# Dual Regulation of Phosphatidylserine Decarboxylase Expression by Envelope Stress Responses

Yasmine Hassoun, Julia Bartoli, Astrid Wahl, Julie Viala, Emmanuelle Bouveret

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Yasmine Hassoun, Julia Bartoli, Astrid Wahl, Julie Viala, Emmanuelle Bouveret. Dual Regulation of Phosphatidylserine Decarboxylase Expression by Envelope Stress Responses. Frontiers in Molecular Biosciences, 2021, 8, 10.3389/fmolb.2021.665977. [https://doi.org/10.3389/fmolb.2021.665977](https://doi.org/10.3389/fmolb.2021.665977). [CC-BY](https://creativecommons.org/licenses/by/4.0/)

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# Dual Regulation of Phosphatidylserine Decarboxylase Expression by Envelope Stress Responses

Yasmine Hassoun<fnref n="1" />, Julia Bartoli<fnref n="1" />, Astrid Wahl<fnref n="1" />, Julie Pamela Viala<fnref n="1" />* and
Emmanuelle Bouveret<fnref n="2" />*

Bacteria adapt to versatile environments by modulating gene expression through a set of stress response regulators, alternative Sigma factors, or two-component systems. Among the central processes that must be finely tuned is membrane homeostasis, including synthesis of phospholipids (PL). However, few genetic regulations of this process have been reported. We have previously shown that the gene coding the first step of PL synthesis is regulated by σE and ppGpp, and that the BasRS (PmrAB) two component system controls the expression of the DgkA PL recycling enzyme. The gene coding for phosphatidylserine decarboxylase, the last step in phosphatidylethanolamine synthesis is another gene in the PL synthesis pathway susceptible of stress response regulation. Indeed, psd appears in transcriptome studies of the σE envelope stress Sigma factor and of the CpxAR two component system. Interestingly, this gene is presumably in operon with mscM coding for a miniconductance mechanosensitive channel. In this study, we dissected the promoter region of the psd-mscM operon and studied its regulation by σE and CpxR. By artificial activation of σE and CpxRA stress response pathways, using GFP transcriptional fusion and western-blot analysis of Psd and MscM enzyme production, we showed that the operon is under the control of two distinct promoters. One is activated by σE, the second is activated by CpxRA and also responsible for basal expression of the operon. The fact that the phosphatidylethanolamine synthesis pathway is controlled by envelope stress responses at both its first and last steps might be important for adaptation of the membrane to envelope perturbations.

**Keywords:** psd, mscM, SigmaE, CpxRA, envelope stress response, E. coli, phospholipid synthesis

## OPEN ACCESS

**Edited by:**
Heidi Vitrac,
University of Texas Health Science Center at Houston, United States

**Reviewed by:**
Stanley Spinola,
Indiana University-Purdue University Indianapolis, United States
Tracy Raivio,
University of Alberta, Canada

*Correspondence:
Julie Pamela Viala
jviala@imm.cnrs.fr
emanuelle.bouveret@pasteur.fr

**Specialty section:**
This article was submitted to Cellular Biochemistry, a section of the journal Frontiers in Molecular Biosciences

**Received:** 09 February 2021

**Accepted:** 09 April 2021

**Published:** 07 May 2021

**Citation:**
Hassoun Y, Bartoli J, Wahl A, Viala JP and Bouveret E (2021) Dual Regulation of Phosphatidylserine Decarboxylase Expression by Envelope Stress Responses. *Front. Mol. Biosci.* 8:665977.
doi: 10.3389/fmolb.2021.665977

## INTRODUCTION

Phospholipids (PL) are major structural and functional components of biomembranes and play a dynamic role in many regulatory processes. The biochemistry of the PL biosynthesis pathway is well described in bacteria (Parsons and Rock, 2013). It begins by two successive acylations of the positions 1 and 2 of a glycerol 3-phosphate backbone to give phosphatidic acid. Then, the third

<footnote n="1">¹ LISM, Institut de Microbiologie de la Méditerranée, UMR 7255, CNRS and Aix-Marseille Université, Marseille, France,</footnote>

<footnote n="2">² SAMe Unit, UMR 2001, Microbiology Department, Pasteur Institute, Paris, France</footnote>
position of the future polar head group is activated with a CDP
nucleotide. Finally, two separate pathways give rise in *Escherichia
coli* to the zwitterionic phosphatidylethanolamine (PE), or
to the anionic phospholipids phosphatidylglycerol (PG), and
cardiolipin (CL). Bacterial species display specific membrane
compositions, and the less frequent phosphatidylcholine,
phosphatidylinositol, and a variety of other membrane lipids
can be found in addition to the common PE, PG, and CL lipids
(Sohlenkamp and Geiger, 2016).

Though the composition of the membrane is highly stable
in *E. coli*, it can adapt to the growth conditions, through
modification of fatty acid chains, but also of the ratio of the
different lipids (Sohlenkamp and Geiger, 2016). For example, the
proportion of unsaturated fatty acids varies with temperature
to modify the fluidity of the membrane. Another example of
adaptation is the increase of CL following increase of osmotic
pressure. Also, in stationary phase, there is a relative increase
in anionic phospholipids, together with the replacement of fatty
acid unsaturations by cyclopropane groups. This conversion is
performed by the Cfa enzyme, whose expression is activated by
$\sigma^S$ alternative Sigma factor (Wang and Cronan, 1994; Eichel et al.,
1999). In addition, global transcriptome studies had suggested
that expression of PL synthesis genes might be globally affected
during the stringent response (Durfee et al., 2008), and that
specific genes of PL synthesis might be regulated by envelope
stress responses (De Wulf et al., 2002; Rhodius et al., 2006).

In bacteria, several regulating pathways are present to monitor
and respond to envelope perturbations. In *E. coli*, the alternative
Sigma factor $\sigma^E$ responds to the accumulation of unfolded
outer membrane proteins or altered forms of LPS in the
periplasm through a proteolytic signaling cascade (Mitchell
and Silhavy, 2019). In addition, two-component systems such
as CpxRA, RcsBA, or BaeRS respond to various envelope
perturbations such as defects in protein secretion, alterations in
LPS or peptidoglycan, or exposure to toxic compounds, by a
phosphorylation signaling cascade that ends up in the activation
of a transcriptional regulator (Mitchell and Silhavy, 2019). We
have previously shown that *plsB*, coding for the first acylation step
in PL synthesis, is activated by the envelope stress response $\sigma^E$
factor, and that *dgkA*, coding for a diacylglycerol kinase involved
in PL recycling, is controlled by the two-component system
BasRS (Wahl et al., 2011).

The *psd* gene coding for phosphatidylserine decarboxylase, the
last step in phosphatidylethanolamine synthesis, appears in the
regulon of $\sigma^E$ like *plsB*, but also in the regulon of the CpxAR
two-component system (De Wulf et al., 2002; Rezuchova et al.,
2003; Rhodius et al., 2006). The *psd* gene is followed by *mscM*
(previously called *yjeP*), and they probably form an operon.
MscM protein is a miniconductance mechanosensitive channel
of the MscS family, involved in the protection of the physical
integrity of the cell during transitions from high to low osmolarity
(Booth, 2014; Edwards et al., 2012).

In this study, we dissected the organization of the promoter
region of the *psd-mscM* operon, and we studied the dual
regulation of these genes by $\sigma^E$ and the response regulator
CpxR. We showed that the operon is under the control of
two distinct promoters. One is activated by $\sigma^E$, the other by

CpxRA. This second promoter is responsible for basal expression
of the operon. Therefore, the PL synthesis pathway (and more
specifically the PE pathway) is controlled by envelope stress
responses at both its first and last steps, which might be important
for adaptation of the membrane to envelope perturbations.

## MATERIALS AND METHODS

### Plasmid and Strain Constructions

pUA66 and pUA-psdP2 plasmids were obtained from the library
of *E. coli* promoters fused to GFP coding sequence (Zaslaver et al.,
2006). The other *psd* transcriptional fusions were constructed
using primers indicated in **Supplementary Table 1**, and cloned
in *Xho*I/BamHI* sites of pUA66. Expression plasmids for *rpoE*,
*nlpE*, and *nlpEIM* were constructed using primers indicated
in **Supplementary Table 1**, and cloned in *EcoRI/Sal*I sites of
pBAD24 vector (Guzman et al., 1995). A region of 1600 base pairs
encompassing *psd* promoter was cloned in pKO3 vector (Link
et al., 1997). Mutations were introduced in the pKO3-*psd* vector,
in the pBAD-*nlpE* and in the transcriptional fusions by PCR
mutagenesis on plasmid, using the oligonucleotides indicated
(**Table 1 and Supplementary Table 1**).

In order to introduce the sequence coding for the Flag
or SPA tags downstream of the coding sequences of interest
on the chromosome, we amplified the corresponding cassette
from the template plasmid pJL148 (Zeghouf et al., 2004).
MG1655_Psd-Flag and MG1655_MscM-SPA strains were then
constructed by PCR recombination at the locus followed by P1
transduction as described previously (Datsenko and Wanner,
2000) (**Table 2**). When required, the gene conferring resistance to
Kanamycin was removed using the pCP20 plasmid (Cherepanov
and Wackernagel, 1995), allowing the transformation by
transcriptional fusion plasmids. Point mutations were introduced
on the chromosome using the pKO3 vector (Link et al., 1997).

### Transcriptional Fusions with GFP

We used several clones from the *E. coli* transcriptional
fusions library (Zaslaver et al., 2006) and we constructed the
required additional transcriptional fusions (see above for plasmid
construction and **Table 1**). MG1655 wild-type *E. coli* strain
or isogenic mutants were transformed with plasmids carrying
the *gfp* transcriptional fusions and maintained with kanamycin.
For co-transformation, compatible plasmids (pBAD24 and
derivatives) were used, and ampicillin added. Selection plates
were incubated at 37°C for 16 h. A total of 600 µl of LB
medium supplemented with required antibiotics, and with 0.01%
arabinose when necessary for pBAD-driven expression, were
incubated (four to six replicate each assay) and grown for 16 h
at 30°C in 96-well polypropylene plates of 2.2 ml wells under
aeration and agitation. Fluorescent intensity measurement was
performed in a TECAN infinite M200 plate reader. A total of
150 µl of each well was transferred into black Greiner 96-well
plate for reading optical density at 600 nm and fluorescence
(excitation: 485 nm; emission: 530 nm). The expression levels
<table><caption>**TABLE 1** | Plasmids.</caption><thead><tr><th>Lab code</th><th>Name</th><th>Description</th><th>References</th></tr></thead><tbody><tr><td>pEB0794</td><td>pJL148</td><td>ampR, kanaR, SPA-FRT-kanaR-FRT cassette</td><td>Zeghouf et al., 2004</td></tr><tr><td>pEB0267</td><td>pKD46</td><td>ampR, ts, lambdaRed genes</td><td>Datsenko and Wanner, 2000</td></tr><tr><td>pEB0266</td><td>pCP20</td><td>camR, ampR, pSC101 ori, ts</td><td>Cherepanov and Wackernagel, 1995</td></tr><tr><td>pEB0227</td><td>pBAD24</td><td>ampR, colE1 ori, PBAD promoter</td><td>Guzman et al., 1995</td></tr><tr><td>pEB1470</td><td>pBAD-nlpE</td><td>PCR ebm981/982 (EcoRI/Xhol) in pBAD24 (EcoRI/Sall)</td><td>This work</td></tr><tr><td>pEB1966</td><td>pBAD-nlpE<sub>IM</sub></td><td>PCR mutagenesis ebm1785/1786 on pEB1470</td><td>This work</td></tr><tr><td>pEB1102</td><td>pBAD-rpoE</td><td></td><td>Wahl et al., 2011</td></tr><tr><td>pEB0232</td><td>pKO3</td><td>camR, pSC101 ori, sacB</td><td>Link et al., 1997</td></tr><tr><td>pEB1973</td><td>pKO3-psd</td><td>PCR ebm1762/1763 (BglII/Xhol) in pEB0232 (BamHI/Sall)</td><td>This work</td></tr><tr><td>pEB1983</td><td>pKO3-psd_mutexCpxR</td><td>PCR mutagenesis ebm1777/ebm1778 on pEB1973</td><td>This work</td></tr><tr><td>pEB1988</td><td>pKO3-psd_mutexP<sub>σ</sub><sup>E</sup></td><td>PCR mutagenesis ebm1808/ebm1809 on pEB1973</td><td>This work</td></tr><tr><td>pEB2021</td><td>pKO3-psd_mutexP<sub>σ</sub><sup>E</sup>_mutexCpxR</td><td>PCR mutagenesis ebm1777/ebm1778 on pEB1988</td><td>This work</td></tr><tr><td>pEB0067</td><td>pACYC184</td><td>camR, p15A ori</td><td>Chang and Cohen, 1978</td></tr><tr><td>pEB1975</td><td>pACYC-psd-3Flag</td><td>PCR ebm1762/968 on EB1075 (BglII/Xhol) in pACYC184 (BamHI/Sall)</td><td>This work</td></tr><tr><td>pEB2033</td><td>pACYC-psd(S254A)-3Flag</td><td>PCR mutagenesis ebm1911/1912 on pEB1975</td><td>This work</td></tr><tr><td>pEB2099</td><td>pACYC-psd</td><td>PCR ebm1762/1763 (BglII/Xhol) in pACYC184(BamHI/Sall)</td><td>This work</td></tr><tr><td>pEB0898</td><td>pUA66</td><td>kanaR, pSC101 ori, GFPmut2</td><td>Zaslaver et al., 2006</td></tr><tr><td>pEB1120</td><td>psdBis</td><td>PCR ebm435/436 in pUA66 Xhol/BamHI</td><td>This work</td></tr><tr><td>pEB1509</td><td>psdP<sub>σ</sub><sup>E</sup></td><td>PCR ebm435/1023 Xhol/BamHI in pUA66</td><td>This work</td></tr><tr><td>pEB1981</td><td>psdP<sub>σ</sub><sup>E</sup>_mut</td><td>PCR mutagenesis ebm1808/1809 on pEB1509</td><td>This work</td></tr><tr><td>pEB1963</td><td>psdP2</td><td></td><td>Zaslaver et al., 2006</td></tr><tr><td></td><td>psdP2mutexCpxR</td><td>Mutagenesis ebm1777/ebm1778 on pUA-psdP2</td><td>This work</td></tr></tbody></table>

were calculated by dividing the intensity of fluorescence by the optical density at 600 nm, after subtracting the values of blank sample. These results are given in arbitrary units because the intensity of fluorescence is acquired with an automatic optimal gain and hence varies from one experiment to the other.

## SDS-PAGE and Western Blotting

Total cell extracts were prepared by resuspending cell pellets in Laemli buffer 1X at a concentration of 0.3 uOD<sub>600</sub> nm in 10 µl, and then heating for 10 min at 95°C. After separation of 8 µl of total cell extracts on SDS-PAGE, electrotransfer onto nitrocellulose membranes was performed using Trans-Blot turbo transfer system from Bio-Rad. After blocking in PBS 1X + milk 5%, SPA-tagged or 3Flag-tagged proteins were detected with monoclonal anti-Flag M2 antibody purchased from Sigma. IscS protein was used as an internal control and revealed with polyclonal anti-IscS antibodies (Gully et al., 2003). Fluorescent secondary antibodies were respectively IRDye 800 anti-mouse and IRDye 680 anti-rabbit purchased from Li-Cor. Scanning and quantification were performed on a Li-Cor Odyssey-Fc imaging system, reading at 700 nm (for IscS detection) or 800 nm (for Flag detection).

## Reverse Transcriptase - PCR

RNAs were purified using the NEB Monarch Total RNA MiniPrep Kit and further digested with Dnase I followed by clean up with the Qiagen Rneasy Mini kit. Reverse transcription was performed using Invitrogen SuperScript III First-Stand Synthesis

System with random hexamers. Finally, PCR was performed using ebm446 and ebm2079 primers.

## RESULTS

In a global experimental study of all the transcriptional starting sites of *E. coli* (Thomason et al., 2015), two putative promoters were identified upstream the *psd* ORF (Figure 1). The first one, that we will call psdP<sub>σ</sub><sup>E</sup>, had been already reported as a promoter activated by the alternative sigma factor σ<sup>E</sup> (Rezuchova et al., 2003; Rhodius et al., 2006). The second one, that we will call psdP2, is located 197 nucleotides downstream psdP<sub>σ</sub><sup>E</sup>. It has already been reported that *psd* expression was activated by CpxR, and a putative CpxR box was proposed (De Wulf et al., 2002). This CpxR box is located 41 nucleotides upstream the predicted transcription start site of the psdP2 promoter, which is a distance in perfect agreement with CpxR acting as an activator of the psdP2 promoter (Busby, 2019).

## Dissection of the Promoter Region

In order to dissect the promoter region of *psd* and to study the genetic regulation of *psd* expression, we first used transcriptional fusions with GFP, expressed from the low copy vector pUA66, which enable to follow promoter activity by measuring fluorescence directly in living cells (Zaslaver et al., 2006). A transcriptional fusion containing the upstream region of *psd* ORF was already available from a library of *E. coli* promoters

<footnote><sup>a</sup>The limits of the transcriptional fusions are given relative to the start codon of psd.</footnote>
<table><caption>TABLE 2 | *Escherichia coli* strains.</caption><thead><tr><th>Lab code</th><th>Name</th><th>Description</th><th>References</th></tr></thead><tbody><tr><td>EB758</td><td>BW25113 Δ<i>cpxR</i>:kana<sup>R</sup></td><td></td><td>Baba et al., 2006</td></tr><tr><td>EB944</td><td>MG1655</td><td>Wild-type reference. F- λ- rph-1</td><td>Lab stock</td></tr><tr><td>EB425</td><td>MG1655 ppGpp°</td><td>Δ<i>relA</i>Δ<i>spoT</i>::cat</td><td>Wahl et al., 2011</td></tr><tr><td>EB559</td><td>MG1655Δ<i>dksA</i></td><td></td><td>Wahl et al., 2011</td></tr><tr><td>EB544</td><td>MG1655 ppGpp+</td><td>MG1655Δ<i>relA</i> <i>spoT</i>203 zib563:tetra</td><td>My et al., 2013</td></tr><tr><td>EB780</td><td>MG1655 Δ<i>cpxR</i></td><td>P1 transduction from EB758 in MG1655. kanaR cassette removed using pCP20.</td><td>This work</td></tr><tr><td>EB533</td><td>EH150</td><td>Mutation Gly280Asp. <i>psd</i>-2<sup>ts</sup></td><td>Hawrot and Kennedy, 1975</td></tr><tr><td>EB1075</td><td><i>psd</i>-3Flag</td><td>PCR ebm472/448 on pJL148 recombined in MG1655pKD46, then P1 transduction in MG1655</td><td>This work</td></tr><tr><td>EB1078</td><td>Δ<i>cpxR</i> <i>psd</i>-3Flag</td><td>P1 transduction from EB1075 in EB780</td><td>This work</td></tr><tr><td>EB1098</td><td><i>Psd</i>-3Flag<sub>mut</sub><i>CpxR</i></td><td>Recombination pEB1983 in EB1075</td><td>This work</td></tr><tr><td>EB1085</td><td><i>Psd</i>-3Flag<sub>mut</sub><i>Pσ</i><sup>E</sup></td><td>Recombination pEB1973 in EB1075</td><td>This work</td></tr><tr><td>EB1113</td><td><i>Psd</i>-3Flag<sub>mut</sub><i>CpxR</i><i>mut</i><i>Pσ</i><sup>E</sup></td><td>Recombination pEB2021 in EB1075</td><td>This work</td></tr><tr><td>EB1095</td><td>MscM-SPA</td><td>PCR ebm488/489 on pJL148 recombined in MG1655pKD46, then P1 transduction in MG1655</td><td>This work</td></tr><tr><td>EB1081</td><td><i>Psd</i><sub>mut</sub><i>CpxR</i></td><td>Recombination pEB1983 in MG1655</td><td>This work</td></tr><tr><td>EB1103</td><td><i>Psd</i><sub>mut</sub><i>Pσ</i><sup>E</sup></td><td>Recombination pEB1988 in MG1655</td><td>This work</td></tr><tr><td>EB1112</td><td><i>Psd</i><sub>mut</sub><i>CpxR</i><i>mut</i><i>Pσ</i><sup>E</sup></td><td>Recombination pEB2021 in MG1655</td><td>This work</td></tr></tbody></table>

<figure><img src="image_3.png" /></figure>

(Zaslaver et al., 2006), but it only contained the putative psdP2 promoter. Therefore, we constructed additional transcriptional fusions, containing both promoters (called psdBis) or only the psdPσE promoter (Figure 1A).

We first studied the control of *psd* expression by σE. We artificially induced the σE response, by overproducing the σE factor from an inducible pBAD-*rpoE* plasmid as previously described (Wahl et al., 2011). We co-transformed an *E. coli* wild-type strain with the different GFP transcriptional fusions

and with the pBAD-*rpoE* plasmid. Upon σE overproduction, only the transcriptional fusions containing the psdPσE promoter (psdBis and psdPσE, but not psdP2) showed a strong induction (Figure 2). We then mutated two nucleotide positions in the predicted -10 box of the psdPσE promoter. These mutations completely abolished the induction of the psdPσE transcriptional fusion by pBAD-*rpoE* (Figure 2).

We then tested if CpxR was a transcriptional activator of the psdP2 promoter. We first compared the activity of the
<figure><img src="image_4.png" /></figure>

**FIGURE 2** | Effect of $\sigma^E$ overproduction on the psdP$\sigma^E$ promoter. MG1655 strain was transformed with plasmids carrying the transcriptional fusions pUA66, pUA-psdP2, pUA-psdBis, pUA-psdP$\sigma^E$, or pUA-psdP$\sigma^Emut$ together with plasmids pBAD24 or pBAD-rpoE. Cultures were grown overnight at 30°C in LB supplemented with ampicillin, kanamycin, and 0.01% arabinose. The values show the mean ratio of GFP fluorescence over optical density at 600 nm, after subtraction of the pUA66 negative control, in arbitrary units (A.U.). The values are the mean of six replicas and the error bars show the SEM (standard error of the mean). ns, non-significant, \*\*\*p < 0.0001 in a two-way ANOVA statistical analysis.

psdP2 transcriptional fusion in wild-type and $\Delta cpxR$ strains. This activity was reduced in the $\Delta cpxR$ strain (Figure 3A), suggesting a potential role of CpxR in the regulation of psd expression, even in balanced growth conditions. In reverse, in order to induce the CpxR response, we overproduced the NlpE lipoprotein or its NlpE<sub>IM</sub> variant that triggers an even stronger response (Delhaye et al., 2016). We co-transformed an *E. coli* wild-type strain by the different transcriptional fusions and by the pBAD-*nlpE* or-*nlpE*<sub>IM</sub> plasmids. The psdP2 transcriptional fusion showed an activation when NlpE was overproduced, and the effect was reinforced with NlpE<sub>IM</sub> (Figure 3B). Furthermore, the activation was abolished when the experiment was repeated in a $\Delta cpxR$ strain (Figure 3C), which showed that this was indeed the result of an activation of the CpxR pathway. Finally, to prove that the activation was due to direct binding of CpxR response regulator to the psdP2 promoter, we introduced point mutations that destroy the CpxR binding motif in the psdP2 transcriptional fusion (Figure 1B). With this mutated transcriptional fusion, the activation of psdP2 promoter was abolished (Figure 3B).

In conclusion, psd expression (and potentially mscM expression) is under the control of two distinct promoters. One is activated by $\sigma^E$, the other by CpxRA. Given that this promoter organization is very similar to the one we described previously for *plsB* (Wahl et al., 2011), we hypothesized that this second promoter might be regulated by ppGpp as shown for *plsB* and suggested by global study of the stringent response (Durfee et al., 2008). We tested this by measuring the activity of the transcriptional fusions in strains with modified ppGpp levels as previously described (Wahl et al., 2011). The ppGpp° ($\Delta relA \Delta spoT$) strain is devoid of ppGpp, the DksA cofactor is necessary for ppGpp action on RNAP hence a $\Delta dksA$ mutant mimics the absence of ppGpp, and finally the *spoT203* mutant is impaired in ppGpp degradation, leading to ppGpp accumulation in the cell. The psdP2 promoter did not show a strong dependence on ppGpp levels (Supplementary Figure 1). The psdP2 expression did slightly increase in a $\Delta dksA$ mutant, but

not in the ppGpp° mutant, and it did not show a corresponding repression in the ppGpp+ mutant. Interestingly, the activity of the psdP$\sigma^E$ transcriptional fusion increased in the ppGpp accumulating mutant (Supplementary Figure 1), which is in agreement with the known positive effect of ppGpp on $\sigma^E$ (Costanzo et al., 2008).

## Impact of $\sigma^E$ or CpxR Regulation on Psd and MscM Protein Amounts

Because psd expression is controlled by two promoters, we then wanted to study the relative involvement of these two promoters in the control of the amounts of Psd in the cell. For that, we constructed a strain where Psd is fused at its C-terminus with the 3Flag tag, in order to detect the protein by western blot with a monoclonal antibody. Psd is produced as a proenzyme, which after endoproteolysis gives rise to a mature two-domains enzyme (Li and Dowhan, 1990; Choi et al., 2015). Because the tag is fused to the C-terminus of the protein, we detected as expected the small C-terminal Psd$\alpha$-3Flag subdomain, indicating that the cleavage occurred normally (it migrated at a higher position than the expected 12 kDa, but this is not exceptional, and might be influenced by the pyruvoyl N-terminal modification). Furthermore, the tagged strain did not display any obvious growth defect, suggesting that the Psd-3Flag fusion was functional. However, in order to prove the functionality of the Psd-3Flag tagged protein, we also cloned this construction or the wild-type psd gene under its own promoter in a plasmid, and tested the complementation of a psd-ts mutant (Hawrot and Kennedy, 1975). Expression of psd-3Flag or wild-type psd expressed from a plasmid similarly restored growth of the psd<sup>ts</sup> mutant at 42°C, showing that the recombinant Psd-3Flag protein was functional (Figure 4). We then tested the effect of overproducing $\sigma^E$ or inducing the CpxR response through NlpE expression. The levels of Psd-3Flag protein were clearly increased when $\sigma^E$ was overproduced (Figure 5A). Similarly,
<figure><img src="image_5.png" /><figcaption>**FIGURE 3** | Control of the psdP2 promoter by CpxR. (A) MG1655 or Δ*cpxR* strains were transformed with the indicated transcriptional fusions. Cultures were grown overnight at 30°C in LB supplemented with kanamycin. (B,C) MG1655 or Δ*cpxR* strains were transformed with the indicated transcriptional fusions together with pBAD24, pBAD-*nlpE*, or pBAD-*nlpE_IM* plasmids. Cultures were grown overnight at 30°C in LB supplemented with ampicillin, kanamycin and 0.01% arabinose. The values show the mean ratio of GFP fluorescence over optical density at 600 nm, after subtraction of the pUA66 negative control, in arbitrary units (A.U.). The values are the mean of 12 (A) or 4 (B,C) replicas and the error bars show the SEM (standard error of the mean). ns, non-significant, \*\*\*p < 0.001, \*\*\*p < 0.0001 in a two-way ANOVA statistical analysis.</figcaption></figure>

levels of Psd-3Flag increased when NlpE or NlpE IM proteins were overproduced (Figure 5B). The increases observed when the CpxRA pathway was artificially induced were not as strong as the ones observed when $\sigma^E$ was overproduced. However, they were clearly abolished when the experiment was repeated in a Δ*cpxR* genetic context (Figure 5B). It has to be noted that when Psd-3Flag amounts were strongly increased, a band at approximately 45 kDa was also detected (Figure 5A). We hypothesized that this band might correspond to the accumulation of an unmatured

full length Psd-3Flag proenzyme. In order to test this, we introduced the point mutation S254A, which is known to prevent the maturation of Psd (Li and Dowhan, 1990), in the plasmid expressing *psd*-3Flag. As expected, this mutant form was detected at 45 kDa (Supplementary Figure 2). Interestingly, when we compared the production of Psd-3Flag with or without induction of *rpoE*, the band at 45 kDa accumulating when *rpoE* is induced migrated at the exact same size than the unmatured Psd(254A)-3Flag (Supplementary Figure 2), suggesting that endoproteolytic cleavage is indeed impaired when Psd-3Flag is overproduced.

On the genome of *E. coli*, *psd* ORF is followed by *mscM* ORF, suggesting an organization of these two genes as an operon. To study the involvement of $\sigma^E$ and CpxR on *mscM* expression, we constructed a strain where MscM is fused at its C-terminus with the SPA tag. MscM-SPA was faintly detected on Western blot at the expected size of 130 kDa, and MscM protein levels were increased when *rpoE* or *nlpE* were artificially expressed, similarly as for Psd-3Flag (Figure 6A). We then performed an RT-PCR experiment, which confirmed that *psd* and *mscM* are expressed as an operon, controlled by the same two promoters (Figure 6B).

## Characterization of Strains Devoid of *psd-mscM* Control by $\sigma^E$ and CpxR

The above experiments using transcriptional fusions or following the amount of Psd and MscM proteins demonstrated the control of *psd* and *mscM* expression by the two envelope stress response pathways mediated by $\sigma^E$ or CpxR. The next question was naturally to understand the physiological role of these regulations. To address this question, we chose to introduce point mutations in the promoters of *psd*, on the chromosome, in order to prevent specifically the activation of *psd-mscM* expression by $\sigma^E$, CpxR or both stress responses at the same time. We have shown above that the mutations chosen in the $\sigma^E$ promoter and CpxR box completely abolished the induction of the transcriptional fusions by pBAD-*rpoE* or pBAD-*nlpE*, respectively (Figures 2, 3). Note that both mutations are localized inside the ORF of the upstream *rsgA* gene. The mutation in the Psd$\sigma^E$ promoter does not modify the amino acid sequence of RsgA, while the mutation in the CpxR box does modify residue Lys340 of RsgA in Glutamine. We introduced the mutations in wild-type MG1655 strain and in the Psd-3Flag strain using homologous recombination with the pKO3 plasmid (Link et al., 1997). The three types of mutants were readily obtained in the two strains (Table 2). The mutant strains did not display any obvious growth phenotype in LB (Supplementary Figure 3) showing that $\sigma^E$ and CpxR regulations are not essential in balanced growth conditions. In the Psd-3Flag background strains, we observed that mutations in the $\rho\sigma^E$ promoter and CpxR box had no effect on the basal amounts of Psd-3Flag protein (Figure 5C and data not shown). This suggests that the decreased expression observed with the psdP2muCpxR transcriptional fusion (Figure 3A) does not impact protein level, and shows that these regulations are not important to control basal Psd and MscM protein levels. However, when $\sigma^E$ was induced, the amounts of Psd-3Flag protein did not increase anymore in the strains containing the $\rho\sigma^E$ mutation (Figure 5C, first and
<figure><img src="image_6.png" /><figcaption>**FIGURE 4** | Strain EH150 *psd2*-ts (Hawrot and Kennedy, 1975) was transformed by pACYC184, pACYC-*psd*, or pACYC-*psd*-3Flag plasmids and the selection was performed at 30°C. Then, colonies were spread out on LB chloramphenicol plates at 30 or 42°C for 24 h.</figcaption></figure>

third panel), while Psd-3Flag still increased in the CpxRbox mutant (**Figure 5C**, second panel). When the CpxRA pathway was induced, with pBAD-*nlpE* or pBAD-*nlpE_IM* plasmids, Psd-3Flag protein amounts did not increase anymore in the strains containing the mutation in the CpxR binding site (**Figure 5C**, second and third panel), while Psd-3Flag still increased in the PoE mutant (**Figure 5C**, first panel). These results confirmed what we observed with the transcriptional fusions (**Figure 3A**), and showed that the σE and CpxR control of *psd* promoters impact the amount of proteins produced *in vivo* upon stress induction.

DISCUSSION

The goal of this work was to decipher the genetic control
of *psd-mscM* operon and its regulation by the two envelope
stress responses mediated by the alternative Sigma factor σE
and by the two-component system CpxRA. We found that *psd*
promoter region comprises two promoters, confirming the +1
transcription start sites identified previously (Thomason et al.,
2015): a distal σE promoter and a proximal P2 promoter, activated
by CpxRA, which is likely to be also responsible for the basal
expression of *psd*. This organization is very similar to what we
observed previously for the promoter of *plsB*, which comprises a
distal σE promoter and a proximal P2 promoter, responsible for
the basal expression of *plsB* (Wahl et al., 2011). However, *plsB*
expression was not controlled by CpxR (data not shown). We also
systematically tested if other genes of PL synthesis were regulated
by σE or CpxR but it was not the case (data not shown). Therefore,
it appears that envelope stress responses control the first step of
PL synthesis (*plsB*) and the last step of PE synthesis (*psd*). This

suggests that in response to envelope stress, an increase in the
pathway for PE might be important, in order to help in envelope
biogenesis processes such as LPS or outer membrane proteins
trafficking. An enhanced PE biosynthesis might be obtained by
"pushing" from the top with PlsB and "pulling" from the bottom
of the pathway with Psd. However, enzymes of PL synthesis
are believed to be present in excess and it is not clear how
increasing enzyme amounts might have an effect on the flux in
the PL synthetic pathway. This regulation might be a long-term
adaptation rather than an immediate response to the encountered
stress. An alternative hypothesis is that the enzymes might be
damaged during envelope stress and need to be replaced.

We previously showed that the basal promoter of the *plsB*
gene was repressed by ppGpp (Wahl et al., 2011). Furthermore,
it was proposed that the genes for PE pathway, including
*psd*, might be downregulated during the stringent response,
while the genes for PG/CL were upregulated (Durfee et al.,
2008), which would be consistent with an increase in PG and
CL in the membrane during growth arrest. However, we did
not detect a strong regulation of psdP2 promoter by ppGpp
(Supplementary Figure 1).

It is striking to find *psd* in operon with a gene encoding a
mechanosensitive channel. There is no obvious direct functional
link between these two genes, apart from a function in membrane
homeostasis. Mechanosensitive channels open in response to
transition from high to low osmolarity in order to protect cells
from bursting (Booth, 2014). There are seven mechanosensitive
channels in *E. coli*, MscL and six channels of the MscS family,
including MscM. This redundancy might be explained by a
need to respond to different rates or intensities in osmolarity
variations (Booth, 2014). As for *psd*, a genetic regulation might
<figure><img src="image_7.png" /></figure>

**FIGURE 5** | Psd protein levels are controlled by σ<sup>E</sup> and CpxR. (A) *E. coli* Psd-3Flag strain was transformed by pBAD24 or pBAD-rpoE plasmids. Two replicas of each transformation were grown in LB supplemented with ampicillin until OD<sub>600</sub> nm = 0.8, then cultures were induced with 0.2% arabinose for 2 h. (B) Psd-3Flag and ΔcpxR-Psd-3Flag strains were transformed by pBAD24, pBAD-nlpE, or pBAD-nlpE_IM plasmids. Three replicas of each transformation were grown in LB supplemented with ampicillin until OD<sub>600</sub> nm = 0.8, then cultures were induced with 0.2% arabinose for 2 h. (C) Psd-3Flag_mutexE, Psd-3Flag_mutexCpxR, and Psd-3Flag_mutexCpxR_mutexE strains containing mutations in the promoter region were transformed by pBAD24, pBAD-rpoE, pBAD-nlpE, or pBAD-nlpE_IM plasmids. Two replicas of each transformation were grown in LB supplemented with ampicillin until OD<sub>600</sub> nm = 0.8, then cultures were induced with 0.2% arabinose for 2 h. Proteins were separated by SDS-PAGE 12% and detected by western blot using anti-Flag monoclonal antibody to detect Psd-3Flag or anti-IscS polyclonal antibody as an internal loading control. Quantification of the bands are shown in **Supplementary Figure 4**.

appear irrelevant for a rapid response to physical stress. However,
the number and types of channels are important factors to
determine the protective ability and cell fate (Bialecka-Fornal
et al., 2015), and it has been shown that over-expression of
MscM confers protection to hypo-osmotic shock in a strain
otherwise devoid of all the other mechanosensitive channels
(Edwards et al., 2012). Therefore, increased expression of mscM
by σE and CpxR in response to envelope stress might be a
bet hedging strategy, as it might confer adaptation for future
additional envelope injuries. Similarly, mscL and mscS genes
regulated by the alternative Sigma factor σS (Stokes et al., 2003).
Also, in Pseudomonas aeruginosa, the cmpX gene coding for a
putative mechanosensitive channel is part of the SigX envelope
stress response regulon (Gicquel et al. 2013). Furthermore,
these mechanosensitive channels possess diverse periplasmic and
cytoplasmic domains that might play additional functions in

stress responses, which for example was suggested recently for
YbdG in *E. coli* (Amemiya et al., 2019).

Unfortunately, we were not able yet to find growth conditions or stress conditions where the σE or CpxRA regulations on plsmB and psd-mscM had a role for cell physiology. We tried to subject the mutant strains to stresses known to induce the σE or CpxRA pathways, i.e., osmotic stress, alkaline stress, envelope porin misfolding or mislocalization, or heat shock, but we did not detect any effects in these experiments. As mentioned above, these regulations are certainly involved in long-term adaptation rather than being an immediate response to the encountered stress. This might be why we did not detect any obvious phenotypic defects in the regulatory mutant strains when testing for immediate resistance to stress. Combining stresses, long-term, or competition experiments might be needed to be able to unravel a phenotypic advantage of the wild-type over the
<figure><img src="image_8.png" /></figure>

**FIGURE 6** | *mscM* is in operon with *psd*, under control of the σE and CpxR dependent promoters. (A) *E. coli* MscM-SPA strain was transformed by pBAD24, pBAD-*rpoE*, pBAD-*nlpE*, or pBAD-*nlpE_IM* plasmids. Two replicas of each transformation were grown in LB supplemented with ampicillin until OD600 nm = 0.8, then cultures were induced with 0.2% arabinose for 2 h. Proteins were separated by SDS-PAGE 10% and detected by western blot using anti-Flag monoclonal antibody for the detection of MscM-SPA or anti-IscS polyclonal antibodies as an internal loading control. (B) RT-PCR was performed on MG1655 strain transformed by pBAD or pBAD-*rpoE* induced in the above conditions. The black arrows indicate the positions of the primers used for the amplification. PCR controls on water, genomic DNA, and equivalent amounts of RNA as in the RT-PCR reaction were performed.

regulatory mutant strains. Interestingly, and in a reversed way, several studies have shown that PL synthesis mutants, which affect membrane composition, activate envelope stress responses, including σE and CpxRA signaling pathways (Mileykovskaya and Dowhan, 1997; Itou et al., 2012; Rowlett et al., 2017; Nepper et al., 2019). Therefore, the σE or CpxRA regulations of *plsB* and *psd-mscM* might also be part of a feedback loop involved in maintaining an optimum PL synthesis balance for membrane homeostasis.

## DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

## AUTHOR CONTRIBUTIONS

YH, EB, and JV contributed to conception and design of the study. YH, JB, and AW performed experiments. EB wrote the

manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

## FUNDING

This work was funded by the Centre National de la Recherche Scientifique (CNRS), the Pasteur Institute, and grant ANR-19-CE13-0009-02. YH was supported by a doctoral fellowship from the French Ministry of Higher Education and Research.

## ACKNOWLEDGMENTS

We thank Celia Torrielli for the construction of *E. coli* strains.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmolb.2021.665977/full#supplementary-material
REFERENCES

Amemiya, S., Toyoda, H., Kimura, M., Saito, H., Kobayashi, H., Ihara, K., et al. (2019). The mechanosensitive channel YbdG from *Escherichia coli* has a role in adaptation to osmotic up-shock. *J. Biol. Chem.* 294, 12281–12292. doi: 10.1074/jbc.ra118.007340

Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., et al. (2006). Construction of *Escherichia coli* K-12 in-frame, single-gene knockout mutants: the Keio collection. *Mol. Syst. Biol.* 2:2006.0008.

Bialecka-Fornal, M., Lee, H. J., and Phillips, R. (2015). The rate of osmotic downshock determines the survival probability of bacterial mechanosensitive channel mutants. *J. Bacteriol.* 197, 231–237. doi: 10.1128/jb.02175-14

Booth, I. R. (2014). Bacterial mechanosensitive channels: progress towards an understanding of their roles in cell physiology. *Curr. Opin. Microbiol.* 18, 16–22. doi: 10.1016/j.mib.2014.01.005

Busby, S. J. W. (2019). Transcription activation in bacteria: ancient and modern. *Microbiology* 165, 386–395. doi: 10.1099/mic.0.000783

Chang, A. C., and Cohen, S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. *J. Bacteriol.* 134, 1141–1156. doi: 10.1128/jb.134.3.1141-1156. 1978

Cherepanov, P. P., and Wackernagel, W. (1995). Gene disruption in *Escherichia coli*: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. *Gene* 158, 9–14. doi: 10.1016/0378-1119(95) 00193-a

Choi, J. Y., Duraisingh, M. T., Marti, M., Ben Mamoun, C., and Voelker, D. R. (2015). From Protease to Decarboxylase: the molecular metamorphosis of phosphatidylserine decarboxylase. *J. Biol. Chem.* 290, 10972–10980.

Costanzo, A., Nicoloff, H., Barchinger, S. E., Banta, A. B., Gourse, R. L., and Ades, S. E. (2008). ppGpp and DksA likely regulate the activity of the extracytoplasmic stress factor sigmaE in *Escherichia coli* by both direct and indirect mechanisms. *Mol. Microbiol.* 67, 619–632. doi: 10.1111/j.1365-2958.2007.06072.x

Datsenko, K. A., and Wanner, B. L. (2000). One-step inactivation of chromosomal genes in *Escherichia coli* K-12 using PCR products. *Proc. Natl. Acad. Sci. U.S.A.* 97, 6640–6645. doi: 10.1073/pnas.120163297

De Wulf, P., McGuire, A. M., Liu, X., and Lin, E. C. (2002). Genome-wide profiling of promoter recognition by the two-component response regulator CpxR-P in *Escherichia coli*. *J. Biol. Chem.* 277, 26652–26661. doi: 10.1074/jbc.m203487200

Delhaye, A., Collet, J. F., and Laloux, G. (2016). Fine-tuning of the Cpx envelope stress response is required for cell wall homeostasis in *Escherichia coli*. *mBio* 7:e00047-16.

Durfee, T., Hansen, A. M., Zhi, H., Blattner, F. R., and Jin, D. J. (2008). Transcription profiling of the stringent response in *Escherichia coli*. *J. Bacteriol.* 190, 1084–1096. doi: 10.1128/jb.01092-07

Edwards, M. D., Black, S., Rasmussen, T., Rasmussen, A., Stokes, N. R., Stephen, T. L., et al. (2012). Characterization of three novel mechanosensitive channel activities in *Escherichia coli*. *Channels* 6, 272–281. doi: 10.4161/chan.20998

Eichel, J., Chang, Y. Y., Riesenberg, D., and Cronan, J. E. (1999). Effect of ppGpp on *Escherichia coli* cyclopropane fatty acid synthesis is mediated through the RpoS sigma factor (sigmaS). *J. Bacteriol.* 181, 572–576. doi: 10.1128/jb.181.2. 572-576.1999

Gicquel, G., Bouffartigues, E., Bains, M., Oxaran, V., Rosay, T., Lesouhaitier, O., et al. (2013). The extra-cytoplasmic function sigma factor sigX modulates biofilm and virulence-related properties in *Pseudomonas aeruginosa*. *PLoS One* 8:e80407. doi: 10.1371/journal.pone.0080407

Gully, D., Moinier, D., Loiseau, L., and Bouveret, E. (2003). New partners of acyl carrier protein detected in *Escherichia coli* by tandem affinity purification. *FEBS Lett.* 548, 90–96. doi: 10.1016/s0014-5793(03)00746-4

Guzman, L. M., Belin, D., Carson, M. J., and Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. *J. Bacteriol.* 177, 4121–4130.

Hawrot, E., and Kennedy, E. P. (1975). Biogenesis of membrane lipids: mutants of *Escherichia coli* with temperature-sensitive phosphatidylserine decarboxylase. *Proc. Natl. Acad. Sci. U.S.A.* 72, 1112–1116. doi: 10.1073/pnas.72.3.1112

Itou, A., Matsumoto, K., and Hara, H. (2012). Activation of the Cpx phosphorelay signal transduction system in acidic phospholipid-deficient pgsA mutant cells of *Escherichia coli*. *Biochem. Biophys. Res. Commun.* 421, 296–300. doi: 10.1016/j.bbrc.2012.04.003

Li, Q. X., and Dowhan, W. (1990). Studies on the mechanism of formation of the pyruvate prosthetic group of phosphatidylserine decarboxylase from

*Escherichia coli*. *J. Biol. Chem.* 265, 4111–4115. doi: 10.1016/s0021-9258(19) 39709-1

Link, A. J., Phillips, D., and Church, G. M. (1997). Methods for generating precise deletions and insertions in the genome of wild-type *Escherichia coli*: application to open reading frame characterization. *J. Bacteriol.* 179, 6228–6237. doi: 10. 1128/jb.179.20.6228-6237.1997

Mileykovskaya, E., and Dowhan, W. (1997). The Cpx two-component signal transduction pathway is activated in *Escherichia coli* mutant strains lacking phosphatidylethanolamine. *J. Bacteriol.* 179, 1029–1034. doi: 10.1128/jb.179. 4.1029-1034.1997

Mitchell, A. M., and Silhavy, T. J. (2019). Envelope stress responses: balancing damage repair and toxicity. *Nat. Rev. Microbiol.* 17, 417–428. doi: 10.1038/ s41579-019-0199-0

My, L., Rekoske, B., Lemke, J. J., Viala, J. P., Gourse, R. L., and Bouveret, E. (2013). Transcription of the *Escherichia coli* fatty acid synthesis operon fabHDG is directly activated by FadR and inhibited by ppGpp. *J. Bacteriol.* 195, 3784–3795. doi: 10.1128/jb.00384-13

Nepper, J. F., Lin, Y. C., and Weibel, D. B. (2019). Rcs phosphorelay activation in cardiolaripin-deficient *Escherichia coli* reduces biofilm formation. *J. Bacteriol.* 201:e00804-18.

Parsons, J. B., and Rock, C. O. (2013). Bacterial lipids: metabolism and membrane homeostasis. *Prog. Lipid Res.* 52, 249–276. doi: 10.1016/j.plipres.2013.02.002

Rezuchova, B., Miticka, H., Homerova, D., Roberts, M., and Kormanec, J. (2003). New members of the *Escherichia coli* sigmaE regulon identified by a two-plasmid system. *FEMS Microbiol. Lett.* 225, 1–7. doi: 10.1016/s0378-1097(03) 00480-4

Rhodius, V. A., Suh, W. C., Nonaka, G., West, J., and Gross, C. A. (2006). Conserved and variable functions of the sigmaE stress response in related genomes. *PLoS Biol.* 4:e2. doi: 10.1371/journal.pbio.0040002

Rowlett, V. W., Mallampalli, V. K. P. S., Karlstaedt, A., Dowhan, W., Taegtmeyer, H., Margolin, W., et al. (2017). Impact of membrane phospholipid alterations in *Escherichia coli* on cellular function and bacterial stress adaptation. *J. Bacteriol.* 199:e00849-16.

Sohlenkamp, C., and Geiger, O. (2016). Bacterial membrane lipids: diversity in structures and pathways. *FEMS Microbiol. Rev.* 40, 133–159. doi: 10.1093/ femsre/fuv008

Stokes, N. R., Murray, H. D., Subramaniam, C., Gourse, R. L., Louis, P., Bartlett, W., et al. (2003). A role for mechanosensitive channels in survival of stationary phase: regulation of channel expression by RpoS. *Proc. Natl. Acad. Sci. U.S.A.* 100, 15959–15964. doi: 10.1073/pnas.2536607100

Thomason, M. K., Bischler, T., Eisenbart, S. K., Förstner, K. U., Zhang, A., Herbig, A., et al. (2015). Global transcriptional start site mapping using differential RNA sequencing reveals novel antisense RNAs in *Escherichia coli*. *J. Bacteriol.* 197, 18–28. doi: 10.1128/jb.02096-14

Wahl, A., My, L., Dumoulin, R., Sturgis, J. N., and Bouveret, E. (2011). Antagonistic regulation of dgaK and plsB genes of phospholipid synthesis by multiple stress responses in *Escherichia coli*. *Mol. Microbiol.* 80, 1260–1275. doi: 10.1111/j. 1365-2958.2011.07641.x

Wang, A. Y., and Cronan, J. E. (1994). The growth phase-dependent synthesis of cyclopropane fatty acids in *Escherichia coli* is the result of an RpoS(KatF)-dependent promoter plus enzyme instability. *Mol. Microbiol.* 11, 1009–1017. doi: 10.1111/j.1365-2958.1994.tb00379.x

Zaslaver, A., Bren, A., Ronen, M., Itzkovitz, S., Kikoin, I., Shavit, S., et al. (2006). A comprehensive library of fluorescent transcriptional reporters for *Escherichia coli*. *Nat. Methods* 3, 623–628. doi: 10.1038/nmeth895

Zeghouf, M., Li, J., Butland, G., Borkowska, A., Canadien, V., Richards, D., et al. (2004). Sequential peptide affinity (SPA) system for the identification of mammalian and bacterial protein complexes. *J. Proteome Res.* 3, 463–468. doi: 10.1021/pr034084x

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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