Characterization and Complementation of Mutants of Methylophilus Methylotrophus that have Thermolabile Forms of Proteins Involved in C1 Metabolism

Abstract

Two temperature-sensitive mutants of MethylophiZus methylotrophus have been isolated and characterized. The first, NH-4, had a temperature-sensitive defect in methylamine oxidation and was unable to utilize methylamine as sole carbon and nitrogen source at 42 OC. The activities of the methylamine oxidation system and methylamine dehydrogenase in cells grown on methylamine at 30 "C were much more thermolabile than those of the wild-type. Furthermore, the affinity of the mutant enzyme for methylamine was lower than that of the wild-type enzyme. These results suggest that NH-4 produces a mutant enzyme with an altered conformation which is more susceptible to thermal denaturation than the wild-type enzyme. Surprisingly, this mutant could grow at 42 "C on media containing methylamine if an alternative carbon or nitrogen source was available. The methylamine oxidation system of whole cells grown under these conditions was not inactivated at 42 OC. The second mutant, NH-7, was unable to grow at the restrictive temperature on media containing either methanol or methylamine as sole carbon source. It contained thermolabile forms of methanol and methylamine dehydrogenases and cytochrome cL. This phenotype could be due to a mutation in a gene essential for the production of mature forms of these periplasmic proteins, which are involved in C, metabolism. Cosmid pAD833, which has previously been shown to carry genes involved in methanol oxidation (mox genes), complemented both NH-4 and NH-7. Subcloning indicated that the gene which complemented NH-7 was within a 3 kbp region which contained at least two mox genes. This region was near an 8 kbp region containing the gene which complemented mutant NH-4.

Investigation of Thermolabile Variants of the Methanol and Methylamine Dehydrogenases of Methylophilus Methylotrophus and the Effect of an 11{middle dot}5 kbp Region of the Chromosome on the Stability of these Enzymes

“…In contrast, the methylamine oxidation activity in mutant NH-4 grown on methanol plus methylamine or on methylamine plus NH,+ was 1.5-to 2-fold greater than in wild-type cells when assayed at 30°C and it was not thermolabile, confirming results previously obtained using cells grown at 42 "C (Hutchinson & Goodwin, 1993). The therrnolability of the methylamine oxidation activity of mutant NH-7, unlike that of NH-4, did not depend on the combination of carbon and nitrogen sources in the growth medium.…”

“…The methylamine oxidation activity of whole cells of mutant NH-4 grown on methylamine (as sole carbon and nitrogen source) and assayed at 30 "C was slightly higher than that observed in wild-type cells (Table 1); however, as described previously (Hutchinson & Goodwin, 1993), the ability of the mutant cells to oxidize methylamine was reduced almost fourfold when the assay was done at 42 "C, indicating that this activity was thermolabile. In contrast, the methylamine oxidation activity in mutant NH-4 grown on methanol plus methylamine or on methylamine plus NH,+ was 1.5-to 2-fold greater than in wild-type cells when assayed at 30°C and it was not thermolabile, confirming results previously obtained using cells grown at 42 "C (Hutchinson & Goodwin, 1993).…”

“…As shown previously, the enzyme in extracts of methylamine-grown cells was about fourfold less active at 42 "C than at 30 "C (Hutchinson & Goodwin, 1993). When cells were grown at 30 "C on methylamine plus NH,+ the methylamine dehydrogenase activity of NH-4 assayed at 30°C was very low compared with the other strains, although it decreased by less than 50% at 42 "C. In contrast, in extracts of NH-4 grown on methanol plus methylamine the specific activity was three times greater at 42 "C than at 30 "C. However, our previous results indicated that the specific activity of methylamine dehydrogenase in extracts of mutant NH-4 grown on methanol plus methylamine at 42 "C was low and that the activity was similar at 30°C and 42°C (Hutchinson & Goodwin, 1993). Thus the temperature of growth, as well as the growth substrates, influenced the specific activity of the …”

“…The bacterial strains and cosmids used are shown in Table 1 of the preceding paper (Hutchinson & Goodwin, 1993). Bacteria were grown in S2 salts medium as described by Southgate & Goodwin (1989) and .…”

“…We have recently isolated two temperature-sensitive mutants of Methylophilus methylotrophus with defects in the oxidation of C, compounds (Hutchinson & Goodwin, 1993). One (mutant NH-7) was unable to grow on either methanol or methylamine at the restrictive temperature (42 "C); it grew slowly on methanol at 30 "C, and the * Author for correspondence.…”