New Type of Osmoregulated Solute Transporter Identified in Halophilic Members of theBacteriaDomain: TRAP Transporter TeaABC Mediates Uptake of Ectoine and Hydroxyectoine inHalomonas elongataDSM 2581^T

Abstract

The halophilic bacterium Halomonas elongata synthesizes as its main compatible solute the aspartate derivative ectoine. We constructed a deletion mutant of H. 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 of teaC was determined, and two further ORFs of 603 bp (teaB) and 1,023 bp (teaA) were identified. Deletion of teaA and teaB proved 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 M), 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 of teaC and teaBC in 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.Halophilic organisms living in saline environments such as salt lakes, coastal lagoons, and man-made salterns are challenged by two stress factors, the high inorganic ion concentration and the low water potential. A nonadapted organism exposed to such an environment must cope with its cytoplasmic water having a higher water potential than the water of the surrounding environment. Water always flows from a high to a low potential until the potential gradient is abolished. Thus, the cytoplasm, which is surrounded by a membrane freely permeable to water, will lose its free cytoplasmic water, resulting in cell shrinkage (4). The loss of water will cause cessation of growth, possibly due to molecular crowding, and thus reduced diffusion rates of proteins and metabolites.Halophilic prokaryotes have developed two principal mechanisms to lower the potential of cytoplasmic water, avoiding the loss of water from the cell and achieving a cytoplasm of osmotic strength similar to that of the surrounding medium. These two mechanisms, which allow osmotic adaptation, are the salt-in-cytoplasm mechanism and the organic-osmolyte mechanism.The salt-in-cytoplasm mechanism is considered the typical archaeal strategy of osmoadaptation (e.g., halobacteria). However, members of the Bacteria domain such as Salinibacter ruber and a specialized group of g...

A mathematical model for growth and osmoregulation in halophilic bacteria

“…Ectoine is taken up by the TeaABC system in H. elongata (Grammann et al, 2002). Since cells growing under constant conditions maintain a Fig.…”

“…Dö tsch and others constant level of compatible solutes, the kinetics of ectoine uptake can be derived from the whole-cell growth kinetics of an ectoinesynthesis-deficient mutant, whose growth is limited by ectoine uptake (Grammann et al, 2002). The uptake rate of ectoine is thenanalogous to equation 10 -given by:…”

“…Mutants of H. elongata lacking a functional TeaABC uptake system accumulate ectoine in the medium. It is therefore believed that the main function of TeaABC is the recovery of ectoine leaked into the medium (Grammann et al, 2002), since wild-type cultures do not show this extracellular accumulation. How ectoine leaks through the cytoplasmic membrane…”

“…Assuming full upregulation of ectoine synthesis immediately after the osmotic upshift, we calculated a value of 1.25 mmol (mg B) 21 h 21 from the initial slope (first 30 min) of the ectoine increase. Grammann et al (2002) measured the growth rate of a strain that is deficient in ectoine synthesis (KB 1) under ectoine-limiting conditions. Since all intracellular ectoine in this strain must be taken up from the medium via the TeaABC transporter, it is possible to estimate ectoine uptake kinetics from the whole-cell growth kinetics determined in this experiment.…”

“…It is assumed that ectoine synthesis is triggered by the initial accumulation of K + -glutamate after osmotic upshifts (Kraegeloh & Kunte, 2002). The pathway of ectoine synthesis from aspartate (Peters et al, 1990), and specific uptake systems for ectoine (Grammann et al, 2002) and potassium (Kraegeloh et al, 2005), have been described.…”