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.
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.
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.
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.
Citation: Reshetnikov AS, Rozova ON, Trotsenko YA, But SY, Khmelenina VN, Mustakhimov II (2020) Ectoine degradation pathway in halotolerant methylotrophs. PLoS ONE 15(4): e0232244. https://doi.org/10.1371/journal.pone.0232244
Editor: Ivan A. Berg, University of Münster, GERMANY
Received: July 5, 2019; Accepted: April 10, 2020; Published: April 30, 2020
Copyright: © 2020 Reshetnikov et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This research was supported by the Russian Science Foundation (#18-14-00326). PI - Yuri A. Trotsenko.
Competing interests: The authors have declared that no competing interests exist.
Microorganisms living in saline environments are enforced to synthesize highly water-soluble organic compounds and accumulate them at concentrations sufficient to maintain cell turgor. Ectoine (1,4,5,6,tetra-2-methyl-4-pyrimidinecarboxylic acid) and its derivative hydroxyectoine are compatible solutes in many halophilic bacteria and some Archaea [1–6]. In addition to balancing the osmotic pressure between the cytoplasm and its surroundings, ectoines can exert beneficial effects on microbial metabolism as they stabilize protein folding and protect whole cells against various stresses such as UV radiation, freezing, drying, and high temperatures [7–9]. Ectoine accumulation by de novo synthesis is an essential trait of halotolerant methylotrophs, the bacteria utilizing methane or its oxidized or substituted derivatives as carbon and energy sources [10, 11]. The aerobic methanotroph Mm. alcaliphilum 20Z isolated from a Siberian soda lake is a halotolerant bacterium able to grow at salinity up to 10% NaCl . It synthesizes ectoine, glutamate and sucrose as the main compatible solutes. The genes encoding the enzymes for ectoine biosynthesis are localized in the ectABC-ask operon transcribed from two σ70-dependent promoters under the control of the MarR-family regulator EctR . The biosynthesis of ectoine can also be regulated at the level of enzyme activities .
Fluctuation in salinity is a ubiquitous stress factor in many natural habitats of microorganisms. When exposed to low external osmolarity, microbial cells can modulate turgor by the expulsion of previously accumulated compatible solutes through the transient opening of mechanosensitive channels .
Many microorganisms can use the released compatible solutes as osmostress protectants or as nutrients . In the Gram-negative bacteria Chromohalobacter salexigens, Sinorhizobium meliloti and Ruegeria pomeroyi DSS-3, utilization of ectoine and hydroxyectoine has been found to proceed via the pathway where the key constituents are the enzymes (EutABCDE; eut: ectoine utilization) [16–18] catalyzing the conversion of hydroxyectoine to ectoine (EutABC) and ectoine decomposition to acetate and diaminobutyrate (EutDE). It also includes the genes for specific diaminobutyrate aminotransferase and aspartate semialdehyde dehydrogenase catalyzing the synthesis of aspartate. The genes for these enzymes are co-transcribed with the genes for transport systems: either a binding-protein-dependent ABC (EhuABCD; ehu: ectoine-hydroxyectoine uptake) transporter or a 5-hydroxyectoine/ectoine-specific TRAP transporter [18–21]. In the salt-tolerant bacterium Halomonas elongata, a gene cluster related to those previously involved in ectoine degradation in S. meliloti and R. pomeroyi DSS-3 has been identified . Four enzymes of the pathway encoded by the doeAB and doeCD genes (doe: degradation of ectoine) catalyze ectoine hydrolysis to N-acetyl-DAB and further deacetylation of the latter with the formation of diaminobutyrate and acetate . DAB can either flow off to aspartate or re-enter the ectoine synthesis pathway, forming a cycle of ectoine synthesis and degradation.
However, the ectoine degradation ability in methylotrophic ectoine producers has not been investigated thus far. In this paper we have shown, on the basis of genomic and mutational analysis, that the ectoine degradation pathway is functional in the halotolerant methanotroph Mm. alcaliphilum 20Z.
Materials and methods
Bacteria and growth conditions
Methylotuvimicrobium alcaliphilum 20Z (earlier Methylomicrobium) (NCIMB 14124T = VKM B-2133T) was routinely cultivated at 30°C in a methane–air atmosphere (1 : 1) or in the presence of 0.3% methanol (v/v) in the nitrate-containing 2×P medium supplemented with 0.1 M NaHCO3 and different amounts of NaCl as described . Escherichia coli S17-1 and Rosetta (DE3) obtained from Stratagene (USA) were routinely grown at 37°C in the LB medium . Gentamicin (10 μg/ml) or kanamycin (100 μg/ml) was added to the growth medium if required.
Cloning and expression of the DoeA and DoeB enzymes in E. coli
The DNA from Mm. alcaliphilum was prepared with a ZymoResearch Fungal/Bacterial DNA MiniPrep™ kit (Irvine, USA). The genes coding for the putative DoeA (doeA, MEALZ_3978) and DoeB (doeB, MEALZ_3976) were amplified using the primers listed in S1 Table. The PCR-products were treated with endonucleases NdeI and HindIII and ligated in expression vector pET22b opened at the respective restriction sites. The resulting plasmids were transferred into E. coli Rosetta (DE3) and protein expression was induced by 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma-Aldrich, USA) added in the logarithmic growth phase (OD600 = 0.6–0.8). After 18-h incubation at 18°C, cells were collected by centrifugation (5,000 g for 30 min, 4°C) and disrupted in an ultrasonic disintegrator (Misonix, USA) with 1-min cooling in ice after each 10-s sonication. The suspension was centrifuged for 30 min at 11,000 g and 4°C and the soluble fraction was analyzed by SDS-polyacrylamide gel electrophoresis.
The phylogenetic analysis
The nucleotide sequences of the doe genes were obtained from the NCBI database (http://www.ncbi.nlm.nih.gov) by BLAST searches. The alignments of amino acid sequences were generated with ClustalW of the MEGA 6 program . The minor corrections in sequence alignments were made manually. Phylogenetic analysis was carried out using the MEGA 6 program and the Maximum Likelihood model.
Insertions into the doeA and doeB genes were introduced using the suicide vector pCM184  as described . Briefly, the 3′- and 5′-fragments of each gene to be mutated were PCR-amplified using the appropriate primers (S1 and S2 Tables) and inserted into pCM184 upstream and downstream of the kanamycin (Km) resistance gene. The cells of E. coli S17-1  were transformed by the resultant plasmids, and the donor strains were obtained and mated with the wild-type Mm. alcaliphilum 20Z. Mutants were selected on the plates with a mineral medium containing 0.5% methanol (v/v) and 100 μg/ml kanamycin. Double-crossover mutants were identified by diagnostic PCR tests. The Mm. alcaliphilum strain ΔectBC deficient in two enzymes for ectoine biosynthesis, i.e., diaminobutyrate aminotransferase and ectoine synthase, was obtained as described .
Homologous overexpression of the ectABC-operon
The ectABC gene cluster was amplified from Mm. alcaliphilum DNA and overexpressed in the wild type strain and in the strain with disrupted doeA by using the previously designed vector pMHA200_Pmxa_cat . The catalase-encoding cat gene was replaced by the ectABC cluster inserted between the SacI and VspI sites. In the resultant construct pMHA200_Pmxa_ectABC, the kanamycin resistance gene was replaced by the gentamicin resistance gene amplified from the vector p34S-Gm employing the restriction sites for PstI (S1 and S2 Tables). E. coli S17-1 cells were transformed by the resultant plasmid pMHA300_Pmxa_ectABC and transferred into Mm. alcaliphilum strains by conjugation. Conjugants were selected on the plates with the mineral medium P containing 10 μg/ml Gm (if the cells of the wild-type strain were transformed) or 100 μg/ml Km and 10 μg/ml Gm (if the cells of the ΔdoeA mutant were transformed). The presence of the pMHA300_Pmxa_ectABC plasmid in the cells of Mm. alcaliphilum 20Z was identified by diagnostic PCR tests.
Expression of the doeA gene in E. coli. To identify the product of DoeA reaction, the doeA gene was expressed in E. coli XL1-Blue from the Plac promoter. A PCR fragment containing the doeA sequence was amplified from the Mm. alcaliphilum DNA using primers DoeA_f and DoeA_r (S1 and S2 Tables) and cloned in the low-copy-plasmid pHSG575 opened by endonucleases EcoRI and HindIII . E. coli cells transformed by the pHSG575_doeA plasmid and those without the plasmid were grown in the mineral medium M9 containing glucose, 3% NaCl and 4 mM ectoine. 10 ml of each culture was harvested by centrifugation: (i) E. coli harboring the plasmid pHSG575_doeA grown without IPTG; (ii) E. coli harboring the plasmid pHSG575_doeA grown in the presence of 0.5 mM IPTG and (iii) E. coli not harboring the plasmid. The methanol extract from the cells of each culture was analyzed by HPLC to identify intracellular metabolites. The Nα-acetyl-DAB and Nγ-acetyl-DAB standards were obtained by the alkaline hydrolysis of pure ectoine .
Analytical methods. The methanol/chloroform extraction procedure was used to isolate organic solutes from cells as described . The excretion of ectoine into the growth medium was checked by analyzing freeze-dried culture liquid re-dissolved in an acetonitrile/water mixture (70:30 v/v). Ectoine concentration was measured by isocratic HPLC (LC-20 Prominence, Shimadzu) in a Reprosil 100 NH2 column (4×150 mm, 3 μm) using acetonitrile/water (70:30 v/v) as the mobile phase and UV detection at 210 nm. The commercially available ectoine (Sigma) was used for the standard curve preparation. Quantification of glutamate was performed with a glutamate assay kit (Sigma) according to the manufacturer's instructions. Sucrose was analyzed by HPLC (LC-20 Prominence, Shimadzu) with a refractive index (RI) detector (RID-20A, Shimadzu) using a ReproGel H+ column (length, 300 mm; particle size, 7.8 mm). The samples were separated at 50 ⁰C using an isocratic elution program at a flow rate of 0.5 ml/min; mobile phase was 1 mM H2SO4. The commercially available sucrose (Sigma) was used for standard curve preparation. Nα-acetyl-DAB and Nγ-acetyl-DAB were identified by HPLC (LC-20 Prominence, Shimadzu) in a Reprosil OPA column (4.6×150 mm, 3 μm) with o-phthalaldehyde (OPA) pre-column derivatization and detection at 330 nm. The samples were separated at 25 ⁰C with a gradient elution program at a flow rate of 1 ml/min. The mobile phase was potassium phosphate buffer (pH 7.2) (solvent A) or potassium phosphate buffer–methanol–acetonitrile mixture (50:35:15, v/v) (solvent B). The gradient elution program was as follows: 100–0% A (0–40 min), 100% B (40–50 min), 100–0% B (55–60 min) and 100% A (60–75 min).
Plasmid isolation, digestion by restriction enzymes, agarose gel electrophoresis, ligation and transformation of E. coli cells were performed according to . Restriction enzymes, T4 DNA ligase, Pfu DNA polymerase and dNTPs were purchased from Thermo Scientific (USA).
Osmotic down-shock experiments
100 ml of the ΔdoeB culture grown exponentially in the presence of 6% NaCl was diluted four times by the mineral medium P without NaCl and incubated under the optimal temperature and aeration. The cells were harvested by centrifugation from 100 ml of each culture: (i) grown at 6% NaCl but not exposed to down shock; (ii) diluted and incubated for 40 and (iii) for 90 min. The intracellular metabolites were identified in methanol extracts by HPLC.
Identification of the genes encoding ectoine degradation enzymes
The genes potentially involved in ectoine degradation were identified by comparing the genomes of Mm. alcaliphilum 20Z and H. elongata. In the methanotroph, four open reading frames showing moderate similarities to the doeA, doeB, doeС and doeD genes coding for the enzymes of ectoine degradation in H. elongata (42, 22, 36 and 49% identities) were adjacently located (Fig 1, S3 Table). These genes are oriented in the same direction, making up the doeBDAC cluster. The organization of the cluster differs from the H. elongata ectoine degradation operon, which additionally codes for the AsnC/Lrp-like DNA-binding protein, the MocR/GabR-type transcriptional regulator EnuR, and the two enzymes involved in the conversion of hydroxyectoine into ectoine [6, 18].
Heterotrophic bacteria with the established ectoine degradation pathway are presented. doeA, ectoine hydrolase; doeB, Na-acetyl-L-2,4-diaminobutyric acid deacetylase; doeX, AsnC/Lrp-like DNA-binding protein; doeC, aspartate-semialdehyde dehydrogenase; doeD, L-2,4-diaminobutyrate transaminase. Only ectoine degradation genes, without transporter genes and regulatory proteins, are indicated for the heterotrophic bacteria, since they are absent in the considered clusters of methylotrophs.
We failed to obtain functional recombinant DoeA and DoeB enzymes from Mm. alcaliphilum 20Z by heterologous expression of the doeA or doeB genes in E. coli, since E. coli Rosetta (DE3) transformed by the pET22b_doeA or pET22b_doeB plasmids synthesized mostly insoluble protein forms. The variation of cultivation temperature, the replacement of the T7 promoter by a weaker arabinose promoter, or the use of the pHUE plasmid for expression of proteins with an additional ubiquitin peptide was ineffective. Nevertheless, Nα-acetyl-DAB was found in the E. coli cells transformed by the low-copy plasmid pHSG575_doeA and grown in the presence of 4 mM ectoine and 3% NaCl (Fig 2). On the contrary, two isomers, Nα- and Nγ-acetyl-DAB, have been found in E. coli expressing doeA from H. elongata . Thus, it might be supposed that DoeA from Mm. alcaliphilum 20Z catalyzes a more specific reaction of ectoine hydrolysis, generating Nα-acetyl-DAB but not forming Nγ-acetyl-DAB.
The chromatograms of methanol extracts from E. coli cells transformed by plasmids pHSG575 (A) and pHSG575_doeA carrying the doeA gene from Mm. alcaliphilum (B, C). E. coli cells were grown in the presence of 3% NaCl and 4 mM ectoine. B, cells grown without IPTG; C, cells grown in the presence of 0.5 mM IPTG; D, standards of Nγ-acetyl-DAB and Nα-acetyl-DAB obtained by the alkaline hydrolysis of pure ectoine . Intracellular metabolites were analyzed by HPLC.
Phenotypic characterization of the ΔdoeA and ΔdoeB mutants
To check the function of the putative doeA gene in Mm. alcaliphilum 20Z, this gene was disrupted by insertion of a kanamycin cassette using the suicidal vector pCM184. The growth rate of the knockouted strain was not changed, but the mutant accumulated a higher level of ectoine compared to the wild-type strain when grown at 1 or 3% NaCl (Table 1).
The pMHA300_Pmxa_ectABC plasmid carrying the ectABC gene cluster under the control of the constitutive methanol dehydrogenase promoter Pmxa was introduced into both the wild type strain Mm. alcaliphilum and its ΔdoeA mutant. Such transformation was performed in order to reduce the contribution of EctR to the transcriptional regulation of expression of the ect-genes . During the growth at a low salinity (1% NaCl), the cells of the wild type strain transformed by the pMHA300_Pmxa_ectABC plasmid accumulated fivefold more ectoine compared to the strain without this plasmid. The ΔdoeA mutant transformed by the same plasmid had a threefold higher ectoine level than the wild type strain (Table 1). The differences in ectoine contents between the wild type strain and the ΔdoeA mutant growing at a salinity of 3% were less pronounced. These data implied the participation of DoeA in the regulation of intracellular ectoine level. In both strains, the intracellular glutamate content gradually increased in response to an increase in salinity of the medium, but intracellular glutamate levels did not show any noticeable dependence on strain modification.
The strain impaired in the doeB gene (ΔdoeB) was obtained by the insertion of kanamycin cassette. Methanol extracts from cells of the ΔdoeB strain grown at 6% NaCl had a peak corresponding to Nγ-acetyl-DAB. However, the peak of Nγ-acetyl-DAB gradually disappeared after osmotic down-shock as a result of fourfold dilution of the culture with the growth medium without NaCl and 40-min incubation under the optimal growth conditions (Fig 3). The peak of Nα-acetyl-DAB (a substrate for DoeB) was detected after further 50-min incubation in the diluted medium (Fig 3). Therefore, we assume that the osmotic down-shock induced ectoine cleavage into Nα-acetyl-DAB and this isomer is a substrate for DoeB enzyme. In the wild type cells, the peak of Nα-acetyl-DAB was found but did not increase substantially during exposure in the diluted medium (S1 Fig).
The culture was grown under methane in a mineral salt medium in the presence 6% NaCl (A), diluted with the medium without NaCl to a final concentration of 1.5% NaCl (B, C) and incubated for 40 min (B) or 90 min (C) under the optimal growth conditions. D, the standards of Nγ-acetyl-DAB and Nα-acetyl-DAB obtained by the alkaline hydrolysis of pure ectoine .
Genomic analysis of the ectoine degradation pathway in methylotrophs
The homologs of ectoine degradation genes were revealed in all halophilic/halotolerant aerobic methylotrophs capable of ectoine biosynthesis (Fig 1). In most methylotrophs, these genes are organized in the doeBDAC cluster. The exception is Methylohalobius crimeensis possessing the doeCD cluster and individual doeA and doeB genes, as well as methanotrophic representatives of the genus Methylobacter (M. marinus, M. whittenburyi and Methylobacter sp. BBA5.1), where these genes are scattered on chromosomes (Fig 1). The differences in the genetic organization correlated with the level of sequence divergence of the proteins. For example, the proposed enzymes from all non-methanotrophic methylotrophs of the genus Methylophaga encoded by the doeBDAC cluster are most similar to the Mm. alcaliphilum enzymes (80–81% identities). On the contrary, DoeA of Mm. alcaliphilum exhibited only a 30–31% identity with DoeA from methanotrophs of the genus Methylobacter and from Mh. crimeensis (Fig 1). In addition, DoeA of Mm. alcaliphilum (phylum Proteobacteria) shared much higher similarity (~70% identity) with DoeA from Tsukamurella paurometabola (the phylum Actinobacteria) possessing the doeBDAC cluster than with DoeA from the proteobacterial Rhodobacter sphaeroides and Nitrosococcus halophilus harboring separately located genes of ectoine catabolism.
In Mm. alcaliphilum, the cluster of ectoine catabolic genes lacks the genes encoding the AsnC/Lrp-like transcriptional repressor and the MocR/GabR-type activator EnuR found in the H. elongata operon [6, 18]. The operon of H. elongata also possesses two genes involved in the conversion of hydroxyectoine into ectoine . Though the sequence homologous to the ectoine hydroxylase encoding gene is present in the Mm. alcaliphilum genome, we failed to prove its functionality and hydroxyectoine accumulation in the methanotroph .
Study of ectoine uptake by methanotrophic cells
The genetic determinants potentially responsible for the transport systems of ectoine were searched in Mm. alcaliphilum 20Z on the basis of sequences coding for the specific transporters of ectoine and hydroxyectoine in Gram-negative bacteria . We have failed to find sequences homologous to the genes coding for the EhuABC transporter  in the genome of the methanotroph. The sequences slightly similar (15–20% identities) to the genes encoding hydroxyectoine/ectoine-specific Ueh TRAP transporter were found in the bacteria utilizing ectoines as nutrients  (S4 Table). Moreover, while growing in the medium with 5% NaCl and 1 mM ectoine, the cells of both the wide type strain 20Z and the strain ΔectBC deficient in the ability to synthesize ectoine accumulated exogenous ectoine (Table 2). In the mutant strain ΔectBC, intracellular ectoine content was twice lower than in the wild type strain. The cells of the strain with impaired ectBC genes had a higher level of sucrose (Table 3) thus balancing intracellular osmotic pressure. The addition of 1 mM trehalose to the growth media of both strains as a negative control had no effect on intracellular sucrose level.
The cells were grown to the early stationary stage (OD600 = 2.1) at a salinity of 3%, 5% NaCl and 5% NaCl in the presence of 1 mM ectoine. Sucrose content was measured by HPLC as indicated in Materials & Methods.
Here we have shown for the first time that adaptation of the halotolerant methanotroph Mm. alcaliphilum 20Z to the changes in external osmolarity includes intracellular degradation of ectoine. In the methanotroph, four putative enzymes catalyzing ectoine catabolism (ectoine hydrolase, N-acetyl-DAB deacetylase, diaminobutyrate deaminase and aspartate-semialdehyde dehydrogenase) are encoded by the single operon doeBDAC (Figs 1 and 4). In vivo operation of at least two enzymes (ectoine hydrolase DoeA and N-acetyl-DAB deacetylase DoeB) was confirmed by mutational analysis. Disruption of the gene of ectoine hydrolase, the starting enzyme of the pathway, did not change the growth rate of the mutant strain, suggesting that the carbon flux through this pathway did not generate significant metabolic imbalance in the adapted culture. Nevertheless, the deletion of this gene led to an increase in the intracellular ectoine content. Only a slight increase in ectoine content in the mutant strain could be explained by the presence of a mechanism regulating ectoine biosynthesis via the transcriptional repressor EctR.
The scheme of the degradation pathway was constructed on the basis of the genetic and chromatographic analysis performed in this work. Ask: aspartate kinase; AsdH: b-aspartate-semialdehyde-dehydrogenase; Hdh: homoserine dehydrogenase; EctB: L-2,4-diaminobutyrate transaminase; EctA: L-2,4-diaminobutyrate Nγ-acetyltransferase; EctC: ectoine synthase; EctD: ectoine hydroxylase; DoeA: ectoine hydrolase; DoeB: Nα-acetyl-L-2,4-diaminobutyrate deacetylase; DoeD: L-2,4-diaminobutyrate transaminase; DoeC: aspartate-semialdehyde dehydrogenase.
Our results demonstrate some distinctive features of ectoine metabolism in the obligate methanotroph and the previously studied heterotrophic bacteria. In Mm. alcaliphilum, Nα-acetyl-DAB is a major product of the ectoine hydrolase DoeA. In H. elongata, DoeA produced both isomers of N-acetyl-DAB. It is not improbable that Nγ-acetyl-DAB can also be formed during ectoine degradation in the methanotroph, but in low amounts that could not be identified due to the low activity of DoeA in both strain 20Z and recombinant E. coli cells. However, chromatographic analysis of cytoplasm of the Mm. alcaliphilum ΔdoeB mutant and E. coli expressing methanotrophic doeA indicates that Nγ-acetyl-DAB is formed, if at all, in a much lower amount compared to the enzyme from H. elongata producing Nγ- and Nα-acetyl-DAB at the ratio of 1:2. If this ratio is true for the enzymes of both bacteria, we could detect Nγ-acetyl-DAB in our experiments. In H. elongata, N-γ-acetyl-DAB, the substrate of ectoine synthase EctC, is also an inducer of the EnuR protein, which controls transcription of the ectoine/hydroxyectoine biosynthetic pathway . Diaminobutyric acid in this heterotrophic bacterium can either flow off to aspartate or re-enter the ectoine synthesis pathway, forming a cycle of ectoine synthesis and degradation. In the genome of the methanotroph, we did not find any sequences for the EnuR-like genes and the genes encoding the AsnC/Lrp-like transcriptional repressor. In our experiments, Nα-acetyl-DAB (a substrate for DoeB) was detected in response to osmotic down-shock of ΔdoeB cells, thus indicating the non-constitutive character of ectoine catabolism in the methanotroph. Disappearance of the Nγ-acetyl-DAB peak from the cells of the ΔdoeB strain exposed to osmotic down-shock also implies that the ectoine synthase (EctC) could remain functional under these conditions when the ectione degradation pathway operates. Moreover, the intracellular concentration of ectoine did not always clearly correspond to the osmotic pressure of the medium and significantly increased under certain conditions (i.e., in the presence of ectoine in the medium or overexpression of the enzymes involved in ectoine synthesis). We believe that the increase in intracellular osmolarity due to the excess of ectoine does not initiate the degradation mechanism, since it could be compensated by changes in the levels of other metabolites (sucrose). It is also interesting that ectoine degradation was activated upon a mild dilution stress, indicating that cells preserve the previously invested carbon and nitrogen resources via ectoine synthesis for metabolism under the conditions when very high cellular pool of ectoine is no longer needed. Mm. alcaliphilum is an obligate C1-utilizer incapable of using any organic compound as a single growth substrate; nevertheless, acetate and aspartate, the products of the ectoine degradation route, can enter the central biochemical pathways in the methanotroph. Acetate can enter the tricarboxylic acid (TCA) cycle after the activation by acetate kinase . In addition, the oxaloacetate derived from aspartate can be reduced to malate, which is subsequently converted to pyruvate via the malic enzyme . The special role of the NAD+-malic enzyme in the withdrawal of excessive C4 intermediates from the TCA cycle has been proposed recently .
Since no ectoine was found in the growth medium after fourfold dilution of the culture (from 6% NaCl to 1.5% NaCl), we can assume the absence of ectoine extrusion from cells, at least under the conditions tested. The genome sequence analysis showed that Mm. alcaliphilum codes for at least four proteins (WP_014150036, WP_014148079, WP_014150313, WP 014148079) with identities to MscL/MscS mechanosensitive channel proteins from E. coli. The proteins of MscS family make water-filled open channel pores of ~13–16 Å in diameter . The MscL proteins make very large pores in the membrane that are ∼30 Å in diameter (the largest gated pore known) and, if this channel is gated inappropriately, its presence is devastating for the cell . An absence of ectoine in the medium indicates that moderate osmotic down-shock does not initiate the pore opening (by MscL homolog) in the methanotroph, that would be sufficient for ectoine excretion, and the pores created by the MscS family proteins are too small.
Here we have shown that Mm. alcaliphilum is able to take up ectoine from the medium simultaneously with synthesis of osmoprotectant. The candidate for the respective transporter is the TRAP-type transporter, which is considered to use electrochemical sodium gradients to energize this consumption. However, we did not estimate the affinity and specificity of these systems to ectoine. In contrast to many Gram-negative bacteria utilizing ectoine as a growth substrate, the genes encoding putative solute transporters in methylotrophs are located outside the cluster of ectoine catabolism. We have also shown that the cytoplasmic glutamate content increases upon the increase in salinity of the medium, thus confirming the osmoprotective role of the amino acid . However, no noticeable correlation between the glutamate and ectoine contents in the mutant strains has been detected.
Although ectoine was first detected in the extremely halophilic phototrophic bacterium Ectothiorhodospira halochloris , the metabolism of this osmoprotector has been studied mainly in heterotrophic microorganisms. In this work we have considered for the first time some aspects of ectoine catabolism in bacteria utilizing only C1 compounds as carbon sources. The differences in ectoine degradation pathways in heterotrophs and obligate methylotrophs are apparently related to their physiological traits. Heterotrophic bacteria have developed a more complex regulatory mechanism, which allows them to switch from the use of ectoine as an osmoprotector to its use as a substrate for growth. In methanotrophs, the function of ectoine is predominantly associated with osmoadaptation and the pathway of ectoine degradation serves for preservation of carbon, nitrogen and energy accumulated in the osmoprotector molecule. This correlates with the differences in genetic organization of the ectoine degradation pathway in methylotrophs and heterotrophs.
S1 Fig. The chromatograms of intracellular solutes in the wild type strain of Mm. alcaliphilum 20Z in the Reprosil OPA column with o-phthalaldehyde (OPA) pre-column derivatization.
A, the glutamate standard; B, the standards of Nγ-acetyl-DAB and Nα-acetyl-DAB obtained by the alkaline hydrolysis of pure ectoine . The culture was grown under methane in a mineral salt medium in the presence of 6% NaCl (C) diluted with the medium without NaCl to a final concentration of 1.5% NaCl (D, E) and incubated for 40 min (D) or 90 min (E) under the optimal growth conditions.
S2 Fig. The phylogenetic tree of the putative DoeA proteins from methylotrophic bacteria and DoeA from heterotrophic bacteria with the established ectoine-degradation pathway.
S2 Table. Bacterial strains and plasmids used in this study.
S3 Table. The putative proteins encoding by doeBDAC gene cluster detected in the genome of Mm. alcaliphilum 20Z.
S4 Table. Sequences encoding putative solute transporters found in genomes of Methylomicrobium alcaliphilum 20Z and their identity (%) to the respective genes of the 5-hydroxyectoine/ectoine TRAP transporters found in the Ruegeria pomeroyi DSS-3  and Halomonas elongata DSM 2581 .
- The authors are grateful to all members of the Organization for Methanotroph Genome Analysis for collaboration (OMeGA) and to the U.S. Department of Energy Joint Genome Institute and Genoscope for the access to methanotrophic genomes for comparative analyses.
- 1. Galinski EA. Osmoadaptation in bacteria. Adv Microb Physiol. 1995; 37:272–328. pmid:8540423
- 2. Galinski EA, Pfeiffer HP, Trüper HG. 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira. Eur J Biochem. 1985; 149:135–139. pmid:3838936
- 3. Pastor JM, Salvador M, Agrandoňa M, Bernal V, Reina-Bueno M, Csonka LN, et al. Ectoines in cell stress protection: uses and biotechnological production. Biotechnol Adv 2010; 28 (6):782–801. pmid:20600783
- 4. Czech L, Hermann L, Stoveken N, Richter AA, Hoppner A, Smits SHJ, et al. Role of the extremolytes ectoine and hydroxyectoine as stress protectants and nutrients: genetics, phylogenomics, biochemistry, and structural analysis. Genes (Basel), 2018; 9 (4): 117. pmid:29565833
- 5. Widderich N, Czech L, Elling FJ, Könneke M, Stöveken N, Pittelkow M, et al. Strangersin the archaeal world: osmostress-responsive biosynthesis of ectoine and hydroxyectoine by the marine thaumarchaeon Nitrosopumilus maritimus. Env Microbiol. 2015; 18: 1227–1248.
- 6. Schwibbert K, Marin-Sanguino A, Bagyan I, Heidrich G, Lentzen G, Seitz H. et al. A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581 T. Environ Microbiol. 2011; 13:1973–1994. pmid:20849449
- 7. Lippert K, Galinski EA. Enzyme stabilization by ectoine-type compatible solutes: protection against heating, freezing and drying. Appl Microbiol Biotechnol. 1992; 37:61–65.
- 8. Bolen DW, Baskakov IV. The osmophobic effect: natural selection of a thermodynamic force in protein folding. J Mol Biol. 2001; 310:955–963. pmid:11502004
- 9. Graf R, Anzali S, Buenger J, Pfluecker F, Driller H. The multifunctional role of ectoine as a natural cell protectant. Clinics Dermatol. 2008; 26:326–333.
- 10. Khmelenina VN, Kalyuzhnaya M, Sacharovski VG, Suzina NE, Trotsenko YA, Gottschalk G. Osmoadaptation in halophilic and alkaliphilic methanotrophs. Arch Microbiol. 1999; 172:321–329. pmid:10550474
- 11. Reshetnikov AS, Khmelenina VN, Mustakhimov II, Kalyuzhnaya M, Lidstrom M, Trotsenko YA. Diversity and phylogeny of the ectoine biosynthesis genes in aerobic, moderately halophilic methylotrophic bacteria. Extremophiles. 2011; 15:653–663. pmid:21971967
- 12. Mustakhimov II, Reshetnikov AS, Glukhov AS, Khmelenina VN, Kalyuzhnaya MG, Trotsenko YA. Identification and characterization of EctR1, a new transcriptional regulator of the ectoine biosynthesis genes in the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. J Bacteriol. 2010; 192:410–417. pmid:19897647
- 13. Reshetnikov AS, Khmelenina VN, Mustakhimov II, Trotsenko YA. Genes and enzymes of ectoine biosynthesis in halotolerant methanotrophs. In: Methods in Enzymology. 2011; 495: Ch. 216–230.
- 14. Booth IR. Bacterial mechanosensitive channels: progress towards an understanding of their roles in cell physiology. Curr Opin Microbiol. 2014; 18:16–22. pmid:24607989
- 15. Welsh DT. Ecological significance of compatible solute accumulation by micro-organisms: from single cells to global climate. FEMS Microbiol Rev. 2000; 24: 263–290. pmid:10841973
- 16. Vargas C, Jebbar M, Carrasco R, Blanco C, Calderon MI, Iglesias-Guerra F, et al. Ectoines as compatible solutes and carbon and energy sources for the halophilic bacterium Chromohalobacter salexigens. J Appl Microbiol. 2006; 100: 98–107. pmid:16405689
- 17. Rodriguez-Moya J, Argandona M, Reina-Bueno M, Nieto JJ, Iglesias-Guerra F, Jebbar M, et al. Involvement of EupR, a response regulator of the NarL/FixJ family, in the control of the uptake of the compatible solutes ectoines by the halophilic bacterium Chromohalobacter salexigens. BMC Microbiol 2010; 10: 256. pmid:20942908
- 18. Schulz A, Stöveken N, Binzen IM, Hoffmann T, Heider J, Bremer E. Feeding on compatible solutes: A substrate-induced pathway for uptake and catabolism of ectoines and its genetic control by EnuR. Environ Microbiol. 2017; 19(3):926–946. pmid:27318028
- 19. Jebbar M, Sohn-Bosser L, Bremer E, Bernard T, Blanco C. Ectoine-induced proteins in Sinorhizobium meliloti include an ectoine ABC-type transporter involved in osmoprotection and ectoine catabolism. J Bacteriol. 2005; 187:1293–1304. pmid:15687193
- 20. Lecher J, Pittelkow M, Zobel S, Bursy J, Bonig T, Smits SH, et al. The crystal structure of UehA in complex with ectoine-A comparison with other TRAP-T binding proteins. J Mol Biol. 2009; 389: 58–73. pmid:19362561
- 21. Hanekop N, Hoing M, Sohn-Bosser L, Jebbar M, Schmitt L, Bremer E. Crystal structure of the ligand-binding protein EhuB from Sinorhizobium meliloti reveals substrate recognition of the compatible solutes ectoine and hydroxyectoine. J Mol Biol 2007; 374: 1237–1250. pmid:17996893
- 22. Sambrook J, Russell DW. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 2001.
- 23. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007; 24:1596–1599 pmid:17488738
- 24. Marx CJ, Lidstrom ME. Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. Bio Techniques. 2002; 33:1062–1067.
- 25. Simon R, Priefer U, Pühler A. A broad host-range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Nature Biotech. 1983; 1:784–791.
- 26. But SY, Khmelenina VN, Mustakhimov II, Trotsenko YA. Construction and characterization of Methylomicrobium alcaliphilum 20Z knockout mutants defective in sucrose and ectoine biosynthesis genes. Microbiology (Russian). 2013. 82(2): 253–255.
- 27. Mustakhimov II, But SY, Reshetnikov AS, Khmelenina VN, Trotsenko YA. Homo- and heterologous reporter proteins for evaluation of promoter activity in Methylomicrobium alcaliphilum 20Z. Appl Bioche Microbiol (Russian). 2016; 52:279–286.
- 28. Takeshita S, Sato M, Toba M, Masahashi W, Hashimoto-Gotoh T. High-copy-number and low-copy-number vectors for lacZα-complementation and chloramphenicol- or kanamycin-resistance selection. Gene. 1987; 61: 63–74. pmid:3327753
- 29. Kraegeloh A, Kunte HJ. Novel insights into the role of potassium for osmoregulation in Halomonas elongata. Extremophiles. 2002; 6:453–462 pmid:12486453
- 30. Mustakhimov II, Reshetnikov AS, But SY, Rozova ON, Khmelenina VN, Trotsenko YA. Engineering of hydroxyectoine production based on the Methylomicrobium alcaliphilum. Appl Biochem Microbiol (Russian). 2019; 55: 626–630.
- 31. Rozova ON, Khmelenina VN, Gavletdinova JZ, Mustakhimov II, Trotsenko YA. Acetate kinase—an enzyme of the postulated phosphoketolase pathway in Methylomicrobium alcaliphilum 20Z. Ant Leeuwenhoeck. 2015. 108: 965–974.
- 32. Rozova ON, Mustakhimov II, But SY, Reshetnikov AS, Khmelenina VN. Role of the malic enzyme in metabolism of the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z. PLOS ONE. 2019; 14(11): e0225054. pmid:31738793
- 33. Cox C, Nomura T, Ziegler C. et al. Selectivity mechanism of the mechanosensitive channel MscS revealed by probing channel subconducting states. Nat Commun. 2013; 4: 2137). pmid:23842527
- 34. Booth IR, Blount P. The MscS and MscL families of mechanosensitive channels act as microbial emergency release valves. J Bacteriol. 2012; 194:184802–4809.