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1.
Nat Struct Biol ; 8(12): 1031-6, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11685226

ABSTRACT

Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease. This specialized secretion process requires customized chaperones specific for particular effector proteins. The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers. Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein. Isothermal titration calorimetry studies of Tir-CesT and enzymatic activity profiles of SigD-SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Salmonella enterica/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Calorimetry , Crystallography, X-Ray , Dimerization , Light , Models, Molecular , Molecular Sequence Data , Protein Denaturation , Protein Folding , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Scattering, Radiation , Sequence Alignment , Solvents , Substrate Specificity , Ultracentrifugation
2.
J Biol Chem ; 274(17): 12103-7, 1999 Apr 23.
Article in English | MEDLINE | ID: mdl-10207036

ABSTRACT

FlgM is an anti-sigma factor of the flagellar-specific sigma (sigma) subunit of RNA polymerase in Bacillus subtilis, and it is responsible of the coupling of late flagellar gene expression to the completion of the hook-basal body structure. We have overproduced the protein in soluble form and characterized it. FlgM forms dimers as shown by gel exclusion chromatography and native polyacrylamide gel electrophoresis and interacts in vitro with the cognate sigmaD factor. The FlgM.sigmaD complex is a stable heterodimer as demonstrated by gel exclusion chromatography, chemical cross-linking, native polyacrylamide gel electrophoresis, and isoelectric focusing. sigmaD belongs to the group of sigma factors able to bind to the promoter sequence even in the absence of core RNA polymerase. The FlgM.sigmaD complex gave a shift in a DNA mobility shift assay with a probe containing a sigmaD-dependent promoter sequence. Limited proteolysis studies indicate the presence of two structural motifs, corresponding to the N- and C-terminal regions, respectively.


Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Amino Acid Sequence , Bacterial Proteins/metabolism , Base Sequence , Cloning, Molecular , DNA Primers , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Hydrolysis , Molecular Sequence Data , Protein Binding
3.
Nature ; 390(6657): 249-56, 1997 Nov 20.
Article in English | MEDLINE | ID: mdl-9384377

ABSTRACT

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.


Subject(s)
Bacillus subtilis/genetics , Genome, Bacterial , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Cloning, Organism , DNA, Bacterial , Molecular Sequence Data
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