Grinnell Poster

Caulobacter as an alternative
chassis organism for high
output protein secretion
Undergraduates: Nora Kostow,
Alexander Aaring, Qimeng Gao
Advisor: Dr. Lisa Bowers
Abstract
BACKGROUND
We used the robust type I secretion
system of Caulobacter crescentus
to secrete enzymes Esp and DspB,
which degrade S. aureus biofilms.
Biofilms:
Bacteria
•  Cells within a biofilm are frequently found to be more
resistant to antimicrobials compared to planktonic cells7.
CONSTRUCTS
Promoter
Outer Membrane RsaA C-­‐terminal Periplasmic Space Inner Membrane ADP+Pi Heterologous protein   Perpetual motion—RsaA is expressed
constitutively1
 Flexible—Tolerates heterologous fusions of
4-80 kDa2 3
RsaA:
N-­‐terminus RsaA C-­‐terminus • N-terminus: association signal
• C-terminus: secretion tag
• < 82 amino acids are required for secretion4
Thanks to sponsors:
Grinnell College and Integrated DNA
Technologies, Inc. RBS
Coding Region
Promoter/RBS:
Coding:
• Pxyl Inducible
Promoter
(xylose)
• Esp
3 B.
4 Figure 1. Expression and secretion of chimeric proteins from
Caulobacter. We collected supernatants from equal numbers of
cells of each Caulobacter secretion strain, precipitated the
proteins with TCA, separated them on a 6-18% polyacrylamide
gel, and stained with silver. Results: Protein of expected size
was secreted from constructs: Pxyl-dspB-rsaA, Pxyl-esp-rsaA,
BBa_K081005-esp-rsaA and PrsaA-esp-rsaA.
DISCUSSION &
FUTURE DIRECTIONS C-­‐terminal Secre*on Tag
• Proteins fused to the RsaA C-terminal secretion
tag are secreted from Caulobacter crescentus and
retain activity.
•  Pxyl and PrsaA are effective promoter-RBS
combination parts in Caulobacter crescentus
RsaA C-terminal
Secretion Tag
• PrsaA
Constitutive
Promoter
Figure 2. Relative S. aureus biofilm growth after treatment
with engineered Caulobacter strains. Abbreviations: B for
BBa_K081005, R for PrsaA, X for Pxyl, O for optimized, W for
wild type, E for Esp, D for DspB, and CC for WT Caulobacter). • Esp secreted from Caulobacter crescentus
degrades Staphylococcus aureus biofilms.
•  Genes codon optimized for Caulobacter
crescentus are expressed better than WT genes.
•  Caulobacter secretion could be optimized (in
terms of promoters, RBS’s, size of the sec tag, scar,
new strain (knockouts, etc))
• BBa_K081005
Esp
•  Predicted serine protease secreted by S. epidermidis
•  Degrades fibrinogen, complement protein C5, and
other proteins8.
•  Degrades S. aureus biofilms by changing S. aureus
from the sessile to the planktonic form9.
Figure 3. The three different promoters had no significant
difference in their effects on biofilm inhibition.
•  Predicted N-acetylglucosaminidase secreted by
Aggregatibacter actinomycetemcomitans10.
•  Causes the detachment of cells from biofilm colonies
by cleaving the 1→ 4 glycosidic bond of β-substituted
N-acetylglucosamine10.
REFERENCES
1. 
Nomellini, J., M. Toporowski and J. Smit. 2004. Secre*on or Presenta*on of Recombinant Proteins and Pep*des Mediated by the S-­‐layer of Caulobacter crescentus. in Protein Expression Technologies: Current Status and Future Trends F. Baneyx Edit. 477-­‐524. Koronakis, V. andersen, C. and Hughes, C. 2001. Channel-­‐tunnels. Curr. Opin. Struct. Biol. 11: 403-­‐407. Koronakis, V., Sharff, A., Koronakis, E,. Luisi, B., and Hughes, C. 2000. Crystal structure of the bacterial membrane protein TolC central to mul*drug efflux and protein export. Nature. 405: 914-­‐919. 4.  Bingle, W.H., J.F. Nomellini, and J. Smit. 2000. Secre*on of the Caulobacter crescentus S-­‐layer protein: further localiza*on of the C-­‐terminal secre*on signal and its use for secre*on of recombinant proteins. J. of Bacteriol. 182(11):3298-­‐301. 5.  Lu, T., and J. Collins. 2007. Dispersing biofilms with engineered enzyma*c bacteriophage. PNAS 104(27):
11197-­‐11202. 6.  Foley & Gilbert 1996. An*bio*c resistance of biofilms. Biofouling. 10(16):331-­‐346. 7.  Simões and M.J. Vieira. 2009. Persister cells in Pseudomonas fluorescens biofilms treated with a biocide. In Proceedings of the interna*onal conference processes in biofilms: Fundamentals to applica*ons 58–62, Davis, CA. 8.  Moon et al. 2001. Isola*on and Characteriza*on of a Highly Specific Serine Endopep*dase from an Oral Strain of Staphylococcus epidermidis. Biological Chemistry. 382(7):1095-­‐1099. 9.  Iwase, Uehara, Shinji, Tajima, Seo, Takada, Agata & Mizunoe 2010. Staphylococcus epidermidis EspInhibits Staphylococcus aureus Biofilm Forma*on and Nasal Coloniza*on. Nature 465: 346-­‐351. 10.  Kaplan, J., C. Ragunath, N. Ramasubbu, and D. Fine. 2003. Detachment of Ac8nobacillus ac8nomycetemcomitans Biofilm Cells by an Endogenous beta-­‐Hexosaminidase Ac*vity. J. of Bacteriol. 185
(16):4693-­‐4698.
2. 
3. 
DspB
Figure 4. The Caulobacter strain with optimized esp codons was
more effective at inhibiting S. aureus biofilms than the strain
expressing WT esp (with the same BBa_K081005 promoter,
p=0.002). It is plausible that Caulobacter expresses higher G/C
content genes better than lower G/C content genes.
5 S. epidermidis usually grows slowly and forms relatively small
isolated colonies. A similar growth phenotype was seen in
Caulobacter strains that were producing Esp protein. Esp
protein may hinder the growth or viability of the bacteria that
secrete it.
BIOFILM DISRUPTION
• DspB
S. epidermidis S. aureus Figure 5. Streak plates with strains used in this study.
A.  Caulobacter secretion strains. (1) WT, (2) PrsaA esp
(3) PrsaA dspB, (4) Pxyl esp (no xylos), (5) Pxyl esp (2% xylose)
(6) Pxyl esp (20% xylose). B. S. epidermidis and S. aureus.
Surface
•  Biofilms can be protective barriers against adverse conditions
and can aid in the communication between cells6.
Outer Membrane Protein (OMP) RsaA C-­‐terminal recogni*on site 1 hydrated extracellular polymeric substance (EPS) matrix5.
Type I Secretion:
2 Dispersal
•  Biofilms are a community of microorganisms encased in a
 Aquatic gram-negative
bacterium
 Secretes S-layer protein
RsaA (10-12% total protein)
 Non-infectious with
100x less endotoxin than
E. coli1)
 Genetically tractable and
easy to culture
ATP-­‐ Binding Casse^e Transporter (ABC) ATP A.
Growth
6 EPS
Caulobacter crescentus
GROWTH PHENOTYPES
Pioneer
BACKGROUND
Membrane Fusion Protein (MFP) Attachment
PROTEIN SECRETION