Prototype of a heme chaperone essential for cytochrome c maturation.

Heme, the iron-containing cofactor essential for the activity of many enzymes, is incorporated into its target proteins by unknown mechanisms. Here, an Escherichia coli hemoprotein, CcmE, was shown to bind heme in the bacterial periplasm by way of a single covalent bond to a histidine. The heme was then released and delivered to apocytochrome c. Thus, CcmE can be viewed as a heme chaperone guiding heme to its appropriate biological partner and preventing illegitimate complex formation.

Protein disulphide oxidoreductases in bacteria.

Thioredoxins and eukaryotic protein disulphide isomerases were, until recently, the only enzymes known to catalyse reversible oxidation and reduction of cysteine residues of a wide spectrum of protein substrates. Genetic and biochemical investigations on different bacterial systems have now led to the discovery of novel prokaryotic protein disulphide oxidoreductases that are located either in the periplasm or in the cytoplasmic membrane.

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.

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.

The -24/-12 promoter comes of age.

A new bacterial promoter type has been identified in the last few years. Originally designated as nif (= nitrogen fixation) or ntr (= nitrogen regulation) consensus promoter, it is now evident that this promoter occurs in many different bacterial species and is used not only for genes involved in nitrogen assimilation but also for genes determining many other unrelated metabolic functions. The general features of this type of promoter are (i) the conserved -24(GG)/-12(GC) consensus sequence, (ii) its recognition by a specific RNA polymerase sigma factor, sigma 54, which is encoded by the ntrA gene (synonyms: glnF, rpoN, rpoE), and (iii) the requirement for a transcriptional regulatory protein to activate the expression of the associated genes. In addition, many (but not all) of these genes possess a promoter-upstream activator sequence (enhancer) which is the target site for the binding of the activating protein and is required for maximal expression. In some cases, in which gene expression does not appear to be dependent on the presence of upstream binding sites, the activating protein may interact directly with the RNA polymerase-promoter complex. In conclusion, the expression from all -24/-12 consensus promoters known to date is positively controlled.

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 biosynthesis of nitrogenase MoFe protein polypeptides in free-living cultures of Rhizobium japonicum.

The biosynthesis of the constituent polypeptides of nitrogenase component I (Rj 1) in free-living cultures of Rhizobium japonicum (strain 110) was investigated under different growth conditions. Cells were pulse-labelled and the proteins analysed by one and two-dimensional gel electrophoresis. The positions of the constituent Rj 1 polypeptides were identified by co-electrophoresis with purified Rj 1 isolated from bacteroids of soybean nodules, and by comparison with an immunoprecipitate from a culture induced for nitrogenase. The synthesis of the proteins preceded any detectable enzyme activity and increased with time, reaching a maximum after 3 days. At this time, between 6 and 8% of the total sodium dodecyl sulfate-soluble protein synthesized was Rj 1. Exposure to air led to a dramatic decrease in the rate of Rj 1 synthesis, with almost complete regression after 20 min. In the presence of KNO3, there was no nitrogenase activity, but the proteins were present in similar amounts (7%) as the control culture. When mannitol and glycerol were used as the sole carbon sources, the amount of Rj 1 synthesized was extremely low.

Bacterial genes and proteins involved in the biogenesis of c-type cytochromes and terminal oxidases.

A total of nine genes potentially concerned with the biosynthesis of c-type cytochromes have been identified recently in the bacteria Bradyrhizobium japonicum and Rhodobacter capsulatus, and homologous counterparts appear to be present also in Escherichia coli. Most of the respective gene products are membrane-bound, while others are located in the periplasmic space. As inferred from sequence analyses, several of these proteins may play roles in membrane transport or redox processes, both functions being consistent with the required steps in cytochrome c formation (membrane translocation of heme; covalent linkage of protoheme IX to cysteine thiols). Further genes of B. japonicum, E. coli, Bacillus subtilis and Paracoccus denitrificans have been studied whose products are necessary for the formation of intact heme/copper oxidases. Some of them are probably required in protein folding and assembly whereas others appear to be enzymes catalyzing steps in the biosynthesis of the heme cofactors.

Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase altered by distinct mutations.

Neither the tertiary structure nor the location of active sites are known for phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 structure), a member of class II aminoacyl-tRNA synthetases. In an attempt to detect the phenylalanine (Phe) binding site, two Escherichia coli PheRS mutant strains (pheS), which were resistant to p-fluorophenylalanine (p-F-Phe) were analysed genetically. The pheS mutations were found to cause Ala294 to Ser294 exchanges in the alpha subunits from both independent strains. This alteration (S294) resided in the well-conserved C-terminal part of the alpha subunit, precisely within motif 3, a typical class II tRNA synthetase sequence. We thus propose that motif 3 participates in the formation of the Phe binding site of PheRS. Mutation S294 was also the key for proposing a mechanism by which the substrate analogue p-F-Phe is excluded from the enzymatic reaction; this may be achieved by steric interactions between the para-position of the aromatic ring and the amino acid residue at position 294. The Phe binding site model was then tested by replacing the alanine at position 294 as well as the two flanking phenylalanines (positions 293 and 295) by a number of selected other amino acids. In vivo and in vitro results demonstrated that Phe293 and Phe295 are not directly involved in substrate binding, but replacements of those residues affected PheRS stability. However, exchanges at position 294 altered the binding of Phe, and certain mutants showed pronounced changes in specificity towards Phe analogues. Of particular interest was the Gly294 PheRS in which presumably an enlarged cavity for the para position of the aromatic ring allowed an increased aminoacylation of tRNA with p-F-Phe. Moreover, the larger para-chloro and para-bromo derivatives of Phe could interact with this enzyme in vitro and became highly toxic in vivo. The possible exploitation of the Gly294 mutant PheRS for the incorporation of non-proteinogenic amino acids into proteins is discussed.

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.

Identification of nodS and nodU, two inducible genes inserted between the Bradyrhizobium japonicum nodYABC and nodIJ genes.

The so-called common nodulation (nod) gene cluster of Bradyrhizobium japonicum is characterized by a unique composition of genes that are arranged in the following order: nodY, nodA, nodB, nodC, nodS, nodU, nodI, nodJ. As reported here, the identification of the two new genes nodS and nodU resulted from the DNA sequencing of a 4.5-kilobase nodC-downstream region covering nodS, nodU, nodI, and nodJ. The predicted NodS, NodU, NodI, and NodJ proteins had the following respective amino acid (aa) lengths and molecular weights (Mr): 209 aa, Mr 23,405; 569 aa, Mr 62,068; 306 aa, Mr 34,127; and 262 aa, Mr 28,194. The 3' end of nodC overlapped the 5' end of nodS by 71 nucleotides. Using translational fusions of lacZ to nodC, nodS, and nodU, the expression of these genes was shown to be inducible by the isoflavone daidzein and depended on transcription from a DNA region farther upstream. These data and the adjacent location of all genes suggested the existence of a nodYABCSUIJ operon. The nodI and nodJ gene products shared about 70% sequence similarity with the corresponding Rhizobium leguminosarum bv. viciae proteins; NodI belongs to the family of ATP-binding proteins that are constituents of bacterial binding protein-dependent transport systems. By interspecies hybridization, DNA regions homologous to nodSU were detected in other strains of Bradyrhizobium. Likewise, nodS- and nodU-like genes were identified in Rhizobium sp. strain NGR234 (A. Lewin, E. Cervantes, W. Chee-Hoong, and W. J. Broughton, Molecular Plant-Microbe Interactions 3:317-326, 1990) in which nodS confers host specificity for Leucaena leucocephala.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and functional analysis of two different nodD genes in Bradyrhizobium japonicum USDA110.

Bradyrhizobium japonicum has two closely linked homologs of the nodulation regulatory gene, nodD; these homologs are located upstream of and in divergent orientation to the nodYABCSUIJ gene cluster. We report here the nucleotide sequence and mutational analyses of both nodD copies. The predicted NodD1 and NodD2 proteins shared 62% identical amino acid residues at corresponding positions and exhibited different degrees of homology with NodD proteins of other Bradyrhizobium, Azorhizobium, and Rhizobium strains. Induction of the nodYABCSUIJ operon, as measured by expression of a translational nodC'-'lacZ fusion, required the nodD1 gene, but not nodD2. A B. japonicum mutant deleted for both nodD copies (strain delta 1267) still showed residual nodulation activity; however, nodulation of soybean was significantly delayed, and nodulation of mung bean and siratro resulted in strongly reduced nodule numbers. Fully efficient nodulation of mung bean and siratro by strain delta 1267 was restored by genetic complementation with the nodD1 gene, but not with nodD2. We conclude from these data that nodD1 is the critical gene that contributes to maximal nodulation efficiency, whereas the nodD2 gene does not play any obvious role in nodulation of the host plants tested.

A new Bradyrhizobium japonicum gene required for free-living growth and bacteroid development is conserved in other bacteria and in plants.

In the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, a new DNA region, orf74, was discovered which is required for optimal free-living growth and, by consequence, also necessary for the formation of an effective symbiosis. A Tn5-233 insertion of orf14 resulted in a mutant, strain 74, that has a reduced growth rate in free-living cultures under all conditions tested and less than 1% residual symbiotic nitrogen fixation activity as compared with the wild type. Nodule distribution and nodule morphology are severely affected similarly as in a previously characterized B. japonicum nifA mutant. Protein databank searches revealed that the 142-amino-acid protein encoded by orf74 is homologous to a 161-amino-acid protein encoded by orf17 of Bacillus subtilis (approximately 46% identity; J. C. R. Struck, R. Kretschmer-Kazemi Far, W. Schröder, F. Hucho, H. Y. Toschka, and V. A. Erdmann, Biochim. Biophys. Acta, 1050:80-83, 1990) as well as to a 178-amino-acid protein encoded by orf178 of Escherichia coli (approximately 45% identity; K. L. Poulsen, N. W. Larsen, S. Molin, and P. Andersson, Mol. Microbiol., 6:895-905, 1992). Significant similarity was also found with unknown ORFs of two plant species. When expressed from a strong constitutive promoter, orf17 of B. subtilis could partially complement B. japonicum mutant 74. By contrast, orf74 of B. japonicum was unable to functionally complement an E. coli orf178 mutant. The conservation of this DNA region in gram-negative and gram-positive bacteria suggests that the gene is essential for a fundamental cellular process which is required in B. japonicum for both free-living and symbiotic growth.

A novel cloning vector for the direct selection of recombinant DNA in E. coli.

A new plasmid cloning vector (pHE3) is described carrying dominant p-fluorophenylalanine-sensitivity (pheS) and chloramphenicol-resistance markers. This vector is used in combination with a p-fluorophenylalanine-resistant Escherichia coli recipient (strain RR28). Foreign DNA can be cloned into pHE3 leading to insertional inactivation of pheS. Transformation of RR28, and plating on minimal medium with chloramphenicol plus p-fluorophenylalanine (pfp) directly selects for colonies containing recombinant DNA.

Use of a promoter-probe vector system in the cloning of a new NifA-dependent promoter (ndp) from Bradyrhizobium japonicum.

Many of the symbiotic nitrogen-fixation genes in the soybean root nodule bacterium, Bradyrhizobium japonicum, are transcribed from -24/-12 promoters that are recognized by the sigma 54-RNA polymerase and activated by the transcriptional regulator protein, NifA. Several lines of evidence suggest that the B. japonicum genome has more than those seven NifA-regulated promoters which were characterized previously. Here, we present a strategy aimed at the cloning of new NifA-activated promoters. It makes use of (i) a promoter-probe vector into which random B. japonicum genomic fragments were cloned in front of a promoterless reporter gene and (ii) a screening procedure that allowed us to distinguish constitutive promoters from promoters that were specifically activated by NifA under microaerobic or anaerobic conditions. With certain modifications, the system may be generally applicable to clone positively regulated, anaerobically induced genes. A novel NifA-dependent promoter region (ndp) of B. japonicum was found by these means. The transcription start point was mapped, and its 5'-flanking DNA carried a -24/-12-type promoter sequence plus potential binding sites for NifA and integration host factor. Further transcript analyses confirmed that maximal transcription from this promoter occurred only in the presence of NifA and sigma 54 during anaerobic growth of B. japonicum. In Escherichia coli, expression of beta-galactosidase derived from a transcriptional ndp::lacZ fusion was activated 11-fold by B. japonicum NifA, and this activation also required sigma 54 but was independent of NtrC. The DNA around ndp shared no similarity with known sequences in databases.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrogen fixation genes involved in the Bradyrhizobium japonicum-soybean symbiosis.

The symbiotic nitrogen fixation genes (nif, fix) of Bradyrhizobium japonicum, the root nodule endosymbiont of soybean, are organized in at least two separate chromosomal gene clusters. These genes code for proteins of the nitrogenase complex, for proteins involved in their assembly with cofactors and for putative electron transport functions. One gene, nifA, codes for a transcriptional regulatory protein that plays a central role in the control of expression of the other genes in response to the cellular oxygen status. Only at low partial pressures of O2 will the target promoters be activated by NifA.

Relaxing the substrate specificity of an aminoacyl-tRNA synthetase allows in vitro and in vivo synthesis of proteins containing unnatural amino acids.

It has previously been demonstrated that the unnatural amino acid p-Cl-phenylalanine can be attached to tRNA(Phe) by a modified phenylalanyl-tRNA synthetase with relaxed amino acid substrate specificity. We show that this modification to the translational machinery of Escherichia coli is the only requirement for the incorporation of either p-Cl- or p-Br-phenylalanine into full-length luciferase in vitro. The incorporation of p-Cl-phenylalanine was also demonstrated in vivo using a suitably modified host strain. These results represent the first description of the incorporation into a protein in vivo of an unnatural amino acid which is normally rejected by the cellular translational machinery.

Impaired affinity for phenylalanine in Escherichia coli phenylalanyl-tRNA synthetase mutant caused by Gly-to-Asp exchange in motif 2 of class II tRNA synthetases.

Phenylalanyl-tRNA synthetase (PheRS; alpha 2 beta 2 subunit structure) is a member of class II of tRNA synthetases. We report here the genetic analysis of an Escherichia coli mutant strain which is auxotrophic for phenylalanine because it has a PheRS with a decreased affinity for phenylalanine. The mutant pheS gene encoding the PheRS alpha subunit was cloned and sequenced, and the deviation from the wild-type gene was found to result in a Gly191-to-Asp191 exchange. This alteration is located within motif 2, one of 3 conserved sequence motifs characteristic for class II aminoacyl-tRNA synthetases. Motif 2 may thus participate in the formation of the phenylalanine binding site in PheRS.

Cloning and sequence analysis of the phenylalanyl-tRNA synthetase genes (pheST) from Thermus thermophilus.

While crystals suitable for X-ray diffraction analyses are available of phenylalanyl-tRNA synthetase (PheRS) from the thermophilic bacterium Thermus thermophilus, neither the primary structure of its constituent alpha and beta subunits nor the nucleotide sequence of the corresponding pheS and pheT genes were known. Using specific oligonucleotides of conserved pheS regions that were adapted to the T. thermophilus codon usage, we identified, cloned and subsequently sequenced the pheST genes of this bacterium. The sequences reported here will greatly aid in the three-dimensional structure determination of T. thermophilus PheRS, a heterotetrameric (alpha 2 beta 2), class II aminoacyl-tRNA synthetase.

Heme C incorporation into the c-type cytochromes FixO and FixP is essential for assembly of the Bradyrhizobium japonicum cbb3-type oxidase.

The monoheme and diheme c-type cytochromes FixO and FixP are two of the subunits of the respiratory cbb3-type oxidase of Bradyrhizobium japonicum. The cysteines of the respective heme C binding motifs CXXCH were changed to serines by site-directed mutagenesis, which led to inactive oxidases in all mutants. Western blot analyses showed that an intact heme binding site in the FixO polypeptide is a prerequisite not only for the synthesis of holo-FixO protein but also for the formation of the entire cbb3-type oxidase complex. Both heme binding sites of FixP were essential for maturation and assembly of this subunit. It was not possible to create stable FixP variants that contained only one heme C.