Halomonas Elongata Research Articles

Glass-forming property of hydroxyectoine is the cause of its superior function as a desiccation protectant

We were able to demonstrate that hydroxyectoine, in contrast to ectoine, is a good glass-forming compound. Fourier transform infrared and spin label electron spin resonance studies of dry ectoine and hydroxyectoine have shown that the superior glass-forming properties of hydroxyectoine result from stronger intermolecular H-bonds with the OH group of hydroxyectoine. Spin probe experiments have also shown that better molecular immobilization in dry hydroxyectoine provides better redox stability of the molecules embedded in this dry matrix. With a glass transition temperature of 87°C (vs. 47°C for ectoine) hydroxyectoine displays remarkable desiccation protection properties, on a par with sucrose and trehalose. This explains its accumulation in response to increased salinity and elevated temperature by halophiles such as Halomonas elongata and its successful application in ``anhydrobiotic engineering'' of both enzymes and whole cells.

Comparison of ectoine synthesis regulation in secreting and non-secreting strains of Halomonas

Ectoine is an osmotic pressure compatible solute. It is synthesized by Halomonas and other microorganisms in a hypertonic environment. As a stabilizing agent of cells proteins, nucleic acids and other biological products, ectoine has wide applications. Therefore, an efficient production method for ectoine is in great demand. Ectoine is overproduced by Halomonas salina DSM 5928, an ectoine-secreting strain, in which the synthesis of ectoine is not limited by its intracellular threshold concentration. In order to explain the mechanism of secretion of ectoine, the response to NaCl stress, and the release and uptake kinetics of ectoine were compared between H. salina DSM 5928 and Halomonas elongata DSM 2581, a non-ectoine-secreting strain. Moreover, the ectoine binding protein TeaA from each of these two strains was cloned and expressed, and binding abilities were examined in vitro. The results indicated that H. salina DSM 5928 and H. elongata DSM 2581 respond to NaCl in the medium in different ways. Compared with H. elongata DSM 2581, the amount of ectoine released was higher and the uptake of ectoine under NaCl stress was lower in H. salina DSM 5928. In addition, the binding ability of TeaA to ectoine in H. salina DSM 5928 was also lower. These results reveal the secretion mechanism of ectoine as well as critical regulation and control factors involved in ectoine synthesis.

Cloning and identification of a novel NhaD-type Na+/H+ antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake

In this study, metagenomic DNA was screened for the Na(+)/H(+) antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na(+)/H(+) antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na(+)/H(+) antiporter was finally cloned. The presence of Hb_NhaD conferred tolerance of E. coli KNabc to up to 0.5 M NaCl and 0.2 M LiCl, and an alkaline pH. Hb_NhaD has the highest identity (70.6%) with a putative NhaD-type Na(+)/H(+) antiporter from an uncharacterized Clostridiaceae species, and also has lower identity with known NhaD-type Na(+)/H(+) antiporters from Halomonas elongata (20.8%), Alkalimonas amylolytica (19.0%), Vibrio parahaemolyticus (18.9%) and Vibrio cholerae (18.7 %). pH-dependent Na(+)(Li(+))/H(+) antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc carrying Hb_nhaD. Hb_NhaD exhibited very high Na(+)(Li(+))/H(+) antiport activity over a wide pH range from 6.5 to 9.0 with the highest activity at pH 7.0 which is significantly different from those of the above known NhaD-type Na(+)/H(+) antiporters. Also, the apparent K m values of Hb_NhaD for Na(+) and Li(+) at pH 7.0 were determined to be 1.31 and 2.16, respectively. Based on the above results, we proposed that Hb_NhaD should be categorized as a novel NhaD-type Na(+)/H(+) antiporter.

Perchlorate and halophilic prokaryotes: implications for possible halophilic life on Mars

In view of the finding of perchlorate among the salts detected by the Phoenix Lander on Mars, we investigated the relationships of halophilic heterotrophic microorganisms (archaea of the family Halobacteriaceae and the bacterium Halomonas elongata) toward perchlorate. All strains tested grew well in NaCl-based media containing 0.4 M perchlorate, but at the highest perchlorate concentrations, tested cells were swollen or distorted. Some species (Haloferax mediterranei, Haloferax denitrificans, Haloferax gibbonsii, Haloarcula marismortui, Haloarcula vallismortis) could use perchlorate as an electron acceptor for anaerobic growth. Although perchlorate is highly oxidizing, its presence at a concentration of 0.2 M for up to 2 weeks did not negatively affect the ability of a yeast extract-based medium to support growth of the archaeon Halobacterium salinarum. These findings show that presence of perchlorate among the salts on Mars does not preclude the possibility of halophilic life. If indeed the liquid brines that may exist on Mars are inhabited by salt-requiring or salt-tolerant microorganisms similar to the halophiles on Earth, presence of perchlorate may even be stimulatory when it can serve as an electron acceptor for respiratory activity in the anaerobic Martian environment.

Ectoine production from lignocellulosic biomass-derived sugars by engineered Halomonas elongata

In this study, the water-retaining cyclic amino acid ectoine was produced from a variety of sugars, including glucose, xylose, cellobiose, and glucose/xylose mixture using engineered Halomonas elongata. When grown on xylose as the sole carbon source, H. elongata produced 333 mmol/kg fresh cell weight (FW) of ectoine, which was 1.4-fold higher than that produced from glucose. To improve ectoine production, an ectD deficient H. elongata mutant was constructed. The engineered H. elongata produced 377 mmol/kg FW of ectoine from a glucose/xylose mixture. Ectoine was also produced from rice straw hydrolysate. These results show that H. elongata can produce ectoine from a variety of sugars derived from lignocellulosic biomass and thus has tremendous potential as a host for producing useful compounds from biomass resources.

Life at high salt concentrations, intracellular KCl concentrations, and acidic proteomes

Extremely halophilic microorganisms that accumulate KCl for osmotic balance (the Halobacteriaceae, Salinibacter) have a large excess of acidic amino acids in their proteins. This minireview explores the occurrence of acidic proteomes in halophiles of different physiology and phylogenetic affiliation. For fermentative bacteria of the order Halanaerobiales, known to accumulate KCl, an acidic proteome was predicted. However, this is not confirmed by genome analysis. The reported excess of acidic amino acids is due to a high content of Gln and Asn, which yield Glu and Asp upon acid hydrolysis. The closely related Halorhodospira halophila and Halorhodospira halochloris use different strategies to cope with high salt. The first has an acidic proteome and accumulates high KCl concentrations at high salt concentrations; the second does not accumulate KCl and lacks an acidic proteome. Acidic proteomes can be predicted from the genomes of some moderately halophilic aerobes that accumulate organic osmotic solutes (Halomonas elongata, Chromohalobacter salexigens) and some marine bacteria. Based on the information on cultured species it is possible to understand the pI profiles predicted from metagenomic data from hypersaline environments.

ISOLATION AND CHARACTERIZATION OF NOVEL EXTREMELY HALOTHERMOPHILIC BACTERIUM, HALOMONAS CASEINILYTICA WN.1B.S, FROM WADI AN NATRUN, EGYPT

The purpose of the present study was to isolate microbial halothermophiles from hyper saline Al- Hamra Lakeat Wadi An Natrun, Egypt. Twenty eight bacterial isolates were obtained and the morphological and physiological properties in addition to enzyme activities were studied. Amongst those isolates, WN.1B.s was selected as the most potent isolate based on growth at high temperature (up to 65oC) and at high salt concentration (up to 34%, near saturation state).A phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate WN.1B.s had the highest sequence similarity with respect to type strains of Halomonas caseinilytica (97 %), Halomonas elongata (96 %), Halomonas eurihalina (95 %), Halomonas koreensis (95 %) and Halomonas halmophila (95 %).Based on physiological characteristics and16S rRNA sequence analysisthis isolate was identified as Halomonas caseinilyticaWN.1B.s which belonged to bacterial domain, class Gammaproteobacteria, order Oceanospirillales, family Halomonadaceae, Halomonas species. Enzyme screening for strain WN.1B.s showed that, the isolate secrete amylases, lipases, cellulases and pectinase enzymes under harsh conditions that may be useful in different industrial processes.

Acid-shifted isoelectric point profiles of the proteins in a hypersaline microbial mat: an adaptation to life at high salt concentrations?

In a metagenomic analysis of a stratified hypersaline (9 % salt) microbial mat in Guerrero Negro, Mexico, Kunin et al. (Mol Systems Biol 4:198, 2008) detected a significantly acid-shifted proteome, and concluded that adaptation by enriching proteins with acidic amino acids is more widespread than previously assumed. We here reevaluate these data and conclusions by comparing the isoelectric point profiles of the Guerrero Negro microbial mats (average isoelectric point 6.8) with those of the proteins encoded by the genomes of prokaryotes adapted to different salt concentrations ranges and belonging to different phylogenetic and physiological groups. Average isoelectric points below 6.8 were found not only in the proteomes of the moderately halophilic aerobic bacteria Halomonas elongata and Chromohalobacter salexigens, but even in common types of marine bacteria of the genera Alteromonas and Aliivibrio. We did not find clear evidence that the isoelectric point profile of the Guerrero Negro microbial mat can be considered to be the result of species-independent molecular convergence of the members of the microbial community determined by the salinity of the overlaying brine.

Vom Genom zum Produkt: Herstellung von Schutzstoffen mit Halophilen

The compatible solute ectoine protects cells from damages caused by heat, UV radiation or toxins. Ectoine is marketed in health care products and produced on a scale of tons in a biotechnological process with the halophile Halomonas elongata. The H. elongata genome was sequenced leading to the elucidation of the entire ectoine metabolism. Based on the genome data a metabolic model of ectoine metabolism was built, which resulted in a strain with a three times increased volumetric productivity.

Use of Raman spectroscopy for identification of compatible solutes in halophilic bacteria

We explored the use of Raman spectroscopy to detect organic osmotic solutes as biomarkers in the moderately halophilic heterotrophic bacterium Halomonas elongata grown in complex medium (accumulation of glycine betaine) and in defined medium with glucose as carbon source (biosynthesis of ectoine), and in the anoxygenic phototrophic Ectothiorhodospira marismortui known to synthesize glycine betaine in combination with minor amounts of trehalose and N-α-carbamoyl glutamineamide. We tested different methods of preparation of the material: lyophilization, two-phase extraction of water-soluble molecules, and perchlorate extraction. Raman signals of glycine betaine and ectoine were detected; perchlorate extraction followed by desalting the extract on an ion retardation column gave the best results. Lyophilized cells of E. marismortui showed strong signals of carotenoid pigments, and glycine betaine could be detected only after perchlorate extraction and desalting. The data presented show that Raman spectroscopy is a suitable tool to assess the mode of osmotic adaptation used by halophilic microorganisms.

Complete genome sequence of the halophilic and highly halotolerant Chromohalobacter salexigens type strain (1H11(T)).

Chromohalobacter salexigens is one of nine currently known species of the genus Chromohalobacter in the family Halomonadaceae. It is the most halotolerant of the so-called ‘moderately halophilic bacteria’ currently known and, due to its strong euryhaline phenotype, it is an established model organism for prokaryotic osmoadaptation. C. salexigens strain 1H11T and Halomonas elongata are the first and the second members of the family Halomonadaceae with a completely sequenced genome. The 3,696,649 bp long chromosome with a total of 3,319 protein-coding and 93 RNA genes was sequenced as part of the DOE Joint Genome Institute Program DOEM 2004.

Unexpected property of ectoine synthase and its application for synthesis of the engineered compatible solute ADPC

A new cyclic amino acid was detected in a deletion mutant of the moderately halophilic bacterium Halomonas elongata deficient in ectoine synthesis. Using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) techniques, the substance was identified as 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC). We were able to demonstrate that ADPC is the product of a side reaction of lone ectoine synthase (EC 4.2.1.108), which forms ADPC by cyclic condensation of glutamine. This reaction was shown to be reversible. Subsequently, a number of ectoine derivatives, in particular 4,5-dihydro-2-methylimidazole-4-carboxylate (DHMICA) and homoectoine, were also shown to be cleaved by ectoine synthase, which is classified as a hydro-lyase. This study thus reports for the first time that ectoine synthase accepts more than one substrate and is a reversible enzyme able to catalyze both the intramolecular condensation into and the hydrolytic cleavage of cyclic amino acid derivatives. As ADPC supports growth of bacteria under salt stress conditions and stabilizes enzymes against freeze-thaw denaturation, it displays typical properties of compatible solutes. As ADPC has not yet been described as a natural compound, it is presented here as the first man-made compatible solute created through genetic engineering.

Enhanced Substrate Release from a TRAP Transporter Binding Protein by Remote Modulation of its Intrinsic Conformational Dynamics

The tripartite ATP-independent periplasmic (TRAP) transporter TeaABC enables Halomonas elongata to uptake ectoine, a compatible solute protective under osmotic stress. TeaABC consists of three proteins: the Na+-coupled transporter TeaC; the transmembrane protein TeaB; and the periplasmic binding-protein TeaA. Like the binding proteins in ABC transporters, TeaA contains two globular domains, in-between which the substrate binds with high affinity. This notwithstanding, TeaA must somehow release ectoine upon interaction with TeaC. By analogy with similar transport systems, it is plausible that release is facilitated by a conformational change in TeaA, induced by TeaC. However, direct evidence and structural detail of such a mechanism are still unknown.Here, we use experimental and theoretical methods to demonstrate that modulation of the conformational dynamics of TeaA, as may occur by association with TeaC, results in an enhanced propensity for ectoine release. First, a novel crystal structure of apo TeaA reveals a pronounced domain rearrangement and opening of the binding cleft, relative to the known substrate-bound structure. Extensive molecular simulations and free-energy calculations are then employed to elucidate the mechanism by which the conformational dynamics of TeaA is modulated not only by ectoine binding and release, but also by structural elements remote from the binding cleft. This insight allows us to design a triple mutation whose influence on the dynamics of the protein may resemble that of the transporter domain TeaC, while not directly affecting the binding site. Calorimetric studies of mutant and wild-type TeaA demonstrate how this conformational modulation results in a reduction of its substrate affinity. Altogether, this data provides compelling support for the view that ectoine release from TeaA onto TeaC may be induced by the latter through a mechanism of conformational modulation of the former.

Genome-scale reconstruction of metabolic network for a halophilic extremophile, Chromohalobacter salexigens DSM 3043.

BackgroundChromohalobacter salexigens (formerly Halomonas elongata DSM 3043) is a halophilic extremophile with a very broad salinity range and is used as a model organism to elucidate prokaryotic osmoadaptation due to its strong euryhaline phenotype.ResultsC. salexigens DSM 3043's metabolism was reconstructed based on genomic, biochemical and physiological information via a non-automated but iterative process. This manually-curated reconstruction accounts for 584 genes, 1386 reactions, and 1411 metabolites. By using flux balance analysis, the model was extensively validated against literature data on the C. salexigens phenotypic features, the transport and use of different substrates for growth as well as against experimental observations on the uptake and accumulation of industrially important organic osmolytes, ectoine, betaine, and its precursor choline, which play important roles in the adaptive response to osmotic stress.ConclusionsThis work presents the first comprehensive genome-scale metabolic model of a halophilic bacterium. Being a useful guide for identification and filling of knowledge gaps, the reconstructed metabolic network iOA584 will accelerate the research on halophilic bacteria towards application of systems biology approaches and design of metabolic engineering strategies.

A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T

The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at high salinity by synthesizing and accumulating the compatible solute ectoine. Ectoine levels are highly regulated according to external salt levels but the overall picture of its metabolism and control is not well understood. Apart from its critical role in cell adaptation to halophilic environments, ectoine can be used as a stabilizer for enzymes and as a cell protectant in skin and health care applications and is thus produced annually on a scale of tons in an industrial process using H. elongata as producer strain. This paper presents the complete genome sequence of H. elongata (4 061 296 bp) and includes experiments and analysis identifying and characterizing the entire ectoine metabolism, including a newly discovered pathway for ectoine degradation and its cyclic connection to ectoine synthesis. The degradation of ectoine (doe) proceeds via hydrolysis of ectoine (DoeA) to Nα-acetyl-l-2,4-diaminobutyric acid, followed by deacetylation to diaminobutyric acid (DoeB). In H. elongata, diaminobutyric acid can either flow off to aspartate or re-enter the ectoine synthesis pathway, forming a cycle of ectoine synthesis and degradation. Genome comparison revealed that the ectoine degradation pathway exists predominantly in non-halophilic bacteria unable to synthesize ectoine. Based on the resulting genetic and biochemical data, a metabolic flux model of ectoine metabolism was derived that can be used to understand the way H. elongata survives under varying salt stresses and that provides a basis for a model-driven improvement of industrial ectoine production.

Structure and Function of the Universal Stress Protein TeaD and Its Role in Regulating the Ectoine Transporter TeaABC of Halomonas elongata DSM 2581T

The halophilic bacterium Halomonas elongata takes up the compatible solute ectoine via the osmoregulated TRAP transporter TeaABC. A fourth orf (teaD) is located adjacent to the teaABC locus that encodes a putative universal stress protein (USP). By RT-PCR experiments we proved a cotranscription of teaD along with teaABC. Deletion of teaD resulted in an enhanced uptake for ectoine by the transporter TeaABC and hence a negative activity regulation of TeaABC by TeaD. A transcriptional regulation via DNA binding could be excluded. ATP binding to native TeaD was shown by HPLC, and the crystal structure of TeaD was solved in complex with ATP to a resolution of 1.9 A by molecular replacement. TeaD forms a dimer-dimer complex with one ATP molecule bound to each monomer, which has a Rossmann-like alpha/beta overall fold. Our results reveal an ATP-dependent oligomerization of TeaD, which might have a functional role in the regulatory mechanism of TeaD. USP-encoding orfs, which are located adjacent to genes encoding for TeaABC homologues, could be identified in several other organisms, and their physiological role in balancing the internal cellular ectoine pool is discussed.

Phylogenetic relationships within the family Halomonadaceae based on comparative 23S and 16S rRNA gene sequence analysis

A phylogenetic study of the family Halomonadaceae was carried out based on complete 16S rRNA and 23S rRNA gene sequences. Several 16S rRNA genes of type strains were resequenced, and 28 new sequences of the 23S rRNA gene were obtained. Currently, the family includes nine genera (Carnimonas, Chromohalobacter, Cobetia, Halomonas, Halotalea, Kushneria, Modicisalibacter, Salinicola and Zymobacter). These genera are phylogenetically coherent except Halomonas, which is polyphyletic. This genus comprises two clearly distinguished clusters: group 1 includes Halomonas elongata (the type species) and the species Halomonas eurihalina, H. caseinilytica, H. halmophila, H. sabkhae, H. almeriensis, H. halophila, H. salina, H. organivorans, H. koreensis, H. maura and H. nitroreducens. Group 2 comprises the species Halomonas aquamarina, H. meridiana, H. axialensis, H. magadiensis, H. hydrothermalis, H. alkaliphila, H. venusta, H. boliviensis, H. neptunia, H. variabilis, H. sulfidaeris, H. subterranea, H. janggokensis, H. gomseomensis, H. arcis and H. subglaciescola. Halomonas salaria forms a cluster with Chromohalobacter salarius and the recently described genus Salinicola, and their taxonomic affiliation requires further study. More than 20 Halomonas species are phylogenetically not within the core constituted by the Halomonas sensu stricto cluster (group 1) or group 2 and, since their positions on the different phylogenetic trees are not stable, they cannot be recognized as additional groups either. In general, there is excellent agreement between the phylogenies based on the two rRNA gene sequences, but the 23S rRNA gene showed higher resolution in the differentiation of species of the family Halomonadaceae.

The Crystal Structure of UehA in Complex with Ectoine—A Comparison with Other TRAP-T Binding Proteins

Substrate-binding proteins or extracellular solute receptors (ESRs) are components of both ABC (ATP binding cassette) and TRAP-T (tripartite ATP-independent periplasmic transporter). The TRAP-T system UehABC from Silicibacter pomeroyi DSS-3 imports the compatible solutes ectoine and 5-hydroxyectoine as nutrients. UehA, the ESR of the UehABC operon, binds both ectoine and 5-hydroxyectoine with high affinity (K(d) values of 1.4+/-0.1 and 1.1+/-0.1 microM, respectively) and delivers them to the TRAP-T complex. The crystal structure of UehA in complex with ectoine was determined at 2.9-A resolution and revealed an overall fold common for all ESR proteins from TRAP systems determined so far. A comparison of the recently described structure of TeaA from Halomonas elongata and an ectoine-binding protein (EhuB) from an ABC transporter revealed a conserved ligand binding mode that involves both directed and cation-pi interactions. Furthermore, a comparison with other known TRAP-T ESRs revealed a helix that might act as a selectivity filter imposing restraints on the ESRs that fine-tune ligand recognition and binding and finally might determine the selection of the cognate substrate.

Biological control of grey mould in strawberry fruits by halophilic bacteria

Grey mould caused by Botrytis cinerea is an economically important disease of strawberries in Tunisia and worldwide. The aim of this study was to select effective halophilic bacteria from hypersaline ecosystems and evaluate the abilities of antifungal bacteria to secrete extracellular hydrolytic enzymes, anti-Botrytis metabolites and volatiles. Grey mould was reduced in strawberry fruits treated with halophilic antagonists and artificially inoculated with B. cinerea. Thirty strains (20.2%) were active against the pathogen and reduced the percentage of fruits infected after 3 days of storage at 20 degrees C, from 50% to 91.66%. The antagonists were characterized by phenotypic tests and 16S rDNA sequencing. They were identified as belonging to one of the species: Virgibacillus marismortui, B. subtilis, B. pumilus, B. licheniformis, Terribacillus halophilus, Halomonas elongata, Planococcus rifietoensis, Staphylococcus equorum and Staphylococcus sp. The effective isolates were tested for antifungal secondary metabolites. Moderately halophilic bacteria may be useful in biological control against this pathogen during postharvest storage of strawberries. The use of such bacteria may constitute an important alternative to synthetic fungicides. These moderate halophiles can be exploited in commercial production and application of the effective strains under storage and greenhouse conditions.

Biotechnological Coproduction of Compatible Solutes and Polyhydroxyalkanoates using the Genus Halomonas

AbstractAn increased usage of poly‐β‐hydroxyalkanoates (PHA), for instance as bulk biodegradable and biocompatible plastics, will require a cheaper production and downstream processing. If the synthesis of this intracellular biopolyester could be combined with the production of another valuable intracellular product, the economic balance of the process could be improved. It was found that the moderately halophilic bacterium Halomonas elongata simultaneously synthesizes PHA and a protector molecule, called ectoine. Whereas the synthesis of PHA is a response to the shortage of nutrients, the production of ectoine counteracts osmotic imbalances. This behavior is in so far surprising as the conditions of a bi‐factorial stress initiate the fast simultaneous synthesis of ectoine and PHA. In the presence of 100 g/L NaCl, Halomonas elongata accumulated up to 50 % w/w PHA and up to 14 % ectoine within 2–3 days under so far non‐optimized conditions. Furthermore, it was found that other Halomonas species (e.g. Halomonas halodenitrificans and own isolates of Halomonas halodeneurihalina and Halomonas salina) were able to produce both ectoine and PHA.