The major goal of this study was to achieve sustained cultivation of virulent T. pallidum in vitro. The putatuive binding of host proteins to the outer membrane (OM) of intact, virulent T. pallidum subsp. pallidum has been investigated. A major breakthrough was the development of a filtration assay, usinglow protein-binding membrane filters, for the measurement of substances bound to or incorporated into th eOM of T. pallidum. This avoided the conventional manipulations which can damage the fragile OM of T. pallidum. Using this filtration assay, studies on the binding of host serum proteins demonstrated that intact treponemes did not bind host proteins as previously reported. It also indicated that previous studies were probably performed with damaged by this research. The studies on the binding of polyclonal and monoclonal antibodies to intact and detergent treated treponemes provided evidence of the low level binding of antibody to intact treponemes which was greatly enhanced but the removal of the outer membrane with 0.1% Triton X. This research research corroborated that of others which suggests that the outer membrane of T. pallidum contains very little protein or surface exposed antigen.

FtsH is an essential AAA family Zn++ metalloprotease of Escherichia coli, possessing ATPase-dependent chaperon activity and ATP-dependent protease activity. Heat shock transcription factor Sigma32, LpxC, SecY, and bacteriophage protein CII are some of the substrates of FtsH. Although FtsH is known to influence several cellular processes, the role of FtsH in bacterial cell division had not been identified. FtsZ is the principal cell division protein that marks the cell division site at mid-cell by forming a ring structure. Using a pair of ftsH-null and isogenic wild type strain of E. coli, earlier studies in the laboratory had demonstrated that proteolytic function of FtsH is required for the presence of FtsZ rings at mid-cell site. It was also shown that FtsZ is not a substrate for FtsH protease in vivo. In view of these observations, using a pair of ftsH-null and isogenic wild type strain of E. coli, experiments were carried out to find out the mechanism behind the requirement for FtsH protease for the presence of FtsZ ring at mid-cell site. Viability of the cells having ftsH-null status was maintained by a suppressor mutation at another locus, and was found to be comparable to that of isogenic wild type cells. Immunostaining for FtsZ showed that only 20% cells of ftsH-null strain of E. coli has FtsZ ring at mid-cell site, On the contrary, more than 90% cells of isogenic wild type cells had FtsZ ring at mid-cell site. Live cell imaging with FtsZ-GFP also showed similar results. Low fraction of ftsH-null cells having FtsZ ring was found to be independent of slow growth rate of the cells. Confocal microscopy revealed that ftsH-null cells lacked the normal helical spiral-type structure of FtsZ, unlike the intact FtsZ helices present in isogenic wild type cells. FtsZ protein levels in the membrane and cytoplasmic fractions of ftsH-null cells were found to be same as those in the isogenic wild type cells. Exogenous expression of wild type FtsH in ftsH-null cells could restore FtsZ ring status to normalcy, similar to that in the isogenic wild type cells. However, this restoration could not be accomplished by FtsH mutants, which were lacking in ATP binding, ATPase, or protease activities. FtsA anchors FtsZ to the membrane and a specific FtsZ/FtsA ratio is known to be critical for cell division. Further, FtsA and/or ZipA are required for the stabilisation of FtsZ ring at mid-cell site. The levels of FtsA were found to be lower by more than 2.5-fold in all the membrane and soluble fractions of ftsH-null cells. The levels of FtsA were found restored to normalcy upon complementation with exogenous expression of FtsH. Low levels of FtsA were not due to the slow growth of ftsH-null cells. Exogenous expression of FtsA or FtsA-GFP restored FtsZ in more than 90% of ftsH-null cells. Moreover, FtsA mutants, which are defective in the interaction with FtsZ, did not restore FtsZ rings to normalcy. The levels of ZipA were found to be same in ftsH-null and isogenic wild type cells. Expression of ZipA or ZipA-GFP could restore FtsZ rings to normalcy in ftsH-null cells. These data showed that low FtsA levels might be the reason for low percentage of cells having FtsZ ring in ftsH-null cells. It implied that ftsH-null cells might have been managing FtsZ ring stabilisation with ZipA, to facilitate septation. Real time RT-PCR showed that the levels of ftsA mRNA and those of all the other fts genes, except ftsZ, in the 16-gene dcw cluster, were found to be low in ftsH-null cells. Moreover, real time RT-PCR using specific primers designed for multiple promoters of ftsZ and for the RNaseE processing site, just upstream of ftsZ, showed that the levels of transcripts of the genes upstream to RNaseE site were significantly low and that the levels of ftsZ transcripts, which were downstream to RNaseE site, were unaffected. On the contrary, the levels of mRNAs of fts genes, such as ftsE, ftsX, ftsN, and zipA that were located at another part of the genome, were normal in ftsH-null cells. These observations suggested that the reason for the low levels of FtsA protein might be low levels of ftsA mRNA. In addition, the low levels of other fts mRNAs from the dcw cluster, and probably of the respective proteins, might contribute to the slow growth of ftsH-null cells. The ftsH null strains also showed less compact nucleoids and the nucleoids did not look bilobular. This data suggested that there may be some defect in the compaction of nucleoids in ftsH-null cells. On the contrary, isogenic wild type cells, when grown slow like the growth of ftsH-null cells, had no defect in nucleoid compaction and looked bilobular. The proper compaction of nucleoids could be restored only by wild type FtsH, but not by the protease mutant of FtsH. These observations suggest that proteolytic activity of FtsH might be required for the proper compaction of nucleoids, which in turn might have influence on the placement of FtsZ ring at mid-cell site. In parallel, different percentage of silver stained single-dimension SDS-PAGE showed conspicuous difference in the protein profiles of the membrane and soluble fractions of ftsH-null cells, in comparison to those of isogenic wild type cells. FtsZ co-immuno precipitation (CoIP) of total cell lysates of ftsH-null and isogenic wild type cells showed differential interaction of two proteins, the outer membrane protein A (OmpA) and a 50 kDa protein, between the two strains. The level of OmpA was 2.5-fold high in ftsH-null cells, in comparison to that in isogenic wild type cells. However, overexpression of ompA in isogenic wild type cells did not have any effect on FtsZ rings in isogenic wild type cells. Two-dimensional gel electrophoresis for membrane and soluble fractions of ftsH-null cells, in comparison with that of isogenic wild type cells, showed that several proteins in each fraction were either present or absent between these two strains. Most of these proteins were then identified using MALDI-TOF / LC –MS methods. Identification of these proteins, which were present differentially between ftsH-null and isogenic wild type cells, has revealed existence of many more hitherto unidentified potential substrates of FtsH and therefore cell processes, which FtsH may influence.

Many Gram-positive and Gram-negative bacteria produce an additional thick layer of carbohydrate polymers on the cell wall surface. These capsules (capsular polysaccharides; CPS) play critical roles in interactions between bacteria and their environments (Whitfield, 2006). This is especially important in infection processes since for both Gram-negative and Gram-positive pathogens CPS is the point of first contact with the host immune system (Whitfield, 2006). However, the details of CPS biosynthesis and assembly mechanisms are still unclear. Therefore, we embarked on structural and kinetic studies of the proteins Wzc, Wza and Wzb/ Cps4B from the Wzy-dependent pathway, as well as the protein WbdD from the ATP-binding cassette (ABC) transporter dependent system. Full-length Wzc failed to crystallise due to the presence of large disordered regions and the overall difficulty of membrane protein crystallisation. A truncated version of Wzc (1-480) without the C-terminal tyrosine kinase domain was crystallised and diffracted to 15 Å in house. A previous study suggested Wza and Wzc form a functional complex (Whitfield, 2006), so Wza was also studied. Since the full-length Wza structure is available (C. Dong et al., 2006), Pulsed electron–electron double resonance spectroscopy (PELDOR) was used to study the conformational change. The PELDOR spectroscopy distance fingerprint of Wza was determined. These data also confirmed that PELDOR is a powerful tool to study large, highly symmetrical membrane proteins and can be used to study other complex membrane protein systems, such as ion channels or transporters. The crystal structure of Wzb the cognate phosphatase of Wzc was determined to 2.2 Å. Also Cps4B, which is a functional homologue of Wzb but has a completely unrelated sequence, was crystallised in two crystal forms. Form I and II Cps4B crystals diffracted to 2.8 Å and 1.9 Å resolution in house, respectively. The full-length WbdD failed to crystallise due to the presence of large disordered regions. Therefore, a shorter construct, WbdD₅₅₆ (1-556) was cloned and crystallised. The structure was determined to 2.2 Å. WbdD is a bifunctional enzyme consisting of a methyltransferase (MTase) and a kinase domain. In order to better understand the function of this protein, a variety of techniques were used, such as the ADP-Glo kinase assay, Nuclear magnetic resonance (NMR) spectroscopy, small angle X-ray scattering (SAXS) and X-ray crystallography. The various findings in the current projects provide meaningful insights towards a better understanding of the CPS biosynthesis and assembly mechanisms, which may contribute to a more intensive study identifying inhibitors and beginning to unravel the mechanism of chain length regulation.

The physiological role of the teichoic acid polymers found in Gram-positive bacterial cell walls is not known. Studies of Bacillus subtilis hybrid strains implicate a defined chromosomal region, which includes the tag-1 locus, as necessary for teichoic acid biosynthesis. A set of ten mutants carrying lesions in this region was identified from among forty-four temperature-sensitive (ts) mutants generated by nitrosoguanidine mutagenesis and bacteriophage 029 selection. This protocol gave a population enriched for ts, versus auxotrophic, mutants. For each of the ten mutants, the frequency of genetic reconstruction, or correction, of the ts phenotype indicated that it was due to change(s) in a single gene. Results of two-factor transformation crosses sorted the mutants into three complementation groups; all ten could complement tag-1. Mutants in two complementation groups were transformed to ts⁺ with cloned rodC DNA. The map order of the newly isolated ts markers was determined from the results of two factor crosses. Orientation with respect to the hisA marker was inferred from transduction experiments. The newly isolated strains were shown to be conditional rod⁻ mutants. Growth at 48°C resulted in reduced growth rates and spherically shaped cells. Additional phenotypes seen for some mutants, namely 029 phage resistance and ts spore outgrowth, appeared closely associated with the ts rod⁻ mutation. Wall phosphate content for two of the mutants, following growth at 48°C, was found to be reduced in comparison to the wild-type control. Taken together these results lend support to the argument that the tag-1 region of the chromosome, which most likely directs teichoic acid biosynthesis, is important for establishment and maintenance of the normal bacillary morphology seen for B. subtilis. The importance of other gene products to the organization of newly synthesized wall was examined using B. subtilis macrofibers. Left- and right-handed macrofibers were converted to spheroplasts and the multi-celled structures regenerated under the two sets of conditions conducive for production of the original, and inverse hand. The helix hands observed for the regenerated structures always corresponded to those expected on the basis of the parental genotype.

<p>FtsZ, a bacterial tubulin homologue, is a cytoskeleton protein that plays key roles in cytokinesis of almost all prokaryotes. FtsZ assembles into protofilaments (pfs), one subunit thick, and these pfs assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane, and also serves as a scaffold to recruit cell-wall remodeling proteins for complete cell division in vivo. FtsZ can be subdivided into 3 main functional regions: globular domain, C terminal (Ct) linker, and Ct peptide. The globular domain binds GTP to assembles the pfs. The extreme Ct peptide binds membrane proteins to allow cytoplasmic FtsZ to function at the inner membrane. The Ct linker connects the globular domain and Ct peptide. In the present studies, we used genetic and structural approaches to investigate the function of Escherichia coli (E. coli) FtsZ. We sought to examine three questions: (1) Are lateral bonds between pfs essential for the Z ring? (2) Can we improve direct visualization of FtsZ in vivo by engineering an FtsZ-FP fusion that can function as the sole source of FtsZ for cell division? (3) Is the divergent Ct linker of FtsZ an intrinsically disordered peptide (IDP)?</p><p> One model of the Z ring proposes that pfs associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of E. coli FtsZ by inserting either small peptides or whole FPs. Of the four lateral surfaces on FtsZ pfs, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174 located on the left and right surfaces, completely blocked function, and were identified as possible sites for essential lateral interactions. Another goal was to find a location within FtsZ that supported fusion of FP reporter proteins, while allowing the FtsZ-FP to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by super-resolution techniques.</p><p> The Ct linker is the most divergent region of FtsZ in both sequence and length. In E. coli FtsZ the Ct linker is 50 amino acids (aa), but for other FtsZ it can be as short as 37 aa or as long as 250 aa. The Ct linker has been hypothesized to be an IDP. In the present study, circular dichroism confirmed that isolated Ct linkers of E. coli (50 aa) and C. crescentus (175 aa) are IDPs. Limited trypsin proteolysis followed by mass spectrometry (LC-MS/MS) confirmed Ct linkers of E. coli (50 aa) and B. subtilis (47 aa) as IDPs even when still attached to the globular domain. In addition, we made chimeras, swapping the E. coli Ct linker for other peptides and proteins. Most chimeras allowed for normal cell division in E. coli, suggesting that IDPs with a length of 43 to 95 aa are tolerated, sequence has little importance, and electrostatic charge is unimportant. Several chimeras were purified to confirm the effect they had on pf assembly. We concluded that the Ct linker functions as a flexible tether allowing for force to be transferred from the FtsZ pf to the membrane to constrict the septum for division.</p> / Dissertation

Peptidoglycan, the major component of the bacterial cell wall, is essential for cell viability. Several important antibiotics disrupt peptidoglycan metabolism, including the β-lactams and vancomycin. There are several bacterial enzymes involved in peptidoglycan metabolism that are not yet the target of antibiotics, such as the lytic transglycosylases (LTs). Relatively little experimental characterization has been done on LTs, due largely to the difficulties of working with insoluble, heterogeneous, and highly variable peptidoglycan. This research develops a method for the generation of a soluble, homogeneous oligosaccharide substrate that can be used to study LTs. The approach taken was based on the enzymatic degradation of peptidoglycan into fragments of a specific nature, and their separation by HPLC. This work identifies the challenges associated with this approach, and discusses the potential flaws in the 'top-down' generation of a soluble substrate. / This thesis was typeset with LaTeX using Minion Pro and Myriad Pro typefaces.

Thesis (Ph.D.)--University of Wollongong, School of Biological Sciences. / Typescript. Includes bibliographical references: leaf 269-331.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Insect-derived baculoviruses have been used extensively as a safe and versatile research model for transgenic protein expression. Preclinical studies have revealed the promising potential of Baculoviruses as a delivery vector for a variety of therapeutic applications, including vaccination, tissue engineering and cancer treatments. Coronaviruses are enveloped viruses containing linear, non-segmented ribonucleic acid. Human coronavirus NL63 was first discovered in the Netherlands in January 2004, where a 7-month-old girl presented with an acute respiratory tract infection that was later established to predominantly infect infants, the elderly and immunocompromised individuals.

Les enzymes de la famille Mur (MurA-MurG) sont essentielles pour la survie bactérienne, car elles catalysent les étapes cytoplasmiques de la biosynthèse du peptidoglycane, la principale composante de la paroi cellulaire. En outre, les Murs métabolisent des molécules qui sont absentes chez les eucaryotes, et ces enzymes sont structurellement et biochimiquement tractables. Cependant, malgré le fait que nombreux inhibiteurs anti-Mur ont été développés, un nombre tres réduit de ces molécules ont montré une activité antibactérienne prometteuse, ce qui a incité l'hypothèse selon laquelle, dans le cytoplasme bactérien, les enzymes Mur peuvent exister dans un complexe où les sites actifs sont à proximité, bloquant donc l'accès de petites molécules venant de l'extérieur. Cette hypothèse est soutenue par l'observation selon laquelle, dans de nombreux organismes, les gènes codant pour les enzymes Mur sont présents dans un seul opéron, souvent dans le même ordre; en outre, souvent des paires de gènes sont fusionnées pour générer un seul polypeptide, préconisant la possibilité que des complexes entre ces enzymes pourraient être formés dès qu'ils sont synthétisés. Nous avons obtenu les premières informations structurales et fonctionnelles sur la forme fusionnée MurE-MurF, présente dans le pathogène humain Bordetella pertussis, et nous avons montré qu'elle interagit avec la glycosyltransférase périphérique MurG, ce qui suggère la présence d'un complexe enzymatique ternaire. De façon intéressante, nous avons constaté que MurG de B. pertussis est capable de s'associer avec elle-même et de former différentes espèces oligomériques. Cette découverte pourrait renforcer le rôle de MurG en tant que protéine agissant comme une plateform capable d'ancrer d'autres enzymes Mur à la face interne de la membrane cytoplasmique bactérienne. Nos resultats pourront également être explorés pour comprendre le rôle potentiel de MurG en tant que régulateur de l'activité des enzymes de synthèse du PG. Ces résultats passionnants ouvriront le chemin vers la compréhension du mecanisme d’interaction des enzymes Mur dans le cytoplasme bactérien et pourraient permettre l'emploi éventuel des Murs comme cibles de facto pour développer de nouveaux antibiotiques. / Enzymes of the Mur family (MurA-MurG) are essential for bacteria, since they catalyse the cytoplasmic steps of peptidoglycan biosynthesis, the major component of bacterial cell wall; they metabolize molecules that do not exist in eukaryotes, and are structurally and biochemically tractable. However, despite the fact that many anti-Mur inhibitors have been developed, few of these molecules have shown promising antibacterial activity, which has prompted the hypothesis that within the bacterial cytoplasm Mur enzymes may exist in a complex where the active sites are in closed proximity, blocking small molecule access from the outside. This suggestion is supported by the observation that in many organisms, genes encoding Mur enzymes are present in a single operon, often in the same order, and often pairs of genes are fused to generate a single polypeptide, advocating the possibility that complexes between these enzymes could be formed as soon as they are synthesized. We have obtained the first structural and functional information on the MurE-MurF fused form, present in the human pathogen Bordetella pertussis, and shown that it interacts with the peripheral glycosyltransferase MurG, suggesting the presence of a ternary enzymatic complex. Interestingly, we have found that B. pertussis MurG is able to self-associate and form different oligomeric species. This finding could strengthen the hypothesis of MurG as a scaffold protein capable of anchoring other Murs to the inner face of bacterial inner membrane, but could be also further explored to understand its potential role as a regulator of the activity of PG synthesis enzymes. These exciting results will open the path towards the understanding of how Mur enzymes interact within the bacterial cytoplasm, and could permit the eventual employment of Mur enzymes as de facto targets for novel antibiotic development.

Many chemicals are released into surface water and biodegradation is one important mechanismto remove the contaminants. Their actual concentrations in water depend on their persistence.The OECD 309 test is the most recognized method to assess the persistence of chemicals in theaquatic environment. However, the quantity of sediment used in the OECD 309 suspensiontests is not clearly defined. To investigate the influence of sediment concentrations and thebacterial cell count on the biodegradation, we compared the biodegradation rate constants of amixture of organic chemicals in suspension tests with 5 different water-sediment ratios. Thesediment concentrations were 125, 50, 20, 8 and 3 g L-1. The 5 test systems were spiked with129 chemicals including pharmaceuticals, agrochemicals, food additives, and cosmetics at aconcentration of 1 μg mL-1 each. The chemical degradation was monitored by analyzing thewater subsamples collected at different time points during the 14-day incubation period. It wasthe first time that the effects of sediment concentrations on biodegradation were systematicallystudied for a broad range of chemicals. The bacterial cell count was measured to test whether itcorrelated with sediment concentration and biodegradation rate constant. In the present study,k could be quantified for at least 2 sediment concentrations for 10 chemicals. The resultsshowed that the biodegradation rate constants of the chemicals, k, were positively correlatedwith sediment concentration for 10 chemicals; 6 of these correlations were significant. The totalbacterial cell count showed a positive correlation with sediment concentration, but it was notsignificant. Also, k was positively correlated with total cell count, but these correlations werenot significant for any of 10 chemicals. It is concluded that sediment plays an important role inchemical biodegradation. Sediment-associated total cell count can be one factor contributing tothis effect. The variable results of chemical biodegradation rate constants caused by differentsediment concentrations in the present suspension tests suggest the need for a standardizedsediment concentration in OECD 309 tests.