Silke I. Patzer

The Zinc-responsive Regulator Zur and Its Control of theznu Gene Cluster Encoding the ZnuABC Zinc Uptake System in Escherichia coli

The synthesis of the Escherichia colizinc transporter, encoded by the znuACB gene cluster, is regulated in response to the intracellular zinc concentration by thezur gene product. Inactivation of the zur gene demonstrated that Zur acts as a repressor when binding Zn2+. Eight chromosomal mutant zur alleles were sequenced to correlate the loss of Zur function with individual mutations. Wild-type Zur and ZurΔ46–91 formed homo- and heterodimers. Dimerization was independent of metal ions since it also occurred in the presence of metal chelators. Using an in vivo titration assay, the znu operator was narrowed down to a 31-base pair region overlapping the translational start site of znuA. This location was confirmed by footprinting assays. Zur directly binds to a single region comprising a nearly perfect palindrome. Zinc chelators completely inhibited and Zn2+ in low concentrations enhanced DNA binding of Zur. No evidence for autoregulation of Zur was found. Zur binds at least 2 zinc ions/dimer specifically. Although most of the mutant Zur proteins bound to the znu operator in vitro, no protection was observed in in vivo footprinting experiments. Analysis of the mutant Zur proteins suggested an amino-terminal DNA contact domain around residue 65 and a dimerization and Zn2+-binding domain toward the carboxyl-terminal end.

Ton-dependent colicins and microcins: modular design and evolution

Ton-dependent colicins and microcins are actively taken up into sensitive cells at the expense of energy which is provided by the proton motive force of the cytoplasmic membrane. The Ton system consisting of the proteins TonB, ExbB and ExbD is required for colicin and microcin import. Colicins as well as the outer membrane transport proteins contain proximal to the N-terminus a short sequence, called TonB box, which interacts with TonB and in which point mutants impair uptake. No TonB box is found in microcins. Colicins are composed of functional modules which during evolution have been interchanged resulting in new colicins. The modules define sites of interaction with the outer membrane transport genes, TonB, the immunity proteins, and the activity regions. Six TonB-dependent microcins with different primary structures are processed and exported by highly homologous proteins. Three of these microcins are modified in an unknown way and they have in common specificity for catecholate siderophore receptors.

Dual Repression by Fe2+-Fur and Mn2+-MntR of the mntH Gene, Encoding an NRAMP-Like Mn2+ Transporter in Escherichia coli

The uptake of Mn(2+), a cofactor for several enzymes in Escherichia coli, is mediated by MntH, a proton-dependent metal transporter, which also recognizes Fe(2+) with lower affinity. MntH belongs to the NRAMP family of eukaryotic Fe(2+) and Mn(2+) transporters. In E. coli strains with chromosomal mntH-lacZ fusions, mntH was partially repressed by both Mn(2+) and Fe(2+). Inactivation of fur resulted in the loss of Fe(2+)-dependent repression of mntH transcription, demonstrating that Fe(2+) repression depends on the global iron regulator Fur. However, these fur mutants still showed Mn(2+)-dependent repression of mntH. The Mn(2+)-responsive transcriptional regulator of mntH was identified as the gene product of o155 (renamed MntR). mntR mutants were impaired in Mn(2+) but not Fe(2+) repression of mntH transcription. Binding of purified MntR to the mntH operator was manganese dependent. The binding region was localized by DNase I footprinting analysis and covers a nearly perfect palindrome. The Fur binding site, localized within 22 nucleotides of the mntH operator by in vivo operator titration assays, resembles the Fur-box consensus sequence.

Crystal Structure of Colicin M, a Novel Phosphatase Specifically Imported by Escherichia coli>

Colicins are cytotoxic proteins secreted by certain strains of Escherichia coli. Colicin M is unique among these toxins in that it acts in the periplasm and specifically inhibits murein biosynthesis by hydrolyzing the pyrophosphate linkage between bactoprenol and the murein precursor. We crystallized colicin M and determined the structure at 1.7Å resolution using x-ray crystallography. The protein has a novel structure composed of three domains with distinct functions. The N-domain is a short random coil and contains the exposed TonB box. The central domain includes a hydrophobic α-helix and binds presumably to the FhuA receptor. The C-domain is composed of a mixed α/β-fold and forms the phosphatase. The architectures of the individual modules show no similarity to known structures. Amino acid replacements in previously isolated inactive colicin M mutants are located in the phosphatase domain, which contains a number of surface-exposed residues conserved in predicted bacteriocins of other bacteria. The novel phosphatase domain displays no sequence similarity to known phosphatases. The N-terminal and central domains are not conserved among bacteriocins, which likely reflect the distinct import proteins required for the uptake of the various bacteriocins. The homology pattern supports our previous proposal that colicins evolved by combination of distinct functional domains.

Gene Cluster Involved in the Biosynthesis of Griseobactin, a Catechol-Peptide Siderophore of Streptomyces sp. ATCC 700974

The main siderophores produced by streptomycetes are desferrioxamines. Here we show that Streptomyces sp. ATCC 700974 and several Streptomyces griseus strains, in addition, synthesize a hitherto unknown siderophore with a catechol-peptide structure, named griseobactin. The production is repressed by iron. We sequenced a 26-kb DNA region comprising a siderophore biosynthetic gene cluster encoding proteins similar to DhbABCEFG, which are involved in the biosynthesis of 2,3-dihydroxybenzoate (DHBA) and in the incorporation of DHBA into siderophores via a nonribosomal peptide synthetase. Adjacent to the biosynthesis genes are genes that encode proteins for the secretion, uptake, and degradation of siderophores. To correlate the gene cluster with griseobactin synthesis, the dhb genes in ATCC 700974 were disrupted. The resulting mutants no longer synthesized DHBA and griseobactin; production of both was restored by complementation with the dhb genes. Heterologous expression of the dhb genes or of the entire griseobactin biosynthesis gene cluster in the catechol-negative strain Streptomyces lividans TK23 resulted in the synthesis and secretion of DHBA or griseobactin, respectively, suggesting that these genes are sufficient for DHBA and griseobactin biosynthesis. Griseobactin was purified and characterized; its structure is consistent with a cyclic and, to a lesser extent, linear form of the trimeric ester of 2,3-dihydroxybenzoyl-arginyl-threonine complexed with aluminum under iron-limiting conditions. This is the first report identifying the gene cluster for the biosynthesis of DHBA and a catechol siderophore in Streptomyces.

Structural and Mechanistic Studies of Pesticin, a Bacterial Homolog of Phage Lysozymes

Background: Pesticin is a protein toxin that is formed by Yersinia pestis to kill related strains. Results: The crystal structure and functional analyses revealed a receptor binding, a translocation, and an activity domain. Conclusion: Folding of the activity domain is very similar to folding of phage T4 lysozyme. Significance: This is the first case that an activity domain is derived from a known enzyme. Yersinia pestis produces and secretes a toxin named pesticin that kills related bacteria of the same niche. Uptake of the bacteriocin is required for activity in the periplasm leading to hydrolysis of peptidoglycan. To understand the uptake mechanism and to investigate the function of pesticin, we combined crystal structures of the wild type enzyme, active site mutants, and a chimera protein with in vivo and in vitro activity assays. Wild type pesticin comprises an elongated N-terminal translocation domain, the intermediate receptor binding domain, and a C-terminal activity domain with structural analogy to lysozyme homologs. The full-length protein is toxic to bacteria when taken up to the target site via the outer or the inner membrane. Uptake studies of deletion mutants in the translocation domain demonstrate their critical size for import. To further test the plasticity of pesticin during uptake into bacterial cells, the activity domain was replaced by T4 lysozyme. Surprisingly, this replacement resulted in an active chimera protein that is not inhibited by the immunity protein Pim. Activity of pesticin and the chimera protein was blocked through introduction of disulfide bonds, which suggests unfolding as the prerequisite to gain access to the periplasm. Pesticin, a muramidase, was characterized by active site mutations demonstrating a similar but not identical residue pattern in comparison with T4 lysozyme.

Activation of Colicin M by the FkpA Prolyl Cis-Trans Isomerase/Chaperone

Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA.