Isolation and characterization of Methanobacterium thermoautotrophicum DeltaH mutants unable to grow under hydrogen-deprived conditions

Abstract

By using random mutagenesis and enrichment by chemostat culturing, we have developed mutants of Methanobacterium thermoautotrophicum that were unable to grow under hydrogen-deprived conditions. Physiological characterization showed that these mutants had poorer growth rates and growth yields than the wild-type strain. The mRNA levels of several key enzymes were lower than those in the wild-type strain. A fed-batch study showed that the expression levels were related to the hydrogen supply. In one mutant strain, expression of both methyl coenzyme M reductase isoenzyme I and coenzyme F420-dependent 5,10-methylenetetrahydromethanopterin dehydrogenase was impaired. The strain was also unable to form factor F390, lending support to the hypothesis that the factor functions in regulation of methanogenesis in response to changes in the availability of hydrogen.

FIG. 1

Dot blot analysis of MCR I mRNA levels in wild-type M. thermoautotrophicum, the chemostat enrichment culture, and mutant strains. Organisms were grown in 100-ml serum bottles under MII medium conditions. The analysis was performed as described in Materials and Methods.

FIG. 2

Fed-batch culture of strain JB2. The cell density is plotted against time. Changes in impeller speed are indicated by arrows. The inset shows mRNA levels (arbitrary units) at the different time points (A, B, and C) estimated by dot blot analyses; 1 AU is defined as the amount of F₄₂₀-MDH RNA at time point A.

FIG. 3

HPLC analyses of coenzyme extracts. (A) Wild-type M. thermoautotrophicum; (B) strain JA1. Cofactor isolation and HPLC analyses were performed as described in the text. The chemostat enrichment culture and the other mutant strains showed patterns similar to that in panel A.