Abstract
Methylated amines are ubiquitous in the environment and play a role in regulating the earth’s climate via a set of complex biological and chemical reactions. Microbial degradation of these compounds is a major sink. Recently we isolated a facultative methylotroph, Gemmobacter sp. LW-1, an isolate from the unique environment Movile Cave, Romania, which is capable of methylated amine utilisation as a carbon source. Here, using a comparative genomics approach, we investigate how widespread methylated amine utilisation trait is within the members of the bacterial genus Gemmobacter. Five genomes of different Gemmobacter species isolated from diverse environments, such as activated sludge, fresh water, sulphuric cave waters (Movile Cave) and the marine environment were available from public repositories and used for the analysis. Our results indicate that some members of the genus Gemmobacter, namely G. aquatilis, G. caeni and G. sp. LW-1 have the genetic potential of methylated amine utilisation while others (G. megaterium and G. nectariphilus) have not. Ancestral state reconstruction analysis also suggested that methylated amine utilisation trait might not be ancestral to members of the genus Gemmobacter and has been gained. Based on our analysis, we suggest that the trait of methylated amine utilisation within the members of the genus Gemmobacter might be independent of their habitat and more randomly distributed.
Importance
Methylated amines are an important carbon and/or nitrogen for microorganisms in the environment. Recent advances in DNA sequencing and availability of bacterial genome sequences have allowed us to perform in silico experiments to understand and compare genetic potential. Using a comparative genomic approach, we show differential methylated amine utilisation trait within a bacterial genus i.e. Gemmobacter and that the trait is independent of the habitat. Ancestral state reconstruction analysis suggests that members within the genus Gemmobacter that can use methylated amines might have gained the trait. Our study also emphasizes the need to use a holistic approach to understand the role of microorganisms in C and N cycling in the environment, rather than methods based on phylogenetic marker genes.
Introduction
Methylated amines (MAs) are ubiquitous in the environment with a range of natural and anthropogenic sources including the oceans, vegetation, sediments and organic-rich soils, animal husbandry, food industry, pesticides, combustion, sewage, and automobiles, to mention only a few (1-3). Methylated amines are also known to influence earth’s climate, via a series of complex biological and chemical interactions (4). Some of the most abundant methylated amines found in the atmosphere are trimethylamine (TMA), dimethylamine (DMA) and monomethylamine (MMA) (1). Microbial metabolism of methylated amines involves aerobic and anaerobic microorganisms, e.g. some methanogenic archaea such as Methanosarcina and Methanomicrobium can use MAs to produce methane (5-7) while Gram-positive and Gram-negative methylotrophic bacteria can use MAs as carbon and nitrogen source (8). Previously, MAs were typically associated with marine ecosystems as they are byproducts of degradation of osmolytic chemicals such as glycine betaine, carnitine, choline and trimethylamine N-oxide (8). However, recent studies have reported the detection and activity of aerobic methylotrophic bacteria that utilize MAs in a variety of natural and anthropogenic environments (1, 9-12) and could play a major role in global C and N budgets.
Aerobic methylotrophs are a polyphyletic group of microorganisms capable of utilizing one-carbon (C1) compounds such as methane, methanol or methylated amines as their sole source of carbon and energy (9, 14, 15). Methylotrophs can degrade TMA to DMA by using the enzymes TMA dehydrogenase, TMA monooxygenase or TMA methyltransferase, encoded by the genes tdm, tmm and mtt, respectively (16-18). The enzymes DMA dehydrogenase (dmd) or DMA monooxygenase (dmmDABC) modulate the conversion of DMA to MMA (15, 16). Two distinct pathways have been characterised for the oxidation of MMA (10). The direct MMA-oxidation pathway mediated by a single enzyme (MMA dehydrogenase (MMADH) in gram negative bacteria and MMA oxidase in gram positive bacteria) converts MMA to formaldehyde and releases ammonium (19, 20). The alternate pathway, referred to as the N-methylglutamate (NMG) pathway or indirect MMA-oxidation pathway, is mediated by three individual enzymes via the oxidation of MMA to gamma-glutamylmethylamide (GMA) and its further degradation to N-methylglutamate (NMG) and 5,10-methylenetetrahydrofolate (CH2 = H4F) (3, 10). A stepwise conversion of MMA in the NMG pathway is modulated by the enzymes GMA synthetase (gmaS), NMG synthase (mgsABC) and NMG dehydrogenase (mgdABCD) (3, 8). The capability to use MMA not only as a source for carbon but also for nitrogen is widespread in bacteria. Notably, the NMG pathway is not only restricted to methylotrophs but also present in non-methylotrophic bacteria that use MMA as a nitrogen but not as a carbon source (16, 21, 22).
In a recent study, we isolated an alphaproteobacterial facultative methylotrophic bacterium, Gemmobacter sp. LW-1 (recently renamed from Catellibacterium (23)) from the Movile Cave ecosystem (24) (Mangalia, Romania) that can use methylated amines as both carbon and nitrogen source (12) and subsequently obtained its genome sequence (25). Using a 13C-MMA DNA based stable-isotope probing (SIP) experiment we also showed that Gemmobacter sp. LW-1 was indeed an active MMA utiliser in microbial mats from this environment (12). This was the first report of methylated amine utilisation in a member of the bacterial genus Gemmobacter. However, growth on C1 compounds (methanol and formate) has been previously reported for the genus Gemmobacter, i.e. for G. caeni (26).
The genus Gemmobacter (family Rhodobacteraceae) currently comprises ten validated species: Gemmobacter megaterium (27), G. nectariphilum (23, 28), G. aquatilis (29), G. caeni (23, 26), G. aquaticus (23, 30), G. nanjingense (23, 31), G. intermedius (32), G. lanyuensis (33), G. tilapiae (34) and G. fontiphilus (23). These species were isolated from a wide range of environments including fresh water environments (freshwater pond (29, 34), freshwater spring (23, 33)), coastal planktonic seaweed (27), white stork nestling (32), waste water and activated sludge (26, 28, 31), suggesting that members of the genus Gemmobacter are widely distributed in anthropogenic and natural environments.
Here, using a comparative genomics approach we study how widespread methylated amine utilisation trait is within the members of the genus Gemmobacter. We used five isolate genomes for members within the genus Gemmobacter (G. sp. LW-1, G. caeni, G. aquatilis, G. nectariphilus and G. megaterium) available from public repositories (Accessed March 2017 and June 2018). These genomes were used for comparative genomics and ancestral state reconstruction analysis to understand patterns of gain/loss of methylated amine utilisation across the Gemmobacter phylogeny.
Materials and Methods
Genome data acquisition
Five Gemmobacter genomes (G. caeni, G. aquatilis, G. nectariphilus, G. megaterium, Gemmobacter sp. LW-1) available through the Integrated Microbial Genomes (IMG) database (https://img.jgi.doe.gov/) were used for comparative genome analysis (35). Accession numbers and genome characteristics are listed in Supplementary Table S1.
Phylogenetic analysis
Phylogenetic relatedness between the different members of the genus Gemmobacter was determined using phylogenetic trees constructed from 16S rRNA gene sequences (nucleotide) and metabolic gene sequences (gmaS and mauA; amino acids) involved in MMA utilisation. RNAmmer (36) was used to retrieve 16S rRNA gene sequences from the genome sequences. Multiple sequence alignment of 16S rRNA gene sequences was performed using the SINA alignment service via SILVA (37, 38) and subsequently imported into MEGA7 (39) to construct a neighbour-joining nucleotide-based phylogenetic tree (40). Bootstrap analysis was performed with 1000 replicates to provide confidence estimates for phylogenetic tree topologies (41).
To determine phylogenetic affiliations for the protein encoding genes gmaS and mauA, gene sequences retrieved from the genome sequences were aligned to homologous sequences retrieved from the NCBI Genbank database using Basic Local Alignment Search Tool (BLAST, blastx) (42) and curated gmaS sequences used for primer design in our previous study (12). Amino acid sequences were aligned in MEGA7 (39) using ClustalW (43) and the alignment was subsequently used to construct maximum likelihood phylogenetic trees based on the JTT matrix-based model (44). Bootstrap analysis was performed with 1000 replicates to provide confidence estimates for phylogenetic tree topologies (41).
Comparative genomic analyses
CGView Comparison Tool (CCT) was used to visually compare the genomes within the genus Gemmobacter (45). CCT utilizes BLAST to compare the genomes and the BLAST results are presented in a graphical map (45). Average Nucleotide Identity (ANI) (46) between different genomes was estimated using one-way ANI (best hit) and two-way ANI (reciprocal best hit) based on Goris et al. (47). In addition the whole-genome based average nucleotide identity (gANI) and the printra-species value were determined for G. sp. LW-1 and G. caeni (these two genomes revealed the closest ANI) based on Konstantinidis and Tiedje (48) via the Joint Genome Institute (JGI) platform (https://ani.jgi-psf.org/html/home.php; Version 0. 3, April 2014). In order to determine if two genomes belong to the same species, the computation of empirical probabilities (printra-species) can be calculated as follows, AF represents alignment fraction.
Pan-genome analysis including average amino acid identity (AAI) analysis, pan-genome tree construction and determination of core and dispensable genes and singletons (unique genes) was carried out using the Efficient Database framework for comparative Genome Analyses using BLAST score Ratios (EDGAR) platform (49).
In order to compare the genetic potential for methylated amine utilisation within the available Gemmobacter genomes, known protein sequences involved in methylated amine utilisation pathways (3, 16) were used as query sequences through the BLAST (blastp) program (42) available within the Rapid Annotation using Subsystem Technology (RAST) server (50). The list of protein queries used is given in Supplementary Table S2.
We examined patterns of gain/loss of methylated amine utilisation along the above 16S rRNA gene phylogeny, by performing ancestral state reconstruction analysis using the phytools package in R (51). We used stochastic character mapping (52) to map presence/absence of methylated amine utilisation, assigning a prior probability of one to species known to utilise methylated amines, zero to those known not to do so, and 0.5 to those where the trait value was unknown. We used MCMC (with the function make.simmap) to simulate 1000 stochastic maps, and then we used the function densityMap (53) to visualise the aggregate result from the stochastic mapping analysis.
Results and discussion
Phylogenetic relatedness based on the 16S rRNA and metabolic gene
The phylogenetic relatedness of the five members within the genus Gemmobacter (G. sp. LW-1, G. caeni, G. aquatilis, G. nectariphilus and G. megaterium) was resolved based on 16S rRNA gene sequences (Figure 1). Three members of the genus Gemmobacter (G. sp. LW-1, G. caeni, and G. aquatilis) clustered together with several other related Gemmobacter and Rhodobacter 16S rRNA gene sequences retrieved from fresh water, soil and sediment and activated sludge environments (Figure 1). G. nectariphilus and G. megaterium sequences clustered together with Paracoccus kawasakiensis and other related Gemmobacter sequences from marine, fresh water and activated sludge environments (Figure 1). Based on the 16S rRNA gene sequences retrieved from public database, we observed that the members of the genus Gemmobacter are widely distributed in anthropogenic (such as activated sludge and clinical environments) and natural environments i.e. fresh water, soil and sediment, and marine environments (Figure 1).
GMA synthetase, a key enzyme in the NMG pathway, is encoded by the gene gmaS. gmaS sequences retrieved from the isolate genomes along with other ratified gmaS sequences were used to construct an amino acid-based phylogenetic tree (Figure 2). Multiple sequence alignment and construction of a phylogenetic tree based on amino-acid sequences clearly separated gmaS from glnA gene sequences, a homolog of the gmaS gene encoding for the enzyme glutamine synthetase functionally unrelated (Figure 2). gmaS gene sequences retrieved from genomes of G. sp. LW-1, G. caeni and G. aquatilis clustered within Group I of alphaproteobacterial gmaS sequences containing sequences from marine and non-marine bacteria within the orders Rhodobacterales and Rhizobiales as described in Wischer et al. (12) and were closely related to Paracoccus yeei, P. sp. 1W-5 and Rhodobacter sp. 1W-5 (Figure 2).
Whilst gmaS gene sequences were detected in three of the five investigated Gemmobacter genomes, mauA gene sequences were identified only in the genomes of G. caeni and G. sp. LW-1 (Supplementary Figure S1). It has been suggested that the NMG pathway for MMA utilisation is more universally distributed and more abundant across proteobacterial methylotrophs than the direct MMA oxidation pathway (13) although the NMG pathway is energetically more expensive compared to the energetically favourable direct MMA oxidation pathway (54). In addition, genes encoding for the enzymes within the NMG pathway (gmaS) can not only be detected in methylotrophs but also in non-methylotrophic bacteria that use MMA as a nitrogen source, but not as a carbon source (12, 16).
A comparative genome analysis of members within the genus Gemmobacter
At the time of the analysis, five Gemmobacter genomes obtained from isolates from different environments were available (Figure 1 and Table 2). Gemmobacter genome sizes range from ~3.96 Mb to ~5.14 Mb with GC contents between 64.71% to 66.19%. Analysis of sequence annotations revealed that on average 91.13% of the genomes consist of coding sequences.
The genomes were compared using the CGView comparison tool (45) was used (Figure 3). Gemmobacter sp. LW-1, isolated from the Movile Cave ecosystem was used as the reference genome and the results of the BLAST comparison with other Gemmobacter genomes are represented as a BLAST ring for each genome (Figure 3). Similarities between segments of the reference genome sequence and the other genome sequences are shown by a coloured arc beneath the region of similarity indicating the percentage of similarity as a colour code. Our analysis (Figure 3) revealed low identity levels (mostly <88%) between Gemmobacter sp. LW-1 and G. aquatilis, G. nectariphilus and G. megaterium across the genomes. Moreover, the analysis suggested several sites of potential insertion/deletion events in the genome of Gemmobacter sp. LW-1. Possible insertion/deletion regions can be identified as those gaps in the map where no homology is detected. For example, the region between 2200-2300 kbp (Figure 3) where a gap can be found in the otherwise contiguous homologous regions between the reference genome G. sp. LW-1 and the first of the query genomes (G. caeni). This might likely be due to a lack of hits or hits with low identity that can be spurious matches. Since it covers a large region we could possibly rule out that it is not an artefact arising from a lack of sensitivity in the BLAST analysis. Instead it could be an insertion or deletion of a large DNA fragment such as a prophage or a genomic island. Even though the genomes of G. sp. LW-1 and G. caeni are closely related, the analysis demonstrates that their genomes are not completely identical. Despite the fact that the majority of their genomes indicate very high identity levels (mostly >96-98% as shown by the dominance of dark red colours of the circle representing the BLAST hit identity between G. sp. LW-1 and G. caeni), many segments appear to be exclusive to G. sp. LW-1.
In order to further resolve the similarity between these genomes we calculated the average nucleotide identity (ANI) (46) (Supplementary Table S3 and Supplementary Figure S2A-D) and the average amino acid identity, short AAI (Supplementary Figure S2E). It is generally accepted that an ANI value of >95-96% can be used for species delineation (55-57). Our analysis revealed that Gemmobacter sp. LW-1 and G. caeni share an ANI value of 98.62 (Supplementary Table S3) implying that both are in fact the same species. The genome-based average nucleotide identity (gANI) between G. sp. LW-1 and G. caeni was calculated as 98.70. The AF was calculated to be 0.91, which would result in a computed probability of 0.98 suggesting that both genomes belong to the same species. However, it should be noted that these are draft genomes and a more in depth characterization of their physiology and phenotype is required to delineate these organisms at the level of strain.
Pan-genome analysis, carried out using the EDGAR platform (49), identified metabolic genes present in all Gemmobacter species (core genes), two or more Gemmobacter species (accessory or dispensable genes), and unique Gemmobacter species (singleton genes). A pan-genome tree was constructed (Figure 4A) based on the pan-genome dataset and neighbor-joining method (40). As with the 16S-based phylogenetic tree (Figure 1), the five Gemmobacter species formed two main clusters in the pan-genome tree analysis (Figure 4A). The pan-genome tree also confirmed the evolutionary closeness between Gemmobacter caeni and Gemmobacter sp. LW-1 (Figure 4A). According to pan-genome analysis of the five Gemmobacter genomes, a total of 9,286 genes were identified, consisting of 1,806 core genes, 3085 dispensable genes and 305, 1,072, 896, 1,165 and 957 singletons for G. sp. LW-1, G. caeni, G. aquatilis, G. nectariphilus and G. megaterium, respectively (Figure 4B). On average 53.3% of singletons were identified as having hypothetical functions (Figure 4C). In addition, the number of singletons did not correlate with the size of the genome, which is in contrast with the correlation between the number of genes and the size of the genome.
Methylated amine utilisation, N assimilation and C1 oxidation
Investigation of the methylated amine utilisation pathways in five Gemmobacter species revealed the presence of the genes encoding the enzymes TMA dehydrogenase (tmd), TMA monooxygenase (tmm), TMA methyltransferase (mttB), TMAO demethylase (tdm) and DMA monooxygenase in genomes of G. sp. LW-1, G. caeni and G. aquatilis while none of these genes were detected in G. nectariphilus or G. megaterium (Figure 5). These findings are supported by results from a previous study which showed growth of G. sp. LW-1 on TMA as a carbon and nitrogen source (12). G. sp. LW-1, G. caeni and G. aquatilis could potentially use the TMA oxidation or demethylation pathway to convert TMA to DMA. Based on the genome sequences, it can be suggested that these three species use the enzyme DMA monooxygenase (dmmDABC) to oxidize DMA to MMA but not the DMA dehydrogenase since the corresponding protein encoding gene (dmd) was not found (Figure 5).
In order to compare the distribution of the direct MMA-oxidation pathway and the NMG pathway, the genomes of five Gemmobacter species were tested for the presence of mauA and gmaS gene sequences (Figure 5). The direct MMA-oxidation pathway (mauA-dependent) is so far only known to be present in methylotrophic bacteria that can use MMA as a carbon source. Whereas the NMG pathway (gmaS-dependent) has been shown to be present in non-methylotrophic bacteria that can use MMA as a nitrogen source (12, 13, 21, 54). Analysis of the genome sequences revealed that both G. sp. LW-1 and G. caeni possess genes for both MMA oxidation pathways (Figure 5). We have previously shown that Gemmobacter sp. LW-1 can use MMA and TMA as both a carbon and nitrogen source (12). Genome sequence of G. aquatilis indicated the presence of genes involved only in the NMG pathway. In the facultative methylotroph Methylobacterium extorquens AM1 it has been shown that the NMG pathway is advantageous compared to the direct MMA-oxidation pathway (54). Particularly under limiting MMA concentrations (<1 mM) the energetically expensive NMG pathway plays a key role during growth with MMA as a sole nitrogen source. NMG pathway enables facultative methylotrophic bacteria to switch between using MMA as a nitrogen source or as a carbon and energy source whereas the direct MMA oxidation pathway allows for rapid growth on MMA only as the primary energy and carbon source (54). This could suggest that G. aquatilis might use the NMG pathway for utilizing MMA as both nitrogen and carbon source. However, growth assays are required to confirm whether G. aquatilis can use MMA as a carbon source. We did not detect genes for either MMA oxidation pathways (mauA- and gmaS-dependent) in the genome sequences of G. nectariphilus and G. megaterium suggesting the lack of genetic potential of these organisms to use MMA as either C or N source.
Gemmobacter sp. LW-1 was isolated from the Movile Cave ecosystem (12). Microbial mats and lake water within the cave have been shown to harbor a wide diversity of methylated amine-utilizing bacteria (12, 58). Whilst the mechanism of MAs production within the system has to be elucidated, it can be speculated that degradation of floating microbial mats (i.e. organic matter) could result in MAs (12). Similarly, G. caeni isolated from activated sludge (26) could possibly use the MAs generated from organic matter degradation. Interestingly, whilst G. megaterium was isolated from a marine environment (seaweed (27)) possibly encountering MAs from the degradation of osmolytes such as glycine betaine (N,N,N-trimethylglycine) we did not detect metabolic genes involved in methylated amine utilisation. Our analysis suggest that the trait for methylated amine utilisation could be independent of the habitat.
The C1 units derived from methylated amines need to be further oxidized when the nitrogen is sequestered without assimilation of the carbon from the methylated amines. Genome analysis confirmed that all five Gemmobacter species possess the genetic capability for C1 oxidation and also indicate that tetrahydrofolate (H4F) is the C1 carrier (Figure 5). The bifunctional enzyme 5,10-methylene-tetrahydrofolate dehydrogenase/ cyclohydrolase, encoded by the gene folD, was detected in all the Gemmobacter genomes (Figure 5/ Table 1). Genes encoding key enzymes in the C1 oxidation pathway via tetrahydromethanopterin (H4MPT) were not detected. (10). The formate-tetrahydrofolate ligase, encoded by the gene fhs (Figure 5), provides C1 units for biosynthetic pathways (16). However, the oxidation of formyl-H4F (CHO-H4F) can also be facilitated by purU, the gene encoding for the formyl-H4F deformylase. The formate dehydrogenase (fdh) mediates the last step of the C1 oxidation pathway, the oxidation of formate to CO2. The genes for the C1 oxidation pathway via H4F were detected in all five Gemmobacter genomes.
The xoxF gene, encoding the lanthanide-dependent methanol dehydrogenase (59-61) and the mxaF gene, encoding the alpha subunit of the calcium-dependent methanol dehydrogenase (62), are responsible for the oxidation of methanol to formaldehyde (63, 64) and were both detected in genomes of G. sp. LW-1, G. caeni and G. aquatilis but not in G. nectariphilus and G. megaterium (Figure 5/ Table 1). The lanthanide-dependent methanol dehydrogenase is thought to be more widespread in bacterial genomes (65). The fae gene, encoding the formaldehyde-activating enzyme that catalyses the reduction of formaldehyde with H4MPT was not detected in any of the five Gemmobacter genomes confirming that these members of the genus Gemmobacter lack the H4MPT pathway for formaldehyde oxidation (Figure 5/ Table 1). However, in all the Gemmobacter genomes we detected genes encoding the glutathione (GSH)-dependent formaldehyde oxidation pathway, including the genes adhI / flhA and fghA coding for the GSH-dependent formaldehyde dehydrogenase and the S-formyl-GSH hydrolase, respectively (Figure 5 and Table 1). Reduction of formaldehyde with GSH can be mediated by the GSH-dependent formaldehyde-activating enzyme, encoded by the gene gfa (66). A Gfa-like protein was detected in the genomes of G. sp. LW-1, G. caeni, G. necariphilus and G. megaterium but not in G. aquatilis. This could perhaps indicate a nonessential role of Gfa in formaldehyde oxidation as suggested by Chen (16) and Wilson et al. (67). Investigation of the nitrogen assimilation pathway revealed the presence of the genes encoding glutamine synthetase (GS; gluL) and glutamine synthase (GOGAT; glxB) in all five Gemmobacter genomes. In bacteria this pathway is essential for glutamate synthesis at low ammonium concentrations (16).
Using comparative genome analysis we provide genome-based evidence that the two Gemmobacter isolates G. sp. LW-1 and G. caeni are capable of generating energy from complete oxidation of methylated amines via the H4F-dependent pathway using either the energetically expensive NMG pathway or the energetically favourable direct MMA oxidation pathway. Gemmobacter aquatilis is genetically capable of methylated amine degradation to yield formaldehyde and only encodes the genes for the energetically expensive NMG pathway, which indicates that G. aquatilis could use this pathway to use MMA as a nitrogen source. Both G. nectariphilus and G. megaterium genomes indicate the lack of potential to use methylated amines (Figure 5/Table 1).
Evolution of methylated amine utilisation in Gemmobacter
Three of the five investigated Gemmobacter species are genetically capable of methylated amine utilisation, of which two (G. sp. LW-1 and G. caeni) possess the genes for both MMA utilisation pathways, whereas in the genome of G. aquatilis we detected only genes involved in the indirect MMA oxidation pathway (NMG pathway). The two other Gemmobacter species (G. megaterium and G. nectariphilus) do not possess the genes for methylated amine utilisation. Therefore, the question arises if the genes for methylated amine utilisation have been acquired or lost during evolution.
Stochastic character mapping along the 16S rRNA gene phylogeny suggested that the ability to use methylated amines has either been gained or lost multiple times (Supplementary Figure S3). Most likely is that methylated amine utilisation is not ancestral in Gemmobacter, and has evolved three times, once in the clade containing G. sp. LW-1, G. caeni and G. aquatilis, once in Haematobacter and once in Paracoccus (Supplementary Figure S3). An alternative, but less plausible, scenario is that methylated amine utilisation is an ancestral trait in Gemmobacter, and has been lost and regained multiple times across the phylogeny.
Conclusions
Three of the five investigated Gemmobacter genomes (G. sp. LW-1, G. caeni and G. aquatilis) indicated metabolic potential to utilize methylated amines, of which only two (G. sp. LW-1 and G. caeni) possess the genes for both the MMA oxidation pathways, the energetically expensive NMG pathway and the energetically favourable direct MMA oxidation pathway. G. sp. LW-1 and G. caeni are facultative methylotrophs which could potentially use these pathways to utilize MMA as both a carbon and nitrogen source, while potentially G. aquatilis could only use the NMG pathway as a nitrogen source. Furthermore, the genomes of G. sp. LW-1 and G. caeni showed a high similarity to each other (>98%) suggesting that both belong to the same species. G. megaterium and G. nectariphilus genomes indicated no metabolic potential to utilize MAs. Phylogenetic analysis (16S tree, Figure 1), pan-genome and ANI analysis revealed that G. sp. LW-1 and G. caeni are closely related, although they were isolated from different environments. Whilst G. caeni and G. nectariphilus were isolated from a similar environment (activated sludge) it revealed a high amount of evolutionary change from the common ancestor. Overall, these results suggest that the trait for methylated amine utilisation could be independent from the habitat and localised factors could influence the ability of these organisms to use methylated amines. Access to Gemmobacter isolates with or without the genetic potential for methylated amine utilisation trait will allow us to perform physiological experiments in future to test how this trait can affect fitness of closely related organisms. Ancestral state reconstruction analysis confirms that across Gemmobacter and related genera, methylated amine utilisation has either evolved or been lost multiple times over the evolutionary history of this group. The adaptive or non-adaptive processes behind this pattern remain to be investigated.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
We thank Dr Felipe Hernandes Coutinho (Federal University of Rio de Janeiro) for a fruitful discussion on genomic regions of insertion and deletion in Gemmobacter sp. LW-1. The EDGAR platform is financially supported by the BMBF grant FKZ 031A533 within the de.NBI network.