Rho-dependent transcriptional switches regulate the
bacterial response to cold shock

Mildred Delaleau¹, Nara Figueroa-Bossi², Thuy Duong Do¹,³, Patricia Kerboriou², Eric Eveno¹,
Lionello Bossi², and Marc Boudvillain¹,³,⁴

¹Centre de Biophysique Moléculaire, CNRS UPR4301, affiliated with Université d'Orléans, rue Charles Sadron, 45071 Orléans cedex 2, France.

²Université Paris-Saclay, CEA, CNRS, Institut de Biologie Intégrative de la Cellule (I2BC), 91190 Gif-sur-Yvette, France.

³ED 549, Sciences Biologiques & Chimie du Vivant, Université d'Orléans, France.

⁴Corresponding author (marc.boudvillain@cnrs.fr)

(Authors preprint version - not peer-reviewed)

Running title: Cold-shock Rho-dependent riboswitches
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ABSTRACT

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Rho-dependent transcription termination plays a pivotal role in the regulation of bacterial gene expression. Binding of the Rho helicase to the nascent transcript triggers termination in response to cellular signals that directly or indirectly modulate mRNA structure and the accessibility of <u>Rho utilization (Rut)</u> sites. Despite the impact of temperature on RNA structure, Rho-dependent termination has never been associated to the bacterial response to temperature shifts. Here, we show that Rho is in fact a central player in the cold shock response (CSR), challenging the conventional view that CSR is primarily a posttranscriptional program. We identify <u>Rut</u> sites in the 5' untranslated regions of key CSR genes/operons (<i>cspA</i>, <i>cspB</i>, <i>cspG</i>, <i>nsrR-rnr-yjfHl</i>) that trigger premature termination by Rho at 37°C but not at 15°C, <i>in vitro</i> and <i>in vivo</i>. High concentration of the RNA chaperone CspA or base-pair mutations in the <i>cspA</i> mRNA leader can shift the balance between termination and antitermination, revealing how Rho can be controlled through RNA restructuring to activate CSR genes during the cold shock, and then to silence these genes during cold acclimation.

Overall, the work presented here sets a new paradigm for how RNA thermosensors can modulate gene expression.
46 INTRODUCTION

The cold shock response (CSR) is a complex and highly orchestrated set of molecular events that enables bacteria to cope with sudden temperature downshifts and to maintain cellular homeostasis under low temperature conditions. In *Escherichia coli* and various other mesophilic bacteria, an abrupt decrease in temperature causes the arrest of cell growth and a drastic reduction of mRNA translation due, among others, to increased structuring of the mRNAs. The CSR proteins escape this general inhibition and are involved in a variety of cold acclimation processes such as the remodeling of cell membrane composition or the facilitation of nucleic acid transactions thermodynamically constrained by the lower temperature. Some CSR proteins are upregulated transiently during cold acclimation while others are produced at high levels for as long as cold persists. Cold acclimation can take several hours during which bulk protein synthesis gradually increases until cells are sufficiently cold-adapted to resume growth, albeit at a slower rate (reviewed in ref. ¹⁻³).

In *E. coli*, two CSR proteins appear to play a central role in cold acclimation by dynamically regulating gene expression at the posttranscriptional level. The RNA chaperone CspA helps unfold mRNA structures that are formed at low temperatures and impede translation while the [3'→5'] exoribonuclease RNase R, which is not inhibited by RNA secondary structures, stimulates the degradation and turnover of mRNAs.⁴ The expression of RNase R (encoded by *rnr*) is induced 8-15 fold upon cold shock, an increase attributed to cold-induced folding and increased mRNA stability against degradation.⁵,⁶ The expression of CspA is also stimulated following a cold shock (by a factor that varies with growth stage), in part through greater protection of the *cspA* mRNA against ribonucleases⁷,⁸, but also largely through the regulation of *cspA* translation.⁴,⁹,¹⁰ Indeed, contrary to most RNAs in the cell, the *cspA* mRNA folds into a structure favoring efficient translation at low temperatures (e.g. 15°C) whereas, at 37°C, the mRNA adopts a conformation inhibiting translation.⁴,⁹ Notably, binding of CspA to the "15°C-conformation" can trigger an mRNA rearrangement less favorable to translation⁴ thereby providing a negative feedback control on CspA expression during the CSR.¹¹ Conversely, Binding of CspA to the "37°C conformation" stimulates *cspA* translation.¹² This may contribute to the basal expression and abundance of CspA during early exponential growth at 37°C.¹³,¹⁴ Sequence elements critical for the posttranscriptional regulation of *cspA*
76 have been identified in its 5' untranslated region (5'UTR) and in the initial section of its coding sequence (CDS).$^{4,9,10,12,15,16}$
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78 There are nine Csp (cold shock protein) homologues in E. coli but only expression of
79 CspA, CspB, CspG, and CspI is induced by cold shock (Supplementary Figure 1).$^{1-3}$ Although
80 some of these proteins share regulatory features and have partially redundant functions$^{4,17-81}$,
81 $CspA$ stands out as the primary contributor to the CSR due to its remarkable abundance.
82 Upon cold shock, CspA production can indeed represent close to 15% of total protein
83 synthesis.$^{11,20}$ Thus, despite a modest affinity for RNA ($K_d$ in µM range)$^{12,21}$, the high
84 concentration of CspA in cold-shocked cells (~100 µM)$^{22}$ and its fairly relaxed sequence
85 specificity$^2$ enables its widespread binding to and unfolding of cellular RNA. This process
86 largely contributes to the reduction of global mRNA structure and to the recovery of bulk
87 translation during the cold acclimation phase.$^4$

88 While the regulatory interplay between mRNA structure, translation, and decay during
89 cold acclimation is relatively well established$^{1-3}$, the contribution of transcription, in particular
90 its elongation phase, remains uncertain despite early work suggesting its potential
91 significance. In one instance, incremental deletions of the long 5'UTR of cspA were shown to
92 strongly stimulate the production of the mRNA independently of cold shock.$^{23}$ Since the
93 deletions did not change the rate of decay of the cspA mRNA, the authors postulated that the
94 5'UTR might be "involved in negative regulation of the cspA gene expression at the level of
95 transcription". Transcription initiation is not the regulated step$^7$ but CspA binding to the 5'-
96 leader of its own mRNA stimulates transcriptional pausing in the 5'UTR, which could play a
97 role.$^{24}$ However, CspA inhibits other pause signals or intrinsic transcription terminators, most
98 likely by preventing the formation of the corresponding RNA hairpin structures$^{25}$, and is thus
99 usually regarded as a positive rather than negative transcription regulator.$^{1-3}$

100 A source of negative transcriptional regulation in bacteria is Rho-dependent
101 termination (Figure 1), which silences hundreds of genomic loci in E. coli$^{26-28}$ and has been
102 implicated in several conditional attenuation mechanisms (reviewed in ref. 29-31). However, to
103 our knowledge, the involvement of Rho-dependent termination in the CSR has not been
104 investigated. This is somewhat surprising given that cold shock is expected to produce
105 widespread decoupling of translation from transcription$^{32,33}$ and that Rho-dependent
106 termination is the main mechanism of surveillance of transcription-translation coupling
(Figure 1, bottom; reviewed in ref. ³⁴,³⁵). In addition, increased mRNA structuring at low
temperature may hide *Rho utilization* (*Rut*) sites and inhibit termination by preventing Rho
access to the transcript (Figure 1, top right) in a manner reminiscent of the riboswitch-like
mechanisms used by some Rho-dependent transcriptional attenuators.³⁶,³⁷

Here, we present comprehensive evidence for the involvement of Rho in the cold shock
response. We identify *Rut* sites in the 5'UTRs of genes or operons involved in the CSR (*cspA*,
*cspB*, and *cspG* genes and *nsrR-rnr-yjfHl* operon) and show that transcription of these
genes/operons is prematurely terminated by Rho at 37°C but not at 15°C, *in vitro* and *in vivo*.
We also demonstrate that CspA can restore Rho-dependent termination at 15°C, implying that
dual-input, antagonistic control (T° vs Csp concentration) of transcriptional attenuation
contributes to the dynamic regulation of cold shock genes during the acclimation phase.
Altogether, our results unveil the existence of an upper layer of regulation in the bacterial
response to cold stress and a novel mechanism through which RNA thermosensors modulate
gene expression.

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## 122 RESULTS AND DISCUSSION

123 Identification of *Rut* sites in the 5’UTRs of cold shock genes

124 In a previous study, we mapped putative *Rut* sites along the genome of *E. coli* using a new
125 global screening approach that we named “Helicase-SELEX”.³⁸ Close examination of the
126 Helicase-SELEX data revealed that a number of putative *Rut* sites localized within the 5'UTRs
127 of genes involved in the CSR, namely *cspA*, *cspB*, *cspG*, *lpxP* (encoding a palmitoleoyl-
128 transferase involved in cold adaptation of the membrane), and *nsrR* (at the start of the *nsrR-
129 rnr-yjfHl* operon) as well as in the initial section of the *nrn* CDS (Figure 2A). Interestingly, these
130 putative *Rut* sites matched some of the so-called bicyclomycin-sensitive regions previously
131 detected in genome-wide analyses of the transcriptional response to Rho inhibitor
132 bicyclomycin (see supporting data in ref. ²⁶,²⁷ and Figure 2A). These regions were not discussed
133 specifically in these previous studies and have remained overlooked until now.

134 To substantiate the presence of Rho-dependent terminators in these regions of the *E.
135 coli* genome, we first calculated the percentages of C and G residues along the coding DNA
136 strand, in search of large C>G biases (aka C>G bubbles) characteristic of Rho-dependent
137 terminators.<sup>39,40</sup> In each case, a C>G bubble overlapping with the putative *Rut* site uncovered
138 by Helicase-SELEX was indeed found (**Figure 2B**). Next, we performed *in vitro* transcription
139 experiments with DNA templates encoding the upstream sections of the relevant transcription
140 units (5'UTR and first ~200-400 nucleotides [nt] of the CDS) under control of the strong T7A1
141 promoter.<sup>40</sup> We also used a DNA template encoding the Rho-dependent λtR1 terminator as
142 positive control.

143 Rho-dependent termination signals were detected in all instances when the
144 transcription reactions were carried out at 37°C in presence of Rho (**Figure 2**, 'Term'). Addition
145 of transcription factor NusG stimulated the production of Rho-dependent truncated
146 transcripts, including at sites closer to the promoter, as observed under similar conditions for
147 other Rho-dependent terminators (e.g. in ref. <sup>37,41-43</sup>). Importantly, the termination regions
148 detected *in vitro* (**Figure 3A-E**) are located immediately downstream from the C>G bubbles
149 and putative *Rut* sites (**Figure 2B**), consistent with Rho-dependent termination initiating in the
150 5'-leader regions of the cold shock genes. A notable exception is *lpxP*, which has a shorter
151 5'UTR (67 base pairs [bp]) than the other cold shock genes (146-161 bp) and for which the
152 termination region starts ~50 bp downstream from the start codon (**Figure 3E**).

153 A strong *in vitro* Rho-dependent termination signal was also detected in the CDS of the
154 *rnr* gene, ~100 bp downstream from the short (38 bp) intergenic *nsrR-rnr* region
155 **(Supplementary Figure 2A)**. This intragenic signal complements the upstream termination
156 signal located in the 5'UTR of the *nsrR-rnr-yjfHl* operon (**Figure 3D**), in good agreement with
157 observations from genome-wide studies (**Figure 2**). Altogether, these data support the
158 presence of Rho-dependent termination signals in the 5'UTRs of *cspA*, *cspB*, *cspG*, and *nsrR*
159 and in the upstream parts of the *lpxP* and *rnr* CDSs.

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161 **Temperature governs Rho-dependent transcriptional attenuation of cold shock genes**

162 Results were radically different for some of the DNA templates when the transcription
163 reactions were carried out at 15°C instead of 37°C. Most notably, the formation of Rho-
164 dependent truncated transcripts was strongly inhibited with templates *cspA*, *cspB*, and *cspG*
165 (**Figure 3A-C**, right gels). With template *nsrR*, termination within the 5'UTR was also strongly
166 inhibited at 15°C while a new site of strong Rho-dependent termination became apparent
farther downstream in the *nsrR* CDS (Figure 3D, star). By comparison, the Rho-dependent termination profiles obtained with templates *lpxP* (Figure 3E), λtR1 (Figure 3F), and *rnr* (Supplementary Figure 2A) were much less affected by the decrease in reaction temperature. Taken together, these data strongly suggest that the 5'-leaders of the *cspA*, *cspB*, *cspG*, and *nsrR* genes adopt structures at 15°C that prevent Rho-dependent termination, most likely because they hide the *Rut* sites from Rho (Figure 1, top right). By contrast, RNA folding at 15°C does not effectively shield the *lpxP*, *rnr*, and λtR1 terminators nor the downstream *nsrR* terminator (*) from Rho action. Thus, temperature may regulate the expression of *cspA*, *cspB*, *cspG*, and *nsrR-rnr-yjfH1* by a conditional Rho-dependent attenuation mechanism. This transcriptional regulation, working alongside the established posttranscriptional mechanisms<sup>4-10</sup>, is expected to ensure the tightest form of control in a manner akin to the multilayered *modus operandi* of some riboswitches.<sup>31</sup> Finally, the experiment in Figure 3 shows that *CspA*, at the sub-saturating concentration of 0.5 µM (*I<sub>low</sub>[CspA]), has no effect on Rho-dependent transcription termination with the tested DNA templates at both 15°C and 37°C (see more about this point below).

To determine if the above findings hold true *in vivo*, we first compared the amounts of *cspA* transcripts formed in WT (*rho*<sup>+</sup>) and *rho*<sup>R66S</sup> strains of *E. coli* MG1655. The *rho*<sup>R66S</sup> mutant is viable but defective in the termination of many transcripts<sup>44,45</sup>, presumably because the R66S mutation weakens Rho binding to *Rut* sites.<sup>46</sup> Total RNA was extracted from log phase cultures of the *rho*<sup>+</sup> and *rho*<sup>R66S</sup> strains after they were either cold-shocked at 15°C or kept at 37°C for 30 min. To detect the full-length *cspA* mRNA (*cspA-FL*, hereafter) and potentially shorter species, we performed northern blots with a <sup>32</sup>P-labeled oligonucleotide probe hybridizing to the +3 to +48 region of the *cspA* 5'leader (+1 being the transcription start site).

Formation of *cspA-FL* was readily detected in the cold-shocked cells, with no significant difference in *cspA-FL* abundance between the *rho*<sup>+</sup> and *rho*<sup>R66S</sup> strains (Figure 4A; *p* = 0.41, unpaired, two-tailed Student's t-test). Results were dramatically different for cells cultured at 37°C throughout. In this case, *cspA-FL* was weakly expressed in the *rho*<sup>R66S</sup> strain and almost undetectable in the *rho*<sup>+</sup> strain (Figure 4A). Overall, *cspA-FL* was around two orders of magnitude more abundant in the *rho*<sup>R66S</sup> mutant than in the *rho*<sup>+</sup> strain at 37°C but still two orders of magnitude less abundant than in the cold-shocked cells (*p* < 0.05; unpaired, two-tailed Student's t-tests). Moreover, the proportions of truncated *cspA* transcripts were
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comparatively much lower in the cold-shocked cells (Figure 4A, graphs). These dramatic
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effects cannot be explained by concomitant changes in the cellular levels of Rho or RNA
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polymerase, which do not vary much upon cold shock (Supplementary Figure 1).⁴ Rather, they
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are consistent with the *in vitro* data from above and support the scenario in which Rho-
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dependent termination downregulates *cspA* in exponentially growing cells at 37°C whereas
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cold shock triggers anti-termination and thus stimulates *cspA* expression at the co-
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transcriptional level.

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Similar Northern Blot experiments were performed with oligonucleotide probes
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hybridizing to the 5'UTRs of cspB and cspG. In the case of cspB, signals were weaker than for
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cspA but the trend was similar, i.e. full-length cspB mRNA was barely detected at 37°C, and
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only in the rho<sup>R66S</sup> mutant, but was present in considerably higher amounts in both rho<sup>+</sup> and
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rho<sup>R66S</sup> strains after cold-shock (Supplementary Figure 3A). Moreover, the signal for the full-
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length cspB mRNA in the rho<sup>R66S</sup> mutant was more easily detected when cells were cultured
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at 30°C instead of 37°C, which is consistent with the lower temperature of induction of cspB
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as compared to cspA.<sup>47</sup> Similarly, we detected a weak signal for the full-length cspG mRNA at
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30°C, but not at 37°C, in the rho<sup>R66S</sup> mutant only, whereas we observed a strong induction of
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cspG after cold-shock in both rho<sup>+</sup> and rho<sup>R66S</sup> strains (Supplementary Figure 3B). Note that in
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contrast to cspA and cspB, the optimal temperature of induction for cspG has not been
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determined.

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To probe the polycistronic *nsrR-rnr-yjfH1* transcript products, we used denaturing 1.5%
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agarose (instead of 8% polyacrylamide) electrophoresis and two distinct oligonucleotide
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probes for Northern blotting: probe A hybridizes to the 5'UTR of *nsrR* while probe B hybridizes
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to the intergenic region between the *nsrR* and *nrn* genes. With both probes, we detected two
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diffuse bands (Figure 4B) reflecting the complex biogenesis and posttranscriptional processing
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of the *nsrR-rnr-yjfH1* transcripts.<sup>5,6</sup> The intensities of the two bands were higher after cold-
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shock (Figure 4B), confirming the stabilization of the transcripts under this condition.<sup>5,6</sup> The
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intensities of the bands were also higher in the *rho*<sup>R66S</sup> mutant than in the *rho*<sup>+</sup> strain,
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supporting Rho involvement in the regulation of *nsrR-rnr-yjfH1* expression. The effect of the
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*rho*<sup>R66S</sup> mutation was particularly marked for the longest transcript species (slowest migrating
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band) and was still noticeable for this species at 15°C. These observations support a scenario
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where Rho-dependent polarity along the *nsrR-rnr-yjfH1* transcription unit is mediated by
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several Rho-dependent signals such as those detected *in vitro* in the 5'UTR and in the CDS of
*nsrR* and that operate at high and low temperatures, respectively (**Figure 3D**). Of note,
translation of the *nsrR* gene is very inefficient⁴⁸ and may thus not adequately mask the
intragenic termination signal (**Figure 3D**, star) *in vivo*. By contrast, the downstream *rnr* gene is
efficiently translated, in particular at low temperature⁴⁹, which should mask the Rho-
dependent signal in the *rnr* CDS (**Supplementary Figure 2A**).

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To confirm our *in vivo* observations, we performed quantitative RT-PCR experiments
with pairs of primers hybridizing in the coding regions of *cspA*, *cspB*, *cspG*, *nsrR*, *rnr*, and *lpxP*.
As a control, we first performed RT-PCR quantification of the *rho* gene, which is regulated by
Rho-dependent attenuation⁵⁰ The RT-qPCR signal for *rho* was indeed higher in the *rho*<sup>R66S</sup>
mutant than in the *rho*<sup>+</sup> strain and this difference was highly significant at both 37°C and 15°C
(**Figure 4C**), suggesting that the Rho-dependent attenuator controlling *rho* expression is not
much affected by cold shock. For each strain, the differences in *rho* signals between 37°C and
15°C were modest (less than 5-fold), confirming that *rho* mRNA levels do not vary much upon
cold shock.⁴ By contrast, we observed dramatic increases of the RT-qPCR signals for *cspA*, *cspB*,
and *cspG* upon cold shock and large differences between the signals for the *rho*<sup>+</sup> and *rho*<sup>R66S</sup>
strains at 37°C (15-40 fold), which however were cancelled out at 15°C (**Figure 4D**). These
results are in good agreement with the Northern blot experiments (**Figure 4A** and
**Supplementary Figure 3**) and further support that Rho-dependent attenuators sensitive to
cold regulate *cspA*, *cspB*, and *cspG*.

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The increases of RT-qPCR signals upon cold shock were smaller for *nsrR* and *rnr*, as
were the differences between *rho*<sup>+</sup> and *rho*<sup>R66S</sup> signals at 37°C (**Figure 4D**). These differences
were further reduced but not fully abrogated at 15°C (*p* < 10<sup>-2</sup>, unpaired, two-tailed Student's
t-tests), which is also consistent with Northern blot observations (**Figure 4B**). Thus, Rho-
dependent attenuation appears to be less important for the temperature-dependent
regulation of the *nsrR* and *rnr* genes than for *cspA*, *cspB*, and *cspG*. Alternatively, a
temperature lower than 15°C may be required to fully switch off Rho-dependent attenuation
of the *nsrR-rnr-yjfHl* operon. Interestingly, RT-qPCR probing of *lpxP* suggests that this gene is
also regulated by Rho in a temperature-dependent manner (**Figure 4D**) even though *in vitro*
Rho-dependent termination in *lpxP* is not strongly affected by temperature (**Figure 3E**). *In vivo*,
however, the intragenic *lpxP* terminator (**Figures 2B and 3E**) should be regulated by cold-shock
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stimulated *lpxP* translation.⁴ Increased translation may also better protect the *lpxP* mRNA from degradation by ribonucleases, thereby further contributing to the higher level of the mRNA at 15°C versus 37°C (Figure 4D).

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Taken together, our *in vitro* and *in vivo* data reveal a new facet of the intricate regulation of cold-shock genes based on the modulation of Rho-dependent attenuation. Two alternative mechanisms appear to be involved. On one hand, an increase in the efficiency of translation upon cold shock contributes to shield intragenic *Rut* sites from Rho action (e.g. as for *lpxP* and, possibly, *rnr*; Figure 1, top left). On the other hand, an increase in the secondary structure of the mRNA at the lower temperature prevents Rho access to a *Rut* site located in the 5'-leader portion of the mRNA (e.g. as for *cspA*, *cspB*, and *cspG*; Figure 1, top right).

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## Mutations that stabilize the cold-shock conformation of the cspA mRNA leader inhibit Rho-dependent attenuation

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Paralogs corresponding to six of the nine Csp proteins found in *E. coli* are also present in *Salmonella enterica*. Notably, *Salmonella* CspA shares 100% identity with its *E. coli* counterpart, and the two genes exhibit a 91% identity in their 5'UTRs, which are 164 and 160 nt long, respectively (Supplementary Figure 4A). Drawing from the analogy with *E. coli*, where the structure of the 5'UTR of *cspA* was determined by DMS mapping *in vivo*⁴, the *Salmonella* *cspA* sequence is predicted to adopt two alternative conformations: a more 'open' conformation at 37°C and a folded one containing a long hairpin-like structure at lower temperature (Supplementary Figure 4B). Conceivably, the latter structure might sequester residues required for Rho binding and thus promote readthrough transcription following the cold shock.

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To evaluate the role of Rho in the cold-shock regulation of *cspA* in *Salmonella*, we engineered a strain carrying a second copy of the 5'UTR of *cspA* fused to the coding sequence of the *lacZ* gene (in a *lacY*⁺ background) at an ectopic location under the control of the TetR-regulated *P*<sup>tet</sup> promoter. An isogenic derivative of this strain carrying *rho* allele Y80C, which causes a termination defect at *Rut* sites<sup>43,51</sup>, was subsequently constructed through phage P22 transduction. Measurement of β-galactosidase activity 30 minutes (Figure 5A) and 180 minutes (Figure 5B) after the addition of the *P*<sup>tet</sup> inducer, anhydrotetracycline (AHTc), revealed

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a significant increase in *lacZ* expression caused by the Rho mutation at 37°C, but not at 15°C (Figures 5A and 5B). This is consistent with the *cspA* leader sequence containing a Rho-dependent terminator active only at 37°C. Notably, the mutant-to-wild-type expression ratio at 37°C exhibited a gradual rise during the 30 to 180-minute interval (from approximately 4-fold to 30-fold), suggesting that termination efficiency is enhanced when the bacteria enter stationary phase. Enhanced termination can explain the early finding that *cspA* mRNA levels undergo an abrupt decline in *E. coli* cells transitioning from mid-to-late exponential growth, as reported in early studies.<sup>13,14</sup>

The P<sub>tet</sub> promoter fusion utilized in these experiments contains only one of the two operator sequences normally found at the 5' end of the *tetA* gene (see Materials and Methods for details).<sup>52</sup> Consequently, TetR-mediated repression exhibits some degree of "leakiness", resulting in low-level transcription in the absence of the inducer. However, due to termination in the *cspA* leader region, expression of the *cspA-lacZ* fusion at 37°C is insufficient to support bacterial growth on a medium containing lactose as the sole carbon source. As a result, the strain is Lac<sup>-</sup>. The introduction of the *rho*<sup>Y80C</sup> mutation confers a Lac<sup>+</sup> phenotype, most likely by allowing transcription to read through the *cspA* leader region (Figure 5C). This observation suggested that the lactose-based medium could be used to positively select mutants evading Rho-dependent termination in the *rho*<sup>+</sup> background. Colonies growing on minimal lactose plates (see example in Figure 5C, upper left spot) were isolated and characterized. This led to the identification of four mutants carrying single base-pair substitutions in the 5'UTR of *cspA*: C41A, A55G, C73U, and C76U. Remarkably, all four changes allow additional base pairing in the hairpin structure predicted to form at low temperature, thereby stabilizing the structure (Figure 5D and Supplementary Figure 4B).

Measurements of *lacZ* expression in strains carrying each of the four *cspA* alleles alone or in combination with *rho*<sup>Y80C</sup> confirmed the *cspA* changes to be completely epistatic to the Rho mutation, indicating that they act in the same pathway as Rho (Figures 5E and 5F). This supports the conclusion that the changes in the *cspA* leader RNA prevent Rho from terminating transcription at 37°C because they enable the hairpin structure to form at this higher temperature.

We selected two of the mutations, C41A and A55G, for further characterization *in vitro*.
We prepared <sup>32</sup>P-labeled *Salmonella cpsA* leader transcripts carrying either mutation, no
mutation (WT), or the G54U substitution expected to destabilize rather than stabilize the hairpin structure (Supplementary Figure 4B). We probed the transcript conformations by native polyacrylamide gel electrophoresis (PAGE). The C41A and A55G transcripts migrated faster in the PAGE gel than the WT transcript (Supplementary Figure 5A), indicating more compact conformations consistent with the stabilization of the hairpin structure by the mutations. By contrast, the G54U mutation markedly reduced the electrophoretic mobility of the transcript, consistent with a more open conformation enacted by hairpin destabilization. Furthermore, we observed different *in vitro* transcription termination profiles with DNA templates encoding the WT *Salmonella cpsA* or mutant sequences. Notably, in defined conditions, the WT and G54U sequences could sustain premature Rho-dependent termination in the 5'UTR whereas the C41A and A55G mutant sequences could not (Supplementary Figure 5B), thereby linking termination efficiency to the stability of the leader hairpin structure. Thus, both our *in vitro* and *in vivo* observations support the conclusion that the temperature-dependent folding pathway of the *cspA* mRNA leader not only governs *cspA* translation⁴,⁹ but also finely controls Rho-dependent attenuation of *cspA* expression.

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337 **CspA stimulates Rho-dependent termination at csp terminators**

338 Since CspA contributes to regulate cold shock genes (including *cspA* itself) during cold acclimation, we wondered if this could proceed, at least in part, through the modulation of Rho-dependent attenuation. To test this possibility, we performed *in vitro* transcription experiments in the presence of CspA. As noted above, we first tested a concentration (0.5 µM) just below the dissociation constants measured for CspA with model oligonucleotides at 20°C (1-10 µM range).¹²,²¹ At this sub-saturating concentration, CspA had no effect on Rho-dependent transcription termination with the tested DNA templates at both 15°C and 37°C (Figure 3). Results were different when we tested CspA at its concentration in cold-shocked cells (~100 µM).²² At this high (saturating) concentration, CspA was able to both stimulate Rho-dependent termination and activate upstream termination sites (Figure 6A, B and Supplementary Figure 2B-D).

349 The upstream shift of the termination window was particularly marked with the *cspA* template for which Rho-dependent termination prevailed in the 5'UTR or farther in the CDS in presence or absence of CspA, respectively (Figure 6A). Although these features are

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reminiscent of the action of NusG, in particular at suboptimal Rho-dependent terminators³⁷,⁴¹-⁴³, CspA and NusG appear to have additive rather than mutually exclusive effects (Figure 6A, B and Supplementary Figure 2B-D). More importantly, CspA was able to activate Rho-dependent termination at 15°C with the *csp* templates and at the upstream *nsrR* terminator, this activation being particularly efficient when NusG is also present (Figure 6A, B and Supplementary Figure 2B, D). CspA had a more modest effect at the *rnr*, *lpxP*, and control λtR1 terminators and did not shift or enlarge the termination window in these cases (Figure 6C and Supplementary Figure 2A, C). Thus, CspA (and possibly other Csp) can stimulate Rho-dependent termination *in vitro*, presumably by disrupting RNA structures that block Rho access to *Rut* sites (Figure 1). This effect seems particularly relevant for cold shock genes as it could contribute to the dynamic regulation of their expression during cold acclimation.

To test this hypothesis in bacterial cells, we performed Northern blots and RT-qPCR analyses of the *cspA* mRNA levels in the *rho*⁺ and *rho*<sup>R66S</sup> strains at different times during the cold acclimation phase. Both analyses confirmed that Rho-dependent attenuation of *cspA* is switched off shortly after cold shock, resulting in similarly high mRNA levels in both *rho*⁺ and *rho*<sup>R66S</sup> strains (Figure 6D, compare 0 h and 0.5 h time points). After 1 h at 15°C, however, *cspA* mRNA became less abundant in the *rho*⁺ strain than in the *rho*<sup>R66S</sup> mutant and this difference remained significant after 2 h of incubation at 15°C despite the decrease in *cspA* mRNA levels in both strains (Figure 6D). After 5 h of incubation at 15°C, *cspA* mRNA levels were even lower and the difference between *rho*⁺ and *rho*<sup>R66S</sup> strains was no longer statistically significant. These data are consistent with Rho-dependent attenuation of *cspA* being reactivated during the cold acclimation phase, a process that is correlated to the sharp increase of the concentrations of Csp proteins during this phase³,⁴,²² and that is congruent with our *in vitro* observations (Figure 6A). Thus, the sequential but antagonistic effects of cold temperature and Csp action on the Rho-dependent attenuator of *cspA* trigger a burst of *cspA* expression upon cold shock (~70-fold burst for *rho*⁺ cells; Figure 6D).⁴⁷ By contrast, *cspA* expression decreases if the exponentially growing cells remain at 37°C (Supplementary Figure 6)¹³,¹⁴, an effect that we ascribe to increased termination by Rho (see discussion of Figure 5A, B above), further accentuating the difference between the low and high temperature situations (compare WT levels in Figures 5A and 5B or in Figures 4A, D and 6D).

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CONCLUSIONS

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Growing evidence marks Rho-dependent termination of transcription as a pivotal mechanism in the bacterial response to environmental changes and stresses. Relevant examples involve Rho in the response to genotoxic and oxidative stresses<sup>53,54</sup>, exposure to toxic substances such as copper<sup>55</sup> or antibiotics<sup>56</sup>, nutriment fluctuations<sup>37,43</sup> or variations in the levels of key metabolites<sup>36,45,57-61</sup>, and even in the synthesis of the general stress sigma factor RpoS.<sup>62</sup> Rho involvement often hinges on a Rho-dependent terminator whose efficiency is tuned by a specific cytoplasmic signal affecting the structure or translatability of the mRNA (Figure 1). Notably, Rho-dependent regulatory mechanisms have been linked to the dynamic remodeling of mRNA leader regions upon binding of small molecule ligands<sup>36,63</sup>, regulatory small RNAs<sup>43,62</sup>, and proteins.<sup>37</sup> Here, we provide evidence that a physical signal – temperature – can also remodel Rho access to mRNAs and regulate transcription termination.

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Our data unveil the existence of thermosensing Rho-dependent terminators in the 5'UTRs of transcripts encoding key CSR proteins, namely CspA, CspB, CspG, and Rnase R. The transcription terminators are functional at 37°C and contribute to repress the synthesis of the CSR proteins alongside the known posttranscriptional mechanisms (Figure 7, left). Upon a temperature downshift, extensive restructuring of the 5'-leader of the mRNA, which is well documented for cspA and cspB<sup>4,9</sup>, hinders Rho action and allows transcription to proceed into the structural genes (Figure 7, right). Once the Csp proteins have reached a sufficiently high concentration, binding of these proteins to the mRNA brings the 5'-leader back to a silencing-prone conformation. This negative feedback mechanism down-regulates csp expression at both transcriptional and posttranscriptional levels during the acclimation phase (Figure 7).

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Previous work supports the implication of transcriptional regulation in the CSR. Early studies suggested that cspA transcription is regulated by sequences present in the leader portion of the gene and by the interaction of the 5'-leader RNA with Csp proteins<sup>17,18,23,24,47</sup>, although the exact mechanism remained unidentified. More recently, genome-wide studies unveiled Rho-dependent signals in the 5'UTRs and CDSs of genes/operons involved in the CSR, including CspA (Figure 2A).<sup>26,27,38</sup> Besides the representative cases studied here, genome-wide studies suggest that several other CSR genes could be regulated in a Rho-dependent manner (Table S4). This regulation may be based on RNA thermosensors located in 5'-leaders, as it is the case for cspA/B/G genes (Figure 7) or on intragenic Rut sites whose accessibility is
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modulated by the efficiency of translation, as for *lpxP* (see above). The presence of *Rut* peaks
upstream from the promoters of some CSR genes (**Table S4**) suggests an additional, yet
untested scenario whereby regulation would stem from read-through of terminators that
normally insulate upstream transcription units (i.e. thermosensing terminators located in
mRNA 3'UTRs).

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The regulation of *rpoS* by the cold-induced sRNA DsrA⁶⁴ provides another compelling example of a Rho-dependent switch operating during cold acclimation. Indeed, DsrA binding to the 5'-leader of *rpoS* enhances the translatability of the mRNA at low temperature⁶⁵ but also inhibits Rho-dependent attenuation of *rpoS*.⁶² The latter is likely to contribute to the increased expression of *rpoS* at low temperatures.⁶⁶ Conversely, one might envision that activation of Rho-dependent termination participates in the repression of non-CSR proteins upon cold shock. Translational repression associated to the increased structuring of bulk mRNA⁴ is thought to trigger widespread decoupling of translation from transcription.³²,³³ Whether this favors Rho-dependent termination (contributing to gene silencing) or is compensated by increased structural shielding of intragenic *Rut* sites (**Figure 1**) awaits experimental testing. Regardless, the work described here defines Rho-dependent transcription termination as an important component of the bacterial response to cold stress and sets a new paradigm for how RNA remodeling by antagonistic factors, here temperature and Csp chaperones, can modulate transcription termination.

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## MATERIALS AND METHODS

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### Materials

436

Unless specified otherwise, chemicals and enzymes were from Merck and New England
Biolabs, respectively. Radionucleotides were obtained from PerkinElmer and synthetic
oligonucleotides (**Table S1**) from Eurogentec or Merck.

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DNA templates for *in vitro* transcription were prepared by standard PCR amplification of genomic DNA⁶⁷ with the forward (fwd) and reverse (rev) primer pairs listed in **Table S1**. In a second round of PCR, the forward primer was replaced by primer T7A1_univ or pT7-univ_A to introduce the full sequence of the T7 φ6.5 (for T7 transcription) or T7A1 promoter (for transcription with *E. coli* RNAP), respectively. Sequences of the DNA templates are provided in **Table S3**.

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Plasmid pET28b-CspA was obtained by subcloning the *E. coli* cspA CDS between the Ncol and Xhol sites of plasmid pET28b(+) (Novagen). The CspA protein was overexpressed in BL21(DE3) cells harboring the pET28b-CspA plasmid and purified as described previously.⁶⁸ The Rho and NusG proteins were prepared as described previously. ⁶⁷ Rho concentrations are expressed in hexamers throughout.

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## Bacterial strains and growth conditions

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Bacterial strains are listed in **Table S2**. *E. coli* MG1655 rho⁺ and rhoR⁶⁶S strains were kindly provided by Pr. Joseph T. Wade (New York state University at Albany). *Salmonella* strains are described in the next sections. For cold shock experiments, bacteria were cultured in LB broth at 37°C until absorbance at 600 nm (A₆₀₀) reached 0.5. Culture aliquots (50 mL) were then withdrawn and either kept at 37°C or transferred to a 15°C water bath. Cultures were further incubated at 37°C or 15°C for the indicated times.

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## Construction of an ectopic cspA-lacZY fusion in *Salmonella*

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*Salmonella* strain MA14009 harbors a tripartite *tetR*-P<sup>tet</sup>-*ccdB*-cat cassette⁶⁹ inserted in the region upstream of the *chiPQ* promoter in the background of *chiP-lacZY* translational fusion.⁴³ This strain was made recombineering-proficient by introduction of lambda *Red* plasmid pKD46.⁷⁰ The resulting strain was transformed with a DNA fragment spanning the cspA leader region amplified from wild-type *Salmonella* with primers AN13 and AN16 (**Table S1**). These primers carry 5' extensions designed to recombine within the P<sup>tet</sup> promoter region on one end (AN13), and with the sequence immediately downstream from the initiation codon of *lacZ* on the opposite end (AN16), so as to replace the entire segment between P<sup>tet</sup> and the beginning of *lacZ* open reading frame, with the cspA sequence. Recombinants were obtained selecting for AHTc-resistance as described.⁶⁹ One of these recombinants, strain MA14627, which thus carries the 5'UTR of cspA directly fused to the *lacZY* operon under the control of the *tetR*-P<sup>tet</sup> module was used for the experiments described here.

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## Isolation of *Salmonella* cspA leader mutants

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Two cultures of strain MA14627 inoculated from separate single colonies were grown overnight at 37°C. 0.1 ml aliquots were spread on the surface of two minimal (NCE) lactose plates⁴³, and incubated at 37°C. Lac⁺ colonies typically developed during a 36-to-48-hour
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incubation. A number of colonies were picked, purified by streaking, and subjected to colony
PCR (primers ppZ18 – AB24). The amplified fragments were subjected to Sanger sequencing.

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Out of 14 clones analyzed, 7 carried changes in the *cspA* leader sequence: the four point-mutants characterized in this study, C41A, A55G, C73U (three occurrences), C76U and one carrying the deletion of the first 80 bp of the 5' UTR. The latter was not analyzed any further.

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RNA extraction and Northern blot analyses

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Total RNA was extracted from *E. coli* cell cultures by the acid hot phenol procedure as described previously⁷¹, with minor modifications. Briefly, bacterial cultures were mixed with one tenth of volume of ice cold Ethanol:Phenol [95:5] solution to quench RNA degradation.

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Bacteria were pelleted, suspended in RNA extraction buffer (20 mM sodium acetate pH 5.2, 2% SDS, and 0.3 M sucrose), and boiled for 2 min. The mixtures were mixed with one volume of acidic phenol, left at room temperature for 30 sec, and incubated on ice for 2 min. Mixtures were then centrifuged at 12,000 g for 5 min. The aqueous phases were collected and extracted

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thrice with a solution of Phenol:Chloroform:Isoamyl Alcohol [25:24:1] and twice with chloroform. RNA preparations were purified on RNA Clean & Concentrator-25 columns following manufacturer (Zymo Research) instructions and stored at -20°C in ME buffer (10 mM MOPS, PH 6.5, 1 mM EDTA) before use. For Northern blotting, total RNA was fractionated on a denaturing 8 % polyacrylamide/urea gel or a 1.5 % agarose MOPS/formaldehyde gel and electro-transferred to a Hybond-N⁺ membrane (Cytiva). Blots were hybridized to ³²P-end labeled DNA oligonucleotide probes complementary to the mRNA region of interest and to 5S ribosomal RNA (Table S1). Hybridization signals were detected by phosphorimaging with a Typhoon FLA-9500 instrument equipped with ImaqeQuant TL v8.1 software (Cytiva). Signals for the 5S rRNA were used for normalization of the mRNA signals.³⁷

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Reverse transcription-quantitative PCR (RT-qPCR) analyses

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Residual DNA was removed from RNA samples by treatment with Turbo DNase I (Thermo
Fisher Scientific), following manufacturer's instructions. Then, 0.5 µg of total RNA was used
for cDNA synthesis with 2.5 µM random hexamer primer, 0.5 mM of each dNTP, and 200 U of
Maxima H-minus reverse transcriptase (Thermo Fisher Scientific) upon incubation for 5 min at
25°C and 1 h at 50°C. For quantitative PCR, each cDNA was mixed with the Power SYBR® Green
PCR Master Mix (Applied Biosystems™) and forward and reverse primers (0.2 µM each; Table

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S1). Mixtures were placed in a LightCycler®480 qPCR instrument (Roche) and amplified with the following conditions: initial denaturation at 95°C for 10 min followed by 40 cycles of 15 s of denaturation at 95°C, and 1 min of hybridization/elongation at 60°C. Sample melting curves were used to monitor the specificity of cDNA amplification at each cycle. The threshold cycle 'Ct' was calculated with the instrument software (LightCycler®480 v1.5.0) using the second derivative maximum method. Data analysis was performed by the relative standard curve method, as recommended in Applied Biosystems User Bulletin #2. Sample signals were then normalized using *rrsB* (which encodes 16S rRNA).⁶¹ Statistical analyses were performed with Kaleidagraph 4.1 (Synergy software) using the unequal variance option of the unpaired two-tailed Student's t-test.

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## Measurement of β-galactosidase activity

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β-galactosidase activity was assayed in toluene-permeabilized *Salmonella* cells as described by Miller⁷² and is expressed in Miller units. All measurements were performed three times, each time on duplicate cultures, as specified in the legend to Figure 5. Statistical significance was determined as specified in the legend to Figure 5 using GraphPad Prism 9 software.

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## Transcription termination experiments

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Standard transcription termination experiments were performed as described⁴⁰, with minor modifications. Briefly, DNA template (0.1 pmol), *E. coli* RNAP (0.3 pmol), Rho (1.4 pmol), NusG (0 or 2.8 pmol), Superase-In (0.5U/µL), and CspA (0, 10, or 2000 pmol) were mixed in 18 µL of transcription buffer (20 mM HEPES, pH 7.5, 150 mM potassium acetate, 10 mM NaCl, 100 nM CaCl₂, 5 mM MgCl₂, 1.5 mM DTT) and incubated for 10 min at 37°C. Then, 2 µL of initiation mix (2 mM ATP, GTP, and CTP, 0.2 mM UTP, 2.5 µCi/µL of ³²P-αUTP, and 250 µg/mL rifampicin in transcription buffer) were added to reaction mixtures before incubation for 20 min at 37°C. Transcription reactions were stopped with 4 µL of EDTA (0.5 M), 6 µL of tRNA (0.25mg/mL), and 80 µL of sodium acetate (0.42 M) before extraction with a phenol:chloroform:isoamyl alcohol [25:24:1] mixture and precipitation at -20°C with 330 µL of ethanol. Reaction pellets were dissolved in denaturing loading buffer (95% formamide, 5 mM EDTA), and analyzed by denaturing 8% PAGE and Typhoon FLA-9500 phosphorimaging.

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## Electrophoretic mobility shift assay (EMSA)
541 DNA templates encoding the wild-type mRNA leader of *Salmonella cspA* (nt 1-164) and single-
542 point mutants C41A, A55G, and G54U were transcribed with T7 RNA polymerase as described
543 previously<sup>73</sup>, in the presence of <sup>32</sup>P-αUTP. The <sup>32</sup>P-labeled transcripts were purified by
denaturing 8% PAGE and stored at -20°C in ME buffer. Trace amounts (~400 β-ray counts per
544 minute) of each transcript were dissolved in EMSA buffer (10 mM MOPS, pH 6.5, 1 mM EDTA,
545 and either 50 mM KCl or 150 mM potassium acetate) and incubated for 3 min at 95°C and
546 then for 15 min at 37°C. Samples were mixed with Ficoll-400 (8% v/v final) and analyzed by
547 native 6% PAGE (4 h migration at 20°C and 10 V/cm) and Typhoon FLA-9500 phosphorimaging.
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549

550 Bioinformatics analyses

551 Percentages of C and G residues along the *E. coli* MG1655 genome were computed with
552 EMBOSS freak 5.0.0 (step: 1; averaging window: 78) and visualized with Kaleidagraph 4.1.
553 Log₁₀ fold enrichment (FE) profiles were determined from Helicase-SELEX data³⁸ deposited in
554 the European Nucleotide Archive under accession #PRJEB50170. Curated reads were mapped
555 on the MG1655 genome (NC_000913.3) with Bowtie2 (v2.4.2, default options). Coverage and
556 RPM normalization were performed with Bedtools v2.29.2. The Log₁₀FE profiles were obtained
557 with MACS2 bdgcmp v2.1.1 using the deposited R10+ (+NusG) or R10- (-NusG) sample as input
558 and R0 sample as control. Peak calling was performed with MACS2 bdgpeakcall v2.1.1 (cutoff:
559 0; Min peak length: 30; Max gap: 30). Log₁₀FE profiles were visualized with the JBrowse
560 genome browser (jbrowse.org).⁷⁴

561

562 AUTHOR CONTRIBUTIONS

563 MD performed Northern blot, RT-qPCR, and *in vitro* transcription experiments; NF-B
564 constructed the *Salmonella* strains used in this study; TDD prepared DNA templates and
565 performed Northern blot and EMSA experiments; PK and LB performed β-galactosidase
566 assays; EE and MB performed computational mining of published Helicase-SELEX and genomic
567 data; LB isolated *Salmonella cspA* leader mutants; MB prepared proteins and wrote the paper
568 with help from MD, NF-B, and LB; MD, NF-B, LB, and MB conceived the study. All authors
569 participated in data analysis.

570

571 AKNOWLEDGMENTS
572 N.F-B, LB, and MB acknowledge support from the French Centre National de la Recherche

573 Scientifique (CNRS). This work was also supported by grants from Agence Nationale de la

574 Recherche (ANR) to M.B. (ANR-15-CE11-0024-2 and ANR-19-CE44-0009-01).

575

576 COMPETING INTERESTS

577 The authors declare no competing interests
578 FIGURE LEGENDS

579 **Figure 1:** Rho-dependent termination. Rho binds to free *Rut* sites within nascent transcripts
580 and then uses its ATP-dependent motor activity to catch up with RNA polymerase (RNAP)
581 along the RNA chain and to trigger the dissociation of the transcription elongation complex
582 (bottom).<sup>75</sup> Rho action is prevented by the presence of translating ribosomes or by secondary
583 structures in the nascent transcript that block *Rut* sites (top). In an alternative termination
584 pathway, Rho binds RNAP early on during transcription and detects accessible *Rut* sites in the
585 nascent transcript from this RNAP-bound position<sup>76</sup>, which is not represented here for
586 simplicity.

587

588 **Figure 2:** Putative *Rut* sites in the 5'-UTRs of *E. coli* genes involved in the CSR. (A) Fold
589 enrichment (FE; log₁₀ scale) profiles for Helicase-SELEX experiments performed with Rho in
590 presence or absence of NusG were determined from published data.<sup>38</sup> Cyan bars highlight the
591 positions of the enrichment peaks (median log₁₀FE ≥ 0.3) suggestive of *Rut* sites in the 5'-UTRs.
592 Regions where transcription is upregulated *in vivo* in presence of the Rho inhibitor
593 Bicyclomycin, as determined by ChIP-chip (orange arrows)<sup>27</sup> or microarrays and RNAseq
594 (purple bars)<sup>26</sup> are also shown. Note that different scales along the genome coordinates (x-
595 axis) are used for the various panels. (B) C>G bubbles (black arrows) overlap with the *Rut* peaks
596 detected by Helicase-SELEX (cyan lines) and are present upstream from the *in vitro*
597 transcription termination regions (brown lines), a feature characteristic of Rho-dependent
598 terminators. Percentages of C and G residues were calculated using a sliding window of 78 nt.
599 Termination boundaries were estimated with the molecular size calibration/calculation
600 function of ImageQuant software using all *in vitro* transcription gel lanes.

601

602 **Figure 3:** Cold shock genes contain Rho-dependent terminators in their 5'UTRs.
603 Representative gel images show the *in vitro* transcription profiles obtained with DNA
604 templates encoding the upstream sections of the *E. coli* cspA, cspB, cspG, lpxP and nsrR
605 transcription units (A-E) or the λtR1 terminator (F). Transcription reactions were performed at
606 15 or 37°C with or without Rho (20 nM), NusG (70 nM), or CspA (0.5 µM), as indicated. Bands
607 corresponding to runoff (RO) and termination (Term) transcripts are indicated. Since
608 transcripts are labeled by incorporation of α³²P-UTP during transcription, band intensities
609

reflect both the amounts and sizes of the transcripts. A downstream *nsrR* termination site (*) is detected at 15°C (panel D).

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**Figure 4:** Transcription of cold shock genes is regulated by Rho *in vivo*. *E. coli* log phase cultures (A<sub>600</sub> = 0.5) were incubated for 30 min at 15°C or 37°C before RNA extraction. (**A**, **B**) Northern blot analysis of the expression of *cspA* and *nsrR* in WT (*rho*<sup>+</sup>) and *rho*<sup>R66S</sup> (R66S) strains. Phosphorimager *cspA* counts were normalized to 5S RNA and the resulting values were divided by that obtained for WT at 37°C. The [*cspA*] values are (mean ± SD) values from three independent biological replicates. Pairwise comparisons of the normalized WT and R66S signals along gel lanes are provided on the right of the *cspA* membrane images (FL: full-length transcripts). Polycistronic *nsrR-rnr-yjfH1* transcripts are subjected to complex posttranscriptional processing<sup>5,6</sup> and were not quantified. (**C**, **D**) RT-qPCR confirms Rho-dependent regulation of cold shock genes. For each gene, signals are normalized to the condition *rho*<sup>+</sup>, 37°C. Four biological replicates were analyzed by RT-qPCR, each time in triplicate (open circles represent the triplicate means). Statistical significance was determined by unpaired two-tailed Student's t-tests (ns, p > 0.05; *, p < 0.05; **, p < 10<sup>-2</sup>; ***, p < 10<sup>-3</sup>; ****, p < 10<sup>-4</sup>).

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**Figure 5.** Mutations in the *cspA* leader RNA prevent Rho-dependent transcription termination in *Salmonella*. (**A**, **B**) Impact of Rho mutation Y80C on *cspA-lacZY* expression. Isogenic strains carrying the *cspA-lacZY* fusion under control of the *P*<sup>tet</sup> promoter and either *rho*<sup>+</sup> or *rho*<sup>Y80C</sup> were cultured in LB at 37°C until reaching mid-exponential phase (DO<sub>600</sub> ≈ 0.4 - 0.5). At this point, expression of the *cspA-lacZY* fusion was induced by adding the *P*<sup>tet</sup> inducer AHTc. The cultures were then divided, with one half transferred to 15°C and the other maintained at 37°C, and both were further incubated for (**A**) 30 and (**B**) 180 min. Cells were then processed for the determination of β-galactosidase activity. (**C**) Lac phenotype of strains carrying the *P*<sup>tet</sup>-*cspA-lacZY* fusion in combination with either *rho*<sup>+</sup> (strain MA14635) or *rho*<sup>Y80C</sup> (strain MA14636). Bacteria were cultured to stationary phase and 10 µl were spotted on minimal-lactose plates ±AHTc (37°C). The strain carrying *rho*<sup>Y80C</sup> is Lac<sup>+</sup> under non-inducing conditions (-AHTc) whereas the *rho*<sup>+</sup> strain is Lac<sup>-</sup>. The yellow arrow highlights a Lac<sup>+</sup> colony forming of the lawn of *rho*<sup>+</sup> cells. (**D**) Position of mutations suppressing the inhibitory effect of Rho on

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639
640 cspA-lacZ expression at 37°C. (E, F) Effect of the mutations in D on the expression of the cspA-
lacZ fusion in the presence or absence of Rho Y80C at 37°C (E) and at 15°(F). Cells were
641 cultured and treated as described in (A, B) with a time of incubation of 30 min after AHTc
642 induction. Throughout this experiment, β-galactosidase activity was assayed with the method
643 of Miller⁷² on three independent replicates of the bacterial cultures, with each assay carried
644 out in duplicate. Statistical significance was determined by one-way ANOVA with Šidák's
645 multiple comparisons test (ns, p > 0.05; ***, p ≤ 10⁻³; ****, p ≤ 10⁻⁴).

647

648 **Figure 6:** A high concentration of CspA stimulates Rho-dependent termination at *E. coli csp* terminators. Transcription experiments were performed with the *cspA* (**A**), *cspB* (**B**) and control λtR1 (**C**) DNA templates as described in the legend to Figure 3, except that a 200-fold higher concentration of CspA was used (100 vs 0.5 µM). (**D**) Northern blot and RT-qPCR analyses of Rho-dependent attenuation of *cspA* as a function of the duration of the incubation at 15°C following cold shock. Four biological replicates were analyzed by RT-qPCR, each time in triplicate (open circles represent the triplicate means). Statistical significance was determined by unpaired two-tailed Student's t-tests (ns, p > 0.05; *, p < 0.05; **, p < 10⁻²; ***, p < 10⁻³).

657

658 **Figure 7:** Model for regulation of *csp* genes. (**Left**) Rho-dependent attenuation of transcription 659 in the 5'UTR inhibits *csp* expression at 37°C. Poor translatability of the transcripts that escape 660 termination further contributes to *csp* down-regulation directly⁴,⁹,¹⁰ and, possibly indirectly, 661 by facilitating intragenic transcription termination or RNA degradation or both.⁷ (**Right**) 662 Restructuring of the 5'-leader upon the cold shock hides the *Rut* site and prevents premature 663 transcription termination, at the same time enhancing the accessibility of the ribosome 664 binding site. Protection of the CDS by translating ribosomes may contribute to stabilize the 665 mRNA. During cold acclimation, the concentration of Csp proteins increases until it becomes 666 sufficient to promote Csp binding to the mRNA. Csp binding drives the mRNA into a 667 conformation that resembles the "37°C-conformation"⁴ and thereby restores attenuation.
669 1. Barria, C., Malecki, M., and Arraiano, C.M. (2013). Bacterial adaptation to cold. Microbiology (Reading) 159, 2437-2443. 10.1099/mic.0.052209-0.

671 2. Zhang, Y., and Gross, C.A. (2021). Cold Shock Response in Bacteria. Annual review of genetics 55, 377-400. 10.1146/annurev-genet-071819-031654.

673 3. Gualerzi, C.O., Giuliodori, A.M., and Pon, C.L. (2003). Transcriptional and post-transcriptional control of cold-shock genes. J Mol Biol 331, 527-539. 10.1016/s0022-2836(03)00732-0.

675 4. Zhang, Y., Burkhardt, D.H., Rouskin, S., Li, G.W., Weissman, J.S., and Gross, C.A. (2018). A Stress Response that Monitors and Regulates mRNA Structure Is Central to Cold Shock Adaptation. Mol Cell 70, 274-286 e277. 10.1016/j.molcel.2018.02.035.

678 5. Cairrao, F., Cruz, A., Mori, H., and Arraiano, C.M. (2003). Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA. Mol Microbiol 50, 1349-1360. 10.1046/j.1365-2958.2003.03766.x.

681 6. Cairrao, F., and Arraiano, C.M. (2006). The role of endoribonucleases in the regulation of RNase R. Biochem Biophys Res Commun 343, 731-737. 10.1016/j.bbrc.2006.03.040.

683 7. Fang, L., Jiang, W., Bae, W., and Inouye, M. (1997). Promoter-independent cold-shock induction of cspA and its derepression at 37 degrees C by mRNA stabilization. Mol Microbiol 23, 355-364. 10.1046/j.1365-2958.1997.2351592.x.

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Gene expression

<figure><img src="image_1.png" /><figcaption>**Figure 1:** Rho-dependent termination. Rho binds to free *Rut* sites within nascent transcripts and then uses its ATP-dependent motor activity to catch up with RNA polymerase (RNAP) along the RNA chain and to trigger the dissociation of the transcription elongation complex (bottom) (Molodtsov et al., 2023). Rho action is prevented by the presence of translating ribosomes or by secondary structures in the nascent transcript that block *Rut* sites (top). In an alternative termination pathway, Rho binds RNAP early on during transcription and detects accessible *Rut* sites in the nascent transcript from this RNAP-bound position (Hao et al., 2021), which is not represented here for simplicity.</figcaption></figure>
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /><figcaption>**Figure 2:** Putative *Rut* sites in the 5'-UTRs of *E. coli* genes involved in the CSR. (A) Fold enrichment (FE; log₁₀ scale) profiles for Helicase-SELEX experiments performed with Rho in presence or absence of NusG were determined from published data (Delaleau et al., 2022). Cyan bars highlight the positions of the enrichment peaks (median log₁₀FE ≥ 0.3) suggestive of *Rut* sites in the 5'UTRs. Regions where transcription is upregulated *in vivo* in presence of the Rho inhibitor Bicyclomycin, as determined by ChIP-chip (orange arrows; (Peters et al., 2009)) or microarrays and RNAseq (purple bars; (Peters et al., 2012)) are also shown. Note that different scales along the genome coordinates (x axis) are used for the various panels. (B) C>G bubbles (black arrows) overlap with the *Rut* peaks detected by Helicase-SELEX (cyan lines) and are present upstream from the *in vitro* transcription termination regions (brown lines), a feature characteristic of Rho-dependent terminators. Percentages of C and G residues were calculated using a sliding window of 78 nucleotides. Termination boundaries were estimated with the molecular size calibration/calculation function of ImageQuant software using *in vitro* transcription gel lanes.</figcaption></figure>
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

**Figure 3:** Cold shock genes contain Rho-dependent terminators in their 5'UTRs. Representative gel images show the *in vitro* transcription profiles obtained with DNA templates encoding the upstream sections of the E. coli *cspA*, *cspB*, *cspG*, *lpxP* and *nsrR* transcription units (A-E) or the λtR1 terminator (F). Transcription reactions were performed at 15 or 37°C with or without Rho (20 nM), NusG (70 nM), or CspA (0.5 µM), as indicated. Bands corresponding to runoff (RO) and termination (Term) transcripts are indicated. Since transcripts are labeled by incorporation of α³²P-UTP during transcription, band intensities reflect both the amounts and sizes of the transcripts. A downstream nsrR termination site (*) is detected at 15°C (panel D).
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 4:** Transcription of cold shock genes is regulated by Rho *in vivo*. *E. coli* log phase cultures (A<sub>600</sub> ~ 0.5) were incubated for 30 min at 15°C or 37°C before RNA extraction. (A, B) Northern blot analysis of the expression of *cspA* and *nsrR* in WT (*rho*<sup>+</sup>) and *rho*<sup>R66S</sup> (R66S) strains. Phosphorimager *cspA* counts were normalized to 5S RNA and the resulting values were divided by that obtained for WT at 37°C. The [*cspA*] values are (mean ± SD) values from three independent biological replicates. Pairwise comparisons of the normalized WT and R66S signals along gel lanes are provided on the right of the *cspA* membrane images (FL: full-length transcripts). Polycistronic *nsrR-rnr-yjfHl* transcripts are subjected to complex posttranscriptional processing (Cairrao and Arraiano, 2006; Cairrao et al., 2003) and were not quantified. (C, D) RT-qPCR confirms Rho-dependent regulation of cold shock genes. For each gene, signals are normalized to the condition *rho*<sup>+</sup>, 37°C. Four biological replicates were analyzed by RT-qPCR, each time in triplicate (open circles represent the triplicate means). Statistical significance was determined by unpaired two-tailed Student's t-tests (ns, p > 0.05; *, p < 0.05; **, p < 10<sup>-2</sup>; ***, p < 10<sup>-3</sup>; ****, p < 10<sup>-4</sup>).</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Figure 5.** Mutations in the cspA leader RNA prevent Rho-dependent transcription termination in *Salmonella*. (A, B) Impact of Rho mutation Y80C on cspA-lacZY expression. Isogenic strains carrying the cspA-lacZY fusion under control of the P<sup>tet</sup> promoter and either rho<sup>+</sup> or rho<sup>Y80C</sup> were cultured in LB at 37°C until reaching mid-exponential phase (DO<sub>600</sub> ≈ 0.4 - 0.5). At this point, expression of the cspA-lacZY fusion was induced by adding the P<sup>tet</sup> inducer AHTc. The cultures were then divided, with one half transferred to 15°C and the other maintained at 37°C, and both were further incubated for (A) 30 and (B) 180 min. Cells were then processed for the determination of β-galactosidase activity. (C) Lac phenotype of strains carrying the P<sup>tet</sup>-cspA-lacZY fusion in combination with either rho<sup>+</sup> (strain MA14635) or rho<sup>Y80C</sup> (strain MA14636). Bacteria were cultured to stationary phase and 10 µl were spotted on minimal-lactose plates ±AHTc (37°C). The strain carrying rho<sup>Y80C</sup> is Lac<sup>+</></sup> under non-inducing conditions (-AHTc) whereas the rho<sup>+</sup> strain is Lac<sup>-</sup>. The yellow arrow highlights a Lac<sup>+</sup> colony forming of the lawn of rho<sup>+</sup> cells. (D) Position of mutations suppressing the inhibitory effect of Rho on cspA-lacZ expression at 37°C. (E, F) Effect of the mutations in D on the expression of the cspA-lacZ fusion in the presence or absence of Rho Y80C at 37°C (E) and at 15°(F). Cells were cultured and treated as described in (A, B) with a time of incubation of 30 min after AHTc induction. Throughout this experiment, β-galactosidase activity was assayed with the method of Miller (Miller, 1992) on three independent replicates of the bacterial cultures, with each assay carried out in duplicate. Statistical significance was determined by one-way ANOVA with *Šidák's* multiple comparisons test (ns, p > 0.05; ***, p ≤ 10<sup>-3</sup>; ****, p ≤ 10<sup>-4</sup>).</figcaption></figure>
A

<figure><img src="image_16.png" /></figure>

C

<figure><img src="image_17.png" /></figure>

B

<figure><img src="image_18.png" /></figure>

D

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

**Figure 6:** A high concentration of CspA stimulates Rho-dependent termination at *E. coli* csp terminators. Transcription experiments were performed with the cspA (A), cspB (B) and control λtR1 (C) DNA templates as described in the legend to Figure 3, except that a 200-fold higher concentration of CspA was used (100 vs 0.5 µM). (D) Northern blot and RT-qPCR analyses of Rho-dependent attenuation of cspA as a function of the duration of the incubation at 15°C following cold shock. Four biological replicates were analyzed by RT-qPCR, each time in triplicate (open circles represent the triplicate means). Statistical significance was determined by unpaired two-tailed Student's t-tests (ns, p > 0.05; *, p < 0.05; **, p < 10⁻²; ***, p < 10⁻³).
<figure><img src="image_21.png" /><figcaption>**Figure 7:** Model for regulation of *csp* genes. (Left) Rho-dependent attenuation of transcription in the 5'UTR inhibits *csp* expression at 37°C. Poor translatability of the transcripts that escape termination further contributes to *csp* down-regulation directly (Giuliodori et al., 2010; Yamanaka et al., 1999; Zhang et al., 2018) and, possibly indirectly, by facilitating intragenic transcription termination or RNA degradation or both (Fang et al., 1997). (Right) Restructuring of the 5'-leader upon the cold shock hides the *Rut* site and prevents premature transcription termination, at the same time enhancing the accessibility of the ribosome binding site. Protection of the CDS by translating ribosomes may contribute to stabilize the mRNA. During cold acclimation, the concentration of Csp proteins increases until it becomes sufficient to promote Csp binding to the mRNA. Csp binding drives the mRNA into a conformation that resembles the "37°C-conformation" (Zhang et al., 2018) and thereby restores attenuation.</figcaption></figure>