Effects of Oxidative Damage on Mitochondria and Yeast Spore Development

TRICIA BROWN* AND STEVEN W. GORSICH
Central Michigan University, Department of Biology, Mt Pleasant, MI.
 
Mitochondria are cellular components that provide large amounts of energy to cells and are essential for the success of sperm and oocyte (eggs) development in mammals. The comparable process in the budding yeast, Saccharomyces cerevisiae, is spore development.  Yeast is an attractive model to study spore development as it is more amendable to laboratory techniques and their mitochondria are easily visualized and manipulated.  Like sperm and oocytes, spores can be irreversibly damaged by various chemicals that cause a specific type of damage known as oxidative damage.  Proper mitochondrial structure is believed to be responsible for protecting yeast cells from oxidative damage during spore development.  The present study demonstrates that known oxidative stress agents cause spore development and mitochondrial defects.  Lacking proper mitochondrial morphology increases these effects.  [Received 10/05/07]

Stress Tolerance – Using Bakers Yeast as a Producer of Bio-ethanol

SANDRA ALLEN*, WILLIAM CLARK, AND STEVEN W. GORSICH
Central Michigan University, Department of Biology, Mt Pleasant, MI.
 
Due to economical, environmental, and political concerns, there is a high demand for an alternative and renewable fuel source.  Bio-ethanol is one alternative that has been proposed and tested, but still contains complications.  Industrial fermentation of bio-ethanol is achieved  by growing baker’s yeast, Saccharomyces cerevisiae, with a cornstarch substrate. While this process is quite efficient, our nation lacks the amount of corn needed to produce the desired amount of bio-ethanol.  As a solution, new alternative substrates that yeast can use to ferment ethanol are being investigated. Unlike cornstarch, alternatives substrates, such as biomass waste, produce multiple growth inhibitors during the fermentation process.  These inhibitors prevent yeast from optimally growing and fermenting ethanol.  The findings below demonstrate that one of these inhibitors, furfural, induces cellular damage.  This damage is partially repaired by the gene, ZWF1, which has a known function in stress tolerance. These data will be applied in an effort to engineer a robust yeast strain capable of tolerating industrial fermentation of biomass waste.  [Received 10/05/07]

Mutated Immunoglobulin-degrading Enzymes of Streptococcus Species and Their Effects on Human IgG

Immunoglobulin-degrading enzymes can cause an increase in virulence in certain pathogenic bacteria.  These enzymes are specifically known for cleaving IgG to create fragments similar to those created by papain digestion. These enzymes are known as IdeS and IdeZ and are originally found in two different Streptococcus species. Though both enzymes have different primary protein sequences, the amino acids coding for the active sites are identical.  In this research, IdeS and IdeZ plasmid DNA was obtained from recombinant E. coli, which could then be mutated using specific PCR primers.  Mutations were focused around the active site, in order to determine amino acids that influenced the structure of the enzymes and their ability to cleave human IgG. In IdeZ, a serine residue that is positioned between two amino acids directly involved in the active site of the enzyme was changed to an alanine residue.  This mutation eliminated IdeZ function as determined by western blot analysis.  Characterizing IdeS and IdeZ IgG-cleavage functions could have medical and veterinary applications. [Received 10/05/07]

in A Hospital Before a UVGI-HVAC Installation

Department of Crop and Soil Science,

Michigan

State

University

,

East Lansing

,

MI

,

48824

The objective of this study was to assess the background microbial concentrations in air and fomites at two campuses of a hospital before installation of the UVGI-HVAC.  Air samples were collected using an impingement method.  Approximately 1500 L (4 impinges x 30 min vacuuming with the flow rate of 12.5 L/min) of air was collected from each of the four rooms (two waiting rooms and two patient areas).  In each room, five commonly touched and five untouched surfaces were also collected using electrostatic wipes.  Samples were assayed with culture methods using selective agars, i.e. Tryptic(ase) Soy Agar (TSA), R2S, Mannitol Salt Agar (MSA) and Malt Extract Agar (MEA).  The results suggested that concentrations of bacteria ranged from 12 to 66 cfu/m³ in the bioaerosols and 0.1 to 64 cfu/cm² on the fomites.  In the indoor air bacteria concentrations were strongly correlated with each other (r = 0.774 to 0.980) while the fungi concentrations showed low correlations with the bacterial levels.  Microorganisms detected from untouched fomites were comparatively higher than the commonly touched surfaces.  It can be hypothesized that microorganisms can survive on fomites for a long period of time and common surfaces in the hospital are most often cleaned by staff or microbials are transferred between different people touching the surfaces frequently.  Untouched surfaces represent accumulation over time from the air.  The effectiveness of the system will be assessed by comparing the results before and after installation. [Received 10/09/07]

Impairment of Water Quality at Silver Lake Sand Dunes Recreational Area, Michigan

SHIKHA SINGH*, SANGEETHA SRINIVASAN*, TOMOYUKI SHIBATA,
MARK V. WONG, THENG THENG FONG AND JOAN B. ROSE
Department of Fisheries and Wildlife, Michigan State University, East Lansing,Michigan- 48824

The Silver Lake Sand Dunes recreational state park has a 2 mile long sand dune, and is an important economic part of the tourism industry in Michigan. About 50% of the shoreline is developed and privately owned, some of which use septic tanks.  Silver lake is located 1.2 miles northeast of Lake
Michigan and drains into Lake Michigan via Silver Lake and is fed by Hunters Creek (also influenced by septic tanks). Recent monitoring at Lake Michigan precipitated a closure due to high E.coli levels.  Eleven sites were tested along the shoreline of Silver Lake, Silver Creek and Lake Michigan using
fecal indicators Escherichia coli, Enterococci, Clostridium perfringens, coliphage virus and human sewage marker esp, human and cow specific Bacteroides marker.  Two bathing sites on Silver Lake and one in the state park site had E. coli levels 10 to 20x higher than the Michigan standard. 
Concentrations of E. coli ranged between 10 to 6467 CFU/100 mL with one sample being too numerous to count. Enterococci concentrations ranged between 0.3 to 443 CFU / 100 mL, and C. perfringens was between <0.3 and 97 CFU / 100 mL.  Coliphage virus concentrations were found to be lower (<1 to 40 PFU/100 mL).  Seven of nine Silver Lake sites failed either the Michigan standard or EPA criteria/Hawaii levels for safe swimming. Human virus samples have also been recently collected.   Public health risks warrant that the state considers assisting the community in finding better sewage treatment options to protect the water quality and the public health in the future. [Received 10/10/07]

ESTABLISHMENT OF A PLANT PATHOGEN EFFECTOR MODEL SYSTEM

Department of Biological Sciences, Western Michigan University, Kalamazoo, MI

Our aim is to create a model system to study the action of plant pathogens when they infect plant cells in furtherance of our desire to find ways to stop bacterial pathogen infections.  We have begun screening effector proteins of Pseudomonas syringae to determine which ones are lethal in Saccharomyces cerevisiae. We have initially picked 20 Pseudomonas syringae pathovar tomato effectors to screen, as homologues exist in common with Pseudomonas syringae pathovar phaseolicola.

We have established an inducible expression system in Saccharomyces cerevisiae to identify which effectors are lethal. The expression vector contains an inducible GAL1 promoter, a V5 epitope that will be used for visualization of the effector protein and 6xHIS tag for purification.

At present, we have PCR amplified and cloned 13 of the 20 effectors into the pCR-Blunt II-TOPO vector.  Each construct has been sequenced and verified correct. Nine effectors have been moved to an intermediate donor vector using the Gateway System. We have successfully transferred 1 of the effectors, HopM1, to the yeast expression plasmid and have transformed it into S. cerevisiae for examination of phenotypes.

HopM1 when expressed in yeast is lethal on solid media. Examination of the lethal effect using a titer assay has not duplicated the effect seen on solid media, suggesting that the effect seen is a delay or arrest of growth but not an outright cytotoxic effect. Results will be presented to show the effect of the growth rates and comparison of death, compared to WT strains containing HopM1. [Received 10/11/07]

Murine dendritic cells produce IL-12 and induce Th1 polarization in response to Campylobacter jejuni infection

Comparative Medicine and Integrative Biology, College of Veterinary Medicine, Michigan StateUniversity, East Lansing, MI-48824

Campylobacter jejuni is a frequent cause of food-borne gastroenteritis. Dendritic cells (DCs) are central to initiating immune responses to pathogens. Interaction of C. jejuni with murine DCs and its impact on T-cell responses are unknown. We hypothesized that C. jejuni activates murine bone marrow-derived DCs (BM-DCs) to undergo maturation, secrete T-helper cell (Th)-polarizing signal(s) and initiate Th1-type responses. To test this hypothesis, immature BM-DCs were infected with C. jejuni and various responses were assessed. BM-DCs were efficient at C. jejuni uptake and killing; they readily internalized C. jejuni by 2 h post-infection (p.i). Viable intracellular bacteria were reduced rapidly and no live intracellular bacteria were recovered by 8 h p.i. Following infection, BM-DCs had significant up-regulation of surface MHC-II, CD40 and costimulatory molecules (CD80 and CD86), thus demonstrating a mature phenotype. C. jejuni-infected BM-DCs secreted tumor necrosis factor-alpha, interleukin (IL)-6 and IL-12p70, which peaked at 24 h p.i. Formalin-killed C. jejuni also induced maturation of and cytokine production by BM-DCs but at a lower magnitude than responses elicited by live bacteria. Various strains of C. jejuni elicited differential cytokine production from BM-DCs. In a DC-CD4⁺ T cell co-culture system, C. jejuni-infected BM-DCs induced high-level interferon-? production from CD4⁺ T cells indicating Th1 polarization. In conclusion, we demonstrate that murine DCs infected with C. jejuni undergo maturation, secrete proinflammatory cytokines, and induce Th1 polarization, thus playing an important role in mounting anti-C. jejuni immunity in C57BL/6 mice. (Funded by NIH NO1-AI-30058) [Received 10/11/07]  NOTE: This poster was presented at ASM’s 107th General Meeting at Toronto in May 2007