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With the rise of antibiotic resistance, there is ongoing need to find new antibiotics. As bacteria develop resistance to the current classes of antibiotics available, it is imperative to discover new ways to target bacteria. In this thesis, I focused on one of the basic components that all bacteria need to survive: a source of carbon. Here, I explore whether we can exploit this aspect for drug discovery. For bacteria to colonize a host and cause an infection, it must first be able to meet its nutritional needs for growth. Different host infection sites will have different carbon sources available. Some sites, like the gut, will have commensal bacteria which will compete with invading pathogens for carbon sources. While we still lack understanding of the specific growth environment bacteria experience during infection, it is important to understand how bacteria grow when given different nutrients. For the first part of my work, I systematically probed the gene essentiality patterns of E. coli grown in different carbon sources. I generated a large dataset of growth phenotypes that I compiled into a user-friendly web-application, Carbon Phenotype Explorer (CarPE). I identified many poorly annotated genes, and further characterized the gene ydhC as an adenosine transporter. After characterizing how the growth of E. coli and the genes essential for survival change depending on each carbon source, I looked at whether antibiotic efficacy changed depending on the carbon source used. I found that growth in oxaloacetate alters the proton motive force and potentiates macrolide antibiotics. I also found that linezolid, a compound that does not work on gram-negative bacteria due to efflux, is more effective when adenosine is the carbon source. Together, this work forms a foundation for future research into studying how carbon sources can be exploited in the field of antibiotic discovery. / Thesis / Candidate in Philosophy / There is an urgent need for new antibiotics. Previous antibiotic discovery has primarily been conducted on bacteria growing in nutrient rich laboratory conditions. This led to antibiotics that targeted the same few bacterial processes. However, since bacteria need to survive in a host to cause an infection, there are targets that may be viable during an infection that we miss by using standard laboratory media. Bacteria need a source of carbon to survive, and each infection site contains different chemicals that bacteria can use as a source of carbon. My work studies how bacteria grow in the presence of different carbon sources. First, I systematically tested which bacterial genes are required for E. coli to grow in 30 different carbon sources. I then examined the effectiveness of antibiotics on bacteria grown using these different carbon sources. Together, this work helps us understand how changing carbon sources in the growth media we use to cultivate bacteria can change which genes are required and how it may change how bacteria survive antibiotic stress. When we discover the specific compositions of host infection environments, we can leverage this knowledge to find antibiotics that target these carbon acquisition pathways in bacteria.

In this dissertation I describe a model system to characterize the response of an oral bacterial pathogen, Aggregatibacter actinomycetemcomitans to the metabolic byproducts of a representative member of the oral flora, Streptococcus gordonii. A. actinomycetemcomitans is a causative agent of periodontal infections in humans. To cause infection, A. actinomycetemcomitans must overcome numerous challenges, including the host immune system and toxic metabolite production from other microbes. The most numerically dominant flora in the oral cavity are oral streptococci, which are well known for their ability to produce copious amounts of lactic acid and H₂O₂. By studying the response to H₂O₂ and lactic acid in pure and co-cultures, I have demonstrated that A. actinomycetemcomitans responds to these metabolites by several novel mechanisms that both enhance its survival in the presence of the host immune system and in the presence of the model oral streptococci S. gordonii. These studies have demonstrated that metabolites produced by normal flora can impact the survival of a single species in vivo as much as previously known virulence factors have done. In addition, I present a new method for measuring metabolite production in an attached cell population. This method is a novel application of scanning electrochemical microscopy (SECM) and I used this technique to study H₂O₂ production in the three dimensional space surrounding a multispecies biofilm in real time. In a related study I present the use of SECM to discover a novel redox chemistry phenomenon in the opportunistic pathogen Pseudomonas aeruginosa. / text

Salmonella enterica is a facultative intracellular pathogen capable of eliciting severe, systemic disease necessitating antibiotic intervention. Systemic infection is facilitated by intracellular replication within host immune cells, which is enabled by complex regulatory networks governed by two-component systems (TCSs). Intracellular-active TCSs sense antimicrobial chemical cues in the microenvironment and respond adaptively through transcriptional regulation to support intracellular survival. SsrA/SsrB and PhoQ/PhoP are two essential TCSs that elicit a robust defense against host immunity by regulating clusters of virulence genes and integrating novel targets to support regulon expansion and enhance pathogenicity. Metabolic adaptation is critical to bacterial survival and can initiate host-pathogen interactions that influence infection outcome. Further, mitigation of host immunity by manipulation of arginine metabolism has been documented in intracellular pathogens. Herein, I investigated TCS-mediated regulatory evolution pertaining to arginine metabolism, hypothesizing that adaptations to metabolic regulation might confer a fitness advantage to Salmonella replicating intracellularly. I explored intracellular regulation of de novo biosynthesis and extracellular import of arginine, establishing PhoP-mediated regulation of arginine transport. I determined that arginine transport contributes to bacterial fitness in macrophages and began to investigate the mechanism by which arginine importation enriches for intracellular replication. This work informs on evolutionary mechanisms that serve to enhance virulence in Salmonella and provides further insight into our understanding of the intracellular lifestyle of infection. / Thesis / Master of Science (MSc) / Salmonella enterica is an intestinal pathogen that survives within host immune cells and causes systemic disease. These bacteria replicate within antimicrobial cells by using sensory networks to detect harmful immune factors and respond adaptively by eliciting change in gene expression to defend against immune-based killing. The amino acid arginine is an important component of host immunity, as well as bacterial antimicrobial defenses; therefore, I hypothesized that bacterial metabolism might be adapted to the host immune cell environment in order to mitigate arginine-dependent antimicrobial activity. Here, I establish that arginine metabolism is controlled by intracellular-specific sensory networks, and demonstrate that this regulation is important for bacterial survival. This work provides evidence for the importance of this amino acid in Salmonella infection, which informs on our overall understanding of systemic disease.

The apoplast is the site of infection for many important bacterial crop pathogens, including the model pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The chemical environment within the plant apoplast can determine the outcome of bacterial infection and the composition of this compartment is known to change in response to the presence of invading organisms. However, this metabolically dynamic environment has received little attention in the literature, and even less is known about how metabolites in the apoplast influence the expression of virulence genes. In this study, several aspects of the metabolic context of virulence were assessed. First, a broad-scale analysis of the tomato apoplast was undertaken, which identified metal ions, sugars, organic acids and amino acids, the most abundant of which was the non-protein amino acid gamma-aminobutyric acid (GABA). The impact these components had on the expression of virulence genes and metabolism in Pst DC3000 were then tested. Components such as fructose and aspartate caused high levels of virulence gene expression which correlated with the accumulation of intracellular glutamate, whereas repressive components, such as GABA and threonine, resulted in lower glutamate levels. Second, metabolic flux analysis showed that Pst DC3000 underwent major changes in central carbon metabolism in response to virulence gene inducing conditions. The identification of altered internal metabolism in Pst DC3000 cells expressing virulence genes led to the conclusion that Pst DC3000 may understand its external environment by sensing intracellular metabolites or metabolic fluxes. Third, the role of GABA assimilation in virulence was explored, and it was found that high internal GABA levels resulted in virulence gene repression. In addition, previously unidentified mechanisms for GABA uptake and transport were detected by the use of a novel ‘unlabelling’ experiment.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-22Bitstream added on 2014-06-13T19:03:37Z : No. of bitstreams: 1 souza_jam_dr_jabo.pdf: 4083420 bytes, checksum: cb86ca179e1196f514509e27854de1c3 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O nitrogênio é o nutriente requerido em maior quantidade para a cultura da soja. Avanços nas pesquisas de melhoramento genético vegetal e microbiologia do solo permitiram expandir o uso de inoculantes comerciais contendo estirpes de Bradyrhizobium japonicum e Bradyrhizobium elkanii. Estas bactérias infectam as raízes da planta e induzem a formação de nódulos, que abrigam a forma bacterióide, diferenciada da bactéria, responsável pela fixação simbiótica do nitrogênio. Informações sobre processos bioquímicos envolvidos no metabolismo da relação simbiótica podem ser adquiridas através de análises globais de expressão gênica. Para esta finalidade, destaca-se a tecnologia de microarranjo de DNA para detecção de genes diferencialmente expressos em larga escala. O objetivo geral deste trabalho foi identificar genes diferencialmente expressos, por meio de microarranjos de DNA, em Bradyrhizobium elkanii SEMIA 587 cultivada em diferentes meios de cultura, RDM (Rhizobia Defined Medium), TY (Triptone-Yeast Medium) e YMB (Yeast-Mannitol Medium), e em bacterióides isolados de nódulos de soja em diferentes períodos de desenvolvimento, 13, 28 e 48 dias após inoculação. Para esta finalidade, a partir do seqüenciamento de DNA genômico de B. elkanii, um microarranjo (Be587) foi gerado contendo 2654 genes. Em meio RDM, a bactéria confrontou-se com a necessidade de se adaptar e sintetizar suas subunidades formadoras de macromoléculas a partir de uma única fonte de carbono, refletindo em um metabolismo mais ativo nas fases lag e log. Por outro lado, em meio TY, as células cultivadas na presença de uma boa fonte de carbono e energia cresceram rapidamente esgotando os recursos disponíveis no meio, 8 o que pode ter causado uma situação de estresse que se refletiu na identificação... / Nitrogen is the most required nutrient by soybean culture. Advanced researches in genetic plant breeding and soil microbiology allowed the expansion in commercial inoculants applications containing strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. These bacteria infect plant roots and induce nodule formation which home the differentiated bacteria, named bacteroid. The bacteroid in turn is responsible for symbiotic nitrogen fixation. Biochemical knowledge about processes of symbiotic regulation can be acquired by global analysis of gene expression. To achieve such information, the DNA microarray technology, used for detection of differentially expressed genes in large scale, was used. The purpose of this work was identificate differentially expressed genes of Bradyrhizobium elkanii SEMIA 587, grown under different media conditions, such as RDM (Rhizobia Defined Medium), TY (Triptone- Yeast Medium) and YMB (Yeast-Mannitol Medium), and in bacteroids from soybean nodules at different developmental stages, 13, 28 e 49 days after inoculation. For this purpose, the DNA microarray Be587 with 2654 genes was generated from B. elkanii genomic DNA. In RDM medium the bacterium was confronted with the need of adaptation and building of macromolecules subunits from a single carbon source, what was reflected in a more active metabolism in lag and log phases. In turn, in TY medium with good carbon and energy sources the cells grew fastly and exhaust the medium sources available. Such condition can submitted the bacterial cells to a stress condition that reflected in the identification of higher number differentially expressed genes. At different bacteroids stages, the analysis detected genes related to nodulation and 10 nitrogen fixation regulation more than structural genes. Inasmuch, an organic nitrogen recycle might be involved... (Complete abstract, click electronic access below)

Made available in DSpace on 2014-12-17T14:33:06Z (GMT). No. of bitstreams: 1 CarolineGBM_DISSERT.pdf: 778935 bytes, checksum: 2a19b59e3c8f14ceef97a9f6a966da73 (MD5) Previous issue date: 2010-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / O aumento da concentra??o de CO2 na atmosfera tem sido observado, principalmente a partir da revolu??o industrial. Uma das causas principais desse comportamento tem sido a queima de combust?veis f?sseis. Isso tem levado a altera??es globais no ciclo do carbono. Desta forma tem sido de suma import?ncia trabalhos que mostrem a influ?ncia dos sistemas em geral e suas contribui??es relativas na din?mica e ciclo do carbono. Dentro deste contexto, os ecossistemas aqu?ticos apresentam import?ncia no processamento da mat?ria org?nica produzida internamente nos sistemas aqu?ticos (aut?ctone), bem como a mat?ria org?nica trazida dos sistemas terrestres (al?ctone). Os principais organismos que metabolizam a mat?ria org?nica dissolvida (carbono org?nico dissolvido COD) presente nos sistemas aqu?ticos s?o as bact?rias. No entanto a qualidade da mat?ria org?nica determina a prefer?ncia e a via metab?lica (produ??o bacteriana - PB ou respira??o bacteriana - RB) pela qual o carbono ser? direcionado quando assimilado pelas bact?rias. Nos sistemas aqu?ticos a diversidade da mat?ria org?nica presente, muitas vezes estimula a produ??o bacteriana. Desta forma, os objetivos deste trabalho foram avaliar os efeitos do COD al?ctone e aut?ctone na PB e RB, al?m de avaliar o efeito da mistura de COD sobre o balan?o de CO2 no experimento de mesocosmo. Para testar os objetivos realizamos um experimento de mesocosmo com o arranjo experimental do tipo (2x2) destinado a simular condi??es onde houvesse o predom?nio de mat?ria org?nica aut?ctone (fitopl?ncton), al?ctone (detrito de vegeta??o terrestre) e ambas combinadas. Consistindo em quatro tratamentos incluindo o Controle. A dura??o do experimento foi de 42 dias. Verificamos no geral que os tratamentos enriquecidos com mat?ria org?nica al?ctone apresentaram as maiores taxas metab?licas (RB, CO2), o que provavelmente esteve relacionado ? qualidade da mat?ria org?nica utilizada. Conclu?mos que o aporte de mat?ria org?nica de origem terrestre resulta em aumento da atividade de decomposi??o resultando na condi??o de heterotrofia nos tanques estudados. Conclu?mos ainda que com o esgotamento da mat?ria, os tanques passaram a apresentarem-se subsaturados em CO2, resultando na condi??o de autotrofia. Conclu?mos tamb?m que nos tanques com mistura de fonte o efeito observado foi antag?nico. / The increased concentration of CO2 in the atmosphere has been observed, mainly from the industrial revolution. One of the main causes of this behavior has been the burning of fossil fuels. This has led to changes in the global carbon cycle. Thus it has been extremely important work showing the influence of systems in general and their contributions on the dynamics and the carbon cycle. Within this context, aquatic ecosystems have importance in the processing of domestically produced organic matter in aquatic systems (indigenous) and the organic matter brought from the terrestrial (allochthonous). The main organisms that metabolize dissolved organic matter (dissolved organic carbon - DOC) present in aquatic systems is bacteria. However the quality of organic matter determines the preference and the metabolic pathway (bacterial production - PB or bacterial respiration - RB) by which carbon will be directed when assimilated by bacteria. In aquatic systems, the diversity of organic matter present, often stimulates bacterial production. Thus, the objectives were to evaluate the effects of allochthonous and autochthonous DOC in the PB and RB, and to evaluate the effect of mixing of DOC on the CO2 balance in the mesocosm experiment. For testing purposes we conducted a mesocosm experiment with the experimental arrangement of type (2x2) to simulate conditions where there was a predominance of autochthonous organic matter (phytoplankton), allochthonous (terrestrial vegetation detritus) and both combined. Consisting of four treatments including control. The experiment lasted 42 days. We note that in general the treatments enriched with allochthonous organic matter showed the highest metabolic rates (RB, CO2), which probably was related to the quality of organic matter used. We conclude that the input of organic matter from terrestrial origin results in increased activity of decomposition resulting in the condition of heterotrophy in the tanks studied. We also concluded that the exhaustion of matter, the tanks began to present themselves in subsaturados CO2, resulting in the condition of autotrophy. We also conclude that the tanks blend with the source of the observed effect was antagonistic.