Biomedical subjects

H Hennecke

Publications and source records attributed to H Hennecke.

At least 55 records · Page 3Linked to original sources

PubMed1996

Requirements for maturation of Bradyrhizobium japonicum cytochrome c550 in Escherichia coli.

Various forms of Bradyrhizobium japonicum cytochrome c550 (the cycA gene product) were overexpressed in Escherichia coli cells grown under different conditions. Antibodies directed against a synthetic cytochrome c550 peptide were used as tools to detect both, apoprotein and holoprotein. Complete maturation of the apoprotein into its holo form with haem covalently bound to the polypeptide was observed only under anaerobic growth conditions and in E. coli K12 derivatives, whereas haem binding did not occur in the E. coli BL21 host. When maturation was complete, holocytochrome c550 was found exclusively in the periplasmic fraction. A cycA-expressing plasmid construct lacking the genetic information for the signal sequence produced apoprotein that was rapidly degraded without further maturation. Mutations in the haem-binding site resulted in products that were translocated through the cytoplasmic membrane, but apparently became degraded. Our results support the view that attachment of haem to the apoprotein is not a prerequisite for cleavage of the signal sequence and occurs on the periplasmic side of the membrane, subsequent to translocation of the apoprotein precursor.

Amino Acid Sequence↗

PubMed1996

Overproduction of phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 in Escherichia coli.

The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus was overproduced in Escherichia coli. Three different promoter systems were used for the overexpression of the pheST genes: the tac, araB, and T7 promoters. Despite several attempts for improvement, the overproduction of the FRS was lower than that found with most of the other T. thermophilus genes. Nevertheless, enzyme amounts sufficient for biochemical and biophysical studies could be obtained more easily from the overproducing E. coli than from T. thermophilus, since at least fivefold higher specific FRS activity was present in the overproducing cells than in T. thermophilus. Also, a simple purification procedure was established. After heat treatment at 70 degrees C to remove thermolabile E. coli proteins, only three chromatographic steps, i.e., Q-Sepharose FF, hydroxyl apatite, and heparin-Sepharose chromatography, were necessary to obtain apparently homogeneous FRS. With a different plasmid construction we introduced six histidine residues at the N terminus of the alpha subunit. Thus, affinity chromatography on a nickel-chelate matrix can be used for the purification of FRS as well as for its mutant variants, which may be less stable than the native FRS and cannot be purified with heat treatment. We also cloned the pheST genes in a phagemid, which will enable mutagenesis studies and overexpression in a one-vector system without any subcloning steps.

Amino Acid Sequence↗

PubMed1996

Bradyrhizobium japonicum possesses two discrete sets of electron transfer flavoprotein genes: fixA, fixB and etfS, etfL.

A group of four co-regulated genes (fixA, fixB, fixC, fixX) essential for symbiotic nitrogen fixation has been described in several rhizobial species, including Bradyrhizobium japonicum. The complete nucleotide sequence of the B. japonicum fixA, fixB and fixC, genes is reported here. The derived amino acid sequences confirmed the previously noted sequence similarity between FixA and the beta-subunit and between FixB and the alpha-subunit of mammalian and Paracoccus denitrificans electron transfer flavoproteins (ETF). Since the classical role of ETF is in beta-oxidation of fatty acids, a process unrelated to nitrogen fixation, we rationalized that B. japonicum ought to contain bona fide etf genes in addition to the etf-like genes fixA and fixB. Therefore, we identified, cloned, sequenced, and transcriptionally analyzed the B. japonicum etfSL genes encoding the beta- and alpha-subunits of ETF. The etfSL genes, but not the fix genes, are transcribed in aerobically grown cells. An amino acid sequence comparison between all available ETFs and ETF-like proteins revealed the existence of two distinguishable subfamilies. Group I comprises housekeeping ETFs that link acyl-CoA dehydrogenase reactions with the respiratory chain, such as in the fatty acid degradation pathway. B. japonicum EtfS and EtfL clearly belong to this group. Group II contains ETF-like proteins that are synthesized only under certain specific growth conditions and receive electrons from the oxidation of specific substrates. The products of the anaerobically induced fixA and fixB genes of B. japonicum are members of that group. B. japonicum is the first example of an organism in which genes for proteins of both groups I and II of the ETF family have been identified.

Amino Acid Sequence↗

PubMed1996

The Bradyrhizobium japonicum fixGHIS genes are required for the formation of the high-affinity cbb3-type cytochrome oxidase.

We report structural and functional analyses of the Bradyrhizobium japonicum fixGHIS genes, which map immediately downstream of the fixNOQP operon for the symbiotically essential cbb3-type heme-copper oxidase complex. Expression of fixGHIS, like that of fixNOQP, is strongly induced in cells grown microaerobically or anaerobically. A fixGHI deletion led to the same prominent phenotypes as those known from a fixNOQP deletion: defective symbiotic nitrogen fixation (Fix-) and decreased cytochrome oxidase activity in cells grown under oxygen deprivation. Only traces, if any, of cytochrome cbb3 subunits were present in membranes isolated from the delta fixGHI strain, as revealed by Western blot analysis with subunit-specific antibodies. This effect was not due to lack of fixNOQP transcription. The results suggested a critical involvement of the fixGHIS gene products in the assembly and/or stability of the cbb3-type heme-copper oxidase. On the basis of sequence similarities between the FixI protein and a Cu-transporting P-type ATPase (CopA) of Enterococcus hirae, and between FixG and a membrane-bound oxidoreductase (RdxA) of Rhodobacter sphaeroides, we postulate that a membrane-bound FixGHIS complex might play a role in uptake and metabolism of copper required for the cbb3-type heme-copper oxidase.

Amino Acid Sequence↗

PubMed1996

Two different mechanisms are involved in the heat-shock regulation of chaperonin gene expression in Bradyrhizobium japonicum.

Heat-shock regulation was detected for three out of the five members of the groESL multigene family in Bradyrhizobium japonicum. The results uncovered the simultaneous presence of two distinct heat-shock control systems which so far have not been reported to co-exist in a single prokaryotic organism. The first system concerns groESL1 whose transcription is controlled in a sigma32-dependent manner similar to that known from work done with Escherichia coli. Heat-shock control of groESL4 is mediated by the second system, which is characterized by an inverted-repeat DNA structure originally described as a heat-shock regulatory element (CIRCE) in Bacillus subtilis. This element represses expression of groESL4 under non-stress conditions, as inferred from the increased expression of a groESL4'-'lacZ fusion suffering a 4 bp deletion within the CIRCE element. The two control systems clearly differ with respect to the temperature dependence and the kinetics of the heat-shock response, and they also respond differently to the stress signal elicited by incorporation of the amino acid analogue p-F-phenylalanine into cellular protein. Knock-out mutations in groEL4 resulted in an increased expression of groESL4, suggesting that repression via CIRCE depends, itself, upon the cellular level of GroEL4 protein.

Base Sequence↗

PubMed1996

The Bradyrhizobium japonicum rpoH1 gene encoding a sigma 32-like protein is part of a unique heat shock gene cluster together with groESL1 and three small heat shock genes.

The heat shock response of Bradyrhizobium japonicum is controlled by a complex network involving two known regulatory systems. While some heat shock genes are controlled by a highly conserved inverted-repeat structure (CIRCE), others depend on a sigma 32-type heat shock sigma factor. Using Western blot (immunoblot) analysis, we confirmed the presence of a sigma 32-like protein in B. japonicum and defined its induction pattern after heat shock. A B. japonicum rpoH-like gene (rpoH1) was cloned by complementation of an Escherichia coli strain lacking sigma 32. A knockout mutation in rpoH1 did not abolish sigma 32 production in B. japonicum, and the rpoH1 mutant showed the wild-type growth phenotype, suggesting the presence of multiple rpoH homologs in this bacterium. Further characterization of the rpoH1 gene region revealed that the rpoH1 gene is located in a heat shock gene cluster together with the previously characterized groESL1 operon and three genes encoding small heat shock proteins in the following arrangement: groES1, groEL1, hspA, rpoH1, hspB, and hspC. Three heat-inducible promoters are responsible for transcription of the six genes as three bicistronic operons. A sigma 32-dependent promoter has previously been described upstream of the groESL1 operon. Although the hspA-rpoH1 and hspBC operons were clearly heat inducible, they were preceded by sigma 70-like promoters. Interestingly, a stretch of about 100 bp between the transcription start site and the start codon of the first gene in each of these two operons was nearly identical, making it a candidate for a regulatory element potentially allowing heat shock induction of sigma 70-dependent promoters.

Amino Acid Sequence↗

PubMed1996

A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum.

It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.

Aerobiosis↗

PubMed1995

A bacterial thioredoxin-like protein that is exposed to the periplasm has redox properties comparable with those of cytoplasmic thioredoxins.

The membrane-anchored thioredoxin-like protein (TlpA) from the Gram-negative soil bacterium Bradyrhizobium japonicum was initially discovered due to its essential role in the maturation of cytochrome aa3. A soluble form of TlpA lacking the N-terminal membrane anchor acts as a protein thiol:disulfide oxidoreductase. TlpA possesses an active-site disulfide bond common to all members of the thiol:disulfide oxidoreductase family. In addition, it contains two non-active-site cysteines that form a structural disulfide bond (Loferer, H., Bott, M., and Hennecke, H. (1993) EMBO J. 12, 3373-3383; Loferer, H., and Hennecke, H. (1994) Eur. J. Biochem. 223, 339-344). Here, we compare the far- and near-UV CD spectra of TlpA before and after reduction of both disulfides by dithiothreitol and show that the non-active-site disulfide bond is not required for the integrity of TlpA's native conformation. In contrast to dithiothreitol, reduced glutathione (GSH) selectively reduces the active-site disulfide and leaves the non-active-site disulfide bond intact, even at high molar excess over TlpA. The selective reduction of the active-site disulfide bond leads to a 10-fold increase of the intrinsic tryptophan fluorescence of TlpA at 355 nm, which may be interpreted as a quenching of tryptophan fluorescence by the active-site disulfide bond. Using the specific fluorescence of TlpA as a measure of its redox state, a value of 1.9 +/- 0.2 M was determined for the TlpA:glutathione equilibrium constant at pH 7.0, demonstrating that TlpA is a reductant, like cytoplasmic thioredoxins. The DsbA protein, which acts as the final oxidant of periplasmic secretory proteins in Escherichia coli, is not capable of oxidizing the active-site cysteines of TlpA. This suggests that TlpA's primary role in vivo is keeping the thiols of certain proteins reduced and that TlpA's active, reduced state may be maintained owing to its kinetically restricted oxidation by other periplasmic disulfide oxidoreductases such as DsbA.

Bacterial Proteins↗

PubMed1995

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.

Chaperonins↗

PubMed1995

The cycHJKL gene cluster plays an essential role in the biogenesis of c-type cytochromes in Bradyrhizobium japonicum.

We present an extended genetic analysis of the previously identified cycH locus in Bradyrhizobium japonicum. Three new open reading frames found in an operon-like structure immediately adjacent to the 3' end of cycH were termed cycJ, cycK and cycL. A deletion mutant (delta cycHJKL) and biochemical analysis of its phenotype showed that the genes of the cluster are essential for the biogenesis of cellular c-type cytochromes. Mutations in discrete regions of each of the genes were also constructed and shown to affect anaerobic respiration with nitrate and the ability to elicit an effective symbiosis with soybean, both phenotypes being a consequence of defects in cytochrome c formation. The CycK and CycL proteins share up to 53% identity in amino acid sequence with the Rhodobacter capsulatus Cc11 and Cc12 proteins, respectively, which have been shown previously to be essential for cytochrome c biogenesis, whereas cycJ codes for a novel protein of 169 amino acids with an M(r) of 17857. Localisation studies revealed that CycJ is located in the periplasmic space; it is probably anchored to the cytoplasmic membrane via an N-terminal hydrophobic domain. Based on several considerations discussed here, we suggest that the proteins encoded by the cycHJKL-cluster may be part of a cytochrome c-haem lyase complex whose active site faces the periplasm.

Amino Acid Sequence↗

PubMed1995

Increased rates of tRNA charging through modification of the enzyme-aminoacyl-adenylate complex of phenylalanyl-tRNA synthetase.

The transfer of amino acid to tRNA by Escherichia coli phenylalanyl-tRNA synthetase (PheRS) was studied using replacements of Ala294 in the alpha subunit previously shown to have modified amino acid specificity. Steady-state analysis of tRNA charging showed little difference between wild-type and mutants, whereas pre-steady-state analysis revealed higher rates of tRNA charging by both the A294S PheRS-phenylalanyl adenylate and the A294G PheRS-p-Cl-phenylalanyl adenylate. The decrease in energy required for the formation of the transition state of amino acid transfer in these mutants could be related to a weaker binding of the amino acid in the aminoacyl adenylate complex. Thus a compromise appears to exist between amino acid activation and tRNA charging, because slowing down the first step increases the rate of the second step, possibly as a result of decreased stability of the PheRS.amino acid-AMP complex.

Adenosine Monophosphate↗

PubMed1995

Escherichia coli genes required for cytochrome c maturation.

The so-called aeg-46.5 region of Escherichia coli contains genes whose expression is induced under anaerobic growth conditions in the presence of nitrate or nitrite as the terminal electron acceptor. In this work, we have examined more closely several genes of this cluster, here designated ccmABCDEFGH, that are homologous to two separate Bradyrhizobium japonicum gene clusters required for the biogenesis of c-type cytochromes. A deletion mutant of E. coli which lacked all of these genes was constructed. Maturation of indigenous c-type cytochromes synthesized under anaerobic respiratory conditions, with nitrite, nitrate, or trimethylamine N-oxide as the electron acceptor, was found to be defective in the mutant. The biogenesis of foreign cytochromes, such as the soluble B. japonicum cytochrome c550 and the membrane-bound Bacillus subtilis cytochrome c550, was also investigated. None of these cytochromes was synthesized in its mature form when expressed in the mutant, as opposed to the situation in the wild type. The results suggest that the E. coli ccm gene cluster present in the aeg-46.5 region is required for a general pathway involved in cytochrome c maturation.

Anaerobiosis↗

PubMed1995

A single rRNA gene region in Bradyrhizobium japonicum.

Bradyrhizobium japonicum contains only a single rRNA (rrn) gene region, despite its comparatively large genome size of 8,700 kb. The nucleotide sequence revealed an organization of rRNA and tRNA genes that is frequently found in bacteria: 5'-rrs (16S rRNA)-ileT (tRNA(Ile))-alaT (tRNA(Ala))-rrl (23S rRNA)-rrf (5S rRNA)-3'. The 5' end of the primary transcript, one of the 16S rRNA processing sites, and the 5' end of the mature 16S rRNA were determined by primer extension. DNA hybridization experiments showed that the slowly growing Bradyrhizobium strains generally have only a single copy of the 16S rRNA gene, whereas the faster-growing Rhizobium species contain three rrs copies.

Base Sequence↗

PubMed1995

Overlapping promoters for two different RNA polymerase holoenzymes control Bradyrhizobium japonicum nifA expression.

The Bradyrhizobium japonicum NifA protein, the central regulator for nitrogen fixation gene expression, is encoded in the fixRnifA operon. This operon is activated during free-living anaerobic growth and in the symbiotic root nodule bacteroid state. In addition, it is expressed in aerobic conditions, albeit at a low level. Here, we report that this pattern of expression is due to the presence of two overlapping promoters: fixRp1, which is of the -24/-12 class recognized by the RNA polymerase sigma 54, and fixRp2, which shares homology with the -35 and -10 regions found in other putative B. japonicum housekeeping promoters. Primer extension analyses showed that fixRp1 directed the synthesis of a transcript, P1, that starts 12 nucleotides downstream of the -12 region. In addition to sigma 54, P1 was dependent on NifA and low oxygen tension. Transcripts originating from fixRp2 started at two sites: one coincided with P1, while the most abundant, P2 initiated just two nucleotides further downstream of P1. Expression from fixRp2 was dependent on the upstream -68 promoter region, a region known to bind a putative activator protein, but it was independent of sigma 54 and NifA. This promoter was expressed in aerobic and anaerobic conditions but was not expressed in 30-day-old bacteroids. Mutations in the conserved 12 region for the sigma 54 promoter did not show any transcript, because these mutations also disrupted the overlapping -10 region of the fixRp2 promoter. Conversely, mutations at the -24 region only affected the sigma 54-dependent P1 transcript, having no effect on the expression of P2. In the absence of omega(54), anaerobic expression from the fixRp(2) promoter was enhanced threefold, suggesting that in the wild-type strain, the two RNA polymerase holoenzymes must compete for binding to the same promoter region.

Bacterial Proteins↗

PubMed1995

Bradyrhizobium japonicum cytochrome c550 is required for nitrate respiration but not for symbiotic nitrogen fixation.

Bradyrhizobium japonicum possesses three soluble c-type cytochromes, c550, c552, and c555. The genes for cytochromes c552 (cycB) and c555 (cycC) were characterized previously. Here we report the cloning, sequencing, and mutational analysis of the cytochrome c550 gene (cycA). A B. japonicum mutant with an insertion in cycA failed to synthesize a 12-kDa c-type cytochrome. This protein was detectable in the cycA mutant complemented with cloned cycA, which proves that it is the cycA gene product. The cycA mutant, a cycB-cycC double mutant, and a cycA-cycB-cycC triple mutant elicited N2-fixing root nodules on soybean (Nod+ Fix+ phenotype); hence, none of these three cytochromes c is essential for respiration supporting symbiotic N2 fixation. However, cytochrome c550, in contrast to cytochromes c552 and c555, was shown to be essential for anaerobic growth of B. japonicum, using nitrate as the terminal electron acceptor.

Amino Acid Sequence↗

PubMed1994

Cloning and sequencing of the pheU gene for tRNA(Phe) of Thermus thermophilus HB8, and genomic mapping of the pheU and pheST genes.

The gene pheU for tRNA(Phe) from the extreme thermophile Thermus thermophilus HB8 was cloned and sequenced. It differed from the published tRNA(Phe) sequence by one nucleotide. Both the pheU gene and the pheST genes encoding the alpha and beta subunits of phenylalanyl-tRNA synthetase were located on the physical map of the T. thermophilus chromosome, where pheU was present on a hitherto unrecognized HpaI DNA fragment.

Base Sequence↗

PubMed1994

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.

Aerobiosis↗

PubMed1994

Regulated expression in vitro of genes coding for formate hydrogenlyase components of Escherichia coli.

Purified FHLA, the transcriptional activator of the formate regulon from Escherichia coli, is able to efficiently stimulate transcription from the sigma 54-dependent promoters of the fdhF, hyp, and hyc transcriptional units. Expression was dependent on the presence of sigma 54, of the upstream activatory sequence (UAS), and of formate. Hypophosphite, a formate analogue, could substitute for formate in vitro suggesting that formate per se was active in regulation. The integration host factor (IHF) had a direct effect on the expression (in vivo and in vitro) of the hyp and hyc genes but not of the fdhF gene. Binding of IHF within the region between the hyp and the hyc operon could be shown. A model is proposed for the transcriptional regulation of the inversely oriented hyp and hyc operons. It involves two upstream regulatory sequences, one between the hyp and the hyc operon (IR1), and the other between hycA and hycB (IR2). The UAS situated within IR1 is responsible for activation of the hyc operon, that within IR2 for activation of the hyp operon. A supramolecular transcription complex is proposed which involves the binding of IHF to a site located between the UAS and the promoter responsible for transcription of the hyc operon.

Bacterial Proteins↗