- Top of page
- Materials and methods
It has previously been shown that myo-inositol hexakisphosphate (myo-InsP6) mediates iron transport into Pseudomonas aeruginosa and overcomes iron-dependent growth inhibition. In this study, the iron transport properties of myo-inositol trisphosphate and tetrakisphosphate regio-isomers were studied. Pseudomonas aeruginosa accumulated iron (III) at similar rates whether complexed with myo-Ins(1,2,3)P3 or myo-InsP6. Iron accumulation from other compounds, notably d/lmyo-Ins(1,2,4,5)P4 and another inositol trisphosphate regio-isomer, d-myo-Ins(1,4,5)P3, was dramatically increased. Iron transport profiles from myo-InsP6 into mutants lacking the outer membrane porins oprF, oprD and oprP were similar to the wild-type, indicating that these porins are not involved in the transport process. The rates of reduction of iron (III) to iron (II) complexed to any of the compounds by a Ps. aeruginosa cell lysate were similar, suggesting that a reductive mechanism is not the rate-determining step.
Most Gram-negative micro-organisms take up physiologically essential iron by synthesizing and utilizing high affinity ferric iron chelators called siderophores. The ferric-siderophore complexes formed with environmental iron are recognized by proteins in the outer membrane that initiate their active transport into the cell. A number of micro-organisms bypass the requirement to synthesize their own siderophores by transporting iron complexed with exogenous chelators. Examples include adaptation to use siderophores produced by other species, such as ferrichrome, coprogen, and rhodotorulic acid transport by Escherichia coli ( Braun et al. 1991 ), and enterobactin transport by Pseudomonas aeruginosa ( Poole et al. 1990 ). Other compounds, such as ferric dicitrate, can also be used ( Harding & Royt 1990; Van Hove et al. 1991 ).
In a previous study, it was shown that iron complexed with myo-inositol hexakisphosphate (phytic acid, InsP6) reversed iron-dependent growth inhibition of Ps. aeruginosa imposed by a non-utilizable chelator ( Smith et al. 1994 ). The only previous association of this molecule with prokaryotic systems has been characterization of phytases from soil organisms such as Bacillus subtilis ( Powar & Jagannathan 1982) and Pseudomonas sp. ( Irving & Cosgrove 1971; Richardson & Hadobas 1997). Myo-InsP6 is found in soil and is abundant in many eukaryotic cells ( Cosgrove 1980), although its precise role within these cells is the subject of some debate. Some workers argue that it is a store either of myo-inositol or phosphate, or both ( Berridge & Irvine 1989). Others suggest a wide variety of roles, including neurotransmission ( Vallejo et al. 1988 ), coated vesicle formation ( Timerman et al. 1992 ; Voglmaier et al. 1992 ; Norris et al. 1995 ), cell cycle progression ( Guse et al. 1993 ), respiratory burst of neutrophils ( Eggelton et al. 1991 ), and reducing inflammation ( Cecconi et al. 1994 ). It may also have an antioxidant effect by virtue of its affinity with iron (III) ( Graf et al. 1987 ), estimated to be in the range 1025–1030 M−1 at neutral pH ( Hawkins et al. 1993 ). The metal ion chelation properties of myo-InsP6 have also been proposed to explain its role in affecting the dietary availability of elements such as iron and zinc ( Hallberg et al. 1987 ; Hurrell et al. 1992 ).
In order to gain a better understanding of the iron transport process mediated by inositol polyphosphates in Ps. aeruginosa, several inositol tetrakis- and trisphosphate regio-isomers have been studied. Iron transport properties in mutants lacking outer membrane porins have also been compared, and the rate of reduction of iron (III) complexed with each inositol polyphosphate by a Ps. aeruginosa cell lysate has been measured.
- Top of page
- Materials and methods
In this study, we have shown that Ps. aeruginosa can accumulate iron complexed with a number of inositol polyphosphates. The differences in transport properties cannot easily be explained by factors such as molecular weight and size, as all inositol tetrakis- and trisphosphate compounds are regio-isomers, respectively. Rather, we believe it reflects the mechanism by which Fe (III) is complexed. We propose that the 1,2,3 vicinal axial-equatorial-axial trisphosphate motif of myo-InsP6 and myo-Ins(1,2,3)P3 accommodates the six co-ordination sites of iron (III) to form a water-excluding octahedral complex ( Fig. 5). Preliminary molecular modelling studies support this proposal (data not shown). This octahedral co-ordination, which does not have an exchangeable site, is also consistent with our observation that free radical formation is inhibited when iron (III) is complexed with these molecules ( Spiers et al. 1996 ). Clearly, the other compounds in this study also chelated iron (III), even though they do not possess 1,2,3 vicinal phosphate groups. Therefore, groups in other positions can participate in chelation. In most cases, notably d-myo-Ins (1,4,5)P3 and d/l-myo-Ins (1,2,4,5)P4, this resulted in a greater rate and accumulation of iron (III) by Ps. aeruginosa. It is noteworthy that these compounds do not inhibit iron-catalysed free radical formation ( Hirst 1996; Spiers et al. 1996 ), indicating that iron complexed by these molecules can redox cycle. We have been unable to predict 1 : 1 fully occupied octahedral complexes with d-myo-Ins(1,3,4)P3 and d-myo-Ins(1,2,4,6)P4, which mediate similarly low rates of iron accumulation as myo-InsP6 and myo-InsP(1,2,3)P3. However, we cannot rule out the possibility of formation of stable bi- or ter-molecular complexes.
Figure 5&. emsp;Model for the proposed interaction of the 1,2,3 axial-equatorial-axial vicinal phosphates of myo-Ins(1,2,3)P3 with iron (III)
Download figure to PowerPoint
Our attempts to understand the molecular nature of the transport process into Ps. aeruginosa have been frustrating. Using iron complexed with myo-InsP6, we did not detect any differences in iron transport profiles using Ps. aeruginosa mutants lacking OprF, OprD or OprP, indicating that these major porins are not involved. Our low temperature and metabolic inhibitor process also argue against a diffusion process. This is in contrast to some ferri-siderophores in which Meyer (1992) reported reduced accumulation of iron in strains lacking OprF, showing that Ps. aeruginosa can take up siderophore-liganded iron through this porin.
We have shown that Ps. aeruginosa isolated cell membranes have a single class of binding site for myo-InsP6 ( Smith et al. 1994 ). In this present study, no compound, even at a concentration of 100 μmol l−1, was able to displace 1 nmol l−1[3H]-myo-InsP6 ( Hirst 1996), indicating that the interaction with the outer membrane is specific for myo-InsP6. This is perhaps not surprising given that this is the major naturally occurring inositol polyphosphate which Ps. aeruginosa is likely to encounter. In an attempt to identify the transport component, we have screened more than 50 000 transposon insertion mutants but have failed to isolate a single Fe (III)-InsP6 transport-deficient mutant.
We have also examined Ps. aeruginosa reductase activity, but did not demonstrate a rate-determining role. Halle & Meyer (1992a, b) have characterized a ferri-pyoverdine reductase activity from Ps. aeruginosa, which is initiated by the enzyme-catalysed reduction of FMN to FMNH2 by NADH; FMNH2 then mediates the reduction of Fe (III) to Fe (II). We demonstrated that reduction of ferri-inositol polyphosphate, like ferri-pyoverdine, is FMN dependent. However, in no case did the rate of reduction correlate with the rate of iron transport into Ps. aeruginosa, indicating that reductase activity is unlikely to be the rate-determining step. Similarly, our mutagenesis screening strategy should have picked up reductase mutants if this activity was involved in transport.
In summary, we have shown that Ps. aeruginosa can transport iron (III) complexed to a number of inositol polyphosphates. It is likely that it is myo-InsP6 which will be encountered by Ps. aeruginosa in environments such as soil. While some of the other compounds tested do occur naturally, notably the intracellular signalling molecule d-myo-Ins(1,4,5)P3 ( Berridge 1995), it is unlikely that they will be encountered by Ps. aeruginosa, even when causing infections. We do however, believe that they shed light on the manner in which myo-InsP6 chelates iron. We believe that the comparatively slow rate of iron (III) accumulation by Ps. aeruginosa when complexed with myo-InsP6 reflects the ability of this molecule to form an octahedral complex with iron (III) occupying all six co-ordination sites. This is consistent with myo-InsP6 being a biologically ‘safe’ form of stored iron, protecting biological systems from the toxic effects of this element, but one which Ps. aeruginosa is able to exploit. Given its abundance in some environments it will be interesting to determine whether other micro-organisms can utilize iron bound by this molecule.