A proteomic view of probiotic Lactobacillus rhamnosus GG

Results and Discussion
bose-5-phosphate, at the beginning of stationary
phase aft er the exhaustion of glucose.
Th e shift in the energy source used was also
refl ected in the fermentation end products. A
shift from homolactic fermentation to mixedacid
fermentation when the culture reached
stationary phase was proposed because the
expressions of pyruvate-formate lyase, aldehyde-alcohol
dehydrogenase, and enzymes
of the pyruvate oxidase pathway were upregulated
during stationary phase, while the
expression of lactate hydrogenase was higher
during exponential growth. Th is conclusion is
also supported by observations made in Lactococcus
strains, which shift from homolactic to
mixed-acid fermentation during limited glucose
availability (Neves et al., 2005). In study
I, L. rhamnosus GG was suggested to prefer
mixed-acid fermentation at both the exponential-
and stationary-growth phases in MRS
medium, when compared to growth in whey
medium. However, the diff erences were less
pronounced at stationary phase. L. rhamnosus
GG thus possibly prefers mixed-acid fermentation
during stationary growth independent
of the growth medium, and in general, MRS
medium supports mixed-acid fermentation
better than whey medium.
The progression of growth was also
refl ected in changes in the expression of components
of the proteolytic system. Several oligopeptide
transport systems and peptidases
showed growth phase-dependent expression,
and a similar phenomenon was seen in studies
I and IV, in which several peptidases showed
growth medium- and growth pH-dependent
abundances, respectively. Th is is presumably
a result of the different specificities of the
transport systems and peptidases towards the
varying selection of peptides available in each
growth condition.
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Other pathways of basic metabolism also
showed growth phase-dependent expression.
Fatty acid metabolism and pyrimidine biosynthesis
were repressed at the end of exponential
growth, and these changes were apparent at
both the transcriptome and proteome levels.
Th e increased need for fatty acid biosynthesis
during exponential growth was also apparent
in study I, in which the fatty acid biosynthesis
proteins were more abundant when cells were
cultured in whey medium than in MRS medium
at the exponential phase; this diff erence
was no longer present in stationary phase.
The expression of common stress
response proteins was mainly elevated when
the cells reached stationary phase, but two
stress proteins, DnaK and HtrA, exhibited
their highest levels in the exponential growth
phase. Th is eff ect could be a response to the
higher protein synthesis rate in the exponential
phase because chaperone proteins are also
required for de novo protein folding, in addition
to folding and degradation of damaged
proteins. Th e need for DnaK for de novo protein
folding is also supported by the results of
studies I and IV, in which the abundance of
DnaK was highest under the most favorable
growth conditions, that is, rich MRS medium
(compared to whey medium) and growth at
pH 5.8 (compared to pH 4.8).
Several genes that mediate potential probiotic-associated
traits in L. rhamnosus GG
were expressed in a growth phase-dependent
manner. Cell surface-exposed pilus has been
suggested to play a key role in the adhesion of
L. rhamnosus GG to human intestinal mucus
(Kankainen et al., 2009), and the transcription
of pilus-encoding genes was most active during
exponential growth. However, the predicted
adhesion factors inlJ and prtP were up-regulated
during stationary growth. Th e expression
of genes involved in exopolysaccharide