Several bacteria were isolated from the bottom sediments of Tyume River and investigated for bioflocculant production potentials. Kaolin clay suspension (4 g/l) was used to measure the flocculating activity and three of the positive isolates were identified by 16S rRNA gene nucleotide sequence analyses and the sequences deposited in GenBank as Streptomyces sp Gansen (accession number HQ537129), Brachybacterium sp UFH (accession number HQ537131.), and Cellulomonas sp Bola (accession number HQ537132). Streptomyces sp Gansen exhibited its maximum flocculating activity using lactose (85% activity), peptone (76.3% activity), Ca2+ as sole sources of carbon, nitrogen and cations respectively, and at a neutral pH of 7.0, while, the bioflocculant produced by Brachybacterium sp UFH with glucose, urea and Ca2+ as carbon, nitrogen and cations sources yielded 82% and 97% flocculation activity respectively at a neutral pH. Also, glucose (73.2% activity), ammonium chloride (78.2% activity) and Ca2+ resulted in optimal production of bioflocculant by Cellulomonas sp Bola, also at a neutral pH. Chemical analysis confirmed that bioflocculant produced by Streptomyces Gansen is a polysaccharide while Brachybacterium sp UFH and Cellulomonas sp Bola produces a glycoprotein compound. This freshwater actinomycetes appears to have a tremendous potential as sou rces of new bioflocculants.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Bacteriophage (phage), virus of bacteria, has been proposed as a mean to inactivate bacteria that are pathogens of humans. Applied prophylatically to food, phage might decrease the numbers of potential pathogens we ingest. Much active research on using the phages of bacteria to control Gram negative foodborne pathogens are described in the literatures, but comparatively little research describes the phages of Gram positive bacteria and their use as biocontrol agents on food. In this work, previous undescribed phages, able to infect Bacillus cereus and Listeria monocytogenes, were isolated from soil and ruminants faecal material, respectively. As the first step in assessing their potential as biocontrol agents, the isolated phages were purified, concentrated and characterised (albeit to different degrees). The Bacillus phages had a narrow host range while the Listeria phages had a broad host range. Listeria phages also infected L. monocytogenes 2000/47, a strain which recurs in New Zealand clinical cases. Both Bacillus and Listeria phages appeared to be of the Myoviridae family judging by their structure in electron micrographs. The Bacillus FWLBc1 and FWLBc2 phages were lytic phages with a latent period of 106 and 102 min at 37°C, and an average burst size of 322 and 300 phages per infected cell, respectively. Moreover, they both had genomes of approximately 134 kb. All newly isolated and characterized phages were chloroform resistant and survived storage better at 4°C than at room or freezing temperatures. Bacillus phages significantly reduced the bacterial population in mashed potatoes within 24 h at room temperature, when applied at a phage to host ratio of 1000. Listeria phages rapidly inactivated the host population to a low optical density. The findings of this thesis will add to the current knowledge of phages in the context of various environmental conditions for different bacteria and will demonstrate the potential of phages as food safety biocontrol agents.

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

Thesis (M. Med. Sc.)--University of Hong Kong, 2008. / Includes bibliographical references (p. 23-34)

Thesis (doctoral)--Universität, Giessen, 2004.

Universiẗat, Diss., 2004--Gießen. / Zsfassung in dt. und engl. Sprache.

The Type VII secretion system (T7SS) is linked to virulence and long-term pathogenesis in a broad range of Gram-positive bacteria, including the human commensal and pathogen Staphylococcus aureus. The Type VIIb secretion system (T7SSb) is responsible for the export of small toxic proteins, which induce antibacterial immune responses and mediate bacterial persistence in the host. In addition, it is also involved in bacterial competition. The T7SSb requires several proteins to build up the secretion machinery. This work focuses on the structural and functional investigation of the motor ATPase EssC and the putative pore forming, multi-pass membrane component EsaA. Both proteins are indispensable for substrate secretion. EssC belongs to the FtsK/SpoIIIE ATPase family and is conserved among the T7SSs. It contains three C-terminal, cytosolic ATPase domains, designated as EssC- D1, -D2 and -D3, whereby EssC-D3 is the most distal one. In this thesis, I am presenting the crystal structure of the EssC-D3 at 1.7 Å resolution. As the deletion of EssC-D3 abrogates substrate export, I have demonstrated that this domain comprises a hydrophobic, surface-exposed pocket, which is required for substrate secretion. More specifically, I have identified two amino acids involved in the secretion process. In addition, my results indicate that not only EssC-D3 is important for substrate interaction but also EssC-D2 and/or EssC-D1. Unlike in the related Yuk T7SSb of Bacillus subtilis, the ATPase activity of D3 domain contributes to substrate secretion. Mutation of the modified Walker B motif in EssC-D3 diminishes substrate secretion completely. The membrane protein EsaA encompasses an extracellular segment spanning through the cell wall of S. aureus. I was able to reveal that this part folds into a stable domain, which was crystallized and diffracted up to 4 Å. The first attempts to dissolve the structure failed due to a lack of homologues structures. Therefore, crystals for single-wavelength anomalous dispersion, containing selenomethionyl-substitutes, were produced and the structure solution is still in progress. Preliminary experiments addressing the function of the extracellular domain indicate an important role in substrate secretion and bacterial competition. / Das Typ VII Sekretionssystem (T7SS) ist wichtig für Virulenz und Langzeit- Pathogenität von Gram-positiven Bakterien. Zu diesen gehört auch Staphylococcus aureus, bekannt als Kommensal und Pathogen im Menschen. Das Typ VIIb Sekretionssystem (T7SSb) exportiert kleine, toxische Proteine, die antibakterielle Immunantworten auslösen und für bakterielle Persistenz verantwortlich sind. Außerdem ist es an dem Konkurrenzkampf zwischen Bakterien beteiligt. Das System benötigt verschiedene Komponenten, um eine Sekretion zu ermöglichen. Diese Doktorarbeit konzentriert sich auf zwei dieser Proteine, die ATPase EssC und das Membranprotein EsaA. Beide Komponenten sind unentbehrlich für eine vollständige Funktionalität. EssC gehört zu der Familie der FtsK/SpoIIIE ATPasen und ist evolutionär in allen T7SSs erhalten. EssC besitzt drei C-terminale, zytosolische ATPase Domänen, bezeichnet als EssC-D1, -D2 und D3, wobei EssC-D3 C-terminal gelegen ist. In dieser Arbeit präsentiere ich die Kristallstruktur der ATPase Domäne EssC-D3, aufgelöst bis zu 1.7 Å. Die Domäne ist unabdingbar für die Sekretion. Durch die Strukturauflösung wurde eine hydrophobe, Oberflächen-exponierte Substrat- Bindetasche bestimmt, die eine essenzielle Rolle für den Export der toxischen Substrate einnimmt. Durch dieses Projekt konnten zwei Aminosäuren in dieser Tasche bestimmt werden, die für den Prozess der Substratsekretion wichtig sind. Weiterhin wurde bewiesen, dass nicht nur EssC-D3, sondern auch die ATPase Domäne EssC-D2 und/oder EssC-D1 mit den Substraten interagieren kann. Im Gegensatz zu dem verwandten T7SSb in Bacillus subtilis, verfügt EssC-D3 über ATPase Aktivität und ermöglicht dadurch den Substratexport. Das Membranprotein EsaA besitzt einen extrazellulären Abschnitt, der sich durch die Zellwand von S. aureus erstreckt. Dieser extrazelluläre Part besteht aus einer stabilen Domäne, welche kristallisiert werden konnte und bis zu 4 Å diffraktiert. Aufgrund von fehlenden homologen Strukturen konnte die Struktur der Domäne noch nicht bestimmt werden. Für die Phasenbestimmung, die wichtig für die Strukturauflösung ist, wurden Kristalle mit Selenomethionyl-Substituten hergestellt. Die Strukturauflösung ist noch nicht beendet. Erste Experimente bezüglich der extrazellulären Domäne zeigen, dass diese ebenfalls wichtig für die Substratsekretion und zusätzlich am Konkurrenzkampf zwischen Bakterien beteiligt ist.

The biosynthetic enzymes involved in wall teichoic acid biogenesis in Grampositive bacteria have been the subject of renewed investigation in recent years with the benefit of modem tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model Gram-positive bacterium, Bacillus subtilis 168, using phage resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE results in the loss of a-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also report the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity ofTagE using a wide variety of acceptor substrates and showed that this enzyme has a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognizes its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies on the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis. / Thesis / Doctor of Philosophy (PhD)

published_or_final_version / abstract / toc / Microbiology / Master / Master of Philosophy