Interchangeability of class I and II fumarases in an obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z.

The methanotrophic bacterium Methylotuvimicrobium alcaliphilum 20Z is an industrially promising candidate for bioconversion of methane into value-added chemicals. Here, we have study the metabolic consequences of the breaking in the tricarboxylic acid (TCA) cycle by fumarase knockout. Two fumarases belonging to non-homologous class I and II fumarases were obtained from the bacterium by heterologous expression in Escherichia coli. Class I fumarase (FumI) is a homodimeric enzyme catalyzing the reversible hydration of fumarate and mesaconate with activities of ~94 and ~81 U mg-1 protein, respectively. The enzyme exhibited high activity under aerobic conditions, which is a non-typical property for class I fumarases characterized to date. The calculation of kcat/S0.5 showed that the enzyme works effectively with either fumarate or mesaconate, but it is almost four times less specific to malate. Class II fumarase (FumC) has a tetrameric structure and equal activities of both fumarate hydration and malate dehydration (~45 U mg-1 protein). Using mutational analysis, it was shown that both forms of the enzyme are functionally interchangeable. The triple mutant strain 20Z-3E (ΔfumIΔfumCΔmae) deficient in the genes encoding the both fumarases and the malic enzyme accumulated 2.6 and 1.1 mmol g-1 DCW fumarate in the medium when growing on methane and methanol, respectively. Our data suggest the redundancy of the metabolic node in the TCA cycle making methanotroph attractive targets for modification, including generation of strains producing the valuable metabolites.

Neurotoxicity of Silver Nanoparticles and Non-Linear Development of Adaptive Homeostasis with Age.

For the first time in the world, the behavioral functions of laboratory mammals exposed to silver nanoparticles were studied with regard to age. Silver nanoparticles coated with polyvinylpyrrolidone with a size of 8.7 nm were used in the present research as a potential xenobiotic. Elder mice adapted to the xenobiotic better than the younger animals. Younger animals demonstrated more drastic anxiety than the elder ones. A hormetic effect of the xenobiotic in elder animals was observed. Thus, it is concluded that adaptive homeostasis non-linearly changes with age increase. Presumably, it may improve during the prime of life and start to decline just after a certain stage. This work demonstrates that age growth is not directly conjugated with the organism fading and pathology development. Oppositely, vitality and resistance to xenobiotics may even improve with age at least until the prime of life.

Enzymes of an alternative pathway of glucose metabolism in obligate methanotrophs.

Aerobic methanotrophic bacteria utilize methane as a growth substrate but are unable to grow on any sugars. In this study we have shown that two obligate methanotrophs, Methylotuvimicrobium alcaliphilum 20Z and Methylobacter luteus IMV-B-3098, possess functional glucose dehydrogenase (GDH) and gluconate kinase (GntK). The recombinant GDHs from both methanotrophs were homotetrameric and strongly specific for glucose preferring NAD+ over NADP+. GDH from Mtm. alcaliphilum was most active at pH 10 (Vmax = 95 U/mg protein) and demonstrated very high Km for glucose (91.8 ± 3.8 mM). GDH from Mb. luteus was most active at pH 8.5 (Vmax = 43 U/mg protein) and had lower Km for glucose (16 ± 0.6 mM). The cells of two Mtm. alcaliphilum double mutants with deletions either of the genes encoding GDH and glucokinase (gdh─/glk─) or of the genes encoding gluconate kinase and glucokinase (gntk─/glk─) had the lower glycogen level and the higher contents of intracellular glucose and trehalose compared to the wild type strain. The gntk─/glk─ knockout mutant additionally accumulated gluconic acid. These data, along with bioinformatics analysis, demonstrate that glycogen derived free glucose can enter the Entner-Doudoroff pathway or the pentose phosphate cycle in methanotrophs, bypassing glycolysis via the gluconate shunt.

Symposium report: emerging threats for human health - impact of socioeconomic and climate change on zooanthroponosis in the Republic of Sakha (Yakutia), Russia.

Population growth, socio-cultural and economic changes as well as technological progress have an immediate impact on the environment and human health in particular. Our steadily rising needs of resources increase the pressure on the environment and narrow down untainted habitats for plants and wild animals. Balance and resilience of ecosystems are further threatened by climate change, as temperature and seasonal shifts increase the pressure for all species to find successful survival strategies. Arctic and subarctic regions are especially vulnerable to climate change, as thawing of permafrost significantly transforms soil structures, vegetation and habitats. With rising temperature, the risk of zoonotic diseases in the Republic of Sakha (Yakutia) has also increased. As vegetation periods prolong and habitats broaden, zoonotic pathogens and their vectors find more favourable living conditions. Moreover, permafrost degradation may expose historic burial grounds and allow for reviving the vectors of deadly infections from the past. To assess the current state of knowledge and emerging risks in the light of the "One Health" concept, a German-Russian Symposium took place on 13 August 2018 in Yakutsk, Russian Federation. This symposium report presents the main findings generated from presentations and discussions.

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.

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.

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 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.

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.

Bifunctional sucrose phosphate synthase/phosphatase is involved in the sucrose biosynthesis by Methylobacillus flagellatus KT.

The aerobic obligate methylotroph Methylobacillus flagellatus KT was shown to synthesize sucrose in the presence of 0.5-2% NaCl in the growth medium. In the genome of this bacterium, an open reading frame (ORF) encoding a predicted 84-kD polypeptide homologous to the plant and cyanobacterial sucrose phosphate synthases (SPSs) was found. Using heterologous expression of the putative sps gene in Escherichia coli, followed by affinity chromatography, pure recombinant protein SPS-His6 was obtained. The enzyme catalyzed two reactions: conversion of fructose 6-phosphate and UDP-glucose into sucrose 6-phosphate and hydrolysis of sucrose 6-phosphate to sucrose. The bifunctional sucrose phosphate synthase/phosphatase (SPS/SPP) was a 340 kDa homotetrameric Mg(2+) -dependent enzyme activated by fructose 1,6-phosphate2 and ATP but inhibited by glucose 6-phosphate, fructose 1-phosphate, AMP and inorganic phosphate. The amino acid sequence of the protein had a C-terminal domain homologous to SPPs. This correlated with the absence of the spp gene in the M. flagellatus chromosome. The ORFs homologous to the M. flagellatus SPS were found in the genomes of another obligate methylotroph Methylovorus glucosetrophus as well as the lithoautotrophic bacteria Acidithiobacillus ferrooxidans, Nitrosomonas europaea and Nitrosospira multiformis whose genomes lacked the spp genes. Thus, data extending the knowledge of biochemical properties of bacterial SPSs have been obtained.

Diversity and phylogeny of the ectoine biosynthesis genes in aerobic, moderately halophilic methylotrophic bacteria.

The genes of ectoine biosynthesis pathway were identified in six species of aerobic, slightly halophilic bacteria utilizing methane, methanol or methylamine. Two types of ectoine gene cluster organization were revealed in the methylotrophs. The gene cluster ectABC coding for diaminobutyric acid (DABA) acetyltransferase (EctA), DABA aminotransferase (EctB) and ectoine synthase (EctC) was found in methanotrophs Methylobacter marinus 7C and Methylomicrobium kenyense AMO1(T). In methanotroph Methylomicrobium alcaliphilum ML1, methanol-utilizers Methylophaga thalassica 33146(T) , Methylophaga alcalica M8 and methylamine-utilizer Methylarcula marina h1(T), the genes forming the ectABC-ask operon are preceded by ectR, encoding a putative transcriptional regulatory protein EctR. Phylogenetic relationships of the Ect proteins do not correlate with phylogenetic affiliation of the strains, thus implying that the ability of methylotrophs to produce ectoine is most likely the result of a horizontal transfer event.

Genes and enzymes of ectoine biosynthesis in halotolerant methanotrophs.

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) is a widely distributed compatible solute accumulated by halophilic and halotolerant microorganisms to prevent osmotic stress in highly saline environments. Ectoine as a highly water keeping compound stabilizing biomolecules and whole cells can be used in scientific work, cosmetics, and medicine. Detailed understanding of the organization/regulation of the ectoine biosynthetic pathway in various producers is an active area of research. Here we review current knowledge on some genetic and enzymatic aspects of ectoine biosynthesis in halophilic and halotolerant methanotrophs. By using PCR methodology, the genes coding for the specific enzymes of ectoine biosynthesis, diaminobutyric acid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA), and ectoine synthase (EctC), were identified in several methanotrophic species. Organization of these genes in either ectABC or ectABC-ask operons, the latter additionally encoding aspartate kinase isozyme (Ask), correlated well with methanotroph halotolerance and intracellular ectoine level. A new gene, ectR1 encoding the MarR-like transcriptional regulatory protein EctR1, negatively controlling transcription of ectoine biosynthetic genes was found upstream of ectABC-ask operon in Methylomicrobium alcaliphilum 20Z. The ectR-like genes were also found in halotolerant methanol utilizers Methylophaga alcalica and Methylophaga thalassica as well as in several genomes of nonmethylotrophic species. The His(6)-tagged DABA acetyltransferases from Mm. alcaliphilum, M. alcalica, and M. thalassica were purified and the enzyme properties were found to correlate with the ecophysiologies of these bacteria. All these discoveries should be very helpful for better understanding the biosynthetic mechanism of this important natural compound, and for the targeted metabolic engineering of its producers.

Identification and characterization of EctR1, a new transcriptional regulator of the ectoine biosynthesis genes in the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.

Genes encoding key enzymes of the ectoine biosynthesis pathway in the halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z have been shown to be organized into an ectABC-ask operon. Transcription of the ect operon is initiated from two promoters, ectAp(1) and ectAp(2) (ectAp(1)p(2)), similar to the sigma(70)-dependent promoters of Escherichia coli. Upstream of the gene cluster, an open reading frame (ectR1) encoding a MarR-like transcriptional regulator was identified. Investigation of the influence of EctR1 on the activity of the ectAp(1)p(2) promoters in wild-type M. alcaliphilum 20Z and ectR1 mutant strains suggested that EctR1 is a negative regulator of the ectABC-ask operon. Purified recombinant EctR1-His(6) specifically binds as a homodimer to the putative -10 motif of the ectAp(1) promoter. The EctR1 binding site contains a pseudopalindromic sequence (TATTTAGT-GT-ACTATATA) composed of 8-bp half-sites separated by 2 bp. Transcription of the ectR1 gene is initiated from a single sigma(70)-like promoter. The location of the EctR1 binding site between the transcriptional and translational start sites of the ectR1 gene suggests that EctR1 may regulate its own expression. The data presented suggest that in Methylomicrobium alcaliphilum 20Z, EctR1-mediated control of the transcription of the ect genes is not the single mechanism for the regulation of ectoine biosynthesis.

Characterization of the pyrophosphate-dependent 6-phosphofructokinase from Methylococcus capsulatus Bath.

An active pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) from the thermotolerant methanotroph Methylococcus capsulatus Bath, containing a six-residue polyhistidine tag, was characterized. The enzyme was homodimeric (2 x 45 kDa), nonallosteric and most active at pH 7.0. PPi-PFK catalyzed reactions of PPi-dependent phosphorylation of fructose-6-phosphate (F-6-P) (K(m) 2.27 mM and V(max) 7.6 U mg(-1) of protein), sedoheptulose-7-phosphate (K(m) 0.027 mM and V(max) 31 U mg(-1)) and ribulose-5-phosphate. In the reaction with F-6-P, the apparent K(m) for PPi was 0.027 mM, while in the reverse reaction, K(m) for orthophosphate was 8.69 mM and that for fructose-1,6-bisphosphate 0.328 mM (V(max) 9.0 U mg(-1)). Phylogenetically, M. capsulatus PPi-PFK was most similar to PPi-PFKs from the lithoautotrophic ammonia oxidizers Nitrosomonas europaea (74.0%), Nitrosospira multiformis (73.6%) and Betaproteobacterial methylotroph Methylibium petroleiphilum PM1 (71.6% identity). Genes coding PPi-PFK and a putative V-type H(+)-translocating pyrophosphatase (H(+)-PPi-ase) were cotranscribed as an operon. The potential significance of the PPi-PFK for regulation of carbon and energy fluxes in M. capsulatus Bath is discussed.

Characterization of the recombinant diaminobutyric acid acetyltransferase from Methylophaga thalassica and Methylophaga alcalica.

Diaminobutyric acid acetyltransferase (EctA) catalyzes the acetylation of diaminobutyric acid to gamma-N-acetyl-alpha,gamma-diaminobutyrate with acetyl coenzyme A. This is the second reaction in the ectoine biosynthetic pathway. The recombinant EctA proteins were purified from two moderately halophilic methylotrophic bacteria: Methylophaga thalassica ATCC 33146T and Methylophaga alcalica ATCC 35842T. EctA found in both methylotrophs is a homodimer with a subunit molecular mass of c. 20 kDa and had similar properties with respect to the optimum temperature for activity (30 degrees C), Km for diaminobutyrate (370 or 375 microM) and the absence of requirements for divalent metal ions. The enzyme from M. thalassica exhibited a lower pH optimum and was inhibited both by sodium carbonates and by high ionic strength but to a lesser extent by copper ions.

Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph "Methylomicrobium alcaliphilum 20Z".

The genes involved in biosynthesis of the major compatible solute ectoine (1,4,5,6-tetrahydro-2-methylpyrimidine carboxylic acid) in halotolerant obligate methanotroph "Methylomicrobium alcaliphilum 20Z" were studied. The complete nucleotide sequences of the structural genes encoding L: -aspartokinase (Ask), L-2,4-diaminobutyric acid transaminase (EctB), L-2,4-diaminobutyric acid acetyltransferase (EctA), and L-ectoine synthase (EctC) were defined and shown to be transcribed as a single operon ectABCask. Phylogenetic analysis revealed high sequence identities (34-63%) of the Ect proteins to those from halophilic heterotrophs with the highest amino acid identities being to Vibrio cholerae enzymes. The chromosomal DNA fragment from "M. alcaliphilum 20Z" containing ectABC genes and putative promoter region was expressed in Escherichia coli. Recombinant cells could grow in the presence of 4% NaCl and synthesize ectoine. The data obtained suggested that despite the ectoine biosynthesis pathway being evolutionary well conserved with respect to the genes and enzymes involved, some differences in their organization and regulation could occur in various halophilic bacteria.