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DEVELOPMENT OF A SIMPLIFIED AND RAPID TWO-DIMENSIONAL THINLAYER CHROMATOGRAPHY DIRECT BIOAUTOGRAPHY BASED ON
Staphylococcus aureus EXPRESSING A BLUE CHROMOPROTEIN
Dulyarit Chindaphun1,*, Nongluksna Sriubolmas2, Suthep Wiyakrutta1,#
1
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400,
Thailand
2
School of Pharmacy, Eastern Asia University, Pathum Thani 12110, Thailand
*e-mail: [email protected], #e-mail: [email protected]
Abstract
Staphylococcus aureus, a Gram-positive bacterial pathogen commonly causes skin
infections has been of much concern because the rise of antibiotic-resistant and more virulent
strains which have emerged and rapidly spread world-wide. There is an urgent need to search
for new and effective antibacterial compounds to cope with this global public health threat.
Here, we report a simplified and rapid two-dimensional thin-layer chromatography (2D-TLC)
direct bioautography for the detection of S. aureus inhibitory compounds in natural product
extracts from plants and micro-organisms. The method is based on the use of a recombinant
S. aureus expressing a blue chromoprotein aeCP597 derived from Actinia equina (a beadlet
sea anemone) as a bioautographic micro-organism. The S. aureus-codon optimized synthetic
aeCP597 gene under the control of SarA P1 constitutive promoter was integrated into the hsa
gene on the S. aureus chromosome by means of the TargeTron pNL9164 plasmid vector
resulted in blue-clored S. aureus cells. In it’s bioautographic application, soft agar medium
seeded with the blue S. aureus was overlaid onto the developed and air-dried 2D-TLC plate.
After incubation at 37ºC for 6 h, clear zone of S. aureus growth inhibition spot could be
visually detected and photo-documented without the use of any chromogenic spray reagent.
The present method is simpler, more rapid, costs less, and not health hazard to the operator,
compared with the conventional bioautography.
Keywords: 2D-TLC, bioautography, chromoprotein, Staphylococcus aureus
Introduction
Staphylococcus aureus is a Gram-positive pathogenic bacterium which is at present
causing public health problem worldwide particularly the methicillin-resistant S. aureus
(MRSA). MRSA can cause a wide array of diseases from infection of skin and soft tissue s to
life-threatening systemic infections. MRSA was formerly considered as a nosocomial
pathogen causing healthcare-associated MRSA (HA-MRSA) infections. However, during the
past 20 years, distinct community-associated MRSA (CA-MRSA) infections affecting
healthy persons have emerged (1-3). Moreover, antimicrobial resistance among these drug
resistant S. aureus is steadily increasing as well as the widening spectrum of invasive
infection. Thus there is an urgent need to search for new and effective anti-S. aureus drugs.
Screening for new antibiotic is usually a starting point of antibiotic drug development
program. Many methods are available for screening of natural products from plants and
microorganisms for new antibiotics. However, improvement of the current screening methods
to make them simpler, more rapid, and more economy can have significant impact on the
success of the project. 2D-TLC bioaugraphy is an effective method for separation and
detection of antimicrobial compounds in complex mixtures such as extracts from plants and
microorganisms as well as from combinatorial synthesis reactions (4-5). However, current
2D-TLC bioaugraphic processes are tedious, low throughput, and of high cost. Here, we
report the development of an improved 2D-TLC bioaugraphic method for screening of anti-S.
aureus compounds in natural product extracts.
Methodology
Bacterial strains, plasmids and culture conditions
TargeTron plasmid pNL9164 purchased from Sigma-Aldrich was used as an
expression vector for the synthetic GFP-like protein aeCP597 gene. Escherichia coli DH5α
was used as a cloning host while Staphylococcus aureus (ATCC 35556) was the host for
aeCP597 gene expression (Table 1). Cultivations of both E. coli DH5α and S. aureus
harboring pNL_aeCP597 were done by growing at 37⁰C in Luria-Bertani medium (LB) and
Brain Heart infusion medium (BHI) supplemented 50µg/ml ampicillin and 10µg/ml
erythromycin, respectively.
Generation of Staphylococcus aureus strain expressing the aeCP597 blue
chromoprotein
To obtain chromoprotein expressed in S. aureus, nucleotide sequence encoding GFPlike protein aeCP597 from Actinia equine (accession no. DQ159069) was codon-optimized
for expression in S. aureus and synthesized by GenScript (Piscataway, NJ, U.S.). The
synthetic aeCP597 gene construct contains the S. aureus SarA P1 promoter and a ribosome
binding site from the superoxide dismutase gene (sod RBS) at the upstream- and a SarA
termination signal at the downstream of the structural gene. The constructed DNA fragment
was cloned into the MluI restriction site of the pUC57 vector resulted in the pUC57_aeCP597
plasmid as shown in Figure 1A which was transformed into from E. coli DH5α. The
pUC57_aeCP597 plasmid was extracted from the host, digested with MluI restriction enzyme
and cloned into the corresponding sites of the TargeTron vector pNL9164 yielding
pNL_aeCP597 (Figure 1B) which was subsequently transformed into E. coli DH5α. The
pNL_aeCP597 plasmid was extracted and purified for transformation into competent S.
aureus ATCC 35556 cells. Transformation by electroporation was done at 2.1 kV, 25 µF, and
100. The transformants were selected on BHI containing 10µg/ml erythromycin. Colony
PCR was applied to screen for the plasmid containing clones (6) by using primers pNL1 (5’AACCAACAATGGCAATTTTAGAAAG-3’)
and
pNL2
(5’CTATAGTGAGTCGTATTAGTCGA-3’) and PCR reaction mix (GoTaq® Green Master
Mix) (Promega, USA) with the following thermal cycling conditions: 95 oC for 5 min; 30
cycles of 95 oC for 30 s, 53 oC for 30 s, 72 oC for 1 min 30 s ; and a final extension at 72 oC
for 5 min. Single colony of transformants containing the pNL_aeCP597 plasmid were
cultured overnight on BHI plate supplemented with 10µg/ml erythromycin and subsequently
inoculated into 1 ml of BHI broth without antibiotic. The cultivation was done by incubating
at 32°C with 300 rpm shaking for 1h. One milliliter of the culture was transferred into 5 ml of
fresh BHI broth containing 10 µg/ml erythromycin and incubated overnight. A 1:100 dilution
of the overnight culture was grown in BHI broth containing 10 µg/ml erythromycin at 32°C
with 300 rpm shaking until an OD595 nm reached 0.5. Then CdCl2 was added to a final
concentration of 10 µM and the culture was further incubated for 90 min to induce intron
mobilization.
Preparation of bioactive compounds
Recently, our group has isolated an endophytic fungus identified as Alternaria sp.
TCHE 202 from a Thai medicinal plant, Terminalia Chebuia Retz. The TCHE 202 fungus
which is known to produce anti-S. aureus metabolite(s) was cultured in Yeast extract sucrose
broth (YES) at 25°C for 21 days under static conditions. The fungal fermentation broth was
harvested by filtration, extracted with dichloromethane and concentrated by rotary
evaporation at 40 C under reduced pressure to yield a semi-solid crude extract free of the
organic solvent.
Two-Dimensional Thin-Layer Chromatography (2D-TLC)
One milligram of crude extracts was spotted on 10×10 cm TLC sheet (silica gel 60,
aluminium sheet, Merck Millipore) and developed in a standard vertical glass chamber using
hexane:ethyl acetate (2:8) and methanol:dichloromethane (1:9) for the first and second
dimension, respectively. The resulting spots on the silica gel were visualized under UV light
at 254 nm and 365 nm. Tetracycline and chloramphenicol were applied as antibacterial
standards for system testing. Twenty micrograms of each antibiotic in 2 µl of absolute
ethanol was spotted on TLC sheet and developed with methanol:dichloromethane (1:9), air
dried, and then with hexane:ethyl acetate (1:1) (5). Handling with techniques to minimize
microbial contamination, the developed TLC sheets were left in a biosafety hood for
complete removal of the organic solvents before subjected to bioautography.
2D-TLC Bioautography
S. aureus (the “blue” or the wild-type strain) was cultured overnight in 100 ml tryptic
soy broth (TSB) at 37°C with 280 rpm shaking. Fresh TSB was inoculated with the overnight
culture at 1:10 dilution and incubated 37°C with 280 rpm shaking for 3 hr. The cells were
collected by centrifugation at 4000×g for 10 min at 25 ºC, then re-suspended gently in sterile
normal saline solution (NSS). The optical densities of the bacterial suspensions were
measured by using a spectrophotometer (Unicam Heλios Alpha) at 580 nm wavelength. The
S. aureus cell suspensions in NSS were prepared at 40 and 25 % transmission (%T). The
bacterial suspensions were mixed well in different proportions with 10 ml of melted (45 ºC)
LB medium containing 0.6% agar and subsequently poured onto the developed TLC sheet to
a thickness of 1 mm. The poured medium was allowed to solidify at room temperature for 15
min and then incubated at 37 ºC for 6 hr.
Results
Expression of aeCP597 blue chromoprotein in Staphylococcus aureus (ATCC
35556)
The codon-optimized synthetic aeCP597 blue chromoprotein gene under the control
of the constitutive S. aureus SarA P1 promoter, the sod RBS, and the SarA termination signal
(see Methodology and Figure 1 for details) was cloned into the group II intron RNA coding
sequence of the pNL9164 TargeTron vector designed for targeted integration of the aeCP597
gene construct at the hsa gene on the S. aureus ATCC 35556 chromosome. Transformants
harboring the pNL_aeCP597 were treated with CdCl2 to induce intron mobility and
chromosomal integration of the aeCP597 gene construct. Constitutive expression of the
aeCP597 blue chromoprotein gene resulted in blue S. aureus cells (Figure 2).
Application of the blue Staphylococcus aureus in 2D-TLC bioautography
Application of the blue S. aureus in 2D-TLC bioautography was first tested with two
standard antibacterial drugs, tetracycline and chloramphenicol. 2D-TLC separation of the two
compounds was performed in the usual way. The mobile phases used for development in the
first and the second could effectively separate the two compounds. In the subsequence direct
bioautography step, it was found that seeding the indicator microorganisms (S. aureus) in soft
(0.6% agar) for direct overlaying onto the developed TLC plate could produce even growth
of the bacterium throughout the plate which facilitate the unambiguous detection of the
inhibition zones of the active compounds on the TLC plate (Figure 3 and 4). Additionally,
seeding the soft agar medium with high inoculum of S. aureus cells, the bioautographic result
could be determined within 6 h. That is the whole 2D-TLC bioautography could be
performed and the result could be obtained in the same day.
Comparing the blue S. aureus bioautography with the conventional chromogenic
reagent spraying method
The 2D-TLC bioautography based on the blue S. aureus was performed in comparison
with the method using chromogenic spraying reagent such as methyl thiazolyl tetrazolium
chloride (MTT) solution. The results are shown in Figure 4. The MTT method gave more
contrast of the inhibition zone (Figure 4D) which became appearance around 30 to 60 min
after spraying. However, the clear zones turned dark relatively rapidly.
Discussion and Conclusion
We have create an S. aureus strain chromosomally integrated with a codon-optimized
blue chromoprotein gene aeCP597 derived from a beadlet sea anemone Actinia equine. The
blue chromoprotein was constitutively expressed without the need for an inducer.
Chromosomal integration makes the aeCP597 gene expression highly stable without the need
for antibiotic selection. Expression of the aeCP597 chromoprotein imparts blue color to the S.
aureus cells. The blue S. aureus has been successfully applied in the bioautograhic detection
of anti-S. aureus compounds in a fungal crude extract separated on 2D-TLC plates. This
bioautography required only 5-6 h before the inhibition zones could be clearly visually
determined and documented without the requirement of special equipment making it the oneday 2D-TLC bioautographic assay. Compared with the conventional bioautography which
use chromogenic spraying reagent, the blue S. aureus based method is as effective in term of
clear zone detection. The omission of the chromogenic reagent spraying step makes the blue
S. aureus method more cost effective and, importantly, the risk of health hazard due to toxic
reagent spraying can be avoided.
Figure 1 Relevant features of the pNL_aeCP597 plasmid. SarA_P1_aeCP597: synthetic gene, pT181 cop634ts repC4 ori: temperature-sensitive origin of replication for Staphylococcus aureus, ErmC:
erythromycin resistance gene for selection in Staphylococcus aureus, Amp: ampicillin resistance gene for
selection in Escherichia coli, ColE1ori: origin of replication for E. coli, Pcad: induced plasmid integration
Figure 2 Color comparison of Staphylococcus aureus strains. A. The wild-type S. aureus ATCC 35556. B.
The ATCC 35556 expressing aeCP597 blue chromoprotein gene (the blue S. aureus).
Figure 3 2D-TLC bioautography of standard antibiotics, tetracycline (1), chloramphenicol (2). A. Standard
antibiotics after 2D-TLC separation and viewed under 254 nm UV light. B. The 2D-TLC plate from A
subjected to bioautographic assay with the “blue” S. aureus after incubation at 37ºC for 6 h. White zones of
growth inhibition due to the action of antibiotic spots are clearly visible against the blue background of cell
growth.
Figure 4 2D-TLC bioautography of crude extract from the endophytic fungus Alternaria sp. TCHE 202. A.
Crude extract after 2D-TLC separation and viewed under 365 nm UV light. B. The 2D-TLC plate from A
subjected to bioautographic assay with the “blue” S. aureus after incubation at 37ºC for 6 h. White zones of
growth inhibition due to the action of antibiotic spots are clearly visible against the blue background of cell
growth. C. A plate of 2D-TLC separation of the TCHE 202 crude extract similar to that in A but was
overlaid with the wild-type S. aureus ATCC 35556. No inhibition zone is visible. D. 2D-TLC plate from C
after spraying with 1mg/ml methyl thiazolyl tetrazolium chloride (MTT) solution. Inhibition zones are
clearly visible but darken rapidly. Spot 1, 2, and 3 are unidentified S. aureus inhibiting compounds.
Table1. Bacteria strains used in this study
Organism/plasmid
S. aureus
E. coli
Strain collection no.
ATCC 35556
DH5alpha
pNL9164
T6701
pUC57
SD1176
Common name
SA113
DH5alpha
TargeTron Vector
pNL9164
pUC57 plasmid DNA
Origin, other information
Derived from NCTC 8325
Genetic transformation
Re-targeted to S. aureus
hsa gene
Plasmid cloning vector in E. coli
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Acknowledgements: This research was financially supported by Mahidol University. DC
thanks Dr. Kanidtha Jariyachawalid and Juntratip Jomrit for their helpful advice.