Next Article in Journal
Comparison of Microbiological and Probiotic Characteristics of Lactobacilli Isolates from Dairy Food Products and Animal Rumen Contents
Next Article in Special Issue
Methane Oxidation and Molecular Characterization of Methanotrophs from a Former Mercury Mine Impoundment
Previous Article in Journal / Special Issue
Parallel and Divergent Evolutionary Solutions for the Optimization of an Engineered Central Metabolism in Methylobacterium extorquens AM1

Microorganisms 2015, 3(2), 175-197; https://doi.org/10.3390/microorganisms3020175

Article
C1-Pathways in Methyloversatilis universalis FAM5: Genome Wide Gene Expression and Mutagenesis Studies
1
Department of Microbiology, University of Washington, Seattle, WA 98195-1700, USA
2
Department of Chemical Engineering, University of Washington, Seattle, WA 98195-7735, USA
3
Science Institute, University of Washington, Seattle, WA 98195-1570, USA
4
Biology Department, San Diego State University, North Life Science Room 401, San Diego, CA 92182-4614, USA
*
Author to whom correspondence should be addressed.
Current address: Department of Microbiology and Molecular Genetics, Michigan State University, 6198 Biomedical Physical Sciences. East Lansing, MI 48824, USA.
Current address: College of Science, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
Academic Editor: Ludmila Chistoserdova
Received: 5 January 2015 / Accepted: 26 March 2015 / Published: 9 April 2015

Abstract

:
Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylamine as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methylamine vs methanol. As expected, all genes for the N-methylglutamate pathway were induced during growth on methylamine. Among other functions responding to the aminated source of C1-carbon, are a heme-containing amine dehydrogenase (Qhp), a distant homologue of formaldehyde activating enzyme (Fae3), molybdenum-containing formate dehydrogenase, ferredoxin reductase, a set of homologues to urea/ammonium transporters and amino-acid permeases. Mutants lacking one of the functional subunits of the amine dehydrogenase (ΔqhpA) or Δfae3 showed no growth defect on C1-compounds. M. universalis FAM5 strains with a lesion in the H4-folate pathway were not able to use any C1-compound, methanol or methylamine. Genes essential for C1-assimilation (the serine cycle and glyoxylate shunt) and H4MTP-pathway for formaldehyde oxidation showed similar levels of expression on both C1-carbon sources. M. universalis FAM5 possesses three homologs of the formaldehyde activating enzyme, a key enzyme of the H4MTP-pathway. Strains lacking the canonical Fae (fae1) lost the ability to grow on both C1-compounds. However, upon incubation on methylamine the fae1-mutant produced revertants (Δfae1R), which regained the ability to grow on methylamine. Double and triple mutants (Δfae1RΔfae3, or Δfae1RΔfae2 or Δfae1RΔfae2Δfae3) constructed in the revertant strain background showed growth similar to the Δfae1R phenotype. The metabolic pathways for utilization of methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed based on these gene expression and phenotypic data.
Keywords:
rhodocyclaceae; methyloversatilis; C1-metabolism; N-methylglutamate pathway; formaldehyde activating enzyme homologues

1. Introduction

Facultative betaproteobacterial methylotrophs, belonging to Burkholderiales and Rhodocyclales, occupy a variety of ecological niches, including soils, sediments, plant biomass, wastewater sludge, hot springs, and oil sands [1,2,3,4,5,6,7]. These bacterial lineages show exceptionally versatile metabolic capabilities, including the ability to utilize single carbon compounds, such as methanol and methylated amines [8,9,10]. Despite the relatively high abundance of these facultative methylotrophic betaproteobacteria in nature, only a few of them have been isolated in pure culture and characterized [4,5,6,9,10]. C1-utilization pathways have been investigated in Methyloversatilis universalis and Methyloversatilis thermotolerans (both are members of the family Rhodocyclaceae) and Methylibium petroleiphilum PM1 (a representative of unclassified Burkholderiales) [11,12,13]. It has been shown that a homolog of the pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase is essential for oxidation of C1 and C2 alcohols during aerobic or anaerobic growth in M. universalis [8,13]. The same enzyme is crucial for methanol utilization even in strains possessing the canonical methanol dehydrogenase, such as Methyloversatilis sp. 18–153 or RZ94 [8,10].
In addition to methanol, all tested members of the genera Methyloversatilis are capable of utilizing methylated amines [6,10]. M. universalis FAM5 oxidizes methylamine via the N-methylglutamate pathway [12]. The pathway includes three enzymes: the N-methylglutamate synthase (NMGS, encoded by mgsABC gene cluster), which can produce N-methylglutamate from glutamate and methylamine; the N-methylglutamate dehydrogenase (NMGDH, encoded by mgdABCD gene cluster), which is predicted to produce methylene-tetrahydrofolate (H4F) and regenerated glutamate from N-methylglutamate; and the gamma-glutamylmethylamine synthetase (GMAS, gmas), a reversible enzyme which produces gamma-glutamylmethylamine (GMA) from glutamate and methylamine using ATP. The gamma-glutamylmethylamine synthetase is predicted to serve as the first enzyme of the pathway, however the exact functional role of the enzyme and its product, gamma-glutamylmethylamine, are not well defined. It has been predicted that the enzyme contributes to methylamine detoxification, by converting the potentially toxic amine into a metabolically neutral form [14]. This vision of the pathway has been supported by a number of studies, including mutagenesis, 13C-labeling and in vivo NMR investigation [12,14,15,16]. The GMA has also been proposed to be a substrate for NMGS instead of methylamine [17,18,19]. Overall, the exact topology of the pathway is still awaiting a careful enzymatic investigation. Nonetheless, several genomic studies suggest that the N-methylglutamate pathway is recruited for methylamine utilization by a number of methylotrophic and non-methylotrophic bacteria [16,17,18,19,20,21,22].
Recently the genomes of seven strains representing three Methyloversatilis species have been sequenced at the University of Washington [23] and in collaboration with the Joint Genomic Institute (JGI) [10]. The C1-pathways were found to be highly conserved in all strains sequenced. It was predicted that formaldehyde, a product of methanol or methylamine oxidation could be converted to formate by a tungsten-dependent aldehyde dehydrogenase in addition to the tetrahydrofolate (H4F) or tetrahydromethanopterin (H4MPT)-pathways [5,13,23]. Formate is further oxidized to CO2 by two NAD-dependent formate dehydrogenases [13]. Two C1-assimilation pathways, the serine cycle and Calvin-Benson-Bassham (CBB) cycle, could be predicted for sequenced Methyloversatilis spp, with the exception of M. thermotolerans 3tT, which possesses only the serine cycle [10,11,23]. Only serine cycle enzymes were induced in M. universalis cells during anaerobic growth on methanol [13]. Genome analyses also indicate that M. universalis FAM5 also possesses a heme-containing amine dehydrogenases (Qhp). It has been shown that the Qhp oxidizes a number of aliphatic and aromatic amines [24,25,26]. Qhp was highly expressed during growth of the Paracoccus denitrificans IFO 12442 on methylamine. However, the enzyme displays relatively low affinity for methylamine and is specific to n-butylamine [25]. It has been speculated that during growth of P. denitrificans on methylamine the enzyme plays a secondary role and might contribute to oxidation/detoxification of the amine [25,26]. The contribution of the enzyme to metabolism of methylamine has not been tested by mutagenesis.
Here we investigate pathways for aerobic utilization of methanol and methylamine in M. universalis FAM5. The predicted end-product of methylamine oxidation via the N-methylglutamate pathways is methylene-H4F [12]. Thus the metabolic arrangement of methylamine utilization was expected to differ from the methanol utilization network, where formaldehyde is the key end product of the methanol oxidation [8].

2. Materials and Methods

2.1. Bacterial Strains, Plasmids and Culture Conditions

The M. universalis sp FAM5 strains used in this study are listed in Table 1. Since the wild type aggregates in liquid culture we selected a strain of the M. universalis (named here FAM5E) that does not clump. No differences in growth rate between strain FAM5 and FAM5E on plates or in liquid culture were observed (data not shown). The main advantage of the strain FAM5E is that it produces reliable reading of optical density and does not require homogenization. The strain FAM5E was used to generate and test the majority of mutants described in this study (Table 1).
Table 1. Growth phenotypes of the Methyloversatilis universalis wild type and mutant strains.
Table 1. Growth phenotypes of the Methyloversatilis universalis wild type and mutant strains.
StrainDescriptionSuccinateMethanolMethylamineSuccinate + C1 *
FAM5Wild type, forms aggregates in liquid culture, KanSTetSCmR+++++++++++++++
FAM5ETrait of FAM5 which does not form aggregates in liquid culture, KanSTetSCmR+++++++++++++++
ΔmgdDFAM5-ΔmgdD:kan, KanRTetSCmR+++++++-NT
ΔmgsCFAM5-ΔmgsC:kan, KanRTetSCmR+++++++-NT
Δfae1FAM5E-Δfae1:kan, KanRTetSCmR++++--NT
Δfae2FAM5E-Δfae2:kan, KanRTetSCmR+++++++++++NT
Δfae3FAM5E-Δfae3:kan, KanRTetSCmR+++++++++++NT
ΔmtdAFAM5E-ΔmtdA:kan, KanRTetSCmR++++--+++ (1)
ΔqhpAFAM5-ΔqhpA, KanSTetSCmR+++++++++++NT
Δfae1ΔqhpAFAM5-ΔqhpAΔfae1:kan, KanRTetSCmR++++-++NT
Δfae2ΔqhpAFAM5-ΔqhpAΔfae2:kan, KanRTetSCmR+++++++++++NT
Δfae3ΔqhpAFAM5-ΔqhpAΔfae3:kan, KanRTetSCmR+++++++++++NT
Δfae1RΔqhpAΔfae3FAM5E-ΔqhpA Δfae1Δfae3:kan, KanRTetSCmR++++--NT
Δfae1RRevertant of FAM5E-Δfae1, KanSTetSCmR++++-++++++ (2)
Δfae1Δfae3FAM5-Δfae1Δfae3:kan, KanRTetSCmR++++--NT
Δfae1RΔ fae3FAM5-Δfae1Δfae3, KanSTetSCmR++++-++++++ (2)
Δfae1Δ fae2FAM5-Δfae1Δfae2:kan, KanRTetSCmR+++++++++++NT
Δfae3FAM5E-Δfae3, KanSTetSCmR+++++++++++NT
Δfae3Δfae2FAM5E-Δfae3 Δfae2:kan, KanRTetSCmR+++++++++++NT
Δfae1Δfae2Δfae3FAM5E-Δfae1Δfae3Δfae2:kan, KanRTetSCmR++++--NT
NT, not tested; “++++” correspond to doubling time of 6 h; “+++” correspond to doubling time of 9 h; “++” correspond to 12 h doubling time; and “-” indicates no growth. Methanol (5 mM) was added to test if a mutant strain is sensitive to formaldehyde (2) or unable to produce methyl-H4folate for purine synthesis (1).
Cells were grown in previously described minimal medium [6] with succinate (20 mM), methylamine (30 mM or 5 mM), and methanol (25 mM) as growth substrates. The following antibiotic concentrations were used for M. universalis: tetracycline (Tet), 1.0 mg L−1; kanamycin (Kan), 100 mg L−1; and chloramphenicol (Cmp), 10–15 mg L−1.
The following cloning vectors were used: pCR2.1 (Invitrogen, Carlsbad, CA, USA) for cloning of PCR products, pCM184 for generation of single, double and triple mutant strains, and the pCM157 system for construction of unmarked mutations (Table 2) [27]. Escherichia coli strains were routinely cultivated at 37 °C in Luria-Bertani medium (BD Difco, Franklin Lakes, NJ, USA). When indicated, antibiotic concentrations were used: Tet, 12.5 mg L−1; Kan, 100 mg L1; and ampicillin (Amp), 100 mg L−1.
Table 2. Bacterial strains and plasmids used in this study.
Table 2. Bacterial strains and plasmids used in this study.
Strain/PlasmidMarkers/DescriptionReference/Source
Escherichia coli One Shot TOP 10F-mcrA Δ (mrr-hsdRMS-mcrBC) Φ80 lacZΔM15 ΔlacX74 recA1 araD139 Δ (ara-leu)7697 galU galK rpsL StrR endA1 nupGInvitrogen
Escherichia coli S17-1recA pro hsdR RP4-2-Tc:Mu-Km:Tn7 TmpR SpcRStrR[28]
pCR2.1KanR, AmpRInvitrogen
pDriveKanR, AmpRQiagen
pCM184Broad-host-range allelic exchange vector, KanR, TetR[27]
pCM157Cre/LoxP, TetR[27]
pMgdDpCM184 with mgdD upstream and downstream flanksThis study
pMgsCpCM184 with mgsC upstream and downstream flanksThis study
pFAE1pCM184 with fae1 upstream and downstream flanksThis study
pFAE2pCM184 with fae2 upstream and downstream flanksThis study
pFAE3pCM184 with fae3 upstream and downstream flanksThis study
pQHNDH1pCM184 with qhpA upstream and downstream flanksThis study
pQHNDH2pCM184 with qhpX upstream and downstream flanksThis study
pMtdApCM184 with mtdA upstream and downstream flanksThis study

2.2. DNA Manipulations

DNA was isolated using the QIAamp DNA Mini kit (Qiagen, Venlo, Netherlands). Plasmid DNA was purified using the Qiagen Miniprep Kit (Qiagen). E. coli transformation, restriction enzyme digestion and ligation reactions were carried out as described by Sambrook et al. [29]. Polymerase chain reaction (PCR) amplifications were performed using hot-start Taq polymerase (Qiagen) in accordance with the manufacturer’s instructions. Primers used for PCR amplification of upstream or downstream regions (approximately 450–600 bp) of each targeted gene are listed in Table S1. Amplified fragments were cloned into pCR2.1, sequenced and then sub-cloned into pCM184 using appropriate restriction sites. After verification of the nucleotide sequence, the plasmids were transformed into E. coli S17-1, and the resulting donor strains were mated with wild-type M. universalis FAM5 via biparental mating as previously described [8]. The identity of the double-crossover mutants was verified by diagnostic PCR with primers specific to the insertion sites. Mutant phenotypes were assessed on solid media and in liquid culture with succinate, pyruvate, methanol or methylamine as carbon sources.

2.3. RNA-seq Experiments

Trascriptomic experiments were carried out with M. universalis strain FAM5. Cells pre-grown on methanol (10 mM) were used for inoculation of methanol (25 mM) or methylamine (35 mM) cultures at 5:100 ratios (inoculum:fresh media). Cultures were grown to mid-exponential phase (OD600 0.4 ± 0.05). The cellular activities were terminated by addition of “stop solution” as described [30]. The cells were collected by centrifugation at 4300× g at 4 °C for 10 min. mRNA samples were isolated and enriched as previously described [30] and submitted to the University of Washington’s High-Throughput Sequencing Solutions Center on dry ice for single-read Illumina® sequencing (Department of Genome Sciences, University of Washington; http://www.htseq.org/). The RNA-Seq sequence data sets ranged from 12.19 to 25.46 million reads (36 bp) per sample. The M. universalis FAM5 genome to be used as the alignment scaffold was downloaded from MaGE (https://www.genoscope.cns.fr/agc/microscope/mage/) on 31 March 2014. The raw reads were aligned to the scaffold using BWA version 0.7.4-r385 with default parameters [31]. The alignments were post-processed and sorted into BAM files with SAMTools version 0.1.19-44428cd [32]. Reads were attributed to open reading frames (ORFs) using the htseq-count tool from the “HTSeq” framework version 0.5.4p5 in the “intersection-nonempty” mode [33]. The ratios of reads mapped per ORF in the two conditions were calculated along with p-values using DESeq2 [33]. The p-values in Table 2 were corrected for multiple testing using the q-value method of Storey [34]. Proportions of reads mapped to rRNA were 78%–90% for RNA-libraries prepared from cells grown on methanol, and 91%–92% for RNA-libraries prepared from cells grown on methylamine. For display and presentation purposes, the Reads per Kilobase per Million reads sequenced (RPKM) were calculated [35].

2.4. Enzyme Assays

All cell extracts (50 mL) for activity were prepared from methylamine-grown cultures. Cells were harvested by centrifugation at 4500× g using a Sorvall RC-5B centrifuge at 4 °C for 10 min. The supernatant was removed and the cell pellet was stored at −80 C. Cell pellets were thawed and resuspended in 1 mL of 100 mM potassium phosphate buffer, pH 7.6 or 7.2 and broken using a French press (three times, 1000 psi). The extracts were centrifuged at 28,000× g for 5 min to remove cell debris. Qhp activity was detected by measuring amine dehydrogenase activity using a spectrophotometric assay measuring the reduction rate of potassium ferricyanide (500 μM, at 420 nm) in the presence of cell free extract (100 μg), and methylamine (25 mM) as substrate at room temperature. The extinction coefficient of potassium ferricyanide is 1.02 mM−1 cm−1 at pH = 7.6. The total volume of the reaction was 200 μL. Activity was measured using a Spectramax 190 plate reader (Molecular Devices, Sunnyvale, CA, USA); a minimum of two biological replicates was assayed.

2.5. Accession Numbers

The RNA-seq data were uploaded into NCBI/GEO under accession number GSE63822.

3. Results

3.1. Gene Expression Profiles: Methanol vs. Methylamine

Whole transcriptome shotgun sequencing data were generated for M. universalis cells grown on methanol or methylamine using a high-throughput sequencing (Illumina) platform. Based on relative expression, genes (omitting rRNAs) could be grouped into six major categories: genes with very high (Reads per Kilobase Million (RPKM) ≥ 10000), high (RPKM ≥ 5000), moderate (5000 > RPKM ≥ 1000), modest (1000 > RPKM ≥ 250), and low (250 > RPKM ≥ 50) expression, and not expressed (RPKM < 50). Annotation of the M. universalis FAM5 genome predicts 4027 coding sequences [23]. Based on the expression data, 950 (24%) genes were not expressed under any condition tested. Among the silent functional modules were genes encoding denitrification reactions (nitrate and nitrite reductases, nitrate transporters), phototrophy (light-harvesting complexes) and pathways for utilization of urea, acetoin, methanesulfonate, and phenolic compounds. The majority of genes fell into low expression categories (45%). About 25% of genes displayed modest expression, 5% of genes showed moderate expression and only a small fraction (1%) of the genome showed very high/high expression.
Most of the previously recognized metabolic modules essential for oxidation of C1-compounds fell into very high to moderate categories. Expression of central metabolic pathways, such as the citric acid cycle, amino acid biosynthesis, gluconeogenesis and energy generating pathway (respiratory chain and ATP-synthase) genes remained mostly unchanged (Table 3). Genes encoding the H4MPT synthesis and the H4MPT-linked C1-transfer enzymes and the serine cycle enzymes also remained unchanged, suggesting that these pathways contribute in a similar fashion to methanol and methylamine utilization (Table 3).
Cells growth on methanol showed a slightly higher (about 1.4 fold) abundance of transcripts for methanol dehydrogenase (mdh2) and the associated cytochrome gene (cyt) (Table 3; Figure 1). The relative abundance of formate-tetrahydrofolate ligase/formyltetrahydrofolate synthetase (ftfL) transcripts, a tungsten-containing aldehyde ferredoxin oxidoreductase, a distant homolog of the PQQ-dependent methanol dehydrogenase (xoxF3G3J3), cytochrome bc1 reductase complex, a putative NADP(H)-dependent aldo/keto reductase and a number of hypothetical proteins were higher (1.6–3 folds) in methanol grown cells (Table 3; Figure 1). Genes encoding the N-methylglutamate pathway enzymes, co-clustered conserved proteins and a putative transcriptional regulator, were up-regulated (6−10-fold) during growth on methylamine (Table 3, Figure 1). Among other genes altered by the shift to methylamine were: a seven gene cluster (qhpRADCBFE) encoding a heme-containing amine dehydrogenase (Qhp, qhpADC), and associated proteins involved in the posttranslational modification and regulation; a ferredoxin; and a distant homolog of the formaldehyde activating enzyme (fae3). The fae3 gene was almost 10 fold higher in cells shifted to methylamine.
Table 3. Gene expression profile in methane or methylamine grown cells of M. universalis FAM5.
Table 3. Gene expression profile in methane or methylamine grown cells of M. universalis FAM5.
Gene ID (Old)Gene ID (New)Gene ProductMethanol (RPKM) *Methylamine (RPKM) *Fold Changeq-Value
Methylamine oxidation
METUNv1_760110METUNv2_580110Gmas, Gamma glutamylmethylamide synthetase2961.8019,440.256.560.65
METUNv1_760111METUNv2_580111MgsA, N-methyl glutamate synthase subunit A2727.4322,221.308.150.73
METUNv1_760112METUNv2_580112MgsB, N-methyl glutamate synthase subunit B2680.7619,852.557.410.69
METUNv1_760113METUNv2_580113MgsC, N-methyl glutamate synthase subunit C4221.5653,956.5512.780.67
METUNv1_760114METUNv2_580114MgdA, N-methyl glutamate dehydrogenase/oxidoreductase subunit A1414.9013,014.459.200.74
METUNv1_760115METUNv2_580115MgdB, N-methyl glutamate dehydrogenase/oxidoreductase subunit B388.003906.3110.070.74
METUNv1_760116METUNv2_580116MgdC, N-methyl glutamate dehydrogenase/oxidoreductase subunit C3389.1026,611.057.850.75
METUNv1_760117METUNv2_580117MgdD, N-methyl glutamate dehydrogenase/oxidoreductase subunit D701.947962.7411.340.71
METUNv1_760127METUNv2_580127QhpB, Quinohemoprotein amine dehydrogenase, beta subunit420.064164.189.910.68
METUNv1_760128METUNv2_580128QhpA, Quinohemoprotein amine dehydrogenase, alpha subunit733.767390.8410.070.65
METUNv1_760130METUNv2_580130QhpC, Quinohemoprotein amine dehydrogenase, SAM-radical dependent activating subunit380.864283.0611.250.00
Methanol oxidation
METUNv1_770214METUNv2_590217XoxF1, PQQ-linked dehydrogenase 2023.283380.161.670.50
METUNv1_770216METUNv2_590218XoxF2, PQQ-linked dehydrogenase 10,962.1716,741.701.530.05
METUNv1_590046METUNv2_420045XoxF3, PQQ-dependent dehydrogenase1213.63659.01−1.840.62
METUNv1_590042METUNv2_420041XoxJ3, Extracellular solute-binding protein family 34399.571403.51−3.130.05
METUNv1_590043METUNv2_420042XoxG3, Cytochrome c class I3156.111089.89−2.900.30
METUNv1_590049METUNv2_420048Mdh2, PQQ-dependent methanol/ethanol dehydrogenase48,100.3534,793.20−1.380.77
METUNv1_590050METUNv2_420049Mdh2J, Extracellular solute-binding protein family2486.88903.00−2.750.69
METUNv1_590051METUNv2_420050Mdh2G, cytochrome c-type protein1732.91595.39−2.910.58
Formaldehyde oxidation
METUNv1_580096METUNv2_410093Fae 1, Formaldehyde-activating enzyme18,235.0011,635.85−1.570.82
METUNv1_660037METUNv2_480039Fae 2, Formaldehyde-activating enzyme561.52642.501.140.89
METUNv1_700516METUNv2_520523Fae 3, Formaldehyde activating enzyme584.516861.8611.740.00
METUNv1_590006METUNv2_420006Orf 9, Involved in biosynthesis of tetrahydromethanopterin. Essential for formaldehyde oxidation.430.06187.51−2.290.21
METUNv1_580095METUNv2_410092Orf 7, Involved in tetrahydromethanopterin-linked formaldehyde oxidation.371.25549.261.480.75
METUNv1_580094METUNv2_410091Orf 5, Involved in biosynthesis of tetrahydromethanopterin489.69493.891.010.93
METUNv1_580093METUNv2_410090Mch, Methenyltetrahydromethanopterin cyclohydrolase620.26736.891.190.87
METUNv1_580092METUNv2_410089OrfY, Involved in tetrahydromethanopterin C1 transfer.896.40819.12−1.090.94
METUNv1_580091METUNv2_410088MtdB, NAD-dependent methylenetetrahydromethanopterin dehydrogenase1295.291379.771.070.88
METUNv1_580088METUNv2_410086FhcB, Formyltransferase/hydrolase complex subunit B587.63515.00−1.140.92
METUNv1_580087METUNv2_410085FhcA, Formyltransferase/hydrolase complex subunit A766.77744.38−1.030.93
METUNv1_580086METUNv2_410084FhcD, Formyltransferase/hydrolase complex subunit D598.76547.64−1.090.94
METUNv1_580085METUNv2_410083FhcC, Formyltransferase/hydrolase complex subunit C634.50467.32−1.360.88
METUNv1_590040METUNv2_420039Ald, Tungsten-containing aldehyde ferredoxin oxidoreductase408.13155.57−2.620.17
METUNv1_490013METUNv2_320014AldB, Aldehyde dehydrogenase B2762.932224.63−1.240.94
Formate oxidation
METUNv1_770385METUNv2_590384Fdh, Putative formate dehydrogenase subunit A634.02668.941.060.92
METUNv1_700257METUNv2_520260FdhD, NAD-linked formate dehydrogenase delta subunit38.0076.632.020.55
METUNv1_700258METUNv2_520261FdhC, Formate dehydrogenase, accessory protein751.501024.071.360.74
METUNv1_700259METUNv2_520262FdhA, NAD-dependent formate dehydrogenase alpha subunit3720.445479.721.470.39
METUNv1_700260METUNv2_520263FdhB, NAD-dependent formate dehydrogenase beta subunit2758.004434.841.610.46
METUNv1_700261METUNv2_520264FdhG, NAD-dependent formate dehydrogenase gamma subunit1127.751882.411.670.67
METUNv1_700262METUNv2_520265FdhR, Formate dehydrogenase regulator210.25231.001.100.90
METUNv1_570005METUNv2_400021FdsA, NAD-dependent, tungsten-containing formate dehydrogenase alpha subunit1438.461527.511.060.91
METUNv1_570006METUNv2_400022FdsB, NAD-dependent, tungsten-containing formate dehydrogenase beta subunit951.261018.181.070.90
H4F-pathway/Serine cycle/Glyoxylate shunt
METUNv1_460318METUNv2_290319FtfL, formate-tetrahydrofolate ligase/synthetase4729.661674.32−2.820.54
METUNv1_460309METUNv2_290310Fch, Methenyltetrahydrofolate cyclohydrolase2091.391541.64−1.360.90
METUNv1_460310METUNv2_290311MdtA, NADP-dependent methylenetetrahydrofolate dehydrogenase4363.442973.78−1.470.93
METUNv1_460311METUNv2_290312Hpr, Hydroxypyruvate reductase, NAD(P)H-dependent.3620.173220.89−1.120.89
METUNv1_460312METUNv2_290313Sga, Serine-glyoxylate aminotransferase10,590.938570.41−1.240.79
METUNv1_460313METUNv2_290314GlyA, Serine hydroxymethyltransferase 6313.105292.54−1.190.87
METUNv1_460314METUNv2_290315MtkA, Malate thiokinase large subunit4955.016292.071.270.73
METUNv1_460315METUNv2_290316MtkB, Malate thiokinase small subunit5081.455363.841.060.73
METUNv1_460316METUNv2_290317Ppc1, phosphoenolpyruvate carboxylase2661.632768.191.040.86
METUNv1_460317METUNv2_290318Mcl, malyl-CoA lyase3260.854275.941.310.58
METUNv1_770329METUNv2_590331Gk, Glycerate kinase1197.011116.38−1.070.93
METUNv1_770169METUNv2_590170Ppc2, Phosphoenolpyruvate carboxylase1034.151074.001.040.91
METUNv1_460302METUNv2_290303Eno, Enolase 2095.131936.51−1.080.92
METUNv1_710053METUNv2_530053Pgm, Phosphoglyceromutase470.57517.141.100.90
METUNv1_620020METUNv2_450021Ms, Malate synthase A353.94269.76−1.310.89
METUNv1_620018METUNv2_450018Icl, Isocitrate lyase12,174.928389.98−1.450.85
CO2 Fixation (CBB cycle)
METUNv1_750044METUNv2_570044CbbR, RuBisCO operon transcriptional regulator195.62189.12−1.030.93
METUNv1_750045METUNv2_570045CbbL, Ribulose-1,5-bisphosphate carboxylase large subunit127.63130.881.030.94
METUNv1_750046METUNv2_570046CbbS, Ribulose bisphosphate carboxylase small subunit41.6247.501.140.92
METUNv1_750047METUNv2_570047CbxX, chromosomal AAA type ATPase8.7511.941.360.89
METUNv1_750048METUNv2_570048CbbY, haloacid dehalogenase7.2514.562.010.73
METUNv1_750049METUNv2_570049CbbE, Ribulose-phosphate 3-epimerase13.8816.131.160.91
METUNv1_750050METUNv2_570050Pgp, phosphoglycolate phosphatase12.5013.001.040.94
METUNv1_750051METUNv2_570051CbbF, Fructose-1,6-bisphosphatase 26.7530.511.140.92
METUNv1_750052METUNv2_570052CbbP, Phosphoribulokinase 26.6932.001.200.92
METUNv1_750053METUNv2_570053CbbT, Transketolase24.8842.561.710.73
METUNv1_750054METUNv2_570054CbbG, Glyceraldehyde-3-phosphate dehydrogenase17.5019.561.120.93
METUNv1_750055METUNv2_570055CbbA, Fructose-bisphosphate aldolase72.7565.50−1.110.93
METUNv1_700111METUNv2_520114CbbR, RuBisCO operon transcriptional regulator360.50255.84−1.410.90
METUNv1_760018METUNv2_580018CbbQ, Post-translational RubisCO activator 16.2519.381.190.91
Sugar Phosphate Interconversions
METUNv1_470279METUNv2_300282Fbp, Fructose-1,6-bisphosphatase403.90440.811.090.91
METUNv1_700104METUNv2_520106Fba, Fructose-bisphosphate aldolase920.681094.831.190.85
METUNv1_700105METUNv2_520107Pyk, Pyruvate kinase II 807.00786.28−1.030.92
METUNv1_700106METUNv2_520108Pgk, Phosphoglycerate kinase693.63684.62−1.010.92
METUNv1_700107METUNv2_520109Gapdh, Glyceraldehyde 3-phosphate dehydrogenase1478.941536.511.040.90
METUNv1_700108METUNv2_520110Tk, Transketolase982.15730.45−1.340.89
METUNv1_700109METUNv2_520111Prk, Phosphoribulokinase380.94483.871.270.82
METUNv1_700110METUNv2_520112Fbp3, Fructose-1,6-bisphosphatase902.64846.62−1.070.94
METUNv1_470090METUNv2_300091Pps, Phosphoenolpyruvate synthase 1610.661360.78−1.180.94
METUNv1_460093METUNv2_290089Pck, Phosphoenolpyruvate carboxykinase (GTP)242.99191.39−1.270.89
METUNv1_580038METUNv2_410037Pfk, Pyrophosphate-dependent phosphofructokinase866.701075.271.240.85
METUNv1_580036METUNv2_410036Pyrophosphate-energized inorganic pyrophosphatase747.52887.261.190.88
METUNv1_580049METUNv2_410048Pyrophosphate phosphohydrolase555.12602.371.090.89
TCA cycle
METUNv1_700127METUNv2_520130E1 component of pyruvate dehydrogenase 959.881210.511.260.80
METUNv1_700126METUNv2_520129E2 component of pyruvate dehydrogenase 217.00302.251.390.77
METUNv1_700125METUNv2_520128E3 component of pyruvate dehydrogenase 482.26555.081.150.88
METUNv1_700186METUNv2_520190Succinyl-CoA synthetase beta subunit705.14556.31−1.270.91
METUNv1_700187METUNv2_520191Succinyl-CoA synthetase alpha subunit622.44414.43−1.500.86
METUNv1_470127METUNv2_300128Fumarate hydratase class I659.82590.00−1.120.93
METUNv1_460167METUNv2_290164Fumarate hydratase class II (fumarase C)185.50141.26−1.310.91
METUNv1_460003METUNv2_290002ME1, Malic Enzyme952.12789.15−1.210.93
METUNv1_520009METUNv2_350011Mdh, Malate dehydrogenase2673.561934.79−1.380.91
METUNv1_520011METUNv2_350013Succinate dehydrogenase cytochrome b556 subunit 324.52191.00−1.700.70
METUNv1_520012METUNv2_350014Succinate dehydrogenase anchor subunit572.13412.89−1.390.88
METUNv1_520013METUNv2_350015Succinate dehydrogenase flavoprotein subunit4086.042492.77−1.640.84
METUNv1_520014METUNv2_350016Succinate dehydrogenase Fe–S protein843.00709.16−1.190.93
METUNv1_520016METUNv2_350018Citrate synthase2249.292218.66−1.010.91
METUNv1_520017METUNv2_350019E1 component of alpha-ketoglutarate dehydrogenase1429.241533.031.070.89
METUNv1_520018METUNv2_350020E2 component of alpha-ketoglutarate dehydrogenase697.75762.991.090.90
METUNv1_520019METUNv2_350021E3 component of alpha-ketoglutarate dehydrogenase1364.021401.041.030.89
METUNv1_620012METUNv2_450011Isocitrate dehydrogenase kinase/phosphatase 241.63308.131.280.84
METUNv1_620013METUNv2_450012Isocitrate dehydrogenase (NADP+)2578.022899.291.120.80
Amino Acid Synthesis
METUNv1_660052METUNv2_480054Glutamate synthase (NADPH) large chain (NADPH-GOGAT)3985.942866.80−1.390.87
METUNv1_660053METUNv2_480055Glutamate synthase (NADPH) small chain (NADPH-GOGAT)1357.89903.01−1.500.91
METUNv1_450044METUNv2_280044Glutamine synthetase (Glutamate-ammonia ligase)2361.782288.13−1.030.56
METUNv1_470103METUNv2_300104Glutamate dehydrogenase, NADP-specific (NADP-GDH)1040.151519.761.460.93
METUNv1_470104METUNv2_300105Aspartate aminotransferase (Transaminase A) (AspAT)1020.04941.84−1.080.92
METUNv1_470184METUNv2_300183Putative aspartate transaminase617.88620.111.000.93
METUNv1_750043METUNv2_570043Serine-pyruvate aminotransferase679.01606.13−1.120.91
Oxidative Phosphorylation
METUNv1_750182METUNv2_570182ATP synthase F0, A chain886.37965.571.090.83
METUNv1_750183METUNv2_570183ATP synthase F0, C chain 1218.521365.891.120.93
METUNv1_750184METUNv2_570184ATP synthase F0, B chain2528.922775.881.100.89
METUNv1_750185METUNv2_570185ATP synthase delta chain2885.172564.77−1.120.86
METUNv1_750186METUNv2_570186ATP synthase subunit alpha subunit 8915.446780.46−1.310.89
METUNv1_750187METUNv2_570187ATP synthase gamma subunit5294.584705.58−1.130.93
METUNv1_750188METUNv2_570188ATP synthase beta subunit9593.016768.24−1.420.92
METUNv1_750189METUNv2_570189ATP synthase epsilon subunit1784.401464.79−1.220.90
METUNv1_770336METUNv2_590337NAD(P) transhydrogenase subunit alpha 858.91858.01−1.000.93
METUNv1_770337METUNv2_590338NAD(P) transhydrogenase subunit beta399.38455.251.140.92
METUNv1_460126METUNv2_290122NADH-quinone oxidoreductase chain A 156.74148.38−1.060.93
METUNv1_460127METUNv2_290123NADH-quinone oxidoreductase subunit B451.50376.26−1.200.94
METUNv1_460128METUNv2_290124NADH (or F420H2) dehydrogenase subunit C388.51353.76−1.100.94
METUNv1_460129METUNv2_290125NADH-ubiquinone oxidoreductase D subunit 754.27681.62−1.110.92
METUNv1_460130METUNv2_290126NADH-quinone oxidoreductase subunit E207.95211.511.020.92
METUNv1_460131METUNv2_290127NADH-quinone oxidoreductase subunit F628.76528.76−1.190.87
METUNv1_460132METUNv2_290128NADH-quinone oxidoreductase subunit G1248.13938.27−1.330.93
METUNv1_460133METUNv2_290129NADH-quinone oxidoreductase subunit H520.13381.75−1.360.93
METUNv1_460134METUNv2_290130NADH-quinone oxidoreductase subunit I327.57337.821.030.94
METUNv1_460135METUNv2_290131NADH-quinone oxidoreductase subunit J 131.50118.13−1.110.88
METUNv1_460136METUNv2_290132NADH-quinone oxidoreductase subunit K58.8860.631.030.91
METUNv1_460137METUNv2_290133NADH-quinone oxidoreductase subunit L637.25465.50−1.370.83
METUNv1_460138METUNv2_290134NADH-quinone oxidoreductase subunit M372.75302.75−1.230.77
METUNv1_460139METUNv2_290135NADH-ubiquinone oxidoreductase, chain N523.88339.07−1.550.64
METUNv1_660132METUNv2_480138Ferredoxin-NADP reductase522.14879.521.680.69
METUNv1_590030METUNv2_420029Ubiquinol-cytochrome c reductase complex, cytochrome c12503.551092.90−2.290.58
METUNv1_590031METUNv2_420030Ubiquinol-cytochrome c reductase complex, cytochrome b2945.381365.76−2.160.81
METUNv1_590032METUNv2_420032Ubiquinol-cytochrome c reductase iron-sulfur subunit (Rieske iron-sulfur protein) (RISP)3125.541240.80−2.520.84
METUNv1_590008METUNv2_420008Peroxidase/catalase (Catalase-peroxidase)3075.821883.61−1.630.84
METUNv1_670031METUNv2_490032Cytochrome c oxidase subunit 15577.294801.32−1.160.93
METUNv1_670030METUNv2_490031Cytochrome c oxidase subunit 24105.553835.175−1.070.87
METUNv1_670034METUNv2_490035Cytochrome c oxidase subunit 32746.542227.3−1.230.89
METUNv1_670033METUNv2_490034Cytochrome c oxidase assembly protein1365.3251525.0351.120.93
METUNv1_580051METUNv2_410050Hemin uptake protein hemP (fragment)1319.891517.791.150.68
METUNv1_580052METUNv2_410051Bacterioferritin-associated ferredoxin930.37739.50−1.260.80
METUNv1_580053METUNv2_410052Bfr, Bacterioferritin 1845.022568.531.390.77
* Each sample represents the average of two biological replicates. Values represent reads per kilobase of coding sequence per million (reads) mapped (RPKM).
Figure 1. The reconstructed pathway of methanol and methylamine utilization in Methyloversaltilis universalis FAM5 based on genomic, transcriptomic and mutagenesis data. Genes mutated in this study are shown in red. Pathways upregulated (6–11 fold) by growth on methylamine are shown in green; Pathways slightly downregulated (1.5–3 fold) by growth on methylamine are shown in orange; Grey lines show pathways whose expression do not change; and Blue lines indicate steps that are not expressed on tested C1-compounds, methanol or methylamine.
Figure 1. The reconstructed pathway of methanol and methylamine utilization in Methyloversaltilis universalis FAM5 based on genomic, transcriptomic and mutagenesis data. Genes mutated in this study are shown in red. Pathways upregulated (6–11 fold) by growth on methylamine are shown in green; Pathways slightly downregulated (1.5–3 fold) by growth on methylamine are shown in orange; Grey lines show pathways whose expression do not change; and Blue lines indicate steps that are not expressed on tested C1-compounds, methanol or methylamine.
Microorganisms 03 00175 g001

3.2. Mutagenesis Studies: Methylamine Oxidation

It has previously been shown that deletion of two subunits of NMGS (encoded by mgsA and mgsB) completely abolished the ability of the strain M. universalis FAM5 to utilize methylamine; however the mutants were still able to generate small amounts of N-methylglutamate [12]. It has been speculated that the third subunit of the protein (encoded by mgsC) can contribute to the residual production. Here we mutated mgsC and showed that the lack of the gene eliminates the ability of the strain to utilize methylamine (Table 1).
We also tested involvement of the gamma subunit of the NMGDH (mgdD) in methylamine utilization. The mgdD is located at the end of the four-gene cluster encoding the NMGDH. The fourth gene has been identified in all microbes possessing the N-methylglutamate pathway; however, in contrast to the catalytic subunits (mgdA and mgdC) and mgdB, which are typically quite conserved, the gamma subunit of the enzyme is surprisingly divergent [12]. Relatively low homology (30%–45% amino acid identity) has been observed between the mgdD subunit from the Methyloversatilis species and the corresponding protein from betaproteobacterial methylotrophs, such as Methylotenera versatilis 301 or Methylophilus methylotrophus DSM 46235. The gene from Methyloversatilis species shared 25% AA identity with the mgdD gene from Agrobacterium tumefaciens. No homology was observed among mgdD genes from betaproteobacteria and gamma subunit of the NMGDH from alphaprotebacterial methylotrophs, such as Methylocellla silvestris, Methylopila spp, and Methylobacterium extorquens spp. The exact function of the mgdD gene product is still not known. We mutated the mgdD gene in M.universalis FAM5, and the mutant strain lost the ability to utilize methylamine (Table 1). These two additional mutations additionally confirmed the importance of the N-methylglutamate pathway genes for methylamine utilization by M. universalis FAM5.
The transcriptomic study presented above revealed a number of additional functions indicating that the metabolic arrangement of the methylamine utilization in M. universalis FAM5 could be more intricate (Figure 1). Two of the most noticeable differences between cells grown on methanol vs. grown on methylamine are the overexpression of the heme-containing amine dehydrogenases gene cluster and fae3 in cells grown on methylamine. We generated a strain of M. universalis FAM5 lacking the alpha subunit of the Qhp gene (ΔqhpA). The activity of the Qhp enzyme was reduced to background level in the mutant strain (Table 4). However, the strain lacking Qhp gene (ΔqhpA) was able to grow on methylamine similarly to wild type (Table 1). The phenotype of the fae3 mutant is described below.

3.3. Mutagenesis Studies: Metabolic Arrangement Downstream from the Methylamine Oxidation

During growth on methanol, formaldehyde, the end product of alcohol oxidation, is oxidized to formate via the H4MPT pathway. Formate is either oxidized to CO2 to generate energy, or converted to methylene-H4F via an FtfL-Fch-MtdA variant of the H4F pathway. It has been predicted that the end product of the N-methylglutamate pathway is methylene-H4F, which could be incorporated directly into the serine cycle. However it is not known how it is oxidized. The FtfL-Fch-MtdA variant of the H4F pathway is typically linked to assimilation rather that oxidation [36]. In order to investigate the fate of the methylene-H4F in M. universalis FAM5 upon growth on methylamine, two possible scenarios were investigated: (1) The H4F pathway contributes to methylene-H4F oxidation by operating in the reverse direction; (2) The H4F pathway in M. universalis FAM5 contributes only to formate assimilation, and thus it should not be essential for growth on methylamine. However, in order to generate energy and reducing equivalent from C1-oxidation, the M. universalis FAM5 cells should possess an additional system for converting methylene-H4F to formaldehyde, or transfer C1-units from H4F to H4MPT. One candidate for C1-unit decoupling/shuttling is fae3, a homolog of formaldehyde activating enzyme, which has a very strong expression during growth on methylamine, but not methanol.
To verify these scenarios, mutations in H4F (ΔmtdA) and H4MPT pathways (Δfae1) were made. In addition we mutated homologs of the formaldehyde dehydrogenase (Δfae2 and Δfae 3), and generated multiple mutants (Δfae1Δfae2, Δfae1Δfae3, Δfae2Δfae3 and Δfae1Δfae2Δfae3). In addition, a strain lacking fae1 and qhpA was constructed. The mutant phenotypes are shown in Table 1.
Strains lacking mtdA or fae1 genes were not able to grow on methanol or methylamine. Interestingly, revertants were observed for the fae1-mutant. The number of revertants arising on methylamine plates was 10 ± 3 per 109 cells plated. The revertant strains grew on methylamine, albeit with a growth defect compared to the wild type strain (the growth rate was about 30% of the wild type). The revertant strains did not restore the ability to utilize methanol for growth. Single Δfae 2 or Δfae3 mutations, or the double Δfae2Δfae3 mutation displayed a growth rate similar to the wild-type strain on both methanol and methylamine. Δfae1Δfae2 and Δfae1Δfae3 double mutants were not able to grow on C1-compounds, similarly to Δfae1. No revertants were observed for Δfae1Δfae3 mutant upon transfer to methylamine plates. However, the incorporation of the Δfae3 mutation into the revertant Δfae1R strain did not abolish the ability of the strain to utilize methylamine. A similar phenotype was observed for a triple Δfae1RΔfae2Δfae3 mutant. The double Δfae1ΔqhpA mutant phenotype was similar to the single Δfae1 mutant. Like the latter, there were revertants of the double mutant (Δfae1ΔqhpAR) that regained the ability to utilize methylamine for growth. The frequency of reversion was similar to the single fae1-mutant. The triple Δfae3Δfae1ΔqhpAR mutant retained the ability to use methylamine.
Table 4. Activity of the heme-containing amine dehydrogenase in wild type and ΔqhpA-mutant strains upon growth on methylamine.
Table 4. Activity of the heme-containing amine dehydrogenase in wild type and ΔqhpA-mutant strains upon growth on methylamine.
StrainEnzyme Activity (μmol min−1 mg−1 Protein)
FAM5 (WT)30 ± 9
ΔqhpA6 ± 3

4. Discussion

Methylotrophic capabilities of the members of families Rhodocyclaceae/Burholderiaceae have relatively recently been discovered [1,3,4,5,7], and thus not much is known about the pathways for the single carbon utilization in this betaproteobacterial lineage. The M. universalis is the first representative of the family isolated in a pure culture and formally described [6]. The genome of the strain has been sequenced opening up new approaches for investigation of pathways for single carbon or methylated multicarbon compounds investigation [23]. Over the past few years the strain became the model system for understanding molecular mechanisms of C1-carbon utilization and denitrification [8,12,13].
The mutagenesis performed in this study suggests that the N-methylglutamate pathway is the main route for methylamine oxidation. Based on the available genomic and enzymatic data the end product of the N-methylglutamate pathway is methylene-H4F [12,16,20]. Thus it could be predicted that, first, the H4folate pathway contributes to C1-oxidation; and, second, the H4MPT-linked transfer should not be required for growth on methylamine. M. universalis FAM5 possesses the Mtd-Fch-FtfL variant of the H4MPT pathway, which is typically associated with formate assimilation, rather than formaldehyde/methylene-H4F oxidation [25,26]. It has been shown that this portion of the pathway is not essential for growth on methylamine in Methylobacterium spp. [16,20]. We found here that the mtdA mutant is not able to grow on both C1-compounds. The phenotypic data indicate that the enzyme of the pathway can play some additional role in C1-utilization in M. universalis.
We also found that the H4MPT pathway is essential for growth on methylamine. That observation indicates that M. universalis somehow produces methylene-H4MPT during growth on methylamine. Since the heme-dependent amine dehydrogenase (which is predicted to produce formaldehyde) is not essential for growth on methylamine, the C1-units entering H4MPT pathway should come from methylamine oxidation via the N-methylglutamate pathway. Based on the available evidences it could be proposed that the NMGDH releases both methylene-H4F and formaldehyde and that the ratio of the produced compounds depends on the intracellular pool of H4F. It has been shown that the pool of H4F is only a quarter of the pool of H4MPT in facultative methylotrophs [37]. In M.universalis, the relative expression of the dihydrofolate reductase (folA), the only enzyme essential for biosynthesis of H4F (folA) from a supplied source of folate, showed similar levels of expression in methanol or methylamine grown cells. That suggests that the overall capacity of the cells to produce the folate-cofactor did not change. If methylene-H4folate is the only product of methylamine oxidation, the cellular needs for H4F should be much higher upon growth on methylamine. Because formaldehyde is a toxic compound, it is reasonable to speculate that formaldehyde is produced only upon the depletion of the H4F pool. The Fae1 and/or Fae3 enzymes handle the cellular formaldehyde pool, and deliver C1-units into the H4MPT pathway for oxidation. However, while this scenario explains the growth deficiencies of the fae1 mutant, it fails to explain the revertants. It could be speculated that in vivo the N-methylglutamate dehydrogenase uses both H4F and H4MPT cofactors. The preferable compound is H4F, which could be substituted with H4MPT upon depletion of H4F and buildup of H4MPT (due to the lack of Fae enzymes). The latter scenario is also supported by the previous study of methylamine utilization via the NMG-pathway [16], which showed that the H4MPT-biosynthesis, rather than the Fae-driven condensation of formaldehyde, is essential for growth of Methylobacterium extorquens PA1 on methylamine.
The initial reconstruction of the methylotrophy in methylotrophic Rhodocyclaceae/Burholderiaceae has been made based on enzymatic and genetic investigations [13,38,39]. In this study we further evaluated pathways for aerobic utilization of methanol and methylamine via transcriptomics and mutagenesis. The updated reconstruction of the core metabolic pathways in M. universalis FAM5 is shown in Figure 1. It has been demonstrated that methanol oxidation via PQQ-dependent enzymes is operating as a redox arm and is coupled with ATP generation with 0.6–1 mol of ATP produced per 1 mol of methanol oxidized [40]. Thus, the growth on the C1-alcohol is predicted to be reducing-power limited. Interestingly, the transcriptomic studies show that cells of M. universalis FAM5 expressed complex III (cytochrome bc1). As the majority of the cellular energy needs are fulfilled by the first step of methane oxidation, it could be speculated that M.universalis FAM5 uses complex III to supply reducing power for carbon assimilation upon growth on methanol. Methylamine oxidation in M.universalis FAM5 might also be connected to NADPH production, via a ferredoxin-NADP reductase. An uphill electron transfer has been proposed for a number of chemolitotrophs [41,42,43], but it has never been investigated in non-phototrophic methylotrophs. Here we present the first evidence that methylotrophic bacteria might develop a mechanism for restoring reducing power upon growth on C1-compounds. The exact role of the complex III and FNR in cellular energetics will be evaluated in further investigations.

5. Conclusions

Overall, the metabolic arrangement of C1-utilization in M. universalis appears to be quite complex and to some degree redundant. Methylamine utilization is supported by two different enzymatic systems: the N-methylglutamate pathways and heme-dependent amine dehydrogenase. Genes encoding both enzymatic systems are chromosomally co-located and show a very high level of expression upon switch from growth on methanol to growth on methylamine. Like other heme-containing amine dehydrogenases, the enzyme from M. universalis FAM5 is capable of direct conversion of methylamine to formaldehyde. However, from the ΔqhpA phenotypic data it could be suggested that the enzyme is not essential, at least under the growth condition tested, for methylamine utilization.
The functional implication of the enzymatic redundancy of the C1-utilization is not apparent in the experiments described here. It could be speculated, that in the presence of heterogeneous substrates, the simultaneous activation of different enzymatic pathways might provide some benefit. However, it remains to be determined via additional simulation experiments or in-situ investigations.

Supplementary Files

Supplementary File 1

Acknowledgments

This research was supported by a grant (MCB-0604269) from the US National Science Foundation.

Author Contributions

M.G.K. designed the experiments, performed the RNA-seq experiments, compiled and analyzed the data and wrote the paper; A.L. and N.C.M.G. designed and performed mutagenesis experiments; C.M.G performed the enzymatic testing, and D.A.C.B. analyzed the RNA-seq data.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Baytshtok, V.; Kim, S.; Yu, R.; Park, H.; Chandran, K. Molecular and biokinetic characterization of methylotrophic denitrification using nitrate and nitrite as terminal electron acceptors. Water Sci. Technol. 2008, 58, 359–365. [Google Scholar] [CrossRef] [PubMed]
  2. Cai, T.; Qian, L.; Cai, S.; Chen, L. Biodegradation of benazolin-ethyl by strain Methyloversatilis sp. cd-1 isolated from activated sludge. Curr. Microbiol. 2011, 62, 570–577. [Google Scholar] [CrossRef] [PubMed]
  3. Gangwar, P.; Alam, S.I.; Bansod, S.; Singh, L. Bacterial diversity of soil samples from the western Himalayas, India. Can. J. Microbiol. 2009, 55, 564–577. [Google Scholar] [CrossRef] [PubMed]
  4. De Marco, P.; Pacheco, C.C.; Figueiredo, A.R.; Moradas-Ferreira, P. Novel pollutant-resistant methylotrophic bacteria for use in bioremediation. FEMS Microbiol. Lett 2004, 234, 75–80. [Google Scholar]
  5. Doronina, N.V.; Kaparullina, E.N.; Trotsenko, Y.A. Methyloversatilis thermotolerans sp. nov., a novel thermotolerant facultative methylotroph isolated from a hot spring. Int. J. Syst. Evol. Microbiol. 2014, 64, 158–164. [Google Scholar] [CrossRef] [PubMed]
  6. Kalyuzhnaya, M.G.; de Marco, P.; Bowerman, S.; Pacheco, C.C.; Lara, J.C.; Lidstrom, M.E.; Chistoserdova, L. Methyloversatilis universalis gen. nov., sp. nov., a novel taxon within the Betaproteobacteria represented by three methylotrophic isolates. Int. J. Syst. Evol. Microbiol. 2006, 56, 2517–2522. [Google Scholar] [CrossRef] [PubMed]
  7. Ramos-Padron, E.; Bordenave, S.; Lin, S.; Bhaskar, I.M.; Dong, X.; Sensen, C.W.; Fournier, J.; Voordouw, G.; Gieg, L.M. Carbon and sulfur cycling by microbial communities in a gypsum-treated oil sands tailings pond. Environ. Sci. Technol. 2011, 45, 439–446. [Google Scholar] [CrossRef] [PubMed]
  8. Kalyuzhnaya, M.G.; Hristova, K.; Lidstrom, M.E.; Chistoserdova, L. Characterization of a novel methanol dehydrogenase in representatives of Burkholderiales: Implications for environmental detection of methylotrophy and evidence for convergent evolution. J. Bacteriol. 2008, 190, 3817–3823. [Google Scholar] [CrossRef] [PubMed]
  9. Nakatsu, C.H.; Hristova, K.; Hanada, S.; Meng, X.Y.; Hanson, J.R.; Scow, K.M.; Kamagata, Y. Methylibium petroleiphilum gen. nov., sp. nov., a novel methyl tert-butyl ether-degrading methylotroph of the Betaproteobacteria. Int. J. Syst. Evol. Microbiol. 2006, 56, 983–989. [Google Scholar] [CrossRef] [PubMed]
  10. Smalley, N.E.; Taipale, S.; de Marco, P.; Doronina, N.V.; Kyrpides, N.; Shapiro, N.; Woyke, T.; Kalyuzhnaya, M.G. Functional and genomic diversity of methylotrophic Rhodocyclaceae: description of the new species Methyloversatilis discipulorum sp. nov. Int. J. Syst. Evol. Microbiol 2015, in press. [Google Scholar]
  11. Hristova, K.R.; Schmidt, R.; Chakicherla, A.Y.; Legler, T.C.; Wu, J.; Chain, P.S.; Scow, K.M.; Kane, S.R. Comparative transcriptome analysis of Methylibium petroleiphilum PM1 exposed to the fuel oxygenates methyl tert-butyl ether and ethanol. Appl. Environ. Microbiol. 2007, 73, 7347–7357. [Google Scholar] [CrossRef] [PubMed]
  12. Latypova, E.; Yang, S.; Wang, Y.S.; Wang, T.; Chavkin, T.A.; Hackett, M.; Schäfer, H.; Kalyuzhnaya, M.G. Genetics of the glutamate-mediated methylamine utilization pathway in the facultative methylotrophic beta-proteobacterium Methyloversatilis universalis FAM5. Mol. Microbiol. 2010, 75, 426–439. [Google Scholar] [CrossRef] [PubMed]
  13. Lu, H.; Kalyuzhnaya, M.G.; Chandran, K. Comparative proteomic analysis reveals insights into anoxic growth of Methyloversatilis universalis FAM5 on methanol and ethanol. Environ. Microbiol. 2012, 14, 2935–2945. [Google Scholar] [CrossRef] [PubMed]
  14. Suzuki, Y. Metabolism of methylamine in the tea plant (Thea sinensis L.). Biochem. J. 1973, 132, 753–763. [Google Scholar] [PubMed]
  15. Jones, J.G.; Bellion, E. In vivo 13C and 15N NMR studies of methylamine metabolism in Pseudomonas species. J. Biol. Chem. 1991, 266, 11705–11713. [Google Scholar] [PubMed]
  16. Nayak, D.D.; Marx, C.J. Methylamine utilization via the N-methylglutamate pathway in Methylobacterium extorquens PA1 involves a novel flow of carbon through C1 assimilation and dissimilation pathways. J. Bacteriol. 2014, 196, 4130–4139. [Google Scholar] [CrossRef] [PubMed]
  17. Anthony, C. The Biochemistry of Methylotrophs; Academic Press INC: London, UK, 1982; p. 431. [Google Scholar]
  18. Chen, Y.; McAleer, K.L.; Murrell, J.C. Monomethylamine as a nitrogen source for a nonmethylotrophic bacterium, Agrobacterium tumefaciens. Appl. Environ. Microbiol. 2010, 76, 4102–4104. [Google Scholar] [CrossRef] [PubMed]
  19. Chen, Y.; Scanlan, J.; Song, L.; Crombie, A.; Rahman, M.T.; Schäfer, H.; Murrell, J.C. γ-Glutamylmethylamide is an essential intermediate in the metabolism of methylamine by Methylocella silvestris. Appl. Environ. Microbiol. 2010, 76, 4530–4537. [Google Scholar] [CrossRef] [PubMed]
  20. Gruffaz, C.; Muller, E.E.; Louhichi-Jelail, Y.; Nelli, Y.R.; Guichard, G.; Bringel, F. Genes of the N-methylglutamate pathway are essential for growth of Methylobacterium extorquens DM4 with monomethylamine. Appl. Environ. Microbiol. 2014, 80, 3541–3550. [Google Scholar] [CrossRef] [PubMed]
  21. Wischer, D.; Kumaresan, D.; Johnston, A.; Khawand, M.E.L.; Stephenson, J.; Hillebrand-Voiculescu, A.M.; Chen, Y.; Murrell, C.J. Bacterial metabolism of methylated amines and identification of novel methylotrophs in Movile Cave. ISME J. 2015, 9, 195–206. [Google Scholar] [CrossRef] [PubMed]
  22. Beck, D.A.C.; McTaggart, T.L.; Setboonsarng, U.; Vorobev, A.; Goodwin, L.; Shapiro, N.; Woyke, T.; Kalyuzhnaya, M.G.; Lidstrom, M.E.; Chistoserdovaand, L. Multiphyletic origins of methylotrophy in Alphaproteobacteria, exemplified by comparative genomics of Lake Washington isolates. Environ. Microbiol. 2015, 17, 547–554. [Google Scholar] [CrossRef] [PubMed]
  23. Kittichotirat, W.; Good, N.M.; Hall, R.; Bringel, F.; Lajus, A.; Médigue, C.; Smalley, N.E.; Beck, D.; Bumgarner, R.; Vuilleumier, S.; et al. Genome sequence of Methyloversatilis universalis FAM5T, a methylotrophic representative of the order Rhodocyclales. J. Bacteriol. 2011, 193, 4541–4542. [Google Scholar] [CrossRef] [PubMed]
  24. Durham, D.R.; Perry, J.J. Purification and characterization of a heme-containing amine dehydrogenase from Pseudomonas putida. J. Bacteriol. 1978, 134, 837–843. [Google Scholar] [PubMed]
  25. Nakai, T.; Deguchi, T.; Frébort, I.; Tanizawa, K.; Okajima, T. Identification of genes essential for the biogenesis of quinohemoprotein amine dehydrogenase. Biochemistry 2014, 53, 895–907. [Google Scholar] [CrossRef] [PubMed]
  26. Takagi, K.; Torimura, M.; Kawaguchi, K.; Kano, K.; Ikeda, T. Biochemical and electrochemical characterization of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans. Biochemistry 1999, 38, 6935–6942. [Google Scholar] [CrossRef] [PubMed]
  27. Marx, C.J.; Lidstrom, M.E. Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. BioTechniques 2002, 33, 1062–1067. [Google Scholar] [PubMed]
  28. Simon, R.; Priefer, U.; Pühler, A. A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in Gram-negative bacteria. Nat Biotechnol 1984, 1, 784–791. [Google Scholar] [CrossRef]
  29. Sambrook, J.; Fritsch, E.F.; Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, USA, 1989; p. 1626. [Google Scholar]
  30. Matsen, J.B.; Yang, S.; Stein, L.Y.; Beck, D.; Kalyuzhnaya, M.G. Global molecular analyses of methane metabolism in methanotrophic Alphaproteobacterium, Methylosinus trichosporium OB3b. Part I. Transcriptomic study. Front. Microbiol. 2013, 7, 1–13. [Google Scholar]
  31. Li, H.; Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009, 25, 1754–1760. [Google Scholar] [CrossRef] [PubMed]
  32. Li, H.; Handsaker, B.; Wysoker, A.; Fennell, T.; Ruan, J.; Homer, N.; Marth, G.; Abecasis, G.; Durbin, R. The sequence alignment/map format and SAMtools. Bioinformatics 2009, 25, 2078–2079. [Google Scholar] [CrossRef] [PubMed]
  33. Anders, S.; Pyl, T.P.; Huber, W. HTSeq—A Python framework to work with high-throughput sequencing data. Bioinformatics 2014, 31, 166–169. [Google Scholar] [CrossRef] [PubMed]
  34. Storey, J.D.; Tibshirani, R. Statistical significance for genomewide studies. PNAS 2003, 100, 9440–9445. [Google Scholar] [CrossRef] [PubMed]
  35. Mortazavi, A.; Williams, B.A.; McCue, K.; Schaeffer, L.; Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008, 5, 621–628. [Google Scholar] [CrossRef] [PubMed]
  36. Crowther, G.J.; Kosály, G.; Lidstrom, M.E. Formate as the main branch point for methylotrophic metabolism in Methylobacterium extorquens AM1. J. Bacteriol. 2008, 190, 5057–5062. [Google Scholar] [CrossRef] [PubMed]
  37. Hagemeier, C.H.; Chistoserdova, L.; Lidstrom, M.E.; Thauer, R.K.; Vorholt, J.A. Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1. Eur. J. Biochem. 2000, 267, 3762–3769. [Google Scholar] [CrossRef] [PubMed]
  38. Chistoserdova, L. Modularity of methylotrophy, revisited. Environ. Microbiol. 2011, 13, 2601–2622. [Google Scholar] [CrossRef]
  39. Chistoserdova, L.; Kalyuzhnaya, M.G.; Lidstrom, M.E. The expanding world of methylotrophic metabolism. Annu. Rev. Microbiol. 2009, 63, 477–499. [Google Scholar] [CrossRef] [PubMed]
  40. Anthony, C. Methanol oxidation and growth yields in methylotrophic bacteria: A review. Acta Biotechnol. 1983, 3, 261–268. [Google Scholar] [CrossRef]
  41. Elbehti, A.; Brasseur, G.; Lemesle-Meunier, D. First evidence for existence of an uphill electron transfer through the bc1 and NADH-Q oxidoreductase complexes of the acidophilic obligate chemolithotrophic ferrous ion-oxidizing bacterium Thiobacillus ferrooxidans. J. Bacteriol. 2000, 182, 3602–3606. [Google Scholar] [CrossRef] [PubMed]
  42. Osyczka, A.; Moser, C.C.; Daldal, F.; Dutton, P.L. Reversible redox energy coupling in electron transfer chains. Nature 2004, 427, 607–612. [Google Scholar] [CrossRef] [PubMed]
  43. Sievert, S.M.; Scott, K.M.; Klotz, M.G.; Chain, P.S.; Hauser, L.J.; Hemp, J.; Hügler, M.; Land, M.; Lapidus, A.; Larimer, F.W.; et al. Genome of the epsilonproteobacterial chemolithoautotroph Sulfurimonas denitrificans. Appl. Environ. Microbiol. 2008, 74, 1145–1156. [Google Scholar] [CrossRef] [PubMed]
Back to TopTop