Bacillus amyloliquefaciens FZB42 ist ein grampositives Bakterium, das in der Rhizosphäre das Pflanzenwachstum fördert (PGPR - Plant Growth Promotion) und pathogene Organismen hemmt. Abgesehen von dieser Fähigkeit produziert es eine Vielzahl von sekundären Metaboliten, die sowohl ribosomale als auch nicht-ribosomale Peptide umfassen. In dieser Arbeit erfolgte die Untersuchung der transkriptionellen Aktivierung und Regulation der Bacilysin- Biosynthese an den Promotoren der bac- und ywfH- Gene. Durch 5´-Deletionsanalysen wurde der Promotor von Bacilysin identifiziert. Die A (Sigmafaktor A) - abhängige Transkription startet über die konservierten Promotorelemente (-10 und -35) von den bac- und ywfH Genen. Die Untersuchungen der Promotoraktivitäten vom Wildtyp und den erzeugten Regulationsmutanten erfolgten über in vivo ß-Galaktosidase-(Reporter)-Assays. Die Ergebnisse der Reporter-Aktivitäten zeigten, dass Transkriptionsregulatoren die Expression der Bacilysin- Gene aktivieren. Mehrere globale Regulatoren wie DegU, ComA, Hpr und AbrB beeinflussen die Genexpression. In dieser Arbeit wurde mithilfe von DNaseI Footprinting-Analysen die DegU-Bindung an die bac- und ywfH- Promotoren bestätigt.Die negative Regulation der Bacilysin-Biosynthese wird durch den Regulator der transienten Phase Hpr bewerkstelligt. Eine direkte Hpr-Bindung an bac Promotor wurde mit DNaseI Footprint-Analysen gezeigt. Der Promotor des monocistronischen Gens ywfH wurde aber durch Hpr nicht beeinflusst. Die anderen Transkriptionsregulatoren, wie ComA und AbrB, regulieren die Genexpression von Bacilysin indirekt über DegQ und Hpr. In dieser Arbeit konnte demonstriert werden, dass der globale Regulator AbrB den Promotor vom hpr-Gen direkt kontrolliert. Zusammenfassend liefert diese Studie neue Informationen über die genetische Regulation der Bacilysin- Biosynthese in B. amyloliquefaciens FZB42. / Bacillus amyloliquefaciens FZB42 is a Gram-positive, pathogen-suppressing and plant-growth promoting rhizobacterium. Apart from this ability, it produces a vast array of secondary metabolites, which includes both ribosomal and non-ribosomal peptides. In this work, the transcriptional activation and regulation of bacilysin biosynthesis were studied at the promoters of bac and ywfH genes. The promoter of bacilysin was identified using 5''-deletion analysis. Sigma factor A (σA) was found to start transcription via conserved promoter elements (-10 and -35) of bac and ywfH genes. lacZ reporter fusion studies were performed in wild type and regulatory mutants. The results show the involvement of transcriptional regulators to activate the expression of bacilysin genes. Several global regulators such as DegU, ComA, Hpr and AbrB were identified and found to influence gene expression. In particular, I confirmed DegU binding in bac and ywfH promoters using radioactive DNase I footprinting. Furthermore, Hpr, a transition state regulator was found negatively to control bacilysin biosynthesis. Hpr binding to bac promoter was demonstrated using radioactive DNase I footprinting. Remarkably, Hpr does not influence the promoter of the monocistronic gene, ywfH. The other transcriptional regulators, such as ComA and AbrB, were correlated indirectly to affect the gene expression of bacilysin via DegQ and Hpr, respectively. The gene regulation of hpr was studied in this work. It was demonstrated that AbrB, a global regulator, directly controls the promoter of the hpr gene. However, the consensus sequence for AbrB binding was not identified, since it covers the entire promoter region in the DNA-protein interaction study. To conclude, this study provides new information regarding the genetic regulation of bacilysin biosynthesis in B. amyloliquefaciens FZB42.

The members of the genus Bacillus produce a wide variety of secondary metabolites with antimetabolic and pharmacological activities. These metabolites are mostly small peptides and have unusual components and chemical bonds. These metabolites are synthesized nonribosomally by multifunctional enzyme complexes called peptide synthetases. One of those small peptides, bacilysin, is a dipeptide antibiotic composed of L-alanine and L-anticapsin which is produced and excreted by certain strains of Bacillus subtilis. Proteins that are responsible to synthesize bacilysin are encoded by bac operon. It has been shown that the biosynthesis of bacilysin is under the control of quorum sensing global regulatory pathway through the action of ComQ/ComX, PhrC (CSF), ComP/ComA in a Spo0K (Opp)-dependent manner. The objective of the study is to identify the functional roles of bacilysin biosynthesis in the regulatory cascade and idiophase cell physiology operating in B. subtilis by using gel-based and gel-free proteomics techniques. For this, we employed comparative proteome-wide analysis of the bacilysin producer B. subtilis PY79 and its bacilysin nonproducer derivative bacA::lacz::erm OGU1 strain which was recently constructed by our group. Identification via GeLC analysis of 76 differentially expressed proteins from total soluble proteome of wild-type PY79 and bacilysin minus OGU1 strain indicated the direct or indirect multiple effects of bacilysin on metabolic pathways, global regulatory systems and sporulation.

In Bacillus subtilis, genetic competence, sporulation and antibiotic production are controlled by quorum-sensing global regulatory mechanism. Bacilysin, being produced and excreted by certain strains of Bacillus subtilis, is a dipeptide antibiotic composed of L-alanine and L-anticapsin. We showed that the biosynthesis of bacilysin is under the control of quorum sensing global regulatory pathway through the action of ComQ/ComX, PhrC (CSF), ComP/ComA in a Spo0K (Opp)-dependent manner. Recently, the ywfBCDEF genes of B. subtilis 168 were shown to carry biosynthetic core function and renamed as bacABCDE operon. The objective of the present study is to elucidate the effects of previously-identified genes srfA, oppA, comA, phrC, phrF, phrK, comQ (comX), comP, spo0H, spo0A, abrB and codY on the expression of bacilysin biosynthetic operon bacABCDE. In order to monitor the expression of bac operon a B. subtilis strain, namely OGU1, containing a transcriptional bacA-lacZ fusion at bacA locus was constructed. Subsequently, each of the above-mentioned genes of cell density signaling was insertionally inactivated by transforming the competent cells of OGU1 with chromosomal DNA of the corresponding blocked mutant strains. The resulting strains and OGU1 as the control were cultured in PA medium and bacA-directed &amp / #946 / -galactosidase activities were monitored. bacA-lacZ expression was severely impaired in the srfA, oppA, comA, phrC, phrF, phrK, comQ (comX), comP, spo0H and spo0A disrupted mutants. On the other hand, in the abrB single mutant bacA expression level increased nearly 2-fold during exponential growth and in the codY mutant it severely decreased during the stationary phase.

The members of the genus Bacillus produce a wide variety of secondary metabolites with antimetabolic and pharmacological activities. Most of these metabolites are small peptides that have unusual components and chemical bonds and synthesized nonribosomally by multifunctional enzyme complexes called peptide synthetases. Bacilysin, being produced and excreted by certain strains of Bacillus subtilis, is one of the simplest peptide antibiotics known. It is a dipeptide with an N-terminal L-alanine and an unusual amino acid, L-anticapsin, at its C-terminal. Recently, ywfBCDEF operon of B. subtilis 168 was shown to carry bacilysin biosynthesis function, the genes of this operon were renamed as bacABCDE. The first member of bac operon, bacA gene was proved to encode the function of L-alanine &ndash / L-anticapsin amino acid ligation. Bacilysin production is regulated at different levels, negatively by GTP via the transcriptional regulator CodY and AbrB while positive regulation occurs by guanosine 5

Proteins performing catalytic roles predominantly occur in a few protein folds. Functional diversity within a common structural scaffold has been attributed to conformational features that enable exploration of reaction space. In this study, we examined specific aspects of functional diversity in the Double Stranded β-helix(cupin) fold. The cupin domain is a hyper-stable protein fold that can support a variety of functions. Variation in function using a conserved active site in the cupin fold is achieved by changes in the residues that line the active site cavity as well as by the choice of a metal cofactor. Although this appears to be a likely basis for functional diversification, a few exceptions exist. It is thus interesting to examine how enzymes with the same structure, metal cofactor and ligand coordination catalyze a diverse range of reactions. This thesis describes two bi-cupins, BacB (also known as bacilysin synthase, YwfC) and Quercetinase (YxaG). BacB is a part of the protein machinery involved in the synthesis of a di-peptide antibiotic bacilysin. The case of the bicupin protein BacB illustrates the problem of functional annotation of proteins with the cupin fold. None of the predicted functions for this enzyme could be experimentally validated in vitro. The crystal structure, determined by Single-wavelength Anomalous Dispersion (SAD) based on the bound metal-ion at the active site provided a basis to evaluate the catalytic role of this protein. Eventually, the function of this protein could be determined based on characterizing the gene product of bacA, the gene preceding bacB in the B. subtilis bac operon. The crystal structure determination of BacB also led to an analysis of multiple crystal forms, with implications for the role of molecular symmetry in forming protein crystals. The stability of the cupin domain was examined using B. subtilis quercetinase as a model system. The availability of the crystal structure and a robust activity assay enabled us to examine the role of fragment complementation in the stability of the cupin scaffold and its implications for the function of this enzyme. This thesis also has a section on the use of structural homology for function annotation for cupin proteins. The results presented here thus provide a frame-work to understand the structural basis for functional diversity in the cupin family. This thesis is organized as follows: Chapter 1: This chapter provides an introduction to the Double Stranded β-Helix-Helix (DSBH or cupin) fold. Proteins with a cupin scaffold are remarkably diverse - spanning both enzymatic and non-enzymatic functions. This chapter presents a compilation of previous reports encompassing eighteen different functional classes. These functions include seed storage, transcription factors and a host of various enzymatic activities. Cupin proteins can be monocupins, bicupins or multi-domain cupins based on the number of DSBH domains in a single polypeptide chain. Very few multi-domain cupin proteins have been identified and this is generally not considered to be a significant sub-group. The inference that cupin proteins with more than one domain are products of gene duplication events is also examined in detail. The latter part of this chapter aims to provide an introduction to the two model proteins B. subtilis BacB and Quercetinase. Chapter 2: This chapter describes studies on a bi-cupin protein BacB involved in bacilysin synthesis. Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (L-alanine-[2,3-epoxycyclohexano-4]-L-alanine) is achieved by proteins in the bac operon, also referred to as the bacABCDE (ywfBCDEF) gene cluster in B. subtilis. The production of this antibiotic is regulated via a stringent response and branches off the pathway for aromatic amino-acid biosynthesis at prephenate. Extensive genetic analysis from several strains of B. subtilis suggests that the bacABC gene cluster encodes all the proteins that synthesize the epoxyhexanone ring of L-anticapsin. This data, however, could not be reconciled with the putative functional assignments for these proteins whereby BacA, a prephenate hydratase along with a potential isomerase/guanylyl transferase, BacB and an oxidoreductase, BacC, could synthesize L-anticapsin. Here, based on the characterization of the reaction products of BacA and BacB as well as the crystal structure of BacB, we demonstrate that B. subtilis BacB catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to L-anticapsin. The mass and NMR spectra of the reaction product of BacA suggest that BacA is a decarboxylase that acts on prephenate. BacB is an oxidase. This protein is a bi-cupin, with two putative active sites each containing a bound metal ion. Additional electron density at the active site of the C-terminal domain of BacB could be interpreted as a bound phenylpyruvicacid (PPY). A significant decrease in the catalytic activity of a point variant of BacB with a mutation at the N-terminal domain suggests that the N-terminal cupin domain is involved in catalysis. Chapter 3 is based on the crystal packing analysis of three different crystal forms of B. subtilis BacB. BacB is an oxidase that catalyzes the production of the di-peptide antibiotic bacilysin. This protein is a bi-cupin with two double stranded β-helix domains fused in a compact arrangement. BacB crystallizes in three crystal forms, belonging to the triclinic, monoclinic and tetragonal space groups. These different crystal forms could be obtained in similar crystallization conditions. We also note that a slight disturbance to the crystallization droplet results in nucleation events, eventually resulting in a different crystal form. All three crystal forms of BacB diffract to high resolution, thus enabling the structure determination and analysis of the packing arrangements of BacB in different space groups. Metal ions at the lattice interface dominate the different packing arrangements. The crystal packing reveals that a dimer of BacB serves as the template on which higher order symmetrical arrangements are formed. BacB, however, is a monomer in solution. The different crystal forms of BacB thus provide experimental evidence to the hypothesis that molecular symmetry could aid crystallization. Chapter 4 provides a conformational analysis of the cupin fold using B. subtilis quercetinase as a model system to understand the conformational determinants of functional diversity. Controlled proteolysis experiments revealed that this enzyme is active, thermo-stable and maintains its quaternary arrangement even after substantial (ca 33 %) cleavage of the protein. The results presented in this chapter thus show that the cupin scaffold offers a balance between protein stability and function by locating the active site and substrate recognition features in the most stable region of the protein. Chapter 5 is based on the phylogenetic analysis of cupin domains. The members of cupin superfamily exhibit large variations in their sequences, functions, organization of domains, quaternary association and the nature of bound metal ion despite having a conserved β-barrel structural scaffold. Here, an attempt was made to understand structure-function relationships among the members of this diverse superfamily and identify the principles governing functional diversity. The cupin superfamily also contains proteins for which structures are available through world-wide structural genomics initiatives but characterized as “hypothetical”. We have explored the feasibility of obtaining clues to functions of such proteins by means of comparative analysis with cupins of known structure and function. This phylogenetic strategy was applied to BacB leading to clustering with oxidoreductases. BacB was experimentally demonstrated to be an oxidase. Chapter 6 is a summary of the work reported in this thesis and the conclusions that can be drawn based on these studies. The appendix section of this thesis comprises additional experimental details, methodology and aspects of the techniques used in this study. Appendix I contains a description of a methodology for Molecular Replacement (MR) calculations in obtaining phase information for protein crystallography. Appendix II provides additional details of experimental protocols.

The biosynthetic pathway for the production of the dipeptide antibiotic bacilysin has been the subject of intense research over the past three decades. These studies revealed the role of multiple enzymes in the biosynthesis of this antibiotic. The identification of different enzymes was initially guided by genetic studies on different strains of Bacillus. The functional role of some these enzymes have been validated in vitro in the recent past. Despite this, the in vitro synthesis of bacilysin still remains elusive. The focus of this study was on two oxidoreductases - BacC and BacG. In the course of these studies, several variations to conventional oxidoreductase mechanisms were observed. These studies also provided us an opportunity to examine an oxidoreductase, BacC, at atomic resolution. This thesis describes these structural studies alongside efforts to achieve the biosynthesis of bacilysin in vitro. Chapter 1 provides an introduction to the broad goals of this thesis. First, the diversity of naturally occurring antibiotics is described. This is followed by a description of nonribosomal peptides and their preferred route for antibiotic synthesis. A summary of previous work in this area is provided to place this study in perspective. Earlier studies performed in this laboratory and others provided a framework for understanding the role of BacC and BacG. These studies have been described with an emphasis on the pivotal role of oxidoreductases in this process. In this context, known features of oxidoreductases, classification of the enzyme family, known reaction mechanisms, preferred substrates and cofactors of the enzyme have been summarized in this chapter. Chapter 2 describes the structural and biochemical characterization of B.subtilis BacG. The crystal structures of BacG determined in the apo form and ligand bound states could capture different conformational states of this enzyme. These structures revealed a basis to understand the ping-pong reaction mechanism. The catalytic residues Tyr-Ser-Lys-Asn involved in the proton relay were examined by mutational analysis. These biochemical studies could corroborate our observations derived from structural analysis. Put together, these studies suggest synchronized conformational changes in BacG that can rationalize cofactor specificity and catalytic action on di hydroxyphenyl pyruvate to form tetra hydroxyphenyl pyruvate en route to anticapsin biosynthesis. Bacillus subtilis BacC could be structurally characterized at 1.19Å resolution. The atomic resolution structure formed the basis for the analysis reported in this chapter. The structure revealed aspects of non-covalent interactions that could be unambiguously determined due to the high resolution diffraction data. The atomic resolution structure also enabled us to conduct charge density analysis on this protein. Atomic displacement parameters were used as a tool to explore paths of non-covalent interactions. A commercially available substrate, 3-Quinuclidinone, was used to characterize enzymatic activity. We note that this enzyme follows a rapid equilibrium random mechanism. Furthermore, the kinetic profiles were conclusive to draw inferences on allosteric interactions. A comparison between the NADH-complex and the apo enzyme structure suggests aspects of nuanced atomic displacement that governs the intra structural signal transduction. Taken together, this study provided a template to analyze the role of non-covalent interactions in regulating enzymatic activity. Chapter 4 is based on a survey of oxidoreductases that have been previously described in literature. During this study, we collated the extensive structural and biochemical data in this family of enzymes. However, we noted that the data remains disperse thereby limiting efforts to understand the reaction mechanism from a structural perspective. Here we collate information of known sequences, structures, cofactors, ligand preferences, reaction mechanisms and their influence on higher order association and catalytic activity in this class of enzymes. Chapter 5 summarizes the findings on two oxidoreductases (BacC, BacG). These studies on two closely related oxidoreductases BacC and BacG performing different roles in the same biosynthetic pathway revealed aspects biosynthesis that are often poorly recognized in protein engineering. The role of the reaction mechanism and their influence on the cofactor specificity could be inferred from the studies on these two enzymes. These studies also suggest the feasibility of evaluating aspects of enzyme activity and regulation provided the wealth of a priori information that is currently available. Put together, these studies provide a data-set for protein engineering efforts on oxidoreductases with general inferences for other enzymes in the short-chain dehydrogenases/ reductases (SDR) family. Appendix 1 provides a schematic representation of our efforts to biosynthetically obtain bacilysin in vitro. The identification, mass spectrometry of the products and substrates en route to bacilysin biosynthesis are compiled in this section. Appendix 2 describes the preliminary characterization of B.subtilis BacF. This part of the work describes the cloning, expression and purification of BacF and attempts to obtain suitable diffracting crystals for structural analysis.