The concentration of intracellular nickel in Haemophilus influenzae is linked to its surface properties and cell-cell aggregation and biofilm formation.

Of the known proteins which use nickel as a co-factor, Haemophilus influenzae contains only urease and glyoxalase I (gloA). We have recently reported that this pathogen harbours a unique nickel uptake system (nikKLMQO-nimR). Unusually, the disruption of the nickel uptake system (nikQ or nimR mutants) resulted in cells that aggregated and formed an increased biofilm compared to the wild type cells. Using a gloA mutant strain and urease-specific inhibitor we showed that this phenotype is not due to the loss-of-function of these enzymes. By generating H. influenzae "resting cells" which are enzymatically inactive but maintain their structural integrity we have shown that the cell aggregation in the nikQ/nimR mutants is not due to the loss of enzymatic function. The nikQ mutant was unable to accumulate nickel but the addition of excess nickel did restore intracellular nickel levels and this resulted in the nikQ mutant returning to the wild type "free-living" phenotype; cells with no aggregation and no biofilm formation. We used a range of techniques which showed that the nikQ mutant possesses changes to its cell surface properties. The mutant was more negatively charged than wild type cells as well as being more hydrophobic. Analysis of the outer membrane constituents showed that there were molecular differences. Although the nikQ mutant appears to grow the same as its wild type cell we have shown that there is a change in the "lifestyle" of these nickel limited cells and this induces changes to the surface of the cell to promote cell-cell aggregation and biofilm formation.

A novel nickel responsive MerR-like regulator, NimR, from Haemophilus influenzae.

We have identified a novel regulator from the MerR family of transcription factors in the bacterial pathogen Haemophilus influenzae (HI1623; nickel-associated merR-like Regulator--NimR). NimR regulates the expression of a Ni(2+) uptake transporter (NikKLMQO). The promoters for nimR and the nik operon are divergent and overlapping and NimR binds at a site between the promoter elements for nikKLMQO. Expression of this operon requires NimR and depends on Ni(2+). Growth rates of the H. influenzae nimR and nikQ mutants were reduced in chemically defined media compared to the wild type and the mutants were unable to grow in the presence of EDTA. The mutant strains were less tolerant of acidic pH and the wild type Rd KW20 could not tolerate low pH in the presence of fluoramide, a urease specific inhibitor, confirming that both nickel transport and urea hydrolysis are a central process in pH control. H. influenzae nimR and nikQ strains were deficient in urease activity, but this could be specifically restored by the addition of excess Ni(2+). NimR did not directly regulate the expression of urease genes but the activity of urease requires both nimR and nikQ. Purified NimR is a dimer that binds 1 Ni(2+)ion. NimR is the first example of a Ni-dependent regulator from the MerR family and targeting a metal ion uptake system; it is distinct from NikR the Ni-responsive regulators of the ribbon-helix-helix family.