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Abstract:
Bacterial degradation of xylose is sequentially mediated
by two enzymes – an isomerase (XutA) and a
xylulokinase (XutB) – with xylulose as an intermediate.
Pseudomonas fluorescens SBW25, though capable of
growth on xylose as a sole carbon source, encodes
only one degradative enzyme XutA at the xylose utilization
(xut) locus. Here, using site-directed mutagenesis
and transcriptional assays, we have identified two
functional xylulokinase-encoding genes (xutB1 and
xutB2) and further show that expression of xutB1
is specifically induced by xylose. Surprisingly, xyloseinduced
xutB1 expression is mediated by the
mannitol-responsive regulator MtlR, using xylulose
rather than xylose as the direct inducer. In contrast,
expression of the xutA operon is regulated by XutR – a
transcriptional activator of the AraC family – in a
xylose-, xylulose- and ribose-dependent manner.
Detailed genetic and biochemical analyses of XutR,
including DNase I footprinting assays, suggest an
unconventional model of XutR regulation that does not
involve DNA-looping, a mechanism typically found for
AraC-type regulators from enteric bacteria. XutR functions
as a dimer and recognizes two inverted repeat
sequences, but binding to one half site is weak thus
requiring an inducer molecule such as xylose for
activation
