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Regulation of the Ysa Type III Secretion System of Yersinia enterocolitica by YsaE/SycB and YsrS/YsrR. Kimberly A. Walker, 2004.Yersinia enterocolitica biovar 1B contains two type III secretion systems (TTSSs), the plasmid-encoded Ysc-Yop system and the chromosomally encoded Ysa-Ysp system . Proteins secreted from the Ysa TTSS (Ysps) have only been detected in vitro when cells are cultured at 26°C in a high-NaCl medium . However, the exact role of the Ysa TTSS is unclear . Thus, investigations into the regulation of this system may help elucidate the role of the Ysps during the life cycle of Y . enterocolitica . Here we present evidence that the AraC-like regulator YsaE acts together with the chaperone SycB to regulate transcription of the sycByspBCDA operon, a phenomenon similar to that seen in the closely related Salmonella SPI-1 and Shigella flexneri Mxi-Spa-Ipa TTSSs . Deletion of either sycB or ysaE results in a twofold reduction in the activity of a sycB-lacZ fusion compared to the wild type . In a reconstituted Escherichia coli system, transcription of sycB was activated sixfold only when both YsaE and SycB were present, demonstrating that they are necessary for activation . ysrR and ysrS are located near the ysa genes and encode a putative two-component regulatory system . Mutations in either gene indicated that both YsrR and YsrS were required for secretion of Ysps . In addition, transcription from sycB-lacZ and ysaE-lacZ fusions was decreased 6.5- and 25-fold, respectively, in the ysrS mutant compared to the wild type . Furthermore, in the absence of NaCl, the activity of ysaE-lacZ was reduced 25-fold in the wild-type and  ysrS
strains, indicating that YsrS is probably required for the
salt-dependent expression of the ysa locus . These results
suggest that the putative two-component system YsrRS may be a key
element in the regulatory cascade for the Ysa TTSS .
A Membrane-Bound Archaeal Lon Protease Displays ATP-Independent Proteolytic Activity towards Unfolded Proteins and ATP-Dependent Activity for Folded Proteins. Toshiaki Fukui, 2002.In contrast to the eucaryal 26S proteasome and the bacterial ATP-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain, Archaea . We cloned a gene homologous to ATP-dependent Lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal Lon . Lon from Thermococcus kodakaraensis KOD1 (LonTk) is a 70-kDa protein with an N-terminal ATPase domain belonging to the AAA+ superfamily and a C-terminal protease domain including a putative catalytic triad . Interestingly, a secondary structure prediction suggested the presence of two transmembrane helices within the ATPase domain and Western blot analysis using specific antiserum against the recombinant protein clearly indicated that LonTk was actually a membrane-bound protein . The recombinant LonTk possessed thermostable ATPase activity and peptide cleavage activity toward fluorogenic peptides with optimum temperatures of 95 and 70°C, respectively . Unlike the enzyme from Escherichia coli, we found that LonTk showed higher peptide cleavage activity in the absence of ATP than it did in the presence of ATP . When three kinds of proteins with different thermostabilities were examined as substrates, it was found that LonTk required ATP for degradation of folded proteins, probably due to a chaperone-like function of the ATPase domain, along with ATP hydrolysis . In contrast, LonTk degraded unfolded proteins in an ATP-independent manner, suggesting a mode of action in LonTk different from that of its bacterial counterpart .
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