.  MPN values were log transformed and submitted to unpaired t-tests for statistical analysis.  With the exception of one event, initial results have shown no statistically significant differences (p >0.06685, α = 0.05) between the methods suggesting that this novel system may be a possible alternative for rapid detection of Salmonella in surface waters.

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Metagenomic Analysis of Bacteriophages in Raw sewage

Yiseul Kim, Tiong G. Aw, Joan B. Rose, Michigan State University, East Lansing, MI 48824

Background: Viruses are estimated to be the most abundant and diverse biological entities in almost every ecosystem. Bacteriophages play an important role in influencing the diversity and population structure of bacterial communities in both natural environments and engineered systems such as wastewater treatment processes. In this study we used Illumina sequencing technology to characterize the virome, focusing especially on bacteriophages in raw sewage. Ultimately, bacteriophage metagenomics could be used as library independent tools to provide better information about the healthy water and wastewater treatment systems.
Methods: Viruses were concentrated from 2-L of raw sewage sample using polyethylene glycol (PEG) precipitation. The concentrated viral sample was passed through 0.22-μm filters and treated with DNase to remove contaminating cells and free nucleic acids. The extracted viral DNA and RNA were randomly amplified using Phi29 DNA polymerase and Transplex RNA amplification, respectively, to prepare adequate quantities of viral nucleic acids. The amplified products were pooled and processed for sequencing with Illumina Genome Analyzer II at the Research Technology Support Facility (RTSF), Michigan State University (MSU). Metagenomic sequence analysis was carried out using facilities of the High Performance Computer Center (HPCC) at MSU. The resulting reads were assembled, aligned, and annotated for the taxonomic and functional analysis of the viral metagenomes.
Results: Metagenomic analyses revealed that more than 30% of the viruses in raw sewage were novel. Like other biomes that have been studied, the virome of raw sewage was dominated by bacteriophages (48%). The four dominant families were Inoviridae, Myoviridae, Podoviridae, and Siphoviridae. Over half of the reads of these families were related to bacteriophages that infect enterobacteria or lactococci.
Conclusions: (i) This study demonstrated that a large number of bacteriophages have not yet been characterized and raw sewage provided a rich environment for studying bacteriophage diversity. (ii) Future studies will need to investigate the infectivity and host range of these bacteriophages to evaluate the impacts of their use in understanding water quality and treatment.

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Genus-wide Vaccine Candidates: Proof of Principle

Lyndsey Reich, Treesa Antony, Mirabela Hali, Herve Gerard, Alan Hudson and Judith Whittum-Hudson, Wayne State University School of Medicine, Detroit, MI 48201

Background and Significance:  There currently is no chlamydial vaccine approved for human or veterinary use. A genus-wide repertoire of novel peptide candidates would fulfill this long-standing need and offer significant public health benefits worldwide.
Objectives:  To identify new and unique peptides with potential to serve as vaccine(s) against human, livestock and other chlamydial species.
Methods:  BALB/c mice were immunized subcutaneously with peptides in Al(OH)3 or CpG/ Al(OH)3. Peptides were derived from phage display with an mAb to chlamydial exoglycolipid antigen and are mimotopes of the carbohydrate antigen. Serum was tested for chlamydia-specific antibodies against several chlamydial species prior to challenge. Lymphoproliferative responses (LPA) to one or more peptides were tested. After challenge with K/UW-31, vaginal swabs were collected weekly for DFA/culture; tissues were removed for pathology and molecular analyses.
Results:  Several peptides stimulated both B and T cell responses. LPA showed that Pep4 consistently induced positive spleen cell responses to Pep4 in vitro; not all peptides have been similarly tested yet. Peptide-induced antibodies recognized C trachomatis and C pneumoniae, and as predicted, also C muridarum and C abortus; staining intensity was dose-dependent for Pep 4 and Pep 11. In a more recent experiment, clearance of organism was significantly enhanced by several new peptides by day 14 through day 35 p.i. (p<.05).
Conclusions:  Our studies support the feasibility of several novel peptides to serve as (part of) an anti-chlamydial human vaccine. These peptides have added value in that there is potential to serve as vaccines for livestock and other veterinary species.

Note: Poster was presented at Chlamydia Basic Research Society meeting March 2013.

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The Efficacy of a Copper Based Water Disinfectant Under Bench Scale Conditions Over a Forty-Eight Hour Time Interval

Eric Reilly and Madeline Lipp, Michigan State University

This study aimed to determine if ‘Product X’, a copper based disinfectant, could truly be considered a ‘microbial water purifier’ according to NSF guidelines, which call for a 6 log10 (99.9999%) reduction of bacteria and a 4 log10 (99.99%) reduction of virus. To determine the efficacy of the product, sterile water was spiked with R. terrigena, E. coli, and bacteriophage MS2 at concentrations representing typical river conditions. Turbidity, pH, and total organic carbon content of the challenge water were adjusted to meet the conditions of National Sanitization Foundation (NSF) Protocol P231 Microbiological Water Purifiers.  The water was split into two carboys, with one carboy being exposed to ‘Product X’ at a 0.59 ppm copper concentration. Samples were taken from each carboy at specific time intervals between 0 minutes and 48 hours. Bacteriophage assays (USEPA Method 1602) and Colilert™ assays (APHA standard method 9223C) were then performed for each time point to determine the log reduction of bacteria and virus. During the 48 hour study period, log10 reduction for total coliform concentrations ranged between 0.513 and 0.937.  Log10 reduction of E.coli ranged between 0.287 and 0.351.  Log10 reduction of MS2  ranged between 0.187 and 0.056.  Log10 reduction values were not significant when assessed by either time interval (p > 0.05, α = 0.05) or copper concentration (p > 0.05, α = 0.05). Maximum log10 reduction values were less than 6 log10 (99.9999%) reduction for bacteria and 4 log10 (99.99%) for virus.  The copper based disinfectant does not meet the NSF guidelines for a microbial water purifier.

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Analyzing the Role of Two Putative Phosphatase 2A Components on Candida albicans Filamentation

Elizabeth Sommers, I.A.Cleary 2,  S.P.Saville 3, and Derek Thomas, Grand Valley State University, Allendale, MI 49401, 2 University of Tennessee at Martin, Martin, TN 38238, 3 The University of Texas at San Antonio, San Antonio, TX 78249

Candidiasis now represents the fourth most frequent nosocomial infection both in the US and worldwide. The pathogenic potential of C. albicans is intimately related to certain key processes including biofilm formation and filamentation.  Proteomic and microarray analysis has revealed the involvement of the genes 19.1468 and 19.7504, in hypha formation. These two C. albicans genes are predicted to encode components of the protein phosphatase 2A (PP2A) complex. Gene 19.1468 encodes a protein similar to CDC55, one of the two regulatory subunits of the PP2A complex in Saccharomyces cerevisiae. CDC55 is important for cell cycle progression and an S. cerevisiae strain lacking CDC55 is defective for filamentous growth. Gene 19.7504 is predicted to encode a protein most similar to Rts3p in S. cerevisiae, another putative protein phosphatase 2A complex component. While a S. cerevisiae strain lacking RTS3 shows increased sensitivity to the anti-fungal compound rapamycin, C. albicans strains lacking both copies of 19.7504 show no increased sensitivity to rapamycin, but are more sensitive to nourseothricin than a wild-type strain. In tet-NRG1 strain of C. albicans hypha formation in response to embedded conditions is strongly impaired when NRG1 is over-expressed. However, the absence of 19.7504 restores the ability to filament during NRG1 over-expression under embedded growth conditions as does the over expression of CDC55. However, the response is different to simple oxygen limitation. These experiments propose overlapping but distinct roles for these two different putative regulatory subunits of PP2A in the intricate control of hyphal development in C. albicans.

Please note: Some parts of this poster were presented at the Tenth ASM Conference on Candida and Candidiasis. Miami, FL, USA.

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Gull Exclusion at Great Lakes Beaches

Yuanyuan Feng, Michael McNiff, Kara Schultz, and Rebeccah Woodke, and Elizabeth Alm, Institute for Great Lakes Research & Biology Department, Central Michigan University

Gulls at Great Lakes beaches impair water quality and are an emerging public health and economic issue for coastal communities. In particular, Ring-Billed Gull populations have shown a dramatic increase in the Great Lakes region with the population growing an estimated 10% each year since the 1970s. Research suggests that gull feces may be one source of the fecal indicator bacterium E. coli to beach water at times when E. coli levels exceed guidelines, leading to swim advisories and beach closings. In addition, gull feces may contain bacteria with the potential to cause human disease. Therefore we have been investigating the effectiveness of border collies as a non-lethal management tool to reduce gull presence at public beaches.  The study was designed to address two primary questions: 1) are border collies effective for excluding gulls from beaches? 2) will gull exclusion from beaches result in lower levels of E. coli in beach water and sand?  Two hundred meter beach sections along Lake Michigan were randomly assigned as control or dog treatment.  During the 1st half of summer 2012, we recorded the number of gulls at each beach section, with dogs used continuously for 42 days on treatment sections.  A crossover design converted controls to treatments and treatments to controls during the 2nd half of the summer (42-day experimental period).  Gull numbers and E. coli and bacterial pathogens in water and sand and gull feces were compared.

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Analyzing the Interaction between Candida albicans and Pseudomonas aeruginosa and the Effect of Oxygen Limitation

Elizabeth Stinson, C.T. Howard, T.J. Ramnarine, Derek P. Thomas, Grand Valley State University, Allendale, MI 49401

Candidiasis represents is a frequent nosocomial infection both in the US and worldwide. In fact Candida albicans is an increasingly common threat to human health as a consequence of AIDS, steroid therapy, organ and tissue transplantation, cancer therapy, broad spectrum antibiotics and other immune defects. These infections carry unacceptably high morbidity, mortality rates and important economic repercussions (estimated total direct cost of approximately 2 billion dollars in 1998 in US hospitals alone). C. albicans can grow as yeast cells, pseudohyphae or hyphae with its form being dictated by its surrounding conditions. The ability to form hyphae has been fundamentally linked to the disease causing potential of this organism. However, studies have focused on either C. albicans in isolation or whilst it alone is infecting a host. In conditions outside of the laboratory C. albicans is typically surrounded by, and occupying the host with, other non-related microbes that can be a significant part of the environment C. albicans is reacting to. Previous studies have focused on investigating the nature of C. albicans interaction with P. aeruginosa. C. albicans has been shown to produce farnesol whichelicits changes in P. aerugunosa, and P. aeruginosa produces 3-oxo-C12-homoserine lactone which appear to inhibit C. albicans filamentation. In this preliminary study we investigate the interactions between C. albicans and both P. aeruginosa and Salmonella enterica serovar Typhimuriumusing real-time PCR associated with a system which facilitates rapid analysis of interspecies gene regulation. 
 Furthermore, we expand current understanding of this interaction to include oxygen-limiting conditions that may occur in the oral environment. This study verifies C. albicans interactions with Pseudomonas aeruginosa using our system and further analyzes the modulation of C. albicans filamentation by P. aeruginosa.

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Analyzing Interactions Between Candida albicans and Other Microbes

Andrea Engerson and Derek P. Thomas, Grand Valley State University, Allendale, MI 49401

Background: Candidiasis represents the fourth most frequent nosocomial infection in the US and worldwide. These infections carry unacceptably high morbidity, mortality rates and important economic repercussions. C. albicans can grow as yeast cells, pseudohyphae, hyphae or within a biofilm. The ability to form hyphae and biofilms have been fundamentally linked to the disease causing potential of this organism. However, studies have focused on either C. albicans in isolation or whilst it alone is infecting a host. Outside of the laboratory C. albicans is typically surrounded by, and occupying the host with, other non-related microbes that can be a significant part of the environment C. albicans is reacting to. Previous studies have identified a small number of bacteria that influence C. albicans morphology but only the interaction with Pseudomonas aeruginosa is well studied.
Methods: We use a simple well and membrane system to screen bacteria for interactions with C. albicans. Real-time PCR is then used to measure changes in C. albicans hyphal specific genes when exposed to the bacteria in comparison to an unexposed control.
Results: We demonstrate the development of a rapid screening technique that can be used to quickly identify and examine interactions between Candida albicans and other microbes. In addition, we document preliminary studies investigating C. albicans interactions with Acinetobacter baumannii and several Streptococcal species.  We show variations in the influence of A. baumannii associated with changing culture conditions and examine the effect on several C. albicans hyphal specific genes.
Conclusions: This study documents a system to facilitate rapid analysis of interspecies signaling. The system was used for a preliminary investigation of C. albicans interactions with Acinetobacter baumannii. These experiments show such interactions may vary more than previously thought and begins to analyze the varying influence on hyphal specific genes.

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