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Biomedical subjects

W Klingmüller

Publications and source records attributed to W Klingmüller.

At least 19 recordsLinked to original sources
PubMed1992

Direct recovery and molecular analysis of DNA and RNA from soil.

A simple method for the recovery of DNA and direct detection of nif and Tn5 sequences in soil has recently been presented by Selenska and Klingmüller. On the basis of that method we have developed a procedure for the recovery and separation of DNA and RNA from the same soil sample. A 550 bp sequence from the Kmr gene of Tn5 was identified by PCR amplifications in total DNA and RNA, isolated from soil inoculated with a nitrogen-fixing Enterobacter agglomerans 19-1-1, which carries this transposon. Hence, not only the presence of the Kmr gene of Tn5 but also its expression (mRNA synthesis) in the analyzed environmental samples was detected. The authenticity of the products of PCR amplifications of DNA and mRNA was confirmed by the PhotoGene hybridization technique.

Base Sequence↗
PubMed1991

Identification of a promoter dependent on NifA and sigma 54 upstream of nifH in Azospirillum lipoferum.

Southern hybridization experiments strongly indicate that the regulatory region of the Azospirillum lipoferum nifH gene is located on a cloned 1.1 kb BamHI-XhoI restriction fragment. By cloning this fragment into a promoter-probe plasmid in Escherichia coli, a promoter was identified oriented towards the nifH gene. Using a set of several bacterial strains and plasmids, both NifA and the alternative sigma factor, sigma 54, from Klebsiella pneumoniae were shown to be required for the induction of the assumed nifH promoter in this particular heterologous system. However, NtrC from K. pneumoniae did not stimulate this promoter. No other promoter activity was detected in the direction opposite to the identified promoter, indicating that the transcription of the adjacent nifJ gene cannot be initiated from the 1.1 kb BamHI-XhoI fragment. Thus, the genes nifH and nifJ in A. lipoferum cannot be oriented divergently, in contrast to the situation in several other nitrogen-fixing bacteria.

Azospirillum brasilense↗
PubMed1991

DNA recovery and direct detection of Tn5 sequences from soil.

Specific Tn5 sequences inserted in the genome of Enterobacter agglomerans were detected in EcoRI digested DNA directly recovered from soil 70 d after its inoculation with the bacteria, when these were no longer culturable on agar medium. A new method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was needed.

DNA↗
PubMed1991

Cotranscription of the electron transport protein genes nifJ and nifF in Enterobacter agglomerans 333.

A nucleotide sequence showing extensive homology to the nifF gene, which codes for a flavodoxin involved in nitrogen fixation in Klebsiella pneumoniae, was localized on the plasmid pEA3 of Enterobacter agglomerans and determined. The analysis of transcriptional fusions, as well as transcript protection assays, indicated a novel nif gene organization, that is, the cotranscription of nifJ and nifF.

Amino Acid Sequence↗
PubMed1989

Identification and characterization of the nifH and nifJ promoter regions located on the nif-plasmid pEA3 of Enterobacter agglomerans 333.

Small restriction fragments of the plasmid-borne Enterobacter agglomerans 333 nif region were cloned into a promoter probe plasmid as transcriptional fusions with the lacZ gene. Identification of NifA-dependent promoters was accomplished by using a compatible plasmid which constitutively expresses the Klebsiella pneumoniae nifA gene. beta-Galactosidase assays showed strong activation of the cloned E. agglomerans promoters in Escherichia coli by the heterologous K. pneumoniae nifA gene product. The positions of the promoter fragments on the corresponding restriction map were determined by Southern hybridization. As confirmed by sequencing data, the nifH and nifJ promoters are situated at opposite end-points of the nif gene group and their -24 to -12 nucleotide sequences are similar to the consensus sequence of NtrA-dependent promoters. Also, typical NifA-binding motifs are present in both promoters. The agreement of the promoter proximal regions of nifH and nifJ with the corresponding K pneumoniae sequences is about 80%. Also the upstream regions of these genes are in agreement to some extent.

Base Sequence↗
PubMed1989

Identification of a regulatory nifA type gene and physical mapping of cloned new nif regions of Azospirillum brasilense.

Three new Tn5-mutagenized nif genes of Azospirillum brasilense were characterized. The sizes of the restriction fragments and the restriction maps of the cloned nif DNA regions showed that these nif genes are distinct from those reported earlier, e.g. nifHDK, nifE, nifUS, fixABC. The Nif27 mutant was identified as a nifA type regulatory gene of A. brasilense (a) by genetic complementation with nifA of Klebsiella pneumoniae, (b) by the absence of nitrogenase iron protein in western protein blots and (c) by its inability to activate expression of a nifH-lacZ fusion. The growth characteristics of the three mutants showed that none of them is defective in general nitrogen regulatory (ntr) genes. Also, no homology was detected between the three nif DNA regions of the mutants, cloned in pMS188, pMS189 and pMS197, and the K. pneumoniae nif, glnA or ntr genes. In addition, the fixABC genes of Bradyrhizobium japonicum did not show any hybridization with the cloned Azospirillum genes. Unlike the situation in enteric bacteria, the nif genes in A. brasilense are scattered and span a region of about 65 kb.

Azospirillum brasilense↗
PubMed1988

[The release of genetically altered microorganisms].

Microorganisms have been being released into the environment for a long time, e.g., in agriculture or for biotechnological processes. Amongst these microorganisms were some that had been labeled genetically, or improved by selection. In recent years, efforts have been directed towards alteration or optimization of appropriate bacteria and viruses by methods of recombinant DNA technology. In some cases, first releases of such organisms have been undertaken in experimental plots. There is public concern about the environmental risks of such releases, and about possible adverse effects. The present status of research in this controversial field, the regulations available in Germany and the United States, as well as the prospects of such work for the future are presented.

Accident Prevention↗
PubMed1988

Localization and physical mapping of a plasmid-borne 23-kb nif gene cluster from Enterobacter agglomerans showing homology to the entire nif gene cluster of Klebsiella pneumoniae M5a1.

A physical and genetical map of the plasmid pEA3 indigenous to Enterobacter agglomerans is presented. pEA3 is a 111-kb large plasmid containing a 23-kb large cluster of nif genes which shows extensive homology (Southern hybridization and heteroduplex analysis) to the entire nif gene cluster of Klebsiella pneumoniae (Kp) M5a1. All the nif genes on pEA3 are organized in the same manner as in K. pneumoniae, except nifJ, which is located on the left end of pEA3 nif gene cluster (near nifQB). A BamHI restriction map of pEA3 and a detailed restriction map of the 23-kb nif region on pEA3 is also presented. The nif genes of pEA3 showed a low level of acetylene reduction in Escherichia coli, demonstrating that these genes are functional and contain the whole genetic information required to fix nitrogen. The origin of vegetative replication (OriV) of pEA3 was localized about 5.5 kb from the right end of the nif gene cluster. In addition to pEA3, large plasmids from four other strains of E. agglomerans showed homology to all the Kp nif genes tested, indicating that in diazotrophic strains of E. agglomerans nif genes are usually located on plasmids. In contrast, in most of the free-living, nitrogen-fixing bacteria the nif genes are on chromosome.

DNA Restriction Enzymes↗
PubMed1987

Cloning of seven differently complementing DNA fragments with chl functions from Escherichia coli K12.

Seven genomic libraries of chromosomal Escherichia coli K12 wild-type DNA were constructed in plasmid vectors. These were used to transform chl insertion mutants. Selection for growth on nitrate under anaerobic conditions yielded four plasmids which complemented mutants of the chlA, B, E and G types. The chromosomal fragments were mapped with restriction enzymes and subcloned. Three complementation groups were observed among the chlA mutants and two among the chlE mutants. The established complementation groups plus mutants of the chlD type represent eight distinct functions, which are all believed to be required for the molybdenum cofactor activity in the reduction of nitrate to nitrite by E. coli.

Chlorates↗
PubMed1985

Isolation of transfer-negative nif-plasmids (pCE1) and their integration into the chromosome of Escherichia coli with the help of phage Mu.

Strain JC5466 of Escherichia coli K12 harbouring the nitrogen fixation plasmid pCE1 was lysogenized with bacteriophage Mu cts, followed by partial induction and infection with bacteriophage PRD1. This made it possible to obtain transfer-defective derivatives of pCE1, carrying Mu prophage. These derivatives could be mobilized by using the helper plasmid pME400 and it was possible to segregate the helper plasmid from the donor plasmid in the transconjugants. By incubating the strains 302 and 328 at 42 degrees C, for induction of Mu prophage, derivatives with different plasmid contents could be obtained such as strains without plasmids, some with smaller or larger plasmids and others possessing plasmids without any visible alteration in size. Integration of the nitrogen-fixation (nif) genes into the chromosomes of the strains without plasmids and those containing a smaller plasmid, was confirmed by Southern hybridization using radioactive nifKDH DNA. Conjugation assays have shown that the plasmid is integrated into the chromosome as a unit but that it can also be excised.

Chromosomes, Bacterial↗
PubMed1985

Restriction endonucleases in Azospirillum.

Azospirillum brasilense, A. amazonense, and A. lipoferum strains were screened for restriction endonucleases using phage lambda DNA. The extract of A. brasilense 29711 cleaved lambda DNA into specific fragments. It was concluded that this strain possesses a class II restriction endonuclease which was named AbrI. AbrI has a single recognition site on lambda DNA at position of approx. 33 500 bp. AbrI was characterized as an isoschizomer of XhoI, which cuts lambda DNA at 33 498 bp and cleaves double-stranded DNA at the sequence 5'-C TCGAG-3'. From other Azospirilla strains only A. amazonense QRZ42 extracts (AamI activity) cleaved DNA into specific fragments under certain conditions.

Bacteriophage lambda↗
PubMed1983

Nif-hybrids of Enterobacter: selection for nif gene integration with chlorate.

The nif gene group from Klebsiella can be transferred into Enterobacter cloacae by conjugation using Escherichia coli donor cells carrying the composite self-transmissible nif-plasmid pRD1. A small fraction of the hybrids obtained is stable upon prolonged passaging without selection. Their stability is due to integration of pRD1 into the chromosome. Such integration hybrids were chlorate resistant, and nitrate reductase negative, which indicated that integration preferentially occurred within one of the genes for the production or functioning of this enzyme. Chlorate resistance could, therefore, be used to select for additional nitrate reductase-negative sublines with pRD1 in their chromosome. Such sublines have been analyzed further for the presence of nif genes, other pRD1 markers, and for stability. In all except one the complete plasmid seems to have been integrated. Some tend to revert to nitrate utilisation (chlorate sensitivity).

Chlorates↗
PubMed1981

[Prospects and limitations of genetic engineering].

Examples for genetic engineering are the transfer of nuclei between cells of higher animals and the introduction of heterologous DNA into bacteria by means of plasmids. The former approach will help to establish new ways in animal breeding, the latter provides bacterial cells that produce proteins of medical importance. The moral justification of related studies in man is still open, but the possible risks of gene technology can be coped with by adhering to proper safety regulations.

Animals↗