Rational Manipulation of mRNA Folding Free Energy Allows Rheostat Control of Pneumolysin Production by Streptococcus pneumoniae

Abstract

The contribution of specific factors to bacterial virulence is generally investigated through creation of genetic "knockouts" that are then compared to wild-type strains or complemented mutants. This paradigm is useful to understand the effect of presence vs. absence of a specific gene product but cannot account for concentration-dependent effects, such as may occur with some bacterial toxins. In order to assess threshold and dose-response effects of virulence factors, robust systems for tunable expression are required. Recent evidence suggests that the folding free energy (ΔG) of the 5' end of mRNA transcripts can have a significant effect on translation efficiency and overall protein abundance. Here we demonstrate that rational alteration of 5' mRNA folding free energy by introduction of synonymous mutations allows for predictable changes in pneumolysin (PLY) expression by Streptococcus pneumoniae without the need for chemical inducers or heterologous promoters. We created a panel of isogenic S. pneumoniae strains, differing only in synonymous (silent) mutations at the 5' end of the PLY mRNA that are predicted to alter ΔG. Such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. These studies provide proof-of-principle for further investigation of mRNA ΔG manipulation as a tool in studies of bacterial pathogenesis.

Full text

RESEARCH ARTICLE
Rational Manipulation of mRNA Folding Free
Energy Allows Rheostat Control of
Pneumolysin Production by Streptococcus
pneumoniae
Fábio E. Amaral
1,4,5
, Dane Parker
1
, Tara M. Randis
1
, Ritwij Kulkarni
1
, Alice S. Prince
1,2
,
Mimi M. Shirasu-Hiza
3
, Adam J. Ratner
1
*
1Department of Pediatrics, Columbia University, New York, NY United States of America, 2Department of
Pharmacology, Columbia University, New York, NY, United States of America, 3Department of Genetics &
Development, Columbia University, New York, NY, United States of America, 4Life and Health Sciences
Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal, 5ICVS/3B's, PT
Government Associate Laboratory, Braga/Guimarães, Portugal
*ar127@columbia.edu
Abstract
The contribution of specific factors to bacterial virulence is generally investigated through
creation of genetic “knockouts”that are then compared to wild-type strains or comple-
mented mutants. This paradigm is useful to understand the effect of presence vs. absence
of a specific gene product but cannot account for concentration-dependent effects, such as
may occur with some bacterial toxins. In order to assess threshold and dose-response ef-
fects of virulence factors, robust systems for tunable expression are required. Recent evi-
dence suggests that the folding free energy (ΔG) of the 5’end of mRNA transcripts can
have a significant effect on translation efficiency and overall protein abundance. Here we
demonstrate that rational alteration of 5’mRNA folding free energy by introduction of synon-
ymous mutations allows for predictable changes in pneumolysin (PLY) expression by
Streptococcus pneumoniae without the need for chemical inducers or heterologous pro-
moters. We created a panel of isogenic S.pneumoniae strains, differing only in synonymous
(silent) mutations at the 5’end of the PLY mRNA that are predicted to alter ΔG. Such manip-
ulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole
S.pneumoniae on primary and immortalized human cells. These studies provide proof-of-
principle for further investigation of mRNA ΔG manipulation as a tool in studies of
bacterial pathogenesis.
Introduction
Delineating specific bacterial factors involved in interaction with the host is crucial to under-
standing mechanisms of pathogenesis and developing targeted therapies. Classically, such
PLOS ONE | DOI:10.1371/journal.pone.0119823 March 23, 2015 1/11
OPEN ACCESS
Citation: Amaral FE, Parker D, Randis TM, Kulkarni
R, Prince AS, Shirasu-Hiza MM, et al. (2015) Rational
Manipulation of mRNA Folding Free Energy Allows
Rheostat Control of Pneumolysin Production by
Streptococcus pneumoniae. PLoS ONE 10(3):
e0119823. doi:10.1371/journal.pone.0119823
Academic Editor: Indranil Biswas, University of
Kansas Medical Center, UNITED STATES
Received: November 7, 2014
Accepted: January 16, 2015
Published: March 23, 2015
Copyright: © 2015 Amaral et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was supported by the National
Institutes of Health (www.nih.gov) (R01 AI092743 and
R21 AI111020 to A.J.R.). F.E.A. was supported by the
Portuguese Foundation for Science and Technology
(www.fct.pt) SFRH/BD/33901/2009 and the Luso-
American Development Foundation (www.flad.pt).
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.

investigations involve construction of bacteria with disruptions in the genes encoding candi-
date factors (“knockouts”; KO) and comparison with wild-type (WT) or complemented strains.
However, the level of expression of bacterial genes may also play a role in pathogenesis. The
KO vs. WT paradigm cannot account for such differences, and although systems exist for in-
ducible expression of specific bacterial genes, these generally rely on exogenous activators such
as tetracycline that may have variable delivery to relevant sites during infection [1]. Thus, it is
desirable to develop strategies for genetically encoded, tunable, rheostat-like control of bacterial
gene expression.
A number of factors determine the efficiency of protein production. As the genetic code is
degenerate, choice among synonymous codons may play a role in translational efficiency [2].
Early models postulated a direct relationship between tRNA availability and protein produc-
tion, implying that use of non-optimal codons (i.e. those that are rarely used among other
genes in the species being studied) would lead to decreased protein production on the basis of
tRNA scarcity [2–4]. Codon-optimization has been used as a means to increase production of
heterologous genes in E.coli and other host species [5]. Conversely, deoptimization of codons
or codon pairs has been employed to rationally decrease protein production from natively tran-
scribed genes [6,7]. Kudla et al. examined a library of synonymous codon substitutions in het-
erologously expressed green fluorescent protein (GFP) in E.coli and demonstrated that the
mRNA folding free energy (ΔG), particularly at the 5’terminus, correlates strongly with trans-
lational efficiency and with overall protein production [8]. Goodman et al. investigated the role
of both codon bias and mRNA ΔG in synthetic reporters and confirmed a prominent role for
ΔG in shaping expression levels of individual genes [9].
We used Streptococcus pneumoniae (pneumococcus), a major human pathogen with robust
tools for genetic manipulation [10], as a model organism. We constructed a panel of isogenic
pneumococcal strains differing only in synonymous codons at the 5’end of the gene encoding
pneumolysin (PLY), an established virulence factor [11,12], without antibiotic selection cas-
settes or exogenous promoters. These modifications altered the predicted mRNA ΔG and re-
sulted in graded PLY production, thus affecting host-bacterial interactions and providing
proof-of-principle for the use of rational modification of mRNA ΔG as a means to control
protein production.
Materials and Methods
Ethics statement
The use of primary human erythrocytes following written informed consent was approved by
the Institutional Review Board of Columbia University Medical Center. The human cell line
A549 was obtained from ATCC (catalog number CCL-185).
Calculation of ΔG and codon adaptation index
Sequence folding free energies were calculated using the DINAMelt/Quikfold web server
(http://mfold.rna.albany.edu/?q=DINAMelt/Quickfold)[13]. The software uses predicted free
energies at 37°C and enthalpies from the Turner laboratory at the University of Rochester in
Rochester, NY. For ΔG calculations we used version 3.0 free energies. Codon adaptation index
(CAI) based on the set of highly expressed genes from Streptococcus pneumoniae strain D39
was calculated using the CAIcal server (http://genomes.urv.es/CAIcal/)[14].
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Competing Interests: A.J.R. is a Section Editor at
PLOS ONE. A.S.P. is an Editorial Board Member at
PLOS Pathogens. This does not alter the authors'
adherence to PLOS ONE policies on sharing data
and materials.

Bacterial strains, primers, plasmids, transformation and growth
conditions
We used the Janus cassette [10], to create a panel of isogenic S.pneumoniae R6 derivatives with
a wide range of ΔG values (-17.5 to -3.4 kcal/mol) for the 5’end of ply gene (-4 to +38 bp, with
1 being the A in the first ATG). To control for unexpected effects of transformations we recre-
ated, and sequenced, a strain with the WT ply allele for comparison. Each of the new ply con-
structs was inserted into the native chromosomal locus, without resistance markers or
alteration in predicted primary amino acid sequence. All transformed loci were confirmed by
sequencing. All constructs were first assembled in E.coli TOP10 using plasmid pCR2.1-TOPO
(Invitrogen), amplified by PCR, and transformed into pneumococcus. Forward primers dif-
fered in nucleotides encoding the initial PLY region according to Fig. 1, consistent with the
Fig 1. Production of PLY increases with increasing ply 5’mRNA folding free energy. (A) Summary of the silent mutations engineered in the 5’-mRNA
region of the ply gene. IUPAC Nucleic Acid Codes: Y (pYrimidine); R (puRine); N (aNy); H (not G). (B) Increasing levels of predicted ply 5’mRNA ΔG
correlate with increased PLY production as determined by ELISA normalized to total pneumococcal protein. Data are shown as mean of 4
experiments ±SEM. *,P<0.05; **,P<0.01; ***,P<0.001 (ANOVA). (C) Western blot (top, anti-PLY; bottom, anti-PsaA loading control) demonstrates
different levels of PLY production in engineered strains (lanes 1–5) compared to recombinant PLY (lane 6) as a control. Numbers beneath each lane indicate
the ratio of PLY to PsaA band intensity, normalized to the value for the wild-type (ΔG = -7.3) strain in lane 4. (D) Measurement of turbidity (OD
600
) and
calculated doubling times shows identical growth kinetics among isogenic S.pneumoniae strains with altered ply 5’sequences. (N.S. = not significant;
ANOVA)
doi:10.1371/journal.pone.0119823.g001
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following degenerate sequence: 5’-GAAR ATG GCN AAY AAR GCN GTN AAY GAY TTY
ATH YTN GCN ATG AAT TAC G-3’(start codon in bold), and the reverse primer was 5’-
CTA GTC ATT TTC TAC CTT ATC TTC T-3’. Constructs for pneumococcal transformation
were assembled by overlap-extension PCR and included 570 bp upstream of ply (forward prim-
er 5’-GGT TAT TGG CGA CAA GCA TT-3’) and 769 bp downstream (reverse primer 5’-CCT
GCT AAG ATG GTC TTG CC-3’).
Briefly, pneumococcal transformation was achieved with use of semisynthetic casein plus
yeast extract (C+Y) medium [15]. Cultures were grown at 37°C in C+Y pH 6.8 to 0.15 OD
600
,
then diluted 1:20 fold into C+Y pH 8.0 (total volume 1 mL) at 30°C, supplemented with 1 μgof
CSP-1 and 11 μLof1%CaCl
2
and incubated for 12 minutes. After adding 1 μg DNA, cultures
were incubated for 40 minutes at 30°C, followed by 90 minutes at 37°C, followed by appropri-
ate selection in agar plates incubated overnight at 37°C, 5% CO
2
.
E.coli were grown in lysogeny broth (LB) or agar supplemented with appropriate antibiotics
for plasmid selection (ampicillin 200 μg/mL or kanamycin 50 μg/mL). S.pneumoniae were
grown in tryptic soy broth (TSB) or agar supplemented with 200 U/mL of catalase (Worthing-
ton Biochemical), and appropriate antibiotics for strain selection (streptomycin 150 μg/mL or
kanamycin 200 μg/mL).
Western blotting and ELISA
Protein separation, transfer, and immunoblotting were performed as previously described [16].
In blots, PLY was detected using 1:1000 dilution of mouse monoclonal anti-pneumolysin
(1F11), PsaA was detected using 1:25000 dilution mouse monoclonal PsaA antibody (8G12)
[17]. Densitometry was performed using the gel analysis plugin implemented in ImageJ (ver-
sion 1.45s; National Institutes of Health). PLY was detected by indirect ELISA using monoclo-
nal anti-pneumolysin (1F11; 1:1000 dilution).
qRT-PCR and mRNA half-life
S.pneumoniae were grown to early exponential stage (OD
600
= 0.3) and stored in RNAlater
(Ambion). RNA was isolated using the RiboPure-Bacteria Kit (Ambion) with DNase treat-
ment. cDNA was made using the SuperScript III RT Kit (Invitrogen), qRT-PCR was performed
using Quanta SYBR Green PCR Master Mix in a StepOne Plus thermal cycler (Applied
Biosystems).
The primers for the ply gene of S.pneumoniae (GenBank accession no. M17717) for real-
time reverse-transcription PCR were previously described [18]. Samples were normalized to
S.pneumoniae 16S rRNA detected with forward (5’-GCC TAC ATG AAG TCG GAA TCG-3’)
and reverse (5’-TAC AAG GCC CGG GAA CGT-3’) primers. In separate experiments, tran-
scription was arrested by addition of rifampicin to 10 μg/mL and mRNA abundance and decay
were assessed by qRT-PCR of serially collected samples normalized to the 30 sec time point.
mRNA half-life was calculated from best-fit exponential decay curves.
Hemolysis assay
Primary human erythrocytes (0.5% final concentration) were incubated for 30 min at 37°C
with an equal volume of heat-killed (52°C, 20 min) log-phase (OD
600
= 0.25) S.pneumoniae R6
strains suspended in PBS. Intact erythrocytes were pelleted by centrifugation, and hemolysis
was measured by assaying hemoglobin concentration in the supernatant (OD415 of a 100μl
sample). Hemolysis values were normalized to 0% (vehicle control) and 100% lysis (1% Triton
X-100) controls as previously described [16,19].
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Cell culture and cytotoxicity assay
A549 respiratory epithelial cells (obtained from ATCC, catalog number CCL-185) were grown
in minimum essential medium (MEM) with 10% fetal bovine serum as described [20]. Cell
monolayers (80–90% confluent in sterile 24-well plates) were incubated in MEM without
serum overnight prior to stimulation with ~10
8
cfu of the indicated S.pneumoniae strains for
12 hrs. LDH cytotoxicity assay (Roche) was performed as per the manufacturer’s instructions,
and values were normalized to 0% (vehicle control) and 100% lysis (1% Triton X-100) controls
as previously described [21].
Statistical analysis
Statistics were performed with Prism 5 software (GraphPad, Inc.). Data were analyzed by anal-
ysis of variance followed by post-tests to account for multiple comparisons, as appropriate. In
vitro experiments were performed at least three times with at least three technical replicates.
Bars represent SEM. Values of P0.05 were considered significant.
Results
Production of PLY increases with increasing ΔG
Using a degenerate nucleotide sequence derived from the S.pneumoniae R6 PLY protein se-
quence, we designed several candidate 5’mRNA regions for the ply gene (1416 nucleotides
total) that differed in predicted minimum mRNA folding ΔG (calculated using the standard
mfold algorithm over the region from-4 to +38 nucleotides [13]). Each of these genes only con-
tained synonymous mutations, leading to the production of identical amino acid sequences,
but covering a wide range of predicted ΔG values. Because only a small number of codons was
altered in each strain (4–7 codons/strain), the CAI for the full length ply gene based on highly
expressed genes of S.pneumoniae D39 was unchanged (~0.38 in all strains). The two strains
with the lowest 5’mRNA ΔG (-17.5 and -12.8) also had lower CAI over the first 39 bp (0.227
and 0.236, respectively) than the other three strains (0.404, 0.365, and 0.375 for the ΔG = -9.5,
-7.3, and -3.4 strains, respectively). The predicted ΔG of the native ply 5’mRNA is-7.3 kcal/
mol, and this strain was included as a control in all experiments (Fig. 1A). These alterations re-
sulted in differential expression of PLY protein. With increasing ΔG values, we observed higher
PLY levels as measured by both ELISA (Fig. 1B) and western blot (Fig. 1C). As an important
control for changes in bacterial growth that would obviously impact total protein levels, we
confirmed that all strains exhibited similar growth rates and doubling times (Fig. 1D). Thus al-
terations of the ply DNA sequence that alter the predicted ΔG of its resulting mRNA led to pre-
dictable and quantitative alterations in translated protein levels.
Translation kinetics drive the relationship between ΔG and protein
abundance
In order to understand the mechanisms underlying the relationship between mRNA ΔG and
protein production in S.pneumoniae, we tested whether differences in ply mRNA levels corre-
lated with predicted ΔG values for the 5’mRNA and could account for differences in PLY pro-
tein levels. We used real-time reverse transcriptase PCR to quantify ply mRNA normalized to
16S rRNA for each strain. There were no statistically significant differences between wild-type
and engineered strains in mRNA abundance (Fig. 2). Thus the absolute levels of mRNA abun-
dance do not correlate with predicted ΔG values or with differences in PLY protein levels.
Decay kinetics of mRNA can also alter overall protein production. To test whether altered
decay kinetics correlated with differences in PLY protein levels, we next assessed mRNA decay
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at several time points following arrest of transcription with rifampicin (Fig. 3A). Decay curves
were similar and did not differ statistically. We also calculated mRNA half-lives based on
mRNA abundance following transcription arrest. mRNA half-lives for each ply sequence were
all approximately 1.5 min, and there were no statistically significant differences among groups
(Fig. 3B). Taken together, these findings suggest that mRNA synthesis and decay are not al-
tered and do not account for differences in PLY protein levels. Thus our results are consistent
with the hypothesis that changes in mRNA ΔG lead to changes in post-transcriptional process-
ing and changes in protein abundance.
ply mRNA ΔG affects host-pathogen interactions in vitro
The previous experiments were performed using bacteria in culture. To determine whether al-
tered 5’mRNA ΔG also resulted in altered PLY production and cytotoxicity during infection of
human cells, we examined PLY protein levels in two model systems: primary human erythro-
cytes and immortalized epithelial cells. PLY protein causes lysis of infected cells. We found that
primary human erythrocytes exposed to S.pneumoniae strains exhibited lysis rates that corre-
lated with predicted ply mRNA ΔG(Fig. 4A), suggesting that PLY protein levels expressed dur-
ing infection correlate with predicted 5’mRNA ΔG. We confirmed these differences in PLY
protein levels by direct measurement using ELISA (Fig. 4B). In parallel, we found that measure-
ment of A549 epithelial cell cytotoxicity demonstrated a similar positive correlation between
Fig 2. Engineered differences in 5’mRNA ΔG do not alter ply mRNA quantity. Fold difference in ply
mRNA levels (y axis) was calculated relative to wild-type (ΔG = -7.3) at early exponential phase. Relative
mRNA amounts were calculated by ΔΔC
T
normalized to pneumococcal 16S rRNA. (N.S. = not significant;
ANOVA)
doi:10.1371/journal.pone.0119823.g002
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ply mRNA ΔG and cellular damage (Fig. 5). Thus predicted differences in ply mRNA ΔG posi-
tively correlate with differences in lysis rates and differences in PLY protein expression levels
during active infection of human tissue culture cells.
Discussion
There is an emerging literature on the importance of 5’mRNA folding ΔG to efficiency of
translation and to overall levels of protein production [8,9,22–24]. Manipulation of codon bias,
which may also impact protein levels, has been widely used as a method to enhance expression
of genes in heterologous systems [5,25,26]. Rational alteration of 5’mRNA ΔG has been sug-
gested as a technique that might further aid such production [8,9]. Most studies of the
ΔG/protein abundance relationship have focused on either synthetic constructs driving marker
genes such as GFP [8,9] or on examination of naturally occurring coding sequences in model
organisms [22,23,27,28]. In these studies, we rationally manipulated the predicted 5’mRNA
ΔG of a known pneumococcal virulence factor in order to determine concentration-dependent
effects. Such investigations are not possible using traditional KO vs. WT approaches, and deter-
mining the relationship between protein abundance and virulence may drive more detailed un-
derstanding of pathogenic mechanisms.
Using unmarked, isogenic pneumococcal strains, we showed that rheostat-like control of
PLY production is feasible and that changes in ΔG are sufficient to alter host-pathogen interac-
tions. Alteration in expression did not depend on mRNA abundance or stability but occurred
Fig 3. Differences in PLY production do not correlate with mRNA decay kinetics. (A) Abundance of ply
mRNA following transcriptional arrest with rifampicin. mRNA quantities were measured by qRT-PCR and
normalized to 16S rRNA. (N.S. = not significant; ANOVA). (B) Differences in ply 5’mRNA ΔG do not alter
mRNA half-life values, as calculated from best-fit exponential decay curves from data in (A). (N.S. = not
significant; ANOVA)
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at the level of translation. As predicted, there was a direct correlation between PLY production
and target cell lysis using simplified in vitro systems, and we provided proof-of-principle for
the use of such systems to assist in the study of host-pathogen interactions. Expanded testing
of modified strains in animal models of will allow assessment of the durability of altered PLY
regulation during S.pneumoniae infection. Such models will facilitate understanding of the
role of PLY expression levels at different stages and sites of infection.
Our findings are in agreement with those of Coleman et al., who used rational alteration of
codon pair bias to alter PLY expression and found attenuation of inflammatory responses in
vivo [7]. In contrast to that work, we alter PLY expression not by altering codon pair bias but
by altering mRNA ΔG. Moreover, rather than altering the entire sequence, we were able to
alter a short and specific part of the 5' region to alter PLY protein levels and lysis activity. The
Fig 4. Differential PLY production by S.pneumoniae strains is directly related to PLY production.
Lysis of primary human erythrocytes as assessed by hemoglobin release assay correlates with (A) ply 5’
mRNA ΔG and (B) PLY as measured by ELISA (R
2
= 0.95; outside lines represent 95% CI). *,P<0.05;
**,P<0.01 (ANOVA).
doi:10.1371/journal.pone.0119823.g004
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ability to alter a short portion of genetic sequence provides a potentially simple and straightfor-
ward tool for precisely regulating any bacterial protein involved in pathogenesis without having
to alter large sections of the coding sequence and also without a priori knowledge of the host
cell's codon preferences.
The correlation between 5’mRNA ΔG and overall protein production is not perfectly linear,
and other factors, including codon bias, GC content, and alteration of folding by other seg-
ments of the mRNA may have substantial effects on efficiency. Because of the limited number
of strains investigated, we were unable to formally evaluate the role of 5’mRNA ΔG while rig-
orously controlling each of these other variables, and, as noted above, our lowest expressing
strains (ΔG = -17.5 and -12.8) had low 5’CAI in addition to low 5’mRNA ΔG. However, the
other three strains (ΔG = -9.5, -7.3, and -3.4) did not differ substantially in 5’CAI and had al-
tered PLY production consistent with a role for 5’mRNA ΔG. Whether this technique is widely
applicable to alter protein production across a range of target genes and hosts must be deter-
mined experimentally. Despite these limitations, rational alteration of 5’mRNA ΔG has poten-
tial as an important new tool in bacterial pathogenesis research. Given the increasing
availability of whole genome sequences, much emphasis has been placed on the presence or ab-
sence of specific factors as markers of potential virulence in individual strains. Much less is
known about the impact of relative expression levels on pathogenesis. The ability to investigate
threshold and concentration-dependent effects of specific virulence factors without a require-
ment for non-native promoters, chemical inducers, or selective markers is extremely valuable
and merits further exploration in a variety of systems.
Fig 5. Lysis of human A549 respiratory epithelial cells as measured by LDH release correlates with
predicted ply 5’mRNA ΔG(R
2
= 0.91; outside lines represent 95% CI; P<0.0001, ANOVA for
linear trend).
doi:10.1371/journal.pone.0119823.g005
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Acknowledgments
We thank Max Gottesman and Timothy LaRocca for helpful discussions.
Author Contributions
Conceived and designed the experiments: AJR FEA DP TMR ASP MMS. Performed the experi-
ments: FEA DP TMR RK. Analyzed the data: FEA MMS AJR. Contributed reagents/materials/
analysis tools: FEA ASP MMS AJR. Wrote the paper: FEA MMS AJR.
References
1. Shockett PE, Schatz DG. Diverse strategies for tetracycline-regulated inducible gene expression. Proc
Natl Acad Sci USA. 1996; 93:5173–5176. PMID: 8643548
2. Hershberg R, Petrov DA. Selection on codon bias. Annu Rev Genet. 2008; 42:287–299. doi: 10.1146/
annurev.genet.42.110807.091442 PMID: 18983258
3. Ikemura T. Codon usage and tRNA content in unicellular and multicellular organisms. Mol Biol Evol.
1985; 2:13–34. PMID: 3916708
4. Stoletzki N, Eyre-Walker A. Synonymous codon usage in Escherichia coli: selection for translational ac-
curacy. Mol Biol Evol. 2007; 24:374–381. PMID: 17101719
5. Sharp PM, Li WH. The codon Adaptation Index—a measure of directional synonymous codon usage
bias, and its potential applications. Nucleic Acids Res. 1987; 15:1281–1295. PMID: 3547335
6. Coleman JR, Papamichail D, Skiena S, Futcher B, Wimmer E, Mueller S. Virus attenuation by genome-
scale changes in codon pair bias. Science. 2008; 320:1784–1787. doi: 10.1126/science.1155761
PMID: 18583614
7. Coleman JR, Papamichail D, Yano M, García-Suárez MDM, Pirofski L-A. Designed reduction of Strep-
tococcus pneumoniae pathogenicity via synthetic changes in virulence factor codon-pair bias. J Infect
Dis. 2011; 203:1264–1273. doi: 10.1093/infdis/jir010 PMID: 21343143
8. Kudla G, Murray AW, Tollervey D, Plotkin JB. Coding-sequence determinants of gene expression in
Escherichia coli. Science. 2009; 324:255–258. doi: 10.1126/science.1170160 PMID: 19359587
9. Goodman DB, Church GM, Kosuri S. Causes and effects of N-terminal codon bias in bacterial genes.
Science. 2013; 342:475–479. doi: 10.1126/science.1241934 PMID: 24072823
10. Sung CK, Li H, Claverys JP, Morrison DA. An rpsL cassette, janus, for gene replacement through nega-
tive selection in Streptococcus pneumoniae. Appl Environ Microbiol. 2001; 67:5190–5196. PMID:
11679344
11. Berry AM, Yother J, Briles DE, Hansman D, Paton JC. Reduced virulence of a defined pneumolysin-
negative mutant of Streptococcus pneumoniae. Infect Immun. 1989; 57:2037–2042. PMID: 2731982
12. Los FCO, Randis TM, Aroian RV, Ratner AJ. Role of Pore-Forming Toxins in Bacterial Infectious Dis-
eases. Microbiol Mol Biol Rev. 2013; 77:173–207. doi: 10.1128/MMBR.00052-12 PMID: 23699254
13. Markham NR, Zuker M. DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res.
2005; 33:W577–581. PMID: 15980540
14. PuigbòP, Bravo IG, Garcia-Vallve S. CAIcal: a combined set of tools to assess codon usage adapta-
tion. Biol Direct. 2008; 3:38. doi: 10.1186/1745-6150-3-38 PMID: 18796141
15. Lacks S, Hotchkiss RD. A study of the genetic material determining an enzyme in Pneumococcus. Bio-
chim Biophys Acta. 1960; 39:508–518. PMID: 14413322
16. Rampersaud R, Planet PJ, Randis TM, Kulkarni R, Aguilar JL, Lehrer RI, et al. Inerolysin, a Cholester-
ol-Dependent Cytolysin Produced by Lactobacillus iners. J Bacteriol. 2011; 193:1034–1041. doi: 10.
1128/JB.00694-10 PMID: 21169489
17. Crook J, Tharpe JA, Johnson SE, Williams DB, Stinson AR, Facklam RR, et al. Immunoreactivity of five
monoclonal antibodies against the 37-kilodalton common cell wall protein (PsaA) of Streptococcus
pneumoniae. Clin Diagn Lab Immunol. 1998; 5:205–210. PMID: 9521144
18. Johansson N, Kalin M, Giske CG, Hedlund J. Quantitative detection of Streptococcus pneumoniae
from sputum samples with real-time quantitative polymerase chain reaction for etiologic diagnosis of
community-acquired pneumonia. Diagn Microbiol Infect Dis. 2008; 60:255–261. PMID: 18036762
19. Gelber SE, Aguilar JL, Lewis KLT, Ratner AJ. Functional and phylogenetic characterization of Vaginoly-
sin, the human-specific cytolysin from Gardnerella vaginalis. J Bacteriol. 2008; 190:3896–3903. doi:
10.1128/JB.01965-07 PMID: 18390664
Rheostat Protein Expression
PLOS ONE | DOI:10.1371/journal.pone.0119823 March 23, 2015 10 / 11

20. Aguilar JL, Kulkarni R, Randis TM, Soman S, Kikuchi A, Yin Y, et al. Phosphatase-dependent regula-
tion of epithelial mitogen-activated protein kinase responses to toxin-induced membrane pores. PLOS
ONE. 2009; 4:e8076. doi: 10.1371/journal.pone.0008076 PMID: 19956644
21. Randis TM, Kulkarni R, Aguilar JL, Ratner AJ. Antibody-based detection and inhibition of vaginolysin,
the Gardnerella vaginalis cytolysin. PLOS ONE. 2009; 4:e5207. doi: 10.1371/journal.pone.0005207
PMID: 19370149
22. Zur H, Tuller T. Strong association between mRNA folding strength and protein abundance in S. cerevi-
siae. EMBO Rep.; 2012; 13:272–277. doi: 10.1038/embor.2011.262 PMID: 22249164
23. Shah P, Ding Y, Niemczyk M, Kudla G, Plotkin JB. Rate-limiting steps in yeast protein translation. Cell.
2013; 153:1589–1601. doi: 10.1016/j.cell.2013.05.049 PMID: 23791185
24. Tuller T, Girshovich Y, Sella Y, Kreimer A, Freilich S, Kupiec M, et al. Association between translation
efficiency and horizontal gene transfer within microbial communities. Nucleic Acids Res. 2011;
39:4743–4755. doi: 10.1093/nar/gkr054 PMID: 21343180
25. Supek F, Šmuc T. On relevance of codon usage to expression of synthetic and natural genes in Escher-
ichia coli. Genetics. 2010; 185:1129–1134. doi: 10.1534/genetics.110.115477 PMID: 20421604
26. Lee SF, Li Y-J, Halperin SA. Overcoming codon-usage bias in heterologous protein expression in
Streptococcus gordonii. Microbiology. 2009; 155:3581–3588. doi: 10.1099/mic.0.030064-0 PMID:
19696103
27. Tuller T, Waldman YY, Kupiec M, Ruppin E. Translation efficiency is determined by both codon bias
and folding energy. Proc Natl Acad Sci USA. 2010; 107:3645–3650. doi: 10.1073/pnas.0909910107
PMID: 20133581
28. Roller M, Lucic V, Nagy I, Perica T, Vlahovicek K. Environmental shaping of codon usage and function-
al adaptation across microbial communities. Nucleic Acids Res. 2013; 41:8842–8852. doi: 10.1093/
nar/gkt673 PMID: 23921637
Rheostat Protein Expression
PLOS ONE | DOI:10.1371/journal.pone.0119823 March 23, 2015 11 / 11