The gatekeeper of Yersinia type III secretion is under RNA thermometer control

Abstract

Many bacterial pathogens use a type III secretion system (T3SS) as molecular syringe to inject effector proteins into the host cell. In the foodborne pathogen Yersinia pseudotuberculosis , delivery of the secreted effector protein cocktail through the T3SS depends on YopN, a molecular gatekeeper that controls access to the secretion channel from the bacterial cytoplasm. Here, we show that several checkpoints adjust yopN expression to virulence conditions. A dominant cue is the host body temperature. A temperature of 37°C is known to induce the RNA thermometer (RNAT)-dependent synthesis of LcrF, a transcription factor that activates expression of the entire T3SS regulon. Here, we uncovered a second layer of temperature control. We show that another RNAT silences translation of the yopN mRNA at low environmental temperatures. The long and short 5’-untranslated region of both cellular yopN isoforms fold into a similar secondary structure that blocks ribosome binding. The hairpin structure with an internal loop melts at 37°C and thereby permits formation of the translation initiation complex as shown by mutational analysis, in vitro structure probing and toeprinting methods. Importantly, we demonstrate the physiological relevance of the RNAT in the faithful control of type III secretion by using a point-mutated thermostable RNAT variant with a trapped SD sequence. Abrogated YopN production in this strain led to unrestricted effector protein secretion into the medium, bacterial growth arrest and delayed translocation into eukaryotic host cells. Cumulatively, our results show that substrate delivery by the Yersinia T3SS is under hierarchical surveillance of two RNATs.

Full text

RESEARCH ARTICLE
The gatekeeper of Yersinia type III secretion is
under RNA thermometer control
Stephan Pienkoß
1
, Soheila JavadiID
1
, Paweena ChaoprasidID
2
, Thomas Nolte
1
,
Christian Twittenhoff
1,3
, Petra DerschID
2
, Franz NarberhausID
1
*
1Microbial Biology, Ruhr University Bochum, Bochum, Germany, 2Institute of Infectiology, Center for
Molecular Biology of Inflammation (ZMBE), University of Mu¨nster, Mu¨nster, Germany, 3Rottendorf Pharma
GmbH, Ennigerloh, Germany
*franz.narberhaus@rub.de
Abstract
Many bacterial pathogens use a type III secretion system (T3SS) as molecular syringe to
inject effector proteins into the host cell. In the foodborne pathogen Yersinia pseudotubercu-
losis, delivery of the secreted effector protein cocktail through the T3SS depends on YopN,
a molecular gatekeeper that controls access to the secretion channel from the bacterial
cytoplasm. Here, we show that several checkpoints adjust yopN expression to virulence
conditions. A dominant cue is the host body temperature. A temperature of 37˚C is known to
induce the RNA thermometer (RNAT)-dependent synthesis of LcrF, a transcription factor
that activates expression of the entire T3SS regulon. Here, we uncovered a second layer of
temperature control. We show that another RNAT silences translation of the yopN mRNA at
low environmental temperatures. The long and short 5’-untranslated region of both cellular
yopN isoforms fold into a similar secondary structure that blocks ribosome binding. The hair-
pin structure with an internal loop melts at 37˚C and thereby permits formation of the transla-
tion initiation complex as shown by mutational analysis, in vitro structure probing and
toeprinting methods. Importantly, we demonstrate the physiological relevance of the RNAT
in the faithful control of type III secretion by using a point-mutated thermostable RNAT vari-
ant with a trapped SD sequence. Abrogated YopN production in this strain led to unre-
stricted effector protein secretion into the medium, bacterial growth arrest and delayed
translocation into eukaryotic host cells. Cumulatively, our results show that substrate deliv-
ery by the Yersinia T3SS is under hierarchical surveillance of two RNATs.
Author summary
Temperature serves as reliable external cue for pathogenic bacteria to recognize the entry
into or exit from a warm-blooded host. At the molecular level, a temperature of 37˚C
induces various virulence-related processes that manipulate host cell physiology. Here, we
demonstrate the temperature-dependent synthesis of the secretion regulator YopN in the
foodborne pathogen Yersinia pseudotuberculosis, a close relative of Yersinia pestis. YopN
blocks secretion of effector proteins through the type III secretion system unless host cell
contact is established. Temperature-specific regulation relies on an RNA structure in the
PLOS PATHOGENS
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 1 / 27
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Citation: Pienkoß S, Javadi S, Chaoprasid P, Nolte
T, Twittenhoff C, Dersch P, et al. (2021) The
gatekeeper of Yersinia type III secretion is under
RNA thermometer control. PLoS Pathog 17(11):
e1009650. https://doi.org/10.1371/journal.
ppat.1009650
Editor: Rene
´e M. Tsolis, University of California,
Davis, UNITED STATES
Received: May 12, 2021
Accepted: October 27, 2021
Published: November 12, 2021
Copyright: ©2021 Pienkoß 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 manuscript and its Supporting
Information files.
Funding: This work was funded by the German
Research Foundation (DFG, grant number NA 240/
10-2) to F.N. The funder had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.

5’-untranslated region of the yopN mRNA, referred to as RNA thermometer, which allows
ribosome binding and thus translation initiation only at an infection-relevant temperature
of 37˚C. A mutated variant of the thermosensor resulting in a closed conformation pre-
vented synthesis of the molecular gatekeeper YopN and led to permanent secretion and
defective translocation of virulence factors into host cells. We suggest that the RNA ther-
mometer plays a critical role in adjusting the optimal cellular concentration of a surveil-
lance factor that maintains the controlled translocation of virulence factors.
Introduction
Numerous gram-negative plant-or animal pathogens deploy a type III secretion system (T3SS)
to translocate effector proteins into eukaryotic host cells [1,2]. This molecular syringe or
“injectisome” is evolutionary related to flagella. The complex nanomachines cross the bacterial
envelope and the host membrane for cross-kingdom transfer of effectors into the host cell
cytosol [3]. While the overall architecture and the structural components of the T3SS are con-
served, the cocktail of secreted proteins varies in order to execute diverse species-specific activ-
ities. Among other processes, effector proteins can target the host cytoskeleton, autophagy and
innate immune response [4–7].
T3SS components in members of the genus Yersinia are encoded on the virulence plasmid
[8]. This genus consists of the human pathogens Yersinia pestis, the causative agent of bubonic
and pneumonic plague [9], and of the enteropathogens Yersinia enterocolitica and Yersinia
pseudotuberculosis [10]. The T3SS is composed of more than 15 different proteins and the bio-
genesis of this more than 15 MDa-large apparatus is a strictly regulated hierarchical process
that is controlled by internal and external cues at various transcriptional and posttranscrip-
tional levels [11,12]. The basal body is composed of more than a dozen so-called Ysc proteins
and spans the bacterial inner and outer membranes. It is followed by the needle structure, a
polymer of the YscF protein. The distal end of the needle serves as platform for the pore com-
plex comprised of the LcrV-containing tip and YopBD, the translocation pore [13]. The for-
mation of the needle and the pore complex as well as the secretion of effector proteins follow a
specific secretion order. First, substrates consisting of the needle subunit YscF and the ruler
protein YscP, which determines the length of the needle, are secreted [3,14–16]. Once the con-
tact with the host cell is established, proteins of the pore complex pass through the T3SS and
integrate into the host membrane to allow the passage of effector proteins [17–19].
Yet another checkpoint regulates the fidelity of type III secretion (T3S) and prevents the
release of Yersinia outer proteins (Yops) prior to host cell contact (Fig 1). YopN forms a het-
erodimer with TyeA that together with the SycN/YscB chaperones plugs the secretion channel
until secretion is desired [20,21]. While TyeA binds to the C-terminus of YopN, the chaperone
complex SycN/YscB binds to the N-terminus ensuring attachment to the T3SS [21–23]. Once
contact with the host cell is made, YopN-TyeA dissociates and SycN/YscB facilitates the export
of YopN through the T3SS. Translational +1 frameshifting near the 3’ end of the yopN mRNA
in Y.pestis and Y.pseudotuberculosis occasionally generates a YopN-TyeA hybrid protein that
maintains secretion control suggesting that the reversible interaction between YopN and TyeA
is not a functional prerequisite [24,25].
Virulence conditions can be mimicked in the laboratory by decreasing the calcium concen-
tration in the surrounding medium, which is why the YopN complex has been described as
calcium plug [26,27]. YopN export subsequently enables the secretion of other Yops [22,28].
Deletion of yopN results in a temperature-sensitive phenotype at 37˚C characterized by
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 2 / 27

PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 3 / 27

continuous secretion of effector proteins associated with growth impairment [27]. YopN
might serve additional functions within the host cell. Recently, it was shown to affect systemic
infection in mice and to adjust the secretion of the effector proteins YopE and YopH [29,30].
Temperature is a major stimulus for Yersinia T3SS gene expression both under in vitro lab-
oratory conditions and in the host [31–33]. The pathogen interprets a temperature of 37˚C as
signal that it has been taken up by a warm-blooded host. Temperature-dependent transcrip-
tion initiation of a large array of virulence genes is accomplished by the AraC-type regulator
LcrF, whose expression is under the influence of an RNA thermometer (RNAT) [34]. RNATs
are thermolabile RNA structures in the 5’-untranslated region (5’-UTR) that sequester the
Shine Dalgarno (SD) sequence and/or the start codon. At ambient environmental tempera-
tures, ribosome binding to the SD sequence is prevented by the double-stranded RNA. The
metastable structure melts in the host at about 37˚C, which liberates the ribosome binding site
and permits translation initiation [35,36]. Upon return to lower temperatures, the reversible
zipper-like structure returns to the inhibitory state [37].
In search of new temperature-responsive RNA structures in Y.pseudotuberculosis, we used
two global RNA structuromics approaches and identified numerous RNAT candidates that
undergo a conformational change from 25 to 37˚C [38,39]. Several of them are located
upstream of genes critical for various aspects of bacterial virulence. A recently documented
example is the cnfY thermometer controlling translation of the cytotoxic necrotizing factor
that enhances inflammation and Yop delivery by activation of Rho GTPases in the host [40–
42]. In contrast to Yops, the CnfY toxin is not secreted by the T3SS but delivered to host cells
via outer membrane vesicles [43].
Another promising RNAT candidate was identified in the 5’-UTR of yopN coding for the
T3SS regulator described above. In this study, we show the functionality of this RNAT in both
naturally occurring short and long transcripts of yopN by various in vitro and in vivo assays at
different temperatures. Most importantly, by using thermostable RNAT variants with a
sequestered SD sequence, we demonstrate the physiological relevance of the RNAT in the
proper control of type III secretion.
Results
A putative RNAT in the short and long 5’-UTR of yopN
Y.pseudotuberculosis yopN is the first gene of the heptacistronic virA operon composed of
yopN,tyeA,sycN,yscX,yscY,yscV and lcrR [44]. Two alternative transcription start sites
upstream of yopN result in a short and a long 5’-UTR of 37 and 102 nucleotides, respectively
(Fig 2A). Previous RNA-Seq analyses showed that the short transcript is more highly expressed
at 37˚C than the long transcript [32,38]. We examined the expression of both yopN isoforms
by qRT-PCR and, consistent with the previous studies, saw an over-abundance of the short
transcript (Fig 2B). The amount of the short isoform increased about 22-fold at 37˚C under
non-secretion conditions (+ Ca
2+
) and even further to 63-fold under secretion-mimicking
conditions (- Ca
2+
) compared with 25˚C. The long transcript was poorly transcribed and
three-fold induced at 37˚C in the presence of Ca
2+
. Overall, these results show that the short
transcript is the major isoform responsible of yopN expression.
Fig 1. Schematic overview of YopN-regulated Yop secretion in Yersinia.The secretion of effector proteins (Yops)
that modulate the host immune system are controlled by the YopN-TyeA-SycN-YscB complex. YopN and TyeA block
secretion in concert with the chaperone complex SycN/YscB [20,22]. Dissociation of the plug is assumed to occur
through depletion of calcium and after host cell contact resulting in the SycN/YscB-controlled secretion of YopN itself
[23,26,27]. HM: host membrane; OM: outer membrane; IM: inner membrane.
https://doi.org/10.1371/journal.ppat.1009650.g001
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 4 / 27

Fig 2. Expression and RNA structures of 5’-UTRs of yopN.(A) RNA-seq results of the yopN locus at 37˚C and identification of two transcriptional start sites
from [32] and [38] visualized by the Artemis genome browser (s: short transcript; l: long transcript). (B) Comparison of the relative transcript levels of both
yopN 5’-UTRs under non-secretion (+ Ca
2+
) and secretion (- Ca
2+
) conditions at 25 and 37˚C. Samples of Y.pseudotuberculosis YPIII were taken during the
early exponential phase at an OD
600
of 0.5 followed by RNA isolation and qRT-PCR. Transcript levels were normalized to the amount of yopN short at 25˚C
under non-secretion conditions and to the reference genes nuoB and gyrB. The mean transcript amounts and standard deviations comprise the results of three
biological replicates. (C,D) PARS-derived RNA secondary structures of the short and long 5’-UTRs of yopN at 37˚C including the first 30 nucleotides of the
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 5 / 27

A striking observation of our previous comparative analysis of the Y.pseudotuberculosis
RNA structurome at 25 and 37˚C [38] was a thermolabile RNA element upstream of yopN.
The SD sequence (5’-AGGGAGU-3’) and the start codon are part of the same hairpin structure
in both the short and the long transcript (Fig 2C and 2D). An internal loop that exposes some
nucleotides of the SD sequence might explain the temperature sensitivity of this structure.
Compared to the short transcript, the longer one features a slightly extended hairpin, two addi-
tional hairpins downstream of it and a base-paired region between the immediate 5’ end and
nucleotides in the early coding region of yopN. A sequence comparison showed that the yopN
5’-UTRs of Y.pseudotuberculosis and Y.pestis are identical over the entire length of the long
transcript and at least 30 nts into the coding region (S1 Fig). Three alterations were found in
the corresponding Y.enterocolitica sequence. In particular, the exchange of residue U6 oppo-
site the first nucleotide of the AUG start codon for a C residue might reduce the overall stabil-
ity of the RNA structure (Fig 2C).
Predictable consequences of point mutations in the yopN RNAT of both
transcripts
The structured 5’-UTRs of the short and long yopN transcripts suggest that the transcriptional
control of yopN expression in response to temperature (Fig 2A and 2B) is complemented by
RNAT-mediated translational control. To investigate the functionality of the putative yopN
RNAT, five variants of the hairpin of the short transcript were generated by site-directed muta-
genesis (Fig 3A). Three variants were designed to result in a putative stable hairpin (referred to
as repressed; R1, R2 and R3). In R1 and R2, the internal loop of the hairpin is reduced (R2) or
completely closed (R1) by residues pairing with the SD sequence. In R3, base pairing with the
AUG start codon is strengthened. Two other variants were expected to result in a less stable
hairpin (referred to as derepressed; D1 and D2). They result in a larger internal loop of the
hairpin and partially (but not completely) liberate the SD sequence.
The functionality of these variants was measured quantitatively by reporter gene assays
using translational fusion constructs consisting of the yopN 5’-UTR (WT), the different yopN
variants and the positive control lcrF fused to bgaB coding for a heat-stable β-galactosidase
(Fig 3B). Compared with the positive control lcrF that exhibited a temperature-dependent
3-fold induction of reporter enzyme activity, the yopN RNAT showed a 6.9-fold increased β-
galactosidase activity at 37˚C compared to 25˚C (Fig 3C). The point-mutated variants behaved
as expected. R1 with the completely paired SD sequence fully repressed expression at both tem-
peratures whereas R2 that contained just one additional base pair in the internal loop reduced
expression at 25˚C but remained inducible at 37˚C. Stabilizing the AUG start codon in R3
almost completely blocked expression. In contrast, variants D1 and D2 already showed ele-
vated β-galactosidase activities at 25˚C, which further increased at 37˚C. The reporter gene
activities were fully reflected in Western blot experiments detecting the His-tagged BgaB
enzyme (Fig 3C). Cumulatively, these results provide support for the existence of a functional
RNAT in the short 5’-UTR of yopN.
We then wondered whether the structure in the long yopN transcript (Fig 2D) is equally
temperature responsive and constructed two corresponding bgaB fusions to the WT region
and a repressed R1 variant. The β-galactosidase experiments showed essentially the same
results for the short and long 5’-UTRs (Fig 3D) strongly suggesting that they both contain
coding region [38]. The putative SD region is highlighted in gray and the start codon in red. Nucleotide exchanges in the Y.enterocolitica sequence are
indicated in (C).
https://doi.org/10.1371/journal.ppat.1009650.g002
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 6 / 27

Fig 3. Schematic representation and functional characterization of the short yopN 5’-UTR and mutated variants. (A) PARS-derived stem-loop RNA
structure of the short yopN 5’-UTR (nucleotides 1 to 43) [38] and predicted stabilized (R1–3) and destabilized (D1–2) variants. The putative SD region is
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 7 / 27

functional RNATs. In contrast to yopN, the first gene of the virA operon, the 5’-UTRs of the
other T3SS-associated operons virB (yscN) and virC (yscA) do not possess functional RNATs
(S3 Fig).
Finally, to support translational control and exclude any transcriptional control by the 5’-
UTR, the short yopN RNAT and the R1 and D2 variants were translationally fused to gfp (Fig
4A) and mRNA and protein levels were recorded (Fig 4B). Importantly, when transcription
was induced from the P
BAD
promoter by arabinose addition, the transcript amounts of each
construct were the same at 25 and 37˚C excluding an influence of temperature on transcrip-
tion and/or transcript stability. In accordance with the BgaB results, GFP production occurred
as predicted, i.e. inducible GFP levels at 37˚C in the WT situation, overall elevated GFP protein
in the D2 strain and fully repressed GFP production in R1. All these results are in favour of a
model, in which the thermolabile 5’-UTR upstream of yopN melts at host body temperature
and thereby facilitates translation initiation.
Melting of the RNA structure liberates the SD sequence and start codon
To lend biochemical support to the gradual melting of the RNAT with elevated temperatures,
we conducted enzymatic structure probing experiments on in vitro-synthesized RNAs (Fig 5).
The short WT and R1 RNATs were treated with RNases T1 and T2 at 25, 37 and 42˚C. RNase
T1 specifically introduces cuts in single-stranded RNA (ssRNA) at the 3’ end of guanines,
whereas T2 prefers ssRNA at the 3’ end of adenines but also cleaves other single-stranded
nucleotides. The presence of cuts at all temperatures in the loop at the top of the hairpin and in
the large loop at the beginning of the coding region (positions 19–25 and 49–64, respectively)
suggested folding of the RNA into the anticipated structure (Fig 5A and 5B). Poor cleavage at
25˚C and gradual heat-induced melting was observed in the region of the SD sequence at
nucleotides 27–32 (5’-AGGGAG-3’) and in the corresponding anti-SD region with several A
residues around position 14 (Fig 5A and 5B; quantification for some of the bands in Fig 5C).
In addition, temperature-modulated cleavage was also observed for nucleotides of the stem
structure at the bottom of the hairpin (position 34–40), which includes the AUG start codon.
The start codon showed the same behavior in the R1 construct. The SD sequence in this RNA
structure, however, was inaccessible to RNases even at high temperatures due to the intro-
duced stabilizing point mutations (Fig 3A).
The yopN RNAT controls ribosome binding
Typical RNATs adjust expression of the downstream gene to the ambient temperature by con-
trolling the access of the 30S ribosomal subunit to the SD sequence. To demonstrate this activ-
ity for the yopN RNAT, the short WT and R1 RNAs were subjected to toeprinting (in vitro
primer extension inhibition) analysis at 25, 37 and 42˚C. Incubation of each RNAT variant
with the 30S subunit at these temperatures was followed by reverse transcription. The presence
of a truncated cDNA product (toeprint) is indicative of successful binding of the 30S ribosome
highlighted in gray, the start codon in red and mutated nucleotides are highlighted in blue. (B) Plasmid-based translational fusions of 5’-UTRs of interest
and bgaB encoding a heat-stable β-galactosidase to test RNA thermometer (RNAT) functionality. Expression of the fusion products is controlled by the
arabinose-inducible promotor P
BAD
. The RNAT of lcrF served as a positive control [34]. (C,D) The β-galactosidase assays of lcrF, the short and long yopN
5’-UTR and the corresponding mutated variants were conducted at 25 and 37˚C. Y.pseudotuberculosis YPIII cells carrying plasmids of the fusion
constructs were grown to an OD
600
of 0.5 at 25˚C. Subsequently, transcription of the reporter gene was induced by 0.1% (w/v) L-arabinose and the cultures
were split to flasks at 25 and prewarmed flasks at 37˚C and incubated for further 30 minutes. Samples were then taken for the β-galactosidase assay. The
mean activities in Miller Units and the mean standard deviations were calculated from nine biological replicates. The representative Western blot displays
the amount of BgaB-His produced. Protein amounts were adjusted to an optical density of 0.5 and detected by Ponceau S staining after blotting onto a
nitrocellulose membrane.
https://doi.org/10.1371/journal.ppat.1009650.g003
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 8 / 27

to the RNA, which generates a roadblock for the reverse transcriptase. Such a toeprint signal at
the appropriate position between nucleotides 12 to 14 downstream of the start codon was
observed for the WT RNA at 37 and 42˚C (Fig 6). Consistent with a stabilized structure, this
toeprint signal was absent when the R1 RNA was assayed. Instead, another cDNA product was
generated under all conditions even in samples without the 30S subunit. Such ribosome-inde-
pendent formation of termination products is frequently observed [34,41,45] and indicates the
presence of a stable hairpin that interferes with reverse transcriptase activity.
Unrestricted secretion and inefficient translocation of effector proteins
when the RNAT is stabilized
To study the relevance of the yopN thermometer on effector protein secretion, we constructed
a nonpolar yopN deletion mutant as previously described in [29]. This ΔyopN strain was
Fig 4. Temperature-controlled expression of gfp by the short yopN RNAT. (A) Plasmid-based translational fusions
of yopN:gfp were cloned to test RNAT functionality at RNA and protein levels. Transcription of the fusion product is
controlled by the arabinose inducible promotor P
BAD
. (B) Determination of transcript and protein levels of yopN:gfp
(WT-gfp) and mutated yopN variants (R1 and D1) by Northern and Western blot analyses, respectively. The RNAT of
lcrF served as a positive control [8]. Y.pseudotuberculosis YPIII cells carrying plasmids of the fusion constructs were
grown to an OD
600
of 0.5 at 25˚C. Transcription was induced by 0.1% (w/v) L-arabinose and the cultures were split to
flasks at 25˚C and prewarmed flasks at 37˚C and incubated for further 30 minutes. Samples were then taken for
Northern and Western blot analyses. The blots shown represent one of three biological replicates. To ensure equal
amounts of RNA, a total of 10 μg of RNA was loaded per sample. Ethidium bromide stained 23S rRNA served as
loading control. Protein amounts were adjusted to an optical density of 0.5 and detected by Ponceau S staining after
blotting onto a nitrocellulose membrane.
https://doi.org/10.1371/journal.ppat.1009650.g004
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 9 / 27

Fig 5. Temperature-dependent melting of the SD sequence and Start codon-containing stem-loop structure of the yopN RNAT. (A) Enzymatic structure
probing of the yopN RNAT (WT) and the stable variant (R1) at 25, 37 and 42˚C. Radioactively labeled RNA was treated with single-strand specific RNases T1
(0.016 U) and T2 (0.025 U) at the different temperatures. L
OH
: alkaline Ladder; L
T1
: T1 treated RNA at 37˚C; C: water control. (B) PARS-derived RNA secondary
structure of the yopN RNAT [38]. White arrows indicate T1 and T2 cleavages at all temperatures while black arrows indicate cleavages with increasing
temperature. The putative SD region is highlighted in gray and the start codon in red. (C) Quantification of band intensities at 25, 37 and 42˚C of selected
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 10 / 27

equipped with plasmids expressing C-terminally Strep-tagged yopN either with its natural
RNAT or with the variants R1 or D2 downstream of an arabinose-inducible promoter. Growth
of these cultures at 25˚C under non-secretion (+Ca
2+
) or secretion (-Ca
2+
) conditions was
indistinguishable from growth of the WT or ΔyopN strain carrying the empty vector pGM930
(Fig 7A). At 37˚C, the previously described temperature-sensitive phenotype of the ΔyopN
mutant [27] was observed and found to be associated with continuous secretion of Yops under
both secretion and non-secretion conditions (Fig 7B). As expected, production of YopN
allowed growth of Y.pseudotuberculosis in the presence of calcium at 37˚C. This was also the
guanines and adenines. Pixel counting was performed using AlphaEaseFC software and values were normalized to intensities at 25˚C. The enzymatic structure
probing shown here represents one of two experiments performed.
https://doi.org/10.1371/journal.ppat.1009650.g005
Fig 6. Temperature-dependent binding of the 30S ribosomal subunit to the yopN RNAT. Primer extension inhibition of the yopN RNAT and the stable
variant R1 was performed at 25, 37 and 42˚C in presence (+) and absence (-) of the 30S ribosomal subunit. A DNA sequencing ladder (ATGC) of the yopN
RNAT serves as orientation. Full-length transcripts, termination and the characteristic toeprint signals (+12–14 nt) after ribosome binding are indicated. The
primer extension inhibition experiment shown here represents one of two experiments performed.
https://doi.org/10.1371/journal.ppat.1009650.g006
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 11 / 27

case for the ΔyopN strain complemented with the yopN expression plasmid carrying either the
native RNAT or the variant D2. In contrast, uncontrolled permanent secretion and tempera-
ture sensitivity of ΔyopN could not be rescued when yopN was preceded by the closed RNAT
variant R1. To ascertain yopN mRNA synthesis, endogenous and plasmid-derived yopN tran-
script levels were quantified by qRT-PCR (S4 Fig). The plasmid-derived yopN levels were gen-
erally higher than the endogenous transcript levels. Western blot analysis taking advantage of
Fig 7. The closed RNAT variant R1 is unable to rescue the temperature-sensitive phenotype of the ΔyopN mutant. (A) Growth experiment of Y.pseudotuberculosis
YPIII wild type (WT) and ΔyopN with the empty vector pGM930 (EV) and ΔyopN with vectors containing arabinose-inducible constructs of yopN with the wild type
RNAT, the stable variant R1 or the open variant D2. The growth experiment was performed in triplicate. The strains were incubated in secretion-induced (-Ca
2+
) and
secretion-noninduced (+Ca
2+
) LB medium at 25 and 37˚C. Growth was measured by optical density at 600 nm. (B) Visualization of secreted effector proteins by
SDS-PAGE using Coomassie blue staining and Western blotting using total-anti-Yop serum. Samples were taken after five hours at 37˚C and secreted proteins were
TCA-precipitated from filtered supernatants. (C) Production of YopN-Strep was visualized by Western blotting using Strep-tag antibody. Samples weretaken as
described for (B) and protein levels were adjusted to an optical density of 0.5.
https://doi.org/10.1371/journal.ppat.1009650.g007
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 12 / 27

the C-terminal Strep-tag of YopN showed that the R1 UTR prevented production of the gate-
keeper protein whereas the WT and D2 UTR allowed its production specifically at host body
temperature (Fig 7C). Notably, YopN production was further elevated under secretion condi-
tions at low Ca
2+
concentration.
Interestingly, YopE translocation assays demonstrated that sustained secretion of Yops in
the ΔyopN mutant and the mutant containing the R1 variant resulted in decreased transloca-
tion of YopE-TEM into HEp-2 cells (Fig 8). HEp-2 cells infected with these strains showed
almost no blue fluorescence as a measure of efficient YopE-TEM translocation. In contrast,
many HEp-2 cells treated with Y.pseudotuberculosis WT or the ΔyopN mutant complemented
with yopN or the D2 variant turned blue. Successful infection of HEp-2 is reflected by their
rounded shape (S5A Fig) as described previously [46,47]. In a time-course experiment measur-
ing the translocation of YopE-TEM over a 120-minute period revealed different translocation
kinetics. As expected, the ΔyopN mutant was unable to infect the eukaryotic cells. The D2
strain translocated YopE-TEM earlier and more efficiently than the other strains. The WT
strains and the ΔyopN mutant with yopN were equally efficient in translocation whereas the
ΔyopN with the R1 construct showed delayed translocation (S5B Fig).
Fig 8. Visualization of YopE translocation into HEp-2 cells. YopE translocation assay of Y.pseudotuberculosis YPIII
wild type (WT) and ΔyopN with the empty vector pGM930 (EV) and ΔyopN with vectors containing arabinose-
inducible complementation constructs of yopN with the wild type RNAT, the stable variant R1 or the open variant D2.
All strains carry plasmid pMK-bla coding for a yopE-bla
TEM
fusion. HEp-2 cells were infected with bacterial strains at
an MOI of 50, labeled with CCF4-AM and analyzed by fluorescence microscopy. Blue fluorescence signals of HEp-2
cells indicate efficient YopE-TEM translocation. scale bars: 50 μm.
https://doi.org/10.1371/journal.ppat.1009650.g008
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 13 / 27

Discussion
Multifactorial control of T3SS in Yersinia
Many bacterial pathogens use a T3SS as effective syringe-type device to inject effector proteins
into eukaryotic cells. The biosynthesis of the individual components, the correct assembly and
dynamic exchange of subunits comes at a considerable cost to the bacterium. Once fully
assembled, traffic through the T3SS must be controlled because the unrestricted translocation
of effector proteins in the absence of host contact leads to a severe growth arrest (Fig 7).
Growth inhibition might also be due to the loss of essential ions and amino acids through the
open T3SS [48–50]. It is therefore not at all surprising that T3S is a tightly regulated process
that responds to environmental and host cues with many checks and balances [51].
Since the entire T3SS is encoded on a 70-kbp virulence plasmid, the copy number of this
plasmid matters. An immediate strategy of Yersinia to boost the expression of T3SS genes
under adequate conditions is by increasing the plasmid copy number [52]. Gene dose elevation
is followed by a multi-faceted regulation of virulence gene expression. Ambient temperature
plays a key role in this process. For intestinal pathogens such as Y.pseudotuberculosis, 37˚C is
a reliable indicator of successful invasion of a mammalian host. The expression of more than
300 genes changes at least four-fold between cultures grown at 25 or 37˚C indicating a major
temperature-dependent reprogramming of bacterial metabolism and physiology [32]. Tem-
perature-responsive virulence gene expression centers around lcrF coding for the primary vir-
ulence transcription factor of the Ysc-T3SS/Yop machinery (Fig 9). Transcription of the yscW-
lcrF operon at low temperatures outside the host is partially repressed by the global histone-
like regulator YmoA. In addition, translation of residual transcripts is inhibited by an RNAT
in the intergenic region between yscW and lcrF [53,54,34]. Three mechanisms contribute to
the induction of LcrF levels at 37˚C: (i) a temperature-dependent topology change of the
yscW-lcrF promoter, (ii) the proteolytic cleavage of YmoA by the Lon and ClpP proteases, and
(iii) melting of the lcrF RNAT [34,52–54]. To make LcrF-mediated virulence gene induction
not solely dependent on the temperature signal, the pathogen has installed additional mecha-
nisms that control the concentration of the transcription factor. Signals indicating host cell
contact are integrated via the translocon protein YopD, which–when not secreted–ultimately
stimulates lcrF mRNA degradation by the degradosome [55].
Another important signal in Yersinia T3SS gene expression is Ca
2+
, a phenomenon known
as low calcium response [60]. The interplay between YopN and its partner proteins (Fig 1) is
responsible for the calcium-controlled secretion of Yops [61]. We observed a combined effect
of temperature and low calcium, which mimics the situation in the mammalian host, on yopN
mRNA (Fig 2B) and on YopN protein even in the context of a foreign promoter (Fig 7C) sug-
gesting that both transcription and translation are positively affected by low calcium by mech-
anisms not yet understood. Host cell contact-dependent transcriptional regulation of the T3SS
and effector proteins involves a number of factors apart from LcrF, including the carbon stor-
age regulator CsrA, the small RNAs CsrB and CsrC and YopD [33,55]. It is possible that this
multifactorial network also has an impact on translation efficiency of the yopN transcript.
Temperature and host cell contact (simulated by calcium depletion in vitro) certainly play a
dominant role in T3SS gene expression, but several other environmental parameters influence
this process and typically converge on lcrF expression. The [2Fe-2S] transcription factor IscR
mediates oxygen and iron regulation of the T3SS [62]. It is thought to repress T3SS expression
in the intestinal lumen and to induce T3SS expression in deeper tissues according to the grad-
ual changes in oxygen tension and iron availability. A role of the CpxAR two-component sys-
tem [63] and the Rcs phosphorelay system [64], which monitor the bacterial cell envelope
integrity, in LcrF induction indicates that various other environmental cues are fed into T3SS
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 14 / 27

regulation. It seems that Yersinia has coopted an array of regulatory pathways to sense and
integrate the overwhelming signal complexity after transition from the outside to the mamma-
lian gastrointestinal tract in order to precisely adjust the synthesis and activity of the costly and
potentially deleterious T3SS to the ambient conditions.
A novel RNAT at a critical checkpoint of T3SS
The contribution of our study to understanding the intricate control of Yersinia virulence lies
in the discovery of yet another layer of temperature regulation of the Yersinia T3SS. Unlike
LcrF-mediated control, this mechanism does not concern the expression of the entire secretion
machinery but addresses a highly specific process, the gating of the secretion channel. We
identified essentially the same RNAT in both biological isoforms of the yopN 5’-UTR, of which
the short one (37 nts) is much more abundant than the longer one (102 nts). A functional
RNAT in both a long and a short UTR also exists upstream of Y.pseudouberculosis cnfY [41].
Roughly equal activity of the RNAT in the short and long yopN transcripts can be explained by
an identical arrangement of the structure that sequesters the translation initiation region.
The architecture of the short yopN RNAT is rather simple and composed of only a single
hairpin (Fig 2B). This makes it one of the shortest known natural RNATs. Many other RNATs
contain several hairpins upstream of the decisive thermolabile structure. The adjoining, often
more stable hairpins are believed to aid in proper folding of the weaker regulatory hairpin
Fig 9. Overview of temperature-dependent regulatory pathways regarding the assembly and functionality of the T3SS. At ambient temperatures (25˚C), Yersinia
downregulates virulence-associated pathways while the flagellar synthesis is induced [56]. The global regulator YmoA represses the expression of the main virulence
regulator lcrF at 25˚C [53,57–59]. Besides, RNA thermometers (RNATs) also contribute to repression of specific genes like lcrF itself or the secretion regulator yopN
[34]. At virulence-relevant temperatures (37˚C), YmoA is degraded by proteases which leads to the derepression of lcrF [59]. Furthermore, melting of the lcrF RNAT
increases the expression induction and synthesis of the virulence regulator and thus induce the expression of T3SS genes and effector protein genes (Yop genes) [34].
The transcript of yopN posseses an RNAT that additionally induces its expression at 37˚C. In calcium-containing environments, YopN together with TyeA and the
chaperone complex SycN/YscB, prevents the secretion of Yops by blocking the T3SS channel in the cytosol. In contrast, the complex dissociates under calcium
deficiency allowing first the secretion of YopN and subsequently the secretion of further Yops into the surrounding medium [20,22,23,26,27]. Blue box: closed RNAT;
red box: open RNAT; red circles: Yops; blue circles: YopN.
https://doi.org/10.1371/journal.ppat.1009650.g009
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 15 / 27

[35]. RNAT-containing 5’-UTRs with a length below 50 nts are exceptional. One example of
such a simple helix with an internal asymmetric loop was found upstream of Synechocystis
hsp17 [37,65]. Sequence-wise, the Yersinia yopN thermometer is unique and bears no resem-
blance to RNATs in Yersinia or other bacteria. This is contrasted by the lcrF RNAT, which
belongs to the fourU thermometer family, in which the SD sequence is paired by four uridines
[34,66]. Structure-wise, the yopN thermometer follows the most common principle. The SD
region folds back onto an upstream region that has already been transcribed and is waiting for
interaction. While this might have considerable advantages in co-transcriptional RNA folding
and instantaneous repression of translation, there are exceptions to this rule. For example, the
SD sequence of the Neisseria meningitidis fHbp transcript coding for the factor H binding pro-
tein folds onto a downstream sequence in the coding region [67].
The necessary temperature-sensitivity in the yopN thermosensor is built in by three mis-
matches opposite the SD sequence. Partial stabilization of this internal loop by a central CG
pair was not sufficient to eliminate temperature responsiveness (R2 in Fig 3). Closing the
entire loop by three nucleotides complementary to the SD sequence impaired melting and
translation of the yopN mRNA at host body temperature (R1 in Figs 3–6) and resulted in mas-
sive leakage of Yops accompanied by growth cessation (Fig 7). In line with other reports
[34,40,68–71], these results show how the manipulation of just a few nucleotides in the non-
coding region of a protein-coding transcript can dramatically influence expression and the
corresponding biological outcome. The results also reinforce the concept that RNAT function
depends on a delicate balance of stabilizing and destabilizing elements that render the RNA
structure responsive to temperature fluctuations within a narrow and physiologically permis-
sive temperature range. Previous NMR studies provided in-depth insights into the critical con-
tribution of destabilizing internal bulges and loops and non-Watson-Crick-type base pairs to
the functional design of various heat shock and virulence thermometers [65,72–74].
The position of the yopN RNAT at the beginning of the multicistronic virA mRNA suggests
that the six downstream genes of the operon are unaffected by this translational control element.
RNAT-mediated differential regulation of the first or second gene of a bistronic operon has
been reported for the Salmonella groESL operon and Yersinia yscW-lcrF operon, respectively
[34,75]. RNATs or other riboregulators, such as riboswitches or small regulatory RNAs, provide
a simple means to differentially control individual genes in complex operon arrangements [76].
Why is it important to have yopN expression under such strict regulation? The conservation
of the 5’-UTR sequence in various Yersinia species strongly supports its functional relevance.
Otherwise, this sequence outside the coding region would be free to evolve. The absence of
RNAT-like elements upstream of the virB and virC operons (S3 Fig) lends further support to
the importance of the conserved RNAT upstream of yopN. It is conceivable that the additional
checkpoint imposed by the RNAT slightly delays the synthesis of YopN in comparison to
other T3SS proteins that are all under the regulatory umbrella of LcrF. Interestingly, the virA
operon is less dependent on LcrF than the other vir operons [77]. In the sequential assembly of
the T3SS it might be counterproductive to have the gatekeeper present when the T3SS is not
ready yet. Despite its limited size, YopN has at least four protein interaction partners, namely
TyeA, SycN and YscB (Fig 1), and YscI, a component of the inner rod of the T3SS [78]. The
appropriate order of intermolecular interactions between these proteins might require the pre-
cise adjustment of the cellular concentration of the individual components. Another possibility
is that YopN has presently undisclosed functions that need to be kept in check when the condi-
tions are not appropriate. Recent studies suggest that YopN has functions beyond its role as
gatekeeper of the T3SS. The centrally located coiled-coil domains of YopN encompassing
amino acids 65–100 provides a virulence-related function. The region contributes to the trans-
location of YopE and YopH, and is required for systemic infection in mice [29,30].
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 16 / 27

Another scenario, in which the yopN RNAT might play a role, is the return to lower tem-
peratures after shedding from the host to the environment. The reversible nature of zipper-like
RNATs allows them to respond to both increasing and decreasing temperatures. Adjusting
translation to the new situation and preventing the overabundance of YopN might be another
reason for the presence of an RNAT.
Our data suggests that YopN is an active rather than passive gatekeeper that exerts a direc-
tional influence on Yop secretion. The absence of YopN triggered an uncontrolled premature
burst of Yops into the medium, which prevented efficient targeting into host cells. Reduced
YopE-TEM translocation by the ΔyopN strain with or without the R1 variant was associated
with less rounded epithelial cells compared to infections with the WT strain and the D2 vari-
ant. Delayed rounding of HeLa cells infected with the ΔyopN strain and a delayed cytotoxic
response has previously been observed [46,79] and let to a model, in which YopN promotes
host cell contact [80]. In combination with the finding that YopN is involved in the secretion
of YopH and YopE [29,30], these results suggest that YopN regulates the secretion hierarchy to
accomplish an ordered translocation of Yops into host cells. Precise timing of the initiation
and termination of T3S is critical to efficiently interfere with deleterious immune cell function
such as phagocytosis after secretion. While several facets of the biological role of YopN remain
unexplored, the cumulative results of the present and other studies suggest functions of this
versatile protein that are worth further exploration. Since the incorrect intracellular concentra-
tion of YopN causes severe phenotypes, any strategy interfering with the provision of this pro-
tein might be suited to combat the pathogenic outcome of Yersinia infections.
Material and methods
Strains, plasmids and oligonucleotides
Bacterial strains and plasmids used in this study are listed in S1 Table and oligonucleotides are
listed in S2 Table. Bacteria were grown in LB medium and incubated on LB plates at the indi-
cated temperatures. For plasmid-carrying bacteria, the following final antibiotic concentra-
tions were applied: ampicillin (100 μg/mL), kanamycin (50 μg/mL), chloramphenicol (20 μg/
mL) and gentamicin (10 μg/mL).
Plasmid construction
The plasmids pBAD2-bgaB and pBAD-gfp served as backbones for reporter gene constructs
(S1 Table). The short 5’-UTR of yopN plus 30 nucleotides of the coding region were amplified
with primers yopN_short_fw and yopN_rv and cloned into NheI and EcoRI digested plasmids
to obtain translational RNAT:bgaB and RNAT:gfp fusion constructs (pBO6202 and pBO6207).
The long 5’-UTR of yopN plus 30 nucleotides of the coding region was cloned into pBAD2-
bgaB using primers yopN_long_fw and yopN_rv according to the cloning strategy described
above (pBO6203).
Plasmids containing a T7 promotor upstream of the short 5’-UTR of yopN were con-
structed for in vitro synthesis for enzymatic structure probing and primer extension inhibition.
For this purpose, the short 5’-UTR of yopN was amplified with primers yopN_rnf_toe_fw and
yopN_rnf_rv (pBO6247), and yopN_rnf_toe_fw and yopN_toe_rv (pBO6275) and cloned into
SmaI digested pK18.
Specific primers were used to introduce mutations into plasmids by site-directed mutagen-
esis to obtain the following RNAT variants: R1 (AAA13-15UCC) using yopN_R1_fw and
yopN_R1_rv (pBO6256, pBO6269, pBO6297, pBO6270, pBO7273 and pBO6297), R2 (A14C)
using yopN_R2_fw and yopN_R2_rv (pBO6257), R3 (UG4-5CA) using yopN_R3_fw and
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 17 / 27

yopN_R3_rv (pBO6258), D1 (C16A) using yopN_D1_fw and yopN_D1_rv (pBO6216) and
D2 (CG16,18AA) using yopN_D2_fw and yopN_D2_rv (pBO6255 and pBO6268).
Generation and complementation of ΔyopN
A nonpolar yopN deletion strain was constructed as described in [29]. In this mutant, a region
corresponding to amino acids 9–272 of YopN is deleted to maintain the function of the down-
stream located tyeA gene. For this purpose, a 512 bp long 5’-flank and a 446 bp 3’-flank were
amplified with primers yopN_5’_fw, yopN_5’_rv, yopN_3’_fw and yopN_3’_rv and recom-
bined by SOE-PCR [81]. The generated deletion fragment was cloned in the suicide plasmid
pDM4 using the SacI restriction site [82]. To obtain yopN deletion mutants, conjugation with
E.coli S17-1 λ-pir (donor strain) and Y.pseudotuberculosis YPIII (recipient strain) was fol-
lowed by sucrose selection. Putative ΔyopN clones were tested by PCR with internal and exter-
nal primer combinations and confirmed by DNA sequencing (S2 Fig).
For complementation of ΔyopN, a construct of the short 5’-UTR, the coding region and a
C-terminal Strep-II tag was amplified with the primers yopN_comp_strep_fw and yopN_-
comp_strep_rv, and ligated into NcoI and PstI digested pGM930 (pBO7423). Using this plas-
mid, expression of yopN is induced by the addition of 0.05% (w/v) L-arabinose [83]. To obtain
the closed yopN RNAT R1 (AAA13-15UCC) and the open variant D2 (CG16,18AA), primers
yopN_R1_fw and yopN_R1_rv and yopN_D2_fw and yopN_D2_rv were used for site directed
mutagenesis (pBO7440 and pBO7801), respectively.
RNA isolation
Cell pellets from 4 mL culture samples were resuspended in 250 μL TE buffer (1 mM EDTA,
10 mM Tris, pH 7.5) and 12.5 μL SDS (10% w/v). Then, 450 μL of phenol was added and the
samples were incubated at 60˚C for 10 min. After 1 h on ice and centrifugation (1 h, 13000
rpm, 4˚C), 450 μL phenol and 43 μL sodium acetate (3 M, pH 5.5) were added to the aqueous
phase followed by centrifugation (5 min, 13000 rpm, 4˚C). The aqueous phase was then mixed
with 450 μL chloroform and centrifuged (5 min, 13000 rpm, 4˚C). This step was repeated.
After ethanol precipitation and drying for 20 min at 30˚C, the RNA pellets were resuspended
in 40 μL sterile water (Carl Roth GmbH, Karlsruhe, Germany).
Quantitative real-time PCR (qRT-PCR)
For the analysis of relative transcript levels of the short and long 5’-UTR of yopN, samples of Y.
pseudotuberculois YPIII cells for RNA isolation were taken during the early exponential phase
at an optical density (OD
600
) of 0.5 under non-secretion (2.5 mM CaCl
2
) and secretion (20
mM MgCl
2
, 20 mM sodium oxalate) conditions in LB medium at 25 and 37˚C. Transcript lev-
els were determined from three independent cultures measured in technical duplicate. RNA
was isolated as described above. For further details on how the qRT-PCR was performed, see
[41]. For the calculation of relative yopN transcript levels, the primer efficiency corrected
method was used [84]. The non-thermoregulated reference genes nuoB and gyrB served for
normalization [32]. Primer efficiencies were calculated by the CFX Maestro software (nuoB:
100.8%, gyrB: 101.3%, yopN short: 95.3%, yopN long: 102.8%).
Northern blot analysis
Northern blot experiments were carried out as described in [85]. pBAD2-gfp served as a DNA
template for the amplification of a 286 bp fragment possessing a T7 RNA promotor. Based on
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 18 / 27

the described fragment, an in vitro produced and DIG-labeled gfp RNA probe (Roche, Mann-
heim, Germany) was prepared for the detection of gfp transcripts.
Reporter gene assays
Y.pseudotuberculosis YPIII cells carrying plasmids of bgaB fusion constructs and mutated vari-
ants were grown in LB with ampicillin to an OD
600
of 0.5 at 25˚C. Subsequently, the transcrip-
tion was induced with 0.1% (w/v) L-arabinose and the cultures were split to flasks at 25˚C and
prewarmed flasks at 37˚C and incubated for further 30 minutes. Samples containing the bgaB
fusions were then taken for the β-galactosidase assay and Western blot analysis. The mean
activities of Miller Units and the mean standard deviations were calculated from nine biologi-
cal replicates. The β-galactosidase activity was measured as described in [86].
For cells carrying plasmids with gfp fusion constructs, the growth and induction was per-
formed as described for bgaB. A total of 4 mL of cell suspension were used for Northern blot
analysis and 2 mL of cell suspension were used for Western blot analysis.
Western blot analysis
For the preparation of crude protein extracts, cell pellets of Y.pseudotuberculosis YPIII and
ΔyopN were resuspended in 1 x SDS sample buffer (2% (w/v) SDS, 12.5 mM EDTA, 1% (v/v)
β-mercaptoethanol, 10% (v/v) glycerol, 0.02% (w/v) bromophenol blue, 50 mM Tris, pH 6.8)
and protein amounts were adjusted by OD
600
(50 μL per OD
600
of 1). Resuspended protein
samples were heated at 95˚C for 10 min, centrifuged (5 min, 13000 rpm) and loaded to a
12.5% SDS polyacrylamide gel. After SDS-PAGE, proteins were blotted onto a nitrocellulose
membrane (Hybond-C Extra, GE Healthcare, Munich, Germany) Ponceau S staining of the
blotted membrane was performed to assure equal amounts of proteins. For the detection of
BgaB-His, a penta-His HRP conjugate was used (1:4000, QIAGEN GmbH, Hilden, Germany).
The detection of GFP was performed with the primary antibody anti-GFP (1:10000, Thermo
Scientific, Waltham, USA) followed by the secondary antibody goat anti-rabbit HRP conjugate
(1:4000, Bio Rad, Munich, Germany). In the case of YopN-Strep, the protein was detected
using the strep-tactin HRP conjugate (IBA Lifesciences, Go¨ttingen, Germany) as specified by
the manufacturer. Furthermore, for the visualization of secreted Yops, a total Yop antiserum
[40] was used (1:20000) followed by the secondary antibody goat anti-rabbit HRP conjugate
(1:4000, Bio Rad, Munich, Germany). Protein signals on the membranes were detected with
Immobilon Forte Western HRP substrate (Merck, Darmstadt, Germany) and the ChemiIma-
ger Ready (Alpha Innotec,San Leandro USA).
Enzymatic structure probing
To enzymatically probe RNA structures at different temperatures, RNA was first synthesized
by in vitro run-off transcription using T7 RNA polymerase (Thermo Scientific, Waltham,
USA) and the EcoRV-linearized plasmids as described in [38]. For structure probing, the plas-
mids pBO6247 and pBO6270 were used to synthesize RNA consisting of the short 5’-UTR of
yopN plus 30 nt of the coding region and the variant R1. Purified and desphosphorylated RNA
was labeled with [
32
P] at the 5’ end as described in [87]. According to [66], limited digestion of
radiolabeled RNA was performed with the ribonuclease T1 (0.005 U) (Thermo Scientific, Wal-
tham, USA) and T2 (0.074 U) (MoBiTec, Go¨ttingen, Germany) in 5 x TN buffer (500 mM
NaCl, 100 mM Tris acetate, pH 7) at 25, 37 and 42˚C. Furthermore, an alkaline hydrolysis lad-
der [87] and a T1 ladder was prepared. For the T1 ladder, 30000 cpm of labeled RNA was
heated with 1 μL sequencing buffer (provided with RNase T1) to 90˚C and then incubated
with the nuclease at 37˚C for additional 5 min.
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 19 / 27

Primer extension inhibition assay (toeprinting)
In vitro transcribed RNA produced with the plasmids pBO6265 and pBO6273 (short 5’-UTR
of yopN plus 60 nt of the coding region and variant R1), 5’-[
32
P]-labeled reverse primer
yopN_short_toe_rv, 30S ribosomal subunits and tRNA
fMet
(Sigma-Aldrich, St. Louis, USA)
were used for the toeprinting assay as described in [88]. First, the annealing mix consisting of
0.08 pmol RNA and 0.16 pmol radiolabeled primer were mixed with 1 x VD-Mg
2+
(60 mM
NH
4
Cl, 6 mM β-mercaptoethanol, 10 mM Tris/HCl, pH 7.4) and incubated for 3 min at 80˚C.
For binding of 30S ribosomal subunits, the annealing mix was mixed with 16 ρmol tRNA
fMet
,
0.16 ρmol radiolabeled primer and 6 ρmol 30S ribosomal subunit in Watanabe buffer (60 mM
HEPES/KOH, 10.5 mM Mg(CH
3
COO)
2
, 690 mM NH
4
COO, 12 mM β-mercaptoethanol, 10
mM spermidine, 0.25 mM spermine) and incubated for 10 min at 25, 37 and 42˚C. Subse-
quently, a M-MLV mix (1 x VD+Mg
2+
(10 mM Mg(CH
3
COO)
2
, 6 μg BSA, 4 mM dNTPs, 800
U M-MLV reverse transcriptase (Thermo Scientific, Waltham, USA)) was added to initiate the
cDNA synthesis for 10 min at 37˚C. After the addition of formamide loading dye, the reaction
was stopped and the samples were separated on an 8% polyacrylamide gel. A sequencing lad-
der was produced with the Thermo Sequenase cycle sequencing Kit (Thermo Scientific, Wal-
tham, USA), the template pBO6265 and the radiolabeled primer yopN_short_toe_rv.
Growth experiments and Yop secretion
Y.pseudotuberculosis YPIII and ΔyopN carrying complementation constructs (pGM930) were
grown under non-secretion (2.5 mM CaCl
2
) and secretion conditions (20 mM MgCl
2
, 20 mM
sodium oxalate) in LB containing 0.05% (w/v) L-arabinose and ampicillin for 10 h at 25 and
37˚C h. After 7 h, a total of 9 mL was collected, adjusted to an OD
600
of 0.8 and centrifuged (10
min, 4000 rpm, 4˚C). To the sterile filtered supernatant, 1 mL of 100% (w/v) TCA was added
to precipitate secreted proteins overnight at 4˚C followed by a centrifugation (20 min, 13000
rpm, 4˚C). Protein pellets were first resuspended in 0.5 mL 2% SDS solution and then mixed
with 1.5 mL of ice-cold 100% acetone. The samples were incubated for 30 min at -20˚C. After
washing twice with 0.5 mL acetone, pellets were dried for 15 min at 25˚C. Finally, pellets were
resuspended in 25 μL of 2 x SDS sample buffer (4% (w/v) SDS, 25 mM EDTA, 2% (v/v) β-mer-
captoethanol, 20% (v/v) glycerol, 0.04% (w/v) bromophenol blue, 100 mM Tris, pH 6.8) and
5μL were loaded to an 12.5% SDS acrylamide gel to separate secreted Yersinia effector proteins
(Yops).
YopE translocation assay
The YopE translocation assay was performed to examine the YopE secretion ability of Y.
pseudotuberculosis strains carrying complementation constructs (pGM930) and pMK-Bla
(YopE-TEM, [89] using LiveBLAzer-FRET B/G Loading Kit (Life Technologies, Carlsbad,
USA). Strains were pregrown in LB containing 0.1% (w/v) L-arabinose, 1 mM CaCl
2
, ampicil-
lin and kanamycin for 2 h at 25˚C. The bacteria were then shifted to 37˚C, incubated for addi-
tional 2 h, washed and adjusted in PBS buffer to an OD
600
of 1. HEp-2 cells (1.7x10
4
) seeded
in μ-Slides (8-well, Ibidi, Gra¨felfing, Germany) were infected with Y.pseudotuberculosis strains
at MOI of 50 and centrifuged for 5 min (400 g, RT). This was followed by incubation for 60
min at 37˚C and three washing steps of the cells with PBS buffer. Then, 200 μL of infection
buffer (RPMI, 20 mM HEPES, 0.4% BSA) containing gentamicin (25 μg/mL) were added and
HEp-2 cells were stained with CCF4-AM loading dye according to the manufacturer’s proto-
col. YopE-TEM translocation was visualized using a fluorescence microscope (BZ-9000 Fluo-
rescence Microscope, Keyence, Osaka, Japan).
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 20 / 27

Supporting information
S1 Table. Bacterial strains and plasmids used in this study.
(DOCX)
S2 Table. Oligonucleotides used in this study.
(DOCX)
S1 Fig. Sequence alignment of the yopN 5’-UTR of different Yersinia species. Sequence
comparison of the yopN 5’-UTRs and 30 nucleotides of the coding region between Y.pseudotu-
berculosis,Y.pestis and Y.enterocolitica. Bold black nucleotides indicate the putative SD region
and start codon while red nucleotides indicate sequence variations between Yersinia species. l:
long transcript; s: short transcript.
(DOCX)
S2 Fig. Confirmation of the ΔyopN mutant by PCR. (A) Altered DNA fragment sizes due to
yopN deletion using primers that bind within yopN (I:internal primers) and up- and down-
stream of the gene locus. Due to yopN deletion, no DNA fragment is produced with internal
primers compared to the wild type (WT). L: DNA ladder; e: external primers; i: internal prim-
ers. (B) Schematic overview of primer localization.
(DOCX)
S3 Fig. A functional RNAT is only present in the 5’-UTR of virA (yopN). (A) PARS-derived
RNA structures of the 5’-UTRs of yscN (virB) and yscA (virC). The putative SD regions are
highlighted in gray, the start codons in red. (B) Plasmid-based translational fusions of 5’-UTRs
of interest and bgaB encoding a heat-stable β-galactosidase to test RNA thermometer (RNAT)
functionality. Transcription of the fusion products is controlled by the arabinose-inducible
promotor P
BAD
. The β-galactosidase assays of the short 5’-UTR of yopN (virA), the 5’-UTR of
yscN (virB) and the short and long 5’-UTRs of yscA (virC) were conducted at 25 and 37˚C. Y.
pseudotuberculosis YPIII cells carrying plasmids of the fusion constructs were grown to an
OD
600
of 0.5 at 25˚C. Subsequently, transcription of the reporter gene was induced by 0.1%
(w/v) L-arabinose and the cultures were split to flasks at 25 and prewarmed flasks at 37˚C and
incubated for further 30 minutes. Samples were then taken for the β-galactosidase assay. The
mean activities in Miller Units and the mean standard deviations were calculated from nine
biological replicates. The representative Western blot displays the amount of BgaB-His pro-
duced. Protein amounts were adjusted to an optical density of 0.5 and detected by Ponceau S
staining after blotting onto a nitrocellulose membrane.
(DOCX)
S4 Fig. Transcript levels of yopN from cultures of the growth experiment shown in Fig 7.
Comparison of relative transcript levels of endogenous expressed yopN in the wild type (WT)
and induced yopN expression for strains carrying plasmid constructs (yopN, R1 and D2)
under non-secretion (+ Ca
2+
) and secretion (- Ca
2+
) conditions at 25 and 37˚C. Samples of Y.
pseudotuberculosis YPIII strains were taken after 5 h followed by RNA isolation and qRT-PCR.
Transcript levels were normalized to the amount of WT at 25˚C under non-secretion condi-
tions and to the reference genes nuoB and gyrB. The mean transcript amounts and standard
deviations comprise the results of three biological replicates. Primer efficiencies were calcu-
lated by the CFX Maestro software (nuoB: 98.1%, gyrB: 95.4%, yopN short: 93.1%).
(DOCX)
S5 Fig. YopE translocation into HEp-2 cells. (A) The images show different cell morpholo-
gies of HEp-2 cells infected with Y.pseudotuberculosis YPIII wild type (WT) and different
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 21 / 27

ΔyopN strains carrying either the empty vector (EV) or arabinose-inducible constructs of
yopN with the wild type RNAT, the stable variant R1 or the open variant D2. Rounded HEp-2
cells indicate cytoskeleton-damaging activity of translocated YopE-TEM. Scale bars: 25 μm.
All strains carry vector pMK-bla coding for a yopE-bla
TEM
fusion. HEp-2 cells were infected
with bacterial strains at an MOI of 50. (B) Time course YopE translocation assay of HEp-2
cells infected with the aforementioned strains (MOI of 50) for 120 min at 37˚C. Cells were
labeled with CCF4-AM and the ratio between blue and green fluorescence (520 nm / 450 nm;
normalized against uninfected cells) was measured every minute using the CLARIOstar Plus
plate reader (BMG Labtech). Therefore, HEp-2 cells (2 x 10
4
) were seeded in 100 μL RPMI
1640 containing 7.5% NCS in 96-well plate and were cultivated in a 5% CO
2
incubator at 37˚C.
Adherent cells were treated with CCF4-AM in the dark for 1 h at room temperature and the
change of fluorescence was immediately measured after infection of Y.pseudotuberculosis
strains at 37˚C.
(DOCX)
Acknowledgments
We thank Johanna Roßmanith for advice in the initial stage of this project, the RNA group for
continuous discussions and Alexander Kraus for critical reading of an earlier version of this
manuscript.
Author Contributions
Conceptualization: Stephan Pienkoß, Christian Twittenhoff, Petra Dersch, Franz Narberhaus.
Data curation: Franz Narberhaus.
Formal analysis: Stephan Pienkoß.
Funding acquisition: Franz Narberhaus.
Investigation: Stephan Pienkoß, Soheila Javadi, Paweena Chaoprasid, Thomas Nolte.
Methodology: Stephan Pienkoß, Soheila Javadi, Paweena Chaoprasid.
Project administration: Franz Narberhaus.
Resources: Petra Dersch, Franz Narberhaus.
Supervision: Petra Dersch, Franz Narberhaus.
Validation: Stephan Pienkoß.
Visualization: Stephan Pienkoß.
Writing – original draft: Stephan Pienkoß.
Writing – review & editing: Paweena Chaoprasid, Petra Dersch, Franz Narberhaus.
References
1. Coburn B, Sekirov I, Finlay BB. Type III Secretion systems and disease. Clin Microbiol Rev. 2007; 20:
535–549. https://doi.org/10.1128/CMR.00013-07 PMID: 17934073
2. Bu¨ttner D. Protein export according to schedule: architecture, assembly, and regulation of type III secre-
tion systems from plant- and animal-pathogenic bacteria. Microbiol Mol Biol Rev. 2012; 76: 262–310.
https://doi.org/10.1128/MMBR.05017-11 PMID: 22688814
3. Deng W, Marshall NC, Rowland JL, McCoy JM, Worrall LJ, Santos AS, et al. Assembly, structure, func-
tion and regulation of type III secretion systems. Nat Rev Microbiol. 2017; 15: 323–337. https://doi.org/
10.1038/nrmicro.2017.20 PMID: 28392566
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 22 / 27

4. Me
´resse S, Unsworth KE, Habermann A, Griffiths G, Fang F, Martı
´nez-Lorenzo MJ, et al. Remodelling
of the actin cytoskeleton is essential for replication of intravacuolar Salmonella. Cell Microbiol. 2001; 3:
567–577. https://doi.org/10.1046/j.1462-5822.2001.00141.x PMID: 11488817
5. Ogawa M, Yoshimori T, Suzuki T, Sagara H, Mizushima N, Sasakawa C. Escape of intracellular Shi-
gella from autophagy. Science. 2005; 307: 727–731. https://doi.org/10.1126/science.1106036 PMID:
15576571
6. Bliska JB, Wang X, Viboud GI, Brodsky IE. Modulation of innate immune responses by Yersinia type III
secretion system translocators and effectors. Cell Microbiol. 2013; 15: 1622–1631. https://doi.org/10.
1111/cmi.12164 PMID: 23834311
7. Santos AS, Finlay BB. Bringing down the host: enteropathogenic and enterohaemorrhagic Escherichia
coli effector-mediated subversion of host innate immune pathways. Cell Microbiol. 2015; 17: 318–332.
https://doi.org/10.1111/cmi.12412 PMID: 25588886
8. Portnoy DA, Wolf-Watz H, Bolin I, Beeder AB, Falkow S. Characterization of common virulence plas-
mids in Yersinia species and their role in the expression of outer membrane proteins. Infect Immun.
1984; 43: 108–114. https://doi.org/10.1128/iai.43.1.108-114.1984 PMID: 6317562
9. Perry RD, Fetherston JD. Yersinia pestis—etiologic agent of plague. Clin Microbiol Rev. 1997; 10: 35–
66. https://doi.org/10.1128/CMR.10.1.35 PMID: 8993858
10. Galindo CL, Rosenzweig JA, Kirtley ML, Chopra AK. Pathogenesis of Y. enterocolitica and Y. pseudotu-
berculosis in human yersiniosis. J Pathog. 2011; 2011: 182051. https://doi.org/10.4061/2011/182051
PMID: 22567322
11. Diepold A. Assembly and post-assembly turnover and dynamics in the type III secretion system. Curr
Top Microbiol Immunol. 2020; 427: 35–66. https://doi.org/10.1007/82_2019_164 PMID: 31218503
12. Volk M, Vollmer I, Heroven AK, Dersch P. Transcriptional and post-transcriptional regulatory mecha-
nisms controlling type III secretion. Curr Top Microbiol Immunol. 2020; 427: 11–33. https://doi.org/10.
1007/82_2019_168 PMID: 31218505
13. Dewoody R, Merritt PM, Marketon MM. Regulation of the Yersinia type III secretion system: traffic con-
trol. Front Cell Infect Microbiol. 2013;3. https://doi.org/10.3389/fcimb.2013.00003 PMID: 23408095
14. Stainier I, Bleves S, Josenhans C, Karmani L, Kerbourch C, Lambermont I, et al. YscP, a Yersinia pro-
tein required for Yop secretion that is surface exposed, and released in low Ca2+. Mol Microbiol. 2000;
37: 1005–1018. https://doi.org/10.1046/j.1365-2958.2000.02026.x PMID: 10972820
15. Journet L, Agrain C, Broz P, Cornelis GR. The needle length of bacterial injectisomes is determined by
a molecular ruler. Science. 2003; 302: 1757–1760. https://doi.org/10.1126/science.1091422 PMID:
14657497
16. Agrain C, Sorg I, Paroz C, Cornelis GR. Secretion of YscP from Yersinia enterocolitica is essential to
control the length of the injectisome needle but not to change the type III secretion substrate specificity.
Mol Microbiol. 2005; 57: 1415–1427. https://doi.org/10.1111/j.1365-2958.2005.04758.x PMID:
16102009
17. Mueller CA, Broz P, Mu¨ller SA, Ringler P, Erne-Brand F, Sorg I, et al. The v-antigen of Yersinia forms a
distinct structure at the tip of injectisome needles. Science. 2005; 310: 674–676. https://doi.org/10.
1126/science.1118476 PMID: 16254184
18. Mueller CA, Broz P, Cornelis GR. The type III secretion system tip complex and translocon. Mol Micro-
biol. 2008; 68: 1085–1095. https://doi.org/10.1111/j.1365-2958.2008.06237.x PMID: 18430138
19. Montagner C, Arquint C, Cornelis GR. Translocators YopB and YopD from Yersinia enterocolitica form
a multimeric integral membrane complex in eukaryotic cell membranes. J Bacteriol. 2011; 193: 6923–
6928. https://doi.org/10.1128/JB.05555-11 PMID: 22001511
20. Iriarte M, Sory M-P, Boland A, Boyd AP, Mills SD, Lambermont I, et al. TyeA, a protein involved in con-
trol of Yop release and in translocation of Yersinia Yop effectors. EMBO J. 1998; 17: 1907–1918.
https://doi.org/10.1093/emboj/17.7.1907 PMID: 9524114
21. Amer AAA, Gurung JM, Costa TRD, Ruuth K, Zavialov AV, Forsberg Å, et al. YopN and TyeA hydropho-
bic contacts required for regulating Ysc-Yop type III secretion activity by Yersinia pseudotuberculosis.
Front Cell Infect Microbiol. 2016;6. https://doi.org/10.3389/fcimb.2016.00006 PMID: 26913242
22. Cheng LW, Kay O, Schneewind O. Regulated secretion of YopN by the type III machinery of Yersinia
enterocolitica. J Bacteriol. 2001; 183: 5293–5301. https://doi.org/10.1128/JB.183.18.5293-5301.2001
PMID: 11514512
23. Ferracci F, Schubot FD, Waugh DS, Plano GV. Selection and characterization of Yersinia pestis YopN
mutants that constitutively block Yop secretion. Mol Microbiol. 2005; 57: 970–987. https://doi.org/10.
1111/j.1365-2958.2005.04738.x PMID: 16091038
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 23 / 27

24. Ferracci F, Day JB, Ezelle HJ, Plano GV. Expression of a functional secreted YopN-TyeA hybrid protein
in Yersinia pestis is the result of a +1 translational frameshift event. J Bacteriol. 2004; 186: 5160–5166.
https://doi.org/10.1128/JB.186.15.5160-5166.2004 PMID: 15262954
25. Amer AAA, Costa TRD, Farag SI, Avican U, Forsberg Å, Francis MS. Genetically engineered frame-
shifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculo-
sis. PLoS One. 2013; 8: e77767. https://doi.org/10.1371/journal.pone.0077767 PMID: 24098594
26. Yother J, Goguen JD. Isolation and characterization of Ca2+-blind mutants of Yersinia pestis. J Bacter-
iol. 1985; 164: 704–711. https://doi.org/10.1128/jb.164.2.704-711.1985 PMID: 2997127
27. Forsberg Å, Viitanen A-M, Skurnik M, Wolf-Watz H. The surface-located YopN protein is involved in cal-
cium signal transduction in Yersinia pseudotuberculosis. Mol Microbiol. 1991; 5: 977–986. https://doi.
org/10.1111/j.1365-2958.1991.tb00773.x PMID: 1857212
28. Day JB, Ferracci F, Plano GV. Translocation of YopE and YopN into eukaryotic cells by Yersinia pestis
yopN,tyeA,sycN,yscB and lcrG deletion mutants measured using a phosphorylatable peptide tag and
phosphospecific antibodies. Mol Microbiol. 2003; 47: 807–823. https://doi.org/10.1046/j.1365-2958.
2003.03343.x PMID: 12535078
29. Bamyaci S, Ekestubbe S, Nordfelth R, Erttmann SF, Edgren T, Forsberg Å. YopN is required for effi-
cient effector translocation and virulence in Yersinia pseudotuberculosis. Infect Immun. 2018;86.
https://doi.org/10.1128/IAI.00957-17 PMID: 29760214
30. Bamyaci S, Nordfelth R, Forsberg Å. Identification of specific sequence motif of YopN of Yersinia
pseudotuberculosis required for systemic infection. Virulence. 2019; 10: 10–25. https://doi.org/10.1080/
21505594.2018.1551709 PMID: 30488778
31. Han Y, Zhou D, Pang X, Song Y, Zhang L, Bao J, et al. Microarray analysis of temperature-induced tran-
scriptome of Yersinia pestis. Microbiol Immunol. 2004; 48: 791–805. https://doi.org/10.1111/j.1348-
0421.2004.tb03605.x PMID: 15557737
32. Nuss AM, Heroven AK, Waldmann B, Reinkensmeier J, Jarek M, Beckstette M, et al. Transcriptomic
profiling of Yersinia pseudotuberculosis reveals reprogramming of the Crp regulon by temperature and
uncovers Crp as a master regulator of small RNAs. PLoS Genet. 2015; 11: e1005087. https://doi.org/
10.1371/journal.pgen.1005087 PMID: 25816203
33. Nuss AM, Beckstette M, Pimenova M, Schmu¨hl C, Opitz W, Pisano F, et al. Tissue dual RNA-seq allows
fast discovery of infection-specific functions and riboregulators shaping host–pathogen transcriptomes.
Proc Natl Acad Sci. 2017; 114: E791–E800. https://doi.org/10.1073/pnas.1613405114 PMID:
28096329
34. Bo
¨hme K, Steinmann R, Kortmann J, Seekircher S, Heroven AK, Berger E, et al. Concerted actions of a
thermo-labile regulator and a unique intergenic RNA thermosensor control Yersinia virulence. PLoS
Pathog. 2012; 8: e1002518. https://doi.org/10.1371/journal.ppat.1002518 PMID: 22359501
35. Kortmann J, Narberhaus F. Bacterial RNA thermometers: molecular zippers and switches. Nat Rev
Microbiol. 2012; 10: 255–265. https://doi.org/10.1038/nrmicro2730 PMID: 22421878
36. Loh E, Righetti F, Eichner H, Twittenhoff C, Narberhaus F. RNA Thermometers in bacterial pathogens.
Microbiol Spectr. 2018. 6:55–73. https://doi.org/10.1128/microbiolspec.RWR-0012-2017 PMID:
29623874
37. Kortmann J, Sczodrok S, Rinnenthal J, Schwalbe H, Narberhaus F. Translationon demand by a simple
RNA-based thermosensor. Nucleic Acids Res. 2011; 39: 2855–2868. https://doi.org/10.1093/nar/
gkq1252 PMID: 21131278
38. Righetti F, Nuss AM, Twittenhoff C, Beele S, Urban K, Will S, et al. Temperature-responsive in vitro
RNA structurome of Yersinia pseudotuberculosis. Proc Natl Acad Sci. 2016; 113: 7237–7242. https://
doi.org/10.1073/pnas.1523004113 PMID: 27298343
39. Twittenhoff C, Brandenburg VB, Righetti F, Nuss AM, Mosig A, Dersch P, et al. Lead-seq: transcrip-
tome-wide structure probing in vivo using lead(II) ions. Nucleic Acids Res. 2020; 48: e71–e71. https://
doi.org/10.1093/nar/gkaa404 PMID: 32463449
40. Schweer J, Kulkarni D, Kochut A, Pezoldt J, Pisano F, Pils MC, et al. The cytotoxic necrotizing factor of
Yersinia pseudotuberculosis (CNFY) enhances inflammation and Yop delivery during infection by acti-
vation of Rho GTPases. PLoS Pathog. 2013; 9: e1003746. https://doi.org/10.1371/journal.ppat.
1003746 PMID: 24244167
41. Twittenhoff C, Heroven AK, Mu¨hlen S, Dersch P, Narberhaus F. An RNA thermometer dictates produc-
tion of a secreted bacterial toxin. PLoS Pathog. 2020; 16: e1008184. https://doi.org/10.1371/journal.
ppat.1008184 PMID: 31951643
42. Chaoprasid P, Lukat P, Mu¨hlen S, Heidler T, Gazdag E-M, Dong S, et al. Crystal structure of bacterial
cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication. EMBO J. 2021; 40:
e105202. https://doi.org/10.15252/embj.2020105202 PMID: 33410511
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 24 / 27

43. Monnappa AK, Bari W, Seo JK, Mitchell RJ. The cytotoxic necrotizing factor of Yersinia pseudotubercu-
losis (CNFy) is carried on extracellular membrane vesicles to host cells. Sci Rep. 2018; 8:14186.
https://doi.org/10.1038/s41598-018-32530-y PMID: 30242257
44. Miller HK, Kwuan L, Schwiesow L, Bernick DL, Mettert E, Ramirez HA, et al. IscR is essential for Yersi-
nia pseudotuberculosis type III secretion and virulence. PLoS Pathog. 2014; 10: e1004194. https://doi.
org/10.1371/journal.ppat.1004194 PMID: 24945271
45. Murphy ER, Roßmanith J, Sieg J, Fris ME, Hussein H, Kouse AB, et al. Regulation of OmpA translation
and Shigella dysenteriae virulence by an RNA thermometer. Infect Immun. 2020;88. https://doi.org/10.
1128/IAI.00871-19 PMID: 31792074
46. Rosqvist R, Magnusson KE, Wolf-Watz H. Target cell contact triggers expression and polarized transfer
of Yersinia YopE cytotoxin into mammalian cells. EMBO J. 1994; 13: 964–972. https://doi.org/10.1002/
j.1460-2075.1994.tb06341.x PMID: 8112310
47. Black DS, Bliska JB. The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is
required for antiphagocytic function and virulence. Mol Microbiol. 2000; 37: 515–527. https://doi.org/10.
1046/j.1365-2958.2000.02021.x PMID: 10931345
48. Brubaker RR. Influence of Na+, dicarboxylic amino acids, and pH in modulating the low-calcium
response of Yersinia pestis. Infect Immun. 2005; 73: 4743–4752. https://doi.org/10.1128/IAI.73.8.4743-
4752.2005 PMID: 16040987
49. Fowler JM, Wulff CR, Straley SC, Brubaker RR. Growth of calcium-blind mutants of Yersinia pestis at
37˚C in permissive Ca2+-deficient environments. Microbiology. 2009; 155: 2509–2521. https://doi.org/
10.1099/mic.0.028852-0 PMID: 19443541
50. Sturm A, Heinemann M, Arnoldini M, Benecke A, Ackermann M, Benz M, et al. The cost of virulence:
retarded growth of Salmonella Typhimurium cells expressing type III secretion system 1. PLoS Pathog.
2011; 7: e1002143. https://doi.org/10.1371/journal.ppat.1002143 PMID: 21829349
51. Nisco NJD, Rivera-Cancel G, Orth K. The biochemistry of sensing: enteric pathogens regulate type III
secretion in response to environmental and host cues. mBio. 2018;9. https://doi.org/10.1128/mBio.
02122-17 PMID: 29339429
52. Wang H, Avican K, Fahlgren A, Erttmann SF, Nuss AM, Dersch P, et al. Increased plasmid copy num-
ber is essential for Yersinia T3SS function and virulence. Science. 2016; 353: 492–495. https://doi.org/
10.1126/science.aaf7501 PMID: 27365311
53. Cornelis GR, Sluiters C, Delor I, Geib D, Kaniga K, Rouvroit CL de, et al. ymoA, a Yersinia enterocolitica
chromosomal gene modulating the expression of virulence functions. Mol Microbiol. 1991;5: 1023–
1034. https://doi.org/10.1111/j.1365-2958.1991.tb01875.x PMID: 1956283
54. Hoe NP, Goguen JD. Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is
thermally regulated. J Bacteriol. 1993; 175: 7901–7909. https://doi.org/10.1128/jb.175.24.7901-7909.
1993 PMID: 7504666
55. Jackson MW, Silva-Herzog E, Plano GV. The ATP-dependent ClpXP and Lon proteases regulate
expression of the Yersinia pestis type III secretion system via regulated proteolysis of YmoA, a small
histone-like protein. Mol Microbiol. 2004; 54: 1364–1378. https://doi.org/10.1111/j.1365-2958.2004.
04353.x PMID: 15554975
56. Kusmierek M, Hoßmann J, Witte R, Opitz W, Vollmer I, Volk M, et al. A bacterial secreted translocator
hijacks riboregulators to control type III secretion in response to host cell contact. PLoS Pathog. 2019;
15: e1007813. https://doi.org/10.1371/journal.ppat.1007813 PMID: 31173606
57. Kapatral V, Olson JW, Pepe JC, Miller VL, Minnich SA. Temperature-dependent regulation of Yersinia
enterocolitica class III flagellar genes. Mol Microbiol. 1996; 19: 1061–1071. https://doi.org/10.1046/j.
1365-2958.1996.452978.x PMID: 8830263
58. Rohde JR, Fox JM, Minnich SA. Thermoregulation in Yersinia enterocolitica is coincident with changes
in DNA supercoiling. Mol Microbiol. 1994; 12: 187–199. https://doi.org/10.1111/j.1365-2958.1994.
tb01008.x PMID: 8057844
59. Rohde JR, Luan X, Rohde H, Fox JM, Minnich SA. The Yersinia enterocolitica pYV virulence plasmid
contains multiple intrinsic DNA bends which melt at 37˚C. J Bacteriol. 1999; 181: 4198–4204. https://
doi.org/10.1128/JB.181.14.4198-4204.1999 PMID: 10400576
60. Schneewind O. Classic spotlight: studies on the low-calcium response of Yersinia pestis reveal the
secrets of plague pathogenesis. J Bacteriol. 2016; 198: 2018. https://doi.org/10.1128/JB.00358-16
PMID: 27413177
61. Plano GV, Joseph SS. The SycN/YscB chaperone-binding domain of YopN is required for the calcium-
dependent regulation of Yop secretion by Yersinia pestis. Front Cell Infect Microbiol. 2013;3. https://doi.
org/10.3389/fcimb.2013.00003 PMID: 23408095
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 25 / 27

62. Hooker-Romero D, Mettert E, Schwiesow L, Balderas D, Alvarez PA, Kicin A, et al. Iron availability and
oxygen tension regulate the Yersinia Ysc type III secretion system to enable disseminated infection.
PLoS Pathog. 2019; 15: e1008001. https://doi.org/10.1371/journal.ppat.1008001 PMID: 31869388
63. Fei K, Chao H-J, Hu Y, Francis MS, Chen S. CpxR regulates the Rcs phosphorelay system in controlling
the Ysc-Yop type III secretion system in Yersinia pseudotuberculosis. Microbiology. 2021; 167: 000998.
https://doi.org/10.1099/mic.0.000998 PMID: 33295859
64. Li Y, Hu Y, Francis MS, Chen S. RcsB positively regulates the Yersinia Ysc-Yop type III secretion sys-
tem by activating expression of the master transcriptional regulator LcrF. Environ Microbiol. 2015; 17:
1219–1233. https://doi.org/10.1111/1462-2920.12556 PMID: 25039908
65. Wagner D, Rinnenthal J, Narberhaus F, Schwalbe H. Mechanistic insights into temperature-dependent
regulation of the simple cyanobacterial hsp17 RNA thermometer at base-pair resolution. Nucleic Acids
Res. 2015; 43: 5572–5585. https://doi.org/10.1093/nar/gkv414 PMID: 25940621
66. Waldminghaus T, Heidrich N, Brantl S, Narberhaus F. FourU: a novel type of RNA thermometer in Sal-
monella. Mol Microbiol. 2007; 65: 413–424. https://doi.org/10.1111/j.1365-2958.2007.05794.x PMID:
17630972
67. Loh E, Lavender H, Tan F, Tracy A, Tang CM. Thermoregulation of meningococcal fHbp, an important
virulence factor and vaccine antigen, is mediated by anti-ribosomal binding site sequencesin the open
reading frame. PLoS Pathog. 2016; 12: e1005794. https://doi.org/10.1371/journal.ppat.1005794 PMID:
27560142
68. Loh E, Kugelberg E, Tracy A, Zhang Q, Gollan B, Ewles H, et al. Temperature triggers immune evasion
by Neisseria meningitidis. Nature. 2013; 502: 237–240. https://doi.org/10.1038/nature12616 PMID:
24067614
69. Weber GG, Kortmann J, Narberhaus F, Klose KE. RNA thermometer controls temperature-dependent
virulence factor expression in Vibrio cholerae. Proc Natl Acad Sci. 2014; 111: 14241–14246. https://doi.
org/10.1073/pnas.1411570111 PMID: 25228776
70. Blanka A, Du¨vel J, Do
¨tsch A, Klinkert B, Abraham W-R, Kaever V, et al. Constitutive production of c-di-
GMP is associated with mutations in a variant of Pseudomonas aeruginosa with altered membrane
composition. Sci Signal. 2015; 8: ra36–ra36. https://doi.org/10.1126/scisignal.2005943 PMID:
25872871
71. Brewer SM, Twittenhoff C, Kortmann J, Brubaker SW, Honeycutt J, Massis LM, et al. A Salmonella
Typhi RNA thermosensor regulates virulence factors and innate immune evasion in response to host
temperature. PLoS Pathog. 2021; 17: e1009345. https://doi.org/10.1371/journal.ppat.1009345 PMID:
33651854
72. Chowdhury S, Maris C, Allain FH-T, Narberhaus F. Molecular basis for temperature sensing by an RNA
thermometer. EMBO J. 2006; 25: 2487–2497. https://doi.org/10.1038/sj.emboj.7601128 PMID:
16710302
73. Rinnenthal J, Klinkert B, Narberhaus F, Schwalbe H. Direct observation of the temperature-induced
melting process of the Salmonella fourU RNA thermometer at base-pair resolution. Nucleic Acids Res.
2010; 38: 3834–3847. https://doi.org/10.1093/nar/gkq124 PMID: 20211842
74. Barnwal RP, Loh E, Godin KS, Yip J, Lavender H, Tang CM, et al. Structure and mechanism of a molec-
ular rheostat, an RNA thermometer that modulates immune evasion by Neisseria meningitidis. Nucleic
Acids Res. 2016; 44: 9426–9437. https://doi.org/10.1093/nar/gkw584 PMID: 27369378
75. Cimdins A, Roßmanith J, Langklotz S, Bandow JE, Narberhaus F. Differential control of Salmonella
heat shock operons by structured mRNAs. Mol Microbiol. 2013; 89: 715–731. https://doi.org/10.1111/
mmi.12308 PMID: 23802546
76. Krajewski SS, Narberhaus F. Temperature-driven differential gene expression by RNA thermosensors.
Biochim Biophys Acta. 2014; 1839: 978–988. https://doi.org/10.1016/j.bbagrm.2014.03.006 PMID:
24657524
77. Rouvroit CL de, Sluiters C, Cornelis GR. Role of the transcriptional activator, VirF, and temperature in
the expression of the pYV plasmid genes of Yersinia enterocolitica. Mol Microbiol. 1992; 6: 395–409.
https://doi.org/10.1111/j.1365-2958.1992.tb01483.x PMID: 28776801
78. Cherradi Y, Schiavolin L, Moussa S, Meghraoui A, Meksem A, Biskri L, et al. Interplay between pre-
dicted inner-rod and gatekeeper in controlling substrate specificity of the type III secretion system. Mol
Microbiol. 2013; 87: 1183–1199. https://doi.org/10.1111/mmi.12158 PMID: 23336839
79. Sundberg L, Forsberg A. TyeA of Yersinia pseudotuberculosis is involved in regulation of Yop expres-
sion and is required for polarized translocation of Yop effectors. Cell Microbiol. 2003; 5: 187–202.
https://doi.org/10.1046/j.1462-5822.2003.00267.x PMID: 12614462
80. Cornelis GR, Wolf-Watz H. The Yersinia Yop virulon: a bacterial system for subverting eukaryotic cells.
Mol Microbiol. 1997; 23: 861–867. https://doi.org/10.1046/j.1365-2958.1997.2731623.x PMID:
9076724
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 26 / 27

81. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restric-
tion enzymes: gene splicing by overlap extension. Gene. 1989; 77: 61–68. https://doi.org/10.1016/
0378-1119(89)90359-4 PMID: 2744488
82. Milton DL, O’Toole R, Horstedt P, Wolf-Watz H. Flagellin A is essential for the virulence of Vibrio anguil-
larum. J Bacteriol. 1996; 178: 1310–1319. https://doi.org/10.1128/jb.178.5.1310-1319.1996 PMID:
8631707
83. Delvillani F, Sciandrone B, Peano C, Petiti L, Berens C, Georgi C, et al. Tet-Trap, a genetic approach to
the identification of bacterial RNA thermometers: application to Pseudomonas aeruginosa. RNA. 2014;
20: 1963–1976. https://doi.org/10.1261/rna.044354.114 PMID: 25336583
84. Pfaffl MW. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids
Res. 2001; 29: e45–e45. https://doi.org/10.1093/nar/29.9.e45 PMID: 11328886
85. Klinkert B, Cimdins A, Gaubig LC, Roßmanith J, Aschke-Sonnenborn U, Narberhaus F. Thermogenetic
tools to monitor temperature-dependent gene expression in bacteria. J Biotechnol. 2012; 160: 55–63.
https://doi.org/10.1016/j.jbiotec.2012.01.007 PMID: 22285954
86. Gaubig LC, Waldminghaus T, Narberhaus F. Multiple layers of control govern expression of the Escher-
ichia coli ibpAB heat-shock operon. Microbiology. 2011; 157: 66–76. https://doi.org/10.1099/mic.0.
043802-0 PMID: 20864473
87. Brantl S, Wagner E g. Antisense RNA-mediated transcriptional attenuation occurs faster than stable
antisense/target RNA pairing: an in vitro study of plasmid pIP501. EMBO J. 1994; 13: 3599–3607.
https://doi.org/10.1002/j.1460-2075.1994.tb06667.x PMID: 7520390
88. Hartz D, McPheeters DS, Traut R, Gold L. Extension inhibition analysis of translation initiation com-
plexes. Methods Enzymol. 1988; 164:419–25. https://doi.org/10.1016/s0076-6879(88)64058-4 PMID:
2468068
89. Ko
¨berle M, Klein-Gu¨nther A, Schu¨tz M, Fritz M, Berchtold S, Tolosa E, et al. Yersinia enterocolitica tar-
gets cells of the innate and adaptive immune system by injection of Yops in a mouse infection model.
PLoS Pathog. 2009; 5: e1000551. https://doi.org/10.1371/journal.ppat.1000551 PMID: 19680448
PLOS PATHOGENS
RNA thermometer controls YopN, the gatekeeper of Yersinia type III secretion
PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009650 November 12, 2021 27 / 27