Linda Tombras Smith

Molecular Biology of Osmoregulation

The drought of 1983 resulted in some 10 billion dollars in agricultural losses and has focused attention on the vulnerability of our major crops to this devastating form of environmental stress. This article is concerned with the molecular biology of a new class of genes, called osm (osmotic tolerance) genes, that protect bacteria like Escherichia coli against osmotic stress and may work in a similar manner in plants and animals. Osm genes govern the production of a class of molecules, such as betaine and proline, that protect the cell and its constituents against dehydration. These osmoprotectant molecules have been known for many years to accumulate in plants but have only recently been shown to have potent antistress activity for bacteria.

Osmosensing and osmoregulatory compatible solute accumulation by bacteria.

Bacteria inhabit natural and artificial environments with diverse and fluctuating osmolalities, salinities and temperatures. Many maintain cytoplasmic hydration, growth and survival most effectively by accumulating kosmotropic organic solutes (compatible solutes) when medium osmolality is high or temperature is low (above freezing). They release these solutes into their environment when the medium osmolality drops. Solutes accumulate either by synthesis or by transport from the extracellular medium. Responses to growth in high osmolality medium, including biosynthetic accumulation of trehalose, also protect Salmonella typhimurium from heat shock. Osmotically regulated transporters and mechanosensitive channels modulate cytoplasmic solute levels in Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Lactobacillus plantarum, Lactococcus lactis, Listeria monocytogenes and Salmonella typhimurium. Each organism harbours multiple osmoregulatory transporters with overlapping substrate specificities. Membrane proteins that can act as both osmosensors and osmoregulatory transporters have been identified (secondary transporters ProP of E. coli and BetP of C. glutamicum as well as ABC transporter OpuA of L. lactis). The molecular bases for the modulation of gene expression and transport activity by temperature and medium osmolality are under intensive investigation with emphasis on the role of the membrane as an antenna for osmo- and/or thermosensors.

Molecular characterization of the bet genes encoding glycine betaine synthesis in Sinorhizobium meliloti 102F34

As a first step towards the elucidation of the molecular mechanisms responsible for the utilization of choline and glycine betaine (betaine) either as carbon and nitrogen sources or as osmoprotectants in Sinorhizobium meliloti, we selected a Tn5 mutant, LTS23-1020, which failed to grow on choline but grew on betaine. The mutant was deficient in choline dehydrogenase (CDH) activity, failed to oxidize [methyl-14C]choline to [methyl-14C]betaine, and did not use choline, but still used betaine, as an osmoprotectant. The Tn5 mutation in LTS23-1020 was complemented by plasmid pCHO34, isolated from a genomic bank of S. meliloti 102F34. Subcloning and DNA sequencing showed that pCHO34 harbours two ORFs which showed 60% and 57% identity with the Escherichia coli betB gene encoding betaine-aldehyde dehydrogenase (BADH) and betA gene encoding CDH, respectively. In addition to the homology with E. coli genes, the deduced sequence of the sinorhizobial BADH protein displays consensus sequences also found in plant BADHs. The deduced sequence of the sinorhizobial CDH protein shares only 21% identical residues with choline oxidase from Arthrobacter globiformis. The structural organization of the betBA genes in S. meliloti differs from that described in E. coli: (i) the two ORFs are separated by a 210 bp sequence containing inverted repeats resembling a putative rho-independent transcription terminator, and (ii) no sequence homologous to betT (high-affinity choline transport system) or betI (regulator) was found in the vicinity of the sinorhizobial betBA genes. Evidence is also presented that the S. meliloti betBA genes are not located on the megaplasmids.

Characterization of Glycine Betaine Porter I from Listeria monocytogenes and Its Roles in Salt and Chill Tolerance

ABSTRACT Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na+-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12°C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and γ-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.

Osmotic and Chill Activation of Glycine Betaine Porter II in Listeria monocytogenes Membrane Vesicles

Listeria monocytogenes is a foodborne pathogen known for its tolerance to conditions of osmotic and chill stress. Accumulation of glycine betaine has been found to be important in the organisms tolerance to both of these stresses. A procedure was developed for the purification of membranes from L. monocytogenes cells in which the putative ATP-driven glycine betaine permease glycine betaine porter II (Gbu) is functional. As is the case for the L. monocytogenes sodium-driven glycine betaine uptake system (glycine betaine porter I), uptake in this vesicle system was dependent on energization by ascorbate-phenazine methosulfate. Vesicles lacking the gbu gene product had no uptake activity. Transport by this porter did not require sodium ion and could be driven only weakly by artificial gradients. Uptake rates could be manipulated under conditions not affecting secondary transport but known to affect ATPase activity. The system was shown to be both osmotically activated and cryoactivated. Under conditions of osmotic activation, the system exhibited Arrhenius-type behavior although the uptake rates were profoundly affected by the physical state of the membrane, with breaks in Arrhenius curves at approximately 10 and 18 degrees C. In the absence of osmotic activation, the permease could be activated by decreasing temperature within the range of 15 to 4 degrees C. Kinetic analyses of the permease at 30 degrees C revealed K(m) values for glycine betaine of 1.2 and 2.9 microM with V(max) values of 2,200 and 3,700 pmol/min. mg of protein under conditions of optimal osmotic activation as mediated by KCl and sucrose, respectively.

Mechanism of osmotically regulated N-acetylglutaminylglutamine amide production inRhizobium meliloti

Rhizobium meliloti adapts to environments of high osmolarity by accumulating glutamate, trehalose, and the dipeptide N-acetylglutaminylglutamine amide (NAGGN) intracellularly. In this study, the mechanism of NAGGN production and accumulation was examined. NAGGN was produced in osmotically shocked cultures after a lag period of more than one hour, and NAGGN was undetectable in cultures treated with chloramphenicol, indicating that genetic induction is required for NAGGN accumulation.In vitro radiolabeling experiments demonstrated that the peptide synthesis step in NAGGN production did not occur ribosomally. Rather, it was catalyzed by an ATP-dependent enzyme that appeared to be both induced by high osmolarity and activated by K+. Also, a mutant analysis suggested that NAGGN may be partly responsible for the osmotic tolerance observed inR. meliloti. 36% of mutants that were characterized as osmotically sensitive compared to the parent strain, were also found to contain reduced levels of NAGGN. The phenomenon of osmolyte accumulation as it relates to adaptation to other environmental stresses is discussed.

Identification of OpuC as a Chill-Activated and Osmotically Activated Carnitine Transporter in Listeria monocytogenes

ABSTRACT The food-borne pathogen Listeria monocytogenes is notable for its ability to grow under osmotic stress and at low temperatures. It is known to accumulate the compatible solutes glycine betaine and carnitine from the medium in response to osmotic or chill stress, and this accumulation confers tolerance to these stresses. Two permeases that transport glycine betaine have been identified, both of which are activated by hyperosmotic stress and one of which is activated by low temperature. An osmotically activated transporter for carnitine, OpuC, has also been identified. We have isolated a Tn917-LTV3 insertional mutant that could not be rescued from hyperosmotic stress by exogenous carnitine. The mutant, LTS4a, grew indistinguishably from a control strain (DP-L1044) in the absence of stress or in the absence of carnitine, but DP-L1044 grew substantially faster under osmotic or chill stress in the presence of carnitine. LTS4a was found to be strongly impaired in KCl-activated as well as chill-activated carnitine transport. 13C nuclear magnetic resonance spectroscopy of perchloric acid extracts showed that accumulation of carnitine by LTS4a was negligible under all conditions tested. Direct sequencing of LTS4a genomic DNA with a primer based on Tn917-LTV3 yielded a 487-bp sequence, which allowed us to determine that the opuC operon had been interrupted by the transposon. It can be concluded that opuC encodes a carnitine transporter that can be activated by either hyperosmotic stress or chill and that the transport system plays a significant role in the tolerance of L. monocytogenes to both forms of environmental stress.

Elevated carnitine accumulation by Listeria monocytogenes impaired in glycine betaine transport is insufficient to restore wild-type cryotolerance in milk whey.

Listeria monocytogenes accumulates low molecular weight compounds (osmolytes, or compatible solutes) in response to chill stress. This response has been shown to be responsible, in part, for the chill tolerance of the species. Among the osmolytes tested to date, glycine betaine, gamma-butyrobetaine and carnitine display the strongest cryoprotective effect. These osmolytes are not synthesized in the cell and must be transported from the medium. In this study, the compatible solute accumulation profile of the food-borne pathogen L. monocytogenes was determined in balanced growth and stationary phase cultures grown in milk whey at 7 and 30 degrees C. In balanced growth cultures at 7 degrees C, glycine betaine (720 nmol/10(10) cfu) and carnitine (130 nmol/10(10) cfu) were the major osmolytes accumulated by wild-type L. monocytogenes 10403S, whereas carnitine (490 nmol/10(10) cfu) was the dominant osmolyte and glycine betaine was present in smaller amounts (270 nmol/10(10) cfu) in a mutant (L. monocytogenes LTG59) blocked in the major glycine betaine uptake system, glycine betaine porter II. In strain 10403S, glycine betaine and carnitine were present in eightfold and twofold excess at 7 degrees C compared to 30 degrees C; the respective ratios for strain LTG59 were 6 and 8. The intracellular concentration of osmolytes in stationary phase cultures at 7 degrees C was markedly reduced compared to that during balanced growth. Furthermore, at 4 degrees C, small but highly significant differences in growth were observed between strains. Strain LTG59 grew with a lag phase that was significantly longer, a generation time that was significantly greater and reached a final cell yield that was significantly lower than that of strain 10403S. The elevated accumulation of carnitine in the absence of glycine betaine porter II was insufficient to confer the magnitude of the cryoprotective effect displayed by the wild type.

Gbu Glycine Betaine Porter and Carnitine Uptake in Osmotically Stressed Listeria monocytogenes Cells

ABSTRACT The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a Km of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 μM. This porter has a Km for glycine betaine uptake of about 6 μM. The dedicated carnitine porter, OpuC, has a Km for carnitine uptake of 1 to 3 μM and a Vmax of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by γ-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected.