The Roseobacter clade is an abundant and biogeochemically relevant group of marine bacteria. Physiological and ecological traits identified in specific representatives of the clade are often universally attributed to all Roseobacter group members, however, culture-dependent studies utilizing phylogenetically distinct members are rare. Other attributes often associated with this clade include motility, biofilm formation and surface attachment, chemotaxis and quorum sensing. This study compared a collection of 13 diverse Roseobacter strains both pheno- and genotypically on the basis of these traits. Motility was determined for seven previously uncharacterized strains, with five of the strains demonstrating motility. Microscopic analysis using both phase contrast and transmission electron microscopy supported this finding. A crystal violet assay was used to assess biofilm formation on plastic and glass surfaces with a range of surface properties and yielded a wide array of phenotypic responses. Taking into account the variety of surface types and media types tested approximately half (54%) of the strains showed pronounced biofilm formation and all motile strains were capable of forming biofilms. Degenerate primer sets were designed to probe strains for which no genome sequence is currently available for genes involved in flagellar synthesis and chemotaxis. Two strains that demonstrated no signs of motility in the laboratory were found to possess a necessary gene for flagellar formation and a flagellar-associated chemotaxis gene. Genome analysis including other sequenced Roseobacter strains revealed that flagellar, chemotaxis and quorum sensing operons are abundant in members of this lineage, with 89% possessing flagellar and chemotaxis operons and 78% possessing genes believed to be involved in quorum sensing. This study underscores the diversity of this clade and emphasizes the difficulty of assigning phenotypic capabilities to all lineage members.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN. / CHAIRMAN: WALTER J. LOESCHE.

We have previously identified σS, an ECF sigma factor that is important in the virulence and stress response of S. aureus. Transcriptional profiling of sigS revealed that it is differentially regulated in a variety of laboratory and clinical strains of S. aureus, suggesting that there exists a regulatory network that modulates its expression. In order to identify direct regulators of sigS expression, we performed a biotin pull down assay in tandem with mass spectrometry. We identified CymR as a direct regulator and observed that sigS expression is increased in cells lacking cymR. In addition, transposon mutagenesis was performed to identify regulators of sigS expression. We identified insertions in genes that are transcriptional regulators, and elements involved in amino acid biosynthesis and DNA replication, recombination and repair as influencing sigS expression. Finally, methyl nitro-nitrosoguanidine mutagenesis in conjunction with whole genome sequencing was employed and revealed mutations in the lactose repressor, lacR, and the membrane sensor histidine kinase, kdpD, as negatively effecting sigS expression. EMSAs revealed that LacR is an indirect regulator of sigS expression, while the response regulator KdpE is a direct repressor. These results indicate that a complex regulatory network is in place for sigS that modulates its expression. In a continuation of studies on σS regulation, we next explored interplay with the products of genes conserved within the sigS locus. We determined that this region is conserved amongst all the sequenced staphylococci, and includes four genes: SAUSA300_1721 (a conserved hypothetical protein), as well as sigS, ecfX, and ecfY. In order to investigate the relationship between EcfX and σS we performed protein pull down assays and observed that these two protein interact. Further to this, transcriptional analysis of sigS in an ecfX mutant reveal that expression of sigS is decreased, indicating that it is an activator. Architectural analysis of the sigS locus via RNAseq revealed that the majority of transcription in this region comes from ecfY, a gene that is downstream and divergent to sigS. We demonstrate that inactivation of ecfY leads to a significant increase in sigS expression, and that ecfY null strains are more resistant to DNA damaging agents such as UV, H2O2, MMS, and ethidium bromide, which we have previously demonstrated that a sigS mutant is highly sensitive to. Our studies also revealed that an ecfY null strain is better able to survive intracellularly following phagocytosis by RAW 264.7 cell and demonstrates increased survival in whole-human blood, which is again opposed to that previously observed for sigS deficient strains. Because the ecfY null strain overexpresses sigS, we investigated the regulon of this sigma factor using this mutant in conjunction with RNAseq analysis. We identified that genes putatively under the control of σS are involved in DNA damage and repair, virulence, amino acid starvation and nucleic acid biosynthesis. Collectively, our results indicate that σS is regulated via a unique mechanism: positively through an apparent need for an activator protein (EcfX) and negatively via RNA-RNA interaction (the 3’ UTR of ecfY). We suggest that the evidence presented here greatly adds not only to our understanding of the regulatory circuits extant within S. aureus, but also to alternative sigma factor biology in general. Finally, we evaluated the efficacy of a novel library of quinazoline-based compounds against a highly drug resistant strain of S. aureus. We performed structure activity and structure property relationship assays in order to identify lead compounds. These methods lead to the identification of N2,N4-disubstituted quinazoline-2,4-diamines that had low minimum inhibitory concentrations, along with favorable physiochemical properties. Evaluation of their biological activity demonstrated limited potential for resistance of to our quinazoline based compounds, low toxicity to human epithelial cells, and strong efficacy in vivo. Taken together, our findings support the use of quinazoline derivatives as potential new antimicrobials against multidrug resistant S. aureus.

Mycobacterium tuberculosis threatens global health. Its thick, impermeable cell wall renders it tolerant to high doses of many antibiotics. While we understand the biochemical functions of many cell wall-modifying enzymes, we often do not understand their physiological functions: their spatiotemporal roles in the cell cycle, their substrate preferences, or their individual effects on the macromolecular architecture of the cell wall complex. Mycobacterium tuberculosis possesses five peptidoglycan transpeptidases, five lytic transglycosylases, and numerous other peptidoglycan-modifying enzymes that lack precisely-understood roles. The lytic transglycosylases of Mycobacterium tuberculosis are collectively dispensable for in vitro growth. We sought to learn what other classes of peptidoglycan-degrading enzyme might substitute for the lytic transglycosylases or become essential in their absence. A high-throughput chemical screen was performed on a strain lacking all five lytic transglycosylases to identify compounds that specifically killed this strain and not wild-type Mycobacterium tuberculosis. Among the compounds identified were several members of the cephalosporin class of \(\beta\)-lactam antibiotics. It was shown that the cephalosporins had greater access to the periplasmic \(\beta\)-lactamase of Mycobacterium tuberculosis in the lytic transglycosylase-deficient strain and that this strain had enhanced sensitivity to several antibiotics with unrelated mechanisms of action. Together, greater periplasmic access and broadly heightened susceptibility in the deficient strain suggested a role for the lytic transglycosylases in maintaining the mycolic acid permeability barrier. To identify the specific penicillin-binding protein target of the cephalosporins, we isolated spontaneously-occurring resistant mutants. These strains contained polymorphisms in ponA2, a bifunctional penicillin-binding protein. The polymorphisms conferred sensitivity to heat stress, a phenotype associated with ponA2 loss of function. To clarify the relation between loss of function and cephalosporin resistance, a ponA2 deletion strain was created, which exhibited both cephalosporin resistance and sensitivity to carbapenems, another class of \(\beta\)-lactam. Restoration of the wild-type ponA2 allele suppressed both cephalosporin resistance and carbapenem sensitivity. Inactivation of other transpeptidases did not confer resistance to any \(\beta\)-lactams. The association of penicillin-binding protein inactivation with \(\beta\)-lactam resistance is unusual. One model to explain it is that upon deletion of ponA2, Mycobacterium tuberculosis compensates for its loss by upregulating a cephalosporin-resistant, meropenem-sensitive transpeptidase.

In 1809, A. von Thaer wrote in his book "Grundsatz der Rationell Landwirtschaft" that, except for water, the humus in the soil is the sole substance which supplies nutrients for the growth of plants. Since then, the role of humus in soil has been intensively studied and the meaning of humus has varied from the "organic fraction of soil" to "a dark incoherent and heterogeneous colloidal mass in soil" (Buckman and Brady 1960). Based on the latter meaning the term humus does not include any undecayed organic materials or micro-organisms in the soil. However, in the practical consideration of humus, the presence of microorganisms is usually neglected. For exemple, the well known humus fractionation method of Tyurin mentioned by Kononova (1958), does not consider that the treatment with solvent, alkali, and acid will fractionate not only the colloidal organic matter (humus) but also the undecayed organic materials and microorganisms. [...]