Interaction of Bacillus subtilis extracytoplasmic function ECF sigma factors with the Nterminal regi

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Interaction of Bacillus subtilis extracytoplasmic
function (ECF) sigma factors with the N-terminal
regions of their potential anti-sigma factors

• Mika Yoshikawa, Kei Asai, Yoshito Sadaie, and
  Hirofumi Yoshikawa

Presentated by Mayu Ono
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Sigma factors (sigma subunits)

• Specificity factors
• Required for recognition of promoters by RNA
  polymerase
• In B. subtilis
• 17 distinct sigma factors known
• Principle sigma subunit sigma A
• Alternate sigma factors
• - can replace sigma A
• - 7 ECF sigma factors

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Extracytoplasmic function (ECF) sigma factors

• Revealed by genome sequencing
• 7 ECF sigma subfamilies found (B.subtilis)
• Found in a diverse range of bacteria
• Control the uptake or secretion of specific
  molecules or ions and response to a variety of
  extracellular stress signals
• (EX.) in E.coli regulation of citrate uptake
• and heat shock responses
• in S.coelicolor expression of the
• thioredoxin system in response to
• oxidative stress
• Co-transcribed with a cognate anti-sigma factor

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ECF sigma factors VS. All other sigma factors
(Non-ECF sigma factors)

• They differ in
• The structure of region 2
• Recognition helix of the helix-tern-helix motif
• Regulating promoter sequences
• Regulation and response to the extracytoplasmic
  conditions

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ECF anti-sigma factors

• Inner membrane proteins with at least one
  transmembrane domain
• Inhibit transcription from a given set of
  promoters by inhibiting only the sigma factor
  that is required to recognize the particular
  promoter sequence

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ECF anti-sigma factors (contd)

• Conserved amino terminus, which interact with the
  cognate sigma factors (suggested with E.coli RseA
  and P.aeruginose MucA)
• Observed poor homology near the C-terminus

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Gene organization of the ECF sigma operons and
possible membrane spanning domains
for survival at high temp.
induced by nitrogen starvation
required for growth and survival after salt
stress
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SOSUI

• Classification and secondary structure prediction
  system for membrane proteins from amino acid
  sequences

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SOSUI contd
Functions

• The discrimination of membrane proteins from
  soluble ones
• Secondary structure prediction of the
  transmembrane helices
• High accuracy of classification of proteins (
  gt99)

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SOSUI contd.

• 3 basic assumptions in the SOSUI
• Membrane proteins are characterized by at least,
  particularly hydrophobic, primary transmembrane
  helix.
• Hydrophilic transmembrane helices may also exist
  in multi-spanning membrane proteins.
• The primary transmembrane helices are stabilized
  by a combination of amphiphilic side chains at
  the helix ends as well as high hydrophobicity in
  the central region.

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SOSUI http//www.proteome.bio.tuat.ac.jp/sosui_s
ubmit.html
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Disruption of anti-sigma factors, yrhM and YlaD
Effects of anti-sigma disruption on ECF sigma
operons
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Disruption of yxlC or yxlD gene

• RESULT
• Similar abnormal colony morphology

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The effect of induced overexpression of
anti-sigma factor on the cognate sigma factor
gene
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Yeast two-hybrid (Y2H) system
AD-fusion plasmids (pGADT7)
PJ69-4A MATa trp1-901 leu2-3, 112 ura3-52
his-200 gal4? gal80? LYS2GAL1-HIS3 GAL2-ADE2
met2GAL7-lacZ
PJ69-4a MATa trp1-901 leu2-3, 112 ura3-52
his-200 gal4? gal80? LYS2GAL1-HIS3 GAL2-ADE2
met2GAL7-lacZ
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Interactions of ECF sigma factors with potential
anti-sigma factors
Plates SC-LWHA containing 5mM 3-aminotriazole
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Interaction of ECF sigma factors with potential
anti-sigma factor fragments lacking the
membrane-spanning domain
Plates SC-LWHA containing 1mM 3-aminotrizole
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Interaction of YhdL and YhdK
Plates SC-LWHA containing 1mM 3-aminotrizole
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The role of the membrane-spanning domains of YhdL
and YhdK
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Conclusion

• SigV, SigW, and SigX are negatively regulated by
  the second gene products of the bi-cistronic
  operons, sigV, sigW and sigX.
• N-terminal regions of the anti-sigma factors of
  SigV, SigW, SigX, SigY and SigM are located
  intracellularly.
• YhdK has some specific role in the anti-sigma
  function of YhdL (?).

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Conclusion (contd)

• YbbM and YlaD are ZAS family members

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