Expressed Genome of Methylobacillus flagellatus as Defined through Comprehensive Proteomics and New Insights into Methylotrophy

Abstract

In recent years, techniques have been developed and perfected for high-throughput identification of proteins and their accurate partial sequencing by shotgun nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS), making it feasible to assess global protein expression profiles in organisms with sequenced genomes. We implemented comprehensive proteomics to assess the expressed portion of the genome of Methylobacillus flagellatus during methylotrophic growth. We detected a total of 1,671 proteins (64% of the inferred proteome), including all the predicted essential proteins. Nonrandom patterns observed with the nondetectable proteins appeared to correspond to silent genomic islands, as inferred through functional profiling and genome localization. The protein contents in methylamine-and methanol-grown cells showed a significant overlap, confirming the commonality of methylotrophic metabolism downstream of the primary oxidation reactions. The new insights into methylotrophy include detection of proteins for the N-methylglutamate methylamine oxidation pathway that appears to be auxiliary and detection of two alternative enzymes for both the 6-phosphogluconate dehydrogenase reaction (GndA and GndB) and the formate dehydrogenase reaction (FDH1 and FDH4). Mutant analysis revealed that GndA and FDH4 are crucial for the organism's fitness, while GndB and FDH1 are auxiliary.

An Integrated Proteomics/Transcriptomics Approach Points to Oxygen as the Main Electron Sink for Methanol Metabolism in Methylotenera mobilis

“…Such highly expressed genes may correspond to proteins that are difficult to detect directly using mass spectrometry due to poor extraction efficiencies and/or small numbers of tryptic fragments. Indeed, many of these genes are predicted to encode small or transmembrane proteins that are known to have lower levels of detectability with the shotgun proteomics approach (13). Alternatively, some of the genes for proteins annotated as hypothetical proteins (with no homology to known proteins) may encode small RNA molecules.…”

“…A high level of expression of XoxF proteins in M. mobilis JLW8 provides additional evidence for a role in methanol oxidation in this organism. Other highly abundant oxidoreductases included a NAD-dependent aldehyde dehydrogenase of unknown specificity (Mmol_0791), similarly abundant in a related species, Methylobacillus flagellatus KT, previously investigated using shotgun proteomics (13), and a NADH:flavin oxidoreductase (Mmol_0733) that also has unknown substrate specificity. The former was more abundant on methanol, while the latter was present at similar levels on both substrates.…”

“…While considering the most abundant (i.e., most frequently detected) proteins, we separated these into two categories: proteins typically detected at high abundances in bacterial cultures previously investigated using the proteomics approach (3,5,13) (5) and proteins that may be specific to the metabolism of C 1 compounds by M. mobilis JLW8. The latter were investigated in detail and are listed in Table 1 with spectral counts normalized by protein size.…”

“…Three unique peptides were required for positive identification. Reverse database peptide identification (10,26) yielded false discovery rates of Ͻ1% at the protein level (13). Quantitation was done using spectral counts (12,22) to calculate relative-abundance ratios and q values (27,29) derived from t test scores with a q-value cutoff of 0.01, all calculated similarly to the method of Bosch et al (3).…”

EliA is required for inducing the stearyl alcohol-mediated expression of secretory proteins and production of polyester in Ralstonia sp. NT80

“…This observation suggested that stearyl alcohol induces expression of many extracellular proteins and prompted us to investigate the multiple aspects of NT80 cells' response to stearyl alcohol. Several previous studies reported the effects of methanol and ethanol on bacterial cells (Hendrickson et al, 2010;Lu et al, 2012) and several other studies examined the mechanism of assimilation of n-alkanes by bacteria (Bouchez Naïtali et al, 1999;Tani et al, 2001;Throne-Holst et al, 2006). The effects of fatty alcohols on bacterial cells, however, have hardly been explored.…”