Download Different strategies of osmoadaptation in the closely

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). Haliangium
ochraceum gen. nov., sp. nov. and Haliangium tepidum sp. nov.: novel
moderately halophilic myxobacteria isolated from coastal saline
environments. J Gen Appl Microbiol 48, 109–115.
Galinski, E. A., Pfeiffer, H. P. & Trüper, H. G. (1985). 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 149, 135–139.
Galinski, E. A. & Herzog, R. M. (1990). The role of trehalose as a
substitute for nitrogen-containing compatible solutes (Ectothiorhodospira halochloris). Arch Microbiol 153, 607–613.
Grammann, K., Volke, A. & Kunte, H. J. (2002). New type of
osmoregulated solute transporter identified in halophilic members
of the bacteria domain: TRAP transporter TeaABC mediates uptake
of ectoine and hydroxyectoine in Halomonas elongata DSM
2581(T). J Bacteriol 184, 3078–3085.
Haardt, M., Kempf, B., Faatz, E. & Bremer, E. (1995). The
osmoprotectant proline betaine is a major substrate for the
binding-protein-dependent transport system ProU of Escherichia
coli K-12. Mol Gen Genet 246, 783–796.
Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P. &
Kushner, S. R. (1989). New method for generating deletions and
gene replacements in Escherichia coli. J Bacteriol 171, 4617–4622.
Held, C., Neuhaus, T. & Sadowski, G. (2010). Compatible solutes:
thermodynamic properties and biological impact of ectoines and
prolines. Biophys Chem 152, 28–39.
Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R.
(1989). Engineering hybrid genes without the use of restriction
enzymes: gene splicing by overlap extension. Gene 77, 61–68.
Iizuka, T., Jojima, Y., Fudou, R., Hiraishi, A., Ahn, J. W. & Yamanaka, S.
(2003a). Plesiocystis pacifica gen. nov., sp. nov., a marine
myxobacterium that contains dihydrogenated menaquinone, isolated
from the Pacific coasts of Japan. Int J Syst Evol Microbiol 53, 189–195.
Iizuka, T., Jojima, Y., Fudou, R., Tokura, M., Hiraishi, A. &
Yamanaka, S. (2003b). Enhygromyxa salina gen. nov., sp. nov., a
REFERENCES
Brown, A. D. (1976). Microbial water stress. Bacteriol Rev 40, 803–846.
Burg, M. B. & Ferraris, J. D. (2008). Intracellular organic osmolytes:
function and regulation. J Biol Chem 283, 7309–7313.
Bursy, J., Kuhlmann, A. U., Pittelkow, M., Hartmann, H., Jebbar, M.,
Pierik, A. J. & Bremer, E. (2008). Synthesis and uptake of the
compatible solutes ectoine and 5-hydroxyectoine by Streptomyces
coelicolor A3(2) in response to salt and heat stresses. Appl Environ
Microbiol 74, 7286–7296.
Casadaban, M. J. (1976). Transposition and fusion of the lac genes to
selected promoters in Escherichia coli using bacteriophage lambda and
Mu. J Mol Biol 104, 541–555.
da Costa, M. S., Santos, H. & Galinski, E. A. (1998). An overview of
the role and diversity of compatible solutes in Bacteria and Archaea.
Adv Biochem Eng Biotechnol 61, 117–153.
Ellis, L. L., Burcham, J. M., Paynter, K. T. & Bishop, S. H. (1985).
Amino acid metabolism in euryhaline bivalves: regulation of glycine
accumulation in ribbed mussel gills. J Exp Zool 233, 347–358.
slightly halophilic myxobacterium isolated from the coastal areas of
Japan. Syst Appl Microbiol 26, 189–196.
Inbar, L. & Lapidot, A. (1988). The structure and biosynthesis of new
tetrahydropyrimidine derivatives in actinomycin D producer
Streptomyces parvulus. Use of 13C- and 15N-labeled L -glutamate
and 13C and 15N NMR spectroscopy. J Biol Chem 263, 16014–16022.
Kanapathipillai, M., Lentzen, G., Sierks, M. & Park, C. B. (2005).
Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity
of Alzheimer’s beta-amyloid. FEBS Lett 579, 4775–4780.
Kaneshiro, E. S., Holz, G. G., Jr. & Dunham, P. B. (1969).
Osmoregulation in a marine ciliate, Miamiensis avidus. II.
Regulation of intracellular free amino acids. Biol Bull 137, 161–169.
Kimura, Y., Kawasaki, S., Yoshimoto, H. & Takegawa, K. (2010).
Glycine betaine biosynthesized from glycine provides an osmolyte
for cell growth and spore germination during osmotic stress in
Myxococcus xanthus. J Bacteriol 192, 1467–1470.
Kunte, H. J., Lentzen, G. & Galinski, E. A. (2014). Industrial
production of the cell protectant ectoine: protection mechanisms,
processes, and products. Curr Biotechnol 3, 10–25.
Felder, S., Dreisigacker, S., Kehraus, S., Neu, E., Bierbaum, G.,
Wright, P. R., Menche, D., Schäberle, T. F. & König, G. M. (2013a).
Larsen, P. I., Sydnes, L. K., Landfald, B. & Strøm, A. R. (1987).
Salimabromide: unexpected chemistry from the obligate marine
myxobacterium Enhygromxya salina. Chemistry 19, 9319–9324.
Osmoregulation in Escherichia coli by accumulation of organic
osmolytes: betaines, glutamic acid, and trehalose. Arch Microbiol
147, 1–7.
Felder, S., Kehraus, S., Neu, E., Bierbaum, G., Schäberle, T. F. &
König, G. M. (2013b). Salimyxins and enhygrolides: antibiotic,
sponge-related
metabolites
from
the
obligate
marine
myxobacterium Enhygromyxa salina. ChemBioChem 14, 1363–1371.
660
Lippert, K. & Galinski, E. A. (1992). Enzyme stabilization by ectoine-
type compatible solutes: protection against heating, freezing and
drying. Appl Microbiol Biotechnol 37, 61–65.
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
Louis, P., Trüper, H. G. & Galinski, E. A. (1994). Survival of Escherichia
coli during drying and storage in the presence of compatible solutes.
Appl Microbiol Biotechnol 41, 684–688.
May, G., Faatz, E., Lucht, J. M., Haardt, M., Bolliger, M. & Bremer, E.
(1989). Characterization of the osmoregulated Escherichia coli proU
enhance abiotic stress tolerance in plants. Plant Biotechnol 26,
125–134.
Schäberle, T. F., Goralski, E., Neu, E., Erol, O., Hölzl, G., Dörmann, P.,
Bierbaum, G. & König, G. M. (2010). Marine myxobacteria as a source
promoter and identification of ProV as a membrane-associated
protein. Mol Microbiol 3, 1521–1531.
of antibiotics—comparison of physiology, polyketide-type genes and
antibiotic production of three new isolates of Enhygromyxa salina.
Mar Drugs 8, 2466–2479.
McBride, M. J. & Zusman, D. R. (1989). Trehalose accumulation in
Schmitz, R. P. H. & Galinski, E. A. (1996). Compatible solutes in
vegetative cells and spores of Myxococcus xanthus. J Bacteriol 171,
6383–6386.
luminescent bacteria of the genera Vibrio, Photobacterium and
Photorhabdus (Xenorhabdus): occurrence of ectoine, betaine and
glutamate. FEMS Microbiol Lett 142, 195–201.
Meyer, J. D., Nayar, R. & Manning, M. C. (2009). Impact of bulking
agents on the stability of a lyophilized monoclonal antibody. Eur J
Pharm Sci 38, 29–38.
Milner, J. L., Grothe, S. & Wood, J. M. (1988). Proline porter II is
activated by a hyperosmotic shift in both whole cells and membrane
vesicles of Escherichia coli K12. J Biol Chem 263, 14900–14905.
Severin, J., Wohlfarth, A. & Galinski, E. A. (1992). The predominant role
of recently discovered tetrahydropyrimidines for the osmoadaptation
of halophilic eubacteria. Microbiology 138, 1629–1638.
Sleator, R. D. & Hill, C. (2002). Bacterial osmoadaptation: the role of
Moritz, K. D., Amendt, B., Witt, E. M. & Galinski, E. A. (2015). The
osmolytes in bacterial stress and virulence. FEMS Microbiol Rev 26,
49–71.
hydroxyectoine gene cluster of the non-halophilic acidophile
Acidiphilium cryptum. Extremophiles 19, 87–99.
Sydlik, U., Gallitz, I., Albrecht, C., Abel, J., Krutmann, J. & Unfried, K.
(2009). The compatible solute ectoine protects against nanoparticle-
Overbeek, R., Olson, R., Pusch, G. D., Olsen, G. J., Davis, J. J., Disz, T.,
Edwards, R. A., Gerdes, S., Parrello, B. & other authors (2014).
induced neutrophilic lung inflammation. Am J Respir Crit Care Med
180, 29–35.
The SEED and the Rapid Annotation of microbial genomes
using Subsystems Technology (RAST). Nucleic Acids Res 42,
D206–D214.
Tanne, C., Golovina, E. A., Hoekstra, F. A., Meffert, A. & Galinski, E. A.
(2014). Glass-forming property of hydroxyectoine is the cause of
Peters, J. E., Thate, T. E. & Craig, N. L. (2003). Definition of the
its superior function as a desiccation protectant. Front Microbiol
5, 150.
Escherichia coli MC4100 genome by use of a DNA array. J Bacteriol
185, 2017–2021.
Waditee, R., Tanaka, Y., Aoki, K., Hibino, T., Jikuya, H., Takano, J.,
Takabe, T. & Takabe, T. (2003). Isolation and functional
Pikal-Cleland, K. A., Cleland, J. L., Anchordoquy, T. J. & Carpenter, J. F.
(2002). Effect of glycine on pH changes and protein stability during
freeze–thawing in phosphate buffer systems. J Pharm Sci 91, 1969–1979.
characterization of N-methyltransferases that catalyze betaine
synthesis from glycine in a halotolerant photosynthetic organism
Aphanothece halophytica. J Biol Chem 278, 4932–4942.
Reuter, K., Pittelkow, M., Bursy, J., Heine, A., Craan, T. & Bremer, E.
(2010). Synthesis of 5-hydroxyectoine from ectoine: crystal structure
Weber, T., Blin, K., Duddela, S., Krug, D., Kim, H. U., Bruccoleri, R.,
Lee, S. Y., Fischbach, M. A., Müller, R. & other authors (2015).
of the non-heme iron(II) and 2-oxoglutarate-dependent dioxygenase
EctD. PLoS One 5, e10647.
Roberts, M. F. (2005). Organic compatible solutes of halotolerant and
halophilic microorganisms. Saline Syst 1, 5.
Sadeghi, A., Soltani, B. M., Nekouei, M. K., Jouzani, G. S.,
Mirzaei, H. H. & Sadeghizadeh, M. (2014). Diversity of the ectoines
biosynthesis genes in the salt tolerant Streptomyces and evidence for
inductive effect of ectoines on their accumulation. Microbiol Res
169, 699–708.
Sayyar Khan, M., Yu, X., Kikuchi, A., Asahina, M. & Watanabe, K. N.
(2009). Genetic engineering of glycine betaine biosynthesis to
http://mic.microbiologyresearch.org
antiSMASH 3.0—a comprehensive resource for the genome
mining of biosynthetic gene clusters. Nucleic Acids Res 43,
W237–W243.
Widderich, N., Czech, L., Elling, F. J., Könneke, M., Stöveken, N.,
Pittelkow, M., Riclea, R., Dickschat, J. S., Heider, J. & Bremer, E.
(2016). Strangers in the archaeal world: osmostress-responsive
biosynthesis of ectoine and hydroxyectoine by the marine
thaumarchaeon Nitrosopumilus maritimus. Environ Microbiol 4,
(In press).
Wood, J. M. (2006). Osmosensing by bacteria. Sci STKE 2006, pe43.
Edited by: C. Dahl
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 12:05:08
661