Summary: The halophilic γ‐proteobacterium Halomonas elongata DSM 2581 ^T 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.

The halophilic bacteriumHalomonas elongatasynthesizes as its main compatible solute the aspartate derivative ectoine. We constructed a deletion mutant ofH. elongata, KB1, defective in ectoine synthesis and tolerating elevated salt concentrations only in the presence of external compatible solutes. The dependency of KB1 on solute uptake for growth in high-salt medium was exploited to select insertion mutants unable to accumulate external solutes via osmoregulated transporters. One insertion mutant out of 7,200 failed to accumulate the osmoprotectants ectoine and hydroxyectoine. Genetic analysis of the insertion site proved that the mutation affected an open reading frame (ORF) of 1,281 bp (teaC). The nucleotide sequence upstream ofteaCwas determined, and two further ORFs of 603 bp (teaB) and 1,023 bp (teaA) were identified. Deletion ofteaAandteaBproved that all three genes are mandatory for ectoine uptake. Sequence comparison showed significant identity of TeaA, TeaB, and TeaC to the transport proteins of the recently identified tripartite ATP-independent periplasmic transporter family (TRAP-T). The affinity of the cells for ectoines was determined (K_s= 21.7 μM), suggesting that the transporter TeaABC exhibits high affinity for ectoines. An elevation of the external osmolarity resulted in a strong increase in ectoine uptake via TeaABC, demonstrating that this transporter is osmoregulated. Deletion ofteaCandteaBCin the wild-type strain led to mutants which excreted significant amounts of ectoine into the medium when cultivated at high salt concentrations. Therefore, the physiological role of TeaABC may be primarily to recover ectoine leaking through the cytoplasmic membrane.

The halophilic bacteriumHalomonas elongataaccumulates K⁺, glutamate, and the compatible solute ectoine as osmoprotectants. By functional complementation ofEscherichia colimutants defective in K⁺uptake, we cloned three genes that are required for K⁺uptake inH. elongata. Two adjacent genes, namedtrkA(1,374 bp) andtrkH(1,449 bp), were identified on an 8.5-kb DNA fragment, while a third gene, calledtrkI(1,479 bp), located at a different site in theH. elongatachromosome, was found on a second 8.5-kb fragment. The potential protein expressed bytrkAis similar to the cytoplasmic NAD⁺/NADH binding protein TrkA fromE. coli, which is required for the activity of the Trk K⁺uptake system. The deduced amino acid sequences oftrkHandtrkIshowed significant identity to the transmembrane protein of Trk transporters. K⁺transport experiments with ΔtrkHand ΔtrkImutants ofH. elongatarevealed that TrkI exhibits aK_mvalue of 1.12 mM, while the TrkH system has a half-saturation constant of 3.36 mM. Strain KB12, relying on TrkH alone, accumulated K⁺with a lowerV_maxand required a higher K⁺concentration for growth in highly saline medium than the wild type. Strain KB15, expressing only TrkI, showed the same phenotype and the same K⁺transport kinetics as the wild type, proving that TrkI is the main K⁺transport system inH. elongata. In the absence of both transporters TrkH and TrkI, K⁺accumulation was not detectable. K⁺transport was also abolished in atrkAdeletion mutant, indicating that TrkI and TrkH depend on one type of TrkA protein. Reverse transcriptase PCR experiments and Northern hybridization analyses of thetrkAHlocus revealed cotranscription oftrkAHas well as a monocistronic transcript with onlytrkA.

Environmental Context. Bacteria and Archaea have developed two basic mechanisms to cope with osmotic stress. The ‘salt-in-cytoplasm mechanism’ involves adjusting the salt concentration in the cytoplasm according to the environmental osmolarity and the ‘organic-osmolyte mechanism’ involves accumulating uncharged, highly water-soluble organic compounds in order to maintain an osmotic equilibrium with the surrounding medium. This highlight gives an overview of the osmoadaptation of prokaryotes employing the organic-osmolyte strategy and introduces a model explaining the fine-tuning of osmoregulatory osmolyte synthesis. Bacteria and Archaea have developed two basic mechanisms to cope with osmotic stress, the salt-in-cytoplasm mechanism, and the organic-osmolyte mechanism. Organic osmolytes or so-called compatible solutes can be accumulated in molar concentration in the cytoplasm and allow for the adaptation of bacterial cells to varying salt concentrations. The biosynthetic pathways of compatible solutes and different compatible solute transport systems are described. A model for osmoregulatory compatible solute accumulation is introduced.