Biomedical subjects

H Hennecke

Publications and source records attributed to H Hennecke.

At least 37 records · Page 2Linked to original sources

PubMed1999

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).

ATP-Dependent Proteases↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

The Bradyrhizobium japonicum phoB gene is required for phosphate-limited growth but not for symbiotic nitrogen fixation.

We identified by cloning and DNA sequence analysis the phosphate regulatory gene phoB of Bradyrhizobium japonicum. The deduced gene product displayed pronounced similarity to the PhoB protein of Sinorhizobium meliloti (71.4% identical amino acids). Escherichia coli (50.2%) and other bacterial species. Insertion of a kanamycin resistance cassette into phoB led to impaired growth of the B. japonicum mutant in media containing approximately 25 microM phosphate or less. A standard plant infection test using wild-type and phoB-defective B. japonicum strains showed that the phoB mutation had no effect on the symbiotic properties of B. japonicum with its soybean host plant.

Amino Acid Sequence↗

PubMed1998

How replacements of the 12 conserved histidines of subunit I affect assembly, cofactor binding, and enzymatic activity of the Bradyrhizobium japonicum cbb3-type oxidase.

Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb3-type oxidases show 12 conserved histidines. Six of them are diagnostic of heme-copper oxidases and are thought to bind the following cofactors: the low spin heme B and the binuclear high spin heme B-CuB center. The other six are FixN(CcoN)-specific and their function is unknown. To analyze the contribution of these 12 invariant histidines of FixN in cofactor binding and function of the Bradyrhizobium japonicum cbb3-type oxidase, they were substituted by valine or alanine by site-directed mutagenesis. The H131A mutant enzyme had already been reported previously to be defective in oxidase assembly and function (Zufferey, R., Th¿ny-Meyer, L., and Hennecke, H. (1996) FEBS Lett. 394, 349-352). Four of the remaining histidines were not essential for activity or assembly (positions 226, 246, 333, and 457); by contrast, histidines 331, 410, and 418 were required both for activity and stability of the enzyme. The last group of mutant enzymes, H420A, H280A, H330A, and H316V, were assembled but not functional. To purify the latter mutant proteins and the wild-type enzyme, a six-histidine tag was added to the C terminus of subunit I. The His6-tagged cbb3-oxidase complexes were purified 20-fold by a three-step purification protocol. With the exception of the H420A mutant oxidase, the mutant enzymes H280A, H316V, and H331A contained normal amounts of copper and heme B, and they displayed similar visible light spectroscopic characteristics like the wild-type His6-tagged enzyme. The His6-tagged H420A mutant oxidase differed from the His6-tagged wild-type protein by showing altered visible light spectroscopic characteristics. No stable mutant oxidase lacking copper or heme B was obtained. This strongly suggests that copper and heme B incorporations in subunit I are prerequisites for assembly of the enzyme.

Amino Acid Sequence↗

PubMed1998

Identification of the Bradyrhizobium japonicum degP gene as part of an operon containing small heat-shock protein genes.

A degP (htrA)-like gene of Bradyrhizobium japonicum was identified immediately downstream of two genes (hspB and hspC) coding for small heat-shock proteins. All three genes are oriented in the same direction and are separated by only 85 and 72 bp, and a heat-inducible transcript covering hspB, hspC, and degP was detected by RT-PCR. These results show that the genes are organized in an operon. Two mutants, a degP insertion mutant and a DeltahspBCdegP mutant, were constructed by marker replacement mutagenesis. Immunoblot analysis performed with a serum raised against the amino-terminal end of IbpA, an HspB homolog of Escherichia coli, identified three heat-inducible protein bands in B. japonicum extract, one of which was missing in the deletion mutant. None of the mutants showed an obvious defect during growth at different temperatures, after heat-shock treatment, or in the presence of solvents. Moreover, they were not affected in root-nodule symbiosis, indicating that the small heat-shock proteins HspB and HspC and the DegP homolog of B. japonicum are not required under a wide range of growth conditions.

Amino Acid Sequence↗

PubMed1998

A novel DNA element that controls bacterial heat shock gene expression.

The hspArpoH1 and hspBCdegP heat shock operons of Bradyrhizobium japonicum are preceded by a novel, conserved DNA element of approximately 100 bp, which is responsible for the temperature-regulated transcription of their sigma70-type promoters. We designated this motif ROSE for repression of heat shock gene expression and found additional ROSE elements upstream of two newly identified heat shock operons. A critical core region in the hspA-associated ROSE1 was defined by introducing insertions or deletions. While four mutants retained the ability to repress transcription of the hspArpoH1 operon, five deletion mutants produced elevated hspA mRNA levels under low-temperature growth conditions. Derepression was confirmed by increased RpoH1 levels in non-heat-shocked cells from one of these mutants and by strains that contained a translational hspA-lacZ fusion associated with mutated ROSE1 elements. The hspArpoH1 operon was efficiently transcribed in vitro, and a deletion of ROSE1 did not impair this activity. Gel retardation experiments demonstrated that a protein in non-heat-shocked cells specifically binds to the intact ROSE1 element but not to a mutated element lacking the core region. Taken together, these results indicate that a central region of ROSE serves as a binding site for a repressor protein under standard growth conditions in order to prevent the undesired transcription of heat shock genes.

Bacterial Proteins↗

PubMed1998

Expression of the fixR-nifA operon in Bradyrhizobium japonicum depends on a new response regulator, RegR.

Many nitrogen fixation-associated genes in the soybean symbiont Bradyrhizobium japonicum are regulated by the transcriptional activator NifA, whose activity is inhibited by aerobiosis. NifA is encoded in the fixR-nifA operon, which is expressed at a low level under aerobic conditions and induced approximately fivefold under low-oxygen tension. This induction depends on a -24/-12-type promoter (fixRp1) that is recognized by the sigma54 RNA polymerase and activated by NifA. Low-level aerobic expression and part of the anaerobic expression originates from a second promoter (fixRp2) that overlaps with fixRp1 and depends on an upstream DNA region (UAS) located around position -68 (H. Barrios, H. M. Fischer, H. Hennecke, and E. Morett, J. Bacteriol. 177:1760-1765, 1995). A protein binding to the UAS was previously postulated to act as an activator. This protein has now been purified, and the corresponding gene (regR) has been cloned. On the basis of the predicted amino acid sequence, RegR belongs to the family of response regulators of two-component regulatory systems. We identified upstream of the regR gene an additional gene (regS) encoding a putative sensor kinase. A regR mutant was constructed in which neither a specific UAS-binding activity nor fixRp2-dependent transcript formation and fixR'-'lacZ expression was detected in aerobically grown cells. Anaerobic fixR'-'lacZ expression was also decreased in regR mutants to about 10% of the level observed in the wild type. Similarly, regR mutants showed only about 2% residual nitrogen fixation activity, but unlike nodules induced by nifA mutants, the morphology of those nodules was normal, displaying no signs of necrosis. While regR mutants grew only slightly slower in free-living, aerobic conditions, they displayed a strong growth defect under anaerobic conditions. The phenotypic properties of regS mutants differed only marginally, if at all, from those of the wild type, suggesting the existence of a compensating sensor activity in these strains. The newly identified RegR protein may be regarded as a master regulator in the NifA-dependent network controlling nif and fix gene expression in B. japonicum.

Aerobiosis↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

The active-site cysteines of the periplasmic thioredoxin-like protein CcmG of Escherichia coli are important but not essential for cytochrome c maturation in vivo.

A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome c maturation in Escherichia coli. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in ccmG resulted in the complete absence of all c-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels of Bradyrhizobium japonicum holocytochrome c550 expressed in E. coli. This defect, but not that of the ccmG null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG.

Binding Sites↗

PubMed1998

Promoter selectivity of the Bradyrhizobium japonicum RpoH transcription factors in vivo and in vitro.

Expression of the dnaKJ and groESL1 heat shock operons of Bradyrhizobium japonicum depends on a sigma32-like transcription factor. Three such factors (RpoH1, RpoH2, and RpoH3) have previously been identified in this organism. We report here that they direct transcription from some but not all sigma32-type promoters when the respective rpoH genes are expressed in Escherichia coli. All three RpoH factors were purified as soluble C-terminally histidine-tagged proteins, although the bulk of overproduced RpoH3 was insoluble. The purified proteins were recognized by an anti-E. coli sigma32 serum. While RpoH1 and RpoH2 productively interacted with E. coli core RNA polymerase and produced E. coli groE transcript in vitro, RpoH3 was unable to do so. B. japonicum core RNA polymerase was prepared and reconstituted with the RpoH proteins. Again, RpoH1 and RpoH2 were active, and they initiated transcription at the B. japonicum groESL1 and dnaKJ promoters. In all cases, the in vitro start site was shown to be identical to the start site determined in vivo. Promoter competition experiments revealed that the B. japonicum dnaKJ and groESL1 promoters were suboptimal for transcription by RpoH1- or RpoH2-containing RNA polymerase from B. japonicum. In a mixture of different templates, the E. coli groESL promoter was preferred over any other promoter. Differences were observed in the specificities of both sigma factors toward B. japonicum rpoH-dependent promoters. We conclude that the primary function of RpoH2 is to supply the cell with DnaKJ under normal growth conditions whereas RpoH1 is responsible mainly for increasing the level of GroESL1 after a heat shock.

Bacterial Proteins↗

PubMed1997

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.

Bacterial Proteins↗

PubMed1997

Replacement of Pro109 by His in TlpA, a thioredoxin-like protein from Bradyrhizobium japonicum, alters its redox properties but not its in vivo functions.

TlpA, the membrane-anchored, thioredoxin-like protein from Bradyrhizobium japonicum, is essential for cytochrome aa3 biogenesis. The periplasmic domain of TlpA was previously shown to have protein thiol:disulfide oxidoreductase activity and reducing properties similar to those of cytoplasmic thioredoxins. Here, we replaced the proline-109 in its active-site sequence C107 V108 P109 C110 by a histidine residue. The resulting active-site motif (CVHC) resembles that of oxidizing thiol:disulfide oxidoreductases such as protein disulfide isomerase (PDI) and DsbA. Indeed, the TlpA variant P109H was by 66 mV more oxidizing than the wild-type protein. Nevertheless, the altered protein was even more efficient in catalyzing the reduction of insulin disulfides by dithiothreitol than the wild-type due to a faster recycling of its catalytically active, reduced form. Cells of B. japonicum expressing only the mutated tlpA gene had the same phenotypes as wild-type cells, suggesting that the change in the redox potential of TlpA was not critical for its in vivo function.

Amino Acid Sequence↗

PubMed1997

The dnaKJ operon belongs to the sigma32-dependent class of heat shock genes in Bradyrhizobium japonicum.

The dnaKJ genes of Bradyrhizobium japonicum were cloned and sequenced. They map adjacent to each other, as in other proteobacteria of the alpha and gamma subgroups. Primer extension experiments identified two strongly heat-inducible transcripts starting 99 bp (T1) and 204 bp (T2) upstream of dnaK. Synthesis of the shorter transcript T1 in Escherichia coli required the presence of a recently characterized sigma32 homologue (RpoH1) from B. japonicum. The -35 and -10 regions of the promoters associated with the transcription start sites T1 and T2 displayed nucleotide sequence motifs that are characteristic for sigma32-dependent promoters in E. coli and alpha-proteobacteria. Heat shock regulation of dnaK expression was confirmed by immunoblot analysis of DnaK protein. All of these results put dnaK into the sigma32-dependent class, not the CIRCE-dependent class, of heat shock genes in B. japonicum. At normal growth temperature dnaK was expressed at a significant basal level. All attempts to eliminate dnaK function by insertion or deletion mutagenesis failed. By contrast, dnaJ null mutants and insertions in the dnaKJ intergenic region were easily obtained. The growth rate of dnaJ mutants was reduced but the final cell density reached in rich medium and their symbiotic properties were indistinguishable from the wild type.

Amino Acid Sequence↗

PubMed1997

Characterization of the Bradyrhizobium japonicum CycY protein, a membrane-anchored periplasmic thioredoxin that may play a role as a reductant in the biogenesis of c-type cytochromes.

A new member of membrane-anchored periplasmic thioredoxin-like proteins was identified in Bradyrhizobium japonicum. It is the product of cycY, the last gene in a cluster of cytochrome c biogenesis genes. Mutational analysis revealed that cycY is essential for the biosynthesis of all c-type cytochromes in this bacterium. The CycY protein was shown to be exported to the periplasm by its N-terminal signal sequence-like domain. Results from Western blot analyses of membrane and soluble fractions indicated that the CycY protein remains bound to the membrane. A soluble version of the protein devoid of its N-terminal membrane anchor (CycY*) was expressed in Escherichia coli and purified to homogeneity from the periplasmic fraction. The protein showed redox reactivity and properties similar to other thioredoxins such as fluorescence quenching in the oxidized form. Its equilibrium constant with glutathione was determined to be 168 mM, from which a standard redox potential of -0.217 V was calculated, suggesting that CycY might act as a reductant in the otherwise oxidative environment of the periplasm. This is in agreement with our hypothesis that CycY is required, directly or indirectly, for the reduction of the heme-binding site cysteines in the CXXCH motif of c-type apocytochromes before heme attachment occurs.

Amino Acid Sequence↗

PubMed1997

Three disparately regulated genes for sigma 32-like transcription factors in Bradyrhizobium japonicum.

Bradyrhizobium japonicum possesses a subclass of heat-shock genes whose members are transcribed from a sigma 32 consensus promoter. Having identified previously one gene (rpoH1) encoding a sigma 32-like RNA polymerase transcription factor, we report here the characterization of two additional rpoH-like genes (rpoH2 and rpoH3). B. japonicum thus represents the first example of an organism possessing an rpoH multigene family. All three rpoH genes encode functional proteins that are able to initiate transcription from the Escherichia coli groE promoter. Each rpoH gene is apparently regulated by a different mechanism. Although both rpoH1 and rpoH2 are transcribed from sigma 70-type promoters, transcription of the rpoH1 operon was found to be heat inducible by an unknown mechanism, whereas the level of rpoH2 mRNA decreased after heat shock. At extreme temperatures (48 degrees C), rpoH2 was transcribed from a second promoter that resembled the E. coli sigma E-type promoter. The rpoH3 gene was found to be associated with two upstream genes, ragA and ragB, coding for a classical two-component regulatory system. Transcription initiated from a promoter that mapped in front of the putative response regulator gene ragA, suggesting that ragA, ragB and rpoH3 are organized in an operon. The ragA promoter was similar to a sigma 32 consensus promoter. The three B. japonicum rpoH genes also varied in their significance to support growth of the organism. While the rpoH2 gene could not be eliminated by mutation, knock-out mutants of rpoH1 and/ or rpoH3 were readily obtained and shown to be indistinguishable from the wild type under aerobic growth conditions or during root-nodule symbiosis. We conclude that rpoH2 is essential for the synthesis of cellular proteins under physiological growth conditions, whereas rpoH1, and probably also rpoH3, are involved in their synthesis during the stress response.

Amino Acid Sequence↗

PubMed1997

Dissection of the transcription machinery for housekeeping genes of Bradyrhizobium japonicum.

By using a PCR approach, the Bradyrhizobium japonicum sigA gene, which encodes the primary RNA polymerase sigma factor, sigma80, was cloned and its nucleotide sequence was established. The deduced protein is highly homologous to the SigA protein of Rhizobium meliloti (72% amino acid sequence identity) but less so to RpoD of Escherichia coli (51% identity). Well conserved is the C-terminal end of the protein, which is probably involved in promoter recognition and binding of the RNA polymerase core enzyme. A remarkable feature of the primary sequence is an alanine- and proline-rich segment of 24 amino acids between conserved regions 1 and 2, which might function as an interdomain linker. We purified the B. japonicum RNA polymerase holoenzyme. One of the subunits had an apparent molecular mass of 90 kDa and corresponded to the sigA gene product, as judged by N-terminal amino acid sequencing. The purified RNA polymerase was used in an in vitro transcription system to determine the transcription start sites of the rrn and groESL4 operons. They were identical to those previously identified in vivo. The rrn promoter was cloned upstream of a rho-independent terminator, yielding a transcript of about 240 bases. This served as a suitable template to analyze promoter activity. Then mutant derivatives of the rrn promoter were constructed and tested in in vitro transcription experiments. Several base pairs essential for promoter activity were thus identified. The results suggest that the well-characterized -35/-10 promoter class is predominantly used in B. japonicum for the expression of "housekeeping" genes.

Amino Acid Sequence↗

PubMed1996

Histidine 131, not histidine 43, of the Bradyrhizobium japonicum FixN protein is exposed towards the periplasm and essential for the function of the cbb3-type cytochrome oxidase.

In subunit I (FixN) of the Bradyrhizobium japonicum cbb3-type oxidase, only five instead of the normal six strictly conserved histidines (H) could be unambiguously assigned as the putative heme or copper ligands. The ambiguity concerned H43 or H131 as the presumptive N-terminal ligands of the low-spin heme B. We report here that a H43A replacement had a wild-type phenotype, whereas the H131A mutant was defective in oxidase function and subunit assembly or stability, suggesting that H131 serves as the N-terminal low-spin heme ligand. Topological studies revealed that H131 resides on the periplasmic side of helix 2, where one of the low-spin heme ligands is normally found in conventional heme-copper oxidases.

Bacterial Proteins↗

PubMed1996

Assembly and function of the cytochrome cbb3 oxidase subunits in Bradyrhizobium japonicum.

The Bradyrhizobium japonicum cbb3-type cytochrome oxidase, which supports microaerobic respiration, is a multisubunit enzyme encoded by the genes of the fixNOQP operon. We investigated the contribution of the individual subunits to function and assembly of the membrane-bound complex. In-frame deletion mutants of fixN, fixO, and fixQ, and an insertion mutant of fixP were constructed. All mutants, except the fixQ mutant, showed clearly altered absorption difference spectra of their membranes and decreased oxidase activities, and they were unable to fix nitrogen symbiotically. The presence of the individual subunits was assayed by Western blot analysis, using subunit-specific antibodies, and by heme staining of the c-type cytochromes FixO and FixP. These analyses led to the following conclusions: (i) FixN and FixO are necessary for assembly of the multimeric oxidase, (ii) FixN and FixO assemble independently of FixP, and (iii) FixQ is not required for complex formation and, therefore, does not seem to be an essential subunit. The possible oxidase biogenesis pathway involves the formation of a primary core complex consisting of FixN and FixO, which allows the subsequent association with FixP to form the complete enzyme.

Bacterial Proteins↗