World Journal of Microbiology & Biotechnology (2006) 22:299303

DOI 10.1007/s11274-005-9036-x

! Springer 2005

Confirmation of the mode of action of an antibacterial inhibitor using regulated

antisense RNA
Junshu Yang, Li Zheng and Yinduo Ji*
Department of Veterinary and Biomedical Sciences, University of Minnesota, 1971 Commonwealth Ave, St. Paul, MN,
55108, USA
*Author for correspondence: Tel.: +1-612-624-2757, Fax: +1-612-625-5203, E-mail: jixxx002@umn.edu
Received 29 March 2005; accepted 2 August 2005

Keywords: Antisense RNA , Pdf, Staphylococcus aureus

Summary
We previously demonstrated that regulated antisense RNA technology enables us to validate and identify the mode
of action for some antibiotics. In this study, we have expanded the application of the regulated antisense approach
to track the mode of action for a novel inhibitor of polypeptide deformylase (Pdf), which is an attractive target for
the development of novel classes of antibacterial agents. We created a pdf antisense isogenic strain in Staphylococcus
aureus using a TetR-regulated expression system. We demonstrated that the partial inhibition of pdf expression
significantly increased the susceptibility of S. aureus to Pdf-specific inhibitor. This result provides further evidence
that the TetR-regulated antisense technology is a robust tool for tracking the mode of action of novel antibacterial
agents.

Introduction
The recent emergence of multi-drug resistant bacterial
pathogens is generating enormous public health concern. Many of these pathogens are capable of causing
severe and even fatal infections with limited options for
therapy. The slowdown in the discovery of novel classes
of antibiotics in past several decades and the recent
downturn in antibacterial discovery and development in
the pharmaceutical industry is making us more
vulnerable to emerging resistance (Shlaes et al. 2004).
Therefore, there is an urgent need to develop novel,
preventive, and/or therapeutic agents for combating
multi-drug-resistant pathogens. One strategy is to
identify and explore novel molecular targets of the
pathogens (Ji 2002).
Different conditional lethal mutants provide powerful
tools for identifying and characterizing genes essential
for viability in different bacterial systems (Schmid et al.
1989; Geissendorfer & Hillen 1990; Guzman et al. 1995;
Stieger et al. 1999). Regulated promoter-controlled gene
expression strategies have been successfully used to
evaluate drug targets and the mode of action of antibacterial compounds (Zhang et al. 2000; Chan et al.
2003). However, the construction of a regulated promoter-controlled strain in Staphylococcus aureus is very
time-consuming. Recently, DNA microarray techniques

have increasingly been used to characterize the mode of

action of drugs in different bacterial pathogens
(Khodursky et al. 2000) and proteomic approaches have
been used to elucidate the cellular responses of bacteria
to antimicrobial compounds (Bandow et al. 2003).
Nevertheless, it is very difficult to determine a direct
target of a given inhibitor due to its secondary effects on
the bacterial cells.
Regulated gene expression and antisense RNA technology have been combined to rapidly identify and
characterize essential genes (as well as those causing
growth defects) from S. aureus. Therefore we possess a
set of defined strains exhibiting conditional growth
phenotypes (Ji et al. 2001; Forsyth et al. 2002). This set
of conditional mutants has been successfully used to
identify potential targets for certain antibacterial agents
(Ji et al. 2004; Yin et al. 2004).
In this study, we chose the novel target Pdf, an
essential enzyme that removes the N-formyl group from
newly synthesized polypeptides (Chang et al. 1989), and
constructed a TetR-regulated pdf antisense isogenic
strain in S. aureus. We provide a detailed evaluation of
this antisense isogenic strains usefulness in determining
the mode of action of compounds directed to a specific
target. We used Western blot analysis to confirm the
specificity of inhibition for the molecular target by the
induced antisense RNA. Furthermore, we present data

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J. Yang et al.

to demonstrate the utility of the regulated antisense

approach to track the MOA of a novel Pdf inhibitor.

performed as described (Ji et al. 2004). Western blots

were scanned by using Eagle Eye-II software (Stratagene) to quantitate protein bands.

Materials and methods

Northern blot analysis

Bacterial strains and media

Staphylococcus aureus strains were grown in TSB with

appropriate antibiotics to OD600nm of !0.3 with and
without Tc (500 ng/ml), and total RNA was extracted
by using a Qiagen RNAEasy mini protocol kit. Ten lg
of total RNA sample was separated by electrophoresis
on a 1.2% agarose gel containing 1.8% formaldehyde
gel and blotted onto a nylon membrane. The RNA was
cross-linked to the membrane by UV irradiation. Blots
were assayed as previously described (Ji et al. 2004)
using a DIG-labeled pdf238 fragment specifically
hybridized with antisense RNA.

We used Escherichia coli DH10B as a host for recombinant plasmids. E. coli DH10B carrying plasmids
pYH4 or pYJ20010 were incubated at 37 "C in LB
medium containing erythromycin (Erm; 300 lg/ml) for
selection. S. aureus RN4220::tetM was used as a host
and grown at 37 "C in tryptic soy broth (TSB)
containing chloramphenicol (10 lg/ml) and tetracycline
(Tc, 0.5 lg/ml). The S. aureus control strain RN4220/
tetM/pYH4 and the antisense isogenic strain JSAS20010
were incubated at 37 "C in TBS containing
erythromycin (5 lg/ml) for selection.
Construction of S. aureus pdf antisense strains
The oligonucleotide primers pdffor (5-TTGGCGC
GCCTGTATGAGTATCTTAACAACCTTTTTAC-3)
and pdfrev (5-TAAATAGGCTTCTTGAACGC-3)
specific for pdf (SA0942 in S. aureus strain N315) were
used to amplify the pdf fragment from S. aureus RN4220
genomic DNA. The bold face sequence in the oligonucleotide primers is the AscI recognition sequence. The
resulting DNA fragments were cloned into pYH4 DNA
in an antisense orientation and resulting in construction
pYJ20010 (pdffor/pdfrev, data not shown). The recombinant plasmid DNA was electroporated into
RN4220/tetM and denoted JSAS20010.

Staphylococcus aureus susceptibility assay

Staphylococcus aureus strains were grown in TSB with
appropriate antibiotics at 37 "C overnight and were
diluted to !105 c.f.u./ml as cultures for MIC assays with
a 96-well microtiter format. Serial dilutions of 40 standard compounds were prepared in TSB-Erm in a final
assay volume of 100 ll. Fifty ll of 105 c.f.u./ml bacteria
was added to the serially diluted antibiotics. The MIC
was considered to be the concentration at which the
antibiotic prevented turbidity in the well after incubation for18 h at 37 "C. The MIC assay was repeated three
times, and the table in the results section represented one
experiment.

Results and discussion

Growth curves of S. aureus pdf antisense strain
Construction of specific antisense RNA isogenic strains
Staphylococcus aureus cell growth curves were obtained
using an automated microtiter plate format. S. aureus
strains were incubated at 37 "C overnight in TSB with
appropriate antibiotics. The cultures were diluted to
!104 c.f.u./ml with TSB containing appropriate antibiotics and tetracycline at concentrations of 0, 10, 50, 100,
250, 500 ng/ml. The cell growth was monitored at 37 "C
by measuring OD600nm every 15 min with 1 min mixing
before each reading.
Western blot assay
Staphylococcus aureus strains were incubated in TSB
with appropriate antibiotics, in the absence or presence
500 ng/ml of inducer Tc at 37 "C overnight. Cell pellets
from 1 ml of culture at 1.0 of OD600nm were collected by
centrifugation and washed once with 1 ml PBS and
resuspended in 1 ml of PBS. Lysostaphin (100 lg/ml)
was used to lyse the cells. Protein concentrations were
determined by using Bio-Rad protein assay reagent.
Equal amounts of protein samples were loaded onto a
SDS-PAGE gel and Western immunoblotting was

Regulated promoter approaches have been successfully

used to confirm antibiotic mode of action in whole
bacterial cells (Zhang et al. 2000; Chan et al. 2003).
However, to construct such regulated strains, especially in S. aureus is time-consuming. Therefore, we
tested the use of the alternative regulated antisense
RNA approach to down-regulate target gene expression and generate bacterial cells sensitized to antibiotics with the same mode of action. As proof of
concept, we have demonstrated that the downregulation of FabI expression with induced fabI antisense
RNA can cause lethal effects and selectively increase a
bacterial cells susceptibility to FabI inhibitors (Ji
et al. 2004). However, we did not know whether an
antisense RNA that cannot induce an obvious change
of phenotype is able to increase a bacterial cells
sensitivity to its specific inhibitor. To address this
question, we chose to construct an antisense strain
regulating the potential target, Pdf, in S. aureus as
described in Materials and methods. We evaluated the
requirement of Pdf for cell viability by observing the

Use of regulated antisense RNA

consequences of titrating down its expression level.
Conditional phenotypes of pdf antisense isogenic
strains were compared on TSA-Erm plates in the
presence of the same concentration of antisense inducer (Tc) after overnight incubation. As a control,
we monitored the growth of S. aureus carrying empty
vector pYH4 with and without Tc induction phenotype and found it to be unaffected. Very surprisingly,
no obvious difference of phenotypes and colony
forming unit was observed, even after full induction
(500 ng/ml of Tc) of pdf antisense RNA (data not
shown).
Growth inhibition of strain JSAS20010 was also
examined in a liquid medium containing various
amounts of inducer, Tc. The results indicated that the
extent of bacterial cell growth was not limited by the
highest level of antisense RNA in this strain. The control, RN4220/tetM/pYH4, did not show a significant
difference in growth (Figure 1a). Cultures of JSAS20010
did show, however, a small but noticeable decrease in
the cultures growth rates and a growth delay of
approximately 2 h upon induction of pdf antisense RNA
(Figure 1b).

301
Characterization of S. aureus pdf antisense strain
In order to determine whether induced pdf antisense
RNA is sufficient to downregulate endogenous pdf
expression, we characterized the production of transcripts of the pdf fragment in response to Tc induction
and assessed the effects of this induction on Pdf gene
expression.
First, we analysed RNA production from the pdf
antisense strain before and after induction. We found
that in JSAS20010 no detectable pdf antisense RNA was
generated without Tc induction (Figure 2). However,
the pdf antisense strain produced many more pdf antisense RNA products in response to Tc induction
(Figure 2).
Second, we examined the effect of induced pdf antisense RNA on the endogenous pdf expression in order
to determine whether the induced pdf antisense RNA
could cause a significant decrease in the number of Pdf
protein molecules. The whole cell lysate of JSAS20010
without the induction of pdf antisense RNA reacted
strongly with the anti-Pdf antisera (Figure 3); in contrast, following Tc induction, JSAS20010 demonstrated
approximately a10-fold lower reactivity as measured by
densitometer scanning. In addition, the amount of Pdf
in the whole cell lysate of the control strain, RN4220/
pYH4, in the presence of the inducer was similar to that
in the absence of the inducer. These studies demonstrated that induced pdf antisense RNA can significantly
inhibit pdf expression even though the pdf antisense
RNA is not sufficient to cause obvious change of cell
phenotype and growth. One possible explanation is that
levels of the Pdf enzyme significantly lower than those
normally occurring are sufficient for maintaining bacterial survival. The exact reason why a decrease in Pdf
production only delays bacterial growth is unclear; it
might be due to the cumulative effect of Pdf in the
surviving bacterial cells.

Figure 2. Northern blot analysis of pdf antisense RNA expression.

Lane 1, 2: RN4220/pYH4; Lane 3, 4: JSAS20010. The 23S and 16S
RNA were stained as control (data not shown).

Figure 1. Growth curves of control strain RN4220/tetM carrying

pYH4 (a) and JSAS20010 (b) in TSB containing 5 lg/ml of Erm and
varying concentrations of Tc (0, 10, 50, 100, 250, 500 ng/ml). This
experiment was repeated three times, and the growth curves
represented one experiment.

Figure 3. Western blot analysis of Pdf expression during induction of

antisense RNA. Proteins from S. aureus strains whole cell lysis probed
with rabbit anti-Pdf antibodies. Lane 1, 2: RN4220/pYH4; Lane 3, 4:
JSAS20010; Lane 5: standard Pdf.

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J. Yang et al.

Determination of antibiotic mode of action

Our previous studies (Ji et al. 2004; Yin et al. 2004)
demonstrated that titrating down the expression of a
drug target gene by using TetR-regulated antisense
RNA selectively increases bacterial sensitivity to inhibitors of the target protein. Therefore, the regulated
antisense approach could be used to determine whether
the cellular activity of an antibiotic derived from a discovery screen functions through its proposed molecular
target.
Because induced pdf antisense RNA had no significant effect on bacterial growth, the next question was

whether the downregulation of pdf expression by induced pdf antisense RNA is sufficient to increase bacterial susceptibility to Pdf inhibitors. To address this
question, we tested the MIC changes for a synthetic
compound SB220334 using pdf antisense strain
JSAS20010. SB220334 is an actinonin-like molecule
known to inhibit Pdf (Chen et al. 2000) and exhibit
antibacterial activity against S. aureus. As shown in
Table 1, we found that the MIC value of SB220334
significantly decreased 16-fold for JSAS20010 after the
induction of pdf antisense RNA. In contrast, the MIC
values for 35 independent control antimicrobial agents
known not to target Pdf were not changed significantly

Table 1. Antimicrobial activity (MIC values, lg/ml) of antimicrobial compounds against S. aureus with pdf antisense RNA expression
Compound

DNA synthesis inhibitor

Chloroquine diphosphate
Quinacrine 2HCl
Ciprofloxacin
Novobiocin
Trimethoprim
Transcription inhibitor
Actinomycin D
Rifampicin
Translation inhibitor
Mupirocin
Streptomycin
Chloramphenicol
Gentamicin
Kanamycin
Erythromycin
Linezolid
Tiamulin
Virginiamycin
Tetracycline
Cell wall biosynthesis inhibitor
Cycloserine
Phosphomycin
Imipenem
Aztreonam
Ceftazidime
Piperacillin
Vancomycin
Bacitracin
Membrane permeability inhibitor
Polymyxin B
Lipid biosynthesis inhibitor
Cerulenin
Triclosan
Electron transport inhibitor
Oligomycin
Non-specific MOA inhibitor
CCCP
Crystal violet
Dequalinium chloride
Ethidium bromide
Paraquat
SDS
Lead compound
SB-220334
Note: Tc (ng/ml).

RN4220/tetM/pYH4 (control)

JSB20010 (pdf antisense)

Tc=0

Tc=500

Tc=1000

Tc=0

Tc=500

Tc=1000

>64
>64
0.5
0.125
1

>64
>64
0.5
0.125
1

>64
>64
0.5
0.125
1

>64
>64
0.25
0.125
1

>64
>64
0.25
0.125
1

>64
>64
0.25
0.125
1

0.125
0.06

0.125
0.06

0.125
0.06

0.125
0.06

0.125
0.06

0.125
0.06

0.125
2
16
0.125
1
>64
1
0.125
2
32

0.125
2
16
0.125
1
>64
1
0.125
1
32

0.125
2
16
0.125
1
>64
1
0.125
1
32

0.125
2
16
0.125
1
>64
0.5
0.125
1
32

0.125
2
8
0.125
1
>64
0.5
0.125
1
32

0.06
2
8
0.125
1
>64
0.25
0.06
0.5
32

64
4
0.015
>64
4
0.25
0.5
32

64
2
0.015
>64
4
0.5
0.5
32

64
1
0.015
>64
4
0.5
0.5
32

64
0.5
0.007
>64
4
0.25
0.5
32

64
0.5
0.007
>64
4
0.25
0.5
32

32
0.25
0.007
>64
4
0.25
0.5
32

32

32

32

32

32

32

64
0.125

64
0.125

64
0.125

64
0.125

64
0.125

64
0.125

>64

>64

>64

>64

>64

>64

1
0.06
0.25
2
1
>64

1
0.06
0.25
2
4
>64

1
0.06
0.25
2
8
>64

1
0.06
0.25
2
1
>64

1
0.06
0.25
2
4
>64

1
0.06
0.25
2
8
>64

16

16

16

16

Use of regulated antisense RNA

by the induction of pdf antisense RNA (Table 1).
Moreover, the magnitude of increased sensitivity to the
Pdf inhibitor is corresponding with the magnitude of the
decrease in number of the Pdf molecules. Therefore, we
determined that downregulated pdf expression causes a
significant increase in sensitivity to Pdf inhibitors, despite the fact that the pdf antisense RNA is not sufficient
to change the cell phenotype.
In summary, to develop novel antibacterial agents,
pharmaceutical companies have to screen millions of
compounds a year to identify hundreds of compounds
which need different secondary screens. We have demonstrated that regulated antisense RNA can be more
easily and quickly constructed as compared to a promoter-regulated mutant in S. aureus. In this study, we
have demonstrated that the TetR-regulated antisense
RNA system provides a robust tool for secondary
screens to defining and tracking the mode of action of
antibiotics in whole cells.

Acknowledgements
We thank Dr. Martin Burnham for his critical review
and suggestions, Dr. F. Fan for strain RN4220/tetM,
Dr. J. Huang for plasmid DNA pYH4, and Dr. L.
Volker for providing compounds.

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