Reply to “Concerns about Recently Identiﬁed Widespread Antisense
Transcription in Escherichia coli”
n our recent article, “Widespread Antisense Transcription in
Escherichia coli” (1), we describe the identiﬁcation of ~1,000
antisense RNAs (aRNAs) in the model bacterium Escherichia coli.
This ﬁnding is signiﬁcant because it suggests that aRNAs represent
a major class of regulators in bacteria. Slonczewski expressed con-
cerns regarding (i) the documentation of methods used, (ii) pos-
sible contamination of the cDNA library, (iii) insufﬁcient con-
trols, and (iv) failure to consider alternative interpretations of the
Due to strict space limitations for Observations, we provided
only the most relevant aspects of the methods used. A detailed
description of the library construction is included at the end of
Library construction involved ligation of an RNA linker, re-
verse transcription, and two rounds of nested PCR. This method
effectively eliminates the possibility of contamination by exoge-
nous DNA or RNA. We attempted to construct a control cDNA
library from a mock sample lacking RNA. We cloned and se-
quenced several individual fragments from this library, all of
which were products formed by ligation of multiple mutant RNA
linker oligonucleotides. Hence, we did not subject this library to
high-throughput sequencing. This observation supports the idea
that contamination by exogenous DNA or RNA was not a con-
As described in the article, we validated the aRNAs using bioin-
formatic and experimental approaches. Crucially, we demon-
strated that (i) putative aRNAs are signiﬁcantly enriched for “A” at
the initiating nucleotide (P ⬍ 1e
) and are indistinguishable in
this respect from published RNA 5= ends (P ⫽ 0.40); (ii) ⫺10
hexamers for putative aRNAs have signiﬁcantly better matches to
the consensus sequence than random sequences do (P ⫽
) and are indistinguishable from those of published RNAs
(P ⫽ 0.49); (iii) 9 of 10 putative aRNA promoters tested resulted
in signiﬁcant expression of a lacZ reporter gene; in all nine cases,
expression was dependent upon an intact ⫺10 hexamer, indica-
tive of a functional promoter; and (iv) one of two putative aRNAs
tested repressed expression of the overlapping protein-coding
Our data are most easily explained by the presence of a large
number of aRNAs and are not consistent with any other reason-
able interpretation. We note that no other possible interpretations
and no other controls were suggested by Slonczewski.
Lastly, our conclusions are consistent with those of many other
studies. In particular, a study in which the authors identiﬁed a
similar number of aRNAs in Helicobacter pylori (2) was published
while our work was in press.
For cDNA library construction, MG1655 E. coli cells were
grown in LB to an optical density at 600 nm (OD
) of 0.7. RNA
was puriﬁed using the hot-phenol method (3) and treated with
DNase I. rRNA was removed by two rounds of treatment with the
MICROBExpress kit (Ambion). RNA was treated with tobacco
acid pyrophosphatase (TAP). TAP-treated RNA was puriﬁed by
phenol extraction and ethanol precipitation. An RNA oligonucle-
U-3=) was ligated to the RNA 5= ends using T4 RNA ligase 1 (NEB)
for1hat37°C. RNA was gel puriﬁed to remove unligated 5= linker
oligonucleotide. RNA was reverse transcribed with 5=-GTTTCCC
AGTCACGATCNNNNNNNNN-3= and SuperScript III reverse
transcriptase (Invitrogen). cDNA was ampliﬁed by PCR (25 cy-
cles) using primers 5=-GTTTCCCAGTCACGATC-3= and 5=-AC
ACTCTTTCCCTACACG-3=. DNAs of lengths between 80 and
200 bp were gel puriﬁed and reampliﬁed by PCR using primers
5=-GTTTCCCAGTCACGATC-3= and 5=-ACGCTCTTCCGATC
T-3=. One-hundredth of the DNA sample was ampliﬁed by PCR
using the primers 5=-AATGATACGGCGACCACCGAGATCTAC
ACTCTTTCCCTACACGACGCTCTTCCGATCT-3= and 5=-CA
1. Dornenburg, J. E., A. M. DeVita, M. J. Palumbo, and J. T. Wade. 2010.
Widespread antisense transcription in Escherichia coli. mBio 1(1):e00024-
2. Sharma, C. M., S. Hoffmann, F. Darfeuille, J. Reignier, S. Findeiss, A.
Sittka, S. Chabas, K. Reiche, J. Hackermüller, R. Reinhardt, P. F. Stadler,
and J. Vogel. 2010. The primary transcriptome of the major human patho-
gen Helicobacter pylori. Nature 464:250–255.
3. Rhodius, V. A., W. C. Suh, G. Nonaka, J. West, and C. A. Gross. 2006.
Conserved and variable functions of the sigmaE stress response in related
genomes. PLoS Biol. 4(1):e2. doi:10.1371/journal.pbio.0040002.
4. Slonczewski, J. L. 2010. Concerns about recently identiﬁed widespread
antisense transcription in Escherichia coli. mBio 1(2):e00106-10. doi:10
Joseph T. Wade
James E. Dornenburg
Anne M. DeVita
Michael J. Palumbo
Wadsworth Center, New York State Department of Health, Albany, New York,
Published 18 May 2010
Citation Wade, J. T., J. E. Dornenburg, A. M. DeVita, and M. J. Palumbo. 2010. Reply to
“Concerns about recently identiﬁed widespread antisense transcription in Escherichia
coli.” mBio 1(2):e00119-10. doi:10.1128/mBio.00119-10.
Copyright © 2010 Wade et al. This is an open-access article distributed under the terms
of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License,
which permits unrestricted noncommercial use, distribution, and reproduction in any
medium, provided the original author and source are credited.
Address correspondence to Joseph T. Wade, firstname.lastname@example.org.
May/June 2010 Volume 1 Issue 2 e00119-10