The Ni 2؉ -dependent transcription factor NikR is widespread among microbes. The two experimentally characterized NikR orthologs, from Helicobacter pylori and Escherichia coli, display vastly different regulatory capabilities in response to increased intracellular Ni 2؉ . Here, we demonstrate that the nine-residue N-terminal arm present in H. pylori NikR plays a critical role in the expanded regulatory capabilities of this NikR family member. Specifically, the N-terminal arm is required to inhibit NikR binding to low affinity and nonspecific DNA sequences and is also linked to a cation requirement for NikR binding to the nixA promoter. Site-directed mutagenesis and arm-truncation variants of NikR indicate that two residues, Asp-7 and Asp-8, are linked to the cation requirement for binding. Pro-4 and Lys-6 are required for maximal DNA binding affinity of the full-length protein to both the nixA and ureA promoters. The N-terminal arm is highly variable among NikR family members, and these results suggest that it is an adaptable structural feature that can tune the regulatory capabilities of NikR to the nickel physiology of the microbe in which it is found.

Summary Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, while their close relative B. thailandensis is nonpathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection.

We examined the effects of urease and hydrogenase assembly gene deletions on NikR activation in H. pylori strains 26695 and G27. The loss of any component of urease assembly increased NikR activity under Ni 2؉ -limiting conditions, as measured by reduced transcript levels and 63 Ni accumulation. Additionally, SlyD functioned in urease assembly in strain 26695.A diverse complement of proteins is dedicated to the acquisition, trafficking, and regulation of intracellular transition metal ions. The mechanisms by which these activities are integrated to allocate the appropriate proportion of metal to different metal-binding proteins are not yet understood. Additionally, studies of the equilibrium metal-binding properties of transcriptional regulatory proteins important for metal homeostasis have revealed that they avidly bind their cognate metal ions (10 8,9,22,33,42]). These observations suggest that competition may exist between metalloenzyme assembly and metalloregulation. Detailed investigations of this hypothesis are encumbered by the presence of numerous essential metalloenzymes for metals such as zinc and iron. Microbial nickel physiology provides an ideal system for studying intracellular metal competition due to the small number of enzymes that require nickel ions (30) and their nonessentiality under laboratory growth conditions.We have studied the effect of disrupting Ni 2ϩ -dependent enzyme assembly pathways on nickel-dependent gene regulation in the gram-negative gastric pathogen Helicobacter pylori (3). The two Ni 2ϩ -dependent enzymes of H. pylori, urease and hydrogenase, are required for efficient colonization of animal models of infection (15,16,31). Both enzymes require conserved, GTP-dependent pathways for metal cofactor assembly that include an absolute requirement for nickel insertion chaperones under metal-limiting conditions (30). Hausinger and coworkers identified UreE as the Ni 2ϩ -binding protein required for urease assembly in Klebsiella aerogenes (10,38 before NikR, and the subsequent repression of nickel uptake genes. Such competition, if present, would be manifested as a change in NikR activity independently of a change in total nickel levels. In the absence of competition, NikR activity would correlate with a fixed total nickel concentration, independent of Ni 2ϩ -dependent enzyme expression or biosynthesis. Competition between metalloenzymes and metalloregulatory proteins has not been tested. Demonstration of the nature of such competition would facilitate subsequent studies to understand the molecular basis of metal ion partitioning within cells.We examined the effects of Ni 2ϩ -dependent enzyme assembly pathway gene deletions on NikR activity using several assays. In each case, cells were grown under identical conditions and manipulated in the same way for the same length of time. Cells were grown for 20 h (26695) or 24 h (G27) to an optical density at 600 nm of 1.0 in brucella broth (BD Difco) with 5% fetal bovine serum (Sigma) and then exposed to either 100 M dimethylglyoxime (DMG),...

Nickel is an important cofactor for several microbial enzymes. The ATP-dependent NikABCDE transporter is one of several types of uptake pathways known to be important for nickel acquisition in microbes. The Escherichia coli NikA periplasmic binding protein is structurally homologous to the di- and oligopeptide binding proteins, DppA and OppA. This structural similarity raises interesting questions regarding the evolutionary relationships between the recognition of nickel ions and short peptides. We find that in defined minimal growth medium NikABCDE transports nickel ions in the presence of exogenously added L-histidine (L-His), but not D-histidine. Both nickel uptake in cells and nickel binding to purified NikA showed an L-His concentration dependence consistent with recognition of a Ni-(L-His)₂ complex. This discovery reveals parallels to the transport of other metal complexes, notably iron, and suggests the structural diversity of nickel transporters may arise from the need to recognize extracellular nickel complexed with different organic ligands, whether they be exogenously or endogenously produced. Further, these results suggest that experiments examining the physiology and ecology of nickel-requiring microbes should account for the possibility that the growth medium may not support nickel uptake.

Helicobacter pylori requires nickel ions as cofactors for the urease and hydrogenase enzymes, which are important for the colonization of animal models of infection (1-3). This virulence requirement has motivated numerous studies of nickel-dependent gene regulation in H. pylori. The Ni 2ϩ -responsive transcription factor NikR from H. pylori (HpNikR) 3 regulates the expression of multiple genes by directly binding to their promoters in response to increasing nickel (4 -10). HpNikR is an exquisitely sensitive nickel sensor with a K D,Ni of ϳ2 pM (4, 5); however, how that affinity translates to gene regulation in cells remains unclear (e.g. in rich media, changes in gene expression are observed in response to ϳ100 M) (6, 9 -11). From these studies, it appears that the recognition sequences in each promoter are defined by a series of poorly conserved 6-bp imperfect inverted repeat half-sites separated by a 15-bp spacer (5,7,12). HpNikR binds to these promoters with a range of affinities (5, 12). The promiscuity of DNA binding by HpNikR contrasts with high sequence selectivity of the NikR proteins from Escherichia coli (EcNikR (13, 14)) and Geobacter uraniireducens (GuNikR (15)), which recognize highly conserved inverted repeats found in only one or two promoter regions per genome. The NikR protein family represents an excellent system with which to understand the role of protein and DNA sequence in the expansion of prokaryotic regulatory networks.NikR proteins are ribbon-helix-helix (RHH) transcription factors. RHH family members are present in bacteria, archaea, and bacteriophages (16). These proteins display diverse regulatory functions, such as bacteriophage gene regulation (17), plasmid maintenance and segregation (18,19), plasmid antitoxin and repressor functions (20), and metabolite-dependent (21) and metal-dependent (13, 22) gene regulation.The archetypical RHH fold (ϳ45-50 amino acids) consists of a single ␤-strand followed by two ␣-helices, with the two ␤-strands of each obligate RHH dimer forming an antiparallel ␤-sheet motif (23). Structural studies of the Arc and MetJ repressors first demonstrated that the RHH ␤-sheet motif sits in the major groove of DNA, with three solvent-exposed residues making base-specific hydrogen bonds via their side chains (23)(24)(25). Nonspecific DNA phosphate contacts were also observed in the structures, some by tandem turn regions N-terminal to the ␤-sheets and others by the N terminus of helix ␣2 of the RHH domain (23-25). These protein-phosphate interactions are hypothesized to attach the N terminus and helix ␣2 to the DNA backbone and link these structural elements to the ␤-sheet (23). The helix ␣2-phosphate backbone interactions are shared among all of the RHH protein-DNA co-crystal structures (16, 24 -30