Reduction-sensitive and cysteine residue-mediated Streptococcus pneumoniae HrcA oligomerization in vitro.
In both gram-positive and several gram-negative bacteria, the transcription of dnaK and groE operons is negatively regulated by HrcA; however, the mechanism modulating HrcA protein activity upon thermal stress remains elusive. Here, we demonstrate that HrcA is modulated via reduction and oligomerization in vitro. Native-PAGE analysis was used to reveal the oligomeric structure of HrcA. The oligomeric HrcA structure became monomeric following treatment with the reducing agent dithothreitol, and this process was reversed by treatment with hydrogen peroxide. Moreover, the mutant HrcA C118S exhibited reduced binding to CIRCE elements and became less oligomerized, suggesting that cysteine residue 118 is important for CIRCE element binding as well as oligomerization. Conversely, HrcA mutant C280S exhibited increased oligomerization. An HrcA double mutant (C118S, C280S) was monomeric and exhibited a level of oligomerization and CIRCE binding similar to wild type HrcA, suggesting that cysteine residues 118 and 280 may function as checks to one another during oligomer formation. Biochemical fractionation of E. coli cells overexpressing HrcA revealed the presence of HrcA in the membrane fraction. Together, these results suggest that the two HrcA cysteine residues at positions 118 and 280 function as reduction sensors in the membrane and mediate oligomerization upon stress.
Kwon
Hyog-Young
HY
College of Pharmacy, Sungkyunkwan University, Suwon, 440-746, Korea.
Kim
Eun-Hye
EH
Tran
Thao Dang Hien
TD
Pyo
Suhk-Neung
SN
Rhee
Dong-Kwon
DK
D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D018869:Heat-Shock Response; D066298:In Vitro Techniques; D009154:Mutation; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013296:Streptococcus pneumoniae / Q000502:physiology*
D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012097:Repressor Proteins; D003545:Cysteine