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Article

Amplifying and Fine-Tuning Rsm sRNAs Expression and Stability to Optimize the Survival of Pseudomonas brassicacerum in Nutrient-Poor Environments

by
David Lalaouna
1,2,
Sylvain Fochesato
1,
Mourad Harir
3,4,
Philippe Ortet
1,
Philippe Schmitt-Kopplin
3,4,
Thierry Heulin
1 and
Wafa Achouak
1,*
1
Aix Marseille Univ, CEA, CNRS, BIAM, Lab Microbial Ecology of the Rhizosphere (LEMiRE), F-13108 Saint-Paul-Lez-Durance, France
2
Université de Strasbourg, CNRS, ARN UPR 9002, F-67000 Strasbourg, France
3
Research Unit Analytical BioGeoChemistry, Helmholtz Zentrum München, 85764 Neuherberg, Germany
4
Technical University of Munich, Maximus-von-Imhof-Forum 2, 85354 Freising, Germany
*
Author to whom correspondence should be addressed.
Microorganisms 2021, 9(2), 250; https://doi.org/10.3390/microorganisms9020250
Submission received: 1 January 2021 / Revised: 13 January 2021 / Accepted: 19 January 2021 / Published: 26 January 2021
(This article belongs to the Special Issue Plant-Associated Pseudomonads)
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Abstract

:
In the beneficial plant root-associated Pseudomonas brassicacearum strain NFM421, the GacS/GacA two-component system positively controls biofilm formation and the production of secondary metabolites through the synthesis of rsmX, rsmY and rsmZ. Here, we evidenced the genetic amplification of Rsm sRNAs by the discovery of a novel 110-nt long sRNA encoding gene, rsmX-2, generated by the duplication of rsmX-1 (formerly rsmX). Like the others rsm genes, its overexpression overrides the gacA mutation. We explored the expression and the stability of rsmX-1, rsmX-2, rsmY and rsmZ encoding genes under rich or nutrient-poor conditions, and showed that their amount is fine-tuned at the transcriptional and more interestingly at the post-transcriptional level. Unlike rsmY and rsmZ, we noticed that the expression of rsmX-1 and rsmX-2 genes was exclusively GacA-dependent. The highest expression level and longest half-life for each sRNA were correlated with the highest ppGpp and cyclic-di-GMP levels and were recorded under nutrient-poor conditions. Together, these data support the view that the Rsm system in P. brassicacearum is likely linked to the stringent response, and seems to be required for bacterial adaptation to nutritional stress.

1. Introduction

The RNA-binding protein CsrA (carbon storage regulator) and its homolog RsmA (regulator of secondary metabolites) regulate a variety of genes at the post-transcriptional level. Their regulatory activity is modulated by regulatory small RNA (sRNA) antagonists of CsrA/RsmA. Functional homologs of the Csr/Rsm system have been discovered in many Gram-negative genera, including Escherichia [1], Salmonella [2], Vibrio [3], Legionella [4], Yersinia [5] and Pseudomonas [6,7,8], as well as in some Gram-positive bacteria such as the Bacillus genus [9].
The wide distribution of the Csr/Rsm regulatory system in Eubacteria, including animal/plant pathogenic species as well as plant growth-promoting rhizobacteria (PGPR), illustrates that this well-conserved system is involved in various essential functions [10,11]. Indeed, small RNAs positively controlled by either the BarA/UvrY or GacS/GacA two-component systems have been shown to regulate various functions by coordinating the expression of a set of target genes post-transcriptionally [12,13]
The number and size of sRNAs involved in this system are variable. In Escherichia coli, the protein repressor CsrA is regulated by two sRNAs, CsrB and CsrC. These sRNAs sequester CsrA by mimicking its recognition sequence [14,15]. Here, CsrB sRNA, a 360-nucleotide long RNA, works in concert with CsrC sRNA (250 nts). Two homologs were found in Pseudomonas aeruginosa and named RsmY (124 nt) and RsmZ (115 nt) [7]. A third Rsm sRNA, RsmX (119 nt), was discovered in Pseudomonas protegens (formerly Pseudomonas fluorescens) CHA0 [6] and predicted to be found in other species including P. fluorescens Pf-5, P. fluorescens Pf0-1, Pseudomonas putida KT2440 and Pseudomonas syringae pv. tomato DC3000. Surprisingly, up to five RsmX encoding genes have been described in P. syringae [8]. Moreover, experiments with artificial RsmY-like sRNAs have demonstrated that conservation of size and/or primary sequence is not essential for Rsm functions [16]. In fact, the regulatory mechanism is linked to appropriate exposure of mimicked CsrA/RsmA recognition motif.
In general, Csr and Rsm sRNAs seem to be redundant [7,17,18]. However, although all Rsm sRNAs are positively controlled by GacA, additional regulators have been identified, such as PsrA, which activates rsmZ in P. protegens CHA0 [19] and HptB, which exclusively controls rsmY in P. aeruginosa [20]. These additional regulators certainly enable a fine-tuning control by integrating various signals.
We have previously, shown that spontaneous mutations in either gacS or gacA in P. brassicacearum can alter many phenotypes typically associated with phytobeneficial traits, as well as root colonization and modification of the root architecture [18,21,22,23]. These traits include secretion of exoenzymes such as protease and lipase [24], production of antifungal metabolites (DAPG and cyanide) and N-acyl-homoserine lactones, the phytohormone auxin, biofilm formation, alginate production, chemotaxis, and type VI secretion [18]. In addition, overexpression of either rsmX, rsmY, or rsmZ fully suppressed the pleiotropic gacA or gacS mutations and restored the WT phenotypes in P. brassicacearum variants [18].
The primary goal of this study was to determine whether P. brassicacearum genome encodes other Rsm sRNAs and whether their overexpression could override the gacA or gacS mutation. The second part of our work aimed at evaluating the expression and stability of Rsm sRNAs under conditions of different nutrient levels. Finally, to establish the reliability of the results, the analysis of metabolites involved in stringency and biofilm formation was carried out.

2. Experimental Procedures

2.1. Bacterial Strains, Plasmids and Growth Conditions

The bacterial strains and plasmids used in this study are listed in Table S1. P. brassicacearum NFM 421 and gacA mutant were grown as described in Lalaouna et al. [18]. Escherichia coli GM2163, TOP10 and S17-1 strains were grown in LB medium at 37 °C.
In order to test influences of carbon availability, bacterial strains were grown in TSB (BD), TSB/10 and TSB/20 media. For growth on plates, media were solidified with 15 g/L agar (Sigma, St. Louis, USA). Pseudomonas agar F (PAF) (Difco) was used to compare bacterial colony morphology of NFM421 WT, gacS and gacA mutants.

2.2. DNA Manipulation

Chromosomal DNA from P. brassicacearum NFM421 was prepared by phenol/chloroform extraction. Plasmid extraction using the “QIAprep Spin Miniprep Kit” (Qiagen, Hilden, Germany), and purification of DNA fragments from agarose gels with the “QIAquick Gel extraction Kit” (Qiagen, Hilden, Germany) were performed according to the manufacturer’s instructions.

2.3. RNA Manipulation

For RNA extractions, we used “RNAprotect Bacteria Reagent” and “RNeasy Mini Kit” (Qiagen, Hilden, Germany). RT-PCR assays were performed with the “Transcriptor First Strand cDNA Synthesis Kit” (Roche, Manheim, Germany). Gene-specific primers for real-time PCR were designed based on P. brassicacearum NFM421 rsmX-1, rsmX-2, rsmY and rsmZ sequences to obtain predicted PCR products of 200–250 bases (Table S1). Amplifications were performed according to the real Q-PCR Light Cycler 480 SYBR Green I Master kit instructions for the Light Cycler 480 Real Time PCR System (Roche). Real-time PCR was performed in triplicate, and mRNA relative expression was normalized to the 16S reference gene.

2.4. 5′ RACE

The 5′-end of the rsmX-2 transcript was mapped by RACE. Total RNA from P. brassicacearum strain NFM421 isolated in late stationary phase was used for a reverse transcription reaction with the Transcriptor First Strand cDNA Synthesis Kit (Roche, Manheim, Germany). The 5′-phosphorylated, 3′-end cordecypin-blocked oligonucleotide DT88 [25] was ligated to the single-strand cDNA with T4 RNA ligase (New England Biolabs, Ipswich, USA). The anchor-ligated cDNA was first amplified with primers DT89 (anchor-specific primer) and a rsmX-2-specific primer (rsmX-2-R1; Table S1). Next, a nested PCR was performed using DT89 and a second rsm-specific internal primer (rsmX-2-R2). Finally, the PCR product was cloned into the cloning vector pCR®4Blunt-TOPO® (Thermo Fisher Scientific, Waltham, USA). Three independent clones were sequenced using T7 primer (GATC Biotech, Konstanz, Germany).

2.5. Overexpression of sRNAs

To overexpress rsmX-2, we used the pME6032 plasmid [26]. The Shine-Dalgarno sequence was removed, as it is unsuitable for the expression of small RNAs. The rsmX-2 gene was amplified from chromosomal DNA by PCR using primers rsmX-2-PstI and rsmX-2-KpnI (Table S1), digested by PstI and KpnI, and inserted into PstI/KpnI-cut pME6032. The introduction of the PstI restriction site adds 5 nt and modifies 1 nt of the RsmX-2 sRNA at the 5′ end (5′-CTGCAGCCACTG… in place of 5′-TCCACTG). All mutations were verified by sequencing the inserts.

2.6. Construction of Transcriptional lacZ Fusions

To construct the rsmX-2 + 16 transcriptional fusion, we amplified the promoter region and the first 16 nts of rsmX-2 sequence by PCR with primers listed in Table S1. The PCR products were subsequently digested with specific restriction enzymes and cloned into pME6016 plasmids [27] and then sequenced. The pME6016-rsmX-2-lacZ plasmid was introduced into competent cells of NFM421 strain by electroporation ((2500 V for 5 ms using an Eppendorf Multiporator® electroporator, Hambourg, Germany).

2.7. Northern Blot Analysis

RNA was extracted from overnight cultures of NFM421 WT strain and from ∆gacA mutant using “RNeasy Mini Kit” (Qiagen, Hambourg, Germany). The electrophoresis of total RNA was performed on a polyacrylamide gel (5% acrylamide 29:1, 8 M urea). RNAs were then transferred on a Hybond N+ nitrocellulose membrane (GE Healthcare Life Sciences, Uppsala, Swenden). RsmX-2 or 5S-specific digoxygenin-labelled probes were used (Table S1). 5S rRNA was used as loading control. Luminescent detection was carried out as previously described [28].

2.8. Protease Activity Assays

In order to detect the extracellular protease activity, the bacteria are inoculated on TSB/10 solid medium containing 1% skim milk powder (TSA lec). The presence of a halo around the colony attests to protease activity.

2.9. β-Galactosidase Assays

Cultures containing a rsmX-2 + 16-lacZ construct were grown overnight, diluted 1:200 into 8 mL of TSB with tetracycline (20 µg/mL), and grown over a period of 24 h. For the time-course of rsmX-2 expression, 50 mL cultures were sampled at different times and assayed immediately. β-galactosidase activities were quantified as previously described [18]. Experiments were conducted in triplicates.

2.10. Biofilm Assays

As previously described [18], overnight cultures were diluted to OD600nm = 0.05 in K10T-1 medium [29] and 1 mL aliquots were dispensed into glass tubes in triplicate. Following static incubation at 30 °C, the medium was removed, and tubes were washed gently with distilled water. Biofilm formation was visualized by crystal violet staining.

2.11. Stability of Rsm sRNAs

Wild-type cells were grown in TSB/10 or TSB media. To arrest RNA transcription, rifampicin was added at a final concentration of 200 µg/mL, either in early stationary phase (OD600nm 1.5 in TSB and 0.5 in TSB/10) or in late stationary phase (OD600nm 4.5 in TSB and 0.9 in TSB/10). At different time points (1, 2, 5, 10, 15, 20, 30, 45, 60, 90 and 120 min), 0.5 mL from each sample was mixed with 1 mL of RNA protect (Qiagen, Hilden, Germany), and total RNAs were extracted as described above. TURBO DNase treatment following reverse transcription, TaqMan real-time PCR analysis was used to determine the amount of each Rsm.
TaqMan PCR reactions were performed as described in the “LightCycler® 480 Probes Master” (Roche) protocol, with double-labelled oligonucleotide probe (FAM and TAMRA dyes in 5′ and 3′ ends, respectively; Eurogentec). Reactions were run in a LightCycler® 480 System. Threshold detection parameters were determined using the second derivative method. PCR efficiencies were determined by amplification of four 10-fold serial dilutions of all target sequences. Triplicated biological samples were quantified, and mean values were used to express Rsm sRNAs abundance relative to 16S RNA.

2.12. High Resolution Mass Spectrometry Analysis

WT cells were grown in TSB/20, TSB/10 or TSB media. Cells were harvested in late stationary phase and washed with ultrapure water. The lyophilized samples were extracted in methanol water (v:v) in an ultrasonic bath for 15 min. The pellets were centrifuged at 14,000 rpm for 5 min and the supernatants were analyzed in negative mode using 12 Tesla SOLARIX Fourier transform ion cyclotrom mass spectrometer (FT-ICR/MS) from Bruker Daltonics, Bremen, Germany. The injections were performed using a micro-liter pump at a liquid flow rate of 120 μL h−1. Nitrogen was used for both sheath gas as well as curtain gas. A source heater temperature of 200 °C was maintained to ensure rapid solvent evaporation in the ionized droplets. Data acquisition and handling were performed by using Data Analysis Software from Bruker (Bruker Daltonics, Bremen, Germany).

2.13. MassTRIX Metabolite Annotation

Samples were annotated to possible metabolites of P. brassicacearum using the KEGG metabolome database. The exact mass lists (asc files) were uploaded and compared with a 1.0 ppm accuracy window to the metabolome mass translator into pathways (MassTRIX) [30].

3. Results

3.1. Identification of a Fourth Rsm sRNA in P. brassicacearum

We identified a fourth Rsm sRNA by performing a BLAST search against the P. brassicacearum NFM421 genome and using the upstream activating sequence (UAS; TATAGCGAAACGCTTACA) recognized by GacA as query sequence. This revealed an additional UAS located upstream the gene encoding a putative sRNA (Figure 1A), which shares 75% similarity with rsmX gene, while the UAS sequence is almost identical (Figure 1B). We renamed RsmX to RsmX-1, and the novel Rsm sRNA was named RsmX-2. The secondary structure of RsmX-2 was predicted with RNAfold and visualized using vaRNA software [31]. At least three potentially mimicked recognition motifs of RsmA/E (i.e., ANGGA or AGGA motifs) [32], are exposed within a hairpin loop, suggesting that RsmX-2 could sequester these proteins (Figure 1C).
To determine, the relationship and evolutionary history, we performed a phylogenetic analysis of Rsm sRNAs genes. Phylogenetic tree Unweighted Pair Group Method with Arithmetic Mean (UPGMA) tree was constructed using MAFFT toolkit with a modified version of UPGMA. Rsm sRNAs exhibit a remarkably uniform distribution among Pseudomonas species. The UPGMA phylogenetic tree shows that rsmX, rsmY and rsmZ are sorted into three clades (Figure 2).
Typically, Pseudomonads contain single copies of RsmY and RsmZ, however, the copy number of RsmX is variable. P. protegens CHA0 possesses one copy of RsmX [6], while P. syringae contains up to five copies [8]. In this study we identified a fourth Rsm RNA in P. brassicacearum NFM421 found in tandem with rsmX with which it shares 75% of sequence identity. We also show a potential fourth Rsm sRNA in P. fluorescens F113 and Pseudomonas stutzeri, which are in tandem with rsmX and share 75% and 81% sequence identity respectively (Table 1).

3.2. rsmX-2 Expression Is Exclusively GacA-Dependent

The Northern blot analysis (Figure 3A) and 5′ end determination of the transcript by 5′RACE method confirmed that the novel 110-nt long Rsm sRNA is transcribed only in WT and not in the ∆gacA mutant. This was confirmed by the analysis of rsmX-2 expression in wild-type and ∆gacA mutant cells during growth in 10-fold diluted TSB medium, by measuring β-galactosidase activity of the transcriptional rsmX-2 + 16-lacZ fusion (Figure 3B).
Moreover, the overexpression of rsmX-2 from a vector under the control of Ptac promoter restored the wild-type phenotype in gacA mutant cells (Figure 4), indicating that the overall function of RsmX-2 is likely similar to that of RsmX-1, RsmY and RsmZ [18]. This is illustrated by the restoration of protease activity (Figure 4A), colony morphology (Figure 4B) and biofilm formation in a gacA mutant (Figure 4C). These findings indicate that RsmX-2 is part of the Gac-Rsm regulatory system.

3.3. Stringent Conditions Activate rsm sRNAs Genes Expression

The expression of the four rsm genes rsmX-1, rsmX-2, rsmY and rsmZ increases over time until a maximum is reached in stationary phase (24 h of growth), when nutrients become limited. We therefore wondered whether carbon deprivation could activate the rsm expression, as previously reported for the Csr system [33]. To assess the biological relevance of these observations, we monitored the expression level of the four rsm genes under different nutrient level conditions. Here, bacteria were grown in either undiluted TSB medium (considered as a rich medium), 10-fold diluted TSB (TSB/10), or 20-fold diluted TSB (TSB/20) (which was considered as a nutrient-poor medium in which P. brassicacearum growth may occur). Using transcriptional fusions for the promoter from each rsm gene, we found a greater increase in rsm expression when nutrient availability declines (Figure 5). Thus, even if growth decreases when nutrients are scarce, as indicated by OD600 nm monitoring in tables beneath each graph of Figure 5, the expression of the four rsm genes highly increases. The greatest effect was observed for rsmZ (Figure 5). Nutrient starvation conditions influenced the expression levels of rsm genes and also certain targets of RsmA/E synthesis, as indicated by an enhancement of protease activity (Figure S1).

3.4. Stability of Rsm sRNA

If most sRNAs indeed regulate translational efficiency, their turnover in varying environments deserves a closer examination. For this, we examined the stability and steady state level of Rsm sRNAs by quantitative real-time reverse transcription polymerase chain reactions (qRT-PCR). We used two different media: TSB/10 and undiluted TSB medium. To determine the in vivo stability of Rsm sRNAs, exponentially phase and late stationary phase growing bacteria were treated with rifampicin to block any further initiation of transcription. Samples were taken at 1, 2, 5, 10, 15, 20, 30, 45, 60, 90 and 120 min and DNase-treated total RNA was used for qRT-PCR (Table 2, Figure S2). The half-life of each Rsm sRNA was calculated, revealing that these sRNAs are not equally stable. Overall, the half-lives of RsmY and RsmZ are much longer than those of RsmX-1 and RsmX-2 (Table 2, Figure S2).
The half-life of the four Rsm sRNAs increased during growth to reach their maxima during the stationary phase. For example, RsmX-1 and RsmX-2 stability increased ~8-fold in TSB/10 in the stationary phase compared to the exponential phase. RsmY and RsmZ were observed to be highly stable with a half-life of at least 60 min (Table 2, Figure S2).
Moreover, the choice of nutrients condition alters their stability; the four Rsm sRNAs were observed to be more stable in TSB/10 medium at stationary phase (e.g., half-life of RsmY and RsmZ is >60 min and ~50 min in TSB/10 and TSB, respectively). A similar effect was observed for RsmX-1 and RsmX-2, whereas, they both exhibited half-lives of 30–34 min in TSB/10 medium, which were reduced in TSB medium to 23 and 16 min, respectively.
In P. brassicacearum, we hypothesize a correlation between the number of GGA motifs and the stability of the four Rsm sRNAs transcripts conferred by RsmA/E. RsmZ, which showed the highest stability, possesses the highest number of GGA motifs (10 with 4–8 exposed); in contrast, RsmX-1 and RsmX-2 have the lowest number of GGA motifs (6 with 3–4 exposed) and were less stable. It should be noted that RsmY is moderately stable, although it only contains 7 GGA motifs (4–5 exposed) (Table 3).

3.5. Stringent Conditions and Sedentary Lifestyle Signalling Molecules

Given that the Rsm sRNAs expression and stability were increased under starvation conditions and that the bacterial stringent response, is mediated by the ppGpp, we evaluated the production of ppGpp under these culture conditions. Spectra were acquired with a time domain of 4 MW and a total number of 750 scans were accumulated (Figure 6A). As expected, the level of ppGpp is inversely proportional to the availability of nutrients (Figure 6B), while it evolves in the same way as the expression of Rsm sRNAs and in particular rsmZ [34].
Moreover, since Rsm sRNAs promote sedentary state and multicellularity through biofilm formation, a process also coordinated by the bacterial second messenger, cyclic diguanylate monophosphate (c-di-GMP), we also measured its cellular level. The same trend as for ppGpp and Rsm sRNAs was observed, suggesting a link between Gac-Rsm system and these two signal molecules (Figure 6C) as previously shown for other Pseudomonas species [35,36,37].

4. Discussion

4.1. P. brassicacearum Rsm sRNA Amplification by a Duplication of rsmX

The evolutionary relationships between the rsmX genes indicate the duplication events that occurred in different Pseudomonas species (Figure 2) with up to 5 copies in P. syringae was reported by Moll et al. [8], who correlated the number of Csr/Rsm sRNAs with the number of Csr/Rsm proteins. In E. coli and in most Pseudomonas species, there are two sRNAs for one or two regulatory proteins. However, three to five RsmA homologs are predicted in P. syringae, where up to seven Rsm sRNAs are found [8]. In P. brassicacearum, three RsmA homologs are found: RsmA, RsmE and a third putative RsmA-like protein. Recently, Sobrero and Valverde [11] performed a comparative genomics and evolutionary analysis of Rsm RNAs-binding proteins of the CsrA family in the genus Pseudomonas and suggested that the presence of redundant Rsm proteins that can replace or by-pass each other’s activities could help bacteria achieve greater plasticity via post-transcriptional regulation and better noise control in gene expression.
The amplification of regulatory RNAs is not exclusive to Rsm sRNAs. In Vibrio cholerae, four Qrr (quorum regulatory RNA), which are almost 80% identical in sequence and predicted to have similar secondary structures [3], are involved in virulence and biofilm formation [38]. Furthermore, five Qrr sRNAs are involved in the Vibrio harveyi quorum-sensing cascade [39]. In different bacterial species, two quite identical sRNAs that control iron homeostasis, as PrrF1 and PrrF2 in Pseudomonas [40] and, RyhB1 and RyB2 in Salmonella [41].
Gene duplication is an important feature in evolution because it provides raw material for adaptation to environmental challenges [42]. This phenomenon is also implicated in enabling gene amplification, and allowing cells to proliferate under growth-limiting conditions [43]. Any environmental condition favouring cells with more copies of a gene would permit them to outgrow the rest of the population in a short time; this includes duplications of rRNA genes that might confer a selective advantage under fast growth rate conditions [44]. Other cases of gene amplification are found in response to antibiotics [45].
Gene duplication-amplification is a frequent process in bacterial genomes, that often disappears after only a few generations of growth in the absence of selection pressure. However, gene copy (ies) may be preserved if they confer an adaptive evolution by increasing gene dosage in response to certain environmental constraints. We previously showed the key role of Rsm sRNAs in activating beneficial traits in plant-associated bacteria as well as virulence genes in phytopathogens [10,18].

4.2. RsmX-2 Is Part of Gac-Rsm System under Exclusive Control of GacA

Despite the similarity between the rsmX-1 and rsmX-2 sequences, the expression of rsmX-2 differs from that of rsmX-1. The few mutations in the UAS (Figure 1B) of rsmX-2 may explain the 8-fold decrease in the expression level. Our data confirm that RsmX-2 sRNA is a member of the Rsm sRNAs family. Indeed, the exclusive overexpression of rsmX-2 in the gacA mutant suppresses the effect of gacA deletion, suggesting that the four Rsm sRNAs share a redundant function, which appears to be the sequestration of RsmA/E proteins.
In the absence of GacA in P. aeruginosa and P. brassicacearum, transcription of rsmY and rsmZ is still achieved but to a lesser degree, suggesting the involvement of additional regulatory pathways [7,18]. We previously demonstrated the activation of rsmZ expression, which depends on an enhancer sequence located in the rpoS coding region. This suggested the role of additional transcriptional factors as part of the complex network controlling the expression of rsmZ. We showed that the conserved palindromic UAS required for GacA-controlled sRNA genes in Gammaproteobacteria is essential but not sufficient for the full expression of the rsmZ gene in P. brassicacearum NFM 421 [46].
Finally, unlike the two novels recently discovered sRNAs, RsmV [47] and RsmW [48], in P. aeruginosa, which transcription is independent of the GacS-GacA, rsmX-1 and rsmX-2 expression is exclusively GacS-GacA-dependent.

4.3. Regulation of the Amount of RNA by Degradation

sRNAs often modulate mRNA targets stability, notably by recruitment of RNases [49]. This indicates that degradation is a regulatory process, by which bacteria adjust the translation of certain transcripts in response to changing environments. However, turnover of Csr/Rsm sRNAs has not been well-reported in the literature, with the exception of a study reported by Suzuki et al. [50]. These authors identified a regulatory protein (CsrD) that targets the global regulatory RNAs CsrB and CsrC for degradation by RNase E. Investigation of Rsm sRNAs degradation in P. brassicacearum indicates that the half-lives of the four Rsm sRNAs increase in the stationary phase and when nutrients are limited. The rate of turnover for Rsm sRNAs thus seems to be related to growth conditions and cell physiology. Furthermore, the four Rsm sRNAs are not equally stable, with RsmZ appearing to be extremely stable even during the exponential phase (Table 2).
Regulation of RNA amount by degradation is achieved by ribonucleases (RNases), whose activity is dependent on the sequence and/or the structural elements of the RNA molecule. In Salmonella enterica serovar Typhimurium, the endoribonuclease RNase III has been shown to regulate MicA (an sRNA involved in porin regulation) in a target-coupled way, whereas RNase E is responsible for the control of free MicA levels in the cell [51].
We hypothesize that the observed differences in Rsm sRNAs are mainly due to the sequence and structural elements of each Rsm sRNA. In P. protegens CHA0, the estimated half-lives of RsmY and RsmZ are >20 min in the wild-type and <10 min in the rsmA rsmE double mutant [52]. RsmA/E appears to stabilize Rsm sRNAs in vivo probably by protecting them from degradation by RNases. In Erwinia carotovora subsp. carotovora, RsmA also increases the half-life of the RsmB riboregulator [53]. Higher levels of RsmB RNA in the rsmA+ strain than in the rsmA− strain were shown to be due to the increase in RsmB stability and not to an increase in transcription.
It has been suggested that RNase E and Hfq have similar (AU-rich) target sequences that should permit Hfq to protect sRNA from RNase E attack [54]. In P. aeruginosa, RsmY has been described as stabilized by Hfq, which sequesters RNase E cleavage sites [55], and thus enhances RsmA sequestration by RsmY [56]. In P. brassicacearum, an interaction of Hfq with RsmY and RsmZ may explain the extreme stability of these two sRNAs. Nevertheless, in P. aeruginosa, Hfq doesn’t affect RsmZ stability, whereas in P. brassicacearum, RsmZ exhibits an A-U rich loop, which could interact with Hfq.

4.4. Modulation of Rsm sRNAs Expression in Response to Nutrient-Poor Conditions

The stimuli that activate the Gac-Rsm system are still unknown [57]. In this study, we demonstrated that rsm genes are highly up-regulated under nutrient-poor conditions. Even if Rsm sRNAs are principally synthesized in the stationary phase, our results indicate that their expression is not only related to cellular density, but also and essentially to the physiological and metabolic state of cells. According to carbon source nature and availability, certain metabolites may accumulate within cells and induce specific responses. In P. protegens CHA0, pools of 2-oxoglutarate, succinate, and fumarate were shown to be positively correlated with the expression level of rsm genes [58]. Moreover, the expression of Rsm sRNAs is attenuated in the double mutant relA spoT, for which ppGpp synthesis has been completely abolished [34]. Our data show an activation of (p)ppGpp synthesis in response to nutrient starvation, correlating well with an increase of the four Rsm sRNAs in P. brassicacearum NFM421.
Another signaling molecule, the cyclic-di-GMP second messenger, which modulates the transition from the planktonic to the biofilm state [59], was shown to be positively regulated by GacS-GacA system in P. aeruginosa [35,37]. More recently, Liang et al. [36] showed that the QS system and polyketide antibiotic 2,4-DAPG production are regulated by c-di-GMP through RsmA and RsmE proteins in P. fluorescens 2P24. Our work also suggests a connection between the Gac-Rsm cascade and the c-di-GMP signaling pathway in phytobeneficial Pseudomonas.
Surprisingly, under circumstances that are not advantageous for P. brassicacearum proliferation, such as when nutrients are depleted, bacteria favor the activation of rsm expression and enhance their stability and increase (p)ppGpp and c-di-GMP intracellular levels. Consequently, production of certain secondary metabolites and biofilm formation might be enhanced, suggesting their importance in the adaptive response to environmental constraints.
Based on the present results and those of previous studies [34,36], we propose a model according to which ppGpp as well as c-di-GMP are directly or indirectly positively regulated by GacS/GacA system under nutrient limitations conditions (Figure 7).

5. Conclusions

The amplification of Rsm sRNAs and the preservation of several active copies indicate the importance of this post-transcriptional regulation, which allows the bacterium to increase its plasticity by rapidly adjusting its physiology in response to environmental constraints.
The existence of multiple factors devoted to rsm expression and degradation, illustrates that bacteria have evolved versatile mechanisms to control Rsm sRNAs levels. The higher expression level and the greater stability of the four Rsm sRNAs when nutrient conditions are limited underscore their relevance in the ecological niche of P. brassicacearum, a soil in which nutrients are poorly available. It is particularly interesting to note that this transcriptional activation of the four Rsm sRNAS occurs in concert with the increase in the intracellular level of the two signal molecules, (p)ppGpp and c-di-GMP under stringency conditions.
The differences observed in expression and stability between the four Rsm sRNAs illustrate that the subtleties of their respective roles in bacterial adaptation to challenging conditions remain far from being completely understood, as well as the regulation network involving (p)ppGpp and c-diGMP. This definitively deserves to be investigated in further detail.
Finally, although Rsm sRNA concentrations in the cell vary due to noise in gene expression and transcript stability, the overall combined concentrations of each Rsm sRNA are probably maintained in homeostatic balance.

Supplementary Materials

The following are available online at https://www.mdpi.com/2076-2607/9/2/250/s1. Table S1. Primers: plasmids and strains used in this study. Figure S1. Detection of protease activity under nutrient starvation conditions. (A) Wild-type and gacA strains on TSA/20 lec, on TSA 1/10 lec and on undiluted TSA lec media. (B) Halo thickness (distance from bacteria to halo edge) is expressed as the mean of three measurements, in cm. lec means Skimmed milk (10/%) was added to growth medium. Figure S2. Rsm RNA stability. In vivo assay of Rsm RNA degradation of P. brassicacearum NFM421 culture at exponential and stationary growth phases under nutrient replete and deplete conditions and rifampicin treatment. Samples were taken after 5, 15, 30, 45, 60, 90, 120, 150 and 180 min for RNA extraction and q-RT-PCR analysis. Data were normalized to the values obtained from the analysis of 16S rRNA as an internal control.

Author Contributions

Conceptualization, D.L. and W.A.; D.L. performed DNA and RNA experiments; S.F. contributed to RNA decay experiments; M.H. performed metabolites analysis; P.O. performed phylogeny analysis; writing-original draft preparation, D.L.; writing-review and editing, W.A., T.H., M.H., P.O., P.S.-K. and D.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not Applicable.

Informed Consent Statement

Not Applicable.

Data Availability Statement

All data in this article is openly available without any restrictions.

Acknowledgments

This work was supported by IRTELIS PhD program Grant from CEA.

Conflicts 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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Figure 1. rsmX-2 identification and predicted structure. (A) Organization of the rsmX-1 and rsmX-2 genomic region of P. brassicacearum. The upstream activating sequence (UAS) of the GacA protein is represented within the box. GGA motifs involved in the sequestration of RsmA/E proteins are underlined. The transcriptional start has been determined by 5′RACE method. rsmX-2 gene sequence is indicated in bold; (B) Alignment of rsmX-1 and rsmX-2 UAS sequence; (C) rsmX-2 secondary structure predicted using RNAfold and visualised using vaRNA.
Figure 1. rsmX-2 identification and predicted structure. (A) Organization of the rsmX-1 and rsmX-2 genomic region of P. brassicacearum. The upstream activating sequence (UAS) of the GacA protein is represented within the box. GGA motifs involved in the sequestration of RsmA/E proteins are underlined. The transcriptional start has been determined by 5′RACE method. rsmX-2 gene sequence is indicated in bold; (B) Alignment of rsmX-1 and rsmX-2 UAS sequence; (C) rsmX-2 secondary structure predicted using RNAfold and visualised using vaRNA.
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Figure 2. Phylogenetic analysis of rsm RNA sequences. rsm genes from P. brassicacearum NFM 421 are shown in bold.
Figure 2. Phylogenetic analysis of rsm RNA sequences. rsm genes from P. brassicacearum NFM 421 are shown in bold.
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Figure 3. rsmX-2 expression is GacA dependent. (A) Northern blot analysis of rsmX-2 expression in WT and gacA backgrounds, 5S rRNA is used as loading control; (B) β-galactosidase measurements of a transcriptional rsmX-2 + 16-lacZ fusion in the WT and ∆gacA strains.
Figure 3. rsmX-2 expression is GacA dependent. (A) Northern blot analysis of rsmX-2 expression in WT and gacA backgrounds, 5S rRNA is used as loading control; (B) β-galactosidase measurements of a transcriptional rsmX-2 + 16-lacZ fusion in the WT and ∆gacA strains.
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Figure 4. rsmX-2 restores GacA-dependent phenotypes. Restoration of wild-type phenotype by overexpression of rsmX-2 from a plasmid under the control of Ptac promoter in gacA cells as determined by (A) Protease activity on skimmed milk-containing medium, (B) Colony morphology on PAF medium and (C) Biofilm formation.
Figure 4. rsmX-2 restores GacA-dependent phenotypes. Restoration of wild-type phenotype by overexpression of rsmX-2 from a plasmid under the control of Ptac promoter in gacA cells as determined by (A) Protease activity on skimmed milk-containing medium, (B) Colony morphology on PAF medium and (C) Biofilm formation.
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Figure 5. The expression level of rsmX-1, rsmX-2, rsmY and rsmZ depends on nutrient availability. Activity of a lacZ reporter fused to the promoter regions of rsmX-1, rsmX-2, rsmY and rsmZ were measured in the P. brassicacearum wild-type strain and are reported as Miller units of β-galactosidase activity (± standard deviation). The cultures were grown for 24 h at 30 °C before being tested and the tables below each graph indicate the OD600 of the cultures. Experiments were conducted in triplicates.
Figure 5. The expression level of rsmX-1, rsmX-2, rsmY and rsmZ depends on nutrient availability. Activity of a lacZ reporter fused to the promoter regions of rsmX-1, rsmX-2, rsmY and rsmZ were measured in the P. brassicacearum wild-type strain and are reported as Miller units of β-galactosidase activity (± standard deviation). The cultures were grown for 24 h at 30 °C before being tested and the tables below each graph indicate the OD600 of the cultures. Experiments were conducted in triplicates.
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Figure 6. Signal molecules analysis. (A) Full ICR-FT/mass spectra of the wild type (WT) grown in TSB, tenfold dilutes TSB (TSB/10) and twenty-fold diluted TSB (TSB/20). (B,C) detection of (p)ppGpp and c-di-GMP compounds as annotated using metabolome database (MassTRIX: Suhre and Schmitt-Kopplin, 2008).
Figure 6. Signal molecules analysis. (A) Full ICR-FT/mass spectra of the wild type (WT) grown in TSB, tenfold dilutes TSB (TSB/10) and twenty-fold diluted TSB (TSB/20). (B,C) detection of (p)ppGpp and c-di-GMP compounds as annotated using metabolome database (MassTRIX: Suhre and Schmitt-Kopplin, 2008).
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Figure 7. Model for gene regulation by ppGpp and c-di-GMP in the Gac-Rsm system of P. brassicacearum NFM421. This pathway is supported by evidence from this work and previous studies (Lalaouna et al., 2012, Takeuchi et al., 2012, Liang et al., 2020). ↓, positive effect; ┴, negative effect, dotted lines, indirect effects. The new conclusion of this study is that starvation conditions seem to activate the Gac-Rsm system concomitantly with the production of ppGpp and c-di-GMP.
Figure 7. Model for gene regulation by ppGpp and c-di-GMP in the Gac-Rsm system of P. brassicacearum NFM421. This pathway is supported by evidence from this work and previous studies (Lalaouna et al., 2012, Takeuchi et al., 2012, Liang et al., 2020). ↓, positive effect; ┴, negative effect, dotted lines, indirect effects. The new conclusion of this study is that starvation conditions seem to activate the Gac-Rsm system concomitantly with the production of ppGpp and c-di-GMP.
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Table
Table 1. Tandem copy of rsmX gene in Pseudomonas.
Table 1. Tandem copy of rsmX gene in Pseudomonas.
StrainsGeneBeginEndSize (nt)Sequence Identity (%)Number of rsmX-Like Genes
Pseudomonas brassicacearum NFM421rsmX-148398924840003111752
rsmX-248396814839791110
Pseudomonas fluorescens F113rsmX-1 *21138392113950111752
rsmX-2 *21140522114160108
Pseudomonas stutzeri A150rsmX-1 *35690435701310981.52
rsmX-2 *357143357250107
Pseudomonas syringae pv. phaseolicola 1448ArsmX-4 **160448160336112855
rsmX-3 **160740160627113
Pseudomonas syringae pv. syringae B728arsmX-3 **58671695867282113845
rsmX-4 **58674615867572111
Pseudomonas syringae pv. tomato str. DC3000rsmX-361448306144943113875
rsmX-461451226145235113
* in comparison to P. brassicacearum NFM421. ** in comparison to P. syringae pv. tomato str. DC3000.
Table
Table 2. Stability of Rsm sRNAs. Half-lives of P. brassicacearum NFM421 RsmX-1, RsmX-2, RsmY and RsmZ. Experiments were conducted in triplicates.
Table 2. Stability of Rsm sRNAs. Half-lives of P. brassicacearum NFM421 RsmX-1, RsmX-2, RsmY and RsmZ. Experiments were conducted in triplicates.
MediumPhase Half-Lives in WT (in Min)
OD600nmRsmX-1RsmX-2RsmYRsmZ
TSBExpo1.512 ± 1.15 ± 0.68 ± 0.237 ± 2.6
Stat4.523 ± 5.416 ± 1.545 ± 5.253 ± 0.9
TSB/10Expo0.57 ± 1.55 ± 1.68 ± 1.726 ± 3.6
Stat0.937 ± 4.440 ± 8.6≥60≥90
Table
Table 3. Rsm sRNAs stability is correlated to the number of GGA motifs. Relationships between the number of GGA motifs and Rsm sRNAs half-lives of P. brassicacearum NFM421.
Table 3. Rsm sRNAs stability is correlated to the number of GGA motifs. Relationships between the number of GGA motifs and Rsm sRNAs half-lives of P. brassicacearum NFM421.
Half-Lives (in Min)
GenesGGA MotifsGGA Motifs Exposed *TSB/10 24 hTSB 24 h
RsmX-162–437 ± 4.423 ± 5.4
RsmX-26340 ± 8.616 ± 1.5
RsmY75–4≥6045 ± 5.2
RsmZ108–7≥9053 ± 0.9
* predicted with Mfold and RNAfold softwares.
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MDPI and ACS Style

Lalaouna, D.; Fochesato, S.; Harir, M.; Ortet, P.; Schmitt-Kopplin, P.; Heulin, T.; Achouak, W. Amplifying and Fine-Tuning Rsm sRNAs Expression and Stability to Optimize the Survival of Pseudomonas brassicacerum in Nutrient-Poor Environments. Microorganisms 2021, 9, 250. https://doi.org/10.3390/microorganisms9020250

AMA Style

Lalaouna D, Fochesato S, Harir M, Ortet P, Schmitt-Kopplin P, Heulin T, Achouak W. Amplifying and Fine-Tuning Rsm sRNAs Expression and Stability to Optimize the Survival of Pseudomonas brassicacerum in Nutrient-Poor Environments. Microorganisms. 2021; 9(2):250. https://doi.org/10.3390/microorganisms9020250

Chicago/Turabian Style

Lalaouna, David, Sylvain Fochesato, Mourad Harir, Philippe Ortet, Philippe Schmitt-Kopplin, Thierry Heulin, and Wafa Achouak. 2021. "Amplifying and Fine-Tuning Rsm sRNAs Expression and Stability to Optimize the Survival of Pseudomonas brassicacerum in Nutrient-Poor Environments" Microorganisms 9, no. 2: 250. https://doi.org/10.3390/microorganisms9020250

APA Style

Lalaouna, D., Fochesato, S., Harir, M., Ortet, P., Schmitt-Kopplin, P., Heulin, T., & Achouak, W. (2021). Amplifying and Fine-Tuning Rsm sRNAs Expression and Stability to Optimize the Survival of Pseudomonas brassicacerum in Nutrient-Poor Environments. Microorganisms, 9(2), 250. https://doi.org/10.3390/microorganisms9020250

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