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H Hennecke

Publications and source records attributed to H Hennecke.

At least 73 records · Page 4Linked to original sources
PubMed1994

Expression, purification and functional properties of a soluble form of Bradyrhizobium japonicum TlpA, a thioredoxin-like protein.

The TlpA protein of Bradyrhizobium japonicum was previously identified genetically as a membrane-anchored, periplasmic thioredoxin-like protein. Here we describe the heterologous expression in Escherichia coli, subsequent purification and biochemical characterization of TlpA. A soluble form of TlpA, which lacks its N-terminal membrane anchor, was overexpressed in E. coli and purified by a two-step procedure. Pure TlpA was shown to be a monomer in solution and was active in reducing the disulfides of insulin and in reactivating reduced, denatured RNaseA. Evidence is presented that two non-active-site cysteine residues form an intramolecular disulfide bond, a feature that is not normally found in other prokaryotic thioredoxins.

Amino Acid Sequence↗
PubMed1994

Substrate specificity is determined by amino acid binding pocket size in Escherichia coli phenylalanyl-tRNA synthetase.

Alanine at position 294 (Ala294) within the motif 3 consensus of Escherichia coli phenylalanyl-tRNA synthetase alpha subunit has previously been implicated as a determinant of amino acid specificity. To characterize the role of Ala294, the catalytic effects of amino acid replacements at this position were tested with purified wild-type and mutant phenylalanyl-tRNA synthetases. We show that Ala294 is involved in amino acid binding and that it influences specificity as a determinant of binding pocket size. Replacement of Ala294 by either glycine or serine, thereby increasing or decreasing the size of the binding pocket, respectively, reduces affinity for phenylalanine. The Gly294 mutant shows a relaxed specificity toward synthetic para-halogenated phenylalanine analogues, the apparent dissociation constant Km increasing in direct relation to an increase of the van der Waals radius of the para group, thus confirming the role of position 294 in determining amino acid binding pocket size. For the substrate analogue p-chlorophenylalanine, attachment to tRNA and in vivo incorporation into cellular protein by the Gly294 mutant were demonstrated. Tyrosine activation was also improved with this mutant, but the resulting enzyme-Tyr-adenylate complex was rapidly hydrolyzed, indicating the presence of a proofreading mechanism in E. coli phenylalanyl-tRNA synthetase.

Adenosine Triphosphate↗
PubMed1994

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.

Bacteria↗
PubMed1994

Towards engineering proteins by site-directed incorporation in vivo of non-natural amino acids.

Altering protein structure via the techniques of protein engineering has already allowed the development of proteins displaying both modified and novel activities. The only limitation of conventional site-directed mutagenesis, the cornerstone of protein engineering, is that substitutions are restricted to the 20 naturally occurring, proteinogenic amino acids. However, the discovery of a 21st amino acid, selenocysteine, and the development of novel in vitro translation systems have demonstrated that considerably more substitutions are possible. To this end, a number of experimental approaches have been developed that allow the incorporation of synthetic amino acids into proteins. Some of these have already been successfully applied in vitro and efforts to transfer this technology to in vivo systems are now underway.

Amino Acids↗
PubMed1994

Cytochrome c biogenesis in bacteria: a possible pathway begins to emerge.

Cytochrome c biogenesis describes the posttranslational pathway for the conversion of pre-apocytochrome c into the mature holocytochrome c. It involves an unknown number of consecutive biochemical steps, including translocation of the precursor polypeptide and haem into the periplasm and the covalent linkage between these two molecules. Genetic and molecular analysis of several bacterial mutants suggest that at least eight genes contribute to this process. In this review we summarize the present knowledge of the cytochrome c maturation pathway in bacteria and propose a model in which certain genes and their products are attributed to specific functions.

Amino Acid Sequence↗
PubMed1994

The ccoNOQP gene cluster codes for a cb-type cytochrome oxidase that functions in aerobic respiration of Rhodobacter capsulatus.

The genes for a new type of a haem-copper cytochrome oxidase were cloned from Rhodobacter capsulatus strain 37b4, using the Bradyrhizobium japonicum fixNOQP gene region as a hybridizing probe. Four genes, probably organized in an operon (ccoNOQP), were identified; their products share extensive amino acid sequence similarity with the FixN, O, Q and P proteins that have recently been shown to be the subunits of a cb-type oxidase. CcoN is a b-type cytochrome, CcoO and CcoP are membrane-bound mono- and dihaem c-type cytochromes and CcoQ is a small membrane protein of unknown function. Genes for a similar oxidase are also present in other non-rhizobial bacterial species such as Azotobacter vinelandii, Agrobacterium tumefaciens and Pseudomonas aeruginosa, as revealed by polymerase chain reaction analysis. A ccoN mutant was constructed whose phenotype, in combination with the structural information on the gene products, provides evidence that the CcoNOQP oxidase is a cytochrome c oxidase of the cb type, which supports aerobic respiration in R. capsulatus and which is probably identical to the cbb3-type oxidase that was recently purified from a different strain of the same species. Mutant analysis also showed that this oxidase has no influence on photosynthetic growth and nitrogen-fixation activity.

Aerobiosis↗
PubMed1993

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)

Bacterial Proteins↗
PubMed1993

Genes for a microaerobically induced oxidase complex in Bradyrhizobium japonicum are essential for a nitrogen-fixing endosymbiosis.

We report the discovery of a Bradyrhizobium japonicum gene cluster (fixNOQP) in which mutations resulted in defective soybean root-nodule bacteroid development and symbiotic nitrogen fixation. The predicted, DNA-derived protein sequences suggested that FixN is a heme b and copper-binding oxidase subunit, FixO a monoheme cytochrome c, FixQ a polypeptide of 54 amino acids, and FixP a diheme cytochrome c and that they are all membrane-bound. The isolation and analysis of membrane proteins from B. japonicum wild-type and mutant cells revealed two c-type cytochromes of 28 and 32 kDa as the likely products of the fixO and fixP genes and showed that both were synthesized only under oxygen-limited growth conditions. Furthermore, fixN insertion and fixNO deletion mutants grown microaerobically or anaerobically (with nitrate) exhibited a strong decrease in whole-cell oxidase activity as compared with the wild type. The data suggest that the fixNOQP gene products are induced at low oxygen concentrations and constitute a member of the bacterial heme/copper cytochrome oxidase superfamily. The described features are compatible with the postulate that this oxidase complex is specifically required to support bacterial respiration in endosymbiosis.

Aerobiosis↗
PubMed1993

One member of a gro-ESL-like chaperonin multigene family in Bradyrhizobium japonicum is co-regulated with symbiotic nitrogen fixation genes.

This report is concerned with the structural characterization and genetic regulation of new bacterial groES and groEL chaperonin genes, and presents two novelties. The first is the discovery that the nitrogen fixing soybean root nodule bacterium, Bradyrhizobium japonicum, unlike all other prokaryotes investigated so far, possesses a multigene family consisting of five very similar, though not identical, groESL-like genes. The second novelty relates to the finding that these five homologues are expressed to different degrees and, in particular, that one family member (namely groESL3) is induced by a mechanism that does not involve the well-known heat shock response. By contrast, the groESL3 genes are co-regulated together with symbiotic nitrogen fixation genes, in that they are activated by the nitrogen fixation regulatory protein NifA at low oxygen conditions and transcribed from a -24/-12 promoter by the sigma 54 RNA polymerase. Two other members of the groESL gene family are apparently expressed constitutively at different levels, and yet another one is strongly induced by high temperature. As an attractive hypothesis it follows that B. japonicum may modulate its cellular contents of GroES- and GroEL-like chaperonins in response to specific environmental conditions and physiological needs.

Amino Acid Sequence↗
PubMed1993

Bradyrhizobium japonicum TlpA, a novel membrane-anchored thioredoxin-like protein involved in the biogenesis of cytochrome aa3 and development of symbiosis.

We report the discovery of a bacterial gene, tlpA, that codes for a hitherto unknown type of thioredoxin-like protein. The gene was found in the course of studying a Tn5 insertion mutant of the soybean root nodule symbiont Bradyrhizobium japonicum. The TlpA protein shared up to 31% amino acid sequence identity with various eukaryotic and prokaryotic thioredoxins and protein disulfide isomerases, and possessed a characteristic active-site sequence, Trp-Cys-Val-Pro-Cys. In contrast to all members of the thioredoxin family known to date, TlpA was shown to be anchored to the cytoplasmic membrane by means of an N-terminal transmembrane domain, while the active site-containing part of the protein faced the periplasm. The tlpA mutant had a pleiotropic phenotype in that it was defective in the development of a nitrogen fixing endosymbiosis and exhibited a strongly decreased oxidase activity, as compared with the wild-type. Holocytochrome aa3 was spectroscopically undetectable in the mutant, whereas the apoprotein of subunit one (CoxA) of this oxidase was still synthesized and incorporated into the cytoplasmic membrane. Since cytochrome aa3 is not a prerequisite for the development of symbiosis, the results suggest that TlpA is involved in at least two independent cellular processes, one of which is an essential periplasmic step in the maturation of cytochrome aa3.

Alkaline Phosphatase↗
PubMed1993

A novel response-regulator is able to suppress the nodulation defect of a Bradyrhizobium japonicum nodW mutant.

The two-component regulatory system Nod-VW of Bradyrhizobium japonicum is essential for the nodulation of the legume host plants Vigna radiata, V. unguiculata and Macroptilium atropurpureum. The NodV protein shares homology with the sensor-kinases, whereas the NodW protein is a member of the response-regulator class. We report here the identification of a new B. japonicum DNA region that is able to suppress the phenotypic defect of a nodW mutant, provided that this region is expressed from a foreign promoter. The minimal complementing region, which itself is not essential for nodulation in a nodW+ background, consists of one gene designated nwsB (nodW-suppressor). The deduced amino acid sequence of the nwsB gene product shows a high degree of homology to NodW. The nws B gene is preceded by a long open reading frame, nwsA, whose putative product appears to be a sensor-kinase. Downstream of nwsB, an open reading frame encoding a second putative response-regulator was identified. Interspecies hybridization revealed the presence of nwsAB-like DNA also in other Bradyrhizobium strains. Using nwsB'-'lacZ fusions, the nwsB gene was found to be expressed rather weakly in B. japonicum. This low level of expression is obviously not sufficient to compensate for a nodW- defect, whereas strong overexpression of nwsB is a condition that leads to suppression of the nodW- mutation.

Amino Acid Sequence↗
PubMed1993

Formation of several bacterial c-type cytochromes requires a novel membrane-anchored protein that faces the periplasm.

We report here the discovery of a novel bacterial gene (cycH) whose product is involved in the biogenesis of most of the cellular cytochromes c. The cycH gene was detected in the course of characterizing a cytochrome oxidase-deficient Bradyrhizobium japonicum Tn5 mutant (strain COX3) in which the transposon insertion disrupted cycH. All of the c-type cytochromes detectable in aerobically grown B. japonicum wild-type cells were absent in the COX3 mutant, with the exception of cytochrome c1. A secondary phenotypic effect was the spectroscopic absence of the aa3-type cytochrome c oxidase. The nucleotide sequence of the cloned wild-type cycH gene predicted a membrane-bound 369-amino-acid protein with an M(r) of 39727. Results from studies on its membrane topology suggested that approximately 110 N-terminal amino acids are involved in anchoring the protein in the membrane, whereas the remaining two-thirds of the protein are exposed to the periplasm. We postulate that the CycH protein plays an essential role in an as yet unidentified periplasmic step in the biogenesis of holocytochromes c, except that of cytochrome c1.

Aerobiosis↗
PubMed1993

Correlated physical and genetic map of the Bradyrhizobium japonicum 110 genome.

We describe a compilation of 79 known genes of Bradyrhizobium japonicum 110, 63 of which were placed on a correlated physical and genetic map of the chromosome. Genomic DNA was restricted with enzymes PacI, PmeI, and SwaI, which yielded two, five, and nine fragments, respectively. Linkage of some of the fragments was established by performing Southern blot hybridization experiments. For probes we used isolated, labelled fragments that were produced either by PmeI or by SwaI. Genes were mapped on individual restriction fragments by performing gene-directed mutagenesis. The principle of this method was to introduce recognition sites for all three restriction enzymes mentioned above into or very near the desired gene loci. Pulsed-field gel electrophoresis of restricted mutant DNA then resulted in an altered fragment pattern compared with wild-type DNA. This allowed us to identify overlapping fragments and to determine the exact position of any selected gene locus. The technique was limited only by the accuracy of the fragment size estimates. After linkage of all of the restriction fragments we concluded that the B. japonicum genome consists of a single, circular chromosome that is approximately 8,700 kb long. Genes directly concerned with nodulation and symbiotic nitrogen fixation are clustered in a chromosomal section that is about 380 kb long.

Base Sequence↗
PubMed1992

Prokaryotic polyprotein precursors.

Polyproteins have been found only recently in prokaryotes. The four known examples of single bacterial genes encoding precursors that are posttranslationally processed into two mature proteins are addressed here with respect to (i) their genomic arrangement, (ii) the sites of proteolytic processing, (iii) the relevant proteases, (iv) their maturation pathway, and (v) the function of the mature proteins. How these polyproteins may have evolved is also discussed.

Bacteria↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

Genes for a second terminal oxidase in Bradyrhizobium japonicum.

Bradyrhizobium japonicum possesses a mitochondria-like respiratory chain terminating with an aa3-type cytochrome c oxidase. The gene for subunit I of this enzyme (coxA) had been identified and cloned previously via heterologous hybridization using a Paracoccus denitrificans DNA probe. In the course of these studies, another B. japonicum DNA region was discovered which apparently encoded a second terminal oxidase that was different from cytochrome aa3 but also belonged to the superfamily of heme/copper oxidases. Nucleotide sequence analysis revealed a cluster of at least four genes, coxMNOP, organized most probably in an operon. The predicted coxM gene product shared significant similarity with subunit II of cytochrome c oxidases from other organisms: in particular, all of the proposed CuA ligands were conserved as well as three of the four acidic amino acid residues that might be involved in the binding of cytochrome c. The coxN gene encoded a polypeptide with about 40% sequence identity with subunit I representatives including the previously found CoxA protein: the six presumed histidine ligands of the prosthetic groups (two hemes and CuB) were strictly conserved. A remarkable feature of the DNA sequence was the presence of two genes, coxO and coxP, whose products were both homologous to subunit III proteins. A B. japonicum coxN mutant strain was created by marker exchange mutagenesis which, however, exhibited no obvious defects in free-living, aerobic growth or in root nodule symbiosis with soybean. This shows that the coxMNOP genes are not essential for respiration in the N2 fixing bacteroid.

Amino Acid Sequence↗
PubMed1992

Identification of the pheS5 mutation, which causes thermosensitivity of Escherichia coli mutant NP37.

The pheS5 mutation responsible for the thermosensitive phenylalanyl-tRNA synthetase of the classical Escherichia coli NP37 was cloned by a recombination event and identified by DNA sequence analysis. The mutation was subsequently verified by direct sequencing of amplified NP37 DNA generated by an asymmetric polymerase chain reaction. The resulting amino acid exchange, Gly-98 to Asp-98 in the phenylalanyl-tRNA synthetase alpha subunit, might cause subunit disaggregation due to electrostatic repulsion.

Amino Acid Sequence↗