No description available.

Enterococcus faecalis is a well known nosocomial drug resistant pathogen that is responsible for urinary tract infections, bacteremia, wound infections and endocarditis through the formation of biofilms. It has been shown that 68 genes present within the core genome of E. faecalis are upregulated in biofilm formation. One of those 68 genes is a putative seleniumdependent molybdenum hydroxylase (SDMH). Adjacent to this gene are a series of open reading frames that have been postulated to play a role in the maturation of a labile selenium cofactor. The biosynthesis of this labile cofactor has yet to be studied at either the genetic or biochemical level. The addition of selenium to growth medium caused a significant increase in biofilm density and extracellular hydrogen peroxide by wild type E. faecalis. By site-directed mutagenesis gene products encoded in the SDMH operon were shown to be necessary for the selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. This biofilm and peroxide phenotype is inhibited both by tungsten or auranofin in wild type E. faecalis suggesting the SDMH is a necessary enzyme for selenium-dependent biofilm and peroxide formation. These results show that the gene products encoded within the SDMH operon are necessary for a selenium-dependent biofilm formation as well as extracellular hydrogen peroxide production. These mutants will provide the basis for defining the synthesis of the labile selenium cofactor and allow for an expanded understanding of the biological use of selenium.

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In response to cardiac cell death from an injury, zebrafish, as opposed to mammals, are able to regenerate new heart cells without significant scar tissue. Heart attacks, a leading cause of death in the United States, leave behind substantial scar tissue that weakens the heart and leads to a greater chance of a repeated cardiac event. Many genes and major molecular pathways are highly conserved from fish all the way to humans; thus, understanding how the regenerative process works in zebrafish may provide insight into potential therapies for heart attacks in humans. However, we must first understand how heart regeneration occurs in zebrafish at the molecular level. From the time of injury to a zebrafish heart through the completion of regeneration, we want to build a regulatory network showing which genes are up- or down-regulated and how they are interconnected. Transcription factors, such as tcf21 and tbx20, bind to regulatory elements of DNA and can either upregulate or downregulate nearby genes. To build this gene regulatory network, scientists use a technique called ChIP-seq that can determine where in the genome these transcription factors bind. Nearby genes are potential targets of their regulation, and we can validate these enhancers by testing differences in expression using a fluorescent protein reporter construct. ChIP-seq requires high quality antibodies capable of specifically recognizing the transcription factor of interest. These are rarely available. Because each different antibody that is used requires validation and optimization for ChIP-seq, it is not easy to scale up the collection of data for different transcription factors. One way to get around these problems is to express a tagged version of the transcription factor. The tag is recognizable by the same antibody; however, expressing the tagged transcription factor in this manner almost inevitably results in higher than normal levels of expression, leading to false positives in the ChIP-seq data. Using CRISPR/Cas9 technology to target and modify specific sequences in the genome, we developed a novel method to add an epitope tag to these transcription factors at their endogenous loci. This allows us to run ChIP-seq experiments with the transcription factor at physiological levels of expression. We can also use the same antibody to eliminate repeated validation and optimization steps. We have successfully tagged two genes that may be involved in heart regeneration, tcf21 and tbx20. tcf21 is expressed in the developing epicardium and is required for the proper development of the branchial arches. tbx20 is expressed in the cardiomyocytes and is required for the proper development of the heart, and it has also been shown to be upregulated in response to injury in the zebrafish. With tbx20, we have performed a successful ChIP-seq experiment and have tested several promising target genes. It is difficult to test if either tcf21 or tbx20 is required for regeneration, as both of these genes are essential for development and mutants do not survive. The solution to this problem is to engineer the gene so that it can be turned off at a specific time and in a specific cell type. A common method of this in the mouse model utilizes the Cre/loxP system: two loxP sites flank a required segment of a gene, and the introduction of the enzyme Cre deletes the DNA between them. Until CRISPRs this was not feasible in zebrafish, which lacked an efficient method of targeted modification in the genome. We adapted our method for integrating epitope tags to add the two loxP sites in the genome. We have made and tested a fully conditional mutant for tbx20, and we have put in the first of the two loxP sites for tcf21. / Biology

No description available.

A model describing the potential amino acid ligands to the four 4Fe-4S centers (P-clusters) within the Azotobacter vinelandii nitrogenase MoFe protein is presented. Based on interspecies and intersubunit amino acid comparisons of the α- and ß-subunits of the MoFe protein, and the FeMoco biosynthetic proteins, NifE and NifN, four conserved residues (Cys62, His83, Cys88, Cys154 all proposed P-cluster ligands) within the α- subunit were targeted for site-directed mutagencsis studies. In order to define a range of acceptable substitutions, 35 specific site-mutants have been constructed, each with a different amino acid replacement at one of the four targeted positions. Previous studies indicated that these residues were important for MoFe activity, and may act as metallocenter ligands. Unusual redox and spectroscopic properties of the Fe-S centers suggest the involvement of ligands other than the four typical cysteines, though extrusion requirements indicate that some thiol ligands are likely. Surprisingly, mutants with an Asp, Gly, Thr, or Ser substituted for Cys88 are still capable of diazotrophic growth (Nif+), though whole cell and crude extract acetylene reduction activity is lowered. Several substitutions (Cys, Asp, Phe, Asn, Met, Tyr, Leu) are tolerated at the His83 position, these Nif+ mutant strains also have varying acetylene reduction rates and growth rates. All mutants with substitutions at positions 62, 154, resulted in complete loss of diazotrophic growth. The results could be interpreted by the following explanations: 1) Our proposed model for the P-cluster ligation within the MoFe protein is incorrect. 2) Some substitutions permit P-cluster rearrangement to a semi-functional state. 3) Either, P-clusters are not absolutely essential for diazotrophic growth, or the enzyme can function with a reduced number of these metal centers. / Master of Science / incomplete_metadata

Clostridium perfringens is a Gram-positive anaerobic pathogen of humans and animals. While lacking flagella, C. perfringens cells can still migrate across surfaces using a type of gilding motility that involves the formation of filaments of bacteria lined up in an end to end conformation. To discover the gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. Twenty-four mutants with deficiency in gliding motility were identified and one gene, which encodes a homolog of the SagA cell wall-dependent endopeptidase, was further characterized. We also isolated and characterized two hypermotile variants of strain SM101. Compared to wide type cells, the hypermotile cells are longer and video microscopy of their gliding motility suggests they form long, thin filaments that move rapidly away from a colony, analogous to swarmer cells in bacteria with flagella. Whole genome sequencing analysis showed that both mutants have mutations in cell division genes. Complementation of these mutations with wild-type copies of each gene restored the normal motility phenotype. A model is presented explaining the principles underlying the hypermotility phenotype. Heat resistant spores are the major route for disease transmission for C. perfringens, which cause food poisoning. To elucidate the molecular mechanisms involved in spore germination as well as to identify attractive targets for development of germination inhibitors to kill spores, we combined 1D-SDS-PAGE and MALDI-TOF-MS/MS to map the whole spore inner membrane proteome, both from dormant and germinated spores. As the first comprehensive spore inner membrane proteome study, we identified 494 proteins in total and 119 are predicted to be membrane-associated proteins. Among those membrane-associated proteins, 71 changed at least two-fold in abundance after germination. This study provides the first comprehensive list of the spore inner membrane proteins that may be involved in germination of the C. perfringens spore and their relative levels during germination. / Ph. D.

No / Further evidence for the rodent bone marrow micronucleus assay acting as a sensitive predictor of the possible germ cell mutagenicity of chemicals