A proteomic view of probiotic Lactobacillus rhamnosus GG

Results and Discussion
et al., 2009) and the results produced in study
III suggest a model in which in the absence
of bile, L. rhamnosus GG cells are shielded by
EPS, potentially providing protection against
the harsh conditions of the stomach. The
presence of bile could function as a signal of
gut entrance, resulting in the removal of EPS
and, concomitantly, increased adherence of L.
rhamnosus GG cells to the gut.
Other cell envelope-related functions
that were likely to be affected by bile were
membrane fatty acid composition and cell
envelope charge, which might help L. rhamnosus
GG to survive in the presence of bile.
Th e active removal of bile components from
L. rhamnosus GG cells could be performed by
ABC transporters and the proton-translocating
ATPase, which were up-regulated under
bile stress conditions. Th e bile response of L.
rhamnosus GG also included the up-regulation
of a bile salt hydrolase and several twocomponent
systems, which may be involved in
detoxifi cation and sensing of bile compounds.
In addition, bile induced common stress
responses, including elevated expression of
chaperones and proteases, and aff ected some
central metabolic processes, such as carbohydrate
and nucleotide metabolism.
The surfome analysis revealed a high
number of cytoplasmic proteins, which were
located on the surface of L. rhamnosus GG
cells and the abundance of which changed as
a result of the bile shock. Th ese proteins could
be so-called “moonlighting proteins”, proteins
that can have diff erent functions depending
on their location, as suggested previously by
Jeff ery (Jeff ery, 2003). Th e altered abundance
of these proteins could be related either to
their functions in the bile response, e.g., as
adhesins, or to their altered availability to
the Cy dyes resulting from the bile-induced
changes in the cell envelope EPS layer.
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4.4. Eff ect of growth pH on
L. rhamnosus GG
In study IV, the eff ect of mild acid stress on L.
rhamnosus GG was studied. In industrial production,
lactic acid bacteria are cultivated at
constant pH, e.g., at pH 5.8, but the pH of the
fi nal product in many fermented milk-based
food products is decreased to around pH 4.5
due to fermentation end products, such as lactic
acid. Lactic acid is a weak organic acid and
can easily pass the bacterial cell membrane
in its protonated form at low environmental
pH, thus reducing the internal pH. Intracellular
acidifi cation might aff ect the transmembrane
pH gradient, reduce the activity of acidsensitive
enzymes, and cause damage to proteins
and DNA (van de Guchte et al., 2002).
In study IV, L. rhamnosus GG was grown in
bioreactors in whey medium, as in study II,
but the pH of the medium was adjusted either
to pH 5.8, as in study II, or pH 4.8. Th e acid
stress mechanisms of L. rhamnosus GG were
explored by comparing the transcriptomes
and proteomes under these two growth conditions.
In general, pH 5.8 was clearly more
favorable than pH 4.8 for the growth of L.
rhamnosus GG. Th e growth rate was lower at
pH 4.8, and basic biosynthetic processes, such
as protein synthesis, purine and pyrimidine
biosynthesis, and sugar transport, appeared
to be reduced at pH 4.8. A reduction in the
production of pyrimidine biosynthesis proteins
was also detected in the response to bile
stress in study III, and the down-regulation of
pyrimidine biosynthesis appears to be a common
response of L. rhamnosus GG to adverse
growth conditions. A specifi c response of L.
rhamnosus GG to the acidic growth pH was
the increased expression of proton-translocating
ATPase, which is a multimeric enzyme