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mainly represented enzymes of the various degradation pathways.
Noteworthy, proteins with other predicted or unknown functions
were co-regulated and could therefore play hitherto not conceived
roles in the respective pathways or refl ect a more general stress
response to toxic aromatic substrates (e.g., phenols). In several
cases, the genome-predicted pathway involvement of proteins could
not be confi rmed, e.g., phenylacetaldehyde dehydrogenase (PDH)
in anaerobic phenylalanine degradation. Instead, co-regulation
of previously not considered proteins shed new light on the respec-
tive pathways. For instance, a predicted aldehyde:ferredoxin oxi-
doreductase together with a ferredoxin-regenerating system were
upregulated in phenylalanine-adapted cells and could substitute
PDH. Moreover, strong evidence was obtained for thus far unpre-
dicted degradation pathways of three hitherto unknown substrates
(e.g.,
o
-aminobenzoate, anoxic).
5.6. Solvent Stress
Response
A
.
aromaticum
EbN1 grows anaerobically with the aromatic
solvents ethylbenzene, toluene,
p
-cresol, and phenol, the hydro-
phobicity of which determines their toxic properties. Anaerobic
cultivation of
A
.
aromaticum
EbN1 at semi-inhibitory (about 50%
growth inhibition) concentrations of the four substrates resulted in
impaired growth, which was paralleled by decelerated nitrate-
nitrite consumption. In addition, ethylbenzene- and toluene-
utilizing cultures accumulated poly(3-hydroxybutyrate) (PHB) up
to 10% of the cell dry weight. These physiological responses were
also refl ected by 2D DIGE-resolved protein profi les, e.g., upregu-
lation of PHB granule-associated phasins, cytochrome
cd
1
nitrite
reductase of denitrifi cation, and several proteins involved in oxidative
(e.g., SodB) and general (e.g., ClpB) stress responses. One may
speculate that alkylbenzene-derived acetyl-CoA is rerouted from
the TCA cycle to PHB synthesis, decreasing the NAD(P)H pool
under conditions of impaired denitrifi cation (Fig.
6
). PHB, func-
tioning as sink for reducing equivalents, could thus ensure continuous
alkylbenzene consumption (
58
).
6. Conclusion
2D DIGE-based studies have provided new insights into the
metabolic capacities of
A
.
aromaticum
EbN1 that have not been
recognized during initial genome annotation, i.e., new substrates,
new degradation pathways, and novel effl ux system. To date, 2D
DIGE-derived protein signatures were generated for more than 50
different substrate, redox, and growth conditions. They provide
in-depth insights into regulatory hierarchies and overlaps of
individual degradation pathways and general metabolic responses,
as well as into what can be considered as the constitutive core
proteome of
A
.
aromaticum
EbN1.
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