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Amino Acid Contacts between Sigma 70 Domain 4 and the Transcription Activators RhaS and RhaR. Jason R. Wickstrum, 2004.The RhaS and RhaR proteins are transcription activators thatrespond to the availability of L-rhamnose and activate transcriptionof the operons in the Escherichia coli L-rhamnose catabolicregulon . RhaR activates transcription of rhaSR, and RhaS activatestranscription of the operon that encodes the L-rhamnose catabolic enzymes, rhaBAD, as well as the operon that encodes the L-rhamnosetransport protein, rhaT . RhaS is 30% identical to RhaR at theamino acid level, and both are members of the AraC/XylS familyof transcription activators . The RhaS and RhaR binding sitesoverlap the –35 hexamers of the promoters they regulate,suggesting they may contact the  70
subunit of RNA polymeraseas part of their mechanisms of
transcription activation . Insupport of this hypothesis, our lab
previously identified aninteraction between RhaS residue D241 and
70
residue R599 . Inthe present study, we first identified two
positively chargedamino acids in
70,
K593 and R599, and three negatively chargedamino acids in RhaR,
D276, E284, and D285, that were importantfor RhaR-mediated
transcription activation of the rhaSR operon.Using a genetic
loss-of-contact approach we have obtained evidencefor a specific
contact between RhaR D276 and
70
R599 . Finally,previous results from our lab separately showed that
RhaS D250Aand
70
K593A were defective at the rhaBAD promoter . Our genetic
loss-of-contact analysis of these residues indicates that they
identify a second site of contact between RhaS and
70.
Molecular Characterization of Inulosucrase from Leuconostoc citreum: a Fructosyltransferase within a Glucosyltransferase. Vanesa Olivares-Illana, 2003.The gene coding for inulosucrase in Leuconostoc citreum CW28, islA, was cloned, sequenced, and expressed in Escherichia coli . The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme . Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355 . From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase . Two different forms of the islA gene truncated in the C-terminal glucan binding domain were successfully expressed in E . coli and retained their ability to synthesize inulin but lost thermal stability . This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases . Effect of High-Pressure-Induced Ice I-to-Ice III Phase Transitions on Inactivation of Listeria innocua in Frozen Suspension. C. Luscher, 2004.The inactivation of Listeria innocua BGA 3532 at subzero temperatures and pressures up to 400 MPa in buffer solution was studied to examine the impact of high-pressure treatments on bacteria in frozen matrices . The state of aggregation of water was taken into account . The inactivation was progressing rapidly during pressure holding under liquid conditions, whereas in the ice phases, extended pressure holding times had comparatively little effect . The transient phase change of ice I to other ice polymorphs (ice II or ice III) during pressure cycles above 200 MPa resulted in an inactivation of about 3 log cycles, probably due to the mechanical stress associated with the phase transition . This effect was independent of the applied pressure holding time . Flow cytometric analyses supported the assumption of different mechanisms of inactivation of L . innocua in the liquid phase and ice I (large fraction of sublethally damaged cells due to pressure inactivation) in contrast to cells subjected to ice I-to-ice III phase transitions (complete inactivation due to cell rupture) . Possible applications of high-pressure-induced phase transitions include cell disintegration for the recovery of intracellular components and inactivation of microorganisms in frozen food . patS Minigenes Inhibit Heterocyst Development of Anabaena sp . Strain PCC 7120. Xiaoqiang Wu, 2004.The patS gene encodes a small peptide that is required for normal heterocyst pattern formation in the cyanobacterium Anabaena sp . strain PCC 7120 . PatS is proposed to control the heterocyst pattern by lateral inhibition . patS minigenes were constructed and expressed by different developmentally regulated promoters to gain further insight into PatS signaling . patS minigenes patS4 to patS8 encode PatS C-terminal 4 (GSGR) to 8 (CDERGSGR) oligopeptides . When expressed by PpetE, PpatS, or PrbcL promoters, patS5 to patS8 inhibited heterocyst formation but patS4 did not . In contrast to the full-length patS gene, PhepA-patS5 failed to restore a wild-type pattern in a patS null mutant, indicating that PatS-5 cannot function in cell-to-cell signaling if it is expressed in proheterocysts . To establish the location of the PatS receptor, PatS-5 was confined within the cytoplasm as a gfp-patS5 fusion . The green fluorescent protein GFP-PatS-5 fusion protein inhibited heterocyst formation . Similarly, full-length PatS with a C-terminal hexahistidine tag inhibited heterocyst formation . These data indicate that the PatS receptor is located in the cytoplasm, which is consistent with recently published data indicating that HetR is a PatS target . We speculated that overexpression of other Anabaena strain PCC 7120 RGSGR-encoding genes might show heterocyst inhibition activity . In addition to patS and hetN, open reading frame (ORF) all3290 and an unannotated ORF, orf77, encode an RGSGR motif . Overexpression of all3290 and orf77 under the control of the petE promoter inhibited heterocyst formation, indicating that the RGSGR motif can inhibit heterocyst development in a variety of contexts . Origin of Contamination and Genetic Diversity of Escherichia coli in Beef Cattle. Mueen Aslam, 2003.The possible origin of beef contamination and genetic diversity of Escherichia coli populations in beef cattle, on carcasses and ground beef, was examined by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the fliC gene . E . coli was recovered from the feces of 10 beef cattle during pasture grazing and feedlot finishing and from hides, carcasses, and ground beef after slaughter . The 1,403 E . coli isolates (855 fecal, 320 hide, 153 carcass, and 75 ground beef) were grouped into 121 genetic subtypes by using the RAPD method . Some of the genetic subtypes in cattle feces were also recovered from hides, prechilled carcasses, chilled carcasses, and ground beef . E . coli genetic subtypes were shared among cattle at all sample times, but a number of transient types were unique to individual animals . The genetic diversity of the E . coli population changed over time within individual animals grazing on pasture and in the feedlot . Isolates from one animal (59 fecal, 30 hide, 19 carcass, and 12 ground beef) were characterized by the PCR-RFLP analysis of the fliC gene and were grouped into eight genotypes . There was good agreement between the results obtained with the RAPD and PCR-RFLP techniques . In conclusion, the E . coli contaminating meat can originate from cattle feces, and the E . coli population in beef cattle was highly diverse . Also, genetic subtypes can be shared among animals or can be unique to an animal, and they are constantly changing .
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