Positive transcriptional feedback controls hydrogenase expression in Alcaligenes eutrophus H16

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• HoxA is the key regulator of the Alcaligenes eutrophus H16 hox regulon.
• The hox regulon includes genes for hydrogenases, nickel permease, and accessory proteins for hydrogenase biosynthesis.

Purpose of the Study:

• To analyze the expression of the regulatory gene hoxA.
• To understand the regulatory mechanisms governing the hox regulon in Alcaligenes eutrophus H16.

Main Methods:

• Promoter probe vector screening to identify promoter activities upstream of hoxA.
• Translational fusions to assess hoxA expression levels.
• Immunoblotting to detect HoxA protein fluctuations.
• Quantitative transcript assays for hyp mRNA levels.
• Interposon mutagenesis and site-directed mutagenesis to investigate promoter functions.

Main Results:

• Four promoter activities were identified upstream of hoxA, independent of HoxA and sigma(54).
• hoxA is constitutively expressed at low levels, but HoxA protein levels fluctuate with hox regulon induction.
• Elevated hyp mRNA levels were observed under hydrogenase-derepressing conditions.
• A remote promoter is essential for hydrogenase expression and autotrophic growth.
• P(MBH) contributes to hoxA expression under hydrogenase-derepressing conditions.

Conclusions:

• hoxA expression is regulated by a positive feedback loop that amplifies the HoxA regulator.
• This feedback mechanism is crucial for efficient hydrogenase expression and autotrophic growth.
• Results suggest the existence of a large (ca. 17,000-nucleotide) transcript involved in the hox regulon.