Ectoine degradation pathway in halotolerant methylotrophs.

BACKGROUND: Microorganisms living in saline environments are forced to regulate turgor via the synthesis of organic osmoprotective compounds. Microbial adaptation to fluctuations in external salinity includes degradation of compatible solutes. Here we have examined the biochemical pathway of degradation of the cyclic imino acid ectoine, the major osmoprotector in halotolerant methane-utilizing bacteria. METHODS: The BLAST search of the genes involved in ectoine degradation in the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z was performed with the reference sequences of Halomonas elongata. The genes for the key enzymes of the pathway were disrupted by insertion mutagenesis and the cellular metabolites in the methanol extracts of mutant cells were analyzed by HPLC. The doeA gene from Mm. alcaliphilum 20Z was heterologously expressed in Escherichia coli to identify the product of ectoine hydrolysis catalyzed by ectoine hydrolase DoeA. RESULTS: We have shown that the halotolerant methanotroph Mm. alcaliphilum 20Z possesses the doeBDAC gene cluster coding for putative ectoine hydrolase (DoeA), Nα-acetyl-L-2,4-diaminobutyrate deacetylase (DoeB), diaminobutyrate transaminase (DoeD) and aspartate-semialdehyde dehydrogenase (DoeC). The deletion of the doeA gene resulted in accumulation of the higher level of ectoine compared to the wild type strain. Nγ-acetyl-L-2,4-diaminobutyrate (Nγ-acetyl-DAB), a substrate for ectoine synthase, was found in the cytoplasm of the wild type strain. Nα-acetyl-L-2,4-diaminobutyrate (Nα-acetyl-DAB), a substrate for the DoeB enzyme, appeared in the cells as a result of exposure of the doeB mutant to low osmotic pressure. The genes for the enzymes involved in ectoine degradation were found in all aerobic methylotrophs capable of ectoine biosynthesis. These results provide the first evidence for the in vivo operation of the ectoine degradation pathway in methanotrophs and thus expand our understanding of the regulation mechanisms of bacterial osmoadaptation. CONCLUSIONS: During adaptation to the changes in external osmolarity, halophilic and halotolerant methylotrophs cleave ectoine, thereby entering the carbon and nitrogen of the compatible solute to the central metabolic pathways. The biochemical route of ectoine degradation in the halotolerant methanotroph Mm. alcaliphilum 20Z is similar to that in heterotrophic halophiles. We have shown that ectoine hydrolase DoeA in this methanotroph hydrolyzes ectoine with the formation of the only isomer: Nα-acetyl-DAB. All aerobic methylotrophs capable of ectoine biosynthesis harbor the genetic determinants for ectoine degradation.

Metabolic Features of Aerobic Methanotrophs: News and Views.

This review is focused on recent studies of carbon metabolism in aerobic methanotrophs that specifically addressed the properties, distribution and phylogeny of some of the key enzymes involved in assimilation of carbon from methane. These include enzymes involved in sugar sythesis and cleavage, conversion of intermediates of the tricarboxylic acid cycle, as well as in osmoadaptation in halotolerant methanotrophs.

Methylopila carotae sp. nov., a facultative methylotroph, isolated from a root of Daucus carota L.

An aerobic facultatively methylotrophic bacterium, designated strain Das4.1T, was isolated from a root of Daucus carota L. The cells of this strain were observed to be Gram-stain negative, asporogenous, non-motile short rods multiplying by binary fission. Strain Das4.1T can utilise methanol, methylamine and a variety of polycarbon compounds as carbon and energy sources. C1-compounds were found to be assimilated via the isocitrate lyase-negative variant of the serine pathway. On medium with 0.5% methanol, growth of strain Das4.1T was observed at pH 5.5-9.0 (optimum, pH 6.0-7.0) and 18-37 °C (optimum, 24-29 °C) and in the presence of 0-2% (w/v) NaCl (optimum, 0.05%). Cells are catalase and oxidase positive and synthesise indole from L-tryptophan. The major fatty acids of methanol-grown cells were identified as C18:1ω7c, C18:0 and 11-methyl-C18:1ω7c. The predominant phospholipids were found to be phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The major respiratory quinone was identified as Q-10. The DNA G + C content of strain Das4.1T was determined to be 67.3 mol% (Tm). Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Das4.1T belongs to the genus Methylopila and shows high sequence similarity to Methylopila oligotropha 2395AT (98.4%) and Methylopila capsulata IM1T (98.0%). However, the DNA-DNA relatedness of strain Das4.1T with M. oligotropha 2395AT was only 22 ± 3%. Based on genotypic, chemotaxonomic and physiological characterisation, the isolate can be classified as a novel species of the genus Methylopila, for which the name Methylopila carotae sp. nov. is proposed. The type strain is Das4.1T (= VKM B-3244T = CCUG 72399T).

ATP- and Polyphosphate-Dependent Glucokinases from Aerobic Methanotrophs.

The genes encoding adenosine triphosphate (ATP)- and polyphosphate (polyP)-dependent glucokinases (Glk) were identified in the aerobic obligate methanotroph Methylomonas sp. 12. The recombinant proteins were obtained by the heterologous expression of the glk genes in Esherichia coli. ATP-Glk behaved as a multimeric protein consisting of di-, tri-, tetra-, penta- and hexamers with a subunit molecular mass of 35.5 kDa. ATP-Glk phosphorylated glucose and glucosamine using ATP (100% activity), uridine triphosphate (UTP) (85%) or guanosine triphosphate (GTP) (71%) as a phosphoryl donor and exhibited the highest activity in the presence of 5 mM Mg2+ at pH 7.5 and 65 °C but was fully inactivated after a short-term incubation at this temperature. According to a gel filtration in the presence of polyP, the polyP-dependent Glk was a dimeric protein (2 × 28 kDa). PolyP-Glk phosphorylated glucose, mannose, 2-deoxy-D-glucose, glucosamine and N-acetylglucosamine using polyP as the phosphoryl donor but not using nucleoside triphosphates. The Km values of ATP-Glk for glucose and ATP were about 78 µM, and the Km values of polyP-Glk for glucose and polyP(n=45) were 450 and 21 µM, respectively. The genomic analysis of methanotrophs showed that ATP-dependent glucokinase is present in all sequenced methanotrophs, with the exception of the genera Methylosinus and Methylocystis, whereas polyP-Glks were found in all species of the genus Methylomonas and in Methylomarinum vadi only. This work presents the first characterization of polyphosphate specific glucokinase in a methanotrophic bacterium.

Individual stages of bacterial dichloromethane degradation mapped by carbon and chlorine stable isotope analysis.

The fractionation of carbon and chlorine stable isotopes of dichloromethane (CH2Cl2, DCM) upon dechlorination by cells of the aerobic methylotroph Methylobacterium extorquens DM4 and by purified DCM dehalogenases of the glutathione S-transferase family was analyzed. Isotope effects for individual steps of the multi-stage DCM degradation process, including transfer across the cell wall from the aqueous medium to the cell cytoplasm, dehalogenase binding, and catalytic reaction, were considered. The observed carbon and chlorine isotope fractionation accompanying DCM consumption by cell supensions and enzymes was mainly determined by the breaking of CCl bonds, and not by inflow of DCM into cells. Chlorine isotope effects of DCM dehalogenation were initially masked in high density cultures, presumably due to inverse isotope effects of non-specific DCM oxidation under conditions of oxygen excess. Glutathione cofactor supply remarkably affected the correlation of variations of DCM carbon and chlorine stable isotopes (Δδ13C/Δδ37Cl), increasing corresponding ratio from 7.2-8.6 to 9.6-10.5 under conditions of glutathione deficiency. This suggests that enzymatic reaction of DCM with glutathione thiolate may involve stepwise breaking and making of bonds with the carbon atom of DCM, unlike the uncatalyzed reaction, which is a one-stage process, as shown by quantum-chemical modeling.

Distribution of 1-aminocyclopropane-1-carboxylate deaminase and D-cysteine desulfhydrase genes among type species of the genus Methylobacterium.

The presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase determines the ability of bacteria to increase the resistance of plants to various types of stress. The genes of ACC deaminase (acdS) and the closely related enzyme D-cysteine desulfhydrase (dcyD) were searched in type strains of various representatives of the genus Methylobacterium. Using PCR screening and in silico searching in the available complete genome sequences of type strains, the genes were found in 28 of 48 species of the genus. Phylogenetic analysis of amino acid sequences of proteins revealed two large groups of sequences of the AcdS protein and one of the DcyD protein. The distribution of these groups correlates well with the phylogenetic tree based on the sequences of the 16S rRNA genes, which apparently indicates a different evolutionary adaptation to association with plants in the representatives of these groups. For the first time for aerobic methylotrophs it was demonstrated that the gene dcyD encodes D-cysteine desulfhydrase by cloning and recombinant protein characterization.

The genes and enzymes of sucrose metabolism in moderately thermophilic methanotroph Methylocaldum szegediense O12.

Four enzymes involved in sucrose metabolism: sucrose phosphate synthase (Sps), sucrose phosphate phosphatase (Spp), sucrose synthase (Sus) and fructokinase (FruK), were obtained as his-tagged proteins from the moderately thermophilic methanotroph Methylocaldum szegediense O12. Sps, Spp, FruK and Sus demonstrated biochemical properties similar to those of other bacterial counterparts, but the translated amino acid sequences of Sps and Spp displayed high divergence from the respective microbial enzymes. The Sus of M. szegediense O12 catalyzed the reversible reaction of sucrose cleavage in the presence of ADP or UDP and preferred ADP as a substrate, thus implying a connection between sucrose and glycogen metabolism. Sus-like genes were found only in a few methanotrophs, whereas amylosucrase was generally used in sucrose cleavage in this group of bacteria. Like other microbial fructokinases, FruK of M. szegediense O12 showed a high specificity to fructose.

Ancylobacter sonchi sp. nov., a novel methylotrophic bacterium frоm roots of Sonchus arvensis L.

An aerobic facultatively methylotrophic bacterium was isolated from roots of Sonchus arvensis L. and designated strain OsotT The cells of this strain were Gram-stain-negative, asporogenous, motile short rods multiplying by binary fisson. They utilized methanol, methylamines and a variety of polycarbon compounds as the carbon and energy sources. Methanol was assimilated after sequential oxidation to formaldehyde and CO2 via the ribulose bisphosphate pathway. The organism grew optimally at 22-29 °C and pH 7.5-8.0. The dominant phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and diphosphatidylglycerol (cardiolipin). The major cellular fatty acids of strain OsotT cells grown in R2A medium were C18 : 1ω7c (49.0 %), C19 : 0ω8c cyclo (38.3 %) and C16 : 0 (8.4 %). The major ubiquinone was Q-10. The DNA G+C content of strain OsotT was 66.1 mol% (Tm). On the basis of 16S rRNA gene sequence analysis strain OsotT is phylogenetically related to the members of genus Ancylobacter (97.1-98.8 % sequence similarity). Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (27-29 %) with type strains of the genus Ancylobacter, the novel isolate is classified as a new species of this genus and named Ancylobacter sonchi sp. nov.; the type strain is OsotT (=VKM B-3145T=JCM 32039T).

Draft Genome Sequences of Two Gammaproteobacterial Methanotrophs Isolated from Rice Ecosystems.

The genomes of the aerobic methanotrophs "Methyloterricola oryzae" strain 73aT and Methylomagnum ishizawai strain 175 were sequenced. Both strains were isolated from rice plants. Methyloterricola oryzae strain 73aT represents the first isolate of rice paddy cluster I, and strain 175 is the second representative of the recently described genus Methylomagnum.

Methylobacillus methanolivorans sp. nov., a novel non-pigmented obligately methylotrophic bacterium.

Three strains of obligately methylotrophic Betaproteobacteria (ZT, SP and M3) with the ribulose monophosphate pathway of C1 assimilation are described. The isolates were strictly aerobic, Gram-stain-negative, asporogenous, motile (strains ZT and M3) or non-motile (strain SP) rods that multiplied by binary fisson, and were mesophilic and neutrophilic. All three strains utilized methanol but only strains SP and M3 utilized methylamine as carbon and energy sources. The prevailing cellular fatty acids were straight-chain saturated C16 : 0 and unsaturated C16 : 1ω7c acids. The major ubiquinone was Q-8. The predominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Ammonia was assimilated by glutamate dehydrogenase. The DNA G+C contents of strains ZT, SP and M3 were 51.0, 52.0 and 52.0 mol% (Tm), respectively. Levels of 16S rRNA gene sequence similarity between the three strains were very high (99.9-100 %), and they shared high levels of DNA-DNA relatedness (88-98 %). Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (19-30 %) with the type strains of the genus Methylobacillus, the novel isolates ZT, SP and M3 are classified as representing a novel species of this genus, for which the name Methylobacillus methanolivorans sp. nov. is proposed. The type strain is ZT (=VKM B-3037T=JCM 31401T=CCUG 68999T).

The properties and potential metabolic role of glucokinase in halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z.

Aerobic bacteria utilizing methane as the carbon and energy source do not use sugars as growth substrates but possess the gene coding for glucokinase (Glk), an enzyme converting glucose into glucose 6-phosphate. Here we demonstrate the functionality and properties of Glk from an obligate methanotroph Methylomicrobium alcaliphilum 20Z. The recombinant Glk obtained by heterologous expression in Escherichia coli was found to be close in biochemical properties to other prokaryotic Glks. The homodimeric enzyme (2 × 35 kDa) catalyzed ATP-dependent phosphorylation of glucose and glucosamine with nearly equal activity, being inhibited by ADP (K i = 2.34 mM) but not affected by glucose 6-phosphate. Chromosomal deletion of the glk gene resulted in a loss of Glk activity and retardation of growth as well as in a decrease of intracellular glycogen content. Inactivation of the genes encoding sucrose phosphate synthase or amylosucrase, the enzymes involved in glycogen biosynthesis via sucrose as intermediate, did not prevent glycogen accumulation. In silico analysis revealed glk orthologs predominantly in methanotrophs harboring glycogen synthase genes. The data obtained suggested that Glk is implicated in the regulation of glycogen biosynthesis/degradation in an obligate methanotroph.

Draft Genome Sequence of Methylophaga muralis Bur 1, a Haloalkaliphilic (Non-Methane-Utilizing) Methylotroph Isolated from a Soda Lake.

The draft genome sequence of Methylophaga muralis strain Bur 1 (VKM B-3046T), a non-methane-utilizing methylotroph isolated from a soda lake, is reported here. Strain Bur 1 possesses genes for methanol and methylamine (methylamine dehydrogenase and N-methylglutamate pathway) oxidation. Genes for the biosynthesis of ectoine were also found.

Draft Genome Sequence of Methyloligella halotolerans С2T, a New Halotolerant Methylotroph, Accumulating Poly-3-Hydroxybutyrate and Ectoine.

Methyloligella halotolerans С2T is a moderate halophilic obligate methylotroph, accumulating ultra-high-molecular-weight poly-3-hydroxybutyrate (up to 8 to 10 MDa) from methanol. Here we report a draft genome and annotation of Methyloligella halotolerans C2T (VKM B-2706T = CCUG 61687T = DSM 25045T).

Draft Genome Sequences of Gammaproteobacterial Methanotrophs Isolated from Marine Ecosystems.

The genome sequences of Methylobacter marinus A45, Methylobacter sp. strain BBA5.1, and Methylomarinum vadi IT-4 were obtained. These aerobic methanotrophs are typical members of coastal and hydrothermal vent marine ecosystems.

Acetate kinase-an enzyme of the postulated phosphoketolase pathway in Methylomicrobium alcaliphilum 20Z.

Recombinant acetate kinase (AcK) was obtained from the aerobic haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z by heterologous expression in Escherichia coli and purification by affinity chromatography. The substrate specificity, the kinetics and oligomeric state of the His6-tagged AcK were determined. The M. alcaliphilum AcK (2 × 45 kDa) catalyzed the reversible phosphorylation of acetate into acetyl phosphate and exhibited a dependence on Mg(2+) or Mn(2+) ions and strong specificity to ATP/ADP. The enzyme showed the maximal activity and high stability at 70 °C. AcK was 20-fold more active in the reaction of acetate synthesis compared to acetate phosphorylation and had a higher affinity to acetyl phosphate (K m 0.11 mM) than to acetate (K m 5.6 mM). The k cat /K m ratios indicated that the enzyme had a remarkably high catalytic efficiency for acetate and ATP formation (k cat/K m = 1.7 × 10(6)) compared to acetate phosphorylation (k cat/K m = 2.5 × 10(3)). The ack gene of M. alcaliphilum 20Z was shown to be co-transcribed with the xfp gene encoding putative phosphoketolase. The Blast analysis revealed the ack and xfp genes in most genomes of the sequenced aerobic methanotrophs, as well as methylotrophic bacteria not growing on methane. The distribution and metabolic role of the postulated phosphoketolase shunted glycolytic pathway in aerobic C1-utilizing bacteria is discussed.

Draft genomes of gammaproteobacterial methanotrophs isolated from terrestrial ecosystems.

Genome sequences of Methylobacter luteus, Methylobacter whittenburyi, Methylosarcina fibrata, Methylomicrobium agile, and Methylovulum miyakonense were generated. The strains represent aerobic methanotrophs typically isolated from various terrestrial ecosystems.

Draft Genome Sequence of the Moderately Halophilic Methanotroph Methylohalobius crimeensis Strain 10Ki.

Methylohalobius crimeensis strain 10Ki is a moderately halophilic aerobic methanotroph isolated from a hypersaline lake in the Crimean Peninsula, Ukraine. This organism has the highest salt tolerance of any cultured methanotroph. Here, we present a draft genome sequence of this bacterium.

Methylobrevis pamukkalensis gen. nov., sp. nov., a halotolerant restricted facultative methylotroph isolated from saline water.

An aerobic halotolerant restricted facultatively methylotrophic bacterium was isolated from a saline hot spring in Pamukkale, Turkey, and designated strain PK2(T). The cells of this strain were Gram-stain-negative, asporogenous, motile short rods multiplying by binary fission. They utilized methanol, methylamine and mannitol as carbon and energy sources. The organism grew optimally at 30 °C in media containing 85 mM NaCl and at pH 7.5-8.0. C1 compounds were assimilated via the isocitrate-lyase-positive variant of the serine pathway. Poly-ß-hydroxybutyrate and the compatible solute ectoine were found in the cells. The dominant phospholipids were phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The major cellular fatty acids of methanol-grown cells were C(18 : 1)ω7 and C(16 : 1)ω7c. The main ubiquinone was Q-10. The DNA G+C content was 67.9 mol% (T(m)). The 16S rRNA gene sequence suggests that strain PK2(T) is affiliated with the order Rhizobiales within the class Alphaproteobacteria , being most closely related to Mesorhizobium gobiense CCBAU 83330(T) (94% similarity). A novel genus and species, Methylobrevis pamukkalensis gen. nov., sp. nov., is proposed on the basis of phenotypic and genotypic data, with PK2(T) (VKM B-2849(T) = JCM 30229(T)) as the type strain.

Sucrose metabolism in halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.

Sucrose accumulation has been observed in some methylotrophic bacteria utilizing methane, methanol, or methylated amines as a carbon and energy source. In this work, we have investigated the biochemical pathways for sucrose metabolism in the model halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. The genes encoding sucrose-phosphate synthase (Sps), sucrose-phosphate phosphatase (Spp), fructokinase (FruK), and amylosucrase (Ams) were co-transcribed and displayed similar expression levels. Functional Spp and Ams were purified after heterologous expression in Escherichia coli. Recombinant Spp exhibited high affinity for sucrose-6-phosphate and stayed active at very high levels of sucrose (K i  = 1.0 ± 0.6 M). The recombinant amylosucrase obeyed the classical Michaelis-Menten kinetics in the reactions of sucrose hydrolysis and transglycosylation. As a result, the complete metabolic network for sucrose biosynthesis and re-utilization in the non-phototrophic organism was reconstructed for the first time. Comparative genomic studies revealed analogous gene clusters in various Proteobacteria, thus indicating that the ability to produce and metabolize sucrose is widespread among prokaryotes.

Role of NAD⁺-Dependent Malate Dehydrogenase in the Metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b.

We have expressed the l-malate dehydrogenase (MDH) genes from aerobic methanotrophs Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b as his-tagged proteins in Escherichia coli. The substrate specificities, enzymatic kinetics and oligomeric states of the MDHs have been characterized. Both MDHs were NAD⁺-specific and thermostable enzymes not affected by metal ions or various organic metabolites. The MDH from M. alcaliphilum 20Z was a homodimeric (2 × 35 kDa) enzyme displaying nearly equal reductive (malate formation) and oxidative (oxaloacetate formation) activities and higher affinity to malate (Km = 0.11 mM) than to oxaloacetate (Km = 0.34 mM). The MDH from M. trichosporium OB3b was homotetrameric (4 × 35 kDa), two-fold more active in the reaction of oxaloacetate reduction compared to malate oxidation and exhibiting higher affinity to oxaloacetate (Km = 0.059 mM) than to malate (Km = 1.28 mM). The kcat/Km ratios indicated that the enzyme from M. alcaliphilum 20Z had a remarkably high catalytic efficiency for malate oxidation, while the MDH of M. trichosporium OB3b was preferable for oxaloacetate reduction. The metabolic roles of the enzymes in the specific metabolism of the two methanotrophs are discussed.