Manifold aspects of specificity in a nematode-bacterium mutualism.

Coevolution in mutualistic symbiosis can yield, because the interacting partners share common interests, to coadaptation: hosts perform better when associated with symbionts of their own locality than with others coming from more distant places. However, as the two partners of a symbiosis might also experience conflicts over part of their life cycle, coadaptation might not occur for all life-history traits. We investigated this issue in symbiotic systems where nematodes (Steinernema) and bacteria (Xenorhabdus) reproduce in insects they have both contributed to kill. Newborn infective juveniles (IJs) that carry bacteria in their intestine then disperse from the insect cadaver in search of a new host to infect. We ran experiments where nematodes coinfect insects with bacteria that differ from their native symbiont. In both Steinernema carpocapsae/Xenorhabdus nematophila and Steinernema feltiae/Xenorhabdus bovienii symbioses, we detected an overall specificity which favours the hypothesis of a fine-tuned co-adaptation process. However, we also found that the life-history traits involved in specificity strongly differ between the two model systems: when associated with strains that differ too much from their native symbionts, S. carpocapsae has low parasitic success, whereas S. feltiae has low survival in dispersal stage.

The role of ResA in type II cytochrome c maturation.

Numerous bacterial proteins involved in the nitrogen cycle, and other processes, require c-type haem as a cofactor. c-type cytochromes are formed by covalent attachment of haem to the conserved CXXCH motif. Here, we briefly review what is presently known about cytochrome c maturation in Bacillus subtilis with particular emphasis on the crystal structures of ResA.

When mutualists are pathogens: an experimental study of the symbioses between Steinernema (entomopathogenic nematodes) and Xenorhabdus (bacteria).

In this paper, we investigate the level of specialization of the symbiotic association between an entomopathogenic nematode (Steinernema carpocapsae) and its mutualistic native bacterium (Xenorhabdus nematophila). We made experimental combinations on an insect host where nematodes were associated with non-native symbionts belonging to the same species as the native symbiont, to the same genus or even to a different genus of bacteria. All non-native strains are mutualistically associated with congeneric entomopathogenic nematode species in nature. We show that some of the non-native bacterial strains are pathogenic for S. carpocapsae. When the phylogenetic relationships between the bacterial strains was evaluated, we found a clear negative correlation between the effect a bacterium has on nematode fitness and its phylogenetic distance to the native bacteria of this nematode. Moreover, only symbionts that were phylogenetically closely related to the native bacterial strain were transmitted. These results suggest that co-evolution between the partners has led to a high level of specialization in this mutualism, which effectively prevents horizontal transmission. The pathogenicity of some non-native bacterial strains against S. carpocapsae could result from the incapacity of the nematode to resist specific virulence factors produced by these bacteria.

Susceptibility of natural hybrids between house mouse subspecies to Sarcocystis muris.

A previous study showed that the susceptibility of hybrids between two Mus musculus musculus and Mus musculus domesticus did not apply to every parasite. The authors proposed that only parasites which exerted enough constraints would induce the selection of poorly compatible systems of resistance in the subspecies. This study completes the previous work. Experimental infections of mice of the two subspecies and their hybrids with the tissue-dwelling protozoan Sarcocystis muris show that hybrids are more susceptible to the coccidian than M. m. musculus and M. m. domesticus individuals. This result demonstrates that the hybrids are not only susceptible to intestinal helminths, and confirms the 'constraint hypothesis'.

Genes required for cytochrome c synthesis in Bacillus subtilis.

Cytochromes of c-type contain covalently bound haem and in bacteria are located on the periplasmic side of the cytoplasmic membrane. More than eight different gene products have been identified as being specifically required for the synthesis of cytochromes c in Gram-negative bacteria. Corresponding genes are not found in the genome sequences of Gram-positive bacteria. Using two random mutagenesis approaches, we have searched for cytochrome c biogenesis genes in the Gram-positive bacterium Bacillus subtilis. Three genes, resB, resC and ccdA, were identified. CcdA has been found previously and is required for a late step in cytochrome c synthesis and also plays a role in spore synthesis. No function has previously been assigned for ResB and ResC but these predicted membrane proteins show sequence similarity to proteins required for cytochrome c synthesis in chloroplasts. Attempts to inactivate resB and resC in B. subtilis have indicated that these genes are essential for growth. We demonstrate that various nonsense mutations in resB or resC can block synthesis of cytochromes c with no effect on other types of cytochromes and little effect on sporulation and growth. The results strongly support the recent proposal that Gram-positive bacteria, cyanobacteria, epsilon-proteobacteria, and chloroplasts have a similar type of machinery for cytochrome c synthesis (System II), which is very different from those of most Gram-negative bacteria (System I) and mitochondria (System III).

The iron oxidation and hydrolysis chemistry of Escherichia coli bacterioferritin.

Bacterioferritins are members of a class of spherical shell-like iron storage proteins that catalyze the oxidation and hydrolysis of iron at specific sites inside the protein shell, resulting in formation of a mineral core of hydrated ferric oxide within the protein cavity. Electrode oximetry/pH stat was used to study iron oxidation and hydrolysis chemistry in E. coli bacterioferritin. Consistent with previous UV-visible absorbance measurements, three distinct kinetic phases were detected, and the stoichiometric equations corresponding to each have been determined. The rapid phase 1 reaction corresponds to pairwise binding of 2 Fe(2+) ions at a dinuclear site, called the ferroxidase site, located within each of the 24 subunits, viz., 2Fe(2+) + P(Z) --> [Fe(2)-P](Z) + 4H(+), where P(Z) is the apoprotein of net charge Z and [Fe(2)-P](Z) represents a diferrous ferroxidase complex. The slower phase 2 reaction corresponds to the oxidation of this complex by molecular oxygen according to the net equation: [Fe(2)-P](Z) + (1)/(2)O(2) --> [Fe(2)O-P](Z) where [Fe(2)O-P](Z) represents an oxidized diferric ferroxidase complex, probably a mu-oxo-bridged species as suggested by UV-visible and EPR spectrometric titration data. The third phase corresponds to mineral core formation according to the net reaction: 4Fe(2+) + O(2) + 6H(2)O --> 4FeO(OH)((core)) + 8H(+). Iron oxidation is inhibited by the presence of Zn(2+) ions. The patterns of phase 2 and phase 3 inhibition are different, though inhibition of both phases is complete at 48 Zn(2+)per 24mer, i.e., 2 Zn(2+) per ferroxidase center.

Studies of the cytochrome subunits of menaquinone:cytochrome c reductase (bc complex) of Bacillus subtilis. Evidence for the covalent attachment of heme to the cytochrome b subunit.

The menaquinone:cytochrome c reductase, or bc complex, of Bacillus subtilis belongs to a third class of bc-type complex, distinct from the bc1 and b6f classes. Using a mutagenesis approach, we demonstrate that the cytochrome b (QcrB) and c (QcrC) subunits of the complex give rise to bands at 22 and 29 kDa, respectively, after denaturing electrophoresis; that both subunits are required for proper complex assembly and/or stability; and that both subunits retain one heme molecule under denaturing conditions. This unusual property of a b-type cytochrome was investigated further. We present evidence for the existence of a covalent linkage between the polypeptide and heme bH and of an important role for Cys43 in binding of heme bH. It is proposed that heme is also covalently attached to the cytochrome b subunit of b6f complexes of chloroplasts and cyanobacteria.

Interaction of nitric oxide with non-haem iron sites of Escherichia coli bacterioferritin: reduction of nitric oxide to nitrous oxide and oxidation of iron(II) to iron(III).

The bacterioferritin (BFR) of Escherichia coli consists of 24 identical subunits, each containing a dinuclear metal-binding site consisting of two histidines and four carboxylic acid residues. Earlier studies showed that the characterization of iron binding to BFR could be aided by EPR analysis of iron-nitrosyl species resulting from the addition of NO to the protein [Le Brun, Cheesman, Andrews, Harrison, Guest, Moore and Thomson (1993) FEBS Lett. 323, 261-266]. We now report data from gas chromatographic head space analysis combined with EPR spectroscopy to show that NO is not an inert probe: iron(II)-BFR catalyses the reduction of NO to N2O, resulting in oxidation of iron(II) at the dinuclear centre and the subsequent detection of mononuclear iron(III). In the presence of excess reductant (sodium ascorbate), iron(II)-BFR also catalyses the reduction of NO to N2O, giving rise to three mononuclear iron-nitrosyl species which are detectable by EPR. One of these, a dinitrosyl-iron complex of S = 1/2, present at a maximum of one per subunit, is shown by EPR studies of site-directed variants of BFR not to be located at the dinuclear centre. This is consistent with a proposal that the diferric form of the centre is unstable and breaks down to form mononuclear iron species.

Spectroscopic studies of cobalt(II) binding to Escherichia coli bacterioferritin.

The iron storage protein bacterioferritin (BFR) consists of 24 identical subunits, each containing a dinuclear metal binding site called the ferroxidase center, which is essential for fast iron core formation. Cobalt(II) binding to wild-type and site-directed variants of Escherichia coli BFR was studied by optical and magnetic techniques. Data from absorption spectroscopy demonstrate the binding of two cobalt(II) ions per subunit of wild-type and heme-free BFR, each with a pseudotetrahedral or pentacoordinate geometry, and EPR studies show that the two cobalt(II) ions are weakly magnetically coupled. Studies of variants of BFR in which a single glutamic acid residue at the ferroxidase center is replaced by alanine confirm that this is the site of cobalt(II) binding, since the altered centers bind only one cobalt(II) ion. This work shows that the electroneutrality of the ferroxidase center is preserved on binding a pair of divalent metal ions. Optical and EPR data show that cobalt(II) binding to BFR exhibits positive cooperativity, with an average Kd of approximately 1 x 10(-5) M. The favored filling of the ferroxidase center with pairs of metal ions may have mechanistic implications for the iron(II) binding process. Discrimination against oxidation of single iron(II) ions avoids odd electron reduction products of oxygen.

Charge compensated binding of divalent metals to bacterioferritin: H+ release associated with cobalt(II) and zinc(II) binding at dinuclear metal sites.

Divalent metal ion binding to the bacterial iron-storage protein, bacterioferritin (BFR), which contains a dinuclear metal binding site within each of its 24 subunits, was investigated by potentiometric and spectrophotometric methods. Cobalt(II) and zinc(II) were found to bind at both high- and low-affinity sites. Cobalt(II) binding at the high-affinity site was observed at a level of two per subunit with the release of approximately 1.6 protons per metal ion, thus confirming the dinuclear metal centre as the high-affinity site. Zinc(II) binding at the dinuclear centre (high-affinity site) resulted in the release of approximately 2 protons per metal ion, but exhibited a binding stoichiometry which indicated that not all dinuclear centres were capable of binding two zinc(II) ions. Competition data showed that binding affinities for the dinuclear centre were in the order zinc(II) > cobalt(II), and also confirmed the unexpected stoichiometry of zinc(II) binding. This work emphasises the importance of charge neutrality at the dinuclear centre.

Identification of the ferroxidase centre of Escherichia coli bacterioferritin.

The bacterioferritin (BFR) of Escherichia coli takes up iron in the ferrous form and stores it within its central cavity as a hydrated ferric oxide mineral. The mechanism by which oxidation of iron (II) occurs in BFR is largely unknown, but previous studies indicated that there is ferroxidase activity associated with a site capable of forming a dinuclear-iron centre within each subunit [Le Brun, Wilson, Andrews, Harrison, Guest, Thomson and Moore (1993) FEBS Lett. 333, 197-202]. We now report site-directed mutagenesis experiments based on a putative dinuclear-metal-ion-binding site located within the BFR subunit. The data reveal that this dinuclear-iron centre is located at a site within the four-alpha-helical bundle of each subunit of BFR, thus identified as the ferroxidase centre of BFR. The metal-bound form of the centre bears a remarkable similarity to the dinuclear-iron sites of the hydroxylase subunit of methane mono-oxygenase and the R2 subunit of ribonucleotide reductase. Details of how the dinuclear centre of BFR is involved in the oxidation mechanism were investigated by studying the inhibition of iron (II) oxidation by zinc (II) ions. Data indicate that zinc (II) ions bind at the ferroxidase centre of apo-BFR in preference to iron (II), resulting in a dramatic reduction in the rate of oxidation. The mechanism of iron (II) oxidation is discussed in the light of this and previous work.

MCD, EPR and NMR spectroscopic studies of rabbit hemopexin and its heme binding domain.

Heme binding to rabbit hemopexin and its domain I, obtained by proteolytic cleavage of intact hemopexin, was studied by EPR, MCD and 1H-NMR spectroscopies. The data obtained support the proposal that the heme Fe(III) is coordinated by two histidine ligands (Morgan et al. (1988) J. Biol. Chem. 263, 8220-8225; Muster et al. (1991) J. Protein Chem. 10, 123-128) and are inconsistent with recently reported mutagenesis studies indicating that bis-histidine ligation is unlikely (Satoh et al. (1994) Proc. Natl. Acad. Sci. USA 91, 8423-8427). Although the MCD data are consistent with both bis-histidine and histidine/lysine ligation, the EPR spectra are typical of bis-histidine ligation. Overall the magneto-optical spectra are characteristic for bis-histidine ligation. The EPR and NMR data indicate that there is a difference in the heme environments of the intact hemopexin and its domain I but overall the spectroscopic information suggests heme bound to domain I has the same ligands as intact hemopexin. The 1H-NMR studies indicate that heme binding to domain I perturbs at least 4 of the 5 histidines. This is consistent with axial ligation of the heme by two histidines, and a conformational change induced by heme binding affecting two more. Interestingly, resonances of the carbohydrate bound to intact hemopexin and domain I were also perturbed by heme binding. pH dependence studies showed that heme remained bound to intact hemopexin over the pH range 6.5-10.0 without any major change in the ligation or environment of the heme.

Site-directed replacement of the coaxial heme ligands of bacterioferritin generates heme-free variants.

The bacterioferritin (BFR) of Escherichia coli is a heme-containing iron storage molecule. It is composed of 24 identical subunits, which form a roughly spherical protein shell surrounding a central iron storage cavity. Each of the 12 heme moieties of BFR possesses bis-methionine axial ligation, a heme coordination scheme so far only found in bacterioferritins. Members of the BFR family contain three partially conserved methionine residues (excluding the initiating methionine) and in this study each was substituted by leucine and/or histidine. The Met52 variants were devoid of heme, whereas the Met31 and Met86 variants possessed full heme complements and were spectroscopically indistinguishable from wild-type BFR. The heme-free Met52 variants appeared to be correctly assembled and were capable of accumulating iron both in vivo and in vitro. No major differences were observed in the overall rate of iron accumulation for BFR-M52H, BFR-M52L, and the wild-type protein. The iron contents of the Met52 variants, as isolated, were at least 4 times greater than for wild-type BFR. This study is consistent with the reported location of the BFR heme site at the 2-fold axis and shows that heme is unnecessary for BFR assembly and iron uptake.

Hybrid vigour against parasites in interspecific crosses between two mice species.

The resistance and susceptibility to the intestinal pinworm Aspiculuris tetraptera, a natural parasite of the house mouse Mus musculus, is experimentally analysed using both the F1 from wild-type mice of the two subspecies (M. m. domesticus and M. m. musculus) and the F1 from different laboratory inbred mice. The results show that: (i) the F1 from wild-type mice harbour a lower parasite load than the parental mice, suggesting a phenomenon of hybrid vigour; and (ii) the F1 from inbred mice harbour parasite loads similar to the resistant parent, suggesting that resistance is inherited as a dominant feature in these laboratory mice. This analysis supports the hypothesis that recombinations occurring between the two mouse genomes (i.e. M. m. domesticus and M. m. musculus) are responsible for the hybrid dysgenesis observed in the natural hybrid zone between the two mice subspecies.

Structural heterogeneity of Pseudomonas aeruginosa bacterioferritin.

The subunit composition, amino acid sequence and haem-binding characteristics of bacterioferritin (BFR) from Pseudomonas aeruginosa have been studied. Unlike other BFRs, P. aeruginosa BFR was found to contain two subunit types, designated alpha and beta, which differed considerably in their amino acid sequences. The N-terminal 69 and 55 amino acids of the alpha and beta subunits respectively were determined. The alpha subunit differed most from other BFRs. The two subunits were present in variable proportions in different preparations. The maximum stoichiometry of haem binding was found to be sample-dependent and to be different from the previously reported one per subunit [Kadir and Moore (1990) FEBS Lett. 271, 141-143]. This previous haem-binding study was shown to have been carried out with damaged protein, which contained both normal alpha and beta subunits and shorter versions of these that appeared to have been produced by cleavage of the normal subunits. The possibility that aging processes degrade ferritins and affect their haem-binding characteristics is discussed.

Replacement of methionine as the axial ligand of Achromobacter cycloclastes cytochrome c554 at high pH values revealed by absorption, EPR and MCD spectroscopy.

Cytochrome c554 from the denitrifying bacterium Achromobacter cycloclastes is a monoheme class II c-type cytochrome with a His-Met axial coordination at neutral pH. The amino acid composition and the N-terminal sequence of the cytochrome have been determined. Subsequent determination of the pH-dependence of the redox potential and examination of the EPR and MCD spectra of ferricytochrome c554 revealed a new form at high pH values made apparent with both spectroscopies. These observations are consistent with the presence of lysine as the axial ligand for which methionine substitutes at high pH values.

Kinetic and structural characterization of an intermediate in the biomineralization of bacterioferritin.

The mechanism by which iron-storage proteins take up and oxidise iron(II) is not understood. We show by rapid-kinetic and EPR measurements that iron uptake, in vitro, by a bacterial iron-storage protein, bacterioferritin, involves at least three kinetically distinguishable phases: phase 1, the binding of Fe(II) ions, probably at a dimeric iron ferroxidase centre; phase 2, oxidation of the Fe(II) dimer and production of mononuclear Fe(III); and phase 3, iron core formation.