Page 11 - U. Protein structure and function

Polaribacter irgensii KOPRI 22228 cspB expression greatly

increases freeze-tolerance of the host

*
Hana Im andYoun Hong Jung
Department of Integrative Bioscience and Biotechnology, Sejong University
BACKGROUND AIM

Cold temperatures, especially in polar regions, pose serious challenges to living To elucidate the detailed mechanisms involved in the extraordinary freeze-
organisms: decreased membrane fluidity impedes crucial membrane functions such as tolerance conferred by cspB Pi , mutations in the well-conserved C-terminal cold
transport and protein movement, metabolic rates are reduced to harmful levels, protein shock domain (CSD), that are crucial for binding RNA or single-stranded DNA, were
folding is retarded, intracellular ice formation damages cell membranes and also causes introduced. These mutations did not hamper the ability of the host to survive
dehydration, and unintended formation of stable secondary structures in DNA and RNA freezing stress. When the effects of domain-deletion and domain-shuffling of cspB Pi
may inhibit transcription and translation (Phadtare, 2004). Since psychrophilic organisms were analyzed, all cspB Pi variants containing the unique N-terminal domain retained
from cold ecosystems must have a mechanism to survive extremely cold temperatures, the ability to confer the prominent cold-resistance. Far-UV circular dichroism
the roles played by cold shock proteins (Csps) from polar bacteria have been studied to spectra and slow electrophoretic mobility suggested that the N-terminal domain is
understand the mechanism of cold adaptation. Overexpression of cspB Pi from the Arctic intrinsically disordered. The N-terminal domain bound to lipid vesicles in vitro, that
bacterium Polaribacter irgensii KOPRI 22228 conferred extraordinary freezing tolerance is a characteristics shared with other intrinsically disordered proteins known to
to its host (Jung et al., 2018). confer cold-tolerance.
METHODS AND RESULTS

Figure 1. Site-directedmutagenesis of cold shock domain (CSD) of P. irgensii CspB. Figure 2 . Cold-survival ratio of Csp-expressing E. coli cells.
(A)
(A) (B)
N-domain CSD N-domain CSD Cold resistance 100
Cold resistance 100
CspB Pi (B) CspA Pi R1 R2 + 10
WT +++ +++
R1 R2 10 CspB Pi + Cell survival rate (%) 1 pAED4
CspC Pi
CspB Pi
CspB Pi Cell survival rate (%) 1 CspA Pi
CspC Pi
F15R +++ CspBA Pi +++ 0.1 CspBA Pi
CspBC Pi
+++
CspBN Pi
CspB Pi 0.1 pAED4 CspBC Pi +++ 0.01 0 1 2 3
CspB Pi WT
CspBN Pi
F28R +++ CspB Pi F15R Freeze and thaw cycle
CspB Pi F28R
0.01 (A) E. coli BL21(DE3) pLysS cells carrying pAED-csps (pAED vector; Doering, 1992) were incubated at 37 o C until OD 600 reaches ≈ 0.2, and Csp
overexpression was induced by addition of 0.1 mM IPTG for 2 h. After freezing at -20 o C for 2 h, cells were thawed on ice. Numbers of surviving cells
0 1 2 3 were counted as CFU (colony forming unit) at each cycle of freezing and thawing, as discribed (Kim et al., 2006). (B) To identify the domain
Freeze and thaw cycle responsible for the high cold resistance induced by P. irgensii CspB, domain-shuffling between Csps were conducted. Any construct containing N-
(A) To evaluate the importance of CSD for the high cold resistance induced by P. irgensii CspB, Phe15 and Phe28, which are known critical in RNA terminal domain of CspB conferred extraordinary cold-resistance to its host. Mutations on cold shock-domain did not decrease cold-resistance by CspB.
chaperone function of Csp (Phadtare et al., 2002), are changed to Arg by site-directed mutagenesis. (B) E. coli BL21(DE3) pLysS cells carrying
pAED-mutant CspB were incubated at 37 o C until OD 600 reaches ≈ 0.2 , and Csp overexpression was induced by addition of 0.1 mM IPTG for 2 h.
After freezing at -20 o C for 2 h, cells were thawed on ice. Numbers of surviving cells were counted as CFU (colony forming unit) at each cycle of Fig. 4. Purified CspBN Pi binds to phospholipid vesicles.
freezing and thawing, as discribed (Kim et al., 2006). Mutations on CSD of CspB did not show significant changes in cold-resistance.
(A) (B)
Phospholipid only CspBN only
Fig. 3. CspBN Pi protein is intrinsically unstructured. 4000 160 140
(A) Phospholipid (µg/ml) 3000 120 100 80 Proteins (µg/ml)
(A) Secondary structure 2000 60
prediction of CspBN Pi protein. 1000 40
Amino acid sequence of 20
CspBN Pi was analyzed using the 0 0 10 20 30 40 50 0 10 20 30 40 50 0
IUPred program (Dosztanyi et Fraction number Fraction number
al., 2005). A disorder tendency (C) Phospholipid + CspBN (D) Phospholipid + Carbonic anhydrase
higher than 0.5 suggests
unstructured nature for the MW I B 160 160
(B) 5000 protein. (B) The CD spectra of 4000 140 4000 140
CspBN Pi protein. The protein 50 - 120 120
37 -
0 concentration was 790 µg/ml in 3000 25 - 100 3000 100
20 mM Tris-HCl, pH 8.0. The Phospholipid (µg/ml) 2000 20 - 80 Protein (µg/ml) Phospholipid (µg/ml) 2000 80 Protein (µg/ml)
15 -
CD spectra were obtained using
10 -
-5000
Molar ellipticity -10000 a Jasco-720 spectropolarimeter 1000 60 40 1000 60 40
with a 1 mm path-length cell at 0 20 0 0 20 0
25°C
-15000
30
20
30
10
0
Fraction number 40 50 0 10 20 Fraction number 40 50
-20000
Phospholipid vesicles were incubated with 100 µg of purified CspBN Pi protein in 20 mM Tris, pH8.0 at 4°C overnight. The reaction mixture was then

-25000 loaded onto a Superose 6 gel filtration column. (A) Phospholipid vesicles incubated alone. (B) CspBN Pi protein incubated alone. (C) Phospholipid
. 200 210 220 230 240 250 vesicles incubated with CspBN Pi protein. Inlet: 15% SDS-PAGE showing CspBN Pi protein co-eluted with phospholipid vesicles. MW, Precision plus
protein standards (Bio-Rad Laboratories Inc.). Size of each protein band is shown in kDa at left of the gel; I, CspBN Pi input; and B, CspBN Pi protein co-
Wavelength (nm)
eluted with phospholipid vesicles in a fraction indicated by an arrow. (D) Phospholipid vesicles incubated with carbonic anhydrase protein used at the
same concentration.
Fig. 5. The cold-sensitive phenotype of the E. coli quadruple csp deletion strain (∆cspA, ∆cspB, ∆cspG, ∆cspE), BX04, is suppressed by overexpression of Csps from P.
irgensii..
BX04 CspA BX04 CspA
BX04 cells harboring pAED-csps was grown in the liquid culture until a density of OD 600 ≈ 0.4. The cells were then streaked on LB medium plates containing 0.1 mM IPTG, and incubated at various
temperatures ranging from 16 to 37°C. As described before (Xia et al., 2001), BX04 cells were able to form colonies at 37°C, but not at 16°C. P. irgensii Csps complemented the cold sensitivity of
BX04, allowing growth of the host at 16 °C.
CspC CspB CspC CspB
37 ℃ 16 ℃
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
• Several csp genes have been cloned from polar microorganisms, 1. Phadtare, S. (2004). Curr. Issues Mol. Biol. 6: 125- This work was supported by the Korea Research
including P. irgensii. 136. Foundation Grant funded by the Korean Government
• Overexpression of P. irgensii Csps increased cold-resistance of 2. Jung, Y.H., Lee, Y.K., Lee, H.K., Lee, K., and Im, H. (KRF-2014-011146 & KRF-2015R1D1A1 A01058206).
(2018) Brazilian J. Microbiol. 49: 97-103.
host cells. 3. Phadtare, S., Tyagi, S., Inouye, M., and Severinov, K.
• Csps of P. irgensii bind to oligo-dT cellulose beads, suggesting (2002) J. Biol. Chem. 277: 46706-46711.
their single-stranded DNA binding activity. 4. Doering, D. (1992). Functional and structural
• The superior ability of CspB in conferring cold resistance to its studies of a small F-actin binding domain, Ph.D. Contact information
host is due to the unique N-terminal domain, not to the cold- thesis, Massachusetts Institute of Technology, MA.
shock domain. 5. Kim, M.-J., Lee, Y. K., Lee, H. K., and Im, H. (2006) E-mail address: hanaim@sejong.ac.kr (H. Im).
Prot. J. 26: 51-59.
• N-terminal domain of CspB binds to phospholipid vesicles. 6. Dosztanyi, Z., Csizmok, V., Tompa, P., Simon, I.
• Csps suppressed cold-sensitive phenotype of csp quadruple- (2005) Bioinformatics 21: 3433-3434. Department of Integrative Bioscience and Biotechnology,
deletion strain, BX04. 7. Xia, B., Ke, H., and Inouye, M. (2001). Mol. Sejong University, 209 Neungdong-ro, Gunja-dong,
• Manufacturing cspBN Pi -fusion proteins may aid cold production Microbiol. 40: 179-188. Gwangjin-gu, Seoul 05006, Republic of Korea.
of valuable heat-labile proteins.