Nitrogen-fixing Bacteroids Research Articles

An interdisciplinary look at legumes and their bacterial symbionts: some thoughts from Big Sky

An interdisciplinary look at legumes and their bacterial symbionts: some thoughts from Big Sky

An Alternative Succinate (2-Oxoglutarate) Transport System in Rhizobium tropici Is Induced in Nodules of Phaseolus vulgaris

The rhizobial DctA permease is essential for the development of effective nitrogen-fixing bacteroids, which was correlated with its requirement for growth on C(4)-dicarboxylates. A previously described dctA mutant of Rhizobium tropici CIAT899, strain GA1 (dctA), however, was unexpectedly still able to grow on succinate as a sole carbon source but less efficiently than CIAT899. Like other rhizobial dctA mutants, GA1 was unable to grow on fumarate or malate as a carbon source and induced the formation of ineffective nodules. We report an alternative succinate uptake system identified by Tn5 mutagenesis of strain GA1 that was required for the remaining ability to transport and utilize succinate. The alternative uptake system required a three-gene cluster that is highly characteristic of a dctABD locus. The predicted permease-encoding gene had high sequence similarity with open reading frames encoding putative 2-oxoglutarate permeases (KgtP) of Ralstonia solanacearum and Agrobacterium tumefaciens. This analysis was in agreement with the requirement for this gene for optimal growth on and induction by 2-oxoglutarate. The permease-encoding gene of the alternative system was also designated kgtP in R. tropici. The dctBD-like genes in this cluster were found to be required for kgtP expression and were designated kgtSR. Analysis of a kgtP::lacZ transcriptional fusion indicated that a kgtSR-dependent promoter of kgtP was specifically induced by 2-oxoglutarate. The expression of kgtPp was found in bacteroids of nodules formed with either CIAT899 or GA1 on roots of Phaseolus vulgaris. Results suggested that 2-oxoglutarate might be transported or conceivably exported in nodules induced by R. tropici on roots of P. vulgaris.

Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation

Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO(2) to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an alpha-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active alpha-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and delta(13)C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH(3) protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule delta(13)C abundance suggest the recycling of at least part of the HCO(3)(-) produced by CAA1.

Rhizobial secreted proteins as determinants of host specificity in the rhizobium–legume symbiosis

Rhizobia are Gram-negative bacteria than can elicit the formation of specialized organs, called root nodules, on leguminous host plants. Upon infection of the nodules, they differentiate into nitrogen-fixing bacteroids. An elaborate signal exchange precedes the symbiotic interaction. In general, both rhizobia and host plants exhibit narrow specificity. Rhizobial factors contributing to this specificity include Nod factors and surface polysaccharides. It is becoming increasingly clear that protein secretion is important in determining the outcome of the interaction as well. This paper discusses our current understanding of the symbiotic role played by rhizobial secreted proteins, transported both by secretion systems that are of general use, such as the type I secretion system, and by specialized, host-targeting secretion systems, such as the type III, type IV and type VI secretion systems.

Malic Enzyme Cofactor and Domain Requirements for Symbiotic N 2 Fixation by Sinorhizobium meliloti

The NAD(+)-dependent malic enzyme (DME) and the NADP(+)-dependent malic enzyme (TME) of Sinorhizobium meliloti are representatives of a distinct class of malic enzymes that contain a 440-amino-acid N-terminal region homologous to other malic enzymes and a 330-amino-acid C-terminal region with similarity to phosphotransacetylase enzymes (PTA). We have shown previously that dme mutants of S. meliloti fail to fix N(2) (Fix(-)) in alfalfa root nodules, whereas tme mutants are unimpaired in their N(2)-fixing ability (Fix(+)). Here we report that the amount of DME protein in bacteroids is 10 times greater than that of TME. We therefore investigated whether increased TME activity in nodules would allow TME to function in place of DME. The tme gene was placed under the control of the dme promoter, and despite elevated levels of TME within bacteroids, no symbiotic nitrogen fixation occurred in dme mutant strains. Conversely, expression of dme from the tme promoter resulted in a large reduction in DME activity and symbiotic N(2) fixation. Hence, TME cannot replace the symbiotic requirement for DME. In further experiments we investigated the DME PTA-like domain and showed that it is not required for N(2) fixation. Thus, expression of a DME C-terminal deletion derivative or the Escherichia coli NAD(+)-dependent malic enzyme (sfcA), both of which lack the PTA-like region, restored wild-type N(2) fixation to a dme mutant. Our results have defined the symbiotic requirements for malic enzyme and raise the possibility that a constant high ratio of NADPH + H(+) to NADP in nitrogen-fixing bacteroids prevents TME from functioning in N(2)-fixing bacteroids.

Structural Characterization of a K-antigen Capsular Polysaccharide Essential for Normal Symbiotic Infection in Rhizobium sp. NGR234: DELETION OF THE rkpMNO LOCUS PREVENTS SYNTHESIS OF 5,7-DIACETAMIDO-3,5,7,9-TETRADEOXY-NON-2-ULOSONIC ACID

Many early molecular events in symbiotic infection have been documented, although factors enabling Rhizobium to progress within the plant-derived infection thread and ultimately survive within the intracellular symbiosome compartment as mature nitrogen-fixing bacteroids are poorly understood. Rhizobial surface polysaccharides (SPS), including the capsular polysaccharides (K-antigens), exist in close proximity to plant-derived membranes throughout the infection process. SPSs are essential for bacterial survival, adaptation, and as potential determinants of nodulation and/or host specificity. Relatively few studies have examined the role of K-antigens in these events. However, we constructed a mutant that lacks genes essential for the production of the K-antigen strain-specific sugar precursor, pseudaminic acid, in the broad host range Rhizobium sp. NGR234. The complete structure of the K-antigen of strain NGR234 was established, and it consists of disaccharide repeating units of glucuronic and pseudaminic acid having the structure -->4)-beta-d-glucuronic acid-(1-->4)-beta-5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid-(2-->. Deletion of three genes located in the rkp-3 gene cluster, rkpM, rkpN, and part of rkpO, abolished pseudaminic acid synthesis, yielding a mutant in which the strain-specific K-antigen was totally absent: other surface glycoconjugates, including the lipopolysaccharides, exopolysaccharides, and flagellin glycoprotein appeared unaffected. The NGRDeltarkpMNO mutant was symbiotically defective, showing reduced nodulation efficiency on several legumes. K-antigen production was found to decline after rhizobia were exposed to plant flavonoids, and the decrease coincided with induction of a symbiotically active (bacteroid-specific) rhamnan-LPS, suggesting an exchange of SPS occurs during bacterial differentiation in the developing nodule.

Sinorhizobium meliloti Differentiation During Symbiosis with Alfalfa: A Transcriptomic Dissection

Sinorhizobium meliloti is a soil bacterium able to induce the formation of nodules on the root of specific legumes, including alfalfa (Medicago sativa). Bacteria colonize nodules through infection threads, invade the plant intracellularly, and ultimately differentiate into bacteroids capable of reducing atmospheric nitrogen to ammonia, which is directly assimilated by the plant. As a first step to describe global changes in gene expression of S. meliloti during the symbiotic process, we used whole genome microarrays to establish the transcriptome profile of bacteria from nodules induced by a bacterial mutant blocked at the infection stage and from wild-type nodules harvested at various timepoints after inoculation. Comparison of these profiles to those of cultured bacteria grown either to log or stationary phase as well as examination of a number of genes with known symbiotic transcription patterns allowed us to correlate global gene-expression patterns to three known steps of symbiotic bacteria bacteroid differentiation, i.e., invading bacteria inside infection threads, young differentiating bacteroids, and fully differentiated, nitrogen-fixing bacteroids. Finally, analysis of individual gene transcription profiles revealed a number of new potential symbiotic genes.

Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium –legume symbiosis

Symbiosis between legumes and Rhizobium bacteria leads to the formation of root nodules where bacteria in the infected plant cells are converted into nitrogen-fixing bacteroids. Nodules with a persistent meristem are indeterminate, whereas nodules without meristem are determinate. The symbiotic plant cells in both nodule types are polyploid because of several cycles of endoreduplication (genome replication without mitosis and cytokinesis) and grow consequently to extreme sizes. Here we demonstrate that differentiation of bacteroids in indeterminate nodules of Medicago and related legumes from the galegoid clade shows remarkable similarity to host cell differentiation. During bacteroid maturation, repeated DNA replication without cytokinesis results in extensive amplification of the entire bacterial genome and elongation of bacteria. This finding reveals a positive correlation in prokaryotes between DNA content and cell size, similar to that in eukaryotes. These polyploid bacteroids are metabolically functional but display increased membrane permeability and are nonviable, because they lose their ability to resume growth. In contrast, bacteroids in determinate nodules of the nongalegoid legumes lotus and bean are comparable to free-living bacteria in their genomic DNA content, cell size, and viability. Using recombinant Rhizobium strains nodulating both legume types, we show that bacteroid differentiation is controlled by the host plant. Plant factors present in nodules of galegoid legumes but absent from nodules of nongalegoid legumes block bacterial cell division and trigger endoreduplication cycles, thereby forcing the endosymbionts toward a terminally differentiated state. Hence, Medicago and related legumes have evolved a mechanism to dominate the symbiosis.

The pea nodule environment restores the ability of a Rhizobium leguminosarum lipopolysaccharide acpXL mutant to add 27-hydroxyoctacosanoic acid to its lipid A.

Members of the Rhizobiaceae contain 27-hydroxyoctacosanoic acid (27OHC(28:0)) in their lipid A. A Rhizobium leguminosarum 3841 acpXL mutant (named here Rlv22) lacking a functional specialized acyl carrier lacked 27OHC(28:0) in its lipid A, had altered growth and physiological properties (e.g., it was unable to grow in the presence of an elevated salt concentration [0.5% NaCl]), and formed irregularly shaped bacteroids, and the synchronous division of this mutant and the host plant-derived symbiosome membrane was disrupted. In spite of these defects, the mutant was able to persist within the root nodule cells and eventually form, albeit inefficiently, nitrogen-fixing bacteroids. This result suggested that while it is in a host root nodule, the mutant may have some mechanism by which it adapts to the loss of 27OHC(28:0) from its lipid A. In order to further define the function of this fatty acyl residue, it was necessary to examine the lipid A isolated from mutant bacteroids. In this report we show that addition of 27OHC(28:0) to the lipid A of Rlv22 lipopolysaccharides is partially restored in Rlv22 acpXL mutant bacteroids. We hypothesize that R. leguminosarum bv. viciae 3841 contains an alternate mechanism (e.g., another acp gene) for the synthesis of 27OHC(28:0), which is activated when the bacteria are in the nodule environment, and that it is this alternative mechanism which functionally replaces acpXL and is responsible for the synthesis of 27OHC(28:0)-containing lipid A in the Rlv22 acpXL bacteroids.

Accumulation of an Aromatic Amine, β-Phenethylamine, in Root Nodules of Adzuki Bean Vigna angularis

Neuroactive aromatic amines acting on the central nervous system are widespread in the plant kingdom. We have previously found β-phenethylamine (β-PHA), one of the aromatic alkaloids, in root nodules of various annual legume crops. The present study was undertaken to determine the site of β-PHA accumulation within root nodules of the adzuki bean Vigna angularis. High concentrations of β-PHA were always detected in the alkaloid fraction of adzuki bean root nodules. Related aromatic amines such as tyramine, dopamine, and other β-PHA derivatives, which are found in various medicinal plants, were not detected in adzuki bean root nodules. The amounts of β-PHA in root nodules varied not only with the growth stage of the host plant, but also with nodule age; β-PHA levels increased with nodule development, but declined with nodule senescence. Adzuki bean nodules, after crushing with a grinding medium, were separated into bacteroids and a nodule cytosol fraction. A large amount of β-PHA was detected in the bacteroids, while a very small amount was prsent in the nodule cytosol fraction derived from plant cells. The bacteroids in the mature nodules contained considerably higher amounts of β-PHA than did those in immature or senescent nodules. The formation of β-PHA in root-nodule bacteria was then tested using eight strains of Rhizobiaceae (Rhizobium, Bradyrhizobium and Sinorhizobium), including a strain isolated from root nodules of field-grown adzuki bean plant. None of the cultured cells produced β-PHA in liquid media in the presence or absence of phenylalanine, a putative precursor of β-PHA. Nitrogen-fixing bacteroids within nodules are the cells uniquely differentiated from root-nodule bacteria. The present results suggest that β-PHA is formed associated with the differentiation of vegetative bradyrhizobia into nitrogen-fixing bacteroids with the plant host cells.

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

Rhizobium (Sinorhizobium) sp. strain NGR234 contains three replicons, the smallest of which (pNGR234a) carries most symbiotic genes, including those required for nodulation and lipo-chito-oligosaccharide (Nod factor) biosynthesis. Activation of nod gene expression depends on plant-derived flavonoids, NodD transcriptional activators, and nod box promoter elements. Nod boxes NB6 and NB7 delimit six different types of genes, one of which (fixF) is essential for the formation of effective nodules on Vigna unguiculata. In vegetative culture, wild-type NGR234 produces a distinct, flavonoid-inducible lipopolysaccharide (LPS) that is not produced by the mutant (NGRomegafixF); this LPS is also found in nitrogen-fixing bacteroids isolated from V. unguiculata infected with NGR234. Electron microscopy showed that peribacteroid membrane formation is perturbed in nodule cells infected by the fixF mutant. LPSs were purified from free-living NGR234 cultured in the presence of apigenin. Structural analyses showed that the polysaccharide portions of these LPSs are specialized, rhamnose-containing O antigens attached to a modified core-lipid A carrier. The primary sequence of the O antigen is [-3)-alpha-L-Rhap-(1,3)-alpha-L-Rhap-(1,2)-alpha-L-Rhap-(1-]n, and the LPS core region lacks the acidic sugars commonly associated with the antigenic outer core of LPS from noninduced cells. This rhamnan O antigen, which is absent from noninduced cells, has the same primary sequence as the A-band O antigen of Pseudomonas aeruginosa, except that it is composed of L-rhamnose rather than the D-rhamnose characteristic of the latter. It is noteworthy that A-band LPS is selectively maintained on the P. aeruginosa cell surface during chronic cystic fibrosis lung infection, where it is associated with an increased duration of infection.

CDNA Macroarray Analysis of Gene Expression in Ineffective Nodules Induced on the Lotus japonicus sen1 Mutant

The Lotus japonicus sen1 mutant forms ineffective nodules in which development is arrested at the stage of bacterial differentiation into nitrogen-fixing bacteroids. Here, we used cDNA macroarray systems to compare gene expression in ineffective nodules induced on the sen1 mutant with gene expression in wild-type nodules, in order to identify the host plant genes that are involved in nitrogen fixation. Macroarray analysis coupled with Northern blot analysis revealed that the expression of 18 genes was significantly enhanced in ineffective sen1 nodules, whereas the expression of 30 genes was repressed. Many of the enhanced genes encoded hydrolase enzymes, such as cysteine proteinase and asparaginase, that might function in the early senescence of sen1 nodules. By contrast, the repressed genes encoded nodulins, enzymes that are involved in carbon and nitrogen metabolism, membrane transporters, enzymes involved in phytohormone metabolism and secondary metabolism, and regulatory proteins. These proteins might have a role in the establishment of nitrogen fixation. In addition, we discovered two novel genes that encoded glutamate-rich proteins and were localized in the vascular bundles of the nodules. The expression of these genes was repressed in the ineffective nodules, which had lower levels of nitrogenase activity.

The Lotus japonicus Sen1 gene controls rhizobial differentiation into nitrogen-fixing bacteroids in nodules.

A Lotus japonicus mutant, Ljsym75, which forms ineffective symbiotic nodules and defines a new locus involved in the process of nitrogen fixation, was characterized in detail in order to identify the stage of developmental arrest of the nodules. No nitrogen-fixing activity was detectable in Ljsym75 nodules at any stage during plant development, and plant growth was markedly retarded. Ljsym75 plants formed twice as many nodules as the wild-type Gifu, and this phenotype was not influenced by the application of low concentrations of nitrate. Although the ineffective nodules formed on Ljsym75 were anatomically similar to effective Gifu nodules, Ljsym75 nodules senesced prematurely. Microscopic examination revealed that bacteria endocytosed into Ljsym75 nodules failed to differentiate into bacteroids. Moreover, the bacteria contained no nitrogenase proteins, whereas leghemoglobin was detected in the cytosol of the nodules. These results indicate that Ljsym75 is required for bacterial differentiation into nitrogen-fixing bacteroids in nodules, and thus the Ljsym75 gene was renamed sen1 (for stationary endosymbiont nodule). Linkage analysis using DNA markers showed that Sen1 is located on chromosome 4.

Crinkle, a novel symbiotic mutant that affects the infection thread growth and alters the root hair, trichome, and seed development in Lotus japonicus.

To elucidate the mechanisms involved in Rhizobium-legume symbiosis, we examined a novel symbiotic mutant, crinkle (Ljsym79), from the model legume Lotus japonicus. On nitrogen-starved medium, crinkle mutants inoculated with the symbiont bacterium Mesorhizobium loti MAFF 303099 showed severe nitrogen deficiency symptoms. This mutant was characterized by the production of many bumps and small, white, uninfected nodule-like structures. Few nodules were pale-pink and irregularly shaped with nitrogen-fixing bacteroids and expressing leghemoglobin mRNA. Morphological analysis of infected roots showed that nodulation in crinkle mutants is blocked at the stage of the infection process. Confocal microscopy and histological examination of crinkle nodules revealed that infection threads were arrested upon penetrating the epidermal cells. Starch accumulation in uninfected cells and undeveloped vascular bundles were also noted in crinkle nodules. Results suggest that the Crinkle gene controls the infection process that is crucial during the early stage of nodule organogenesis. Aside from the symbiotic phenotypes, crinkle mutants also developed morphological alterations, such as crinkly or wavy trichomes, short seedpods with aborted embryos, and swollen root hairs. crinkle is therefore required for symbiotic nodule development and for other aspects of plant development.

Transcriptome analysis of Sinorhizobium meliloti during symbiosis

The recently determined Sinorhizobium meliloti genome sequence was used to construct nylon macroarrays, to search for genes potentially relevant to symbiosis. Genes related to invasion, oxidative stress protection, iron mobilization, and signaling, were found emphasizing possible common mechanisms between symbiosis and pathogenesis.

A homolog of the CtrA cell cycle regulator is present and essential in Sinorhizobium meliloti.

During development of the symbiotic soil bacterium Sinorhizobium meliloti into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes. Regulatory genes controlling the early stages of this process have not been identified. As a first step in the search for regulators of these events, we report the isolation and characterization of a ctrA gene from S. meliloti. We show that the S. meliloti CtrA belongs to the CtrA-like family of response regulators found in several alpha-proteobacteria. In Caulobacter crescentus, CtrA is essential and is a global regulator of multiple cell cycle functions. ctrA is also an essential gene in S. meliloti, and it is expressed similarly to the autoregulated C. crescentus ctrA in that both genes have complex promoter regions which bind phosphorylated CtrA.

The Rhizobium leguminosarum glnB gene is down-regulated during symbiosis.

Symbiotic nitrogen fixation involves the development, on the legume plant root, of specialised organs called nodules, within which plant photosynthates are exchanged for combined nitrogen of bacterial origin. The glnB gene encodes a signal transduction protein (P(II)) which is a component of the bacterial nitrogen regulation (Ntr) system and an essential regulator of ammonium assimilation. We demonstrate that in Rhizobium leguminosarum the glnB promoter is strongly regulated by nitrogen and NtrC, but still shows a significant level of activity in conditions of nitrogen excess. Expression of genes involved in nitrogen assimilation has been shown to be absent in nitrogen-fixing bacteroids, and, in agreement with this, we find that the glnB promoter is down-regulated during bacteroid differentiation at a time coincident with the arrest of bacterial division in the nodule. This pattern is common to other bacterial genes involved in nitrogen assimilation and it is noteworthy that the zone where the glnB promoter is active is coincident with the region in which NtrC is expressed.

Critical protective role of bacterial superoxide dismutase in rhizobium-legume symbiosis.

In nitrogen-poor soils, rhizobia elicit nodule formation on legume roots, within which they differentiate into bacteroids that fix atmospheric nitrogen. Protection against reactive oxygen species (ROS) was anticipated to play an important role in Rhizobium-legume symbiosis because nitrogenase is extremely oxygen sensitive. We deleted the sodA gene encoding the sole cytoplasmic superoxide dismutase (SOD) of Sinorhizobium meliloti. The resulting mutant, deficient in superoxide dismutase, grew almost normally and was only moderately sensitive to oxidative stress when free living. In contrast, its symbiotic properties in alfalfa were drastically affected. Nitrogen-fixing ability was severely impaired. More strikingly, most SOD-deficient bacteria did not reach the differentiation stage of nitrogen-fixing bacteroids. The SOD-deficient mutant nodulated poorly and displayed abnormal infection. After release into plant cells, a large number of bacteria failed to differentiate into bacteroids and rapidly underwent senescence. Thus, bacterial SOD plays a key protective role in the symbiotic process.

Immunolocalization of a cysteine protease in vacuoles, vesicles, and symbiosomes of pea nodule cells.

PsCYP15A is a cysteine protease from pea (Pisum sativum L.). It was first recognized as an up-regulated transcript in wilted shoots and subsequently in root nodules containing Rhizobium. Proteolytic activity of PsCYP15A in nodule extracts is now reported following immunopurification with polyclonal antiserum raised against recombinant antigen. Western-blot analysis indicated two forms of PsCYP15A, a pro-form (approximately 38 kD) and a mature form (approximately 30 kD). Both forms were present in most tissue samples, but only the mature form was isolated from cell-fractionated symbiosomes containing nitrogen-fixing bacteroids. Immunolabeling of nodule sections showed localization of PsCYP15A antigen in large vacuolar bodies, cytoplasmic vesicles, and the perisymbiont space. Immunolabeling of tissue sections from wilted shoots also indicated the presence of PsCYP15A in vacuoles and cytoplasmic vesicles. This protease may be involved in the adaptation to changes in cell turgor, both in wilted shoots and in nodule tissue. Additionally, the protease may be involved in protein turnover in the symbiosome compartment.

Analysis of the Rhizobium leguminosarum siderophore-uptake gene fhuA: differential expression in free-living bacteria and nitrogen-fixing bacteroids and distribution of an fhuA pseudogene in different strains.

A mutation was isolated in the Rhizobium leguminosarum gene fhuA, which appears to specify the outer-membrane receptor for the siderophore vicibactin. The mutant was defective in iron uptake and accumulated the siderophore vicibactin in the extracellular medium. Expression of fhuA was regulated by Fe3+, transcription being higher in iron-depleted cells. Transcription of fhuA was independent of a functional copy of rpol, a neighbouring gene that specifies a putative ECF sigma factor of RNA polymerase and which is involved in siderophore production in Rhizobium. Mutations in fhuA did not detectably affect symbiotic N2 fixation on peas. An fhuA::gus fusion was expressed by bacteria in the meristematic zone of pea nodules but not in mature bacteroids. Some other strains of R. leguminosarum also contain a pseudogene version of fhuA. The sequences of some of these and the 'real' fhuA genes were determined.