Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

This thesis describes the target selection, cloning, expression, purification, crystallization, structure and biochemical characterization of five essential Mycobacterium tuberculosis (Mtb) proteins. The search for drugs against the causal agent of tuberculosis is urgently needed and the targeting of essential genes is necessary to fulfill this goal. The crystal structures of carbonic anhydrases (CA) Rv1284 and Rv3588c have been determined to 2.0 and 1.7 Å resolution, respectively. Rv3588c, in contrast to Rv1284, is an active β-CA that shows two different active site conformations and pH-dependent oligomerization states. Rv1295 is an active threonine synthase with an unusually high pH optimum; the structure has been solved to 2.5 Å resolution, based on which a modification to the reaction mechanism published previously is proposed. Mtb has a thick and impermeable cell envelope that constitutes an efficient barrier against drugs. One of the essential components of the envelope is mycolic acid (MA). The inhibition of enzymes participating in its synthesis would be lethal for Mtb. Rv0636, a formerly unknown-function protein has β-hydroxyacyl-ACP dehydrase activity which is essential for MA synthesis. Co-expression with partners notably improves its solubility. Around 55% of Mtb proteins have unknown function. Rv3778c is one of them and its three-dimensional structure has been determined to 1.8 Å resolution. Studies aimed at the elucidation of its biochemical function are shown. A pathway not yet reported in Mtb is also suggested.