A comparison of the predicted protein sequence set of H. elongata
with the NR database (downloaded from NCBI October 4, 2009) emphasizes a very close relationship of H. elongata
with C. salexigens
, a halophilic γ-proteobacterium of the family Halomonadaceae (Arahal et al., 2001
). About half of the proteins could be reliably assigned at the species level (1672 out of 3473) using protein BLAST (Altschul et al., 1997
) and MEGAN (Huson et al., 2007
). The vast majority of the assigned proteins are related to C. salexigens
(1544 assignments, corresponds to 92%). The few remaining MEGAN assignments are relatively equally distributed among a number of different species with Marinobacter
sp. ELB17 (2.6%) dominating any other species (< 1%). For an overview, see Fig. S1
. For 290 sequences, no assignment was reported to any taxon by MEGAN because no significant hit to the NR database was found.
Approximately two-thirds of the H. elongata
proteins have an ortholog in C. salexigens
as indicated by bidirectional best BLAST results (2367 proteins, 68.1%). They are very closely related, with an average of 69% sequence identity. Gene order is also well conserved. On closer examination of the set of H. elongata
proteins that have an ortholog in C. salexigens
, it was found that when two genes encoding such orthologous proteins are the nearest genetic neighbours in H. elongata
then their corresponding partners in C. salexigens
are also the nearest genetic neighbours in 70% of the cases. This high level of synteny is also evident from a MUMmer alignment of the two chromosomal sequences (Fig. S2
). This results in a prominent X-alignment. Such X-alignments have been described for several interspecies comparisons (Eisen et al., 2000
). The prominence of the X-alignment may indicate a close relationship between the two species, which is astonishing as both organisms are classified into distinct genera.
We used the Metanor tool of the GenDB genome annotation system (Meyer et al., 2003
) for automatic function prediction. For enzymes, these were cross-checked with the data obtained by the PRIAM program (Claudel-Renard et al., 2003
). A total of 1265 complete or partial EC numbers were assigned. Central metabolism and the biosynthetic pathways related to ectoine biosynthesis and degradation were manually curated in detail.
Halomonas elongata contains a complete set of ribosomal proteins. There are tRNA ligases for 19 of the 20 canonical amino acids. Asparagine (Asn) is not loaded as Asn but as aspartate with subsequent amidation to Asn by a gatABC-encoded enzyme.
To further analyse the protein set from H. elongata
, we assigned cluster of orthologous groups of proteins (COGs) using the eggNOG 2.0 dataset as a reference (Muller et al., 2010
). The assignment procedure, using an in-house script to analyse BlastP results, is described in Experimental procedures
and in Supporting information
We found a number of high-occupancy COGs that contain many different proteins from H. elongata
). Six COGs have more than 20 proteins of which COG0583 is most highly occupied with 57 members. The 20 most highly occupied COGs belong to four functional classes: (i) transcription regulators (six COGs), (ii) ‘general function’ enzymes (five COGs), (iii) transporters (seven COGs) and (iv) two-component systems (two COGs).
The most highly occupied COG0583 contains lysR family transcription regulators. The five high-occupancy COGs with ‘general function’ enzymes code for short-chain alcohol dehydrogenases, aldehyde dehydrogenases, acetyltransferases, methyltransferases and FAD-dependent oxidoreductases. One of the acetyltransferases in the frequent COG0454 is EctA, the first enzyme of the ectoine biosynthesis pathway. The seven high-occupancy COGs with transporters include the three subunits of TRAP transporters (COG1638, COG1593, COG3090, TRAP-type C4-dicaroxylate transport system, periplasmic component, large and small permease component). The teaABC ectoine transporter belongs to this set of COGs. The other frequently occurring transporters are MFS superfamily permeases and DMT superfamily permeases as well as two subunits of ABC-type transporters (ATPase, permease).
We searched for COGs, which are preferentially carried by halophilic/marine bacteria by comparing the proteins of 3 halophiles and 10 marines to 14 non-halophilic species (Supporting information, Table S2
). Among the 97 COGs identified by this approach were many secondary transport systems that are thought to be dependent on Na+
symport, and sodium-proton exchangers. The transport systems that can be mainly found in halophiles belong to the NSS family and the TRAP family of transporters. The distribution of TRAP transporters was analysed by Mulligan and colleagues (2007
) and they found that these uptake systems are extensively used in marine bacteria. There is evidence that TRAP transporters are powered by sodium symport (Mulligan et al., 2007
) and perhaps the utilization of sodium-dependent transporters could be advantageous for halophilic bacteria that have a sodium gradient across their membrane. Members of the NSS family of transporters can be found in Eukarya
and transport nitrogenous substances (Beuming et al., 2006
; Quick et al., 2006
). In bacteria, NSS transporters catalyse the high-affinity uptake of amino acids by a sodium-symport mechanism (Androutsellis-Theotokis et al., 2003
). Again, the preference of marine bacteria for these transporters can be explained by their dependency on sodium for transport. Both the NSS and TRAP transporter, also have a high affinity for their substrates (Androutsellis-Theotokis et al., 2003
; Chae and Zylstra, 2006
; Kuhlmann et al., 2008
), which might be required in the marine environment with sometimes low solute concentrations.
High-salt adaptation may result in protein adaptation, e.g. by adjusting protein pI values. We analysed whether H. elongata
has an unusual average pI when compared with each of the 27 organisms selected for identification of halophile-specific COGs (Supporting information, Table S3
). If there is any pI shift in H. elongata
proteins, the shift is only very slight and towards the acidic direction. These results indicate that an overall acidic proteome is not required for salt adaptation of halophilic bacteria employing the organic osmolyte mechanism.
synthesizes ectoine (1,4,5,6,tetra-2-methyl-4-pyrimidonecarboxylic acid) as its main compatible solute (Severin et al., 1992
). Ectoine is synthesized from aspartate-semialdehyde, the central intermediate in the synthesis of amino acids belonging to the aspartate family (). Ectoine formation comprises three enzymatic steps (Peters et al., 1990
; Ono et al., 1999
). First, aspartate-semialdehyde is transaminated to 2,4-diaminobutyric acid (DABA) with glutamate as amino-group donor. The transamination is catalysed by DABA transaminase (EctB). Then, an acetyl group is transferred to DABA from acetyl-CoA by DABA-Nγ-acetyltransferase (EctA) in order to synthesize Nγ-acetyl-l
-2,4-diaminobutyric acid. Finally, ectoine synthase (EctC) catalyses the cyclic condensation of Nγ-acetyl-l
-2,4-diaminobutyric acid, which leads to the formation of ectoine. Under certain stress conditions (e.g. elevated temperatures) H. elongata
converts some of the ectoine to 5-hydroxyectoine by ectoine hydroxylase (EctD) (Inbar and Lapidot, 1988
; Wohlfarth et al., 1990
Fig. 1 Metabolic pathway of the compatible solute ectoine in H. elongata. The degradation pathway is based on genetic and chromatographic analysis carried out in this study. Shown here is the hydrolysis of ectoine that leads directly to Nγ- and Nα-acetyl- (more ...)
The genes encoding the enzymes for ectoine de novo
synthesis in H. elongata
were identified by transposon mutagenesis, and the nucleotide sequence of ectAB
as well as a partial sequence of ectC
were published in 1998 (Göller et al., 1998
). The genomic region containing these ectoine biosynthesis genes (ectA: Helo_2588, ectB: Helo_2589, ectC: Helo_2590
) is shown in . The ectD
) encoding the hydroxylase for hydroxyectoine synthesis (Prabhu et al., 2004
; Bursy et al., 2007
) is located apart from the ectABC
cluster. Immediately downstream of the 414 nt comprising ectC
gene a further ORF is located (Helo_2591
) that is predicted to encode a transcriptional regulator of the AraC family.
Fig. 2 Ectoine synthesis gene organization in H. elongata and other prokaryotes. Shown are the ect genes from the subdivisions of the proteobacteria, the phyla Actinobacteria, Firmicutes, Planctomycetes and Thaumarchaeota. Only ectoine synthesis genes from prokaryotes (more ...)
gene encodes a 192-residue protein with a calculated molecular mass of 21.2 kDa. According to the studies of Ono and colleagues (Ono et al., 1999
), DABA-acetyltransferase EctA displays a high specificity for its substrate DABA. EctA from H. elongata
is a rather acidic protein with a calculated pI value of 4.8.
Similar acidic EctA proteins can be found in most of the marine and halophilic bacteria (e.g. C. salexigens, pI 5.3; Bacillus halodurans, pI 5.5; Halorhodospira halophila, pI 5.4; Blastospirellula marina pI 4.7) as well as in soil bacteria from the Actinomyces group (e.g. Nocardia farcinica, pI 5.0; Mycobacterium gilvum, pI 5.9; Streptomyces coelicolor, pI 5.0). In contrast, all but one of the remaining non-halophilic bacteria analysed in this study possess EctA proteins with a neutral or alkaline pI (e.g. Bacillus clausii, pI 7.5; Pseudomonas stutzeri, pI 8.0; Wolinella succinogenes, pI 9.0; Bordetella parapertussis, pI 8.4; Phenylobacterium zucineum, pI 8.9).
gene encodes a 421-residue protein with a molecular mass of 46.1 kDa, which requires K+
for its transaminase activity and for protein stability. Gel filtration experiments with purified protein from H. elongata
indicate that the DABA aminotransferase EctB might form a homohexamer in the native state (Ono et al., 1999
). The preferred amino group donor of EctB in the formation of DABA is glutamate, while in the reverse reaction DABA and γ-aminobutyrate are the preferred amino group donors to α-ketoglutarate.
gene encodes ectoine synthase, a 137-residue protein with a calculated molecular weight of 15.5 kDa and a pI value of 4.9. The EctC protein belongs to the enzyme family of carbon-oxygen lyases. In vitro
experiments with purified EctC revealed that ectoine-synthase activity and affinity to its substrate are strongly affected by NaCl (Ono et al., 1999
). N-acetylated amino acids having a carbon skeleton with one (ornithine derivatives) or two (lysine derivatives) atoms more than Nγ-acetyl-diaminobutyric acid are not suitable substrates for EctC. Galinski and co-workers, who described the ectoine biosynthetic pathway for the first time, demonstrated the reversibility of the ectoine synthase reaction when measured in crude cell extracts of Halorhodospira
(Peters et al., 1990
). However, Ono et al
. characterized purified EctC from H. elongata
as an enzyme that is unable to carry out the reverse reaction from ectoine to Nγ-acetyl-diaminobutyric acid when ectoine was offered as substrate in the range of 10 mM to 1 M (Ono et al., 1999
encoded ectoine hydroxylase consists of 332 amino acids and has a molecular weight of 37.4 kDa. The EctD protein is a member of an oxygenase subfamily within the non-heme-containing, iron (II)- and α-ketoglutarate-dependent dioxygenase superfamily. Ectoine hydroxylase was shown to catalyse the direct hydroxylation of ectoine to 5-hydroxyectoine (Bursy et al., 2007
Transcriptional regulation of ectABC
Recent studies on the transcriptional regulation of ectABC
in Bacillus pasteurii
and Halobacillus halophilus
revealed that ectABC
is organized as one operon (Kuhlmann and Bremer, 2002
; Saum and Müller, 2008
), while in the halophilic γ-proteobacterium C. salexigens
the transcriptional organization of the ect
-cluster turned out to be rather complex (Calderón et al., 2004
). Calderón et al
. mapped a total of five promoters regulating ectABC
transcription. Two σ70
-controlled promoters, one σs
-dependent promoter and a promoter of unknown specificity are located upstream of ectA
, while a fifth promoter was found upstream of ectB
To gain further information on the transcriptional regulation of the ectABC
gene-cluster, we mapped the transcriptional initiation sites in H. elongata
by RACE-PCR and found a different, but also complex promoter assembly. Two transcriptional initiation sites could be pinpointed in front of ectA
, and one was mapped immediately upstream of ectC
(). The two transcription initiation sites before ectA
are located 25 and 92 bp, respectively, upstream from the ectA
start codon. Inspection of the DNA sequence upstream of the first ectA
initiation site (25 bp) revealed the presence of putative −10 and −35 sequences that resemble the binding site for the vegetative sigma factor σ70
. The −10 and −35 sequences are separated by 17 bp, a typical spacing for promoters controlled by σ70
. Upstream of the second initiation site (92 bp), a −10 DNA sequence was found that resembles σ38
-controlled promoters. In addition to the −10 region, a so-called G-element exists at position −35. G-elements are characteristic for osmotically induced σ38
promoters (Lee and Gralla, 2004
). RACE-PCR at the ectC
gene mapped a transcription start point 47 bp upstream from the start codon (). Upstream of the initiation site putative −12 and −24 sequences were found that are typical for σ54
-controlled promoters. σ54
-controlled promoters are often involved in transcription of nitrogen-regulated genes (Ausubel, 1984
; Bordo et al., 1998
). The two sequences of the putative σ54
promoter are appropriately spaced by four nucleotides. In addition, a sequence of 18 bp was found −111 to −128 bp upstream of the initiation site that resembles a consensus sequence required for transcription activation of some σ54
promoters controlled by FleQ (Hu et al., 2005
). The transcriptional regulation of ectABC
by an osmoregulated σ38
promoter and a σ54
promoter is in agreement with physiological observations made with other bacteria, such as Corynebacterium glutamicum
and H. halochloris
. In these organisms, it was shown that synthesis of the compatible solutes proline and glycine-betaine, respectively, is not only determined by salinity but also by nitrogen supply (Galinski and Herzog, 1990
; Wolf et al., 2003
Fig. 3 Transcription initiation sites and putative promoters of the ectoine synthesis genes ectABC. Nucleotide sequences of the ectA (A) and ectC (B) promoter regions. Arrows indicate the transcription initiation sites (+ 1), which were mapped by RACE-PCR. The (more ...)
Organization of ectABC in different prokaryotes
genes and the proteins for ectoine synthesis are very conserved among ectoine-producing bacteria. A study published recently by Lo and colleagues (2009
) analysed the phylogenetic distribution of ect
genes and showed that the prevalent organization of these genes is in a single cluster of at least three genes (ectABC
), consistent with the analysis presented in this study. However, comparison of the H. elongata
genome with other genomes revealed that the ectABC
genes are not always organized in this way. A first analysis carried out by Vargas and co-workers with the genome of C. salexigens
came to a similar result (Vargas et al., 2008
). We extended this study and found that in Nitrosococcus oceani, ectC
is located at a site different from ectAB
. Marinobacter hydrocarbonoclasticus
DSM 11845 [formerly aquaeolei
VT8 (Márquez and Ventosa, 2005
)] carries three ectC
ORFs, but these are located at different sites within the genome and none are close to ectAB
. In Alkalilimnicola ehrlichii
, gene ectC
is located downstream of ectAB
but on the opposite strand. In all other genomes that were considered in this study, the ectABC
components were clustered similar to ectABC
from H. elongata
. In summary, the way the ect-
genes are organized in these organisms can be classified as follows ():
- Bacillus halodurans, Ruegeria[formerly Silicibacter (Yi et al., 2007)] sp. TM1040, and all Vibrio species analysed in this study possess only one single ectABC cluster and are missing ectD that encodes the ectoine hydroxylase.
- In H. elongata and two other members of the Oceanospirillales (C. salexigens, Alcanivorax borkumensis SK2), in γ-proteobacterium Saccharophagus degradans, and in B. clausii, the ectABC cluster and gene ectD can be found at separate sites within the genome. C. salexigens differs from H. elongata and the other members of this group in having a second ectD-like ORF named ectE. Similar to ectD, locus ectE is separated from ectABC.
- The majority of genomes that were checked in this study, including the chromosome of the crenarchaeote Nitrosopumilus maritimus, contain a single ect-cluster comprising the genes ectABC and D.
In roughly half of the genomes that were compared with the H. elongata genome an additional ORF (ask) can be found encoding a putative aspartate kinase. The ORF ask is located downstream of ectB, ectC or ectD. All bacterial genomes in this study that contain ask are equipped with at least one further aspartate kinase (LysC). It is therefore tempting to speculate whether ask next to the ect components is coding for a specific kinase involved in ectoine synthesis. H. elongata and its halophilic relative C. salexigens do not possess such an ask. They rely on only one type of aspartate kinase, LysC (Helo_3742), responsible for the synthesis of ectoine and the amino acids lysine, threonine and methionine.
Homology analysis of aspartate kinases by Lo and colleagues (2009
) revealed a separation of two subhomology divisions, which are denoted ASKα and ASKβ. Amino acid sequence analysis showed that LysC from H. elongata
belongs to the ASKβ homology division and is most closely related to the ASKβ aspartate kinase from C. salexigens
. According to the allosteric-specifity grouping of ASKβ enzymes, LysC of H. elongata
is sensitive to the allosteric regulation of Thr and Lys. The gene encoding LysC in H. elongata
is associated with the genes recA recX alaS lysC crsA tRNA
, which is a conserved gene arrangement among γ-proteobacteria. This gene cluster is not associated with genes involved in the amino acid metabolism of the aspartate family (Lo et al., 2009
) and our analysis of the lysC
neighbourhood in H. elongata
could not find any connections with the ectoine metabolism.
In our opinion, allosteric regulation alone is not a suitable mechanism in regulating LysC activity and thereby the internal ectoine concentration. Feedback or allosteric regulation is used in biological systems to achieve a certain optimal concentration of a metabolite. However, the ectoine content has to be adjusted constantly to match the external osmolarity and it is known that there is essentially a linear relationship between compatible solute content and the salt concentration of the medium (Kuhlmann and Bremer, 2002
). Therefore, if an aspartate kinase were involved in controlling ectoine synthesis, then it should be also an osmoregulated enzyme and models explaining the osmoregulatory control of ectoine synthesis have been proposed (Kunte, 2006
Ectoine can be accumulated up to molar concentration by H. elongata
depending on the salinity of the surrounding medium. Furthermore, ectoine can also be utilized as both a carbon and a nitrogen source by H. elongata
and when ectoine is offered as a nutrient, it still serves as compatible solute (Göller, 1999
). In order to find out how ectoine is degraded, the genome of H. elongata
was compared with Sinorhizobium meliloti
In S. meliloti
, a cluster of five ORFs named eutABCDE
was described with hypothetical functions in ectoine catabolism (Jebbar et al., 2005
). No exact function was assigned to any of these five ORFs but their deduced amino acid sequences indicate they are similar to arylmalonate decarboxylases (eutA
), threonine dehydratases (eutB
), ornithine cyclodeaminases (eutC
), aminopeptidases (eutD
) and glutamate-desuccinylases/aspartoacylases (eutE
). A homologue for each of the eutBCDE
genes can be found within the chromosome of H. elongata
(), but no eutA
homologue could be identified. The homologues of eutBC
) and eutDE
(Helo_3665, doeA; Helo_3664, doeB
) are organized in two clusters that are separated by three ORFs (). These three ORFs are homologues of genes annotated as transcriptional regulator (Helo_3663, doeX
), dehydrogenase (Helo_3662, doeC
) and transaminase (Helo_3661, doeD
Fig. 4 Ectoine-degradation gene organization in H. elongata and other bacteria. Ectoine hydrolase genes (doeA) are blue, Nα-acetyl-l-2,4-diaminobutyric acid deacetylase genes (doeB) are red, genes for AsnC/Lrp-like DNA-binding protein DoeX are pink ( (more ...)
The ORFs Helo_3665, Helo_3664, Helo_3662 and Helo_3661 were chosen for mutation experiments, as the predicted enzymatic function of their gene products would make them candidates for the breakdown of ectoine into aspartate (). All four ORFs were deleted (in-frame null mutation) and the resulting mutants were either unable to utilize ectoine as a carbon source or they displayed reduced growth on ectoine. We therefore named the cluster doeABCD (degradation of ectoine ABCD, ). Based on experiments (described below) and sequence homology, we named these ORFs doeA (Helo_3665, ectoine hydrolase), doeB (Helo_3664, Nα-acetyl-l-2,4-diaminobutyric acid deacetylase), doeC (Helo_3662, aspartate-semialdehyde dehydrogenase) and doeD (Helo_3661, diaminobutyric acid transaminase). A fifth ORF belonging to the doe cluster is located between doeB and doeC, and is named doeX (Helo_3663, ). Deletion of Helo_3660 (eutB) and Helo_3659 (eutC) did not impair growth of the corresponding mutants on ectoine and we infer from these results that eutBC does not participate in ectoine degradation.
From the mutational and additional analytical experiments we concluded that the degradation of ectoine proceeds via hydrolysis of ectoine (DoeA) to the novel compound Nα-acetyl-l-2,4-diaminobutyric acid, deacetylation of Nα-acetyl-l-2,4-diaminobutyric acid (DoeB) to l-2,4-diaminobutyric acid, and a transaminase reaction (DoeD) leading to aspartate-semialdehyde. Finally, aspartate-semialdehyde is oxidized by DoeC to aspartate (). The proposed pathway is based on the following experimental and computational data:
) is a homologue to eutD
and codes for a 399 aa protein (44.9 kDa, pI 5.0) that belongs to the peptidase-M24 family. Within that family, DoeA is similar to creatinase (creatine amidinohydrolase), which catalyses the hydrolysis of creatine to sarcosine and urea (Coll et al., 1990
). Deletion of doeA
created a mutant KB41 that was unable to grow on ectoine as carbon source. The doeA+
wild type could be restored in the ΔdoeA
mutant by expressing doeA in trans
from plasmid pKSB7 (pJB3Cm6::doeA
), proving that no polar effect was causing the defect in ectoine catabolism.
To gain information on the enzymatic reaction catalysed by DoeA, the doeA
gene was expressed in E. coli
BL21 cells from plasmid pKSB11. After 16 h of expression, 1 mM ectoine was added to the salt medium (340 mM NaCl). E. coli
is known to accumulate ectoine in the cytoplasm as compatible solute via osmoregulated transporters ProU and ProP but is unable to metabolize ectoine (Jebbar et al., 1992
; Racher et al., 1999
). The cytoplasmic fraction of E. coli
was analysed by HPLC allowing for the detection of N-acetyl-l
-2,4-diaminobutyric acid (N-Ac-DABA), the product of ectoine hydrolysis. Two forms of N-Ac-DABA were detected, which could be distinguished by comparison with a standard of Nγ-acetyl-l
-2,4-diaminobutyric acid (Nγ-Ac-DABA) and Nα-acetyl-l
-2,4-diaminobutyric acid (Nα-Ac-DABA). While neither Nα-Ac-DABA nor Nγ-Ac-DABA was formed in E. coli
, both forms could be detected at a ratio of 2:1 in cells carrying doeA
(). This unambiguously demonstrates that DoeA functions as ectoine hydrolase.
Fig. 5 Chromatographic analysis of the cytoplasm from (A) E. coli expressing recombinant doeA and (B) H. elongata wild type and ΔdoeB-mutant strain KB42.A. Time course of Nα-Ac-DABA and Nγ-Ac-DABA formation from ectoine in E. coli expressing (more ...)
Similar results concerning ectoine hydolysis were obtained with H. elongata
-mutant KB1 after growth with ectoine as carbon source. Although EctA-catalysed acetylation of DABA leading to Nγ-Ac-DABA is blocked, Nγ-Ac-DABA is accumulated in roughly the same concentration as in wild-type cells. In addition, Nα-Ac-DABA is also present although the pathway towards aspartate remained genetically unchanged. Apparently, ectoine hydrolase activity leads to the formation of Nα-Ac-DABA and Nγ-Ac-DABA also in the H. elongata
background. Any significant contribution of ectoine synthase EctC to Nγ-Ac-DABA formation is rather unlikely, as purified EctC from H. elongata
is described as an enzyme with no detectable reverse activity (Ono et al., 1999
The specificity of ectoine hydrolase DoeA with respect to the isoforms of N-Ac-DABA remains somewhat ambiguous. While the results on DoeA expression in E. coli () suggest the formation of both, Nα-Ac-DABA and Nγ-Ac-DABA, upon ectoine hydrolysis (as depicted in ), cleavage of ectoine by DoeA could also produce only one isomer, which subsequently has to be converted into the corresponding isomer by an acetyltransferase. Whatever mechanism is employed by the cell, both are suitable and allow for the formation of Nα-Ac-DABA, which is the essential substrate for the subsequent catabolic enzyme DoeB (see below).
(ii)The 342 aa protein (36.6 kDa, pI 4.6) encoded by doeB
, homologue to eutE
) is closely related to proteins of the succinyl-glutamate desuccinylase/aspartoacylase subfamily, which are part of the M14 family of metallocarboxypeptidases (Makarova and Grishin, 1999
). The desuccinylase is involved in arginine catabolism while the aspartoacylase cleaves N-acetyl-aspartate into aspartate and acetate (Le Coq et al., 2008
). Deletion of doeB
resulted in a mutant KB42 that could not utilize ectoine as carbon and nitrogen source. The doeB+
wild type could be restored in mutant KB42 (ΔdoeB
) by expressing doeB
from plasmid pJSB3 (pJB3Cm6::doeB
). Analysing the cytoplasmic fraction of wild type and ΔdoeB
strain by HPLC revealed that Nα-acetyl-l
-2,4-diaminobutyric acid is accumulated as the predominant amino-reactive solute in mutant KB42 while no Nα-Ac-DABA is detectable in the H. elongata
wild type (). The Nγ-Ac-DABA concentration, however, remains still the same in KB42 and wild-type cells. Based on the results from the feeding experiments (no growth on ectoine as nitrogen-source) and the HPLC analysis (accumulation of Nα-Ac-DABA, unchanged Nγ-Ac-DABA level), we propose that the hydrolysis of ectoine is directly succeeded by DoeB-catalysed deacetylation of Nα-Ac-DABA.
While Nγ-Ac-DABA is not a substrate for DoeB, it serves again as a substrate for ectoine synthase EctC and can be converted back to ectoine. Nα-Ac-DABA, however, is removed from this cycle by deacetylation to DABA, which then can either flow off to aspartate or re-enter the ectoine synthesis pathway in wild-type cells. This closes the cycle of synthesis and degradation, which is powered by acetylation and deacetylation of DABA and Nα-Ac-DABA respectively (). As we will describe below, such a cycle provides a fast mechanism for the cell to regulate the cytoplasmic ectoine concentration.
) encodes a putative aspartate aminotransferase (469 aa, 50.8 kDa, pI 5.6) with similarities to the PLP-dependent aspartate aminotransferase superfamily. Deletion of doeD
resulted in mutant KB48 (ΔdoeD
) that was impaired in growth on ectoine as a sole carbon source. In saline minimal medium (510 mM NaCl), the doeD
deletion reduced the growth rate of strain KB48 threefold down to 0.077 h−1
compared with 0.248 h−1
observed with the wild type indicating a participation of DoeD in ectoine degradation. Deletion of the aminotransferase gene ectB
, resulting in mutant SB1 (ΔectB
), or in doeD
-mutant KB48, resulting in the double knockout mutant SB1.1 (ΔectB
), did not further reduce growth on ectoine. Because both mutant strains were still able to synthesize ectoine other transaminases (or amidases) are still active in H. elongata
, converting DABA to L-aspartate-β-semialdehyde, and likewise, L-aspartate-β-semialdehyde to DABA, which can be readily explained by the rather broad substrate specificity of transaminases (Fotheringham, 2000
(iv)The doeC gene (Helo_3662), located upstream of doeD, encodes a putative dehydrogenase (493 aa, 53.1 kDa, pI 4.8) and is most closely related to those dehydrogenases that act on aldehyde substrates. Knockout of doeC abolished growth of H. elongata strain KB47 (ΔdoeC) on medium containing ectoine as sole carbon source. Growth could be restored in doeC-mutant KB47 when ectoine was offered as nitrogen source in the presence of 10 mM glucose. This finding, together with the results obtained from the doeB mutant KB42, helped to determine the sequential order of the enzymatic reactions as depicted in , in which the dehydrogenase reaction and the transaminase reaction follow after the DoeB catalysed deacetylation of Nα-Ac-DABA.
However, according to the proposed pathway, acetate is split off from Nα-Ac-DABA in the second reaction. Acetate released in this reaction could serve as carbon source and should in principle allow dehydrogenase mutant KB47 to display at least some minimal growth on medium containing ectoine. We were able to prove through feeding experiments, that H. elongata
can indeed utilize acetate as carbon source and that wild-type strain DSM 2581T
, strain KB41 (ΔdoeA
) and strain KB47 (ΔdoeC
) are able to grow on medium containing 10 mM, 20 mM and 40 mM acetate respectively (Fig. S3
). Adding 10 mM ectoine to the acetate medium suppressed growth of dehydrogenase-mutant KB47, while strain KB41 and KB42, as well as wild-type DSM 2581T
, still managed to grow (Fig. S3
). This finding explains why strain KB47 cannot grow on ectoine medium and fails to metabolize the internal acetate, which is split off from Nα-Ac-DABA during ectoine degradation. For now, we can only speculate about the underlying mechanism that causes the inability of strain KB47 to utilize acetate in the presence of ectoine. Internal accumulation of intermediates such as DABA or aspartate-semialdehyde to toxic levels cannot be ruled out as the reason for growth inhibition. However, because mutant KB47 can still feed on ectoine as nitrogen donor in the presence of 10 mM glucose, we favour the idea that some kind of catabolite repression is the reason for the failure of KB47 to grow on medium containing both acetate and ectoine.
Organization of doe genes in bacteria
From comparative genomic data, the ectoine degradation pathway described for H. elongata is mostly employed by non-halophilic organisms that, according to their genetic makeup, are unable to synthesize ectoine de novo. The doeA/eutD and doeB/eutE sites could be found within the genomes of 18 bacteria, all belonging to the proteobacteria (). Besides H. elongata, only two of them are ectoine-synthesizing organisms, namely C. salexigens and Ruegeria (Silicibacter) sp. TM1040. Homologues of both the doeA and doeB sites of H. elongata can be found in many other Bacteria, predominantly in the Rhizobiales of the α-proteobacterial domain and in the Burkholderiales of the β-proteobacteria. For further analysis, we selected only a few representatives from larger sets of highly similar sequences originating from species such as Burkholderia, which appear overrepresented in the list of completely sequenced genomes. The reduced set of sequences contains 18 members, which share a relatively high similarity (48% to > 80% sequence identity) with doeA of H. elongata (Helo_3665). With two exceptions (Ruegeria sp. TM1040 and Burkholderia xenovorans LB400), doeAB is clustered in all these genomes. A second set of nine doeA homologues with a lower similarity to Helo_3665 (30–45% sequence identity) was found within the Firmicutes, Euryarchaeota and the γ-proteobacteria groups. In these genomes, the doeA site is located in a different genetic neighbourhood with no other genes of the ectoine degradation pathway being present. This is illustrated in the computed doeA phylogeny (), where the two sequence sets form separate branches (drawn in black and red colour respectively). The two organisms in which doeA and doeB are not adjacent (Ruegeria sp. TM1040 and B. xenovorans) are highlighted (branches also drawn in red). For reference, organisms carrying the ectABC genes are marked in blue.
Fig. 7 Phylogenetic tree of DoeA homologues and their position in the standard phylogeny (‘tree of life’). Branches marked in black correspond to genomes with the same ‘clustered’ organization of the doeAB genes. Red colour is (more ...)
In most ectoine producers compared in our investigation (), either only doeA-like ORFs could be found, or none of the doe components were present. We therefore assume that either ectoine is not metabolized in these bacteria or that alternative pathways in ectoine degradation must exist with, and without, DoeA participation. In analogy to the glutamate-ornithine pathway, ectoine could be alternatively metabolized to aspartate via Nα-Ac-DABA, Nα-acetyl-aspartate-semialdehyde and Nα-acetyl-aspartate. Also, it cannot be ruled out that in organisms without Doe enzymes, a reverse synthesis pathway degrades ectoine, bypassing the irreversible EctA acetyltransferase reaction.
Ectoine metabolism: reconstruction, modelling and energetic considerations
The metabolic capabilities of an organism are one of the major aspects of cellular physiology. Genome annotation with an emphasis on metabolic reconstruction provides a basis to analyse metabolic capabilities and their impact on the specific adaptation of the organism to its natural environment. The immense amount of data generated, such as the 1265 complete or partial EC numbers assigned for H. elongata
, calls for a computational strategy to drive a comprehensive analysis. As a first step, we have concentrated on the metabolism of ectoine, as this compatible solute is a key metabolite for survival and success of the halophilic bacterium H. elongata
. A Flux Balance Analysis (Varma and Palsson, 1993a
;) approach was selected to create a mathematical model of ectoine metabolism as detailed in Supporting information
. Using this model, we have analysed two aspects of ectoine metabolism: (i) The energy balance of the glucose to ectoine conversion computed and compared with previous calculations reported in the literature (Oren, 1999
). (ii) We attempted to predict the role of the proposed ectoine biosynthesis/degradation cycle.
The metabolic network, reconstructed from the genome of H. elongata, contains a series of enzymes, which are involved in biosynthesis of ectoine from glucose (). Overall, glucose is converted to two molecules of PEP/pyruvate, one of which is converted to acetyl-CoA, the other to aspartate-semialdehyde via oxaloacetate. Ectoine is then produced from one molecule each of aspartate-semialdehyde and acetyl-CoA. The set of available enzymes allows for several distinct pathways, some of which result in an identical overall reaction while others differ with respect to the ATP balance. An exhaustive analysis is required to ensure that all alternatives have been considered. While this is difficult by manual inspection, this becomes a feasible task once a model is available so that mathematically sound techniques can be applied.
Depiction of the pathways considered in the metabolic model for ectoine.
In one of the possible pathways, PEP is converted to oxaloacetate through PEP carboxylase (EC 220.127.116.11). This particular case has been used for the previous calculations on the energetic costs of ectoine synthesis presented by Oren (1999
). Oren's calculations assume nitrogen assimilation via glutamine synthetase (EC 18.104.22.168). With this assumption, the conversion of glucose to ectoine costs 2 ATP. However, an ATP-neutral pathway is possible using glutamate dehydrogenase (EC 22.214.171.124) as a more energy-efficient nitrogen assimilation reaction. This is based on the annotation of a NADH-dependent glutamate dehydrogenase (Helo_3049) in H. elongata
. An ATP-neutral conversion of glucose to ectoine is consistent with a calorimetric analysis carried out by Maskow and Babel (2001
), in which the authors conclude that an efficiency of approximately 100% was reached in the experiment.
The annotated genome allows for yet another solution, in which a total conversion of glucose into ectoine is possible with the simultaneous generation of ATP. The additional ATP can be obtained by using a different pathway, which proceeds via pyruvate to oxaloacetate and is catalysed by the malic enzyme (EC 126.96.36.199) and malate dehydrogenase (EC 188.8.131.52). An estimation of the reaction enthalpy for the conversion of glucose to ectoine shows that this pathway is not only stoichiometrically but is also thermodynamically feasible (see Supporting information). Currently, this hypothetical pathway is awaiting an experimental validation.
In summary, three alternative possibilities for conversion of glucose to ectoine differ in ATP production: (i) the pathway originally proposed by Oren (1999
) requires two ATP for each molecule of ectoine, (ii) the Oren pathway with a modified nitrogen assimilation step is energy-neutral and (iii) the most energy-efficient pathway, which is thermodynamically feasible and has been revealed by modelling of ectoine biosynthesis, allows to generate one ATP per ectoine molecule. This has clear physiological implications. In case (ii), the energy-neutral pathway allows for a 100% ectoine yield only if no other ATP-utilizing process operates. This does not reflect the reality of a living cell, where maintenance processes require additional ATP generation. Only pathway (iii), which is the most energy-efficient, allows a 100% ectoine yield as long as other metabolic processes do not drain more than one ATP per glucose.
The novel degradation pathway of ectoine we introduced here may result in an apparent futile cycle, in which ectoine can be synthesized and degraded simultaneously resulting in a net conversion of acetyl-CoA into acetate. Acetate can be reconverted to acetyl CoA by acetate : CoA ligase (AMP-forming) (EC 184.108.40.206). Therefore, if such a cycle were active, it would result in an increased apparent cost of two ATPs per turn for ectoine synthesis.
The question remains why the cell would invest two high-energy phosphate bonds to run a cycle of ectoine synthesis and degradation. We believe that such a cycle is an elegant mechanism to control the speed of change in internal ectoine concentration as response to external changes in osmolarity. The turnover (or response) time of a metabolite, defined as the ratio between its concentration and the flux through it in the steady state, has been identified as a good indicator of the timescale of its transient responses (Nikerel et al., 2009
). Metabolites with a high turnover tend to complete transitions faster than those with a low turnover. Thus, by keeping a flux through the synthesis/degradation cycle, the cell can achieve fast changes in ectoine levels to quickly respond to changes in external osmolality.
According to our model of a simultaneous activity of ectoine synthesis and degradation (ectoine cycle), disrupting the ectoine degradation pathway should lead to a lower ATP load for cells synthesizing ectoine and thereby result in higher ectoine productivity. In order to verify our proposed ectoine cycle and the mathematical model for ectoine synthesis, the doeA
gene was deleted in the ectoine excreting mutant KB2.11 (ΔteaABC
). Mutants with a dysfunctional or missing TeaABC ectoine transporter were shown to lose ectoine constantly to the medium (Grammann et al., 2002
; Kunte et al., 2002
). As the mathematical model was developed for non-growing cells, ectoine synthesis and export was analysed in minimal medium (510 mM NaCl) with non-growing cells (OD600
= 2.6; 100 µg ml−1
chloramphenicol) of mutant KB2.13 (ΔteaABC
) and the parental strain KB2.11. While KB2.11 accumulated 40 mg ectoine l−1
, the degradation mutant KB2.13 accumulated 50 mg ectoine l−1
, which corresponds to a 20% higher productivity of strain KB2.13. This result supports our proposed regulatory ectoine cycle and demonstrates the usefulness of the mathematical model in predicting the effect of mutations on the bacterial metabolism.
Conclusion and outlook
Determining the complete genome sequence of H. elongata DSM 2581T has increased our understanding of the metabolism of ectoine and will be the first step in our work towards optimizing the industrial production of the compatible solute ectoine. We were able to completely reconstruct ectoine biosynthesis from glucose and ammonia, by annotating the genes encoding for the central metabolic enzymes and the pathways leading to the ectoine precursors aspartate and glutamate, including the enzymes responsible for the metabolite interconversions at the PEP-pyruvate-oxaloacetate node. In addition, we have identified a pathway for ectoine degradation and shown its cyclic connection to ectoine synthesis. On the basis of these data, a Flux Balance Analysis model of ectoine metabolism has been developed.
Initial steps in using this new data for strain optimization have been undertaken and are described briefly in this paragraph. First, the deletion of gene doeA
, encoding the ectoine hydrolase protein, which catalyses the first step in the ectoine degradation pathway, has led to increased volumetric productivity of ectoine as predicted by our metabolic model. Furthermore, the genome sequence has enabled us to identify the gene for polyhydroxyalkanoate synthase phaC
, which is important because Halomonas
species are also known to synthesize polyhydroxyalkanoates in parallel to ectoine (Mothes et al., 2008
). Finally, mutagenesis experiments are now underway to abolish polyhydroxyalkanoate biosynthesis by deleting phaC
, which will hopefully enable new insights into metabolism of this energy storage compound and may also lead to a mutant strain that is able to convert glucose to ectoine more efficiently.
The metabolic model presented in this study only considers ectoine biosynthesis and the influence of different ATP-loads on its synthesis (Fig. S5
). We are currently developing an extended model that will also include growth (increase of biomass). The extended model will help us to evaluate the effect of alternative C-sources on ectoine production, including biologically and environmentally relevant substances such as glutamate or glycerol. In addition, the use of extended models allows us to make specific changes in the metabolism of the organism, resulting in an increase of the carbon flux towards the formation of ectoine. Koffas and co-workers (Koffas et al., 2003
) successfully used this strategy to improve lysine production in C. glutamicum
. By coordinated overexpression of genes encoding two flux-controlling enzymes in central carbon metabolism and the lysine pathway, they were able to enhance the carbon flux towards its product lysine, resulting in an increase in lysine specific productivity by 250%.
The availability of genomic information paves the way for post-genome technologies such as DNA array and proteomics, which can now be employed with H. elongata. These technologies will accelerate the studies of osmoregulation in this halophilic bacterium and will enable us to manipulate its metabolism to create more efficient producer strains for ectoine.