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Functional characterization of key enzymes involved in L-glutamate synthesis and degradation in the thermotolerant and methylotrophic bacterium Bacillus methanolicus.

ABSTRACT: Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter(-1) of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low Km value for ammonium (10 mM) and a high Km value for l-glutamate (250 mM), and the Vmax value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar Km values (1 to 1.4 mM) and Vmax values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.

SUBMITTER: Krog A

PROVIDER: S-EPMC3753979 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Functional characterization of key enzymes involved in L-glutamate synthesis and degradation in the thermotolerant and methylotrophic bacterium Bacillus methanolicus.

Krog Anne A Heggeset Tonje Marita Bjerkan TM Ellingsen Trond Erling TE Brautaset Trygve T

Applied and environmental microbiology 20130628 17

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter(-1) of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. met ...[more]

PMID: 23811508

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Project description:The thermotolerant methylotroph Bacillus methanolicus MGA3 was originally isolated from freshwater marsh soil. Due to its ability to use methanol as sole carbon and energy source, B. methanolicus is increasingly explored as a cell factory for the production of amino acids, fine chemicals, and proteins of biotechnological interest. During high cell density fermentation in industrial settings with the membrane-permeable methanol as the feed, the excretion of low molecular weight products synthesized from it will increase the osmotic pressure of the medium. This in turn will impair cell growth and productivity of the overall biotechnological production process. With this in mind, we have analyzed the core of the physiological adjustment process of B. methanolicus MGA3 to sustained high osmolarity surroundings. Through growth assays, we found that B. methanolicus MGA3 possesses only a restricted ability to cope with sustained osmotic stress. This finding is consistent with the ecophysiological conditions in the habitat from which it was originally isolated. None of the externally provided compatible solutes and proline-containing peptides affording osmostress protection for Bacillus subtilis were able to stimulate growth of B. methanolicus MGA3 at high salinity. B. methanolicus MGA3 synthesized the moderately effective compatible solute L-glutamate in a pattern such that the cellular pool increased concomitantly with increases in the external osmolarity. Counterintuitively, a large portion of the newly synthesized L-glutamate was excreted. The expression of the genes (gltAB and gltA2) for two L-glutamate synthases were upregulated in response to high salinity along with that of the gltC regulatory gene. Such a regulatory pattern of the system(s) for L-glutamate synthesis in Bacilli is new. Our findings might thus be generally relevant to understand the production of the osmostress protectant L-glutamate by those Bacilli that exclusively rely on this compatible solute for their physiological adjustment to high osmolarity surroundings.

Project description:Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5'-ends.Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5'-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5'-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts.The extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism.

Project description:BackgroundThe sulfate-reducing bacterium Desulfatiglans anilini can grow with phenol as sole source of carbon and energy under strictly anaerobic, sulfate-reducing conditions. In the nitrate-reducing bacterium Thauera aromatica, the enzymes involved in phenol degradation have been well elucidated, whereas the anaerobic phenol degradation pathway by D. anilini was not studied in detail yet.ResultsThe pathway of anaerobic phenol degradation by the sulfate-reducing bacterium Desulfatiglans anilini was studied by identification of genes coding for phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) in the genome of D. anilini, by analysis of the transcription and translation of pps-ppc genes, and by measurement of phenylphosphate synthase activity in cell-free extracts of phenol-grown cells. The majority of genes involved in phenol degradation were found to be organized in one gene cluster. The gene cluster contained genes pps? (phenylphosphate synthase alpha subunit), pps? (phenylphosphate synthase beta subunit), ppc? (phenylphosphate carboxylase beta subunit), as well as 4-hydroxybenzoyl-CoA ligase and 4-hydroxylbenzoyl-CoA reductase-encoding genes. The genes pps? (phenylphosphate synthase gamma subunit), ppc? (phenylphosphate carboxylase alpha subunit) and ppc? (phenylphosphate carboxylase delta subunit) were located elsewhere in the genome of D. anilini, and no obvious homologue of ppc? (phenylphosphate carboxylase gamma subunit) was found in the genome. Induction of genes pps and ppc during growth on phenol was confirmed by reverse transcription polymerase chain reaction. Total proteome analysis revealed that the abundance of enzymes encoded by the gene cluster under study was much higher in phenol-grown cells than that in benzoate-grown cells. In in-vitro enzyme assays with cell-free extracts of phenol-grown cells, phenylphosphate was formed from phenol in the presence of ATP, Mg2+, Mn2+, K+ as co-factors.ConclusionsThe genes coding for enzymes involved in the anaerobic phenol degradation pathway were identified in the sulfate-reducing bacterium D. anilini. The results indicate that the first steps of anaerobic phenol degradation in D. anilini are phosphorylation of phenol to phenylphosphate by phenylphosphate synthase and carboxylation of phenylphosphate by phenylphosphate carboxylase.

Dataset Information

Functional characterization of key enzymes involved in L-glutamate synthesis and degradation in the thermotolerant and methylotrophic bacterium Bacillus methanolicus.

Publications

Functional characterization of key enzymes involved in L-glutamate synthesis and degradation in the thermotolerant and methylotrophic bacterium Bacillus methanolicus.

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