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W Klingmüller

Publications and source records attributed to W Klingmüller.

10 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

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↗
PubMed1979

Gentic engineering for practical application.

Genetic engineering has ushered in a new era in biology. Although many problems are still to be solved, there are examples that point to a possible later application for the benefit of mankind: Bacteria can be manipulated to degrade crude-oil spillages, to produce human insulin and to bind nitrogen from the air. If all the bacteria that are indigenous to agricultural soils could be made to bind nitrogen, an increase in soil fertility might well result.

Arthrobacter↗
PubMed1975

[Bacterial resistance factors as vectors for gene manipulation and gene therapy (author's transl)].

Bacterial resistance factors are extrachromosomal elements, consisting of circular closed, double stranded DNA. They may contain genes which make the host bacterium resistant to certain antibiotics. Such resistance factors have become important vectors for selective replication of specific segments of all kinds of DNA. Suitable DNA segments are obtained by treating DNA with restriction endonucleases of defined cutting properties. They can then be linked to the respective vector. It was thus possible to anneal DNA segments originating from the same, but also from different bacterial resistance factors. They gave biologically functional units. The annealed products can be propagated in bacteria. The relevant experiments are described. Their potential dangers for man, and efforts to agree on suitable safety regulations are discussed.

Bacteriophages↗