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Penicillin-binding proteins (PBPs) are required in the synthesis of the cell wall of bacteria. In Bacillus subtilis, PBPs play important roles in the life cycle, including both vegetative growth and sporulation, and contribute to the formation of the different structures of vegetative cell wall and spore cortex. The B. subtilis genome sequencing project revealed there were two uncharacterized genes, ykuA and yrrR, with extensive sequence similarity to class B PBPs. These two genes are renamed and referred to henceforth as pbpH and pbpI, respectively. A sequence alignment of the predicted product of pbpH against the microbial protein database demonstrated that the most similar protein in B. subtilis is PBP2A and in E. coli is PBP2. This suggested that PbpH belongs to a group of the genes required for maintaining the rod shape of the cell. Study of a pbpH-lacZ fusion showed that pbpH was expressed weakly during vegetative growth and the expression reached the highest level at the transition from exponential phase to stationary phase. The combination of a pbpA deletion and the pbpH deletion was lethal and double mutant strains lacking pbpH and pbpC or pbpI (also named yrrR) were viable. The viable mutants were indistinguishable from the wild-type except that the vegetative PG of the pbpC pbpH strain had a slightly slightly lower amount of disaccharide tetrapeptide with 1 amidation and higher amount of disaccharide tripeptide tetrapeptide with 2 amidations when compared to others strains. This suggests that PbpC (PBP3) is involved in vegetative PG synthesis but only affects the PG structure with a very low efficiency. A pbpA pbpH double mutant containing a xylose-regulated pbpH gene inserted into the chromosome at the amyE locus was constructed. Depletion of PbpH resulted in an arrest in cell growth and a dramatic morphological change in both vegetative cells and outgrowing spores. Vegetative cells lacking pbpA and pbpH expression swelled and cell elongation was arrested, leading to the formation of pleiomorphic spherical cells and eventual lysis. In these cells, cell septations were randomly localized, cell walls and septa were thicker than those seen in wild type cells, and the average cell width and volume were larger than those of cells expressing pbpA or pbpH. The vegetative PG had an increased abundance of one unidentified muropeptide. Spores produced by the pbpA pbpH double mutant were able to initiate germination but the transition of the oval-shaped spores to rod-shape cells was blocked. The outgrowing cells were spherical, gradually enlarged, and eventually lysed. Outgrowth of these spores in the presence of xylose led to the formation of helical cells. Thus, PbpH is apparently required for maintenance of cell shape, specifically for cell elongation. PbpH and PBP2a play a redundant role homologous to that of PBP2 in E. coli. A sequence alignment of the predicted product of pbpI against the microbial protein database demonstrated that the most similar protein in B. subtilis is SpoVD and in E. coli is PBP3. This suggested that PbpI belongs to the group of the genes required for synthesis of the spore or septum PG. PbpI was identified using radio-labeled penicillin and found to run underneath PBP4 on SDS-PAGE. PbpI is therefore renamed PBP4b. Study of a pbpI-lacZ fusion showed that pbpI was expressed predominantly during early sporulation. A putative sigma F recognition site is present in the region upstream of pbpI and studies using mutant strains lacking sporulation-specific sigma factors demonstrated that the expression of pbpI is mainly dependent on sigma factor F. A pbpI single mutant, a pbpI pbpG double mutant, and a pbpI pbpF double mutant were indistinguishable from the wild-type. The sporulation defect of a pbpI pbpF pbpG triple mutant was indistinguishable from that of a pbpF pbpG double mutant. Structure parameters of the forespore PG in a pbpI spoVD strain are similar to that of a spoVD strain. These results indicate that PBP4b plays a unknown redundant role. / Master of Science

Indiana University-Purdue University Indianapolis (IUPUI) / Borrelia burgdorferi, a bacterium which causes Lyme disease, is maintained in nature through a cycle involving two distinct hosts: a tick vector and a mammalian host. To adapt to these two diverse environments, B. burgdorferi undergoes dramatic alterations in its surface lipoprotein. Two essential lipoproteins, outer surface protein A (OspA) and outer surface protein C (OspC), are reciprocally regulated throughout the B. burgdorferi lifecycle. Very little is known about the regulation of OspA. These studies elucidate the regulatory mechanisms controlling the expression of OspA. Various truncations of the ospA promoter were created and then studied in our novel in vitro model of ospA repression or grown within the host-adapted model. A T-Rich region of the ospA promoter was determined to be a cis-element essential for both the full expression and full repression of ospA.

In Gram-negative bacteria, the three-layered cell envelope, including the cell wall, outer and inner membranes, is essential for cell survival in the changing, and often hostile environments. Conserved in all prokaryotes, the cell wall is incredibly thin, yet it functions to prevent osmotic lysis in diluted conditions. Based on observations obtained by genetic and chemical perturbations, time-lapse live cell imaging, quantitative imaging and statistical analysis, Part I of this dissertation explores the molecular and physical events leading to cell lysis induced by division-specific beta-lactams. We found that such lysis requires the complete assembly of all essential components of the cell division apparatus and the subsequent recruitment of hydrolytic amidases. We propose that division-specific beta-lactams lyze cells by inhibiting FtsI (PBP3) without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. On the other hand, we demonstrated that cell lysis by beta-lactams proceeds through four physical phases: elongation, bulge formation, bulge stagnation and lysis. Bulge formation dynamics is determined by the specific perturbation of the cell wall and outer membrane plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis, and allows escape and recovery upon drug removal. Asymmetrical in structure and unique to Gram-negative bacteria, outer membrane prevents the passage of many hydrophobic, toxic compounds. Together with inner membrane and the cell wall, three layers of the Gram-negative cell envelope must be well coordinated throughout the cell cycle to allow elongation and division. Part II of this dissertation explores the essentiality of the LPS layer, the outer leaflet of the outer membrane. Using a conditional mutant severely defective in LPS transport, we found that mutations in the initiation phase of fatty acid synthesis suppress cells defective in LPS transport. The suppressor cells are remarkably small with a 70% reduction in cell volume and a 50 % reduction in growth rate. They are also blind to nutrient excess with respect to cell size control. We propose a model where fatty acid synthesis regulates cell size in response to nutrient availability, thereby influencing growth rate. / Chemistry and Chemical Biology

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Autolysins are endogenous enzymes responsible for the cleavage of specific bonds in the bacterial sacculus resulting in damage to the integrity and protective properties of the cell wall. The true biological functions of these enzymes are largely unknown. However, they have been implicated in various important biological synthesis processes making their characterization important. Antibiotic susceptibility testing showed these streptococcal strains to have broad spectrum inhibitory concentrations. The major autolysins of selected streptococcal strains were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymograms). The autolysins were isolated from the specific culture supematants using 4% SDS precipitation and were shown to have apparent molecular masses ranging from 60kDa to 20kDa. Four major autolysins named A, B, C, and D from the Streptococcus milleri 77 strain were characterized. Lytic enzymes were blotted onto polyvinylidene difluoride (PVDF) membrane and N-terminally sequenced. Sequences showed between 100% and 80% similarity to that of a muramidase, glucosaminidase and a peptidase from S. mutans, S. pyogenes and S. pneumonia respectively. Biochemical characterization confirmed autolysin A to exhibit muramidase activity with both autolysin Band C exhibiting endopeptidase activity. Autolysin D showed an 80% N-terminal sequence similarity to Millericin B, a peptidoglycan hydrolase that is known to exhibit peptidase activity. Autolysis was determined using different buffers at two optimal pHs. Assaying for autolytic activity at different growth stages showed autolysis to be moderate during the lag and early exponential phases of the growth cycle. The activities of autolysins were the highest in the late exponential phase and the stationary phase of growth. Zymogram analysis showed that the Streptococcal milleri strains had moderate autolytic expression during the early and late exponential phases of the growth cycle. Control regulatory mechanisms of autolysins were determined in the presence or absence of specific charged groups, such as teichoic acids. In each case the absence of these charged groups inhibited the rate of autolysis, suggesting that the absence of teichoic acids could play a role in the regulation of the autolysins. Two-dimensional-SDS and zymographic-electrophoresis was used to determine total protein profiles for each strain. This is the first report using twodimensional zymography. Specific proteins which were either up- or down-regulated were identified. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

The rivers, lakes and seas are the major water sources for the animal and plant kingdom in this earth. In recent times, the usage and wastage of water have been increasing due to the uncontrolled population growth. In addition to that, the rapid industrialization over the years has led to the gradual depletion of the natural resources like water, soil and air. Some of these industries discharge contaminants like organic products and inorganic (or) toxic heavy metals without treatment into the environment, leading to its degradation. Zinc is the 24th most abundant element present in the earth crust, amounting 75 ppm (0.0075%). The concentration of zinc present in the soil and seawater is about 64 ppm and 30 ppb respectively (Emsley, 2001). Generally, the zinc is found with the base metals such as copper and lead and it has less affinity with oxides and strong affinity with sulphides. Sphalerite, a zinc sulphide ore, is majorly containing 60-62% of zinc. The other sources of zinc from the minerals are smithsonite, hemimorphite, quartzite, and hydro zincate. The major sources of zinc contamination arise from several industrial activities such as mining, coal, waste combustion and steel and iron processing. Drinking water also contains certain amounts of Zn, which may be higher when it is stored in metal tanks. The acute toxicity arises from the ingestion of excessive amounts of zinc salts, either accidentally or as dietary supplement. Vomiting, nausea and stomach cramps usually occur after the consumption of more than 500 mg of zinc sulfate. In addition to that, the higher amounts of zinc affect gastrointestinal tract, liver, bone and prostate glands. Finally, Zn can interrupt the activity in soils, as it negatively influences the activity of microorganisms and earthworms, thus retarding the breakdown of organic matter. To combat this problem, techniques such as chemical precipitation, ion exchange, reverse osmosis, etc. are adopted, but these processes result in a huge amount of secondary sludge formation, inefficient removal of metals and are not cost effective. In recent times, an innovative, eco-friendly, cost-effective method has been introduced to treat the toxic heavy metals namely bioremediation. ―Bioremediation‖ is a process of removal of organic or inorganic contaminants by using bacteria, fungi, algae and its metabolites In this research work, the potential of four bacterial strains of the Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens and the extracellular proteins secreted by these four species for the bio-sorption of zinc has been investigated through batch experiments. The mechanisms of interaction between the zinc ion and the bacterial biomass as well as with the extracellular proteins have been elucidated. Additionally, a carbon paste electrode has been modified by using Pseudomonas sp. and its metabolites to develop biosensors for zinc and the lower limit of detection of zinc in aqueous solution has been determined. The major objectives of this research work are specified below: • To study the potential of Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens for the bio sorption of zinc, in batch systems. • To determine the speciation of zinc with respect to pH in the growth medium and the maximum inhibitory effect of zinc on the growth of the four chosen Pseudomonas sp. • To isolate and characterize the extracellular proteins from the four Pseudomonas sp. such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens. • To study the biosorption of zinc by extracellular proteins secreted by the Pseudomonas sp. • To elucidate the mechanisms involved in the biosorption of zinc at the microbe- metal interface and protein-metal ion interface for all the four systems by different characterization studies such as zeta potential, FTIR analysis and EDAX analysis. • To develop a biomass modified CPE using bacterial cells and extracellular protein to detect the concentration of zinc in aqueous solutions adopting voltammetric techniques. The significant results obtained from this research work are summarized as follows: The initial studies were concentrated on the bio sorption of zinc by using four Pseudomonas species such as P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens. The various factors affecting the bio sorption of zinc by these species were investigated by varying the contact time (10-80 min), pH (2-5±0.2), biomass concentration of the four species in the range of 108- 1011 cells / mL, and the initial zinc concentration from 5 mg/L to 80 mg/L respectively, keeping other parameters such as temperature and agitation speed constant in all the experiments. From the results obtained, the maximum percentage of biosorption achieved by the P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens was found to be 60%, 93%, 70% and 65% respectively for 25 mg/L at pH 5±0.2. The equilibrium time taken by the four species to achieve maximum biosorption was about 10 min and the biosorption kinetics adhered to pseudo-second order reaction and the rate constants were determined for different concentrations of zinc. The biosorption isotherm followed both the Langmuir and Freundlich isotherm models. The Gibbs free energy (ΔG) values determined from the Langmuir isotherm model for all the four systems were found to be -26, -32, -30 and -28 kJ /mole respectively. The Gibbs free energy values indicate that the biosorption of zinc ions onto the bacterial surface is a chemi-sorption process involving co-ordination, complexation or chelation. The characterization studies, namely zeta potential, FTIR analysis and SEM-EDX were also carried out on the bacterial cells before and after interaction with zinc. These studies also provide evidence in support of the complexation of zinc with the functional groups on the bacterial cell surface apart from electrostatic interaction. In the second part of the investigation, the inhibitory effect of zinc on the growth of four Pseudomonas sp. was investigated by varying the concentration of zinc from 50 mg/L to 1000 mg/L and the stability of zinc was analysed with respect to pH (2-12) with different concentrations from 50 - 1700 mg/L. It was found that in the absence of zinc the time taken to reach the exponential phase and the specific growth were almost the same for all the four systems. However, in the presence of zinc ions, the growth of the four Pseudomonas sp. was suppressed beyond 50mg/L of zinc. A control study on the stability of zinc in Luria broth medium showed that zinc was highly stable up to 200 mg/L from pH 2-8. However, the stability of zinc in the growth medium decreased beyond that concentration Additionally, studies on the biosorption of zinc were performed using extracellular proteins isolated from the four Pseudomonas sp. The amount of protein was estimated by the Bradford protein assay method at 594 nm. The biosorption experiments were carried out by varying the protein concentration from 50 to 1000µg/mL and the zinc concentration from 50-1000 mg/L and keeping other parameters fixed, namely such as pH at 5±0.2, reaction time of 20 min, temperature at 30±0.2 and the speed of rotation of 200 rpm. It was found that the maximum percentage of zinc biosorbed by the proteins isolated from P.putida was found to be 91% at 500µg/mL of protein concentration and from the other three species, it was found to be about 60% of biosorption at the same protein concentration. The biosorption isotherms of zinc for extracellular protein adhered to the Giles H1 type for all the four systems. The maximum amount of zinc biosorbed by the protein isolated from P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens was found to be 35.6, 19,18.3 and 10 mg/µg respectively and the Gibbs free energy values were found to be -32, -22,-22 and -23 kJ/mole. The mechanisms involved in protein-zinc interaction were elucidated using FTIR analysis and EDX analysis. The FTIR analysis revealed, that the zinc ions were complexed with carboxylic and amine functional groups. Further, the potential of P.putida, P.alcaligenes, P.aeruginosa and P.fluorescens and their extracellular proteins of P.putida as biosensors for detecting zinc ions in aqueous solutions, using electrochemical methods such as, Cyclic Voltammetry and Differential pulse anodic stripping voltammetry, was assessed. The developed carbon paste electrode coated by the biomass showed an approximately 3-fold increase in the sensing of Zn2+ ion in comparison with the bare electrode. The lower limit of detection of the biosensor for zinc ions by Cyclic voltammetry was found to be 10-6 M, and in case of DPASV the lower limit of detection was about 10-7M. The lower limit of detection of the protein modified biosensor for zinc ions by cyclic voltammetry was found to be 10-7M and in the case of DPASV method the lower limit of detection was found to be 10-9 M.

Mycobacterium tuberculosis (Mtb) is one of the most virulent and prevalent bacterial pathogens across the world. As Mtb infects millions of people a year, it remains essential to study its physiology with the goal of developing new therapeutic interventions. A critical part of the bacteria’s ability to propagate is through successful cell division. Although the process of bacterial cell division and the key proteins therein are well understood in Escherichia coli, much remains to be understood about division in mycobacteria. Genetic and cell biological approaches have recently begun to identify key divisome components in Mycobacterium smegmatis. However, questions remain regarding the role and function of one divisome protein in particular, the DNA translocase FtsK. In this dissertation, I investigated the necessity of FtsK for the growth of mycobacteria. Using an inducible knockdown of FtsK, I present evidence that complete loss of FtsK is required to inhibit growth in both Mtb and M. smegmatis, and that these orthologs share a homologous function. Additional work suggests extended loss of FtsK may be lethal to bacteria. These observations support that FtsK is an essential member of the divisome in mycobacteria, facilitating the processes of growth and division.

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