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Microbiology (2016), 162, 651–661 DOI 10.1099/mic.0.000250 Different strategies of osmoadaptation in the closely related marine myxobacteria Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1 Jamshid Amiri Moghaddam,13 Nils Boehringer,13 Amal Burdziak,2 Hans-Jörg Kunte,3 Erwin A. Galinski2 and Till F. Schäberle1 Correspondence T. F. Schäberle 1 Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany 2 Institute of Microbiology & Biotechnology, University of Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany 3 Bundesanstalt für Materialforschung und -prüfung (BAM), Unter den Eichen 87, 12205 Berlin, Germany [email protected] Received 5 December 2015 Accepted 29 January 2016 Only a few myxobacteria are known to date that are classified as marine, owing to their salt dependency. In this study, the salt tolerance mechanism of these bacteria was investigated. To this end, a growth medium was designed in which the mutated Escherichia coli strain BKA13 served as sole food source for the predatory, heterotrophic myxobacteria. This enabled measurement of the osmolytes without any background and revealed that the closely related strains Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1 developed different strategies to handle salt stress. Ple. pacifica SIR-1, which was grown between 1 and 4 % NaCl, relies solely on the accumulation of amino acids, while Enh. salina SWB007, which was grown between 0.5 and 3 % NaCl, employs, besides betaine, hydroxyectoine as the major compatible solute. In accordance with this analysis, only in the latter strain was a locus identified that codes for genes corresponding to the biosynthesis of betaine, ectoine and hydroxyectoine. INTRODUCTION Micro-organisms living in a highly osmotic environment have to deal with the problem that water follows the osmotic gradient. Cells unable to cope with osmotic stress will become dehydrated. This will eventually disrupt cellular metabolism, and so is used in food conservation by pickling. One strategy to thrive in such environments involves the production of so-called osmolytes to maintain osmotic equilibrium across the cytoplasmic membrane. Osmolytes are organic compounds of low molecular mass that have no influence on cellular metabolism and are non-toxic. Therefore, these highly water-soluble molecules are also called compatible solutes (Brown, 1976; Held et al., 2010). Bacteria accumulate many different organic osmolytes in response to hypertonicity, including some amino 3These authors contributed equally to this article. Abbreviation: SOE, splicing-by-overlap-extension. The GenBank/EMBL/DDBJ accession number for the Enhygromyxa salina SWB007 gene locus harbouring genes related to betaine, ectoine and hydroxyectoine biosynthesis identified in this work is KU237243. Six supplementary figures and two supplementary tables are available with the online Supplementary Material. 000250 G 2016 The Authors acids, e.g. proline and glutamate, and some specialized compatible solutes, e.g. betaine and ectoine (da Costa et al., 1998; Burg & Ferraris, 2008). Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was discovered first in Ectothiorhodospira halochloris (Galinski, et al. 1985) and later on in many bacterial species (Severin et al., 1992; Roberts, 2005). The derivative hydroxyectoine was first discovered in Streptomyces parvalus (Inbar & Lapidot, 1988), before its presence was proven in a wide range of bacteria, and recently also in Archaea (Widderich et al., 2015). These molecules either are synthesized de novo or are taken up from the medium. The biosynthesis is well studied and the underlying genes are known (Sadeghi et al., 2014). In addition to their role in osmoregulation, compatible solutes are known to stabilize cell components under abiotic stress conditions, e.g. temperature, desiccation or oxidative stress. Their properties render them interesting for application in biotechnological research, e.g. for the stabilization of cryocultures/proteins, and as additives for PCR enhancement. Owing to the same effect, they are also applied in formulations of fragile drugs and medical products. Furthermore, compatible solutes display some biological effects. Thus, ectoine was reported to act as an anti-inflammatory upon particleinduced lung inflammation (Sydlik et al., 2009), and inhibited the aggregation of b-amyloid peptides that are Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 12:05:08 Printed in Great Britain 651 J. A. Moghaddam and others involved in senile diseases (Kanapathipillai et al., 2005). Ectoine is already on the market, owing to its moisturizing and protecting properties, in different skin care and cosmetic products, e.g. sun blockers (Kunte et al., 2014). Despite the relatively moderate osmotic stress of marine habitats, which usually provide a salt concentration of about 3 % NaCl, the chance of finding producers of organic osmolytes is high. Marine Vibrio species, for example, are known for their ability to produce ectoine. In recent years the first halophilic myxobacteria, which are not able to grow in the absence of NaCl, have been isolated. Their terrestrial counterparts instead grow without salt and can usually not grow at NaCl concentrations higher than 1.0 %. However, both have the ability to lyse a variety of bacteria and fungi to obtain nutrients. The few genera of halophilic marine myxobacteria isolated to date, i.e. Haliangium, Plesiocystis and Enhygromyxa (Fudou et al., 2002; Iizuka et al., 2003a, b; Schäberle et al., 2010), are of special interest, owing to their ability to produce unprecedented natural products like the antibiotically active salimabromide (Felder et al., 2013a). Despite this interest in these organisms, their salt tolerance mechanism remains to be investigated. This study of the closely related marine myxobacteria Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1 is aimed at investigating their osmo-adaptation mechanisms using analytical experiments and comparative genomics. METHODS Bacterial strains. Enh. salina SWB007 (from the strain collection of the Institute for Pharmaceutical Biology, University of Bonn) was isolated from marine sediments from the German coastline near Prerow (Felder et al., 2013a, b). Ple. pacifica SIR-1 (DSM 14875, type strain) was isolated from a Japanese coastal sea grass (Zostera) (Iizuka et al., 2003a). Escherichia coli BKA13 is a derivative of E. coli MKH13 (Haardt et al. 1995) and cannot synthesize the compatible solute trehalose (DotsB, otsA’). Like its parental strain, E. coli BKA13 is missing all transporters for compatible solute accumulation and the genes for the conversion of choline into glycine betaine: genotype D( putPA)101 D( proP)2 D( proU)608 betTIBA. Generation of otsB gene deletion. DNA sequences upstream and downstream from the gene otsB were joined by applying the splicingby-overlap-extension (SOE) PCR technique (Horton et al., 1989) using a set of specific primers (Table S1, available in the online Supplementary Material). The resulting PCR fragments were ligated into temperature-sensitive plasmid pMAK705 (Hamilton et al., 1989) and transferred into E. coli by transformation. After cultivation for 24 h at 30 uC, mutants carrying chromosomally integrated pMAK705 were selected on Luria–Bertani (LB) medium containing chloramphenicol (50 mg ml21) at 43 uC. These mutants were then cultivated in LB liquid medium; chloramphenicol-sensitive otsB mutants, arising after double cross-over, were identified on LB solid medium by in situ PCR. Media and growth conditions. To prepare a medium for the marine myxobacteria with no background of organic osmolytes, first an E. coli BKA13 culture was prepared. To this end, E. coli BKA13 cells 652 were grown in the minimal medium MM63 including 0.5 % NaCl (Larsen et al., 1987; Grammann et al., 2002) for 20 h, with shaking at 37 uC. The OD600 was measured and the culture was precipitated by centrifugation for 10 min at 8873 g. The cell pellet was washed twice with tap water and subsequently resuspended in tap water and used for the preparation of ASW-Coli medium as described below. The amount of tap water used for resuspension was calculated by the following equation: (OD600 | initial culture volume)/50. Enh. salina SWB007 and Ple. pacifica SIR-1 were grown in ASW-Coli medium containing 75 % artificial seawater (ASW) and 3 % E. coli BKA13 suspension in MilliQ water. The pH of the medium was adjusted to 7.5 with NaOH. After sterilization by autoclaving, the medium was supplemented with trace element solution (1 ml l21) and vitamin B12 (1 ml l21). Standard ASW (100 %) contains KBr (0.1 g l21), MgCl2.6H2O (10.61 g l21), CaCl2.2H2O (1.47 g l21), KCl (0.66 g l21), SrCl2.6H2O (0.04 g l21), Na2SO4 (3.92 g l21), NaHCO3 (0.19 g l21) and H3BO3 (0.03 g l21). The trace element solution consists of 10 mg ZnCl2, 50 mg MnCl2.4H2O, 5 mg H3BO3, 5 mg CuSO4, 10 mg CoCl2, 2.5 mg SnCl2.2H2O, 2.5 mg LiCl, 10 mg KBr, 10 mg KI, 5 mg Na2MoO4.2H2O and 2.6 g Na2EDTA.2H2O dissolved in 1 l distilled water; NaCl was added to reach the desired final concentration, and the solution was sterilized by filtration. Stock solution of vitamin B12 was 0.5 mg ml21 cyanocobalamine in water; the solution was sterilized by filtration. To determine the salt tolerance range of Enh. salina SWB007, 100 ml Erlenmeyer flasks containing 30 ml ASW-Coli medium with different NaCl concentrations (from 0 to 5 % NaCl, in 0.5 % steps) were prepared. The cultures were inoculated with 100 ml of dispersed fresh fruiting bodies. The dispersed fruiting body suspension was prepared beforehand by manually collecting fruiting bodies from another liquid culture using a pipette. Growth was determined by measuring the decrease in OD600, since the E. coli cells served as prey and were lysed by the myxobacteria, which resulted in clearing of the medium. For identification of compatible solutes, the myxobacteria were grown in 5 l Erlenmeyer flasks containing 1 l ASW-Coli medium with 0.5 and 3 % NaCl for Enh. salina SWB007, and 1.0 and 3.5 % NaCl for Ple. pacifica, respectively. Two independent experiments were performed and the mean values are given in Table 1. Incubation was performed at 30 uC at 140 r.p.m., using rotary shakers. After lysis of all E. coli cells (4 days, except for Enh. salina in 3.5 % NaCl medium, which was incubated for 5 days), the fruiting bodies were collected and precipitated by centrifugation at 17 000 g for 1 min. The supernatant was discarded and the pellet was freeze-dried using a lyophilizer (Christ Beta 1-16; Martin Christ). Extraction of intracellular solutes. The extraction of cytoplasmic solutes was performed following the Bligh & Dyer extraction method, as modified by Galinski & Herzog (1990). In brief, 500 ml Bligh & Dyer solution (10/5/4, by volume, methanol/chloroform/demineralized H2O) was added to 30 mg dry material. After shaking for 5 min, 130 ml chloroform and 130 ml demineralized H2O were added and the mixture was shaken again for 5 min. Finally, the mixture was centrifuged at 8000 g for 3 min and the hydrophilic top layer, containing the compatible solutes, was separated for subsequent analyses. HPLC and LCMS analysis for compatible solutes and free amino acids. The aqueous phase was analysed by HPLC using a refractive index detector RI-71 (Shodex) and a UV detector UV1000 (Thermo Scientific) with the following HPLC conditions: column, LiChrospher 100-NH2 5 mm (Merck); isocratic flow of 80 % acetonitrile and 20 % water at 1 ml min21. The samples were diluted 1:4 with the solvent before measurement. Additionally, reference compounds were run to identify the compatible solutes. The evaluation was performed with ChromQuest5 (ThermoQuest). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 12:05:08 Microbiology 162 Different strategies of osmoadaptation Table 1. Content of compatible solutes and free amino acids in Enh. salina SWB007 and Ple. pacifica SIR-1 Enh. salina Osmolytes/amino acid Hydroxyectoine (mmol g21) Betaine (mmol g21) Glutamine (mmol g21) Glutamate (mmol g21) Glycine (mmol g21) Alanine (mmol g21) Proline (mmol g21) Hydroxyectoine (mg g21) Betaine (mg g21) Glutamine (mg g21) Glutamate (mg g21) Glycine (mg g21) Alanine (mg g21) Proline (mg g21) 0.5 % NaCl* ND ND 6.91 76.75 ND ND ND ND ND 1.01 11.29 Ple. pacifica 3% 1% NaCl* NaCl* 97.56 48.38 49.92 272.63 33.24 ND ND 40.94 11.99 15.42 5.31 7.30 40.11 ND ND ND ND ND ND 8.24 295.97 124.89 3.11 13.06 ND ND ND ND ND ND 4.89 ND ND ND ND 3.65 1.38 ND ND *Chromatograms are given in Figs S1–S6. ND , 3.5 % NaCl* ND 1.20 43.55 9.38 0.28 1.50 Not detected. MS analysis was performed using an ESI-MS microTOF spectrometer (Bruker Daltonik) coupled to an HPLC. The same parameters as described above were applied, and the sample was split before transfer to ESI-MS. Free amino acids were analysed by FMOC/ADAM HPLC using a fluorescence detector (SpectraSYSTEM FL2000, 254 nm excitation, 315 nm emission; Thermo separation products); the HPLC column used was a Merck Superspher 60 RP-8, 4 mm|125 mm|4 mm LiChroCART system (Alltech Grom). Amino acids were derivatized with 9-fluorenylmethoxycarbonyl (FMOC) by adding 40 ml boric acid buffer (0.5 M boric acid and 25 mM norvalin) and 80 ml FMOC reagent (1 mM FMOC in acetone) to 40 ml 1:50-diluted sample material. After shaking for 45 s, 100 ml 1-aminoadamantane (ADAM) reagent (50 % 40 mM ADAM in boric acid buffer, 50 % acetone) was added and the mixture was shaken again for 45 s. Finally, 140 ml solvent A [80 % 50 mM sodium acetate in H2O, 20 % 0.5 % tetrahydrofuran in acetonitrile (v/v)] was added. Solvent B consisted of 20 % sodium acetate buffer and 80 % acetonitrile. The HPLC was run at 1.25 ml min21 using the following gradient: 100 % A to 91 % A in 15 min, 91 % A to 70 % A in the next 15 min, 70 % A to 40 % A in the next 10 min, 40 % A to 0 % A (i.e. 100 % B) in the next 2 min, maintained at 100 % B for the next 7 min, 100 % B to 100 % A in the next 2 min, and maintained at 100 % A for the next 2 min. The evaluation was performed with ChromQuest5 (ThermoQuest). RESULTS Generation and characterization of the otsB-deletion mutant E. coli BKA13 Marine myxobacteria are usually cultivated using a medium containing autoclaved baker’s yeast. Such media are not suitable for the analysis of osmoregulated compatible solute synthesis because compatible solutes originating from yeast cells can be accumulated by the myxobacteria and falsify the results. Therefore, we established a novel medium – named ASW-Coli – which is based on E. coli cells as sole food source. E. coli can synthesize the compatible solute trehalose de novo and can convert choline to glycine betaine. If provided with the medium, E. coli is able to transport and amass a variety of different compatible solutes, such as ectoine, proline and glycine betaine, with the help of the transporters ProP and ProU (Milner et al., 1988; May et al., 1989). In order to feed myxobacteria without contaminating compatible solutes, we designed the trehalose-free E. coli strain BKA-13, which is based on E. coli MHK-13 (Haardt et al., 1995). E. coli MHK13 is devoid of the compatible solute transporters ProP and ProU and the proline transporter PutP. Like its parental strain, MC4100 (Casadaban, 1976; Peters et al., 2003), MHK13 is unable to transport and convert choline into glycine betaine (betTIBA). Strain MHK13 still possesses the genes otsB and otsA for trehalose synthesis. To obtain a trehalose-free E. coli, we deleted the gene otsB, which encodes trehalose-6phosphate phosphatase. The DNA regions upstream and downstream of otsB were joined by applying the SOE-PCR technique. The resulting DotsB fragment was cloned into the temperature-sensitive plasmid pMAK705, which facilitated the selection for DotsB mutants. The gene otsB overlaps with ORF otsA, which is located downstream of otsB. By deleting otsB (in-frame null mutation), 26 bp of the 59 region of otsA were removed as well (DotsB otsA’) (Fig. 1). The newly designed otsBA mutant BKA-13 was further characterized, and compared to MHK-13. Both strains were cultivated on minimal medium containing 2.0, 2.5, 2.7, 2.8 and 3.0 % NaCl. While strain MHK-13 grew at all salinities within 2 days, strain BKA-13 failed to grow on medium containing 2.8 and 3.0 % NaCl. On medium with 2.7 % NaCl, small colonies of strain BKA-13 were only visible after 6 days of incubation. Cell extract of E. coli BKA-13 was further analysed by HPLC for the presence of trehalose. No trehalose was detectable in any of the BKA-13 cells grown in up to 2.5 % NaCl. Bioinformatics analyses. The genome of Ple. pacifica SIR-1 (NCBI reference sequence NZ_ABCS00000000.1) and a draft genome of Enh. salina SWB007 (unpublished) were analysed using the bioinformatics program antiSMASH 3.0 (Weber et al., 2015). In the genomic data of Enh. salina SWB007 a gene locus was identified harbouring genes related to betaine, ectoine and hydroxyectoine biosynthesis. The gene cluster was subsequently annotated using the RAST annotation program (version 2.0) (Overbeek et al., 2014) and the UniProt Basic Local Alignment Search Tool (BLAST ). The contig was submitted to GenBank (accession number KU237243). http://mic.microbiologyresearch.org Growth and salt tolerance of myxobacteria The salt tolerance ranges of the two myxobacterial strains Enh. salina SWB007 and Ple. pacifica SIR-1 were determined in ASW-Coli medium. To enable a fast and reliable method for growth determination, an indirect measurement was performed; such a method was required since the myxobacteria rapidly form fruiting bodies instead of Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 12:05:08 653 J. A. Moghaddam and others 5′ ATG 2881 NdeI 1 kb 2106 E. coli MKH13 NdeI 2906 otsB otsA 801 bp 1425 bp NdeI 4305 NdeI 2106 3504 E. coli BKA13 otsA 1399 bp Fig. 1. Comparison of genetic organization of otsAB operons in E. coli MKH-13 and E. coli BKA-13. The deletion of ,800 bp (between positions 2106 and 2906) removed otsB completely and, owing to its overlap with otsA, also part of the beginning of otsA, including its start codon, and resulted in genotype DotsB otsA’. disperse growth. Thus, the decrease in OD600 was measured. OD600 is directly related to lysis and consumption of the prey cells. Therefore, a decrease in OD600 indicates growth of Enh. salina SWB007 or Ple. pacifica SIR-1. Enh. salina SWB007 was able to grow in media containing 0.5 to 3 % NaCl. The fastest lysis rate of the prey cells was in the salinity range 0.5 to 2 % (Fig. 2). After 7 days all E. coli cells were consumed and orange-coloured fruiting bodies appeared. Cultures supplemented with 2.5 and 3 % NaCl took 2 days longer to achieve similar results. With 0 % NaCl the culture initially seemed to grow, but after 4 days the OD600 increased again. In cultures containing more than 3 % NaCl no growth was observed. Ple. pacifica SIR-1 showed no growth in medium containing 0.5 % NaCl. The lower limit was 1 % NaCl and growth was observed up to a concentration of 4 % NaCl in ASWColi medium. However, the growth was decelerated and therefore a concentration of 3.5 % NaCl was used for subsequent analyses. Identification of the compatible solutes of Enh. salina SWB007 and Ple. pacifica SIR-1 Both strains, Enh. salina SWB007 and Ple. pacifica SIR-1, could grow in a relatively high NaCl concentration range, i.e. 0.5–3 % NaCl for Enh. salina SWB007 and 1–4 % NaCl for Ple. pacifica SIR-1. Such a range is usually an indication that the organisms use the organic osmolyte strategy to deal with the salt stress of the environment. To prove this hypothesis, we identified the compatible solutes produced 654 by these organisms under different salt concentrations. Thus, two 1 l cultures of each strain were grown in ASWColi medium: (i) 0.5 and 3 % NaCl for Enh. salina SWB007 and (ii) 1 and 3.5 % NaCl for Ple. pacifica SIR-1. These cultures were incubated until total clearing of the medium before the cells were harvested. In total, 92.9 mg (0.5 % NaCl) and 134.3 mg (3 % NaCl) dry cell mass were obtained from Enh. salina SWB007, and 180 mg (1 % NaCl) and 251 mg (3.5 % NaCl) from Ple. pacifica SIR-1 cultures. Subsequently, 30 mg dried cell masses were extracted and the aqueous phase was analysed by HPLC. Clear differences indicating accumulation of organic osmolytes were observed in Enh. salina SWB007 grown in 0.5 and 3 % NaCl (Fig. 3). The new peaks at high salt concentration were putatively identified as betaine and hydroxyectoine, by comparison to standard substances. To confirm these results, HPLC-MS experiments followed. These confirmed the presence of betaine and hydroxyectoine, as well as minor amounts of ectoine (Fig. 4). The most abundant compatible solute was hydroxyectoine. The peak at 20 min retention time, which also increased with salinity, however, could not be identified. The peak that eluted at 12 min was not dependent on the salt concentration, since it was present in both the 0.5 and the 3 % NaCl cultures at the same order of magnitude. In contrast, the HPLC analysis of Ple. pacifica SIR-1 cultures grown in medium supplemented with either 1 or 3.5 % NaCl revealed no differences concerning the specialized osmolytes (Figs S1 and S2) when analysed under these conditions. Interestingly, this closely related organism Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 12:05:08 Microbiology 162 OD600 (%) Different strategies of osmoadaptation 100 NaCI (%) 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 80 Myxobacterial growth 60 40 20 0 0 1 2 3 4 5 6 7 8 9 Time (days) Fig. 2. Growth of Enh. salina SWB007 in ASW-Coli-Medium at different salt concentrations. Growth was measured by the decrease in OD600 due to lysis of E. coli cells. For better visualization of the data sets, only the mean values are given, and the data points are connected by a dotted line. The original data, as well as the SD values, can be found in Table S2. Each point in this graph represents the mean value of three independent cultures. The OD600 at day 1 was set to 100 %. The fastest lysis of prey cells was obtained between 0.5–2 % NaCl (indicated by squares), slower lysis between 2.5 and 3 % NaCl (circles), and no lysis was observed at 3.5 % or higher concentrations of NaCl (triangles). did not produce any of the typical organic osmolytes, e.g. betaine, ectoine or hydroxyectoine, even at a high salt concentration. Free amino acid analysis In addition to specialized compounds, common substances like amino acids may also serve as compatible solutes. Thus, the content of free amino acids in the cells was determined using FMOC/ADAM HPLC analysis, revealing significant changes (Table 1). Enh. salina SWB007 grown under high-salt conditions accumulated the amino acids alanine, glutamate, glutamine and proline (Table 1, Figs S5 and S6). The level of glutamine increased sevenfold and the level of glutamate fourfold at elevated salinity. Furthermore, alanine and proline were accumulated whereas both amino acids were below the detection limit under low-salt conditions. Free amino acid analysis of Ple. pacifica SIR-1 revealed the accumulation of alanine, glutamate, glutamine, glycine and proline at high salinity (Table 1, Figs S3 and S4). The level of glutamate showed a ninefold increase compared with the level at low salinity (296 mmol g21 versus 33 mmol g21). Alanine, glutamine, glycine and proline were also significantly accumulated under high-salt conditions, while they were below the detection limit at lowsalt conditions. Glycine was identified as the second major amino acid, contributing 28 % of the Ple. pacifica http://mic.microbiologyresearch.org SIR-1 organic osmolyte pool. Comparing the two strains, one common feature could be detected, i.e. glutamate was the major player, making up 52 and 66 % of the total organic osmolyte pool in Enh. salina SWB007 and Ple. pacifica SIR-1, respectively. In Enh. salina SWB007, alanine and glutamine followed, with amounts between 40 and 50 mmol g21, comprising 19 % of the organic osmolyte pool. In Ple. pacifica SIR-1 in contrast these two amino acids were only present in minor amounts, comprising 2.6 % of the organic osmolyte pool. In contrast, glycine was identified as the second major amino acid used by Ple. pacifica SIR-1, resulting in a proportion of 28 % within the organic osmolyte pool. Proline was accumulated by both organisms only in minor amounts, contributing less than 3 % to the organic osmolytes. Genome sequencing, annotation and bioinformatics analysis for compatible solutes biosynthesis To elucidate the reason for the observed differences between these closely related myxobacterial strains, in silico analysis of the genomes was performed. The available draft genome of Ple. pacifica SIR-1 was screened in respect of gene clusters associated with betaine, ectoine and hydroxyectoine biosynthesis. However, no such gene cluster was identified, supporting the previous results. Instead, many solute transporters such as the Na+/proline Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 12:05:08 655 J. A. Moghaddam and others 100 90 80 UV (mAU) 70 60 50 40 * 30 20 10 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 20 18 NaCI 16 Refractive index 14 ** Hydroxyectoine 12 10 8 Betaine 6 4 2 0 –2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Time (min) Fig. 3. Comparison of the compatible solute content of Enh. salina SWB007 in medium containing either 0.5 or 3 % NaCl. Black line, 0.5 % NaCl; red line, 3.0 % NaCl. Betaine and hydroxyectoine were present in cells grown in 3 % NaCl, while in 0.5 % NaCl these substances were not detectable. The peak at 20 min (**) could not clearly be assigned. The peak at 12 min (*) was present in both cultures in a similar amount, and therefore this peak was not dependent on the salt concentration. The peak at 6 min, which was only detectable in the refractive index plot, corresponds to NaCl. AU, Absorbance units. (solutes) symporter (GenBank accession numbers EDM75864.1, EDM79015.1, EDM74523.1, EDM80875.1, EDM77400.1), proton/sodium:glutamate symporter (EDM74633.1), sodium:alanine symporter (EDM78123.1), choline/glycine betaine transporter (EDM75025.1) and many ABC transporters were detected in the genome. In Enh. salina SWB007, biosynthetic gene clusters for the organic osmolytes had been expected, owing to the confirmation of these compounds in the extracts. The draft genome of strain SWB007 was screened for the presence of genes corresponding to organic osmolyte synthesis. Hence, a gene locus associated with ectoine biosynthesis was identified (accession number KU237243). In addition to ectA, ectB, ectC and ectD (Fig. 5, Table 2), further ORFs putatively coding for enzymes involved in organic osmolyte biosynthesis and for osmolyte transporters are present in this gene cluster, i.e. a sensor histidine kinase (ectR) and an aspartokinase (ectAsk). These genes represent 656 the complete biosynthetic gene cluster for the synthesis of the osmolyte hydroxyectoine. Comparison of this gene cluster to reported ectoine/hydroxyectoine gene loci revealed that Enh. salina SWB007 possesses a specific gene order (Fig. 6). A putative regulatory gene, i.e. ectR, was found in about one-quarter of the putative ectoine/ hydroxyectoine producers (Widderich et al., 2016). The functional association of an ectR gene with ectoine biosynthesis was first demonstrated in the halotolerant alkaliphilic Methylomicrobium alcaliphilum. This cluster, including ectR, shows the highest overall identity to Enh. salina SWB007 (Fig. 6). However, the grade of homology varies between the different genes; ectA, ectB and ectC show 35–56 % identity at the protein level, while ectR has only about 10 % identity. This might be an indication that the regulatory mechanism differs from that of Met. alcaliphilum, in which it acts as a transcriptional repressor (Overbeek et al., 2014). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 12:05:08 Microbiology 162 A210 (mAU) Different strategies of osmoadaptation (a) 1000 500 Intensity (×106) Intensity (×104) Intensity (×106) 0 118.0869 [M+H]+ (b) 1.0 140.0646 [M+Na]+ 156.0398 [2M+H]+ 235.1609 [M+K]+ [2M+Na]+ [2M+K]+ 9.8 min 0.5 [3M+H]+ 352.2355 0 8 143.0776 193.9691 [M+H]+ 219.0177 125.0313 177.9918 230.0911 (c) 6 13.2 min 4 2 252.0528 366.0503 0 159.0824 (d) 1.5 [M+H]+ 14.7 min 1.0 [2M+H]+ 317.1936 [2M+Na]+ 0.5 0 0 5.0 10.0 15.0 20.0 Time (min) 100 150 Elution time (min) 200 250 300 350 EIC (×106) m/z Fig. 4. HPLC-MS analysis of compatible solutes produced by Enh. salina SWB007. On the left side, the UV chromatogram (a), as well as the extracted ion counts (EIC) for betaine (b), ectoine (c) and hydroxyectoine (d), are given. AU, Absorbance units. On the right side, the mass spectra of betaine (m/z 118.09, [M+H]+) (b), ectoine (m/z 143.08, [M+H]+) (c) and hydroxyectoine (m/z 159.08, [M+H]+) (d) are shown. The different adducts are indicated. Of special interest are the two methyltransferases (MTs), i.e. glycine/sarcosine N-MT (GS-MT) and sarcosine/ dimethylglycine N-MT (SD-MT), and a betT homologue, which follow directly downstream of the ectoine-related genes, oriented in the same direction. These MTs are required for betaine synthesis from glycine, while betT codes for the transporter. Thus, the genes corresponding to the biosynthesis of betaine and hydroxyectoine are clustered together. DISCUSSION Marine myxobacteria, e.g. Enh. salina strains, are becoming more and more the focus of research owing to their biosynthetic potential (Felder et al., 2013a, b). To adapt these slow-growing organisms to growth under laboratory conditions, insights into their ecology, e.g. analysis of their salt tolerance mechanism, will be beneficial. To overcome the hurdle that Enh. salina strains are routinely grown in media containing a yeast cell suspension, which provides an unfavourable background, the medium ASW-Coli, based on E. coli BKA13 instead of baker’s yeast as nutrient source, was developed. This enabled the growth of marine http://mic.microbiologyresearch.org myxobacteria to be followed by measuring OD600. Hence, the OD decrease due to the consumption of the E. coli cells by the predatory bacteria was measured. The values for the salt tolerance of these strains (0.5–3 % NaCl for Enh. salina SWB007; 1–4 % NaCl for Ple. pacifica SIR-1) determined with this method are in accordance with previous results (Iizuka et al., 2003a; Schäberle et al., 2010). Such a relatively high degree of flexibility to environmental salt concentrations indicates an adaptation strategy involving organic osmolyte accumulation, rather than the salt-in strategy (Sleator & Hill, 2002). At high salt concentrations, a total of 521 mmol g21 solutes was detected in Enh. salina SWB007. The biggest part of this consisted of glutamate (52 %), followed by hydroxyectoine and betaine, which accumulated to 19 and 10 %, respectively. Hence, glutamate, hydroxyectoine and betaine seem to be the main players in osmo-adaptation of Enh. salina. A comparison to marine Vibrio fischeri species revealed that the observed total solute pool falls within expectations (451 mmol g21 for Vib. fischeri DSM 7151). Here too, the major compound was glutamate (approximately 50 %), followed by ectoine (approximately 24 %) (Schmitz & Galinski, 1996). Accumulation of both glutamate and hydroxyectoine is fully congruous with metabolism and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 12:05:08 657 J. A. Moghaddam and others (a) ectR (b) ect A ectASK EctB H H H Glutamate COO 2-Oxoglutarate – gsmt CoA – H COO H3N H H N – GSMT OH N H CH3 COO SAM C C H2 Sarcosine N SAH H3C Dimethylglycine CH3 H3C + N C C H2 – Succinate + CO2 H3C H OH N N O SAH H3C O COO – H H Ectoine OH SAM H N betT H Ng -Acetyl-L-2,4-diaminobutyrate SDMT H 2-Oxoglutarate + O2 Fe2+ H2O H3C sdmt EctD H H H3C SAH Glycine mep H H N L-2,4-Diaminobutyrate SAM NH2 O COO H3N EctC H H Acetyl-CoA H3N H OH EctA H L-Aspartate b-semialdehyde (c) symp ectD O O H3N ect C ectB Hydroxyectoine – C C H2 O Glycine betaine Fig. 5. Hypothesis for organic osmolyte biosynthesis in Enh. salina SWB007. (a) Gene locus coding for genes involved in organic osmolyte biosynthesis. Genes from left to right: ectR, sensor histidine kinase; orf2, hypothetical protein; orf3, hypothetical protein; ectAsk, aspartokinase; ectA, diaminobutyric acid (DABA) acetyltransferase; ectB, DABA aminotransferase; ectC, ectoine synthase; ectD, ectoine hydroxylase; symp, Na/solute symporter; mep, membrane protein (MarC family); gsmt, glycine/sarcosine N-methyltransferase; sdmt, sarcosine/dimethylglycine N-methyltransferase; betT, high-affinity choline uptake transporter BetT. It can be seen that the genes for ectoine/hydroxyectoine (ectAskABCD) and for betaine (gsmt and sdmt) biosynthesis are clustered in Enh. salina SWB007. (b) Hydroxyectoine biosynthetic pathway (modified from Bursy et al., 2008). (c) Glycine betaine biosynthetic pathway through methylation steps of glycine (modified from Sayyar Khan et al., 2009). SAM, S-adenosyl-L -methionine; SAH, S-adenosyl-L -homocysteine. other cellular functions (Reuter et al., 2010). However, it seems that this mechanism cannot be regarded as a general rule for myxobacteria. The terrestrial myxobacterium Myxococcus xanthus, the model organism for myxobacteria, was shown to produce the compatible solutes betaine and trehalose under salt stress conditions (McBride & Zusman, 1989; Kimura et al., 2010). Our results indicate that in addition to betaine the marine strain acquired, or retained, the ability to use hydroxyectoine as the main compatible solute. Hydroxyectoine, a derivative of ectoine, is widespread among halophilic bacteria, and compared with ectoine it provides better protection against various stress conditions, especially desiccation and heat (Lippert & Galinski, 1992; Louis et al., 1994; Tanne et al., 2014). In the intertidal zones of the littoral environment, desiccation is at least a temporary stress factor. This might explain why the organism favours hydroxyectoine as compatible solute. The preferential accumulation of hydroxyectoine has also been observed in other salt-stressed bacteria, such as Streptomyces coelicolor A3(2) and Virgibacillus salexigens (Sadeghi et al., 2014). Recently the ability to synthesize hydroxyectoine was also demonstrated in the acidophilic Acidiphilium cryptum, which displays only limited salt tolerance (Moritz et al., 2015). The authors concluded 658 that hydroxyectoine, besides osmoadaptation, may serve other, hitherto unknown, functions. In Ple. pacifica SIR-1, the total amount of solutes detected was 445 mmol g21. The major solute at high salt concentrations was also glutamate, contributing 66 % of the composition, followed by the amino acid glycine, constituting 28 %. Thus, Ple. pacifica SIR-1, the closest relative to the Enhygromyxa clade, did not produce any of the wellestablished compatible solutes under high-salt conditions. This marine myxobacterium instead accumulated the amino acids glutamate, glycine and proline. Glycine and proline can be enzymically synthesized or may be taken up from the environment. The accumulation of glutamate as primary response was the same as in Enh. salina SWB007. However, glycine has so far not been reported as a major bacterial osmolyte. The few reports available refer to its osmotic use in marine mussels and ciliates, in combination with alanine and taurine in the former and alanine and proline in the latter (Kaneshiro et al., 1969; Ellis et al., 1985). It is worthy of note that glycine has been applied empirically for protein stabilization during freeze–thawing of phosphate buffer systems and as a bulking agent during lyophilization of monoclonal antibodies (Pikal-Cleland et al., 2002; Meyer et al., 2009). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 12:05:08 Microbiology 162 Different strategies of osmoadaptation Table 2. Genes encoded in the gene locus putatively corresponding to organic osmolytes biosynthesis in Enh. salina SWB007 ORF Gene Length (bp) Highest homology Identity (%) Histidine kinase (Marichromatium purpuratum 984) Uncharacterized protein (Sorangium cellulosum) Outer-membrane protein and related peptidoglycan-associated (lipo)protein (Comamonadaceae bacterium A1) Aspartate kinase (Rhodothermus marinus SG0.5JP) Diaminobutyrate acetyltransferase (Alkalilimnicola ehrlichii) Diaminobutyrate aminotransferase apoenzyme (Streptomyces sp. SM8) L -ectoine synthase (Pontibacillus yanchengensis) Ectoine hydroxylase (Castellaniella defragrans 65) SSS sodium solute transporter superfamily (Rubinisphaera brasiliensis) UPF0056 membrane protein (Lyngbya sp. strain PCC 8106) SAM-dependent methyltransferase (Thioploca ingrica) Dimethylglycine methyltransferase (Marichromatium purpuratum 984) Choline transporter (Sphingomonas sp. Ant20) 43.3 61.3 52.6 1 2 3 ectR orf2 orf3 1899 126 153 4 5 6 7 8 9 10 11 12 13 ectAsk ectA ectB ectC ectD solute transporter mep GSMT SDMT betT 1458 537 1335 381 897 1446 633 861 864 1530 Therefore, the fact that glycine is one of the dominant amino acids in Ple. pacifica deserves further investigation. level is threefold more prominent on ectD transcription level than on ectC (Sadeghi et al., 2014). Furthermore, it was suggested that the localization of ectD at the 39-end of the ectABCD mRNA contributes positively to its stability. A putative regulator, encoded by ectR, is clustered with the ectABCD genes. EctR is expected to be responsible for the regulation of hydroxyectoine synthesis, dependent on natural stimuli. It was shown previously that in Gram-negative and Gram-positive bacteria the genes are induced by osmotic or temperature stress (Widderich et al., 2016). The presence of genes for several different osmolyte transporters in the genome can be regarded as a complementary mechanism for osmo-adaptation in Enh. salina SWB007. A Na+/proline symporter is encoded In accordance with the above observations, a gene locus associated with solute biosynthesis could be identified in Enh. salina SWB007 (Fig. 5a). The biosynthesis of ectoine and hydroxyectoine has been firmly established for many micro-organisms (Sadeghi et al., 2014). Accordingly, a biosynthetic hypothesis was deduced for hydroxyectoine and betaine in Enh. salina (Fig. 5b, c). It is striking that only hydroxyectoine is accumulated and not the direct precursor ectoine. Thus, the EctD-catalysed step has to be very efficient. In Streptomyces rimosus it was shown that the effect of salinity on ectD transcription ect A ectB ect C 10 35 56 55 20 7 35 55 54 18 42 48 45 43 Kytococcus sedentarius 40 60 49 46 Streptomyces anulatus 34 52 50 Demetria terragena 33 57 54 Methylomicrobium kenyense 30 54 53 Sporosarcina pasteurii 32 52 54 Methylobacter marinus ectAsk ectR 20 Asd 22 46.6 53.7 63.4 59.2 57.8 58.5 53.8 71.4 54.3 56.6 ectD Enhygromyxa salina SWB007 46 Methylomicrobium alcaliphilum Methylophaga alcalica Fig. 6. Genetic organization of ectoine/hydroxyectoine biosynthesis gene clusters. The Enh. salina SWB007 gene locus is shown in comparison to closely related gene loci; related genes are shown in the same colour. The sequence homology at the protein level is given as a percentage. http://mic.microbiologyresearch.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 12:05:08 659 J. A. Moghaddam and others directly downstream of ectD, and such transporters can mediate the accumulation of organic osmolytes, e.g. betaine, ectoine and proline, at high osmotic pressure (Wood, 2006). Furthermore, two N-methyltransferase genes are clustered with the hydroxyectoine gene locus of Enh. salina SWB007 (Fig. 5a). It can be assumed that these genes are linked to betaine biosynthesis (Waditee et al., 2003). Downstream of these two methyltransferase genes, there is a gene putatively coding for the high-affinity betaine/carnitine/choline transporter, BetT. In contrast, none of the established compatible solutes was detected in Ple. pacifica SIR-1. This is in accordance with the genome analysis, providing no gene cluster corresponding to their biosynthesis. However, the presence of many osmolyte transporters and symporters indicates that this bacterium is reliant on uptake to cope with salt stress. Such an uptake of osmolytes, e.g. amino acids, as a major strategy against the osmotic gradient represents an intelligent ploy for a predatory bacterium, which lyses prey cells and thereby creates for itself a source of these molecules. It is therefore at present unclear whether the markedly increased glycine level (28 % of total solutes) is a result of uptake or genuine osmoregulated biosynthesis. ACKNOWLEDGEMENTS We are very grateful to Dr Marianne Engeser and her team for the high-resolution HPLC-MS measurements. We also thank Erhard Bremer (Philipps University, Marburg) for kindly providing E. coli MKH13. This work was supported by the Ministry of Science, Research and Technology of Iran (scholarship to J. A. M.) and the German Federal Ministry of Education and Research (BMBF). Fudou, R., Jojima, Y., Iizuka, T. & Yamanaka, S. (2002). 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