Significance Pyrrolysine, the 22nd amino acid, is found in few proteins. One, the trimethylamine methyltransferase MttB, forms a small portion of a large family of proteins. Most in this family lack pyrrolysine and have no known activity. We show that one such protein, MtgB, is a glycine betaine methyltransferase, providing functional context that may explain the relationship between family members with and without pyrrolysine. Close relatives of MtgB are encoded in many of the abundant bacteria in the oceans, as well as different microbes undertaking symbioses ranging from plants to humans. This finding implies that MtgB might partake in a widespread and underappreciated pathway of GB metabolism contributing significantly to global carbon and nitrogen cycling as well as human health.

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that causes a wide range of infections including pneumonia, septicemia, necrotizing fasciitis and severe wound and urinary tract infections. Analysis of A. baumannii representative strains grown in Chelex 100-treated medium for hemolytic activity demonstrated that this pathogen is increasingly hemolytic to sheep, human and horse erythrocytes, which interestingly contain increasing amounts of phosphatidylcholine in their membranes. Bioinformatic, genetic and functional analyses of 19 A. baumannii isolates showed that the genomes of each strain contained two phosphatidylcholine-specific phospholipase C (PC-PLC) genes, which were named plc1 and plc2. Accordingly, all of these strains were significantly hemolytic to horse erythrocytes and their culture supernatants tested positive for PC-PLC activity. Further analyses showed that the transcriptional expression of plc1 and plc2 and the production of phospholipase and thus hemolytic activity increased when bacteria were cultured under iron-chelation as compared to iron-rich conditions. Testing of the A. baumannii ATCC 19606T plc1::aph-FRT and plc2::aph isogenic insertion derivatives showed that these mutants had a significantly reduced PC-PLC activity as compared to the parental strain, while testing of plc1::ermAM/plc2::aph demonstrated that this double PC-PLC isogenic mutant expressed significantly reduced cytolytic and hemolytic activity. Interestingly, only plc1 was shown to contribute significantly to A. baumannii virulence using the Galleria mellonella infection model. Taken together, our data demonstrate that both PLC1 and PLC2, which have diverged from a common ancestor, play a concerted role in hemolytic and cytolytic activities; although PLC1 seems to play a more critical role in the virulence of A. baumannii when tested in an invertebrate model. These activities would provide access to intracellular iron stores this pathogen could use during growth in the infected host.

Two novel strains of methanogens were isolated from an estuarine sediment with the capability to utilize quaternary amines. Based on the 16S rRNA analysis, strain B1d shared 99 % sequence identity with Methanolobus vulcani PL-12/M(T) and strain Q3c shared 99 % identity with Methanococcoides sp. PM1 and PM2, but our current isolates display clearly different capabilities of growth on quaternary amines and were isolated based on these capabilities. Strain Q3c was capable of growth on tetramethylammonium and choline, while strain B1d was capable of growth on glycine betaine. Ml. vulcani PL-12/M(T) was incapable of growth on glycine betaine, indicating an obvious distinction between strains B1d and PL-12/M(T). Strain Q3c now represents the only known tetramethylammonium-utilizing methanogen in isolation. Strain B1d is the first quaternary amine-utilizing methanogen from the genus Methanolobus. This study suggests that quaternary amines may serve as ready precursors of biological methane production in marine environments.

Recent studies indicate that environmentally abundant quaternary amines (QAs) are a primary source for methanogenesis, yet the catabolic enzymes are unknown. We hypothesized that the methanogenic archaeon Methanolobus vulcani B1d metabolizes glycine betaine (GB) through a corrinoid-dependent GB:coenzyme M (CoM) methyl transfer pathway. The draft genome sequence of M. vulcani B1d revealed a gene encoding a predicted non-pyrrolysine MttB homolog (MV8460) with high sequence similarity to the GB methyltransferase encoded by Desulfitobacterium hafniense Y51. MV8460 catalyzes GB-dependent methylation of free cob(I)alamin indicating it is an authentic MtgB enzyme. Proteomic analysis revealed that MV8460 and a corrinoid binding protein (MV8465) were highly abundant when M. vulcani B1d was grown on GB relative to growth on trimethylamine. The abundance of a corrinoid reductive activation enzyme (MV10335) and a methylcorrinoid:CoM methyltransferase (MV10360) were significantly higher in GB-grown B1d lysates compared to other homologs. The GB:CoM pathway was fully reconstituted in vitro using recombinant MV8460, MV8465, MV10335, and MV10360. Demonstration of the complete GB:CoM pathway expands the knowledge of direct QA-dependent methylotrophy and establishes a model to identify additional ecologically relevant anaerobic quaternary amine pathways.

Microorganisms in the environment do not exist as the often-studied pure cultures but as members of complex microbial communities. Characterizing the interactions within microbial communities is essential to understand their function in both natural and engineered environments. In this study, we investigated how the presence of a nitrite-oxidizing bacterium (NOB) and heterotrophic bacteria affect the growth and proteome of the chemolithoautotrophic ammonia-oxidizing bacterium (AOB) Nitrosomonas sp. strain Is79. We investigated Nitrosomonas sp. Is79 in co-culture with Nitrobacter winogradskyi, in co-cultures with selected heterotrophic bacteria, and as a member of the nitrifying enrichment culture G5-7. In batch culture, N. winogradskyi and heterotrophic bacteria had positive effects on the growth of Nitrosomonas sp. Is79. An isobaric tag for relative and absolute quantification (iTRAQ) liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach was used to investigate the effect of N. winogradskyi and the co-cultured heterotrophic bacteria from G5-7 on the proteome of Nitrosomonas sp. Is79. In co-culture with N. winogradskyi, several Nitrosomonas sp. Is79 oxidative stress response proteins changed in abundance, with periplasmic proteins increasing and cytoplasmic proteins decreasing in abundance. In the presence of heterotrophic bacteria, the abundance of proteins directly related to the ammonia oxidation pathway increased, while the abundance of proteins related to amino acid synthesis and metabolism decreased. In summary, the proteome of Nitrosomonas sp. Is79 was differentially influenced by the presence of either N. winogradskyi or heterotrophic bacteria. Together, N. winogradskyi and heterotrophic bacteria reduced the oxidative stress for Nitrosomonas sp. Is79, which resulted in more efficient metabolism. IMPORTANCEAerobic ammonia-oxidizing microorganisms play an important role in the global nitrogen cycle, converting ammonia to nitrite. In their natural environment, they coexist and interact with nitrite oxidizers, which convert nitrite to nitrate, and with heterotrophic microorganisms. The presence of nitrite oxidizers and heterotrophic bacteria has a positive influence on the growth of the ammonia oxidizers. Here, we present a study investigating the effect of nitrite oxidizers and heterotrophic bacteria on the proteome of a selected ammonia oxidizer in a defined culture to elucidate how these two groups improve the performance of the ammonia oxidizer. The results show that the presence of a nitrite oxidizer and heterotrophic bacteria reduced the stress for the ammonia oxidizer and resulted in more efficient energy generation. This study contributes to our understanding of microbemicrobe interactions, in particular between ammonia oxidizers and their neighboring microbial community.