Analysis of the PHA granule‐associated proteins GA20 .and GA11 in Methylobacterium extorquens and Methylobacterium rhodesianum

Abstract

Electrophoretic analysis of the proteins bound to poly(3-hydroxybutyric acid), PHB-, granules in Methylobacterium extorquens, M. rhodesianum as well as the PHB-leaky mutants Mu 1 and Mu 11, which were isolated from the latter, resulted in two dominant low-molecular weight proteins, which were referred to as GA11 and GA20. After purification of these proteins antibodies against the GA11 and GA20 protein of M. extorquens were obtained. Both proteins bound to the surface of PHB granules as revealed by immunoelectron microscopy of whole cells of M. extorquens and M. rhodesianum. With cells of the PHB-leaky mutants Mu 1 and Mu 11 no specific labeling was observed. The N-terminal amino acid sequences of the GA11 and the GA20 protein were determined. We found significant homologies between the sequences of the investigated strains. The use of oligonucleotide probes based on the N-terminal sequences of the GA20 protein from M. rhodesianum to identify the corresponding structural genes in various genomic libraries failed.

Poly-β-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11 , gap20 , and phaR

“…gap11 and gap20 are not linked on the chromosome to each other or to other genes known to be involved in PHB biosynthesis in M. extorquens AM1. The molecular masses deduced for the polypeptides translated from gap11 and gap20 (12.8 and 19.1 kDa, respectively) were in agreement with the molecular masses experimentally determined for GA20 and GA11 from M. extorquens MB371, and the N-terminal amino acid sequences translated from gap11 and gap20 were identical to the experimentally determined sequences of GA11 and GA20 (13,14). Gap20 and Gap11 showed high identity to each other (42%), and they revealed similarity with GAPs from Zoogloea ramigera (17) and unknown proteins from PHB-synthesizing ␣-proteobacteria Rhodopseudomonas palustris, Mesorhizobium loti MAFF303099, and S. meliloti (http://jgi.doe.gov /JGI_microbial/html/index.html).…”

“…To identify genes potentially encoding GAPs in M. extorquens AM1, we used the N-terminal amino acid sequences for two GAPs, of 11 and 20 kDa (GA11 and GA20, respectively), previously identified for a similar strain, M. extorquens MB371 (14), as queries to search the genomic database of M. extorquens AM1 (http: //vixen.microbiol.washington.edu/). We identified the two respective genes and designated them gap11 and gap20.…”

“…The C-2 atom, however, is converted to CO 2 in the TCA cycle. Therefore, the production of 14 14 C]acetate utilized that was detected in CO 2 was less than 0.5%, confirming that the TCA cycle functions at very low activity in the wild-type strain during methylotrophic metabolism (42). In the PhaR mutant, the proportion of 14 CO 2 production from the total 2-14 C-labeled acetate utilized was about 11%, indicating that in this mutant the TCA cycle activity is greatly increased compared to that in the wild type.…”

“…Methylobacterium extorquens AM1 accumulates PHB and forms PHB granules like other PHB-producing bacteria (13,14). PHB content in M. extorquens AM1 cells varies depending on growth substrate (23).…”

Connection between Poly-β-Hydroxybutyrate Biosynthesis and Growth on C 1 and C 2 Compounds in the Methylotroph Methylobacterium extorquens AM1

“…Methylotrophic bacteria employing the serine cycle for formaldehyde assimilation are able to accumulate up to 80% of PHB by dry weight, while methylotrophic bacteria with the Calvin-Benson-Bassham cycle of C 1 assimilation can synthesize up to 20% of PHB by dry weight (14). In obligate methanol utilizers, which use the ribulose monophosphate pathway for assimilation of reduced C 1 compounds, PHB has not been detected (14).…”