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Molecular Characterization of a High-Affinity Xylobiose Transporter of Streptomyces thermoviolaceus OPC-520 and Its Transcriptional Regulation. Hiroshi Tsujibo, 2004. Streptomyces thermoviolaceus OPC-520 secretes two types of xylanases [StxI and StxII], an acetyl xylan esterase [StxIII], and an  -L-arabinofuranosidase
[StxIV] in the presence of xylan . Xylandegradation products [mainly
xylobiose] produced by the actionof these enzymes entered the cell
and were then degraded toxylose by an intracellular ß-xylosidase
[BxlA] . Agene cluster involved in xylanolytic system of the strain
wascloned and sequenced upstream of and including a BxlA-encoding
gene [bxlA] . The gene cluster consisted of four different open
reading frames organized in the order bxlE, bxlF, bxlG,
andbxlA . Reverse transcriptase PCR analysis revealed that the
genecluster is transcribed as polycistronic mRNA . The deduced gene
products, comprising BxlE [a sugar-binding lipoprotein], BxlF
[an integral membrane protein], and BxlG [an integral membrane
protein], showed similarity to components of the bacterial ATP-binding
cassette [ABC] transport system; however, the gene for the ATP
binding protein was not linked to the bxl operon . The soluble
recombinant BxlE protein was analyzed for its binding activityfor
xylooligosaccharides . The protein showed high-level affinityfor
xylobiose [Kd = 8.75 x 10-9
M] and for xylotriose [Kd =8.42
x 10-8 M] . Antibodies raised
against the recombinant BxlErecognized the detergent-soluble BxlE
isolated from S . thermoviolaceusmembranes . The deduced BxlF
and BxlG proteins are predictedto be integral membrane proteins .
These proteins contained theconserved EAA loop [between the fourth
and the fifth membrane-spanningsegments] which is characteristic of
membrane proteins frombinding-protein-dependent ABC transporters . In
addition, thebxlR gene located upstream of the bxl
operon was cloned andexpressed in Escherichia coli . The
bxlR gene encoded a 343-residuepolypeptide that is highly
homologous to members of the GalR/LacIfamily of bacterial
transcriptional regulators . The purifiedBxlR protein specifically
bound to a 4-bp inverted sequenceoverlapping the -10 region of the
bxl operon . The binding ofBxlR to the site was inhibited
specifically by low concentrationsof xylobiose . This site was also
present in the region locatedbetween stxI and stxIV
and in the upstream region of stxII.BxlR specifically bound
to the regions containing the invertedsequence . These results
suggest that BxlR might act as a repressorof the genes involved not
only in the uptake system of xylandegradation products but also in
xylan degradation of S . thermoviolaceusOPC-520.
Pathways Leading from BarA/SirA to Motility and Virulence Gene Expression in Salmonella. Max Teplitski, 2003.The barA and sirA genes of Salmonella enterica serovar Typhimurium encode a two-component sensor kinase and a response regulator, respectively . This system increases the expression of virulence genes and decreases the expression of motility genes . In this study, we examined the pathways by which SirA affects these genes . We found that the master regulator of flagellar genes, flhDC, had a positive regulatory effect on the primary regulator of intestinal virulence determinants, hilA, but that hilA had no effect on flhDC . SirA was able to repress flhDC in a hilA mutant and activate hilA in an flhDC mutant . Therefore, although the flhDC and hilA regulatory cascades interact, sirA affects each of them independently . A form of BarA lacking the two N-terminal membrane-spanning domains, BarA198, autophosphorylates in the presence of ATP and transfers the phosphate to purified SirA . Phosphorylated SirA was found to directly bind the hilA and hilC promoters in gel mobility shift assays but not the flhD, fliA, hilD, and invF promoters . Given that the CsrA/csrB system is known to directly affect flagellar gene expression, we tested the hypothesis that SirA affects flagellar gene expression indirectly by regulating csrA or csrB . The sirA gene did not regulate csrA but did activate csrB expression . Consistent with these results, phosphorylated SirA was found to directly bind the csrB promoter but not the csrA promoter . We propose a model in which SirA directly activates virulence expression via hilA and hilC while repressing the flagellar regulon indirectly via csrB .
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