A multitude of CRP/FNR-like transcription proteins in Bradyrhizobium japonicum.

In Bradyrhizobium japonicum, the nitrogen-fixing soya bean endosymbiont and facultative denitrifier, three CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein)-type transcription factors [FixK(1), FixK(2) and NnrR (nitrite and nitric oxide reductase regulator)] have been studied previously in the context of the regulation of nitrogen fixation and denitrification. The gene expression of both fixK(1) and nnrR depends on FixK(2), which acts as a key distributor of the 'low-oxygen' signal perceived by the two-component regulatory system FixLJ. While the targets for FixK(1) are not known, NnrR transduces the nitrogen oxide signal to the level of denitrification gene expression. Besides these three regulators, the complete genome sequence of this organism has revealed the existence of 13 additional CRP/FNR-type proteins whose functions have not yet been studied. Based on sequence similarity and phylogenetic analysis, we discuss in this paper the peculiarities of these additional factors.

Design and validation of a partial-genome microarray for transcriptional profiling of the Bradyrhizobium japonicum symbiotic gene region.

The design and use of a pilot microarray for transcriptome analysis of the symbiotic, nitrogen-fixing Bradyrhizobium japonicum is reported here. The custom-synthesized chip (Affymetrix GeneChip) features 738 genes, more than half of which belong to a 400-kb chromosomal segment strongly associated with symbiosis-related functions. RNA was isolated following an optimized protocol from wild-type cells grown aerobically and microaerobically, and from cells of aerobically grown regR mutant and microaerobically grown nifA mutant. Comparative microarray analyses thus revealed genes that are transcribed in either a RegR- or a NifA-dependent manner plus genes whose expression depends on the cellular oxygen status. Several genes were newly identified as members of the RegR and NifA regulons, beyond genes, which had been known from previous work. A comprehensive transcription analysis was performed with one of the new RegR-controlled genes (id880). Expression levels determined by microarray analysis of selected NifA- and RegR-controlled genes corresponded well with quantitative real-time PCR data, demonstrating the high complementarity of microarray analysis to classical methods of gene expression analysis in B. japonicum. Nevertheless, several previously established members of the NifA regulon were not detected as transcribed genes by microarray analysis, confirming the potential pitfalls of this approach also observed by other authors. By and large, this pilot study has paved the way towards the genome-wide transcriptome analysis of the 9.1-Mb B. japonicum genome.

A mRNA-based thermosensor controls expression of rhizobial heat shock genes.

Expression of several heat shock operons, mainly coding for small heat shock proteins, is under the control of ROSE (repression of heat shock gene expression) in various rhizobial species. This negatively cis-acting element confers temperature control by preventing expression at physiological temperatures. We provide evidence that ROSE-mediated regulation occurs at the post-transcriptional level. A detailed mutational analysis of ROSE(1)-hspA translationally fused to lacZ revealed that its highly conserved 3'-half is required for repression at normal temperatures (30 degrees C). The mRNA in this region is predicted to form an extended secondary structure that looks very similar in all 15 known ROSE elements. Nucleotides involved in base pairing are strongly conserved, whereas nucleotides in loop regions are more divergent. Base substitutions leading to derepression of the lacZ fusion at 30 degrees C exclusively resided in potential stem structures. Optimised base pairing by elimination of a bulged residue and by introduction of complementary nucleotides in internal loops resulted in ROSE elements that were tightly repressed not only at normal but also at heat shock temperatures. We propose a model in which the temperature-regulated secondary structure of ROSE mRNA influences heat shock gene expression by controlling ribosome access to the ribosome-binding site.

Structure of the soluble domain of a membrane-anchored thioredoxin-like protein from Bradyrhizobium japonicum reveals unusual properties.

TlpA is an unusual thioredoxin-like protein present in the nitrogen-fixing soil bacterium Bradyrhizobium japonicum. A hydrophobic N-terminal transmembrane domain anchors it to the cytoplasmic membrane, whereby the hydrophilic thioredoxin domain becomes exposed to the periplasmic space. There, TlpA catalyses an essential reaction, probably a reduction, in the biogenesis of cytochrome aa(3). The soluble thioredoxin domain (TlpA(sol)), devoid of the membrane anchor, was purified and crystallized. Oxidized TlpA(sol) crystallized as a non-covalent dimer in the space group P2(1)2(1)2(1). The X-ray structure analysis was carried out by isomorphous replacement using a xenon derivative. This resulted in a high-resolution (1.6 A) three-dimensional structure that displayed all of the features of a classical thioredoxin fold. A number of peculiar structural details were uncovered: (i) Only one of the two active-site-cysteine sulphurs (Cys72, the one closer to the N terminus) is exposed on the surface, making it the likely nucleophile for the reduction of target proteins. (ii) TlpA(sol) possesses a unique structural disulphide bond, formed between Cys10 and Cys155, which connects an unprecedented N-terminal alpha helix with a beta sheet near the C terminus. (iii) An insertion of about 25 amino acid residues, not found in the thioredoxin prototype of Escherichia coli, contributes only marginally to the thioredoxin fold, but forms an extra, surface-exposed alpha helix. This region plus another surface-exposed stretch (-Ile-Gly-Arg-Ala-), which is absent even in the closest TlpA relatives, might be considered as specificity determinants for the recognition of target proteins in the periplasm. The TlpA(sol) structure paves the way towards unraveling important structure-function relationships by rational mutagenesis.

Discovery of a haem uptake system in the soil bacterium Bradyrhizobium japonicum.

In Bradyrhizobium japonicum, the nitrogen-fixing symbiont of soybeans, we have identified a haem uptake system, Hmu, that comprises a cluster of nine open reading frames. Predicted products of these genes include: HmuR, a TonB-dependent haem receptor in the outer membrane; HmuT, a periplasmic haem-binding protein; and HmuUV, an ABC transporter in the inner membrane. Furthermore, we identified homologues of ExbBD and TonB, that are required for energy transduction from the inner to the outer membrane. Mutant analysis and complementation tests indicated that HmuR and the ExbBD-TonB system, but not the HmuTUV transporter, are essential for haem uptake or haem acquisition from haemoglobin and leghaemoglobin. The TonB system seems to be specific for haem uptake as it is dispensable for siderophore uptake. Therefore, we propose the existence of a second TonB homologue functioning in the uptake of Fe-chelates. When tested on soybean host plants, hmuT-hmuR and exbD-tonB mutants exhibited wild-type symbiotic properties. Thus, haem uptake is not essential for symbiotic nitrogen fixation but it may enable B. japonicum to have access to alternative iron sources in its non-symbiotic state. Transcript analysis and expression studies with lacZ fusions showed that expression of hmuT and hmuR is induced under low iron supply. The same was observed in fur and irr mutant backgrounds although maximal induction levels were decreased. We conclude either that both regulators, Fur and Irr, independently mediate transcriptional control by iron or that a yet unknown iron regulatory system activates gene expression under iron deprivation. An A/T-rich cis-acting element, located in the promoter region of the divergently transcribed hmuTUV and hmuR genes, is possibly required for this type of iron control.

Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant.

Phosphatidylcholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae. For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen-fixing root nodules. A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE). Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N-methyltransferase PmtA. Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE. PmtA-deficient B. japonicum mutants still produced low levels of PC by a second methylation pathway. The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids). Root nodules of soybean plants infected with B. japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild-type level. The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin. Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells. These data suggest that the biosynthesis of wild-type amounts of PC are required to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant.

One of two hemN genes in Bradyrhizobium japonicum is functional during anaerobic growth and in symbiosis.

Previously, we screened the symbiotic gene region of the Bradyrhizobium japonicum chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. Göttfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472-1480, 2000). Here we report more details on one of the genes identified, a hemN-like gene (now called hemN(1)) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that B. japonicum possesses a second hemN-like gene (hemN(2)), which was then cloned by using hemN(1) as a probe. The hemN(2) gene maps outside of the symbiotic gene region; it is located 1.5 kb upstream of nirK, the gene for a Cu-containing nitrite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN(1) and HemN(2), respectively) and share 53% identical (68% similar) amino acids. Expression of both hemN genes was monitored with the help of chromosomally integrated translational lacZ fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (> or = 20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK(2). In addition, maximal anaerobic hemN(1) expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual hemN genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the hemN(1) mutant, strains lacking a functional hemN(2) gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these mutants lacked several c-type cytochromes which are normally detectable by heme staining of proteins from anaerobically grown wild-type cells. Taken together, our results revealed that B. japonicum hemN(2), but not hemN(1), encodes a protein that is functional under the conditions tested, and this conclusion was further corroborated by the successful complementation of a Salmonella enterica serovar Typhimurium hemF hemN mutant with hemN(2) only.

Potential symbiosis-specific genes uncovered by sequencing a 410-kilobase DNA region of the Bradyrhizobium japonicum chromosome.

The physical and genetic map of the Bradyrhizobium japonicum chromosome revealed that nitrogen fixation and nodulation genes are clustered. Because of the complex interactions between the bacterium and the plant, we expected this chromosomal sector to contain additional genes that are involved in the maintenance of an efficient symbiosis. Therefore, we determined the nucleotide sequence of a 410-kb region. The overall G+C nucleotide content was 59.1%. Using a minimum gene length of 150 nucleotides, 388 open reading frames (ORFs) were selected as coding regions. Thirty-five percent of the predicted proteins showed similarity to proteins of rhizobia. Sixteen percent were similar only to proteins of other bacteria. No database match was found for 29%. Repetitive DNA sequence-derived ORFs accounted for the rest. The sequenced region contained all nitrogen fixation genes and, apart from nodM, all nodulation genes that were known to exist in B. japonicum. We found several genes that seem to encode transport systems for ferric citrate, molybdate, or carbon sources. Some of them are preceded by -24/-12 promoter elements. A number of putative outer membrane proteins and cell wall-modifying enzymes as well as a type III secretion system might be involved in the interaction with the host.

An imperfect inverted repeat is critical for DNA binding of the response regulator RegR of Bradyrhizobium japonicum.

RegR is the response regulator of the RegSR two-component regulatory system in Bradyrhizobium japonicum. The only target known so far is the fixR-nifA operon, encoding the redox-responsive transcription factor NifA, which activates many genes required for symbiotic nitrogen fixation in soybean nodules. In previous in vivo studies, we identified a 32 bp upstream activating sequence located around position -68, which is essential for RegR-dependent expression of the fixR-nifA operon. Here, we used an in vitro binding-site selection assay (SELEX) to more precisely define the DNA-binding specificity of RegR. The selected sequences comprised an imperfect inverted repeat (GCGGC-N(5)-GTCGC) which is highly similar to an imperfect inverted repeat in the fixR UAS (GCGAC-N(5)-GACGC). In a parallel approach, band-shift experiments were performed with oligonucleotides comprising defined point or deletion mutations in the fixR UAS. This led to the identification of 11 critical nucleotides within a 17 bp minimal RegR binding site centered at position -64 upstream of the fixR-nifA transcription start site. Notably, all 11 critical nucleotides were located either within the half sites of the inverted repeat (four nucleotides in each half site) or in the 5 bp spacer that separates the half sites (three nucleotides). Based on these results, we defined a DNA motif comprising those nucleotides that are critical for RegR binding (RegR box; 5'-GNG(A)(G)C(A)(G)TTNNGNCGC-3'). A comparison of the RegR box with functional binding sites of the RegR-like regulator RegA of Rhodobacter capsulatus revealed considerable similarities. Thus, the RegR box may assist in the identification of new RegR target genes not only in B.japonicum but also in other alpha-proteobacteria possessing RegR-like response regulators.

Differential degradation of Escherichia coli sigma32 and Bradyrhizobium japonicum RpoH factors by the FtsH protease.

The Escherichia coli heat shock sigma factor sigma32 (RpoH) is rapidly degraded under non-stress conditions. The integrity of the DnaK chaperone machinery and the ATP-dependent FtsH protease are required for sigma32 proteolysis in vivo. Bradyrhizobium japonicum expresses three sigma32-type transcription factors, RpoH1, RpoH2, and RpoH3, which are functional in E. coli. We compared the stability of these sigma factors with E. coli sigma32 stability. In E. coli C600 (wild-type), the half-lives of sigma32, RpoH1, RpoH2 and RpoH3 were 30 s, 7 min, 4 min and 4 min, respectively. The first three proteins were stabilized in ftsH mutant backgrounds, indicating that they are degraded by FtsH in the wild-type. Proteolysis of RpoH3 was FtsH-independent because this sigma factor was not stabilized in ftsH mutants. Interestingly, in a purified in vitro system, all four RpoH proteins were degraded by FtsH, indicating that in vivo protein degradation depends on additional cellular factors. Rationally designed point mutations of sigma32 and RpoH1 suggested that the highly conserved RpoH box does not play a major role in conferring stability to RpoH factors. Presumably, several regions distributed along the primary sequence of the sigma factor are important for FtsH-mediated proteolysis. Finally, we provide evidence that proteolysis of RpoH factors in vivo depends on the DnaK machinery, irrespective of the protease involved.

Periplasmic protein thiol:disulfide oxidoreductases of Escherichia coli.

Disulfide bond formation is part of the folding pathway for many periplasmic and outer membrane proteins that contain structural disulfide bonds. In Escherichia coli, a broad variety of periplasmic protein thiol:disulfide oxidoreductases have been identified in recent years, which substantially contribute to this pathway. Like the well-known cytoplasmic thioredoxins and glutaredoxins, these periplasmic protein thiol:disulfide oxidoreductases contain the conserved C-X-X-C motif in their active site. Most of them have a domain that displays the thioredoxin-like fold. In contrast to the cytoplasmic system, which consists exclusively of reducing proteins, the periplasmic oxidoreductases have either an oxidising, a reducing or an isomerisation activity. Apart from understanding their physiological role, it is of interest to learn how these proteins interact with their target molecules and how they are recycled as electron donors or acceptors. This review reflects the recently made efforts to elucidate the sources of oxidising and reducing power in the periplasm as well as the different properties of certain periplasmic protein thiol:disulfide oxidoreductases of E. coli.

The symbiotically essential cbb(3)-type oxidase of Bradyrhizobium japonicum is a proton pump.

Purified cbb(3)-type oxidase of Bradyrhizobium japonicum was reconstituted into phospholipid vesicles. Tight vesicles were obtained as shown by the disturbance of deltapH with CCCP and the membrane potential with valinomycin, which led to a six-fold increase in cytochrome c oxidase activity. The vesicles were thus suitable for proton translocation experiments. In the presence of valinomycin, a pulse with reduced cytochrome c caused an acidification with a subsequent alkalinization, whereas the same pulse caused only an alkalinization in the presence of valinomycin plus CCCP. We conclude that the cbb(3)-type oxidase of B. japonicum is a proton pump.

Three new NifA-regulated genes in the Bradyrhizobium japonicum symbiotic gene region discovered by competitive DNA-RNA hybridization.

The so-called symbiotic region of the Bradyrhizobium japonicum chromosome (C. Kündig, H. Hennecke, and M. Göttfert, J. Bacteriol. 175:613-622, 1993) was screened for the presence of genes controlled by the nitrogen fixation regulatory protein NifA. Southern blots of restriction enzyme-digested cosmids that represent an ordered, overlapping library of the symbiotic region were competitively hybridized with in vitro-labeled RNA from anaerobically grown wild-type cells and an excess of RNA isolated either from anaerobically grown nifA and rpoN mutant cells or from aerobically grown wild-type cells. In addition to the previously characterized nif and fix gene clusters, we identified three new NifA-regulated genes that were named nrgA, nrgB, and nrgC (nrg stands for NifA-regulated gene). The latter two probably form an operon, nrgBC. The proteins encoded by nrgC and nrgA exhibited amino acid sequence similarity to bacterial hydroxylases and N-acetyltransferases, respectively. The product of nrgB showed no significant similarity to any protein with a database entry. Primer extension experiments and expression studies with translational lacZ fusions revealed the presence of a functional -24/-12-type promoter upstream of nrgA and nrgBC and proved the NifA- and RpoN (sigma(54))-dependent transcription of the respective genes. Null mutations introduced into nrgA and nrgBC resulted in mutant strains that exhibited wild-type-like symbiotic properties, including nitrogen fixation, when tested on soybean, cowpea, or mung bean host plants. Thus, the discovery of nrgA and nrgBC further emphasizes the previously suggested role of NifA as an activator of anaerobically induced genes other than the classical nitrogen fixation genes.

Evidence for a functional similarity between the two-component regulatory systems RegSR, ActSR, and RegBA (PrrBA) in alpha-Proteobacteria.

The symbiotic bacteria Bradyrhizobium japonicum and Sinorhizobium meliloti, and the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodovulum sulfidophilum, Roseobacter denitrificans and Rhodobacter sphaeroides possess homologous two-component regulatory systems, namely RegSR, ActSR, RegBA and PrrBA. The respective response regulators of these bacteria control expression of different regulons that are involved in N2 fixation, CO2 fixation, photosynthesis or acid tolerance. We therefore asked whether the regulators are functionally exchangeable or whether they have disparate functions in the different species, despite the amino acid sequence similarity. In this study, we showed that purified B. japonicum RegR bound in vitro to genuine DNA targets for Rba. capsulatus RegA, and that RegA was phosphorylated in vitro when RegSc (a soluble variant of the sensor kinase RegS) was added to an Escherichia coli extract containing overexpressed RegA. In vivo, RegA and S. meliloti ActR activated transcription of the B. japonicum fixR-nifA operon, normally a target for RegR. The genes for both regulators, regA and actR, were able to complement a B. japonicum regR mutant with respect to the formation of a nitrogen-fixing symbiosis with soybean. Vice versa, RegR activated in Rba. capsulatus the expression of the photosynthesis operon puc, normally a target for RegA. In conclusion, the results show that B. japonicum RegR, Rba. capsulatus RegA, and S. meliloti ActR are functionally similar.

Role of HrcA and CIRCE in the heat shock regulatory network of Bradyrhizobium japonicum.

A large number of bacteria regulate chaperone gene expression by the CIRCE-HrcA system in which a DNA element called CIRCE serves as binding site for the repressor protein HrcA under non-heat-shock conditions. We have cloned the two consecutive genes hrcA and grpE of Bradyrhizobium japonicum by using a complementation approach that screened for GrpE function. In vivo and in vitro transcript mapping demonstrated that both genes are transcribed separately from RpoH (sigma(32))-dependent promoters. To investigate the supposed negative regulatory function of HrcA, we compared the expression of putative target genes in the wild type with that in an hrcA mutant. Transcription of the CIRCE-associated chaperonin operons groESL(4) and groESL(5), as well as the beta-galactosidase activity derived from corresponding groE-lacZ fusions, was strongly elevated in the hrcA mutant even at physiological temperatures. Expression of other heat shock regulons (RpoH or ROSE dependent) was not affected. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein under nondenaturing conditions. Specific binding to the CIRCE element was obtained with a soluble fraction of HrcA in gel retardation experiments.

Classifying symbiotic proteins from Bradyrhizobium japonicum into functional groups by proteome analysis of altered gene expression levels.

The advent of whole genome sequences has brought with it a vast number of new potential proteins whose function is unknown. We describe an approach to sorting proteins into functional groups by comparative two-dimensional (2-D) gel mapping of cells grown under different physiological conditions. Computerized image analysis selects the proteins whose expression levels change significantly for subsequent identification by mass spectrometry. The protein groupings are further subdivided by directed alteration of their expression levels (e.g., by gene inactivation) and following the changes in the expression pattern of the mutants. We have applied this approach to study the regulation of micro- and anaerobically induced genes including the genes involved in nitrogen fixation in the symbiotic bacterium Bradyrhizobium japonicum. The results obtained show that in addition to the two known regulons controlled by the transcription factors NifA and FixK2, a third set of proteins may exist in B. japonicum which are induced by anaerobic conditions and are regulated independently. The approach can be applied generally and can be used to build up functional relationship maps of genomes. Protein identification by mass spectrometry was shown to be vital to the interpretation of the expression analysis since 15% of the 2-D gel spots contained more than one protein.

Characterization of the Bradyrhizobium japonicum ftsH gene and its product.

The Bradyrhizobium japonicum ftsH gene was cloned by using a set of widely applicable degenerated oligonucleotides. Western blot experiments indicated that the FtsH protein was produced under standard growth conditions and that it was not heat inducible. Attempts to delete the ftsH gene in B. japonicum failed, suggesting a pivotal cellular function of this gene. The expression of B. japonicum ftsH in an ftsH-negative Escherichia coli strain significantly enhanced the fitness of this mutant and reduced the steady-state level of sigma(32).

Phosphorylation, dephosphorylation and DNA-binding of the Bradyrhizobium japonicum RegSR two-component regulatory proteins.

Under low oxygen conditions, induction of many genes required for nitrogen fixation in Bradyrhizobium japonicum depends on the redox-responsive transcriptional activator NifA which is encoded in the fixR-nifA operon. Basal expression of this operon depends on the response regulator RegR and a DNA element located around position -68 in the fixR-nifA promoter region. To investigate the functional properties of RegR and the interaction with its putative cognate kinase, RegS, we overproduced and affinity-purified RegR and a truncated soluble variant of RegS (RegS(C)), both as N-terminally His(6)-tagged proteins. RegS(C) autophosphorylated when incubated with [gamma-(32)P]ATP, and it catalyzed the transfer of the phosphoryl label to RegR. The phosphorylated form of RegS(C) exhibited phosphatase activity on RegR-phosphate. Chemical stability tests and site-specific mutagenesis identified amino acids H219 and D63 of RegS and RegR, respectively, as the phosphorylated residues. Competition experiments with isolated domains demonstrated that the N-terminal but not the C-terminal domain of RegR interacts with RegS(C). Band-shift experiments revealed that phosphorylated RegR had at least eightfold enhanced DNA-binding activity compared with dephosphorylated RegR or the mutant protein RegR-D63N, which cannot be phosphorylated. In conclusion, the RegSR proteins of B. japonicum exhibit functional properties in vitro that are typical of two-component regulatory systems.

Heme transfer to the heme chaperone CcmE during cytochrome c maturation requires the CcmC protein, which may function independently of the ABC-transporter CcmAB.

Cytochrome c maturation in Escherichia coli requires the ccm operon, which encodes eight membrane proteins (CcmABCDEFGH). CcmE is a periplasmic heme chaperone that binds heme covalently and transfers it onto apocytochrome c in the presence of CcmF, CcmG, and CcmH. In this work we addressed the functions of the ccmABCD gene products with respect to holo-CcmE formation and the subsequent ligation of heme to apocytochrome c. In the absence of the ccmABCD genes, heme is not bound to CcmE. We report that CcmC is functionally uncoupled from the ABC transporter subunits CcmA and CcmB, because it is the only Ccm protein that is strictly required for heme transfer and attachment to CcmE. Site-directed mutagenesis of conserved histidines inactivates the CcmC protein, which is in agreement with the hypothesis that this protein interacts directly with heme. We also present evidence that questions the role of CcmAB as a heme exporter; yet, the transported substrate remains unknown. CcmD was found to be involved in stabilizing the heme chaperone CcmE in the membrane. We propose a heme-trafficking pathway as part of a substantially revised model for cytochrome c maturation in E. coli.

Bradyrhizobium japonicum FixK2, a crucial distributor in the FixLJ-dependent regulatory cascade for control of genes inducible by low oxygen levels.

Bradyrhizobium japonicum possesses a second fixK-like gene, fixK2, in addition to the previously identified fixK1 gene. The expression of both genes depends in a hierarchical fashion on the low-oxygen-responsive two-component regulatory system FixLJ, whereby FixJ first activates fixK2, whose product then activates fixK1. While the target genes for control by FixK1 are unknown, there is evidence for activation of the fixNOQP, fixGHIS, and rpoN1 genes and some heme biosynthesis and nitrate respiration genes by FixK2. FixK2 also regulates its own structural gene, directly or indirectly, in a negative way.