Identification of the transcriptional activator controlling the butanediol fermentation pathway in Klebsiella terrigena.

The gene budR, whose product is responsible for induction of the butanediol formation pathway under fermentative growth conditions in Klebsiella terrigena, has been cloned and sequenced. This gene is separated from the budABC operon by a nontranslated region of 106 bp and transcribed in the opposite direction. budR codes for a protein of molecular weight 32,124, the sequence of which exhibits characteristics of regulators belonging to the LysR family. When transferred into the heterologous host Escherichia coli, budR activates expression of budA'-lacZ transcriptional and translational fusions with a regulatory pattern identical to that in K. terrigena, namely, induction by acetate, low pH, and anaerobiosis. Induction by acetate was specific, indicating that it is the physiological inducer. Primer extension analysis located the start site of transcription to two positions, 23 and 24 bp upstream of the budR initiation codon, and also showed that BudR strongly autoregulates its own expression. The products of fhlA, arcA, hip, ntrA, and katF did not influence expression of the bud operon. A mutation in fnr, however, led to a threefold increase in expression, indicating that Fnr acts as a repressor. The results support the notion that BudR coordinates the activity of the energy-conserving, nonreductive, but acidifying acetate formation pathway with the expression of the non-energy-conserving, reductive, but nonacidifying butanediol pathway.

Purification and DNA-binding properties of FHLA, the transcriptional activator of the formate hydrogenlyase system from Escherichia coli.

FHLA is the transcriptional activator for the expression of the genes coding for components of the formate hydrogenlyase system of Escherichia coli. The cloned fhlA gene was overexpressed under selected growth conditions, and a purification protocol for the FHLA protein was developed. Purified FHLA in the native state is a homotetramer. It binds to the upstream regulatory sequences of the fdhF gene and of the intergenic region between the divergently transcribed hyc and hyp operons. An additional binding site of FHLA located between the hycA and hycB genes was identified. While binding to the hypA-hycA intergenic region is responsible for activation of expression of the hyc operon, the newly identified site is required for the activation of the sigma 54-dependent promoter located upstream of the hyp operon. Formate seems to have no effect on the DNA-binding properties of FHLA. The binding sites of FHLA were characterized by DNase I footprinting; sequence motifs putatively involved in the interaction with FHLA are described.

Regulated expression in vitro of genes coding for formate hydrogenlyase components of Escherichia coli.

Purified FHLA, the transcriptional activator of the formate regulon from Escherichia coli, is able to efficiently stimulate transcription from the sigma 54-dependent promoters of the fdhF, hyp, and hyc transcriptional units. Expression was dependent on the presence of sigma 54, of the upstream activatory sequence (UAS), and of formate. Hypophosphite, a formate analogue, could substitute for formate in vitro suggesting that formate per se was active in regulation. The integration host factor (IHF) had a direct effect on the expression (in vivo and in vitro) of the hyp and hyc genes but not of the fdhF gene. Binding of IHF within the region between the hyp and the hyc operon could be shown. A model is proposed for the transcriptional regulation of the inversely oriented hyp and hyc operons. It involves two upstream regulatory sequences, one between the hyp and the hyc operon (IR1), and the other between hycA and hycB (IR2). The UAS situated within IR1 is responsible for activation of the hyc operon, that within IR2 for activation of the hyp operon. A supramolecular transcription complex is proposed which involves the binding of IHF to a site located between the UAS and the promoter responsible for transcription of the hyc operon.

Molecular characterization of an operon (hyp) necessary for the activity of the three hydrogenase isoenzymes in Escherichia coli.

The 58/59 min region of the Escherichia coli chromosome contains two divergently oriented gene clusters coding for proteins with a function in hydrogenase formation. One cluster (the hyc operon), transcribed counterclockwise with respect to the E. coli chromosome, codes for gene products with a structural role in hydrogenase 3 formation (Böhm et al., 1990). The nucleotide sequence of the divergently transcribed operon (hyp) has been determined. It contains five genes, all of which are expressed in vivo in a T7 promoter/polymerase system, and the sizes of the synthesized products correspond with those predicted from the amino acid sequence. Complementation analysis of previously characterized mutants showed that the hypB, hypC and hypD genes have a function in the formation of all three hydrogenase isoenzymes, lesions in hypB being complemented by high nickel ion concentration in the medium. Prevention of hypBCDE gene expression led to an altered electrophoretic pattern of hydrogenase 1 and 2 constituent subunits, indicating increased chemical or proteolytic subunits, Under fermentative growth conditions, operon expression was governed by an NtrA-dependent promoter lying upstream of hypA working together with an fnr gene product-dependent promoter which was localized within the hypA gene. The latter (operon-internal) promoter is responsible for hypBCDE transcription under non-fermentative conditions when the -24/-12 NtrA-dependent promoter upstream of hypA is silent.

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.

Mutations in trans which affect the anaerobic expression of a formate dehydrogenase (fdhF) structural gene.

An operon fusion was constructed in which the chloramphenicol acetyltransferase gene (cat) is under the transcriptional control of the anaerobically-activated formate dehydrogenase (fdhF) gene promoter. It was used as a screening system for mutations in trans which prevent the formate-dependent anaerobic induction of fdhF gene expression. Five classes of mutants were identified. The defect in class I mutants was complemented by a plasmid (pBA11) or subclones thereof, which harbor genes of the Escherichia coli 58 min hyd (hydrogenase) gene cluster. They may comprise regulatory gene mutants. The phenotype of class II mutants was reversed by supplementing the medium with 100 microM MoO4(2-); WO4(2-) could substitute for MoO4(2-) in restoring anaerobic induction by formate. Similarly, class III mutants were phenotypically suppressed by inclusion of 500 microM Ni2+ in the medium; these mutants were shown to carry a defective fnr gene. The mutant of class IV had a defect in a formate dehydrogenase structural gene and that of class V was unable to grow under fermentative conditions while maintaining the capability to grow anaerobically in the presence of electron acceptors.