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The Gram-negative bacterial pathogen Klebsiella pneumoniae forms biofilms to facilitate colonization of biotic and abiotic surfaces. The formation of biofilms by K. pneumoniae requires the expression of type 3 fimbriae: elongate proteinaceous filaments extruded by a chaperone-usher system in the bacterial outer membrane. The expression of the mrkABCDF cluster that encodes this fimbrial system is strongly positively regulated by MrkH, a transcriptional activator that responds to the second messenger, c-di-GMP. In this study, we analyzed the mechanism by which the MrkH protein activates transcriptional initiation from the mrkA promoter. A mutational analysis supported by electrophoretic mobility shift assays demonstrated that a 12-bp palindromic sequence (the MrkH box) centered at −78.5 is the binding site of MrkH. Deletion of half a turn, but not a full turn, of DNA located between the MrkH box and the mrkA promoter destroyed the ability of MrkH to activate mrkA transcription. In addition, a 10-bp AT-rich sequence (the UP element) centered at −63.5 contributed significantly to MrkH-dependent mrkA transcription. In vivo analysis of rpoA mutants showed that the R265 and E273 determinants in the C-terminal domain of RNA polymerase α subunit are needed for MrkH-mediated activation of mrkA transcription. Furthermore, results from mutagenesis of the mrkH gene suggest that the N-terminal region of the protein is involved in transcriptional activation. Taken together, our results suggest that MrkH activates mrkA expression by interacting directly with RNA polymerase, to overcome the inefficient transcriptional initiation caused by the presence of defective core promoter elements. [ABSTRACT FROM AUTHOR]

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a kcat = 22 s-1, KMPYR = 2.55 ± 0.05 mM and KMASA = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (TMapp) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (TMapp = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a KD4→2 = 1.7 nM, which is considerably tighter than its E. coli ortholog (KD4→2 = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å2) compared to its E. coli counterpart (500 Å2). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target. [ABSTRACT FROM AUTHOR]

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a kcat = 22 s-1, KMPYR = 2.55 ± 0.05 mM and KMASA = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (TMapp) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (TMapp = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a KD4→2 = 1.7 nM, which is considerably tighter than its E. coli ortholog (KD4→2 = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å2) compared to its E. coli counterpart (500 Å2). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target. [ABSTRACT FROM AUTHOR]