Structures of Sortase B from Staphylococcus aureus and Bacillus anthracis Reveal Catalytic Amino Acid Triad in the Active Site

Surface proteins attached by sortases to the cell wall envelope of bacterial pathogens play important roles during infection. Sorting and attachment of these proteins is directed by C-terminal signals. Sortase B of S. aureus recognizes a motif NPQTN, cleaves the polypeptide after the Thr residue, and attaches the protein to pentaglycine cross-bridges. Sortase B of B. anthracis is thought to recognize the NPKTG motif, and attaches surface proteins to m-diaminopimelic acid cross-bridges. We have determined crystal structure of sortase B from B. anthracis and S. aureus at 1.6 and 2.0 Å resolutions, respectively. These structures show a β-barrel fold with α-helical elements on its outside, a structure thus far exclusive to the sortase family. A putative active site located on the edge of the β-barrel is comprised of a Cys-His-Asp catalytic triad and presumably faces the bacterial cell surface. A putative binding site for the sorting signal is located nearby

Passage of Heme-Iron Across the Envelope of Staphylococcus aureus

The cell wall envelope of Gram-positive pathogens functions as a scaffold for the attachment of virulence factors and as a sieve that prevents diffusion of molecules. Here the isdgenes (iron-regulated surface determinant) of Staphylococcus aureus were found to encode factors responsible for hemoglobin binding and passage of heme-iron to the cytoplasm, where it acts as an essential nutrient. Heme-iron passage required two sortases that tether Isd proteins to unique locations within the cell wall. Thus, Isd appears to act as an import apparatus that uses cell wall–anchored proteins to relay heme-iron across the bacterial envelope

Passage of Heme-Iron Across the Envelope of Staphylococcus aureus

The cell wall envelope of Gram-positive pathogens functions as a scaffold for the attachment of virulence factors and as a sieve that prevents diffusion of molecules. Here the isdgenes (iron-regulated surface determinant) of Staphylococcus aureus were found to encode factors responsible for hemoglobin binding and passage of heme-iron to the cytoplasm, where it acts as an essential nutrient. Heme-iron passage required two sortases that tether Isd proteins to unique locations within the cell wall. Thus, Isd appears to act as an import apparatus that uses cell wall–anchored proteins to relay heme-iron across the bacterial envelope

Structures of complexes comprised of Fischerella transcription factor HetR with Anabaena DNA targets

HetR is an essential regulator of heterocyst development in cyanobacteria. Many mutations in HetR render Anabaena incapable of nitrogen fixation. The protein binds to a DNA palindrome upstream of hetP and other genes. We have determined the crystal structures of HetR complexed with palindromic DNA targets, 21, 23, and 29 bp at 2.50-, 3.00-, and 3.25-Å resolution, respectively. The highest-resolution structure shows fine details of specific protein–DNA interactions. The lower-resolution structures with longer DNA duplexes have similar interaction patterns and show how the flap domains interact with DNA in a sequence nonspecific fashion. Fifteen of 15 protein–DNA contacts predicted on the basis of the structure were confirmed by single amino acid mutations that abolished binding in vitro and complementation in vivo. A striking feature of the structure is the association of glutamate 71 from each subunit of the HetR dimer with three successive cytosines in each arm of the palindromic target, a feature that is conserved among all known heterocyst-forming cyanobacteria sequenced to date

Expression of Cytosolic and Plastid Acetyl-Coenzyme A Carboxylase Genes in Young Wheat Plants

Expression of cytosolic and plastid acetyl-coenzyme A carboxylase (ACCase) gene families at the mRNA level was analyzed in developing wheat (Triticum aestivum) plants. The major plastid ACCase mRNA level is high in the middle part of the plant and low in roots and leaf blades. An alternative plastid ACCase transcript initiated at a different promoter and using an alternative 5′ splice site for the first intron accumulates to its highest level in roots. Cytosolic ACCase mRNA also consists of two species, one of which is present at approximately a constant level, whereas the other accumulates to a high level in the lower sheath section. It is likely that different promoters are also responsible for the two forms of cytosolic ACCase mRNA. The abundances of cytosolic and plastid ACCase mRNAs in the sheath section of the plant are similar. ACCase protein level is significantly lower in the leaf blades, in parallel with changes in the total ACCase mRNA level. Homoeologous ACCase genes show the same expression patterns and similar mRNA levels, suggesting that none of the genes was silenced or acquired new tissue specificity after polyploidization