ABSTRACT
Cloning and sequencing of a 7.1 kb DNA fragment from Agrobacterium sp IP I-671 revealed seven open reading frames (ORFs) encoding D-hydantoinase, D-carbamoylase and putative hydantoin racemase, D-amino acid oxidase and NAD(P)H-flavin oxidoreductase. Two incomplete ORFs flanking the hydantoin utilization genes showed similarities to genes involved in transposition. Expression of the D-hydantoinase and D-carbamoylase gene in Escherichia coli gave mainly inactive protein concentrated in inclusion bodies, whereas homologous expression on an RSF1010 derivative increased hydantoinase and D-carbamoylase activity 2.5-fold and 10-fold, respectively, in this strain. Inactivation of the D-carbamoylase gene in Agrobacterium sp IP I-671 led to a complete loss of detectable carbamoylase activity whereas the low hydantoinase activity remaining after inactivation of the D-hydantoinase gene indicated the presence of a second hydantoinase-encoding gene. Two plasmids of 80 kb and 190 kb in size were identified by pulsed-field gel electrophoresis and the cloned hydantoin utilization genes were found to be localized on the 190 kb plasmid.
Subject(s)
Genes, Bacterial , Hydantoins/metabolism , Rhizobium/genetics , Rhizobium/metabolism , Amidohydrolases/genetics , Amidohydrolases/metabolism , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Gene Expression , Gene Targeting , Genetic Complementation Test , Molecular Sequence Data , Open Reading Frames , Plasmids/genetics , Restriction MappingABSTRACT
A plasmid with the galK gene under control of the promoter of the mannitol utilization genes (mtl) from Pseudomonas fluorescens DSM 50106 was constructed to isolate the mtl regulatory gene. An Escherichia coli galK- mtl- strain with this plasmid was used to screen a genomic library of P. fluorescens for the presence of the regulatory gene by plating on McConkey agar plates supplemented with galactose and mannitol. Clones carrying the regulatory gene were isolated and by complemention assays, deletion analysis and DNA sequencing an open reading frame (mtlR) of 906nt identified encoding the regulator. The deduced protein MtlR with a calculated molecular mass of 34.7kDa showed a low overall similarity to several other regulatory proteins of the XylS/AraC family. When mtlR was cloned and expressed in E. coli, the protein was produced as inclusion bodies. Complete denaturation followed by subsequent slow refolding led to low amounts of active protein. The activity was shown in gel mobility shift assays by binding of MtlR to a DNA fragment containing the promoter/operator region of the P. fluorescens mtl genes.
Subject(s)
Escherichia coli Proteins , Genes, Bacterial/genetics , Pseudomonas fluorescens/genetics , Repressor Proteins/genetics , Trans-Activators/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/physiology , Cloning, Molecular , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Gene Expression/genetics , Gene Expression Regulation, Bacterial , Mannitol/metabolism , Molecular Sequence Data , Protein Binding , Pseudomonas fluorescens/chemistry , Repressor Proteins/physiology , Restriction Mapping , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
Cytological events in Zea mays root meristem were followed during 26 hr of anaerobic treatment. From 8 to 14 hr, mitochondria swelled drastically, Golgi apparatus actively produced vesicles, endoplasmic reticulum proliferated, and chromatin strongly condensed. Plastids appeared normal. By 26 hr, however, Golgi activity receded, mitochondria assumed long, polymorphic shapes, chromatin partly dispersed, and plastids swelled. ER remained prominent, and the cytoplasm contained long fibrous inclusions. This preliminary study emphasizes the need to examine quantitatively all cellular organelles periodically for longer periods when following events of stress or pathology. Our observations corroborate scattered reports in the literature for single organelles under anaerobic stress and represent the first set of correlated observations on the entire spectrum of cellular events.
