Caseinolytic protease (ClpP) is a cylindrical protease that degrades proteins in the presence of ATPase chaperones. On its own, bacterial ClpP can only degrade small peptides; however, the addition of a novel class of antibiotics, ADEPs, can cause unregulated proteolysis leading to bacterial cell death. Bacterial ClpP is an attractive target for antibiotic development. A high-throughput screen of small molecules identified a group of compounds which are termed Activators of Self-Compartmentalizing Proteases (ACP). A collection of ACP3 and ACP4/5 analogs was synthesized and investigated for biological activity. The project resulted in compounds with greater activity than the lead structures against isolated E. coli ClpP. Also, several analogs possessed bacteriostatic activity against N. meningitidis and S. aureus cell lines.

Caseinolytic protease (ClpP) is a cylindrical protease that degrades proteins in the presence of ATPase chaperones. On its own, bacterial ClpP can only degrade small peptides; however, the addition of a novel class of antibiotics, ADEPs, can cause unregulated proteolysis leading to bacterial cell death. Bacterial ClpP is an attractive target for antibiotic development. A high-throughput screen of small molecules identified a group of compounds which are termed Activators of Self-Compartmentalizing Proteases (ACP). A collection of ACP3 and ACP4/5 analogs was synthesized and investigated for biological activity. The project resulted in compounds with greater activity than the lead structures against isolated E. coli ClpP. Also, several analogs possessed bacteriostatic activity against N. meningitidis and S. aureus cell lines.

The major chaperones identified in Escherichia coli that assist in protein folding include trigger factor (TF), DnaK/DnaJ/GrpE and GroEL/GroES systems. The main ATP-dependent proteases are ClpXP, ClpAP, HslUV, Lon, and FtsH. From detailed sequence analysis, we found that tig (gene for TF), clpX, and clpP genes co-localize next to each other in most examined bacteria. We hypothesized that TF and ClpXP are functionally associated. TF is a ribosome-associated folding chaperone whereas ClpXP is a degradation complex. ClpX serves as the regulatory ATPase that recognizes substrates, unfolds and translocates polypeptides into ClpP for degradation. I found that TF physically interacts with ClpX, and that they collaborate to enhance degradation of certain ClpXP substrates. It is estimated that TF enhances the degradation of about 2% of newly synthesized E. coli proteins. One of the ClpXP substrates with degradation enhanced by TF was λO, the λ phage replication protein. Furthermore, TF also enhanced the degradation of ribosome-stalled λO nascent chains. Experiments suggest that TF transfers ribosome-stalled λO to ClpX for degradation by ClpP, demonstrating the existence of co-translational protein degradation in E. coli. To understand ClpXP mechanism, we had previously proposed that the degraded peptides are released from ClpP through transient equatorial side pores. To further understand ClpP dynamics, we determined the structure of ClpP(Ala153Cys) in its oxidized state. The structure shows that each opposing pair of protomers is linked by a disulfide bond. Unexpectedly, this structure resembles the compact structures of Streptococcus pneumoniae, Mycobacterium tuberculosis, and Plasmodium falciparum ClpPs, rather than the extended states seen in previous E. coli ClpP structures. Normal mode analysis of ClpP structures suggested that the iii compact structure is a naturally sampled conformation of WT ClpP. My findings provide insights for understanding ClpP dynamics as well as reveal a novel association between ClpXP protease and TF folding chaperone.

All organisms within a forested system play a role in the biogeochemical cycle, not only within the forest but also within the global community. Soil microorganisms are a vital part of this cycle, as they sequester or make nutrients available for the development of the forest environment. When a disturbance event occurs, changes to the environment occur; however, it is unclear how these changes affect the soils microbial community. This 2-year (2007 and 2008) study was carried out to obtain a preliminary assessment of the microbial community structure and nutrient (nitrogen and phosphorus) availability within lodgepole pine stands of the Boreal Plain ecozone in west-central Alberta. Six stands of different ages were selected to determine the differences between pre and post harvest. Nutrient flux measurements were conducted using plant root simulator (PRS) probes to investigate the changes in nutrient availability. The microbial community structures were determined using two biochemical methods. The first one was a community level physiological profile (CLPP), which provides information concerning the functional characteristics of the microbial communities. Phospholipid fatty acid (PLFA) analysis provides information about the physiological characteristics of the microbial community. Analysis of the PRS probes results varied for the two nutrients: phosphorus (P) and nitrogen (N). Nitrogen availability was determined by examining the fluxes of ammonium and nitrate to the PRS probes. These did not show a strong relationship between the different aged stands during 2007 or 2008. In addition, no statistical difference was shown between the 2007 and 2008 data compared to the LFH or the mineral soil of the stands. Phosphorus, however, did show a potential trend where there was an initial increase of available P after harvest and then a gradual decrease, as the forest stands matured. This was strongly observed within the LFH, while there was a slight increase in the mineral layer. These trends remained consistent over the two-year period showing a gradual decrease in P flux to the PRS probes as a stand aged even in just one year. The microbial communities did not show a strong change after a forest-harvesting event. When examining the functional groups, there was a drastic shift in the LFH layer microbial community over the first sampling season. This change remained the same within the beginning of the second sampling year. This shift occurred in all stands due to an environmental factor, which was suspected to be the increase in moisture during the season. The change in the microbial communities was not observed, however, in the mineral layer of the soil when the functional structure was examined. When the physiological composition of the microbial communities was observed, though, using PLFA, it was apparent that the physiological characteristics of the microbial community had changed in the mineral soil. Furthermore, no physiological change was observed in the microbial communities of the LFH, only a functional change.

All organisms within a forested system play a role in the biogeochemical cycle, not only within the forest but also within the global community. Soil microorganisms are a vital part of this cycle, as they sequester or make nutrients available for the development of the forest environment. When a disturbance event occurs, changes to the environment occur; however, it is unclear how these changes affect the soils microbial community. This 2-year (2007 and 2008) study was carried out to obtain a preliminary assessment of the microbial community structure and nutrient (nitrogen and phosphorus) availability within lodgepole pine stands of the Boreal Plain ecozone in west-central Alberta. Six stands of different ages were selected to determine the differences between pre and post harvest. Nutrient flux measurements were conducted using plant root simulator (PRS) probes to investigate the changes in nutrient availability. The microbial community structures were determined using two biochemical methods. The first one was a community level physiological profile (CLPP), which provides information concerning the functional characteristics of the microbial communities. Phospholipid fatty acid (PLFA) analysis provides information about the physiological characteristics of the microbial community. Analysis of the PRS probes results varied for the two nutrients: phosphorus (P) and nitrogen (N). Nitrogen availability was determined by examining the fluxes of ammonium and nitrate to the PRS probes. These did not show a strong relationship between the different aged stands during 2007 or 2008. In addition, no statistical difference was shown between the 2007 and 2008 data compared to the LFH or the mineral soil of the stands. Phosphorus, however, did show a potential trend where there was an initial increase of available P after harvest and then a gradual decrease, as the forest stands matured. This was strongly observed within the LFH, while there was a slight increase in the mineral layer. These trends remained consistent over the two-year period showing a gradual decrease in P flux to the PRS probes as a stand aged even in just one year. The microbial communities did not show a strong change after a forest-harvesting event. When examining the functional groups, there was a drastic shift in the LFH layer microbial community over the first sampling season. This change remained the same within the beginning of the second sampling year. This shift occurred in all stands due to an environmental factor, which was suspected to be the increase in moisture during the season. The change in the microbial communities was not observed, however, in the mineral layer of the soil when the functional structure was examined. When the physiological composition of the microbial communities was observed, though, using PLFA, it was apparent that the physiological characteristics of the microbial community had changed in the mineral soil. Furthermore, no physiological change was observed in the microbial communities of the LFH, only a functional change.

No description available.

Acyldepsipeptide (ADEP1) is an antibiotic that binds to Escherichia coli ClpP, mimicking the interaction that the protease typically establishes with ClpA/ClpX ATPases in bacterial cells. Binding of ADEP1 causes the N-terminal end of the ClpP to adopt a structured β-hairpin and triggers opening of the axial gate in the tetradecameric ClpP. Open conformation of the axial gate causes translocation of the substrates into the catalytic chamber of ClpP and the resultant uncontrolled proteolysis renders cellular death making ADEP1 a potent antibiotic. Our current understanding about the ADEP1-induced open conformation of the axial gate is limited. Based on the existing X-ray structures, it is unclear whether the mechanism of ADEP1-mediated activation of ClpP is conserved in Gram-positive and Gram-negative bacteria. To understand the activation mechanism of ClpP by ADEP1, we obtained Bacillus subtilis ClpP variants with amino acid substitutions in the N-terminal region and tested the effect of these mutations on substrate translocation using fluorescence-based proteolytic assays and cryo-electron microscopy. We found that compromising the integrity of the β-hairpin adopted by the N-terminal region prevented translocation of the substrate into the catalytic chamber of B. subtilis ClpP. These results suggest that the structural requirements for a functional axial channel are conserved in Gram-positive and Gram-negative bacteria. This study defines the structural requirements for ADEP1-mediated activation of the ClpP protease and serves as a model for the functioning of ClpP in the context of the ClpAP and ClpXP complexes. / Thesis / Master of Science (MSc)

As Terras Pretas de Índio (TPIs) na Amazônia Brasileira são altamente férteis e o seu conteúdo químico parece não exaurir mesmo em condições de floresta tropical. Por essa razão, são frequentemente procuradas pelas populações locais para o cultivo de subsistência. A importância das comunidades microbianas tem aumentado o interesse em compreender a relação entre o uso da terra, as comunidades de plantas, os micro-organismos e os processos do ecossistema. Portanto, o objetivo principal desta pesquisa foi investigar as comunidades bacterianas sob a influência da cobertura vegetal em sistemas de uso da terra (floresta secundária e plantio de mandioca) e na rizosfera de plantas leguminonas nativas em comunidades de bactéria das TPIs. Além disso, investigou-se também as bactérias desnitrificantes nesses solos. A área de estudo está localizada na Estação Experimental do Caldeirão, pertencente à Embrapa Amazônia Ocidental, no município de Iranduba-AM. A funcionalidade da comunidade bacteriana foi determinada pela Análise de Perfil Fisiológico da Comunidade Microbiana (CLPP), a estrutura da comunidade bacteriana foi acessada por Polimorfismo do Tamanho do Fragmento de Restrição Terminal (T-RFLP), a composição e distribuição das comunidades bacterianas foram determinadas por sequenciamento em larga escala (pirosequenciamento), e para quantificar as bactérias desnitrificantes foi utilizada a técnica de PCR quantitativa (qPCR). Os estudos foram realizados no laboratório de Biologia Celular e Molecular (CENA / USP) e no departamento de Biogeoquímica (Max Planck Institute for Terrestrial Microbiology). A análise de T-RFLP mostrou que o uso da terra e a sazonalidade afetaram as comunidades bacterianas na TPI, e mostrou também um claro efeito da rizosfera nas comunidades bacterianas. CLPP demonstrou que a atividade funcional da TPI não foi afetada pela sazonalidade. Além disso, a tecnologia de pirosequenciamento foi uma ferramenta importante para diferenciar filotipos raros. Diferenças distintas de alguns filos bacterianos da rizosfera foram observadas, indicando que a zona de raiz contribui para moldar essas comunidades. A abundância relativa do gene nirK não foi afetada pelo uso da terra nos dois tipos de solos. Alterações na estrutura das comunidades dos genes nirK e nosZ foram observadas em ambos os tipos de solos. As comunidades desnitrificantes na TPI pareceram ser mais influenciadas pelo uso da terra do que pela sazonalidade, e ACH foi mais influenciada pelas variações de sazonalidade. / Amazonian Dark Earths (ADEs) in the Brazilian Amazon are highly fertile and its chemical content seems not to get depleted even under tropical humid conditions. For this reason, these soils are frequently searched by local population for subsistence farming. The importance of microbial communities has grown the interest in understanding the relationship between land use, plant communities, microorganisms, and ecosystem processes. Therefore, the main objective of this research was to investigate the effect of vegetation cover in land use systems (secondary forest and cassava plantation) and rhizosphere of native leguminous plants on bacterial communities of ADEs. Furthermore, it was also aimed to investigate denitrifying bacteria in these soils. The study area is located at the Experimental Station of Caldeirão, belonging to Embrapa Amazônia Ocidental, Iranduba, AM. The bacterial community function was determined by Community Level Physiological Profile (CLPP), the bacterial community structure was assessed by Terminal Restriction Fragment Length Polymorphism (T-RFLP), the bacterial community composition and distribution by high-throughput sequencing (pyrosequencing), and the quantification of denitrifier bacteria by Quantitative PCR (qPCR). The studies were performed in the Laboratory of Cell and Molecular Biology (CENA/USP) and the Deparment of Biogeochemistry (Max Planck Institute for Terrestrial Microbiology). T-RFLP analysis showed that land use and seasonality affected bacterial communities in ADE, and also showed a clear rhizosphere effect on bacterial communities. CLPP have shown that ADE functional activity was not affected by seasonality. Furthermore, pyrosequencing technology was an important tool to differentiate rare phylotypes. Distinct differences of some rhizosphere bacterial phyla were also observed, indicating that the root zone contributed to shape these communities. The relative abundance of nirK gene was not affected by land use in both studied soils. Alterations in the community structure of nirK and nosZ genes were observed for both soils. ADE denitrifying communities seemed to be more affected by land use than seasonality, and ACH was more influenced by seasonal variations.

As Terras Pretas de Índio (TPIs) na Amazônia Brasileira são altamente férteis e o seu conteúdo químico parece não exaurir mesmo em condições de floresta tropical. Por essa razão, são frequentemente procuradas pelas populações locais para o cultivo de subsistência. A importância das comunidades microbianas tem aumentado o interesse em compreender a relação entre o uso da terra, as comunidades de plantas, os micro-organismos e os processos do ecossistema. Portanto, o objetivo principal desta pesquisa foi investigar as comunidades bacterianas sob a influência da cobertura vegetal em sistemas de uso da terra (floresta secundária e plantio de mandioca) e na rizosfera de plantas leguminonas nativas em comunidades de bactéria das TPIs. Além disso, investigou-se também as bactérias desnitrificantes nesses solos. A área de estudo está localizada na Estação Experimental do Caldeirão, pertencente à Embrapa Amazônia Ocidental, no município de Iranduba-AM. A funcionalidade da comunidade bacteriana foi determinada pela Análise de Perfil Fisiológico da Comunidade Microbiana (CLPP), a estrutura da comunidade bacteriana foi acessada por Polimorfismo do Tamanho do Fragmento de Restrição Terminal (T-RFLP), a composição e distribuição das comunidades bacterianas foram determinadas por sequenciamento em larga escala (pirosequenciamento), e para quantificar as bactérias desnitrificantes foi utilizada a técnica de PCR quantitativa (qPCR). Os estudos foram realizados no laboratório de Biologia Celular e Molecular (CENA / USP) e no departamento de Biogeoquímica (Max Planck Institute for Terrestrial Microbiology). A análise de T-RFLP mostrou que o uso da terra e a sazonalidade afetaram as comunidades bacterianas na TPI, e mostrou também um claro efeito da rizosfera nas comunidades bacterianas. CLPP demonstrou que a atividade funcional da TPI não foi afetada pela sazonalidade. Além disso, a tecnologia de pirosequenciamento foi uma ferramenta importante para diferenciar filotipos raros. Diferenças distintas de alguns filos bacterianos da rizosfera foram observadas, indicando que a zona de raiz contribui para moldar essas comunidades. A abundância relativa do gene nirK não foi afetada pelo uso da terra nos dois tipos de solos. Alterações na estrutura das comunidades dos genes nirK e nosZ foram observadas em ambos os tipos de solos. As comunidades desnitrificantes na TPI pareceram ser mais influenciadas pelo uso da terra do que pela sazonalidade, e ACH foi mais influenciada pelas variações de sazonalidade. / Amazonian Dark Earths (ADEs) in the Brazilian Amazon are highly fertile and its chemical content seems not to get depleted even under tropical humid conditions. For this reason, these soils are frequently searched by local population for subsistence farming. The importance of microbial communities has grown the interest in understanding the relationship between land use, plant communities, microorganisms, and ecosystem processes. Therefore, the main objective of this research was to investigate the effect of vegetation cover in land use systems (secondary forest and cassava plantation) and rhizosphere of native leguminous plants on bacterial communities of ADEs. Furthermore, it was also aimed to investigate denitrifying bacteria in these soils. The study area is located at the Experimental Station of Caldeirão, belonging to Embrapa Amazônia Ocidental, Iranduba, AM. The bacterial community function was determined by Community Level Physiological Profile (CLPP), the bacterial community structure was assessed by Terminal Restriction Fragment Length Polymorphism (T-RFLP), the bacterial community composition and distribution by high-throughput sequencing (pyrosequencing), and the quantification of denitrifier bacteria by Quantitative PCR (qPCR). The studies were performed in the Laboratory of Cell and Molecular Biology (CENA/USP) and the Deparment of Biogeochemistry (Max Planck Institute for Terrestrial Microbiology). T-RFLP analysis showed that land use and seasonality affected bacterial communities in ADE, and also showed a clear rhizosphere effect on bacterial communities. CLPP have shown that ADE functional activity was not affected by seasonality. Furthermore, pyrosequencing technology was an important tool to differentiate rare phylotypes. Distinct differences of some rhizosphere bacterial phyla were also observed, indicating that the root zone contributed to shape these communities. The relative abundance of nirK gene was not affected by land use in both studied soils. Alterations in the community structure of nirK and nosZ genes were observed for both soils. ADE denitrifying communities seemed to be more affected by land use than seasonality, and ACH was more influenced by seasonal variations.

Caseinolytic protease (ClpP) is a cylindrical protease forming the core of protein degradation machinery in eubacteria. ClpP is tightly regulated and is non-functional without a member of the Clp-ATPases. A new class of antibiotics, termed ADEPs, bind to ClpP and allow for activation without the Clp-ATPases; leading to cell death. A more efficient synthetic route to the ADEPs utilizing solid-phase peptide synthesis was investigated. A linear peptide was synthesized, however attempts to close the depsipeptidic macrocycle via macrolactonization failed. Further attempts of assembling a branched depsipeptide for ring closure via a macrolactamization resulted in products that were not stable to cleavage conditions. A group of molecules termed Activators of Self-Compartmentalizing Proteases (ACP) were identified through a screen for activity towards ClpP. Compound ACP1 was synthesized along with twelve analogs and their activity towards ClpP evaluated. The project resulted in a compound with a higher activity than its natural product counterpart.