The discovery of the prokaryotic cytoskeleton has revolutionized our thinking about spatial organisation in prokaryotes. However, the roles different bacterial cytoskeletal proteins play in the localisations of diverse biomolecules are controversial. Bacterial chemotaxis depends on signalling through large protein clusters and each cell must inherit a cluster on cytokinesis. In Escherichia coli the membrane chemosensory clusters are polar and new static clusters form at pre-cytokinetic sites, ensuring positioning at new poles after cytokinesis and suggesting a role for the bacterial FtsZ and MreB cytoskeletons. Rhodobacter sphaeroides has both polar, membrane-associated and cytoplasmic, chromosome-associated chemosensory clusters. This study sought to investigate the roles of FtsZ and MreB in the partitioning of the two chemosensory clusters in R. sphaeroides. The relative positioning between the two chemosensory systems, FtsZ and MreB in R. sphaeroides cells during the cell cycle was monitored using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which gradients of FtsZ extend circumferentially to form the Z-ring. The proposed node-precursor model might represent a common mechanism for the formation of cytokinetic rings. The MreB cytoskeleton continuously reorganizes between patchy and filamentous structures, and colocalises with FtsZ at midcell. Membrane chemosensory proteins form individual dynamic unit-clusters with mature clusters containing about 1000 CheW₃ proteins. These unit-clusters diffuse randomly within the membrane but have a higher propensity for curved regions like cell poles. Membrane clusters do not colocalise with FtsZ and MreB and appear excluded from the Z-ring vicinity. The bipolar localisation of membrane clusters is established after cell division via random diffusion and polar trapping of clusters. The cytoplasmic chemosensory clusters colocalise with FtsZ at midcell in new-born cells. Before cytokinesis one cluster moves to a daughter cell, followed by the second moving to the other cell. FtsZ and MreB do not participate in the positioning of cytoplasmic clusters. Therefore the two homologous chemosensory clusters use different mechanisms to ensure partitioning, and neither system utilizes FtsZ or MreB for positioning.

Bacteria are enclosed by a peptidoglycan sacculus, an exoskeleton-like polymer composed of glycan strands cross-linked by short peptides. The sacculus surrounds the cell in a closed bag-like structure and forms the main structural component of the bacterial cell wall. As bacteria grow and divide, cell wall remodelling by peptidoglycan hydrolases results in the release of peptidoglycan fragments from the sacculus. In Gram-negative bacteria, these fragments are efficiently trapped and recycled. Gram-positive bacteria however shed large quantities of peptidoglycan fragments into the environment. For nearly five decades, Gram-positive bacteria were thus assumed not to recycle peptidoglycan and this process has remained enigmatic until recently. In this thesis, the occurrence and physiological role of peptidoglycan recycling in the Gram-positive pathogen Staphylococcus aureus was investigated. S. aureus is an important pathogen, and is becoming increasingly resistant to many antibiotics. Through bioinformatic and experimental means it was determined that S. aureus may potentially recycle components of peptidoglycan and novel peptidoglycan recycling components were identified and characterised. Though disruption of putative peptidoglycan recycling in S. aureus appears not affect growth or gross morphology of this bacterium, potential roles for peptidoglycan recycling in cell wall homeostasis and in virulence were identified. This is to my knowledge the first demonstration of a potential role of peptidoglycan recycling in either of these aspects of bacterial physiology in any Gram-positive bacterium. This is an important step forward in understanding the basic biology of Gram-positive bacteria, and in understanding the mechanisms of virulence in S. aureus. Future study of this process in S. aureus and other Gram-positive bacteria promises to reveal yet further facets of this process and its functions, potentially leading to the identification of novel therapeutic approaches to combat infections.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 135-149). Also available on the Internet.

Indiana University-Purdue University Indianapolis (IUPUI) / Sepsis is a clinical syndrome due to a systemic inflammatory response to severe microbial infection. Little is known about the changes in the bone marrow (BM) and how they affect the hematopoietic response to bacterial infection. Using an animal model of severe sepsis induced by Pseudomonas aeruginosa, we have previously reported that hematopoietic stem cells (HSC) undergo a significant expansion in the BM accompanied with myeloid suppression. This bone marrow response was Toll-like Receptor 4 (TLR4)-dependent. TLR4 is activated by bacterial lipopolysaccharide (LPS) and signals through two major independent downstream molecules: TRIF and MyD88. In the present study, I found that the TLR4/TRIF and the TLR4/MyD88 pathways contribute in a distinct manner to the BM response to P. aeruginosa's LPS. TRIF plays a major role in the expansion of the HSC pool, whereas MyD88 is required for myeloid suppression. Following LPS stimulation, HSCs enter in the cell cycle, expand and exhaust when transplanted in healthy mice. Loss of TRIF rescued completely the long-term engraftment and multilineage reconstitution potential of septic HSCs, but did not affect myeloid differentiation. Conversely, MyD88 deficiency prevented completely the myeloid suppression in the myeloid progenitors, but conferred limited protective effects on the HSC function. It is of great therapeutic value to identify the downstream molecules involved in TLR4/MyD88 dependent myeloid suppression. I found miR-21, a microRNA that is involved in inflammation, was up-regulated upon LPS challenge in a MyD88-dependent manner. However, deletion of miR-21 in the BM did not rescue LPS-induced bone marrow dysfunction, demonstrating that miR-21 is not a critical regulator in these processes. Further studies are warranted to determine the precise molecular mechanisms involved in the complex pathogenesis of BM response to sepsis. Taken together, my results show for the first time that the TLR4/TRIF signaling as a key mediator of HSC damage during acute LPS exposure and that activation of the TLR4/MyD88 signaling pathway play a dominant role in myeloid suppression. These results provide novel insights into our understanding of the molecular mechanisms underlying bone marrow injury during severe sepsis and may lead to the development of new therapeutic approaches in this disease.

The PhD thesis is on the studsy of the influence of the ftsZ antisense RNA and FtsH protease on the synthesis and function of the Escherichia coli cytokinetic protein, FtsZ, which mediates septation during cell division. Thus, it involves three molecules, FtsZ, ftsZ antisense RNA, and FtsH protease. While the E. coli ftsZ antisense RNA is being identified and structurally and functionally characterised for the first time, there has been some earlier studies in the laboratory in which the FtsH protease was found to have influence on the presence of the FtsZ rings at the mid-cell site. The Chapter 1 is the Introduction to the thesis presented in 3 parts –Part 1A, 1B, and 1C, introducing FtsZ and bacterial cell division, bacterial antisense RNAs, and FtsH protease, respectively. The Chapter 2 gives the description of the Materials and Methods used in the study. The Chapter 3 presents the identification, structural and functional characterisation of the ftsZ cis-antisense RNA, and its role in the regulation of FtsZ protein levels. Initially, the expression of cis-encoded antisense RNA from E. coli ftsZ loci was demonstrated during the different growth phases of the bacterium (RT-PCR/qPCR data). Antisense RNA is expressed from three promoters (primer extension and promoter probe data) on the complementary strand of the ftsZ coding region and terminates at the singletrand te complementary toftsAthegenethat 3’islocatedregionupstreamof theofftsZ the gene. Induced overexpression of a portion (423 bp) of the antisense RNA, spanning the ftsZ AUG codon and the ribosome binding site of ftsZ mRNA, from pBS(KS) could downregulate the synthesis of FtsZ protein to approximately 30%, leading to cell division arrest and filamentation of the cells at 42°C. This effect was less dramatic at 30ºC, probably due to less melting of the antisense RNA. Immunostaining performed on the induced culture did not show FtsZ ring formation after overnight induction whereas reduction in the proportion of the cells carrying FtsZ rings could be clearly observed after 2 hrs of induction. Real time PCR analysis performed for relative quantitation of ftsZ mRNA and ftsZas RNA from different growth phases (0.2 to 2.5 OD600 nm) showed growth phase dependent expression of the antisense RNA. While the levels of ftsZas RNA were found to be high at lower OD cultures or early growth phase cultures, the levels were found to be low at the late log phase and stationary phase cultures. Thus, when the cells are actively dividing and therefore need more FtsZ, the levels of the ftsZas RNA are high, while the cells are not actively dividing and therefore the FtsZ levels are low, the levels of the ftsZas RNA are low. At any phase of the growth, the ratio of the ftsZ mRNA to the ftsZas RNA was always found to be 6:1. Thus, the physiological role the ftsZas RNA is to maintain the availability of the ftsZ mRNA at a level that is commensurate with the requirement for the FtsZ protein during the different stages of the cell growth and division. The Chapter 4 is on the study of the possible mechanism behind the influence of FtsH protease on the presence of FtsZ rings at the mid-cell site during septation in cell division. Immunostaining for FtsZ in the mid-log phase E. coli cells showed that 82% of the AR3289 (ftsH wild type) cells possessed FtsZ rings, while only 18% of the AR3291 (ftsH-null maintained viable by a suppressor mutation) cells showed Z-rings. While the AR3289 cells showed a cell doubling time of 20 min, the AR3291 cells had a cell doubling time of 45 min. The mass doubling time of AR3289 and AR3291 were 24 min and 54 min, respectively. These distinct differences were found in spite of the suppressor mutation suppressing all the deleterious effects of the lack of the essential protease, FtsH. Complementation of the ftsH-null cells (AR3291) with the wild type FtsH but not with the ATP-binding or ATPase, or protease-defective mutants of FtsH, restored the FtsZ ring status to about 80% of the cells. The growth rate of AR3291 was also partly restored to comparable to that of the wild type cells upon complementation. Western blotting for FtsZ, and the FtsZ-stabilising proteins, FtsA and ZipA, showed that the ftsH-null cells have low levels of FtsA, as compared to those in the isogenic wild type cells (AR3289). The levels of FtsZ and ZipA were comparable in both the cells. Quantitative PCR performed for different cell division genes within the dcw cluster showed no sign of change in the ftsA transcript levels in the ftsH-null cells, suggesting that the low levels of FtsA in the ftsH-null cells were not due to transcriptional downregulation. Further experiments showed that the half-life of FtsA protein in the AR3289 cells was 45 min, while that in the AR3291 cells was 24 min. This experiment showed that the low levels of FtsA in the ftsH-null cells was due to the low half-life of FtsA in the cells. Growth synchronisation of the AR3289 and AR3291 cells showed that the levels of FtsA prior to cell division stage do not increase in the ftsH-null cells as much as in the isogenic wild type cells. Thus, the ftsH-null cells must be somehow managing the division through the partial stabilisation of FtsZ rings by ZipA. Interestingly, immunostaining for FtsH in AR3289 cells showed the presence of FtsH at the mid-cell site, as co-localised with FtsZ, for a brief period prior to cell constriction. These observations suggest the involvement of FtsH in cell division process. The faster degradation of FtsA in the absence of FtsH protease implies that another protein, which may be a protease that directly degrades FtsA or a chaperone that helps the unfolding of FtsA for degradation, might be the substrate of FtsH protease. The absence of FtsH protease brings up the levels of this unknown protein, which in turn facilitates (if it is a chaperone) degradation of or directly degrades (if it is a protease) FtsA. This model for the link among FtsH, FtsA levels, and the presence of FtsZ has been proposed based on the observations. Thus, the present study reveals for the first time an FtsA-linked role for FtsH protease in the presence of FtsZ ring at the mid-cell site and hence in bacterial septal biogenesis. The thesis is concluded with the list of salient findings, publications, and references.

No description available.

Bacterial cell wall proteins (S-layer) are - due to both the capability to self-assemble into two-dimensional crystals and their distinct chemical and structural properties - suitable for the deposition of metallic particles at their surface . The cluster growth is subject of this thesis. The binding of metal complexes to S-layers of Bacillus sphaericus and Sporosarcina ureae and their subsequent reduction leads to the formation of regularly arranged platinum or palladium cluster arrays on the biomolecular template. A heterogeneous nucleation mechanism is proposed for this process consisting of the binding of metal complexes and their subsequent reduction. The kinetics of the process and the binding of the complexes to the protein are characterized by UV/VIS spectroscopy. This thesis focuses on structural investigations by means of transmission electron microscopy, electron holography, scanning force microscopy, image analysis, and image processing. Preferred cluster-deposition sites are determined by correlation averaging. A more precise determination and quantification is obtained by Multivariate Statistical Analysis. Furthermore a method for the electron beam induced formation of highly-ordered metallic cluster arrays in the transmission electron microscope and a fast screening method for surface layers of Gram-positive bacteria are presented. / Bakterielle Zellhüllenproteine (S-Layer) eignen sich durch ihre Fähigkeit zur Selbstassemblierung zu zweidimensionalen Kristallen und durch ihre besonderen chemischen und strukturellen Eigenschaften zur Abscheidung regelmäßiger metallischer Partikel auf ihrer Oberfläche. In dieser Arbeit wird das Clusterwachstum auf S-Layern untersucht. Die Anbindung von Metallkomplexen an S-Layer von Bacillus sphaericus und Sporosarcina ureae und deren Reduktion führt zur Abscheidung periodisch angeordneter metallischer Platin- bzw. Palladiumcluster auf dem Biotemplat. Für diese Clusterbildung wird ein heterogener Keimbildungsmechanismus vorgeschlagen, bestehend aus Komplexanbindung und Reduktion. Die Bestimmung der Prozeßkinetik und die Charakterisierung der Anbindung der Komplexe an das Protein erfolgt mittels UV/VIS-Spektroskopie. Den Schwerpunkt dieser Arbeit bilden strukturelle Untersuchungen mit Hilfe der Transmissionselektronenmikroskopie, der Elektronenholographie, der Rasterkraftmikroskopie und der Bildanalyse und Bildverarbeitung. Durch Korrelationsmittelung werden Strukturinformationen gewonnen, die eine Bestimmung der lateral bevorzugten Clusterpositionen ermöglichen. Für die auf S-Layern erzeugten Clusterarrays wird die Belegung der einzelnen Positionen mittels Multivariater Statistischer Analyse genauer quantifiziert. Außerdem werden eine Methode zur Erzeugung hochgeordneter metallischer Partikelarrays unter dem Einfluß des Elektronenstrahles im Transmissionselektronenmikroskop und eine Methode zum schnellen Test Gram-positiver Bakterienstämme auf die Existenz von S-Layern vorgestellt.

No description available.

Bacterial cell wall proteins (S-layer) are - due to both the capability to self-assemble into two-dimensional crystals and their distinct chemical and structural properties - suitable for the deposition of metallic particles at their surface . The cluster growth is subject of this thesis. The binding of metal complexes to S-layers of Bacillus sphaericus and Sporosarcina ureae and their subsequent reduction leads to the formation of regularly arranged platinum or palladium cluster arrays on the biomolecular template. A heterogeneous nucleation mechanism is proposed for this process consisting of the binding of metal complexes and their subsequent reduction. The kinetics of the process and the binding of the complexes to the protein are characterized by UV/VIS spectroscopy. This thesis focuses on structural investigations by means of transmission electron microscopy, electron holography, scanning force microscopy, image analysis, and image processing. Preferred cluster-deposition sites are determined by correlation averaging. A more precise determination and quantification is obtained by Multivariate Statistical Analysis. Furthermore a method for the electron beam induced formation of highly-ordered metallic cluster arrays in the transmission electron microscope and a fast screening method for surface layers of Gram-positive bacteria are presented. / Bakterielle Zellhüllenproteine (S-Layer) eignen sich durch ihre Fähigkeit zur Selbstassemblierung zu zweidimensionalen Kristallen und durch ihre besonderen chemischen und strukturellen Eigenschaften zur Abscheidung regelmäßiger metallischer Partikel auf ihrer Oberfläche. In dieser Arbeit wird das Clusterwachstum auf S-Layern untersucht. Die Anbindung von Metallkomplexen an S-Layer von Bacillus sphaericus und Sporosarcina ureae und deren Reduktion führt zur Abscheidung periodisch angeordneter metallischer Platin- bzw. Palladiumcluster auf dem Biotemplat. Für diese Clusterbildung wird ein heterogener Keimbildungsmechanismus vorgeschlagen, bestehend aus Komplexanbindung und Reduktion. Die Bestimmung der Prozeßkinetik und die Charakterisierung der Anbindung der Komplexe an das Protein erfolgt mittels UV/VIS-Spektroskopie. Den Schwerpunkt dieser Arbeit bilden strukturelle Untersuchungen mit Hilfe der Transmissionselektronenmikroskopie, der Elektronenholographie, der Rasterkraftmikroskopie und der Bildanalyse und Bildverarbeitung. Durch Korrelationsmittelung werden Strukturinformationen gewonnen, die eine Bestimmung der lateral bevorzugten Clusterpositionen ermöglichen. Für die auf S-Layern erzeugten Clusterarrays wird die Belegung der einzelnen Positionen mittels Multivariater Statistischer Analyse genauer quantifiziert. Außerdem werden eine Methode zur Erzeugung hochgeordneter metallischer Partikelarrays unter dem Einfluß des Elektronenstrahles im Transmissionselektronenmikroskop und eine Methode zum schnellen Test Gram-positiver Bakterienstämme auf die Existenz von S-Layern vorgestellt.

No description available.