Mapping of pgi and gpd genes involved in C-1 assimilation in the obligate methylotroph Methylobacillus flagellatum.

Homologous matings with plasmids R68.45 and pULB113, and also with Hfr type donor were employed for mapping pgi and gpd genes involved in C-1 metabolism in the obligate methylotroph Methylobacillus flagellatum. A preliminary map of the late chromosomal region was constructed on the basis of these experimental results. The C-1 markers were linked to methionine and leucine auxotrophy and nalidixic acid resistance markers. The phenomenon of retrotransfer, or shuttle transfer of chromosomal markers by Inc P1 plasmids, revealed earlier, was demonstrated for M. flagellatum.

[Characteristics of Methylobacillus flagellatum KT mutants, deficient for phosphoglucoisomerase, an enzyme of the ribulose monophosphate cycle of obligate methylotrophic bacteria]. / Kharakteristika mutantov Methylobacillus flagellatum KT, defektnykh po fosfogliukoizomeraze, fermentu ribulozomonofosfatnogo tsikla obligatnykh metilotrofov.

The activities of the key enzymes of ribulose monophosphate cycle for formaldehyde oxidation and assimilation were tested in crude extracts from temperature sensitive mutants of obligatemethylotroph M. flagellatum KT. Two mutants deficient in phosphoglucoisomerase activity were identified during this screening. Phosphoglucoisomerase of T525 pgi-1 mutant was active both at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. Complete inactivation of the enzyme at 42 degrees C occurred in 2 h in vitro, while in vivo incubation at nonpermissive temperature for more than 10 h was required for the enzyme inactivation. Phosphoglucoisomerase activity of T566 pgi-2 was 5-fold lower as compared with the one from the parent strain incubated at 30 degrees C. The enzyme was inactivated in 2 min. in crude extract at nonpermissive temperature.

[Temperature-sensitive mutant of the obligate methylotroph Methylobacillus flagellatum KT deficient in glucose-6-phosphate dehydrogenase]. / Temperaturochuvstvitel'nyi mutant obligatnogo metilotrofa Methylobacillus flagellatum KT, defektnyi po gliukozo-6-fosfatdegidrogenaze.

The common approach is developed for isolation of mutants deficient in key enzymes of ribulose monophosphate pathway for formaldehyde oxidation and assimilation by obligate methylotrophic bacteria. The approach is based on total isolation of temperature sensitive mutants and their biochemical characterization. A number of ts- mutants of obligate methylotroph M. flagellatum KT is isolated following nitrosoguanidine induced mutagenesis. The modified screening method was developed and used for identification of mutants deficient in the key enzymes of ribulose monophosphate pathway. The mutant deficient in glucose-6-phosphate dehydrogenase (zwf) was identified. The NAD-dependent activity of glucose-6-phosphate dehydrogenase was not measurable under nonpermissive temperature while the level of NADP-dependent activity was only four-fold less comparing with wild type strain. It was concluded that growth limitation of zwf mutant of M. flagellatum KT (designated T623) at 42 degrees C results from the absence of NAD-dependent activity of glucose-6-phosphate dehydrogenase.