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R Haselkorn

Publications and source records attributed to R Haselkorn.

At least 91 records · Page 5Linked to original sources
PubMed1986

Identification and sequence of a gene required for a developmentally regulated DNA excision in Anabaena.

Vegetative cells of the cyanobacterium Anabaena contain an 11 kb DNA element within the coding region of the nifD gene. This element is excised by site-specific recombination between directly repeated 11 bp sequences at each of its ends during differentiation of nitrogen-fixing cells called heterocysts. Site-specific recombination, leading to the same rejoined nifD gene, was observed during propagation in E. coli of a fragment containing the 11 kb element and flanking sequences. An assay for excision of the element in E. coli was developed, based on mini-Mu-lac transposition into the element. Since the 11 kb element lacks an origin of replication, its excision results in loss of lac and conversion of blue colony-forming cells to white on X-gal plates. Insertion and deletion mutagenesis identified a region of the element needed for excision. Mutations in this region could be complemented by a 6 kb fragment containing an open reading frame that runs counter to those of the nif genes, beginning 240 bp from the recombination site.

Base Sequence↗
PubMed1986

Differences in mRNA levels in Anabaena living freely or in symbiotic association with Azolla.

Azolla is a small water fern in whose leaf cavities the filamentous nitrogen-fixing cyanobacterium Anabaena azollae is symbiotically associated. Using cloned genes from Anabaena 7120 for glutamine synthetase (GS), ribulose-1,5-bisphosphate (RuBP) carboxylase, nitrogenase and the 32-kd protein of photosystem II, mRNA levels of the corresponding genes in the Anabaena endosymbiont were studied by Northern hybridization. In RNA isolated from the endosymbiont there is a 10-fold reduction of GS transcript levels, a greater than 5-fold increase in 32-kd transcript levels and a greater than 5-fold decrease in RuBP carboxylase transcript levels, compared with levels in the free-living Anabaena azollae. In the endosymbiont and in heterocysts of the free-living Anabaena azollae the nif H, nif D, and nif K genes are transcribed from a single nif HDK operon.

Chlorophyll↗
PubMed1986

Expression of a family of psbA genes encoding a photosystem II polypeptide in the cyanobacterium Anacystis nidulans R2.

The genome of the cyanobacterium Anacystis nidulans R2 contains three genes (psbA) for the QB protein of photosystem II. This protein is essential for oxygenic photosynthetic electron tansport, and is the target for several herbicides which act by binding directly to the photosynthetic apparatus. Transcripts from the three Anacystis psbA genes are present in wild-type cells at different steady-state levels. The nucleotide sequences of two of the genes, psbAII and psbAIII, predict a protein having the same amino acid sequence which differs from that of the psbAI gene by 25 (out of 360) residues. Inactivation of each of the psbA genes in the Anacystis chromosome, singly or in pairs, shows that each of the genes is capable of producing sufficient functional QB protein to support normal photoautotrophic growth.

Amino Acid Sequence↗
PubMed1986

Anaerobic regulation of nitrogen-fixation genes in Rhodopseudomonas capsulata.

A Rhodopseudomonas capsulata nifH::lacZ gene fusion was used to isolate constitutive mutants of R. capsulata, unable to repress nif gene transcription anaerobically with every fixed-nitrogen source tested. When these nifc strains were grown aerobically, nif gene transcription was repressed. These results indicate that the regulation of nif gene transcription by fixed nitrogen is different from the regulation by oxygen. Under anaerobic conditions, nif gene transcription in both R. capsulata and Klebsiella pneumoniae is specifically prevented by inhibitors of DNA gyrase [DNA topoisomerase type II (ATP-hydrolyzing), EC 5.99.1.3]. A recent study has shown that anaerobically grown Salmonella typhimurium have high DNA gyrase activity, whereas aerobically grown cells have high DNA topoisomerase type I (EC 5.99.1.2) activity and DNA that is more relaxed [Yamamoto, N. & Droffner, M. L. (1985) Proc. Natl. Acad. Sci. USA 82, 2077-2081]. In view of these results, we suggest that the control of nif gene transcription in response to oxygen is determined by the action of DNA gyrase and DNA topoisomerase I. Thus, although nitrogen control of nif gene expression requires the products of regulatory genes for which constitutive mutations can be isolated, oxygen appears instead to prevent the adoption of a DNA conformation necessary, directly or indirectly, for nif gene transcription.

Anaerobiosis↗
PubMed1985

Mutation to herbicide resistance maps within the psbA gene of Anacystis nidulans R2.

A psbA gene encoding the target of photosystem II herbicide inhibition, the 32,000-dalton thylakoid membrane protein, has been cloned from a mutant of Anacystis nidulans R2, which is resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea-(diuron). A cloned DNA fragment from within the coding region of this gene transforms wild-type cells to herbicide resistance, proving that mutation within psbA is responsible for that phenotype. The mutation consists of a single nucleotide change that replaces serine at position 264 of the wild-type protein with alanine in that of the diuron-resistant mutant.

Amino Acid Sequence↗
PubMed1985

Terminal sequences of the bacteriophage phi 6 segmented dsRNA genome and its messenger RNAs.

The ends of the three dsRNA genome segments (L, M, and S) of bacteriophage phi 6 (strand separated and/or intact) and the 5' ends of the middle and small single-strand messenger RNAs have been sequenced by base-specific partial enzymatic digestion. Terminal sequences for the large and middle dsRNA strands extend about 60 bases. The three dsRNA segments have 18 homologous bases at the left end except for position 2, which differs in the L segment. A 17-base homology defines the right ends of L and M dsRNAs and probably S dsRNA as well. The 5' ends of middle and small messenger RNAs are identical to the corresponding viral (+) strands.

Bacteriophages↗
PubMed1985

Isolation and organization of genes for nitrogen fixation in Rhodopseudomonas capsulata.

A library of Rhodopseudomonas capsulata chromosomal DNA was constructed in the broad host range cosmid vector pLAFR1. The library was used to isolate nitrogen fixation genes by complementation of R. capsulata Nif- mutants. Four complementing regions were localized on different cloned DNA fragments by Tn5 and mini-Mu mutagenesis. Additional nif genes were identified by recombination of transposons from the nif cosmids into the R. capsulata chromosome resulting in the creation of new Nif- mutations. Most of the newly cloned DNA fragments containing nif genes were found to be unlinked to any other by Southern hybridization of the cloned DNA to chromosomal DNA blots. One of the new fragments was linked to the nifHDK genes. Another cluster spanning 10-12 kilobase pairs contained a number of nif genes, possibly as many as eight.

Chromosome Mapping↗
PubMed1985

Characterization of nif regulatory genes in Rhodopseudomonas capsulata using lac gene fusions.

Translational fusions of the Escherichia coli lacZYA operon to Rhodopseudomonas capsulata nif genes were obtained by using mini-MudII1734 [Castilho et al., J. Bacteriol. 158 (1984) 488-495] inserts into cloned fragments of R. capsulata DNA. A lac fusion to the nifH gene, which encodes dinitrogenase reductase, was used to classify Nif- mutations occurring in regulatory genes. Nine mutations were unable to activate nifHDK transcription. The nine mutations define four nif regulatory genes. Three of these genes are located on the same R. capsulata 8.4-kb EcoRI fragment. Each is transcribed independently. One of these (complementing mutant J61) is partially homologous with the ntrC gene of Escherichia coli, based on Southern hybridization. The fourth nif regulatory gene (complementing mutants LJ1, AH1 and AH3) is unlinked to the others. Lac fusions to all four regulatory genes were constructed. Each regulatory gene is weakly expressed compared to derepressed nifH and partially repressed in the presence of ammonia.

Escherichia coli↗
PubMed1984

Cotranscription of genes encoding the small and large subunits of ribulose-1,5-bisphosphate carboxylase in the cyanobacterium Anabaena 7120.

In the filamentous cyanobacterium Anabaena, the gene for the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase is linked to and transcribed together with the gene encoding the large subunit (rbcL) of the same enzyme. The two genes are separated by a spacer of 545 base pairs (bp) that does not contain an open reading frame. Both genes hybridize with a predominant 3.1-kilobase transcript that initiates 414 bp upstream from the rbcL coding region. The nucleotide sequence 14-8 bp preceding the transcription start site resembles a good Escherichia coli promoter, but the sequence in the -35 region does not. There is no obvious relationship between the sequence flanking the amino terminus of the cyanobacterial small subunit gene and the transit peptide of eukaryotic small subunit genes. The Anabaena rbcS gene is 61% homologous at the amino acid level with the gene from the unicellular cyanobacterium Anacystis and 37-38% homologous with the corresponding nuclear genes from eukaryotes. In contrast, the Anabaena rbcL gene is approximately equal to 86% homologous at the amino acid level with the rbcL genes from plant chloroplasts. Cotranscription satisfies one of the requirements for coordinate expression of the two genes whose products are needed in equimolar amounts in the mature enzyme. The rbcL-rbcS transcript is equally abundant in Anabaena azollae grown in the light or on fructose in the dark.

Amino Acid Sequence↗
PubMed1984

Organization and regulation of nitrogen fixation genes of Rhodopseudomonas capsulata.

The nitrogen fixation (nif) genes of Rhodopseudomonas capsulata appear to be much more widely dispersed than those of the well-studied Klebsiella pneumoniae. In both organisms the genes coding for the structural components of the nitrogenase complex, nifHDK, comprise a single unit of transcription. Only in the photosynthetic bacterium, however, are there multiple copies of these genes. The extra copies are normally silent but they can be activated by mutation, detected as nif+ pseudorevertants of nif- deletions of the expressed nifHDK genes. Glutamine is required for the repression of nif gene transcription in R. capsulata.

Chromosome Mapping↗
PubMed1983

Sequence of the nifD gene coding for the alpha subunit of dinitrogenase from the cyanobacterium Anabaena.

The nucleotide sequence of nifD, the structural gene for the alpha subunit of dinitrogenase from Anabaena 7120, has been determined. The coding sequence contains 1,440 nucleotides, which predict an amino acid sequence of 480 residues and M(r) of 54,283. The predicted sequence contains eight cysteines, of which five are conserved with respect to adjoining sequences and position relative to the alpha subunits of dinitrogenase from Azotobacter, Clostridium, and Klebsiella. Because there are also five conserved cysteines in the beta subunit of Anabaena dinitrogenase [Mazur, B. J. & Chiu, C.-F. (1982) Proc. Natl. Acad. Sci. USA 79, 6782-6786], the number of cysteine residues participating as ligands to FeS clusters is likely to be 20 per alpha(2)beta(2) tetramer. This number is sufficient to accommodate the known four Fe(4)S(4) clusters, leaving at least four cysteines to be shared among the two FeMo cofactors and the more poorly characterized two-iron center. Although the alpha- and beta-subunit gene sequences are not recognizably homologous, their secondary structures, predicted from the sequences, indicate similar domains around three of the conserved cysteine residues.

Journal Article↗
PubMed1983

Isolation and sequence of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase from the cyanobacterium Anabaena 7120.

Cloned DNA probes containing genes coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcA) of corn and of Chlamydomonas were used to identify, by heterologous hybridization, DNA fragments from Anabaena 7120 carrying the corresponding gene sequence. The same probes were used to isolate, from a recombinant lambda library, a 17-kilobase-pair EcoRI Anabaena DNA fragment containing the coding sequence for the rbcA gene. The entire coding sequence, as well as 210 base pairs of 5' flanking region and 210 base pairs of 3' flanking region, was determined. Comparison of the nucleotide and amino acid sequences with those of corn, spinach, Chlamydomonas, and Synechococcus rbcA genes revealed homology of 71-77% at the nucleotide level and 80-85% at the amino acid level. Conservation of sequence is lost immediately outside the coding region on either side. Codon usage in the Anabaena rbcA gene is not significantly different from that in the Anabaena genes for nitrogenase reductase and nitrogenase beta subunit.

Journal Article↗
PubMed1983

The wild-type gene for glutamine synthetase restores ammonia control of nitrogen fixation to Gln- (glnA) mutants of Rhodopseudomonas capsulata.

The wild-type glnA gene, coding for glutamine synthetase, was cloned from the photosynthetic bacterium Rhodopseudomonas capsulata by using a cosmid library to complement the Gln- phenotype of an Escherichia coli glnA deletion strain. The original cosmid plasmid contained 37 kilobase pairs (kbp) of R. capsulata DNA, of which only 2 kbp was necessary for Gln complementation in E. coli. A plasmid containing this 2-kbp insert was mobilized into G29, a Gln- mutant of R. capsulata which is also unable to repress nitrogenase in ammonia-containing media (Nifc phenotype). The 2-kbp fragment restored glutamine-independent growth and ammonia repression of nitrogenase, indicating that in R. capsulata, production of the signal for nitrogen repression of nif depends on the activity of the glnA gene. Repression of nitrogenase was shown, by hybridization of RNA to cloned nif DNA, to occur at the level of transcription in the wild-type and the complemented G29 strains.

Ammonia↗
PubMed1983

Genetic and physical map of the structural genes (nifH,D,K) coding for the nitrogenase complex of Rhodopseudomonas capsulata.

Functional genes coding for the structural components of the nitrogenase complex (nifH,D,K) have been cloned on an 11.8-kilobase-pair HindIII fragment of DNA from the photosynthetic bacterium Rhodopseudomonas capsulata. The genes were physically mapped by hybridization of individual cloned nif genes from Klebsiella pneumoniae and Anabaena sp. strain 7120 to Southern blots of HindIII digests of the cloned R. capsulata fragment, after introduction of HindIII sites into the latter at specified locations by insertion of Tn5. Plasmids with the 11.8-kilobase-pair HindIII fragment containing the Tn5 insertions were also used for complementation tests with chromosomal Nif- mutations and for the generation of subfragments to locate those mutations by marker rescue. The R. capsulata nifH,D,K genes comprise a single unit of expression, with the same organization and polarity as found in K. pneumoniae. However, the R. capsulata nifH,D,K fragment did not complement Nif- point mutations in the corresponding Klebsiella genes, and the Klebsiella nif genes did not function in R. capsulata.

Cloning, Molecular↗