Univ., Diss., 2009--Marburg.

Thesis (Doctoral)--Technische Universität Carolo-Wilhelmina zu Braunschweig, 1982.

Univ., Diss., 2009--Marburg.

Thesis supervisor: John R. Kirby. Includes bibliographic references (p. 63-67).

Bacterial physiology and behavior is controlled by complex regulatory networks. Chemosensory systems are sophisticated signal transduction systems that can govern a range of cellular functions beyond that of traditional flagellar-based chemotaxis. The soil bacterium Myxococcus xanthus encodes eight chemosensory systems regulating multiple behaviors, including motility, exopolysaccharide production, and development. This work characterizes the Che7 system and demonstrates a role for Che7 in coupling aggregation and sporulation during multicellular development. The regulation requires an interaction between a single domain response regulator (CheY7) and a HEAT-repeat protein (Cpc7). A fatty acid desaturase, Des7, also impacts development in concert with the Che7 signaling system. Genetic analysis indicates the target of Che7 regulation is in the heme biosynthesis pathway, which is one aspect of iron homeostasis. Finally, characterization of iron and iron-responsive elements during development reveal a novel regulator, Fur2, that controls timing of development as well as che7 transcription. This work provides expands the known network regulating development in M. xanthus.

No description available.

Myxococcus xanthus, a model organism for studying development and Type IV pili (T4P), harbors three Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) on its chromosome. CRISPR systems, which function as an adaptive immune system in prokaryotes, are classified into three types based on CRISPR-associated genes. Evidence suggests that these three types mediate immunity slightly differently. M. xanthus CRISPR1 and CRISPR2 are Type I systems while CRISPR3 is a Type III-B system. In a genetic screen, a mariner transposon insertion in the 13th spacer of CRISPR3 (3SP13) was found to restore exopolysaccharide (EPS) production to a pilA mutant. Since the deletion of CRISPR3 failed to suppress a pilA mutation and expression of CRISPR3 from a heterologous promoter led to pilA suppression, it was concluded that the 3SP13 transposon insertion is a gain-of-function mutation. Deletion of the adjacent Repeat Associated Mysterious Proteins (RAMP) genes indicated that they are essential for the 3SP13 transposon insertion to suppress pilA, providing evidence that Type III-B systems may be involved in the regulation of chromosomal genes. We suggest that one of the spacers, once expressed and processed, may inhibit the expression of a negative regulator of EPS production in M. xanthus. / Master of Science

La bacteria Myxococcus xanthus, responde a la luz azul produciendo carotenoides que la protegen del daño fotooxidativo. En oscuridad la transcripción de la mayoría de los genes implicados en la síntesis de estos pigmentos es reprimida por las proteínas CarA y CarH, parálogas y funcionalmente redundantes. Ambas contienen un dominio N-terminal y otro C-terminal de unión al DNA y a cobalaminas respectivamente. Sorprendentemente, CarH, pero no CarA, depende de B12 para llevar a cabo su función represora. En este trabajo se ha demostrado que CarH y su homólogo en Thermus thermophilus (TtCarH) son fotorreceptores que utilizan la adenosilcobalmina (forma de cobalamina) como grupo cromóforo. La luz desmantela la oligomerización de estas proteínas y su unión al DNA inducidas por la adenosilcobalamina, lo que activa la expresión de los genes implicados en la carotenogénesis. Este hallazgo ha sido publicado en la prestigiosa revista PNAS (Ortiz-Guerrero et al. 2011). / The bacteria Myxocccus xanthus responds to light by producing carotenoid, protecting itself against photooxidative damage. In the dark, most of the genes involved in carotenoid synthesis are repressed by the paralogous and functionally redundant proteins CarA and CarH. Both of them contain a DNA-binding N-terminal domain and a cobalamin-binding C-terminal domain. Surprisingly, CarH, but not CarA, repressive depends on B12. In this work we showed that CarH and its homologous in Thermus thermophilus (TtCarH) are photoreceptors in which adenosylcobalamin plays the role of a chromophore. Light dismantles CarH and TtCarH adenosylcobalamine-induced oligomerization and DNA binding, activating structural genes involved in carotenoid. This finding has been reported in the prestigious journal PNAS (Ortiz-Guerrero et al. 2011)

Se investigó la presencia de microorganismos del Orden Myxococcales en suelos representativos del Departamento de Lima como una contribución a los escasos trabajos realizados en referencia a este grupo microbiano con el fin de sentar las bases para futuros trabajos en el área biotecnológica. Para tal fin, se examinaron suelos de 6 lugares diferentes de Lima: Bosques de Zárate, Jardines de Lima, Lomas de Lachay, Lunahuaná-Cañete, Valle del Río Chillón y los Pantanos de Villa, colectándose un total de 114 muestras. Se utilizaron los medios Krzeminieska and Krzeminieska (K&K), el medio Singh (MSg) y agar pellets (A-P) para el aislamiento y purificación de cepas bacteriolíticas; el medio Stanier modificado (MSt) y el agar Stanier almidón (ASTA) para el aislamiento y purificación de cepas celulolíticas; los medios CT, agar Levadura (AL) y el medio IM-1 fueron utilizados para observar las formas vegetativas y fructificantes de los aislados. La identificación se realizó en base a la clasificación morfológica reportada en el Manual Bergey (Holt et al., 1994). Se confirmó la presencia de Myxococales en suelos de Lima, siendo las cepas bacteriolíticas del género Myxococcus las más predominantes. También se identificaron cepas de los géneros Angiococcus, Archangium, Cystobacter, Sorangium y Stigmatella. La identificación basada en características morfológicas, fue la de mayor utilidad para la identificación de Myxococcales aún cuando pruebas adicionales ayudaron en la identificación de las especies. / --- We investigated the presence of microorganisms in soils representative Myxococcales Order of the Department of Lima as a contribution to the limited work done in reference to this microbial group in order to provide the basis for future work in biotechnology. To this purpose, we examined soils from 6 different places in Lima: Forests of Zárate, Gardens of Lima, Lomas de Lachay Lunahuaná-Cañete, Chillon River Valley and the Villa Wetlands, collecting a total of 114 samples. Were used media Krzeminieska and Krzeminieska (K & K), the Singh medium (MSg) and agar pellet (AP) for the isolation and purification of strains bacteriolytic; the Stanier medium modified (SDM) and the agar Stanier starch (ASTA) for isolation and purification of cellulolytic strains, CT media, agar yeast (AL) and the IM-1 medium were used to observe vegetative and fruiting forms of the isolates. The identification was made based on morphological classification reported in Bergey's Manual (Holt et al., 1994). We confirmed the presence of Myxococales in soils of Lima, with bacteriolytic strains of the genus Myxococcus the most predominant. We also identified strains of the genera Angiococcus, Archangium, Cystobacter, Sorangium and Stigmatella. Identification based on morphological characteristics was the most useful for identification of Myxococcales, even when additional tests aided in identifying the species.

Bacterial type IV pili (T4P) are filamentous surface appendages with a variety of functions including motility, surface attachment, and biofilm formation. In many species of bacteria a clear understanding of how the functions of T4P in lifestyle switching are regulated remains to be elucidated. Here, we focus on understanding the regulation of the T4P assembly ATPase PilB. We examined its interactions with the secondary messenger cyclic-di-GMP (cdG). Specifically we investigated how cdG binding regulates PilB functions not only as the assembly ATPase, but also as an EPS signaling molecule in Myxococcus xanthus biofilm regulation. Chapter 2 focuses on the development of a microplate-based biofilm assay for M. xanthus. This new assay allows for the analysis of the M. xanthus submerged biofilms under vegetative conditions in a high throughput format which has been absent in the published literature. M. xanthus biofilm formation tightly correlates with EPS production, suggesting that the assay can be used as a convenient method of examining EPS production. Chapter 3 examines the regulation of M. xanthus PilB (MxPilB) by cdG binding in vivo. We carried out a mutational analysis of the MshEN cdG binding domain in MxPilB. Mutations were created that either diverge with or converge from the MshEN consensus sequence. These two classes of MxPilB variants are expected to either decrease or increase cdG binding affinity, respectively. We examined the motility, EPS production, and piliation phenotypes of these mutants. Our results were consistent with a model where the function of MxPilB is altered in response to cdG binding, and suggesting that PilB responds to different thresholds of cdG concentration. In Chapter 4, we examine the ligand binding to the N-terminal cdG binding domain and C-terminal ATPase domain of Chloracidobacterium thermophilum PilB (CtPilB) in vitro. Our results confirm that these two domains bind to their respective ligands specifically, and demonstrate these domains communicate with each other in response to ligand binding. The results from all of the studies help us to establish a model where cdG binding fine tunes the functions of PilB to regulate the switch of bacteria between the motile and planktonic states. In addition to their roles in motility and biofilm formation, T4P are key virulence factors in many significant human pathogens. Antivirulence chemotherapeutics are considered to be a promising alternative to antibiotics, as they target disease processes rather than bacterial viability. Because PilB is essential for T4P biogenesis, we sought to identify PilB inhibitors for the development of antivirulence therapies. In Chapter 5, we describe the development of the first high throughput screen (HTS), for PilB inhibitors. This assay is uses the reduction of the binding of a fluorescent ATP analog to CtPilB in vitro, leading to the discovery of the plant flavonoid quercetin as a PilB inhibitor. Using M. xanthus as a model a bacterium, quercetin was found to inhibit T4P-dependent motility and T4P assembly in vivo. Builds on this initial success with CtPilB, Chapter 6 describes the development and implementation of a second HTS based on the inhibition of CtPilB as an ATPase. Screening a large chemical library led to the identification of benserazide and levodopa as CtPilB inhibitors. We show that both compounds inhibit T4P assembly in M. xanthus without any detrimental effects on bacterial growth. Furthermore we demonstrate that both levodopa and benserazide inhibit T4P-mediated motility in Acinetobacter nosocomialis, a human pathogen, providing the first evidence that the compounds identified with CtPilB can be effective against a pathogenic bacterium. Both of these studies validate the effectiveness not only of our HTSs, with of CtPilB as a model protein for the identification of PilB inhibitors. / Doctor of Philosophy / Bacteria can be motile or sessile. Motile bacteria can use hair like structures on their surface, called pili, to move in their natural environment, whereas sessile bacteria produce intricate structures attached to solid surfaces known as biofilms. Bacteria are able to switch between being motile and sessile states depending on their environment conditions. However, it isn't clear how this switch is controlled in bacteria that use pili to move. To answer this question, we studied how PilB the protein that assembles pili, might control this switching process. We specifically investigated PilB because it has two known roles. The first is that it can assemble pili, to enable pili-mediated motility. The second is that it can stimulate or promote biofilm formation. This places PilB at the intersection of these two lifestyles, suggesting that this protein may play a key role in deciding whether a bacterium is to be motile or sessile. Another important reason to understand how PilB functions is because pili are used by some antibiotic resistant pathogenic bacteria. Since PilB is essential for the formation of pili, if the actions of PilB could be blocked, bacteria would be unable to make pili. This could stop bacteria from causing disease. By searching for new chemicals which stop PilB from creating pili, we can potentially identify new drugs to treat bacterial infections.