Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions

The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.     

Regulation of envelope protein composition during adaptation to osmotic stress in Escherichia coli.

Adaptation to osmotic stress alters the amounts of several specific proteins in the Escherichia coli K-12 envelope. The most striking feature of the response to elevated osmolarity was the strong induction of a periplasmic protein with an Mr of 31,000. This protein was absent in mutants with lambda plac Mu insertions in an osmotically inducible locus mapping near 58 min. The insertions are likely to be in proU, a locus encoding a transport activity for the osmoprotectants glycine betaine and proline. Factors affecting the extent of proU induction were identified by direct examination of periplasmic proteins on sodium dodecyl sulfate gels and by measuring beta-galactosidase activity from proU-lac fusions. Expression was stimulated by increasing additions of salt or sucrose to minimal medium, up to a maximum at 0.5 M NaCl. Exogenous glycine betaine acted as an osmoregulatory signal; its addition to the high-osmolarity medium substantially repressed the expression of the 31,000-dalton periplasmic protein and the proU-lac+ fusions. Elevated osmolarity also caused the appearance of a second periplasmic protein (Mr = 16,000), and severe reduction in the amounts of two others. In the outer membrane, the well-characterized repression of OmpF by high osmolarity was observed and was reversed by glycine betaine. Additional changes in membrane composition were also responsive to glycine betaine regulation.     

The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12

The ProP and ProU transport systems of Escherichia coli mediate the uptake of several osmoprotectants including glycine betaine. Here we report that both ProP and ProU are involved in the transport of the potent osmoprotectant proline betaine. A set of isogenic E. coli strains carrying deletions in either the proP or proU loci was constructed. The growth properties of these mutants in high osmolarity minimal media containing 1 mM proline betaine demonstrated that the osmoprotective effect of this compound was dependent on either an intact ProP or ProU uptake system. Proline betaine competes with glycine betaine for binding to the proU-encoded periplasmic substrate binding protein (ProX) and we estimate a K_D of 5.2 μM for proline betaine binding. This value is similar to the binding constant of the ProX protein determined previously for the binding of glycine betaine (K_D of 1.4 μM). Our results thus demonstrate that the binding-protein-dependent ProU transport system of E. coli mediates the efficient uptake of the osmoprotectants glycine betaine and proline betaine.     

Salinity stress responses in the plant growth promoting rhizobacteria,Azospirillum spp

In order to adapt to the fluctuations in soil salinity/osmolarity the bacteria of the genusAzospirillum accumulate compatible solutes such as glutamate, proline, glycine betaine, trehalose, etc. Proline seems to play a major role in osmoadaptation. With increase in osmotic stress the dominant osmolyte inA. brasilense shifts from glutamate to proline. Accumulation of proline inA. brasilense occurs by both uptake and synthesis. At higher osmolarityA. brasilense Sp7 accumulates high intracellular concentration of glycine betaine which is taken up via a high affinity glycine betaine transport system. A salinity stress induced, periplasmically located, glycine betaine binding protein (GBBP) of ca. 32 kDa size is involved in glycine betaine uptake inA. brasilense Sp7. Although a similar protein is also present inA. brasilense Cd it does not help in osmoprotection. It is not known ifA. brasilense Cd can also accumulate glycine betaine under salinity stress and if the GBBP-like protein plays any role in glycine betaine uptake. This strain, under salt stress, seems to have inadequate levels of ATP to support growth and glycine betaine uptake simultaneously. ExceptA. halopraeferens, all other species ofAzospirillum lack the ability to convert choline into glycine betaine. Mobilization of thebet ABT genes ofE. coli intoA. brasilense enables it to use choline for osmoprotection. Recently, aproU-like locus fromA. lipoferum showing physical homology to theproU gene region ofE. coli has been cloned. Replacement of this locus, after inactivation by the insertion of kanamycin resistance gene cassette, inA. lipoferum genome results in the recovery of mutants which fail to use glycine betaine as osmoprotectant.     

Quorum Sensing Regulates the Osmotic Stress Response in Vibrio harveyi

Bacteria use a chemical communication process called quorum sensing to monitor cell density and to alter behavior in response to fluctuations in population numbers. Previous studies with Vibrio harveyi have shown that LuxR, the master quorum-sensing regulator, activates and represses >600 genes. These include six genes that encode homologs of the Escherichia coli Bet and ProU systems for synthesis and transport, respectively, of glycine betaine, an osmoprotectant used during osmotic stress. Here we show that LuxR activates expression of the glycine betaine operon betIBA-proXWV, which enhances growth recovery under osmotic stress conditions. BetI, an autorepressor of the V. harveyi betIBA-proXWV operon, activates the expression of genes encoding regulatory small RNAs that control quorum-sensing transitions. Connecting quorum-sensing and glycine betaine pathways presumably enables V. harveyi to tune its execution of collective behaviors to its tolerance to stress.     

Identification of bacterial periplasmic glycine betaine-binding protein after electrophoresis and affinity labeling

Antibodies were elicited in rabbits against periplasmic proteins obtained by cold osmotic shock from the Gram-negative eubacterium Rhizobium meliloti. When analyzed by crossed immunoelectrophoresis (CIE), the periplasmic proteins gave rise to 20 distinct immunoprecipitates corresponding to the same number of bands in polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions and in SDS-PAGE. The periplasmic glycine betaine-binding protein (GB-BP) was identified by autoradiography after affinity labeling with [14C]glycine betaine in PAGE and in CIE gels. The binding proved to be quite specific to glycine betaine, since the GB-BP was not labeled by choline (a metabolic precursor of glycine betaine in Escherichia coli and Rhizobium meliloti) and 15 distinct L-amino acids, including L-proline which, like glycine betaine is also an osmoprotectant. Affinity labeling of the GB-BP with [14C]glycine betaine after protein separation by PAGE or CIE is a simple and sensitive technique permitting the GB-BP to the unambiguously detected and identified in samples of complex protein mixtures containing down to 2 micrograms of GB-BP in PAGE and only 0.2 micrograms in CIE.     

Distribution of compatible solutes in the halophilic methanogenic archaebacteria.

Accumulation of compatible solutes, by uptake or de novo synthesis, enables bacteria to reduce the difference between osmotic potentials of the cell cytoplasm and the extracellular environment. To examine this process in the halophilic and halotolerant methanogenic archaebacteria, 14 strains were tested for the accumulation of compatible solutes in response to growth in various extracellular concentrations of NaCl. In external NaCl concentrations of 0.7 to 3.4 M, the halophilic methanogens accumulated K+ ion and low-molecular-weight organic compounds. beta-Glutamate was detected in two halotolerant strains that grew below 1.5 M NaCl. Two unusual beta-amino acids, N epsilon-acetyl-beta-lysine and beta-glutamine (3-aminoglutaramic acid), as well as L-alpha-glutamate were compatible solutes among all of these strains. De novo synthesis of glycine betaine was also detected in several strains of moderately and extremely halophilic methanogens. The zwitterionic compounds (beta-glutamine, N epsilon-acetyl-beta-lysine, and glycine betaine) and potassium were the predominant compatible solutes among the moderately and extremely halophilic methanogens. This is the first report of beta-glutamine as a compatible solute and de novo biosynthesis of glycine betaine in the methanogenic archaebacteria.     

Identification of an osmotically induced periplasmic glycine betaine-binding protein from Rhizobium meliloti.

The effect of salt stress on glycine betaine-binding activity has been investigated in periplasmic fractions released from Rhizobium meliloti 102F34 by cold osmotic shock. Binding activity was monitored by three techniques: equilibrium dialysis, filter procedure, and detection of 14C ligand-protein binding by direct non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. The three methods demonstrated the existence of a strong glycine betaine-binding activity, but only in periplasmic fractions from cells grown at high osmolarity. The non-denaturing PAGE of such periplasmic shock fluids mixed with [methyl-14C]glycine betaine showed only one radioactive band, indicating the involvement of one glycine betaine-binding protein. To determine the possible implication of this binding protein in glycine betaine uptake, transport activity was measured with cells submitted to cold osmotic shock. No significant decrease of transport activity was noticed. This lack of effect could be explained by the small quantity of periplasmic proteins released as judged by the low activity of phosphodiesterase, a periplasmic marker enzyme, observed in the shock fluid. The specificity of binding was analysed with different potential competitors: other betaines such as gamma-butyrobetaine, proline betaine, pipecolate betaine, trigonelline and homarine, or amino acids like glycine and proline, did not bind to the glycine betaine-binding protein, whereas glycine betaine aldehyde and choline were weak competitors. Optimum pH for binding was around 7.0, but approx. 90% of the glycine betaine-binding activity remained at pH 6.0 or 8.0. The calculated binding affinity (KD) was 2.5 microM. Both glycine betaine-binding activity and affinity were not significantly modified whether or not the binding assays were done at high osmolarity. A 32 kDa osmotically inducible periplasmic protein, identified by SDS-PAGE, apparently corresponds to the glycine betaine-binding protein.     

Corynebacterium glutamicum Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP

Gram-positive soil bacterium Corynebacterium glutamicum uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A ΔbetP ΔputP ΔproP ΔectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (K_m, 48 μM) to ectoine (K_m, 132 μM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (K_m, 63 μM) to betaine (K_m, 333 μM) and proline (K_m, 1,200 μM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.     

Protection of Bacillus subtilis against cold stress via compatible-solute acquisition

Accumulation of compatible solutes is a strategy widely employed by bacteria to achieve cellular protection against high osmolarity. These compounds are also used in some microorganisms as thermostress protectants. We found that Bacillus subtilis uses the compatible solute glycine betaine as an effective cold stress protectant. Glycine betaine strongly stimulated growth at 15°C and permitted cell proliferation at the growth-inhibiting temperature of 13°C. Initial uptake of glycine betaine at 15°C was low but led eventually to the buildup of an intracellular pool whose size was double that found in cells grown at 35°C. Each of the three glycine betaine transporters (OpuA, OpuC, and OpuD) contributed to glycine betaine accumulation in the cold. Protection against cold stress was also accomplished when glycine betaine was synthesized from its precursor choline. Growth of a mutant defective in the osmoadaptive biosynthesis for the compatible solute proline was not impaired at low temperature (15°C). In addition to glycine betaine, the compatible solutes and osmoprotectants l-carnitine, crotonobetaine, butyrobetaine, homobetaine, dimethylsulfonioactetate, and proline betaine all served as cold stress protectants as well and were accumulated via known Opu transport systems. In contrast, the compatible solutes and osmoprotectants choline-O-sulfate, ectoine, proline, and glutamate were not cold protective. Our data highlight an underappreciated facet of the acclimatization of B. subtilis to cold environments and allow a comparison of the characteristics of compatible solutes with respect to their osmotic, heat, and cold stress-protective properties for B. subtilis cells.     

Osmotic adjustment in the mycelial ascomyceteNeocosmospora vasinfecta (E. F. Smith)

Osmotic adjustment in the ascomyceteNeocosmospora vasinfecta was investigated by determining intramycelial water, mycelial solutes, and total mycelial osmolality. Major organic and inorganic solutes as well as proline and glycine betaine were determined under conditions of osmotic stress and shock, imposed by 0.5M KCl. Comparison to glucose as a nonelectrolytic osmoticum was also made. Results quantitatively implicated the polyhydric alcohols as the osmotic adjusters. Changes in amino acids were due to growth and were not osmoregulatory in nature. The osmoticum was not utilized for osmotic adjustment. The growth ofN. vasinfecta in the presence of KCl indicated that this organism is moderately sensitive to osmotic stress.     

Salt Stress Induced Changes in the Exoproteome of the Halotolerant Bacterium Tistlia consotensis Deciphered by Proteogenomics

The ability of bacteria to adapt to external osmotic changes is fundamental for their survival. Halotolerant microorganisms, such as Tistlia consotensis, have to cope with continuous fluctuations in the salinity of their natural environments which require effective adaptation strategies against salt stress. Changes of extracellular protein profiles from Tistlia consotensis in conditions of low and high salinities were monitored by proteogenomics using a bacterial draft genome. At low salinity, we detected greater amounts of the HpnM protein which is involved in the biosynthesis of hopanoids. This may represent a novel, and previously unreported, strategy by halotolerant microorganisms to prevent the entry of water into the cell under conditions of low salinity. At high salinity, proteins associated with osmosensing, exclusion of Na⁺ and transport of compatible solutes, such as glycine betaine or proline are abundant. We also found that, probably in response to the high salt concentration, T. consotensis activated the synthesis of flagella and triggered a chemotactic response neither of which were observed at the salt concentration which is optimal for growth. Our study demonstrates that the exoproteome is an appropriate indicator of adaptive response of T. consotensis to changes in salinity because it allowed the identification of key proteins within its osmoadaptive mechanism that had not previously been detected in its cell proteome.     

Isolation and cloning of a Azospirillum lipoferum locus that complements Escherichia coli proU mutant

Glycine betaine relieved sodium chloride-mediated inhibition of growth in Azospirillum lipoferum ATCC 29708. ³⁵S-methionine labelling of proteins after salinity up-shock revealed strong induction of a 30 kDa protein which cross-reacted with the anti-glycine betaine binding protein antibody from Escherichia coli. This suggested that A. lipoferum had a salinity-induced ProU-like high-affinity glycine betaine transport system. A genomic library of A. lipoferum ATCC 29708 was screened for the proU-like gene by complementation of a proU mutant of E. coli. Four recombinant cosmids, capable of restoring growth of the proU mutant on plates containing 600 mM NaCl and 1 mM glycine betaine were selected. Selected recombinant cosmids hybridized with a proU gene probe from E. coli. Complementation of E. coli proU mutant with the A. lipoferum genomic DNA was evident by the ability of proU mutant (containing selected recombinant cosmids) to grow on minimal medium supplemented with 600 mM NaCl and 1 mM glycine betaine.     

Glycine betaine is the main organic osmotic solute in a stratified microbial community in a hypersaline evaporitic gypsum crust

We have examined the organic osmotic solutes content within the stratified microbial communities in an evaporitic gypsum crust found in an evaporation pond (~194 g/l total dissolved salts) of the salterns of the Israel Salt Company, Eilat. We extracted intracellular solutes from the upper three pigmented layers of the crust: a yellow-orange layer dominated by unicellular cyanobacteria, a green layer with filamentous cyanobacteria, and a layer colored red-purple by purple sulfur bacteria; dense communities of heterotrophic bacteria were present in all layers. The solutes were analyzed by Raman spectroscopy, ¹H and ¹³C nuclear magnetic resonance, and HPLC. All layers contained glycine betaine as the only detectable osmotic solute; ectoine and other solutes known to be produced by many halophilic and halotolerant prokaryotes were not found. In this first attempt to assess the osmotic solute content within complex natural communities of halophilic microorganisms, the predominant role of glycine betaine as an osmolyte was established. Most heterotrophic bacteria cannot produce glycine betaine but preferentially use it when it is supplied. Presence of glycine betaine produced by the photoautotrophic members of the community, therefore, may relieve the heterotrophs from the need to synthesize other compounds at a high-energy cost.     

Preferential Osmolyte Accumulation: a Mechanism of Osmotic Stress Adaptation in Diazotrophic Bacteria

A common cellular mechanism of osmotic-stress adaptation is the intracellular accumulation of organic solutes (osmolytes). We investigated the mechanism of osmotic adaptation in the diazotrophic bacteria Azotobacter chroococcum, Azospirillum brasilense, and Klebsiella pneumoniae, which are adversely affected by high osmotic strength (i.e., soil salinity and/or drought). We used natural-abundance ¹³C nuclear magnetic resonance spectroscopy to identify all the osmolytes accumulating in these strains during osmotic stress generated by 0.5 M NaCl. Evidence is presented for the accumulation of trehalose and glutamate in Azotobacter chroococcum ZSM4, proline and glutamate in Azospirillum brasilense SHS6, and trehalose and proline in K. pneumoniae. Glycine betaine was accumulated in all strains grown in culture media containing yeast extract as the sole nitrogen source. Alternative nitrogen sources (e.g., NH₄Cl or casamino acids) in the culture medium did not result in measurable glycine betaine accumulation. We suggest that the mechanism of osmotic adaptation in these organisms entails the accumulation of osmolytes in hyperosmotically stressed cells resulting from either enhanced uptake from the medium (of glycine betaine, proline, and glutamate) or increased net biosynthesis (of trehalose, proline, and glutamate) or both. The preferred osmolyte in Azotobacter chroococcum ZSM4 shifted from glutamate to trehalose as a consequence of a prolonged osmotic stress. Also, the dominant osmolyte in Azospirillum brasilense SHS6 shifted from glutamate to proline accumulation as the osmotic strength of the medium increased.     

Purification, reconstitution, and characterization of the CpxRAP envelope stress system of Escherichia coli

In Escherichia coli the Cpx sensor regulator system senses different kinds of envelope stress and responds by triggering the expression of periplasmic folding factors and proteases. It consists of the membrane-anchored sensor kinase CpxA, the response regulator CpxR, and the periplasmic protein CpxP. The Cpx pathway is induced in vivo by a variety of signals including pH variation, osmotic stress, and misfolded envelope proteins and is inhibited by overproduced CpxP. Because it is not clear how the Cpx pathway is able to recognize and correspond to so many different signals we overproduced, solubilized, purified, and incorporated the complete membrane-integral CpxA protein into proteoliposomes to analyze its biochemical properties in more detail. Autokinase and phosphotransfer activities of the reconstituted CpxA-His6 protein were stimulated by KCl. NaCl also stimulated the activities but to a lesser extent. Other osmotic active solutes as glycine betaine, sucrose, and proline had no effect. The system was further characterized by testing for susceptibility to sensor kinase inhibitors. Among these, Closantel inhibited the activities of solubilized but not of the reconstituted CpxA-His6 protein. We further analyzed the effect of CpxP on CpxA activities. Purified tagless CpxP protein reduced the phosphorylation status of CpxA to 50% but had no effect on CpxA phosphotransfer or phosphatase activities. As the in vitro system excludes the involvement of other factors our finding is the first biochemical evidence for direct protein-protein interaction between the sensor kinase CpxA and the periplasmic protein CpxP resulting in a down-regulation of the autokinase activity of CpxA.     

<Emphasis Type="Italic">Marinococcus halophilus</Emphasis> DSM 20408<Superscript>T </Superscript>encodes two transporters for compatible solutes belonging to the betaine-carnitine-choline transporter family: identification and characterization of ectoine transporter EctM and glycine betaine transporter BetM

In response to osmotic stress, the halophilic, Gram-positive bacterium Marinococcus halophilus accumulates compatible solutes either by de novo synthesis or by uptake from the medium. To characterize transport systems responsible for the uptake of compatible solutes, a plasmid-encoded gene bank of M. halophilus was transferred into the transport-deficient strain Escherichia coli MKH13, and two genes were cloned by functional complementation required for ectoine and glycine betaine transport. The ectoine transporter is encoded by an open reading frame of 1,578 bp named ectM. The gene ectM encodes a putative hydrophobic, 525-residue protein, which shares significant identity to betaine-carnetine-choline transporters (BCCTs). The transporter responsible for the uptake of glycine betaine in M. halophilus is encoded by an open reading frame of 1,482 bp called betM. The potential, hydrophobic BetM protein consists of 493 amino acid residues and belongs, like EctM, to the BCCT family. The affinity of whole cells of E. coli MKH13 for ectoine (K_s=1.6 M) and betaine (K_s=21.8 M) was determined, suggesting that EctM and BetM exhibit a high affinity for their substrates. An elevation of the salinity in the medium resulted in an increased uptake of ectoine via EctM and glycine betaine via BetM in E. coli MKH13 cells, demonstrating that both systems are osmoregulated.Communicated by W.D. Grant     

Cation-pi interactions as determinants for binding of the compatible solutes glycine betaine and proline betaine by the periplasmic ligand-binding protein ProX from Escherichia coli

Compatible solutes such as glycine betaine and proline betaine are accumulated to exceedingly high intracellular levels by many organisms in response to high osmolarity to offset the loss of cell water. They are excluded from the immediate hydration shell of proteins and thereby stabilize their native structure. Despite their exclusion from protein surfaces, the periplasmic ligand-binding protein ProX from the Escherichia coli ATP-binding cassette transport system ProU binds the compatible solutes glycine betaine and proline betaine with high affinity and specificity. To understand the mechanism of compatible solute binding, we determined the high resolution structure of ProX in complex with its ligands glycine betaine and proline betaine. This crystallographic study revealed that cation-pi interactions between the positive charge of the quaternary amine of the ligands and three tryptophan residues forming a rectangular aromatic box are the key determinants of the high affinity binding of compatible solutes by ProX. The structural analysis was combined with site-directed mutagenesis of the ligand binding pocket to estimate the contributions of the tryptophan residues involved in binding.     

Characterization of a Snorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake

The symbiotic soil bacterium Sinorhizobium meliloti uses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli (ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed in hut mutants (hutX and hutH2). Expression analysis of the hut operon determined using a hutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.