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Antibodies are an essential part of the human immune system, and antibody mediated immunity has been an area of interest for many researchers for almost a century. An accumulation of knowledge regarding antibody structure, glycosylation and receptor interactions has contributed to the current understanding of antibody mediated immunity. It has more recently become evident how bacteria and other microorganisms evade host recognition and eradication through specific antibody degradation or modification. The importance of antibody glycosylation and how glycan modification can fine-tune the elicited immune response has also contributed to the development of antibody-based drugs with improved clinical efficacy. In turn these insights have paved the way and created a need for the development of biotechnological methods and tools to specifically engineer antibodies with defined properties, for analysis to ensure quality and safety, and for improved antibody purification.This thesis highlights the importance of glycosylation for antibody function and presents different aspects and applications of antibody modifications by bacteria. We show, for the first time, activity of the IgG-specific Streptococcal endoglycosidase EndoS during Streptococcus pyogenes infection, clearly demonstrating that EndoS contributes to S. pyogenes pathogenesis and bacterial survival in the context of adaptive immunity. Further this thesis presents the use of bacterial enzymes as antibody modifying tools and their potential as binding reagents for selective antibody purification. The identification and characterization of two novel proteases, BspK and BspE exhibiting unique IgG and IgA cleavage profiles respectively, from Bdellovibrio bacteriovorus highlights the potential of using Bdellovibrio as a source for the identification of novel enzymes with biotechnological applications. Finally, I present the development of a novel method for selective antibody purification, using the inactive variants of

Complex ecosystems harbor multiple predator and prey species whose direct and indirect interactions are understudied. In particular, the combined effects of predator diversity and resource preference on prey removal are not known. To better understand the effect of interspecies interactions combinations of micro-predators – i.e. protists (generalists), predatory bacteria (semi-specialists), and phages (specialists) – and bacterial prey were tracked over a 72-hour period in miniature membrane bioreactors. While specialist predators alone drove their preferred prey to extinction, the inclusion of a generalist resulted in uniform losses among prey species. Most importantly, presence of a generalist predator enabled coexistence of all predators and prey. As the generalist predator also negatively affected the other predators we suggest that resource partitioning between predators and the constant availability of resources for bacterial growth due to protist predation stabilizes the system and keeps its diversity high. The appearance of resistant prey strains and subsequent evolution of specialist predators unable to infect the ancestral prey implies that multitrophic communities are able to persist and stabilize themselves. Interestingly, the appearance of BALOs and phages unable to infect their prey was only observed for the BALO or phage in the absence of additional predators or prey species indicating that competition between predators might influence coevolutionary dynamics.

Soil is a microenvironment with a fragmented (patchy) spatial structure in which many bacterial species interact. Here, we explore the interaction between the predatory bacterium Bdellovibrio bacteriovorus and its prey Escherichia coli in microfabricated landscapes. We ask how fragmentation influences the prey dynamics at the microscale and compare two landscape geometries: a patchy landscape and a continuous landscape. By following the dynamics of prey populations with high spatial and temporal resolution for many generations, we found that the variation in predation rates was twice as large in the patchy landscape and the dynamics was correlated over shorter length scales.We also found that while the prey population in the continuous landscapewas almost entirely driven to extinction, a significant part of the prey population in the fragmented landscape persisted over time. We observed significant surface-associated growth, especially in the fragmented landscape and we surmise that this sub-population is more resistant to predation. Our results thus show that microscale fragmentation can significantly influence bacterial interactions., Accepted Author Manuscript, BN/Cees Dekker Lab

Soil is a microenvironment with a fragmented (patchy) spatial structure in which many bacterial species interact. Here, we explore the interaction between the predatory bacterium Bdellovibrio bacteriovorus and its prey Escherichia coli in microfabricated landscapes. We ask how fragmentation influences the prey dynamics at the microscale and compare two landscape geometries: a patchy landscape and a continuous landscape. By following the dynamics of prey populations with high spatial and temporal resolution for many generations, we found that the variation in predation rates was twice as large in the patchy landscape and the dynamics was correlated over shorter length scales.We also found that while the prey population in the continuous landscapewas almost entirely driven to extinction, a significant part of the prey population in the fragmented landscape persisted over time. We observed significant surface-associated growth, especially in the fragmented landscape and we surmise that this sub-population is more resistant to predation. Our results thus show that microscale fragmentation can significantly influence bacterial interactions., Accepted Author Manuscript, BN/Cees Dekker Lab

Disease-causing microorganisms that have become resistant to drug therapy are an increasing cause of burn, wound, blast and bone infections, with many traditional antimicrobial agents becoming ineffective. Our main hypothesis is that predatory prokaryotes could serve as a novel therapeutic agent to control wound-related bacterial infections. In a previous study, we confirmed that predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are able to prey on a wide range of pathogens including bacteria isolated from Wounded Warriors. The aim of this proposal is to address key questions regarding the safety and efficacy of predatory bacteria and investigating predator prey interactions and resistance. Using enrichment culturing techniques we have verified that no genetically stable predation resistant phenotype developed in host cells following sequential predation. Our data also confirmed that the predators do not breach their host specificity and attack previously resistant bacteria. Cell toxicity assays, using human cell lines, demonstrated that predatory bacteria are significantly less toxic than the control. Finally, using a mouse wound model we determined that administering live predatory bacteria into exposed wounds did not cause any significant adverse effect to the animal or alter wound clinical score compared to a non-infected wound., The original document contains color images.

Bdellovibrio bacteriovorus is an unusual d-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Bdellovibrio bacteriovorus is an unusual d-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Bdellovibrio bacteriovorus is an unusual d-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Bdellovibrio bacteriovorus is an unusual d-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Bdellovibrio bacteriovorus is an unusual d-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Bdellovibrio bacteriovorus is an unusual d-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Bdellovibrio bacteriovorus is an unusual d-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Bdellovibrio bacteriovorus is an unusual d-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Bdellovibrio bacteriovorus is an unusual d-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Bdellovibrio bacteriovorus is an unusual d-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Disease-causing microorganisms that have become resistant to drug therapy are an increasing cause of burns and wound infections. Bdellovibrio and Micavibrio are Gram-negative obligatory predators that feed on other Gram-negative bacteria. The focus of the study was to evaluate the potential application of predatory bacteria (Pb) to control human pathogens most associated with war-related infections. Our findings demonstrated that Pb are able to attack many of the pathogens tested, including bacteria from the genus Acinetobacter, Aeromonas, Aggregatibacter, Bordetella, Burkholderia, Citrobacter, Eikenella, Enterobacter, Escherichia, Klebsiella, Listonella, Morganella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersinia. Pb were also able to attack A. baumannii drug resistant clinical isolates obtained from Wounded Warriors. Positive predation was measured in single and multi-species microbial cultures as well as on monolayer and multilayer pre-formed biofilms grown in static and flow cell systems. The predators were also able to inhibit biofilm formation and remove metabolically inactive biofilms. Bdellovibrio and Micavibrio were shown to prey at 37 deg C but were unable to prey under oxygen limiting conditions. Our study also established that applying biofilm EPS (extracellular polymeric substance) degrading enzymes, with the Bdellovibrio, could enhance the biofilm removal aptitude of the predators. In conclusion, the work presented here highlights the potential use of predatory bacteria as biological based agent for eradicating infection and will pave the way for future studies in animal and human subjects., The original document contains color images.

Recently, a new group of phytate degrading enzymes has been isolated from anaerobic bacteria from the rumen. This enzyme group is characterized by a protein tyrosine phosphatase (PTP) -like active site signature sequence. Searches of genetic databases have identified over 40 homologues to this group of phytate-degrading enzymes in a wide variety of bacterial species. To further our understanding of this enzyme group, two new representative genes from Bdellovibrio bacteriovorus and Pseudomonas syringae pv. tomato DC3000 were overexpressed and their recombinant gene products examined. B. bacteriovorus is a bacterium that preys on other Gram negative bacteria while P. syringae DC3000 is a phytopathogen that infects a variety of plant species. A combination of biochemical, kinetic and site-directed mutagenesis analyses demonstrated that these enzymes dephosphorylate phytic acid (InsP6) following a classical PTP mechanism, and have a high specificity for InsP6. The biological function of these enzymes was studied by over producing HopAO1 and HopAO1 C378S in Saccharomyces cerevisiae. HopAO1 inhibited growth of the yeast while the catalytically inactive HopAO1 C378S mutant had no effect. Future studies with this system will shed light on the biological function of HopAO1, PhyAbb and other PTP-like phytate degrading enzymes.

Recently, a new group of phytate degrading enzymes has been isolated from anaerobic bacteria from the rumen. This enzyme group is characterized by a protein tyrosine phosphatase (PTP) -like active site signature sequence. Searches of genetic databases have identified over 40 homologues to this group of phytate-degrading enzymes in a wide variety of bacterial species. To further our understanding of this enzyme group, two new representative genes from Bdellovibrio bacteriovorus and Pseudomonas syringae pv. tomato DC3000 were overexpressed and their recombinant gene products examined. B. bacteriovorus is a bacterium that preys on other Gram negative bacteria while P. syringae DC3000 is a phytopathogen that infects a variety of plant species. A combination of biochemical, kinetic and site-directed mutagenesis analyses demonstrated that these enzymes dephosphorylate phytic acid (InsP6) following a classical PTP mechanism, and have a high specificity for InsP6. The biological function of these enzymes was studied by over producing HopAO1 and HopAO1 C378S in Saccharomyces cerevisiae. HopAO1 inhibited growth of the yeast while the catalytically inactive HopAO1 C378S mutant had no effect. Future studies with this system will shed light on the biological function of HopAO1, PhyAbb and other PTP-like phytate degrading enzymes.

Recently, a new group of phytate degrading enzymes has been isolated from anaerobic bacteria from the rumen. This enzyme group is characterized by a protein tyrosine phosphatase (PTP) -like active site signature sequence. Searches of genetic databases have identified over 40 homologues to this group of phytate-degrading enzymes in a wide variety of bacterial species. To further our understanding of this enzyme group, two new representative genes from Bdellovibrio bacteriovorus and Pseudomonas syringae pv. tomato DC3000 were overexpressed and their recombinant gene products examined. B. bacteriovorus is a bacterium that preys on other Gram negative bacteria while P. syringae DC3000 is a phytopathogen that infects a variety of plant species. A combination of biochemical, kinetic and site-directed mutagenesis analyses demonstrated that these enzymes dephosphorylate phytic acid (InsP6) following a classical PTP mechanism, and have a high specificity for InsP6. The biological function of these enzymes was studied by over producing HopAO1 and HopAO1 C378S in Saccharomyces cerevisiae. HopAO1 inhibited growth of the yeast while the catalytically inactive HopAO1 C378S mutant had no effect. Future studies with this system will shed light on the biological function of HopAO1, PhyAbb and other PTP-like phytate degrading enzymes.

Recently, a new group of phytate degrading enzymes has been isolated from anaerobic bacteria from the rumen. This enzyme group is characterized by a protein tyrosine phosphatase (PTP) -like active site signature sequence. Searches of genetic databases have identified over 40 homologues to this group of phytate-degrading enzymes in a wide variety of bacterial species. To further our understanding of this enzyme group, two new representative genes from Bdellovibrio bacteriovorus and Pseudomonas syringae pv. tomato DC3000 were overexpressed and their recombinant gene products examined. B. bacteriovorus is a bacterium that preys on other Gram negative bacteria while P. syringae DC3000 is a phytopathogen that infects a variety of plant species. A combination of biochemical, kinetic and site-directed mutagenesis analyses demonstrated that these enzymes dephosphorylate phytic acid (InsP6) following a classical PTP mechanism, and have a high specificity for InsP6. The biological function of these enzymes was studied by over producing HopAO1 and HopAO1 C378S in Saccharomyces cerevisiae. HopAO1 inhibited growth of the yeast while the catalytically inactive HopAO1 C378S mutant had no effect. Future studies with this system will shed light on the biological function of HopAO1, PhyAbb and other PTP-like phytate degrading enzymes.

Recently, a new group of phytate degrading enzymes has been isolated from anaerobic bacteria from the rumen. This enzyme group is characterized by a protein tyrosine phosphatase (PTP) -like active site signature sequence. Searches of genetic databases have identified over 40 homologues to this group of phytate-degrading enzymes in a wide variety of bacterial species. To further our understanding of this enzyme group, two new representative genes from Bdellovibrio bacteriovorus and Pseudomonas syringae pv. tomato DC3000 were overexpressed and their recombinant gene products examined. B. bacteriovorus is a bacterium that preys on other Gram negative bacteria while P. syringae DC3000 is a phytopathogen that infects a variety of plant species. A combination of biochemical, kinetic and site-directed mutagenesis analyses demonstrated that these enzymes dephosphorylate phytic acid (InsP6) following a classical PTP mechanism, and have a high specificity for InsP6. The biological function of these enzymes was studied by over producing HopAO1 and HopAO1 C378S in Saccharomyces cerevisiae. HopAO1 inhibited growth of the yeast while the catalytically inactive HopAO1 C378S mutant had no effect. Future studies with this system will shed light on the biological function of HopAO1, PhyAbb and other PTP-like phytate degrading enzymes.

Recently, a new group of phytate degrading enzymes has been isolated from anaerobic bacteria from the rumen. This enzyme group is characterized by a protein tyrosine phosphatase (PTP) -like active site signature sequence. Searches of genetic databases have identified over 40 homologues to this group of phytate-degrading enzymes in a wide variety of bacterial species. To further our understanding of this enzyme group, two new representative genes from Bdellovibrio bacteriovorus and Pseudomonas syringae pv. tomato DC3000 were overexpressed and their recombinant gene products examined. B. bacteriovorus is a bacterium that preys on other Gram negative bacteria while P. syringae DC3000 is a phytopathogen that infects a variety of plant species. A combination of biochemical, kinetic and site-directed mutagenesis analyses demonstrated that these enzymes dephosphorylate phytic acid (InsP6) following a classical PTP mechanism, and have a high specificity for InsP6. The biological function of these enzymes was studied by over producing HopAO1 and HopAO1 C378S in Saccharomyces cerevisiae. HopAO1 inhibited growth of the yeast while the catalytically inactive HopAO1 C378S mutant had no effect. Future studies with this system will shed light on the biological function of HopAO1, PhyAbb and other PTP-like phytate degrading enzymes.

Recently, a new group of phytate degrading enzymes has been isolated from anaerobic bacteria from the rumen. This enzyme group is characterized by a protein tyrosine phosphatase (PTP) -like active site signature sequence. Searches of genetic databases have identified over 40 homologues to this group of phytate-degrading enzymes in a wide variety of bacterial species. To further our understanding of this enzyme group, two new representative genes from Bdellovibrio bacteriovorus and Pseudomonas syringae pv. tomato DC3000 were overexpressed and their recombinant gene products examined. B. bacteriovorus is a bacterium that preys on other Gram negative bacteria while P. syringae DC3000 is a phytopathogen that infects a variety of plant species. A combination of biochemical, kinetic and site-directed mutagenesis analyses demonstrated that these enzymes dephosphorylate phytic acid (InsP6) following a classical PTP mechanism, and have a high specificity for InsP6. The biological function of these enzymes was studied by over producing HopAO1 and HopAO1 C378S in Saccharomyces cerevisiae. HopAO1 inhibited growth of the yeast while the catalytically inactive HopAO1 C378S mutant had no effect. Future studies with this system will shed light on the biological function of HopAO1, PhyAbb and other PTP-like phytate degrading enzymes.

Recently, a new group of phytate degrading enzymes has been isolated from anaerobic bacteria from the rumen. This enzyme group is characterized by a protein tyrosine phosphatase (PTP) -like active site signature sequence. Searches of genetic databases have identified over 40 homologues to this group of phytate-degrading enzymes in a wide variety of bacterial species. To further our understanding of this enzyme group, two new representative genes from Bdellovibrio bacteriovorus and Pseudomonas syringae pv. tomato DC3000 were overexpressed and their recombinant gene products examined. B. bacteriovorus is a bacterium that preys on other Gram negative bacteria while P. syringae DC3000 is a phytopathogen that infects a variety of plant species. A combination of biochemical, kinetic and site-directed mutagenesis analyses demonstrated that these enzymes dephosphorylate phytic acid (InsP6) following a classical PTP mechanism, and have a high specificity for InsP6. The biological function of these enzymes was studied by over producing HopAO1 and HopAO1 C378S in Saccharomyces cerevisiae. HopAO1 inhibited growth of the yeast while the catalytically inactive HopAO1 C378S mutant had no effect. Future studies with this system will shed light on the biological function of HopAO1, PhyAbb and other PTP-like phytate degrading enzymes.

Recently, a new group of phytate degrading enzymes has been isolated from anaerobic bacteria from the rumen. This enzyme group is characterized by a protein tyrosine phosphatase (PTP) -like active site signature sequence. Searches of genetic databases have identified over 40 homologues to this group of phytate-degrading enzymes in a wide variety of bacterial species. To further our understanding of this enzyme group, two new representative genes from Bdellovibrio bacteriovorus and Pseudomonas syringae pv. tomato DC3000 were overexpressed and their recombinant gene products examined. B. bacteriovorus is a bacterium that preys on other Gram negative bacteria while P. syringae DC3000 is a phytopathogen that infects a variety of plant species. A combination of biochemical, kinetic and site-directed mutagenesis analyses demonstrated that these enzymes dephosphorylate phytic acid (InsP6) following a classical PTP mechanism, and have a high specificity for InsP6. The biological function of these enzymes was studied by over producing HopAO1 and HopAO1 C378S in Saccharomyces cerevisiae. HopAO1 inhibited growth of the yeast while the catalytically inactive HopAO1 C378S mutant had no effect. Future studies with this system will shed light on the biological function of HopAO1, PhyAbb and other PTP-like phytate degrading enzymes.

Bdellovibrio are predatory bacteria that replicate within the periplasm of their Gram-negative bacterial prey. They exhibit a biphasic lifestyle existing as free-swimming cells and inter-periplasmic growing cells. There is great interest in Bdellovibrio spp. due to their potential as antibacterial therapeutics in the current era of increasing levels of antibiotic resistance. These predators are ubiquitous in nature, being found in environments from freshwater, to soil, to the GI tracts of humans. This study isolated novel Bdellovibrio spp. from the GI tract of farm animals and completed initial characterization of these isolates: one isolate from reindeer was found to have greater predation efficiency in a microaerophilic environment and at 37 oC, which contrasts with the well-characterized strains of Bdellovibrio bacteriovorus which grow best in an aerobic environment at 29 °C. This isolate represents the first reported Bdellovibrio spp. that preferentially undergoes a predatory lifecycle under microaerophilic conditions. This study has led us to hypothesize that isolation of novel Bdellovibrio isolates from the proposed environment for future applications may result in the identification of new strains that may be more optimal for future use because they are more likely to have adapted to the environmental conditions they would encounter. The Bdellovibrio bacteriovorus HD100 genome encodes a large catalogue of hydrolytic enzymes which are hypothesized to play a major role in the degradation of the contents of prey cells for uptake as nutrients for growth by the Bdellovibrio. In this study a bioinformatics approach was used to further classify and group these enzymes by similar domain structure. Potential predatory candidates were identified based on previous transcriptomic studies to determine putative roles of each enzyme within the predatory lifecycle. This study will guide the direction of future molecular studies into the functions of the Bdellovibrio enzyme c

Bdellovibrio bacteriovorus are small, predatory bacteria that grow within the periplasmic space of a host bacterium. Bdellovibrio has a biphasic life-cycle switching from a uni-nucleoid, growth-senescent ‘attack-phase’ to a novel, multi-nucleoid filamentous ‘growth-phase’, which elongates and divides, growing saprophytically within the periplasmic space of their prey. Little is known to date about Bdellovibrio developmental processes and cell division within this periplasmic niche. Recent publications have demonstrated that bacterial cytoplasms house highly organised matrices of protein structures, called the bacterial cytoskeleton. The Bdellovibrio processes of prey-cell entry, filamentous cell growth and division coordination brings cellular morphological changes and challenges that could be coordinated by cytoskeletal elements. Green Fluorescent protein (GFP)-tagging and gene knock out approaches were used to gain insights into the function of these elements including: an Intermediate filament like protein Ccrp, which has a role in the maintenance of cell morphology; two actin homologues, which appear to function at different points in the predatory cycle, MreB1 and MreB2; and a new type of cytoskeletal element designated ‘bactofilin’, which may have a role in cell division control. Recent advances in GFP technologies have led to the development of optimised GFP variants, such as mTFP1 and mCherry. These have been used to reveal previously unseen detail of Bdellovibrio development within prey. Bdellovibrio do not follow the familiar pattern of bacterial cell division by binary fission, instead divide synchronously at multiple sites along their length, once prey resources are depleted. This yields both odd and even numbers of progeny Bdellovibrio.

Bdellovibrio bacteriovorus are small, predatory bacteria that grow within the periplasmic space of a host bacterium. Bdellovibrio has a biphasic life-cycle switching from a uni-nucleoid, growth-senescent ‘attack-phase’ to a novel, multi-nucleoid filamentous ‘growth-phase’, which elongates and divides, growing saprophytically within the periplasmic space of their prey. Little is known to date about Bdellovibrio developmental processes and cell division within this periplasmic niche. Recent publications have demonstrated that bacterial cytoplasms house highly organised matrices of protein structures, called the bacterial cytoskeleton. The Bdellovibrio processes of prey-cell entry, filamentous cell growth and division coordination brings cellular morphological changes and challenges that could be coordinated by cytoskeletal elements. Green Fluorescent protein (GFP)-tagging and gene knock out approaches were used to gain insights into the function of these elements including: an Intermediate filament like protein Ccrp, which has a role in the maintenance of cell morphology; two actin homologues, which appear to function at different points in the predatory cycle, MreB1 and MreB2; and a new type of cytoskeletal element designated ‘bactofilin’, which may have a role in cell division control. Recent advances in GFP technologies have led to the development of optimised GFP variants, such as mTFP1 and mCherry. These have been used to reveal previously unseen detail of Bdellovibrio development within prey. Bdellovibrio do not follow the familiar pattern of bacterial cell division by binary fission, instead divide synchronously at multiple sites along their length, once prey resources are depleted. This yields both odd and even numbers of progeny Bdellovibrio.

Cyclic-di-GMP is a ubiquitous second messenger regulating lifestyles across a wide range of bacteria, most notably motility and virulence. The components of c-di-GMP signalling networks are made up of: diguanylate cyclases (DGCs) that contain a GGDEF domain and make c-di-GMP, phosphodiesterases (PDEs) that contain an EAL or HD-GYP domain and break-down c-di-GMP and PilZ domains (or degenerate DGCs/PDEs) which are receptors for c-di-GMP. Bdellovibrio bacteriovorus HD100 is a predatory Gram-negative bacterium that preys upon other Gram-negative bacteria. B. bacteriovorus HD100 has a complex c-di-GMP signalling network, including four DGCs; one EAL domain protein, six HD-GYP proteins, 19 PilZ domain-proteins, a degenerate DGC and a proposed riboswitch all of which can act as cdi-GMP receptors. Bd0367, a DGC, regulates gliding motility and prey cell exit in B. bacteriovorus. Bd0367 has a GGDEF domain but also a response regulator domain, which suggests that c-di-GMP production by Bd0367 may be regulated by a phosphorylation event by a cognate sensor kinase. This thesis investigated the Bd0367 signalling pathway through analysis of Bd0367 protein interaction networks, Bd0367 DGC c-di-GMP production and searched for the cognate histidine kinase for Bd0367. This study confirmed that a conserved aspartate residue is required for Bd0367 to be an active DGC via mutational studies and cellular cdi-GMP extractions. It also demonstrated that Bd0367 enzymatic activity is essential for prey cell exit, but is less important for gliding motility, this was tested by a Bd0367D63A mutation. Which suggests that localised protein interactions formed by Bd0367 (independent of its enzymatic role) may be more important for gliding motility than the Bd0367 specific c-di-GMP generation. This concept was supported by other data as deletion of a gene encoding a PilZ domain protein that interacted with Bd0367 by bacterial two hybrid analysis, caused hyperreversals during gliding motility. Future

Bdellovibrio bacteriovorus are predatory bacteria, which prey on other Gramnegative bacterial species. Bdellovibrio modifies the cell wall peptidoglycan of its prey at several stages of its predatory life cycle: during invasion and entry into the prey periplasm, during the rounding of the prey into a stabilised bdelloplast (in which Bdellovibrio grow, replicate and divide) and at the end of the cycle, at which point the resulting progeny lyse the bdelloplast before repeating the predation process. Bdellovibrio also modify another source of cell wall peptidoglycan during these processes: their own. During the invasion of prey cells, Bdellovibrio adjust their shape to enter prey cells through the size-limited pore in the prey cell envelope. Following this, Bdellovibrio peptidoglycan must be modified during its filamentous growth, division and possibly during the construction and insertion of flagella prior to progeny release. The modification of two “sets” of peptidoglycan was significant in the considerations behind this project. Pioneering work in the 1970s deduced that the combined actions of Bdellovibrio peptidoglycan-modifying enzymes, including an N-deacetylase, were involved in the prey invasion process. Further work by these researchers also proposed mechanisms of bdelloplast stabilisation following predatory invasion. Since then the Bdellovibrio genome has been sequenced, and transcriptomic data alongside bioinformatic investigation, has provided several potential gene targets which may encode such “invasion-enabling” enzymes. I aimed to investigate the roles of these candidate gene products and determine their role in these predatory processes. Other predicted peptidoglycan-active enzymes have been researched for their roles in either the predatory or non-predatory lifestyles of Bdellovibrio in order to begin to separate prey-active proteins from self-active enzymes. Previous work observed that the prey bdelloplast peptidoglycan is Ndeacetylated at the N-acetyl-mu