Identification and sequence analysis of the gene encoding the transcriptional activator of the formate hydrogenlyase system of Escherichia coli

Through complementation of a trans-acting regulatory mutation a gene has been cloned whose product is required for the formate induction of the anaerobic expression of the formate hydrogenlyase structural genes. By restriction analysis, and from the size of the encoded protein, the gene could be identified as being equivalent to fhlA described by Sankar et al. (1988). The nucleotide sequence of the fhlA gene was determined and it was shown to code for a protein with a calculated Mr of 78,467. Analysis of the derived amino acid sequence showed that the carboxy-terminal domain of FHLA shares considerable sequence similarity with NIFA and NTRC, which are the 'regulators' of two-component regulatory systems. Carboxy-terminal truncation of, and introduction of amino-terminal deletions in, the fhlA gene delivered inactive gene products. When overexpressed, FHLA mediates activation of expression of the formate dehydrogenase and hydrogenase structural genes in the presence of formate also under aerobic growth conditions. FHLA appears to bind to the upstream regulatory sequence (URS) in the 5' flanking region of the fdhF gene since activation of fdhF expression was dependent on the presence of the URS.     

Sequencing, mutational analysis, and transcriptional regulation of the Escherichia coli htrB gene

The Escherichia coli htrB gene was originally discovered because its insertional inactivation led to an exquisitely temperature-sensitive phenotype in rich media, i.e. the ability to form colonies at temperatures below 32 degrees C, but not above 33 degrees C. The htrB gene has been sequenced. It can potentially code for two proteins, with Mr values of 35,407 Da and 8669 Da, that are encoded by overlapping, divergent open reading frames. Our data are consistent with the 35,407 Da protein being HtrB. Northern blot analysis clearly shows that the monocistronic htrB message is not under heat-shock regulation. We have also sequenced the flanking DNA and have discovered a new gene, designated orf39.9, located immediately adjacent to htrB, but divergently transcribed.     

Oral administration of Escherichia coli glutamic acid decarboxylase has immunomodulatory effects in streptozotocin-induced diabetes

The extent of destruction of insulin-secreting beta cells of the Islets of Langerhans was investigated in an animal model using oral administration of glutamic acid decarboxylase (GAD) isolated from Escherichia coli. The extent of lymphocytic infiltration of the pancreatic Islet cells and the severity of diabetes were significantly reduced by oral administration of GAD to rats 14 days before intraperitoneal injections of streptozotocin (STZ, 40 mg/kg body wt on 5 consecutive days). In addition, oral administration of GAD to rats 14 days before or 3 days after STZ treatment significantly (p <0.05) reduced the levels of GAD-specific antibodies and improved the in vitro proliferative response of splenocytes to concanavalin A (Con A). These data demonstrate that oral GAD administration probably generates active cellular mechanisms which suppress the disease and therefore raise the possibility of using E. coli GAD as a new means for the immunomodulation of autoimmune diabetes.     

Mutations in the araC regulatory gene of Escherichia coli B/r that affect repressor and activator functions of AraC protein.

Mutations in the araC gene of Escherichia coli B/r were isolated which alter both activation of the araBAD operon expression and autoregulation. The mutations were isolated on an araC-containing plasmid by hydroxylamine mutagenesis of plasmid DNA. The mutant phenotype selected was the inability to autoregulate. The DNA sequence of 16 mutants was determined and found to consist of seven different missense mutations located within the distal third of the araC gene. Enzyme activities revealed that each araC mutation had altered both autoregulatory and activator functions of AraC protein. The mutational analysis presented in this paper suggests that both autoregulatory and activator functions are localized to the same determinants of the AraC protein and that the amino acid sequence within the carboxy-terminal region of AraC protein is important for site-specific DNA binding.     

Intergeneric homology of the speC gene encoding biosynthetic ornithine decarboxylase in Escherichia coli.

A 32P-labeled fragment of DNA containing the speC gene, which encodes the biosynthetic enzyme ornithine decarboxylase of Escherichia coli, was used as a hybridization probe for homologous sequences in the genomes of gram-negative and gram-positive bacteria. The speC probe detected homologous sequences in the DNA of only four members of the Enterobacteriaceae (Citrobacter freundii, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes); no homology was detected with the DNA of other representative members of the Enterobacteriaceae and gram-negative and gram-positive bacteria.     

Cloning and characterization of livH, the structural gene encoding a component of the leucine transport system in Escherichia coli.

The physical location of the genetically defined livH gene was mapped in the 17-kilobase plasmid pOX1 by using transposon Tn5 inactivation mapping and further confirmed by subcloning and complementation analysis. These results indicated that the livH gene maps 3' to livK, the gene encoding the leucine-specific binding protein. Moreover, the nucleotide sequence of the livH gene and its flanking regions was determined. The livH gene is encoded starting 47 base pairs downstream from the livK gene, and it is transcribed in the same direction as the livK gene. The livK-livH intergenic region lacks promoter sequences and contains a GC-rich sequence that could lead to the formation of a stable stem loop structure. The coding sequence of the livH gene, which is 924 base pairs, specifies a very hydrophobic protein of 308 amino acid residues. Expression of livH-containing plasmids in minicells suggested that a poorly expressed protein with an Mr of 30,000 could be the livH gene product.