University of Groningen
Osmotic regulation of transport processes in Lactobacillus plantarum
Glaasker, Erwin
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1998
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Glaasker, E. (1998). Osmotic regulation of transport processes in Lactobacillus plantarum s.n.
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Chapter 8
SUMMARY
A simplified view of a microorganism (or one cell organism) is that of a
compartment containing a variety of small solutes and biological macromolecules
(proteins and nucleic acids) that is encompassed with a membrane. This membrane
is permeable to water, but it forms an effective barrier for most other solutes.
Outside this membrane, the cell usually has a rigid wall that is readily permeable to
small molecules and offers mechanical stability to the cell. Under normal growth
conditions, the concentration of solutes inside the cell (in the cytoplasm) are higher
than those in the environment. In other words, the cytoplasm has a higher
osmolality, i.e., the amount of “osmotically active” solutes in a solution, than the
outside medium. A high osmolality corresponds to a reduced relative concentration
of water (or water activity). Since only water can move “freely” across the
membrane, a high internal solute concentration will result in the movement of water
from the environment to the inside of the cells. As water moves into the cells, the
cellular volume increases, and the membrane expands, until it is restrained by the
rigid cell wall. Additional influx of water results in the build up of pressure, which
is called the turgor pressure of a cell. Turgor pressure is essential for cell division
and thus for cell growth.
In their natural habitats microorganisms are often exposed to changes in the
concentrations of solutes in their environments, whereas the internal concentrations
of solutes (nutrients) remain essentially the same. A higher osmolality of the
environment results in the movement of water from the cell to the environment,
causing hyper-osmotic stress (loss of turgor pressure, changes in intracellular solute
concentrations, cell volume, etc.). The primary response of bacteria to osmotic
stress involves the accumulation of specific solutes to balance the difference in
solute concentration at either side of the membrane. The accumulated solutes are
termed compatible solutes, because they can be accumulated to very high
intracellular concentrations without noticeable negative effects for the cells, i.e.,
they are compatible with life. Hypo-osmotic stress follows from decreases in
external solute concentrations, thereby causing water to move into the cells. The
cells respond to hypo-osmotic stress by rapidly releasing (compatible) solutes to
prevent them from bursting. In either case of osmotic stress, the stress must be
sensed and converted into an activity change of specific cellular enzymes and
transport proteins and/or it must trigger their synthesis in order to restore the
osmotic imbalance.
This research was aimed at the elucidation of the response of the lactic acid
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Summary and perspectives
bacterium Lactobacillus plantarum towards osmotic stress. Understanding the basic
mechanisms underlying the adaptation to changes in osmolality is of utmost
importance, because high osmolality is often applied as a means to preserve food
products. Lactobacillus plantarum is frequently encountered as spoilage organism
in various dressings (and other high-osmolality foods), which led Unilever Research
Laboratoria to fund a research project on the basic mechanism of osmoregulation
in this organism. We have used Lactobacillus plantarum as a model organism to
study the regulation of transport processes with regard to adaptation to growth under
high and low osmolality conditions.
Lactobacillus plantarum responds to an osmotic upshock by accumulating
compatible solutes from the environment (glutamate, proline, alanine, and glycine)
rather than by synthesizing these compounds (Chapter 3). The quarternary
ammonium compound glycine betaine is preferentially accumulated to high internal
concentrations, resulting in a stimulation of growth under high-osmolality
conditions. Since the internal pools of glycine betaine are of primary importance for
regulating the adaptation of Lactobacillus plantarum to high osmolality conditions,
the focus of our work has been on the osmotic regulation of glycine betaine uptake
and efflux. The regulation of glycine betaine uptake occurred mainly at the protein
level via a direct activation of the transport system, whereas changes in protein
expression (synthesis of more transport protein) played a minor role in the osmotic
response. We observed that the time the uptake system remained in its activated
state (high velocity) correlated with the size of the osmotic upshift, suggesting that
turgor pressure or a related parameter is responsible for the sensing of osmolality
changes.
In sugar-stressed cells, turgor pressure is restored by the uptake of sugars via
systems with a very low apparent affinity for the sugars, i.e., systems that only take
up sugars when they are present in very high concentrations (Chapter 4). The sugar
uptake systems were neither directly regulated (increased velocity) nor induced
(increased synthesis) by high-osmolality conditions.
The regulation of transport by changes in osmolality involves the activation and/or
inhibition of systems for uptake and efflux of glycine betaine in such a way that the
osmotic imbalance is rapidly compensated. The following observations concerning
the uptake and efflux of glycine betaine were made (Chapter 5): (i) in the steady
state (no osmolality changes), a basal flux of glycine betaine (but no net uptake or
efflux) is observed; (ii) upon osmotic upshock, the uptake system for glycine betaine
is activated within seconds, whereas the basal efflux is completely inhibited; (iii)
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Chapter 8
upon osmotic downshock, glycine betaine is rapidly released in a process that has
two kinetic components, whereas the uptake system is completely inhibited. The
half-life of the rapid phase of efflux is smaller than 1 second, unaffected by the
metabolic status of the cells, and inhibited by gadolinium ions. These properties are
consistent with a channel-like activity that is regulated by membrane strain. A
channel protein is best described as a regulated protein-pore in the membrane. The
slower phase of efflux has a half life of 4-5 min and is dependent of metabolic
energy or a related parameter. It most likely corresponds to the activity of a carrier
protein, i.e., a protein that specifically binds its substrate (glycine betaine) and
catalyzes translocation across the membrane. Thus, all the systems involved in
uptake and efflux of glycine betaine have the intrinsic property to sense changes in
medium osmolality, and they are able to adjust their activities. The mechanism of
osmosensing is described in more detail in chapter 6.
A rapid efflux of glycine betaine could also be triggered by the addition of
amphipathic drugs that insert into the membrane. The amphipaths also prevented
activation of the glycine betaine uptake system by an osmotic upshift, but they did
not prevent the cells to restore turgor through prelonged uptake of glycine betaine
(Chapter 6). The regulatory mechanism of the channel protein must therefore be
different from that of the uptake system. Both transport activities sense a turgorrelated parameter: the efflux channel may sense membrane tension (or stretch),
whereas the uptake system may sense pressure across the membrane, e.g., an altered
interaction of the protein with the cell wall. At the same time, the mechanistic basis
for activation of uptake (an increase in uptake rate) may be similar to that of the
opening and closing of the channel protein, and involve changes in membrane strain.
We also showed that the glycine betaine uptake rates are regulated by transinhibition (inhibition by accumulated substrate), which forms an additional level of
control against accumulation of excessive amounts of glycine betaine, carnitine, and
proline. Upon osmotic upshock, the trans-inhibition is relieved, which allows the
cell to restore turgor more rapidly.
To unravel the intricacies of osmotic regulation, it is important to have mutants in
which uptake and efflux can be studied separately. We have succeeded in isolating
such mutants by UV irradiation of Lactobacillus plantarum cells and selection on
the basis of resistance towards dehydroproline, a toxic analogue of proline (Chapter
6). The mutants allowed us to establish that uptake and efflux are mediated by
separate systems. Together with a kinetic analysis of glycine betaine and proline
uptake under various osmolalities, the mutants allowed us to obtain compelling
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Summary and perspectives
evidence that Lactobacillus plantarum accumulates a large variety of quarternary
ammonium compounds and proline via a single system.
Finally, a new pH sensitive fluorescent dyes was developed, which allowed us to
measure the cytoplasmic pH of bacteria under various stress conditions, e.g., low
pH (Chapter 7). The application of this and other pH-sensitive dyes led to the
discovery of different ATP-driven extrusion systems for unmodified and conjugated
dyes. Although the implications of this work fall beyond the scope of this thesis, the
extrusion systems may be related to the multidrug resistance (MDR) proteins found
in prokaryotic and eukaryotic cells.
PERSPECTIVES
The primary response of bacteria to osmolality changes in the environment is often
poorly described, and involves the uptake and excretion of compatible solutes via
a direct activation of transport systems that are already present in the membrane.
Since the (initial) osmotic responses of eukaryotic and prokaryotic cells are likely
to be similar, studies in simple prokaryotes may rapidly lead to the discovery of
universal mechanisms of osmosensing and osmoregulation. This study provides
some insight in the sensing mechanisms that regulate the uptake and efflux of the
compatible solute glycine betaine in Lactobacillus plantarum. A detailed structural
analysis is needed to relate specific domains to functions such as osmosensing.
The most intriguing questions in osmoregulation concern the nature of the osmotic
signal that is sensed, the mechanism by which the signal is sensed, and the
regulation of the immediate uptake or release of compatible solutes? These
questions might also be relevant for secondary osmotic responses, i.e., uptake and
synthesis of other compatible solutes, alterations in membrane lipids, cell wall
composition, and exo-polysaccharides, and/or capsule synthesis. Structure-function
analysis of a good model system may result in the identification of the structural
elements involved in the activation of transport enzymes, thereby providing
information about the signal sensing device. These elements may be introduced into
proteins of biotechnological importance, resulting in enzymes that can be activated
by alterations in the medium osmolality.
Osmotic stress is often applied in food preservation by adding large amounts of salts
and/or sugars to foods or by drying of the products. Spoilage organisms adapt most
efficiently to osmotic stress in the presence of glycine betaine or carnitine, which are
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Chapter 8
constituents of many foods from plant or animal origin, respectively. Therefore,
knowledge about the regulation of the corresponding transport systems is important
for designing optimal food preservation techniques. One may be able to improve the
strategies used to reduce the possibilities for food-poisoning by combining osmotic
stress with other techniques to reduce the outgrowth of spoilage and pathogenic
bacteria. The combinations of stresses are generally referred to as “hurdle
technology”, because the stresses can be regarded as hurdles that should not be
overcome by the contaminating bacteria. However, exposure to one stress may lead
to cross-resistance to another stress, which is a major limitation of hurdle technology
that requires further attention.
Industries may also seek alternatives to harsh preservation methods such as heat
sterilization. In addition to osmotic stress, the use of high hydrostatic pressure to
preserve food products may have enormous potential. The transport systems for
compatible solutes, that are regulated by changes in turgor pressure may also be
targets for inactivation by high hydrostatic pressure, and thereby contribute to the
growth inhibition of microorganisms. Information on the kinetics and mechanisms
of pressure inactivation in relation to the presence of compatible solutes may lead
to a more efficient application of high hydrostatic pressure for the preservation of
food products.
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