BACTERIAL METABOLISM

Author: JUIZ RÍO SANDRA MARIA.
Year: 2005.
University: SANTIAGO DE COMPOSTELA [www.usc.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: FACULTAD DE BIOLOGIA.

Summary: Photobacterium damselae is a gamma-proteobacteria belonging to the family Vibrionaceae and currently includes two subspecies: P.damselae subsp.piscicida (causative agent of the pasteurelosis in fish) and P.damselae subsp.damselae. Among the objectives of this thesis we are genetically characterize some of the factors that contribute to the virulence of P.damselae subsp.piscicida, with a special emphasis on the procurement systems of iron. We have also analyzed genetic diversity between strains of this subspecies, and given some of the genetic differences between strains in the two subspecies of P.damselae. As a starting point we have identified the first gene that encodes a protein Fur (ferric uptake regulator), which has shown a sequence homology of 99.3% between the two subspecies of P.damselae, differing protein is a single amino acid in the 148 consists. Furthermore, we have shown that Fur acts on P.damselae as a transcriptional regulator dependent on the concentration of iron in the middle. Moreover has been identified and characterized a system for capturing hemo groups as a source of iron in the two subspecies of P.damselae composed of proteins HutA, TonBExbBD, HutBCD and HutWXZ. This system showed high similarity to proteins of harvesting systems hemo other members of the family Vibrionaceae, and close to 100% identity between the two subspecies. However, we noticed signs of intraspecific microevolución in this system: the gene receptor outer membrane hutA is interrupted pose a pseudogen in many of the strains piscicida. Curiously, the only strains of subsp.piscida possessing the gene hutA intact constitute a potential line of clonal isolates from Japan. The system catchment hemo is expressed in vivo during infection P.damselae subsp.piscicida in turbot, as is apparent from the analysis of the presence of mRNA of operon tonBexbBexbBexbBhutBCD in samples of infected fish. It has also been shown that there are no redundant systems to capture hemo, since the inactivation of hutCD involves the inability to use hemo groups as the sole source of iron. Applying the technique Subtractive Hybridization, we have identified 21 regions of the genome of a specific virulent strain of piscicida, which were absent in a strain avirulenta. By studying the distribution of these regions of DNA in 28 strains P.damselae subsp.piscicida with different backgrounds and geographical isolation from different hosts, it was found that this is a very heterogeneous microorganism from the genetic point of view. We did not find any cases in two strains showing exactly the same pattern of distribution of the 21 regions analyzed. There have also been identified for the first time in this pathogen genes encoding transposasas. These genes are found in multiple copies, inserted in the genome, forming a pattern that proved to be different in each strain analyzed. In addition, we identified in a single strain of piscicida gene sequences similar to the mobile element SXT described in V.cholerae. This element large (approx 100 kb) is representative of a family of transposons conjugativos and integrativos who carry genes for resistance to multiple antibacterials. Previous studies had shown that each subsp. In P.damselae produces a sideróforo different. The application dela Subtractive Hybridization enabled the isolation and characterization of a region of the genome of approx. 22 kb in subsp.piscicida, which contains a gene operon regulated by iron including a possible membrane receptor for sideróforos and 2 pé 8 ptido-si 6f4 ntetasas not ribosómicas (Irp1 and Irp2) high molecular weight. These proteins appear, in terms of limiting iron preparations membranes total strains of subsp.piscicida, being absent from the strains of subsp.damselae. The inactivation of the gene irp1 ago insertion that P.damselae subsp.piscicida not produce sideróforos and is unable to grow encondiciones limitation iron. In addition, the mutation of the gene irp1 led to an increase in the LD50 in at least two command magnitude with respect to the parental strain, demonstrating that the system mediated iron uptake by sideróforos is a factor contributing to the virulence of P.damselae subsp.piscicida in fish. The analysis of the presence of these genes in a collection of strains showed that the two subspecies are unique to certain strains of subsp.piscicida. Interestingly, the sequences aminoacídicas of the protein encoded by this operon show high similarity with protein synthesis sideróforo yersiniabactinaproducido strains of Yersinia spp, whose genes are grouped to constitute a pathogenicity island. Analysis of the functional domains of proteins Irp1 and Irp2 has provided evidence that the sideróforo produced by P.damselae subsp.piscicida may be structurally related yersiniabactina.

Author: MORÁN ZORZANO MARÍA TERESA.
Year: 2005.
University: PÚBLICA DE NAVARRA [www.unavarra.es].
Place of defense: ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS.
Place of preparation: UNIVERSIDAD PÚBLICA DE NAVARRA.

Summary: Starch is the fundamental way of storing energy in many plant species. It is an essential element in the diet of human beings and is a raw material in many industrial processes. The glycogen in turn, is the main form of energy storage in many bacterial species. Like the starch is a homopolisacárido of glucose molecules. Both bacteria and in plants has been traditionally accepted that the ADPglucosa pirofosforilasas is the only enzyme capable of producing ADPglucosa (ADPG) necessary for the biosynthesis of starch and glycogen. However, our team has accumulated evidence on the existence of other important sources of ADPG plant. That is why I raised in this paper to investigate the possible existence of additional sources of ADPG in producing bacteria. To this end he characterized strains of Escherichia coli and Salmonella enterica mutants in the operon glgCAP whose products are responsible for the synthesis of ADPG, their use for glycogen synthesis and degradation of this poliglucano. This work, set out in chapter 1, presents evidence showing that the Enterobacteriaceae possess several important sources of ADPG. Given that bacterial accumulation of glycogen may be determined not only by the synthesis of ADPG but also for its hydrolysis, I proceeded to characterize the regulatory aspects of the ADPglucosa pirofosfatasa (AspP) from Escherichia coli. Initial studies presented in chapter 2 showing that small increases of this enzyme activity leading to a reduction in the content of glycogen, indicating that unless AspP is regulated, it would accumulate glycogen in the bacterium. Subsequent studies showed that AspP is highly regulated both by macromolecular crowding effects and mechanisms of membrane translocation induced by substances present in bacterial cultures saturated. In Chapter 3 describes the identification and characterization of the protein equivalent to AspP bacterial plant. As a first step to investigate their possible involvement in controlling levels of ADPG associated with the synthesis of starch, produced and caractericé plants on producing AspP. The leaves of these plants showed a significant reduction in levels of both ADPG as starch, indicating that the AspP plant hydrolytic catalyzes the breakdown of a pool of ADPG linked to the synthesis of starch. Finally, due to the fact that our group recently showed for the first time the existence of a mechanism for capturing endocítica sucrose in heterotrophic cells, I have investigated the possibility that this mechanism is directly involved in controlling the levels of ADPG and starch in these cells . To that end, as presented in Chapter 4, I compared the rates of accumulation of these substances in cells sycamora cultured in the presence and absence of inhibitors of endocytosis. The presence of inhibitors leads to reduced levels of starch, sucrose and ADPG, confirming that most of the sucrose apoplástica is picked up by endocytosis to be converted into ADPG-almidón.

Author: JIMÉNEZ ZARCO JOSÉ IGNACIO.
Year: 2005.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of preparation: FACULTAD DE CIENCIAS QUÍMICAS.

Summary: The strain Pseudomonas putida KT2440 is a microorganism model for processes related to the biodegradation of contaminants in the environment, in addition to other applications of biotechnology. In this thesis has made a detailed study of the genes involved in the degradation pathways of aromatic compounds present in the genome of the strain KT2440 allowing catabólico determine the overall potential of the bacterium in front of this type of compounds. In this regard, we identified five routes central degradation, for metabolites protocatecuato (pca genes), catechol (genes cat), phenylacetate (genes pha), homogentisato (genes hmg) and galato (genes gal). In addition, we identified genes that encode routes peripheral degradation of p-hydroxybenzoate, benzoate compounds fenilpropenopides, quinato, feniletilamina, fenilalcanoatos, fenilacetaldehído, phenylalanine and tyrosine. An additional result of the analysis was to identify a new path of degradation of nicotinic acid (genes nic), a compound aromatic N-heterociclo which, in its degradation generates metabolites with high biological activity used in the synthesis of pharmaceuticals. The molecular characterization of genes nic led to the identification of new enzymes involved in mineralization. In a first stage involves a nicotinato hydroxylase (NicAB), a hydroxylase dependent molybdenum which had not been previously characterized, and that transforms the ácidonicotínico acid 6-hidroxinicotínico which is used in the synthesis of insecticides. In a second stage, a 6-hidroxinicotinato hydroxylase (NicC) transforms acid 6-hidroxinicotínico in the intermediary Central 2,5-dihidroxipiridina, which is used in the synthesis of acid aminolevulínico precursor to the synthesis of anti-tumor compounds. The 2,5-dihidroxipiridina suffers the loss of aromatic by the action of NicX, which is the first member of a new family of dioxigenasas related aminopeptidasas. This reaction generates acid N-formilmaleámico which is transformed into acid maleámico by the enzyme NicD, which is the first desformilasa described within the family of a / b -hidrolasas. Lso were also identified genes nicF and nicE involved in the last two stages of the route, obtaining Maleic acid and fumaric acid, IMI respectively. Nic Genes are organized into three operons transcripcionales nicAB, nicXR and nicCDEFTP, which are induced by acid 6-hidroxinicotínico, while only nicotinic acid induces the expression of operon nicAB. NicR acts as a repressor of expression nicXR and nicCDFTP, while serving as inducing the expression of nicAB.