RpoS is a pleiotropic regulator of motility, biofilm formation, exoenzymes, siderophore and prodigiosin production, and trade-off during prolonged stationary phase in Serratia marcescens

Han Qin; Ying Liu; Xiyue Cao; Jia Jiang; Weishao Lian; Dairong Qiao; Hui Xu; Yi Cao

doi:10.1371/journal.pone.0232549

. 2020 Jun 2;15(6):e0232549. doi: 10.1371/journal.pone.0232549

RpoS is a pleiotropic regulator of motility, biofilm formation, exoenzymes, siderophore and prodigiosin production, and trade-off during prolonged stationary phase in Serratia marcescens

Han Qin ¹, Ying Liu ¹, Xiyue Cao ¹, Jia Jiang ¹, Weishao Lian ¹, Dairong Qiao ¹, Hui Xu ^1,^*, Yi Cao ^1,^*

Editor: Marie-Joelle Virolle²

PMCID: PMC7266296 PMID: 32484808

Abstract

Prodigiosin is an important secondary metabolite produced by Serratia marcescens. It can help strains resist stresses from other microorganisms and environmental factors to achieve self-preservation. Prodigiosin is also a promising secondary metabolite due to its pharmacological characteristics. However, pigmentless S. marcescens mutants always emerge after prolonged starvation, which might be a way for the bacteria to adapt to starvation conditions, but it could be a major problem in the industrial application of S. marcescens. To identify the molecular mechanisms of loss of prodigiosin production, two mutants were isolated after 16 days of prolonged incubation of wild-type (WT) S. marcescens 1912768R; one mutant (named 1912768WR) exhibited reduced production of prodigiosin, and a second mutant (named 1912768W) was totally defective. Comparative genomic analysis revealed that the two mutants had either mutations or deletions in rpoS. Knockout of rpoS in S. marcescens 1912768R had pleiotropic effects. Complementation of rpoS in the ΔrpoS mutant further confirmed that RpoS was a positive regulator of prodigiosin production and that its regulatory role in prodigiosin biosynthesis was opposite that in Serratia sp. ATCC 39006, which had a different type of pig cluster; further, rpoS from Serratia sp. ATCC 39006 and other strains complemented the prodigiosin defect of the ΔrpoS mutant, suggesting that the pig promoters are more important than the genes in the regulation of prodigiosin production. Deletion of rpoS strongly impaired the resistance of S. marcescens to stresses but increased membrane permeability for nutritional competence; competition assays in rich and minimum media showed that the ΔrpoS mutant outcompeted its isogenic WT strain. All these data support the idea that RpoS is pleiotropic and that the loss of prodigiosin biosynthesis in S. marcescens 1912768R during prolonged incubation is due to a mutation in rpoS, which appears to be a self-preservation and nutritional competence (SPANC) trade-off.

Introduction

Serratia marcescens, a gram-negative bacterium belonging to the Enterobacteriaceae family, is found in a wide range of ecological niches in the environment [1]. It is known for its ability to synthesize the striking red pigment prodigiosin, which is of great interest for its antimicrobial as well as immunosuppressant and other bioactive properties [2–4]. S. marcescens is a model strain for studying the regulation of prodigiosin production and other secondary metabolites, such as carbapenem and serratamolide [5–7]. The biosurfactant serratamolide, which is called serrawettin W1 and catalyzed by the nonribosomal peptide synthetase SwrW, has antimicrobial and hemolytic properties [8, 9]. S. marcescens is also known to secrete a variety of exoenzymes, such as pectate lyase, lipase and cellulase [10].

Prodigiosin is synthesized by the prodigiosin biosynthesis gene cluster designated pig [6]. The pig clusters of 34 strains were divided into three types [11]: type I pig clusters are flanked by cueR and copA and shared by 29 strains; in type II pig clusters, which have been found in 4 strains, cueR and copA are downstream of the pig cluster and adjacent to each other; in type III pig clusters, which have been identified in Serratia sp. ATCC 39006, pigO is present downstream of the pig cluster. The biosynthesis of prodigiosin is under tight control by factors including Hfq, RpoS, cAMP receptor protein (CRP), EepR, HexS, LuxS, RssAB, SmaI and SpnR [7, 12–18]. The stationary phase sigma factor rpoS or σ^s is a transcriptional factor and considered to be a global regulator that controls approximately 23% of the genes in the E. coli genome [19]. Polymorphisms of RpoS are extremely common in E. coli [19]. In E. coli and Salmonella enterica serovar Typhi, RpoS regulates resistance to stresses such as high osmolarity, oxygen free radicals, acid and other biotic stresses [20–22]. Mutation of rpoS reduces stress resistance but promotes nutritional competence, as bacteria allocate the limited resources to growth rather than self-protection when suffering starvation and vice versa; therefore, there is a self-preservation and nutritional competence (SPANC) trade-off in E. coli [23]. However, the effect of RpoS on the stress resistance and fitness of S. marcescens is unknown and remains to be explored.

In the natural environment, both pigmented and nonpigmented S. marcescens strains exist [24]; moreover, nonpigmented S. marcescens mutants can be easily isolated from WT pigmented strains after prolonged starvation [25]. However, the underlying mechanisms causing the loss of prodigiosin production in prolonged starving environments remain incompletely understood. A particular laboratory model for approximating prolonged starving environments is prolonged stationary phase [26]. Bacteria suffer a range of stresses during prolonged stationary phase in which starvation is the major challenge [27]. In Escherichia coli, a wide range of mutations accumulate in prolonged stationary phase over time due to the selection of the environment [28–30]; spontaneous mutants that confer a growth advantage in stationary phase (GASP) are enriched which is an adaptive evolution by natural selection [31]. The tight regulatory networks and flexible genetic characteristics of E. coli contribute to the survival of the bacteria during prolonged stationary phase [31–33]. Gene regulation provides adequate but limited control over the right level of gene expression in a less stressful environment, but mutational changes offer intensive control for the adaption of bacteria in prolonged stationary phase [27].

In this study, the pigmented WT strain S. marcescens 1912768R was isolated from rhizosphere soil in ginger fields and we also found that mutants defective in prodigiosin production were consistently isolated during prolonged stationary phase. In order to characterize the underlying molecular mechanism of the loss of prodigiosin production, comparative genomic profiling was employed between the WT strain and its prodigiosin defective mutants isolated during prolonged stationary phase. From these analyses, rpoS was identified as a candidate transcriptional regulatory gene causing the loss of prodigiosin biosynthesis. The regulatory role of RpoS in prodigiosin production and other phenotypes was further studied. Additionally, the effects of the mutation in rpoS on the competition of S. marcescens 1912768R during prolonged stationary phase were explored.

Materials and methods

Bacterial strains, plasmids and growth conditions

S. marcescens 1912768R was isolated from rhizosphere soil in ginger fields and collected at the China Center for Type Culture Collection (strain number: CCTCC AB 2019405). Prodigiosin-defective mutants 1912768WR and 1912768W were randomly selected after 16 days of prolonged culture in Lysogeny Broth (LB; 10 g l^-1 tryptone, 5 g l^-1yeast extract, and 10 g l^-1 NaCl) medium without any nutrition supplementation and isolated on LB agar plate by the pink and white color of the bacterial colony, respectively.

Bacterial strains and plasmids used in this study are listed in Table 1. S. marcescens strains were grown at 28°C and E. coli strains were grown at 37°C in LB medium or on solid LB agar plate (LBA) supplemented with 2.0% (w/v) agar. Bacterial growth was measured in a spectrophotometer and expressed as OD₆₀₀. Antibiotics were added at the following final concentration: kanamycin (Kn), 50 μg ml⁻¹ and ampicillin (Ap), 100 μg ml⁻¹.

Table 1. Strains and plasmids used in this study.

Strains or plasmids	Characteristics	Reference or source
*Escherichia coli*
DH5α	supE44 ΔlacU169(φ80 lacZΔM15) recA1 hsdR17 thi-1 relA1	[34]
S17-1(λpir)	recA pro hsdR RP4-2-Tc::Mu-Km::Tn7	[35]
*Serratia marcescens*
1912768R	Isolated from rhizosphere soil in ginger fields	This study
1912768WR	Mutant from 1912768R with reduced production of prodigiosin	This study
1912768W	Mutant from 1912768R which is totally defective in the synthesis of prodigiosin	This study
ΔrpoS	Strain with the deletion of rpoS in 1912768R	This study
Plasmids
pMD19-T	Cloning and sequencing vector, Ap^r	Takara
pK18mobsacB	Allelic replacement vector, Kn^r	[36]
pBBR1MCS-2	Broad-host-range cloning vector, Kn^r	[37]

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Genome sequencing and annotation

Single colonies were picked to inoculate in 5 ml LB medium. These cultures were grown for 18 h at 28°C and cells were then collected by centrifugation at 4000 rpm for 10 min. Genomic DNA was isolated from the cell pellets using the ChargeSwitch® gDNA Mini Bacteria Kit (Life Technologies) according to the manufacturer’s instructions. Purified genomic DNA was quantified by TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA). High quality DNA (OD_260/280 = 1.8~2.0, >10 μg) was used to do further research.

The genome sequence of S. marcescens 1912768R was determined by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using the Pacbio sequencing system. For Pacific Biosciences sequencing, 8-10k insert whole genome shotgun libraries were generated and sequenced on a Pacific Biosciences RS instrument using standard methods. An aliquot of 8 μg DNA was spun in a Covaris g-TUBE (Covaris, MA) at 6,000 rpm for 60 seconds using an Eppendorf 5424 centrifuge (Eppendorf, NY). DNA fragments were then purified, end-repaired and ligated with SMRTbell sequencing adapters following manufacturer’s recommendations (Pacific Biosciences, CA). Resulting sequencing libraries were purified three times using 0.45 x volumes of Agencourt AMPure XP beads (Beckman Coulter Genomics, MA) following the manufacturer’s recommendations. The complete genome of Serratia marcescens 1912768R was deposited in GenBank under the accession number CP040350.

The genome sequence of S. marcescens 1912768WR and 1912768W was determined using the HiSeq2000 sequencing platform (Illumina Inc., San Diego, CA). For Illumina sequencing, at least 5 μg genomic DNA was used for each strain in sequencing library construction. Paired-end libraries with the insert size of ~300 bp were constructed according to the manufacturer’s instructions (Bio-scientific, AIR^™ Paired-End DNA Sequencing Kit). Subsequently, we sequenced 100 bp at each end using Illumina Hiseq 2000. The genomes of Serratia marcescens 1912768WR and 1912768W were deposited in GenBank under the accession number VBRI00000000 and VBRJ00000000, respectively.

The assembly was produced using SOAPdenovo software. The last circular step was checked and finished manually. The final assembly of 1912768R generated a circular genome sequence with no gap existed. Identification of predicted open read frames (ORFs) was performed using Glimmer version 3.02 (http://cbcb.umd.edu/software/glimmer/). ORFs with less than 300 base pairs were discarded. Then remained ORFs were queried against non-redundant (NR in NCBI) database, SwissProt (http://uniprot.org), KEGG (http://www.genome.jp/kegg/), and COG (http://www.ncbi.nlm.nih.gov/COG) to do functional annotation. In addition, tRNA were identified using the tRNAscan-SE (v1.23, http://lowelab.ucsc.edu/tRNAscan-SE) and rRNA were determined using the RNAmmer (v1.2, http://www.cbs.dtu.dk/services/RNAmmer/).

Genetic manipulations and plasmid construction

All primers used were designed based on the genome sequence of S. marcescens 1912768R. The in-frame deletion of rpoS was constructed by two-step allelic replacement [38]. Briefly, two fragments flanking the rpoS gene were amplified by PCR. A DNA fragment containing 125 bp of the 5′ end of rpoS and 367 bp upstream of the ATG initiation codon was amplified from chromosomal DNA by PCR using primers F-rpoS-U1 and R-rpoS-D1. A DNA fragment containing 480 bp of the 3′ end of rpoS was amplified using primers F-rpoS-U2 and R-rpoS-D2. Both fragments were purified and fused in a subsequent PCR using primers F-rpoS-U1 and R-rpoS-D2. The fused segment was sequenced and ligated into the suicide vector pK18mobsacB using T4 DNA ligase (Takara, China). The resulting plasmid, pK18mobsacB-UD, was transferred by S17-1(λpir) into S. marcescens 1912768R by conjugation, and the transconjugants with the plasmid integrated into the chromosome by homologous recombination were selected on LB agar medium containing ampicillin and kanamycin at 28°C. To create the rpoS deletion mutant strain, a second recombination event was counter-selected on LB agar containing 10% sucrose. Two external primers, F-rpoS-W and R-rpoS-W, anchored to the upstream and downstream regions of the rpoS gene, respectively, were adopted for the detection of deletion mutants by PCR. The deletion mutant was further confirmed by subsequent sequencing using the same primer pair. This deletion was in frame, and A126-G421 were removed from the 999 nucleic acid bases of the rpoS gene.

To complement the rpoS mutants, the rpoS gene of 1912768R with its native promotor (300 bp upstream of the rpoS ATG initiation codon) was amplified with primers F-rpoS-c and R-rpoS-c by PCR using the genomic DNA of S. marcescens 1912768R as the template. The PCR product was then cloned into pBBR1-MCS2 to generate pBBR1-rpoS-R. The rpoS genes of Serratia sp. YD25, S. plymuthica AS9, Serratia sp. ATCC 39006 and E. coli K-12 substr. MG1655 together with the native promotor of 1912768R were synthesized and cloned into pBBR1-MCS2. The resulting plasmids were transferred by S17-1(λpir) into ΔrpoS 1912768R by conjugation to construct the rpoS complemented strain. Oligonucleotides used as primers for the rpoS deletion and complementation strains are listed in S1 Table.

Sequence alignment

A homologous sequence database search was executed using BLASTp (https://blast.ncbi.nlm.nih.gov). Multiple sequence alignment was performed with ClustalX2.0 and visualized using ESPript 3.0 (http://espript.ibcp.fr/).

Detection of extracellular enzymes, hemolysin and biofilm formation

Phenotypic assays were performed as described previously [39–42]. In all phenotypic plate assays, overnight cultures of the test strains were adjusted to an OD₆₀₀ of 1.0; 5 μl was spotted on the appropriate agar plates and incubated at 28°C. Prodigiosin was detected according to the method of Slater et al. [43]. Proteolytic activities were assayed on skimmed milk agar plates after 48 h as described [44]. Colloidal chitin was prepared per the method reported by Berger and Reynolds [45]; chitinolytic activities were assayed on 2% colloidal chitin plates after 48 h. Detection of hemolysin was indicated by the transparent zone on LB agar plates with 1% sheep erythrocytes (purchased from Beijing Land Bridge Co., Ltd.) for 72 h. Biofilm formation on 96-well plates was determined as previously described [46, 47] and quantified by measuring the absorbance at 575 nm using a plate reader (BioTek Synergy 2) after 72 h in LB medium.

Motility assays

Swimming and swarming ability were assayed visually on LBA containing 0.35% (w/v) or 0.75% (w/v) agar, respectively. To assess swarming motility, 3 μl of an overnight culture in LB liquid medium was spotted on plates, allowed to dry and incubated for 16 h at 28°C. Transmission electron microscopy (TEM) was used to observe the cell shapes of strains 1912768R and ΔrpoS. Bacterial cells were cultured overnight in LB agar plates for 16 h. Colonies were washed in phosphate buffered saline (PBS) and placed on a carbon film-coated copper grid (230 mesh; Beijing Zhongjing Science and Technology Co., Ltd., Beijing, China). Finally, the bacterial liquid on the film was dried at 25°C and observed by TEM [47, 48]. At least three independent preparations were made for each group.

Stress and competition assays

Bacteria grown overnight in LB medium were adjusted to an OD₆₀₀ of 1.0 with PBS buffer and treated as follows. For acid stress, 1 ml of an overnight culture was resuspended in 1 ml of 0.9% NaCl containing 175 mM acetic acid. Aliquots were removed every 5 to 20 min, diluted and plated on LBA. Oxidative stress was induced by treating a bacterial suspension with 0.9% NaCl containing 120 mM H₂O₂ for 5, 15, 30 and 45 min and subsequently diluting, and the bacterial culture was plated. Bacteria were subjected to osmotic stress by incubating for 0, 2, 4, 6 and 8 h in a 3 M NaCl solution. For heat stress, bacteria were treated at 50°C for 0, 5, 10, 15 and 20 min and plated. All plates were incubated for 24 h at 28°C followed by CFU counting. The results are shown as the percentage of the number of CFU/ml, with the CFU at time 0 being 100%.

Competition assays between the strains 1912768R and ΔrpoS mutants were performed under continuous culture in LB medium and M9 medium supplemented with 0.2% glucose at 28°C for 48 h. The competition was started by mixing equal concentrations of each strain (at an OD₆₀₀ = 0.1). Samples were taken at time zero, 24 h and 48 h, and plated on LBA plates. CFU counts of each strain were determined following overnight incubation at 28°C.

RNA purification and qRT-PCR

Cell cultures (2 ml) were collected and centrifuged after16 h in LB medium and washed with RNase free water. RNA was extracted with the RNA Pure Bacteria Kit (with DNase I) (CWbio.Co., Ltd., Beijing, China). DNase treatment was performed with Cwbio DNase I at 28°C for 15 min prior to the concentration step. RNA was quantified using a Nanodrop ND1000 (NanoDrop Technologies), and the quality was evaluated using an Agilent Bioanalyzer (Agilent Technologies). First-strand cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions [15]. Each qRT-PCR was conducted in a final volume of 25.0 μL that contained 8.5 μL of sterile water, 1 μL of the corresponding primers (10 μM), 2.0 μL of suitable cDNA as the template, and 12.5 μL of SYBR Premix ExTaq II (TaKaRa, Shiga, Japan). All reactions were run for 40 cycles, of 3 s at 95°C and 30 s at 60°C. The melting curve program was set for 5 s at 95°C, 1 min at 60°C, and 15 s at 95°C. Gene expression levels were measured using the 16S gene as a control and normalized to the parental strain 1912768R. Oligonucleotides used as primers for quantitative PCR are listed in S1 Table.

Statistical analysis

Experiments were performed at least twice with a minimum of three biological replicates. GraphPad Prism software was used to perform Student’s t-tests and one-way ANOVA with Tukey’s post-test. Significance was set at p < 0.05.

Results

Selective advantage for nonpigmented mutants during prolonged stationary phase

To verify whether prolonged incubation would enrich the de novo emergence of prodigiosin-defective individuals, we performed a series of long-term incubations of the WT strain 1912768R in lysogeny broth (LB) medium. As shown in Fig 1A, cells incubated in batch culture began to lose viability at day 2, marking the transition from the stationary phase to the death phase. More than 99% of the cells died, and the number of viable cells became stable on day 16. Prodigiosin-deficient mutants were first isolated on day 6 and enriched with the extension of culture time. The arrow indicates the time after which cells expressing the GASP phenotype were observed, and the number of GASP mutants (white strains) reached a maximum around day 16. This result suggested that the loss of prodigiosin production might contribute to the survival of white mutants during prolonged stationary phase. To verify whether the selective advantage for nonpigmented mutants is incidental, 10 single colonies of S. marcescens 1912768R were grown for 16 days. As shown in Fig 1B, all nonpigmented mutants were observed around day 6, and the white strains were enriched during prolonged incubation.

Fig 1 — (A) Changes in the number of all viable strains and percentage of emerged white strains during the prolonged incubation of WT. (B) Frequency of *de novo* emergence of prodigiosin-defective mutants. R1-10 represent ten single colonies of WT 1912768R cultured independently under the same culture conditions. (C) Representative images of the WT strain 1912768R and the two mutants. (D) Quantification of prodigiosin production in LB medium. *, P < 0.05; ***, P< 0.001.

To explore the underlying mechanisms of loss of prodigiosin production, a white colony of S. marcescens 1912768R was randomly selected after 16 days of prolonged incubation of under laboratory conditions. A pigment-defective mutant (pink) was also isolated at the same time. These two mutants were named 1912768WR (pink) and 1912768W (white) (Fig 1C). Prodigiosin was extracted and measured from cultures of the WT and two mutant strains. The prodigiosin levels were significantly lower in the mutants as shown in Fig 1D.

RpoS was screened as a candidate regulator of prodigiosin production

Comparative genomic analysis was performed to further explain the underlying molecular mechanism of the deficiency in prodigiosin production. De novo sequencing of 1912768R was performed and the complete genome of 1912768R (GenBank accession number: CP040350) consists of a 5,117,289 bp (GC content 59.75%) single circular chromosome (S1 Fig). A total of 4927 protein-encoding genes were predicted and annotated in four databases namely the Kyoto Encyclopedia of Genes and Genomes (KEGG), the National Center for Biotechnology Information non-redundant (NR), UniProt, and Gene Ontology (GO) databases. The genomes of 1912768WR (GenBank accession number: VBRI00000000) and 1912768W (GenBank accession number: VBRJ00000000) were sequenced. The general features of the genomes are presented in S2 Table.

Comparative analysis revealed 185 and 95 nonsynonymous SNPs in 1912768WR and 1912768W, respectively, and one deletion in 1912768W (Fig 2). Of the 185 SNPs in 1912768WR, 128 were located in coding DNA sequences (CDSs) and 57 were in intergenic regions in the promoters and terminators around protein-coding genes which could influence the transcriptional level of the corresponding genes [49]; of the 95 SNPs in 1912768W, 38 and 57 were located in CDSs and intergenic regions, respectively. The only deletion was in the rpoS gene of 1912768W, resulting in a frameshift mutation.

BlastP analysis revealed that the pig cluster from 1912768R was a type I pig cluster (S2 Fig). Since none of the SNPs/InDels were mapped in the pig cluster, this meant that mutations in transcriptional regulators that regulate prodigiosin production might lead to the observed pigmentation. The number of nonsynonymous mutations that were located in transcriptional regulators were 8 and 3 for strains 1912768WR and 1912768W, respectively. In addition, one nonsynonymous SNP was found in rpoS at site 62 (T/A→C/G) in 1912768WR, and a deletion was found in rpoS at sites 185–186 (CG, 2-nucleotide deletion) in 1912768W. BlastP analysis of RpoS from different Serratia sp. with different pig cluster types and E coli was performed. The sequences were highly conserved with identities of 99.70% (Serratia sp. YD25), 97.89% (Serratia plymuthica AS9), 91.87% (Serratia sp. ATCC 39006) and 94.58% (Escherichia coli K12). The mutations of RpoS within the two mutants were located at sites 62 and 185–186, and these mutations were highly conserved (Fig 3). RpoS was proven to be a negative regulator in prodigiosin regulation in strain Serratia sp. ATCC 39006 [17]; however, this result suggested that RpoS from S. marcescens 1912768R might be a positive regulator of prodigiosin production, and the mutation in rpoS might be causing the deficiency in prodigiosin production.

Fig 3 — Multiple sequence alignment of RpoS from S. *marcescens* 1912768R, *Serratia* sp. YD25, S. *plymuthica* AS9, E. *coli str*. K-12 substr. MG1655 and *Serratia* sp. ATCC 39006. The alpha helix, random coil and beta turn are represented as α, η and T, respectively. Conserved sequences are indicated with a red box, and similar sequences are indicated using a colored background.

RpoS positively regulates the production of prodigiosin in S. marcescens 1912768R

According to the analysis of the function of RpoS in prodigiosin production above, the regulatory role of RpoS in pigmentation was further characterized. A 296 bp segment (A126-G421) was deleted from the 999 bp rpoS gene of 1912768R. Prodigiosin of the WT strain and ΔrpoS mutant strains with the complementation plasmid pBBR1-rpoS-R or vector negative control was extracted and measured after two days of fermentation. The prodigiosin levels were significantly lower in the ΔrpoS mutant and could be complemented in trans (Fig 4A). This effect could stem from reduced transcriptional expression of the prodigiosin biosynthesis operon. Quantitative reverse transcriptase PCR (qRT-PCR) analysis was used to evaluate the relative transcription of the pig cluster in the WT and ΔrpoS mutant strains. More than a 20-fold and 6-fold reduction in the pigA transcript level was observed in the ΔrpoS mutant, suggesting that the pigment defect of the ΔrpoS mutant is largely due to the decreased transcriptional level of the pig cluster (Fig 4B). In Serratia sp. ATCC 39006, the rpoS mutant was unable to grow in minimal medium (MM) with glucose as the sole carbon source, suggesting that the mutant was auxotrophic for essential amino acids [17]. Growth analysis of the rpoS mutant of S. marcescens 1912768R in LB and MM with glucose as the sole carbon source showed no difference, indicating that prodigiosin differences were not due to growth and that the role of RpoS in growth is different (Fig 4C and 4D).

Fig 4 — (A) The *rpoS* mutant colony and color phenotypes were complemented by *rpoS* from S. *marcescens* 1912768R. (B) qRT-PCR analysis revealed reduced expression from the prodigiosin biosynthetic locus. (C-D) Growth curves of the WT strain 1912768R and the Δ*rpoS* mutant in LB or M9 minimal medium supplemented with 0.2% glucose. (E) Complementation of Δ*rpoS* mutant defects by *rpoS* genes from *Serratia* sp. strains with different *pig* cluster types and E. *coli*.

To further confirm the regulatory effect of RpoS on the production of prodigiosin, rpoS genes from strains with different pig cluster types and E. coli K12 together with the promoter from 1912768R were synthesized. The synthesized segments were introduced into the ΔrpoS mutant strain on a multicopy plasmid. With the expression of rpoS, the pigment and colony morphology phenotypes of the ΔrpoS mutant were restored to those of the WT strain to some extent (Fig 4E). These results support the idea that RpoS is a positive regulator of prodigiosin production in S. marcescens 1912768R.

The disruption of rpoS has pleiotropic effects on S. marcescens physiology

The prodigiosin production regulator, RbsR in S39006, is a pleiotropic regulator of motility, virulence, siderophore, exoenzymes and antibiotic production [50]. MetR controls prodigiosin production, methionine biosynthesis, cell motility, H₂O₂ tolerance, heat tolerance, and exopolysaccharide synthesis in Serratia marcescens JNB5-1 [48]. It seems that the regulator of prodigiosin production has pleiotropic effects on strain physiology in general. As the rpoS mutation regulated prodigiosin production in S. marcescens 1912768R, we postulated that it might also regulate other phenotypes and modes of motility. In addition to the abolishment of prodigiosin production, biofilm formation, hemolysis and proteinase secretion also decreased compared to WT strain 1912768R, and the secretion of lipase, chitinase and siderophores increased (Fig 5A). These observations suggested that prodigiosin production might have a correlation with the different phenotypes and further highlighted the extent of the pleiotropy caused by the rpoS mutation in 1912768R.

Tests were performed to determine whether RpoS played a role in the regulation of motility. As shown in Fig 5B, the swimming and swarming ability of the ΔrpoS mutant increased significantly. TEM analysis of the ΔrpoS mutant cells after 12 h of plate culture also demonstrated an increase in flagella and elongated shape, which contributed to the increased mobility. To further identify the role of RpoS in the regulation of motility, qRT-PCR was performed. The transcript levels of the flhC and flhD genes in the ΔrpoS mutant strain increased by several times compared to those in the WT strain (Fig 5C). This increase in expression of the flhDC operon correlated with an increase in the expression of the flagellar gene fliC based on qRT-PCR; this result is consistent with the increase in mobility of the ΔrpoS mutant.

The ΔrpoS mutant displayed impaired resistance to stresses but higher competence than its isogenic WT strain

The physiological role of bacterial prodigiosin in vivo remains undefined. However, a number of reports have suggested potential functions for prodigiosin that may provide advantages such as antagonistic activity to other strains (biotic stress) [51] and continuously challenging natural environments (abiotic stress) [52]. In E. coli and Salmonella enterica serovar Typhi, rpoS regulates resistance to stresses including high osmolarity, oxygen free radicals, acid and other biotic stresses [20–22]. The attenuation function of rpoS or even rpoS mutants confers a reduction in stress resistant but promots of nutritional competence because bacteria allocate limited resources to growth rather than self-protection when suffering starvation and vice versa [23]; therefore, there is a SPANC trade-off in E. coli, which is an adaptive behavior during prolonged starvation [53].

The role of RpoS in stress resistance was explored. The survival rates of 1912768R and the pigmentless ΔrpoS mutant were characterized after exposure to diverse stress conditions, including 10 mM H₂O₂, 50°C, 175 mM acetic acid and 20% NaCl. Plate counts at different time intervals after exposure to the corresponding stress were deterimined to evaluate the viabilities of the WT 1912768R and ΔrpoS mutant strains. The results showed that the survival rates of 1912768R and the ΔrpoS mutant decreased with increasing environmental pressure; in addition, the ΔrpoS mutant was more sensitive to oxidative, osmotic, heat and acid stress (Fig 6A–6D). These results proved the positive regulatory role of RpoS in stress resistance and indirectly suggested that prodigiosin might provide an advantage for S. marcescens in stress resistance.

Fig 6 — The WT strain 1912768R and in-frame deletion Δ*rpoS* mutant were exposed to 3 M NaCl solution (A), 175 mM acetic acid (B), 50°C (C) and 120 mM H₂O₂ (D). The survival percentage was obtained by dividing the surviving population by the initial population, which corresponded to 100%. Data are mean and SD of three independent experiments.

An indirect but frequently used test for outer membrane permeability is susceptibility to SDS and Cm, as outer membrane exclusion is an important component of protection against these compounds [54–57]. The survival rate of the ΔrpoS mutant decreased compared to that of the WT strain when treated with SDS and Cm, indicating that the permeability of the membrane of the ΔrpoS mutant increased (Fig 7A and 7B), which would promote nutritional competence. The general membrane permeability of E. coli is determined by the type of major porin proteins, OmpC and OmpF, which act as molecular sieves; OmpC channels are more restrictive than OmpF channels [57, 58]. Bacteria containing only OmpC are more resistant to toxic hydrophilic agents than those containing only OmpF, while the transcription of ompF increases under glucose limitation, improving the permeability of substrates at low concentrations [54]. To further investigate membrane permeability, the transcript level of ompF/ompC was identified. Fig 7C shows that the transcript level of ompF increased in the ΔrpoS mutant strain, while that of ompC decreased; which is consistent with the increase in cell membrane permeability. These results suggest that RpoS positively regulates membrane permeability. The higher the cell membrane permeability of the strain is, the more sensitive the strain is to pressure, and the easier it is for nutrients to pass through.

Fig 7 — (A-B) Growth of the 1912768R and Δ*rpoS* mutant strains on 96-well plates containing SDS or Cm. (C) Change in transcript levels of *luxS* and porin-related genes (n = 4). (D-E) Effect of *rpoS* on the fitness of S. *marcescens*. S. *marcescens* 1912768R with theΔ*rpoS* mutant at a 1:1 ratio and grown for 24 and 48 h in LB or M9 medium. Samples were removed, diluted and plated on LBA plates for CFU determination. The number of 1912768R and Δ*rpoS* mutant colonies was verified by the different color of the colonies. Mean and SD are shown. *, P < 0.05; **, P < 0.01.

To evaluate the contribution of rpoS to the fitness of WT 1912768R and the ΔrpoS mutant, competition assays were performed in LB and M9 minimum medium with supplemented with 0.2% glucose. Equal numbers of bacteria in the exponential phase were mixed and incubated with the corresponding medium. By coculturing mixtures of 1912768R and the ΔrpoS mutant and plate counting based on the different color of the colonies on LB agar plate (the nonpigmented strain mutated from 1912768R appeared on the 6^th day), we found that the ΔrpoS mutant performed better than the WT strain in both LB and M9 minimum medium at 24 h and 48 h, as shown in Fig 7D and 7E. Hence, rpoS did have a clear deleterious effect on the fitness of S. marcescens under laboratory conditions.

Discussion

In this study, we identified that RpoS was a positive regulator of prodigiosin biosynthesis in S. marcescens 1912768R; mutation of rpoS introduced pleiotropic phenotypes. We also made the case that mutation of rpoS, which led to the loss of prodigiosin production during prolonged stationary phase, was a trade-off between self-preservation and nutritional competence in S. marcescens 1912768R.

The genetic results presented here indicate that the RpoS protein from several species of Serratia and E. coli is highly structurally conserved. Therefore, it was somewhat surprising that the function of RpoS in the regulation of prodigiosin production is different between S. marcescens 1912768R and Serratia sp. ATCC 39006. Random transposon insertion of a 533 bp fragment after the start codon of rpoS in Serratia sp. ATCC 39006 resulted in increased prodigiosin production [17]. In this paper, deletion of the 296 bp fragment revealed that RpoS from S. marcescens 1912768R was a positive regulator of prodigiosin biosynthesis. Complementation of RpoS from different strains with the native promoter of rpoS from strain 1912768R restored the pigmentation, and it seems that the promoters of the pig cluster are more important than the genes for the regulation of prodigiosin production. Currently, the regulatory pathway of RpoS in the biosynthesis of prodigiosin is unknown. Therefore, some theoretical basis is still needed to fully clarify the reason why the regulatory roles of RpoS in regulating production of prodigiosin are different in Serratia marcescens 1912768R and strain Serratia sp. ATCC 39006.

One reason for the opposite regulatory roles of RpoS may be due to the diverse types of pig clusters. BlastP analysis showed that the pig cluster of 1912768R was a type I pig cluster, which is the main form of the prodigiosin coding cluster existing in the majority of strains and is different from the unique pig cluster that only exists in Serratia sp. ATCC 39006 (S2 Fig). Although the global arrangement of the pig clusters in 1912768R and Serratia 39006 is very similar, the intergenic region upstream of pigA is markedly different. Transposon insertion mutations in the region 200–320 bp upstream of pigA in Serratia 39006 cause hyperpigmentation [59]. The promoter of the pig cluster has been mapped, and a different promotor has been proposed in the 488 bp region upstream of pigA in S. marcescens Sma 274 [60]. In S. marcescens 1912768R, this region is 407 bp compared to 924 bp in Serratia 39006, indicating that promoter regions in pig clusters might be different.

Another conspicuous difference between 1912768R and ATCC 39006 is the quorum sensing (QS) system. In Serratia 39006, transcription of the pig cluster is regulated by QS via the smaI/R genes and the N-acyl homoserine lactones (AHLs) N-butanoyl-L-homoserine lactone (BHL) and N-(hexanoyl)-L-homoserine lactone (HHL) [61], while smaI/R homologues do not exist in strain 1912768R. The enzyme LuxS is responsible for the production of autoinducer-2 (AI-2), a molecule that has been implicated in QS in many bacterial species. Mutation of luxS from Serratia sp. ATCC 39006 had no influence on prodigiosin biosynthesis while the luxS mutant of S. marcescens ATCC 274 showed decreased prodigiosin production [18]; in addition, the decreased transcriptional level of rpoS showed no influence on the transcription of luxS in Serratia sp. ATCC 39006, suggesting that the regulation of RpoS in prodigiosin production is independent of LuxS [16]. In this study, the transcriptional level of luxS decreased significantly in the ΔrpoS mutant compared to the WT strain 1912768R (S3 Fig), indicating that the regulation of prodigiosin production in S. marcescens 1912768R by RpoS might be through LuxS (Fig 8), and this mechanism of regulation is unknown in Serratia sp. ATCC 39006 [17].

Fig 8 — Positive regulation is depicted by pointed arrowheads. Dashed lines indicate putative regulation.

Finally, the different roles of RpoS may be due to the diverse function of RpoS in the regulation of secondary metabolites. In Pseudomonas species, the regulatory effect of RpoS on secondary metabolites is diverse or even opposite in some species [62], illustrating that the diversity of the regulatory effect of RpoS is not unique. As the regulatory pathway of RpoS in the biosynthesis of prodigiosin is unknown and complicated, further research is still needed to illuminate the reasons for the distinct regulatory roles of RpoS in the two strains.

The functional diversity of RpoS in Serratia species can also be observed in the regulation of other phenotypes. The Serratia sp. ATCC 39006 ΔrpoS mutant could not be cultured in minimal medium; on the contrary, the growth rate of the ΔrpoS mutant did not change compared to that of the WT strain 1912768R, which might be due to the functional diversity of RpoS in Serratia species. The ΔrpoS mutant showed increased swimming motility, which is different from the unchanged swimming motility observed in the rpoS mutant of Serratia sp. ATCC 39006. Swarming is dependent on both biosurfactants, such as serratamolide, and flagellar motility [7]. Serratamolide is hemolytic, and we observed an obvious decrease in hemolysis of the ΔrpoS mutant. However, the amount of swarming increased, suggesting that the increase in flhDC expression increases motility to overcome the reduction in serratamolide. The TEM results also revealed that ΔrpoS cells have a swarmer morphology characterized by an elongated shape and additional flagella that could account for the increased zone of swarming even with an apparent reduction in biosurfactant.

The stress resistance of the ΔrpoS mutant in our study decreased, which was similar to that of the ΔrpoS mutant of E. coli [20, 23]. We also observed an increase in membrane permeability as shown in Fig 7. In E. coli, the general membrane permeability is determined by the porin proteins OmpC/OmpF [63]; the difference between them is that OmpC channels are more restrictive, protecting cells against toxic hydrophilic agents, but OmpF channels offer higher open access to diffusible nutrients and are the most open porin in E. coli [64]. In our study, the transcript level of ompF increased in the ΔrpoS mutant strain, while that of ompC decreased. As no research has shown that OmpC and OmpF in S. marcescens act similarly to E. coli porins, we speculate that the increase in cell membrane permeability is due to transcriptional changes in the corresponding porin genes. Reducd stress resistance and increased nutritional competence make the ΔrpoS mutant outcompete its parent strain, indicating an SPANC trade-off in S. marcescens 1912768R driven by the mutation of rpoS.

In summary, this study introduces the novel regulatory role of RpoS from S. marcescens on the secondary metabolite prodigiosin and enhances the knowledge on the regulation by RpoS. Additionally, this study confirms that rpoS mutants emerged during prolonged stationary phase are the major cause of the loss of prodigiosin production, and this appears to be an elaborate evolutionary strategy driven by the trade-off in S. marcescens 1912768R.

Supporting information

S1 Table. Primers used in this study.

(PDF)

Click here for additional data file.^{(139.7KB, pdf)}

S2 Table. General genome features of S. marcescens strains 1912768R, 1912768WR and 1912768W.

(PDF)

Click here for additional data file.^{(136.9KB, pdf)}

S1 Fig. Circular diagrams of S. marcescens 1912768R genome.

The outermost circle is the genome size, each scale is 0.1 Mb; the second and third circles are the CDS on the positive and negative strands, and different colors indicate the different COG functional classification of the CDS; the fourth circle is rRNA and tRNA; the fifth circle is the GC content. The outward red part indicates that the GC content in this area is higher than the average GC content of the whole genome. The higher the peak, the greater the difference from the average GC content. The inward blue part indicates that the regional GC content is lower than the average GC content of the whole genome; the innermost circle is the GC-skew value.

(TIF)

Click here for additional data file.^{(1.1MB, tif)}

S2 Fig. Comparison of pig cluster from S. marcescens 1912768R and Serratia sp. ATCC 39006.

(TIF)

Click here for additional data file.^{(349.3KB, tif)}

S3 Fig. Relative transcription of gene rpoS.

Relative transcription of gene rpoS from WT strain, ΔrpoS and rpoS complemented strain at 6 h (a) and 16 h (b).

(TIF)

Click here for additional data file.^{(881.6KB, tif)}

Data Availability

The complete genome of Serratia marcescens 1912768R was deposited in GenBank under the accession number CP040350. The genomes of Serratia marcescens 1912768WR and 1912768W were deposited in GenBank under the accession number VBRI00000000 and VBRJ00000000, respectively.

Funding Statement

This work was supported by National Natural Science Foundation of China (31670078); Sichuan Science and Technology Program (2018GZ0375 and 2018TJPT0004); Chengdu Science and Technology Program (2017-GH02-00071-HZ and 2018-YF05-00738-SN); National Infrastructure of Natural Resources for Science and Technology Program of China (NIMR-2018-8); Science and Technology Program of Sichuan University (2018SCUH0072).

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PLoS One. doi: 10.1371/journal.pone.0232549.r001

Decision Letter 0

Marie-Joelle Virolle

20 Feb 2020

PONE-D-20-03043

Regulation of RpoS in production of prodigiosin and trade-off during prolonged stationary phase in Serratia marcescens 1912768R

PLOS ONE

Dear Dr Yi Cao,

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Reviewer #1: Review of PONE-D-20-03043 for PLoS One, “Regulation of RpoS in production of prodigiosin an trade-off during prolonged stationary phase in Serratia marcescens 1912768R.

This study describes the enrichment of pigmentless mutants of S. marcescens during prolonged stationary phase. The basis of this loss of pigment was found to be mutation of the rpoS sigma factor gene, which is in opposition to what was previously shown by the Salmond group with another Serratia species. The defect in pigmentation was shown to be due to loss of transcription of the pig operon and not due to changes in growth. The rpoS mutant pigment defect was complemented by wild-type rpoS genes from a variety of Serratia species and Escherichia coli. Mutation in rpoS was demonstrated to have pleiotropic effects including increased susceptibility to a number of stresses. Additional data suggested that there is differential expression of OMPs and a selective growth advantage of the rpoS mutant in LB and minimal media.

On the positive side, the figures are well put together. The data for the most part is good, particularly exciting are figure 1A/B and figure 4. There is a lot of room for improvement, particularly with respect to the major issues in the writing and some concerns with interpretation.

Criticisms:

1. The major issue are the numerous writing issues (grammar and spelling) that weaken the manuscript. I have listed many below, but there are too many to list.

2. Line 21. Please change “which is” to “could be” – this is important because the results of this study suggest that loss of pigmentation will be a problem for industry, but this has not been reported as suggested by the current phrase.

3. Line 97. inconsistent with line 238. LB should be Lysogeny Broth, but Luria-Bertani is also accepted, but please be accepted.

4. Table 1. Inconsistent description of DH5a and S17-1 – please add the genotype for S17-1.

5. Table 1. Cut “widely used” for two plasmids, also replace knockout vector with “allelic replacement vector”

6. Line 148-151. Please be more specific about the mutant allele. For example, what base pairs are left.

7. Line 158-9. Replace “double-crossover recombination” with “second recombination”. Double cross-over is incorrect in this allelic replacement approach.

8. Methods line 183. Please describe what is meant by “treated chitin” and/or add a reference.

9. Line 185. What is meant by “skim sheep blood”?

10. Line 215-20. Please describe whether DNase was used in RNA preparation.

11. Line 234-5. Suggest changing the title to “Selective advantage for non-pigmented mutants during prolonged stationary phase”. Line 236. Change “cause” to “enrich for”

12. Figure 2. Should say “intergenic region” rather than “intergenic gene”

13. Line 289 – change “type to types”. Moreover, give some information on the amino acid identity of the RpoS protein.

14. Line 308 – consider adding the fold-change. It is hard to determine this looking at the linear scale of figure 4B.

15. Line 310. Consider adding “indicating that prodigiosin differences were not due to growth”

16. Line 320. It was very good using the same promoter for each rpoS gene. Please indicate that all genes were expressed from the rpoS promoter from 1912768R.

17. Line 339-340. “increased by several orders of magnitude” – this suggests that the Y-axis of Figure 5C was a log10 scale – please clarify whether it is log10 or linear.

18. Line 359-365. The loss of permeability using these assays is not well described. Are these well-established assays for permeability? I would suggest adding references for these.

19. Line 363-5. Is there any information on the predicted pore size of OmpC and OmpF?

20. Line 400-1. It seems that the promoters are more important than the genes for regulation of prodigiosin production.

21. Discussion – “data not shown” should not be included in the discussion section – this new data should be either moved to the results and shown or kept for a subsequent study. Line 410-414 and 442-445.

22. Figure 1C. The difference between R and WR is not clear in this image. A better picture would be helpful.

23. Figure 1A/B. Rename the Y-axis (left side) to “Surviving CFU/ml” or “Surviving Bacteria (CFU/ml)”

24. not clear whether some of the Y-axes are log scale or linear. Like figure Fig 5C, 7C,

Grammatical issues and writing suggestions:

1. Line 24-5. Please use “Exhibited” rather than exhibiting, add “was” after (named 1912768WR). Cut “in the synthesis of the red pigmented secondary metabolite” as it is possibly confusing.

2. Line 29. Replace “disclosed that RpoS was pleiotropic” with “had pleiotropic effects”

3. Line 32. Replace colon with period.

4. Line 35. add “be” after “might” – also consider that the pig promoter rather than the pig cluster is important.

5. Line 41. cut “with RpoS involving in it”

6. Line Lines 50-55 – giant sentence – please fix.

7. Line 57. Change “secret” to “secrete”

8. Line 70. Cut “simple structured” – it is unnecessary.

9. Lines 74-76. Please fix this sentence.

10. Line 78. Cut “Except for these studies”

11. Line 86. Replace “these” with “this”

12. Line 88. Spelling of regulatory.

13. Line 139. Spelling of queried.

14. Line 146. Spelling of S. marcescens.

15. Line 165. Change “were” to “was”

16. Line 181-2. Remove “detection”, and add “of Slater, et al.” after “method”

17. Lines 193-4. Please fix the sentence.

18. Line 212. Change “was” to “were”

19. Please change “at almost the 16th day” to “sound day 16”.

20. Lines 283-287. Many issues. Add period after type I. Change “besides” to “since or because”, replace “and” on line 284 with a coma. “regulating to regulate”.. change “be the reason causing the loss” to “underly / lead to the observed”. Line 286 add “were” after “that”. Add “for strains WR and W respectively” after “3”.

21. Line 323. Change “impacts” to “effects”

22. Line 342. Change “resulting” with “which is consistent with”

23. Line 344-5. Change “resistant” to “resistance” and cut “ability”

24. Line 366 and 371. Spelling of “membrane permeability” and “medium”

25. Line 373. Space after “colony.”

26. Line 388 and 392. Spelling of “production” and “species”

27. Line 413-420 – add “be” after might on 413 and after may on line 414. Line 415 change rpoS to RpoS, line 418 change “researches are” to “research is”. Line 420, spelling of “strains”.

28. Line 443. Replace “an” with “the”

29. Line 467-8. Cut “in our study”

30. Figure 4B. Y-axis – should be “pigA” rather than PigA.

Reviewer #2: Manuscript Number:

PONE-D-20-03043

Manuscript Title:

Regulation of RpoS in production of prodigiosin and trade-off during prolonged stationary phase in Serratia marcescens 1912768R

This manuscript addresses the problem of loss of prodigiosin production in stationary-phase cultures of Serratia marcescens. Prodigiosin-defective mutants of S. marcescens strain 1912768R were isolated from long-term stationary-phase cultures. Strain 1912768WR was partially deficient in prodigiosin production, while strain 1912768W was entirely deficient in prodigiosin production. The genomes of these two mutants (1912768WR and 1912768W) were sequenced and compared to parent strain 1912768R. Genome analysis revealed that 1912768WR and 1912768W carried mutations in the rpoS gene. An rpoS deletion mutant of parent strain 1912768R was constructed (strain �rpoS) and used in subsequent experiments. Complementation studies were also performed using a plasmid-borne copy of “wild-type” rpoS from parent strain 1912768R. The deletion mutant was defective in prodigiosin production and this phenotype was restored by the complementation plasmid. The authors also show that transcription of the pigment operon (pigA transcript) was downregulated in the deletion mutant. In addition, the authors demonstrate that many other phenotypes were affected by the rpoS deletion including biofilm formation, motility, cellular arrangement, the production of extracellular enzymes, the production of siderophores, and the transcription of genes involved in flagella biosynthesis. The authors also show that the rpoS deletion caused a decrease in resistance to several forms of stress and a general increase in membrane permeability, including changes in OmpC and OmpF levels, and taken together, suggesting a role in the “SPANC balance” of S. marcescens. Lastly, the rpoS deletion strain outcompeted the parent strain in growth studies in both rich and minimal media. The authors conclude that the loss of RpoS is the primary reason for the loss of prodigiosin production in strains 1912768WR and 1912768W.

The data regarding rpoS mutations and their effects on stress resistance, growth, and membrane permeability are not very surprising, as RpoS is a well-known stress-response regulator in the Enterobacteriaceae. The role of RpoS in the stationary-phase-inducible, general-stress-responses of Escherichia coli and Salmonella enterica serovar Typhimurium have been well documented in recent decades, and the authors are missing some important references in this research field. However, the data does provide evidence that prodigiosin is RpoS-regulated in Serratia marcescens. The other data presented in this manuscript provide evidence of an extensive RpoS-regulon in S. marcescens, but how this is relevant to prodigiosin production is less clear. Although the authors show that RpoS-regulated functions are involved in various types of stress resistance and in growth rate, evidence that prodigiosin production is somehow directly connected to the SPANC balance in Serratia marcescens is again unclear. Is a potential connection between prodigiosin and other RpoS-regulated functions being inferred? Does less prodigiosin biosynthesis result in less stress resistance and greater nutritional competence? Perhaps the manuscript should focus on the RpoS regulon of Serratia marcescens instead of just focusing on prodigiosin production. In other words, a different manuscript title might solve some of these issues. Overall, it seems that the study of prodigiosin-deficient mutants led to the discovery of an RpoS regulon in this organism.

Furthermore, the authors begin to discuss possible differences in the RpoS regulation of pig loci (prodigiosin biosynthesis operons) in different S. marcescens strains. This is an interesting point of discussion, but is not explained enough in the manuscript. An additional figure illustrating the potential differences in pig regulation could be helpful. In fact, any potential differences between the RpoS-regulated functions of this organism (1912768R) and those of other S. marcescens strains, or closely related species, should be emphasized more in the body of the manuscript.

The experimental strategies and methods appear to be scientifically sound, and there was sufficient statistical analysis of the data. But again, the authors’ findings with S. marcescens were somewhat predictable based on what is already known about the RpoS regulons of closely related species.

There are extensive English language issues throughout the manuscript. One result of these language problems is that the actual experimental data is not always described sufficiently in a clear manner. Some important experimental results are only described in a couple of sentences, and their impact is lost in the overall body of the manuscript. There are also several typographic errors throughout the manuscript, a few examples of which are listed below:

Line 86: “theses” should be “these”

Line 366: “menbrane” should read “membrane”

Line 388: “produciton” should read “production”

Line 392: “spicies” should be “species”

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Jun 2;15(6):e0232549. doi: 10.1371/journal.pone.0232549.r002

Author response to Decision Letter 0

4 Apr 2020

Dear Editors and Reviewers:

On behalf of my co-authors, we thank you very much for giving us an opportunity to revise our manuscript. We appreciate the editor’s and reviewers’ positive and constructive comments and suggestions on our manuscript entitled “Regulation of RpoS in production of prodigiosin and trade-off during prolonged stationary phase in Serratia marcescens 1912768R” (PONE-D-20-03043). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied the comments carefully and made corrections which we hope meet with approval. Revised portion are marked in red in “Revised Manuscript with Track Changes”.

Academic Editor：

Thanks to academic editor for the positive comments and suggestions on the requirements of PLOS ONE, we have revised the paper accordingly and thanks again for the comments and suggestions.

Response: Thanks for the comments and suggestions. we have revised the paper accordingly following the PLOS ONE style templates. Also, we have used editing service of American Journal Experts (https://www.aje.cn/) to polish the English language of our manuscript (Certificate Verification Key: 7CE0-5AFC-7E2A-0AE2-36FD). We hope the revised manuscript meets with the requirements of PLOS ONE's style requirements.

2. PLOS requires an ORCID ID for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

Response: Special thanks for the positive and constructive comments. We have updated the information and authenticated the pre-existing ID (0000-0002-6216-548X).

Response: Thank academic editor for the positive and constructive comments. We will not change the “Data Availability statement”, and we will provide the relevant accession numbers of the genomes at acceptance.

Response: Special thanks for the positive and constructive comments. We have revised the paper accordingly and added the data that is needed in the research; we also removed the phrase that refers to the data, which is not a core part of the research and revised the Discussion part carefully.

Response: Thank you for your positive and constructive comments. We have revised the Authors’ affiliations with “Microbiology and Metabolic Engineering of Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu, P.R. China.”

6. While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

Response: Thanks for the positive comments and suggestions. We have used the PACE to make ensure that the figures of our manuscript meet PLOS requirements.

Replies to Reviewer #1:

Thanks to reviewer#1 for the positive comments on the article and the figures indicating that it could be a useful and interesting paper.

Criticisms:

1. The major issue are the numerous writing issues (grammar and spelling) that weaken the manuscript. I have listed many below, but there are too many to list.

Response: Special thanks for your careful and patient review of the manuscript. We have revised these these wrong spellings and sentences carefully along with the rest of the paper to read more smoothly and be easier to understand. We have used editing service of American Journal Experts (https://www.aje.cn/) to polish the English language of our manuscript (Certificate Verification Key: 7CE0-5AFC-7E2A-0AE2-36FD).

Response: Thank you for your positive and constructive comments. We have changed “which is” to “could be”. This is really important as the problem in industry has not been reported so far.

3. Line 97. inconsistent with line 238. LB should be Lysogeny Broth, but Luria-Bertani is also accepted, but please be accepted.

Response: It should be “Lysogeny Broth”，the article has been revised accordingly，we thank reviewer for the constructive comments and suggestions.

4. Table 1. Inconsistent description of DH5α and S17-1–please add the genotype for S17-1.

Response: Thank reviewer#1 for the positive and constructive comments. we have added the genotype for S17-1.

5. Table 1. Cut “widely used” for two plasmids, also replace knockout vector with “allelic replacement vector”

Response: Thanks for the positive and constructive comments. We have cut “widely used” for the two plasmid and replaced “knockout vector” with “allelic replacement vector”.

6. Line 148-151. Please be more specific about the mutant allele. For example, what base pairs are left.

Response: Special thanks for your your positive and constructive comments. We have pointed out the mutant allele in detail and the article has been revised accordingly.

7. Line 158-9. Replace “double-crossover recombination” with “second recombination”. Double cross-over is incorrect in this allelic replacement approach.

Response: Special thanks for the corrections. We have corrected the wrong description of the allelic replacement approach and we have replaced “double-crossover recombination” with “second recombination”.

8. Methods line 183. Please describe what is meant by “treated chitin” and/or add a reference.

Response: Special thanks for your your positive and constructive comments. The “treated chitin” is confused and we have replaced “treated chitin” with “colloidal chitin”. We have added the reference of preparation of colloidal chitin.

9. Line 185. What is meant by “skim sheep blood”?

Response: “skim sheep blood” is also confused and have been replaced by “sheep erythrocytes”. Thanks for the positive comments and suggestions.

10. Line 215-20. Please describe whether DNase was used in RNA preparation.

Reponse: Thank reviewer #1 for the positive and constructive comments. It is important to describe whether DNase was used in RNA extraction for the untreated RNA have a great influence on the qRT-PCR results. In this study, DNase treatment was performed with Cwbio DNase I at 28°C for 15 min prior to the concentration step and the article has been revised accordingly.

11. Line 234-5. Suggest changing the title to “Selective advantage for non-pigmented mutants during prolonged stationary phase”. Line 236. Change “cause” to “enrich for”.

Response: Special thanks for your your positive and constructive comments. We have changed the title to “Selective advantage for non-pigmented mutants during prolonged stationary phase” and which we do think is more logical. We have replaced “cause” with “enrich for” and it is more accurate.

12. Figure 2. Should say “intergenic region” rather than “intergenic gene”

Response: It should be “intergenic region” and the annotation of “intergenic gene” is wrong. We have changed “intergenic gene” to “intergenic region” in Figure 2 and the article has been revised accordingly. Thanks for the correction.

13. Line 289 – change “type to types”. Moreover, give some information on the amino acid identity of the RpoS protein.

Response: Special thanks for the corrections. We have changed “type” to “types”. The sequences of RpoS are highly conserved with the identity 99.70% (Serratia sp. YD25), 97.89% (Serratia plymuthica AS9), 91.87% (Serratia sp. ATCC 39006) and 94.58% (Escherichia coli K12). We have revised the article accordingly.

14. Line 308 – consider adding the fold-change. It is hard to determine this looking at the linear scale of figure 4B.

Response: Special thanks for Reviewer#1 of the positive and constructive comments. We have changed the linear scale of figure 4B to fold change of 1912768R to ΔrpoS. This modification does make the results more readable.

15. Line 310. Consider adding “indicating that prodigiosin differences were not due to growth”

Response: Special thanks for the positive and constructive comments. The results did suggest that the prodigiosin differences were not due to growth. We have added “indicating that prodigiosin differences were not due to growth” and this is really helpful to describe the impact of the results.

16. Line 320. It was very good using the same promoter for each rpoS gene. Please indicate that all genes were expressed from the rpoS promoter from 1912768R.

Response: Thank you for your positive and constructive comments. We have revised the paper and indicated that the rpoS promoter from 1912768R was synthesized and cloned with the rpoS from different strains to the vector.

17. Line 339-340. “increased by several orders of magnitude” – this suggests that the Y-axis of Figure 5C was a log10 scale – please clarify whether it is log10 or linear.

18. Line 359-365. The loss of permeability using these assays is not well described. Are these well-established assays for permeability? I would suggest adding references for these.

Response: Special thanks for your positive and constructive comments. It is important to add the detailed description of these assays, which will increase the credibility of the conclusion. An indirect test for outer membrane permeability is susceptibility to SDS and Cm, as outer membrane exclusion is an important component of protection against these compounds [1-4]. We have made the changes in the paper accordingly and added the references.

19. Line 363-5. Is there any information on the predicted pore size of OmpC and OmpF?

Response: Special thanks for your positive and constructive comments. The information of OmpC and OmpF was limited in the article [4, 5]. OmpC channels are more restrictive than OmpF channels. Bacteria containing only OmpC are more resistant to toxic hydrophilic agents than those containing only OmpF while the transcription of ompF increased when suffering glucose limitation which improved permeability for substrates at low concentrations [1]. We have added the information on the the predicted pore size of OmpC and OmpF in the paper accordingly and added the references.

20. Line 400-1. It seems that the promoters are more important than the genes for regulation of prodigiosin production.

Response: Thank you for your positive and constructive comments. Complementation of RpoS from different strains with the native promoter of rpoS from strain 1912768R restored pigmentation and it seems that the promoters are more important than the genes for regulation of prodigiosin production. We have made the changes in the paper accordingly.

Response: Special thanks for Reviewer#1 of the positive and constructive comments. Line 410-414, the results of transcriptional level of luxS in ΔrpoS mutant compared to the WT strain 1912768R have been described in S3 Fig; Line 442-445, the results is incomplete and still need to be studied further, so we have removed the phrase that refers to these data.

22. Figure 1C. The difference between R and WR is not clear in this image. A better picture would be helpful.

23. Figure 1A/B. Rename the Y-axis (left side) to “Surviving CFU/ml” or “Surviving Bacteria (CFU/ml)”

Response: Special thanks for your positive and constructive comments. The difference of the color between 1912768R and 1912768WR is difficult to distinguish and we have changed the picture to make them easier to distinguish. We have renamed Y-axis (left side) to “Surviving CFU/ml”.

24. not clear whether some of the Y-axes are log scale or linear. Like figure Fig 5C, 7C,

Response: Thank you for the correction. We are sorry that we used the wrong expression and this caused the misunderstanding. We have changed “increased by several orders of magnitude” to “increased by several times” in the description of Fig 5C. Fig 7C is the relative transcription of corresponding genes in ΔrpoS compared to WT and the Y-axe is linear. We have revised the paper accordingly.

Grammatical issues and writing suggestions:

Response: Special thanks for your careful and patient review. We have replaced “Exhibited” with “exhibiting”, added “was” after (named 1912768WR), and cut the “in the synthesis of the red pigmented secondary metabolite”.

2. Line 29. Replace “disclosed that RpoS was pleiotropic” with “had pleiotropic effects”

Response: Thank you for your careful and patient review. We have replaced “disclosed that RpoS was pleiotropic” with “had pleiotropic effects” which is more accurate.

3. Line 32. Replace colon with period.

Response: Thank you for the correction. However, we could not find the word “colon” in Line 32. Once again, thank you very much for your comments and suggestions.

4. Line 35. add “be” after “might” – also consider that the pig promoter rather than the pig cluster is important.

Response: Thank you for the positive comments and suggestions. We have added “be”after “might” and revised the paper as recommended.

5. Line 41. cut “with RpoS involving in it”

Response: We have cut “with RpoS involving in it” which is not necessary. Thanks for the the positive comments and suggestions.

6. Line Lines 50-55 – giant sentence – please fix.

Response: Thanks for the positive comments and suggestion. We have fixed the sentence and revised the paper correspondingly.

7. Line 57. Change “secret” to “secrete”

Response: It should be “secrete” and we have replaced “secret” with “secrete”. Thanks for the correction.

8. Line 70. Cut “simple structured” – it is unnecessary.

Response: Thanks for the positive comments and suggestion. We have cut the “simple structured”, which is not necessary.

9. Lines 74-76. Please fix this sentence.

Response: Thank reviewer#1 for the positive comments and suggestion. We have fixed the sentence and revised the paper correspondingly.

10. Line 78. Cut “Except for these studies”

Response: Thanks for the positive comments and suggestions. We have cut “Except for these studies” and revised the paper correspondingly.

11. Line 86. Replace “these” with “this”

Response: It should be “this”, and we have replaced “these” with “this”. Thanks for the correction.

12. Line 88. Spelling of regulatory.

Response: It should be “regulatory”. We have replaced “regularoty” with “regulatory” and checked the spelling of the whole manuscript. Thanks for the careful and patient review of the paper.

13. Line 139. Spelling of queried.

Response: Thanks for the correction. We have revised the word accordingly.

14. Line 146. Spelling of S. marcescens.

Response: It should be “S. marcescens”. We have checked the spelling of the whole manuscript. Thanks for the careful and patient review of the paper.

15. Line 165. Change “were” to “was”

Response: It should be “was” and we have changed “were” to “was”. Thanks for the correction.

16. Line 181-2. Remove “detection”, and add “of Slater et al.” after “method”

Response: Thanks for the positive comments and suggestions. We have removed detection and added “of Slater et al.” after “method”. This makes the sentence more concise and easier to understand.

17. Lines 193-4. Please fix the sentence.

Response: Thank you for the corrections. We have fixed the giant sentence to make it more concise and easier to understand, and we have revised in the paper accordingly.

18. Line 212. Change “was” to “were”

Response: It should be “were” and we have replaced “was” with “were”. Thank reviewer#1 for the correction.

19. Please change “at almost the 16th day” to “sound day 16”.

Response: Thanks for the positive comments and suggestions. We have changed “at almost the 16th day” to “sound day 16”.

20. Lines 283-287. Many issues. Add period after type I. Change “besides” to “since or because”, replace “and” on line 284 with a coma. “regulating to regulate”. change “be the reason causing the loss” to “underly / lead to the observed”. Line 286 add “were” after “that”. Add “for strains WR and W respectively” after “3”.

Response: Thanks for the corrections. We have replaced “besides” with “since”. We have replaced “and” on line 284 with a coma. We have changed “regulating” to “regulate”. We have changed “be the reason causing the loss” to “lead to the observed”. We have added “were” after “that” and added “for strains WR and W respectively” after “3”.

21. Line 323. Change “impacts” to “effects”

Response: Thanks for the correction. It should be “effects” and we have changed “impacts” to “effects” which is more concise.

22. Line 342. Change “resulting” with “which is consistent with”

Response: Thank you for the positive suggestion. We have changed “resulting” with “which is consistent with”.

23. Line 344-5. Change “resistant” to “resistance” and cut “ability”

Response: It should be “resistance” and we have changed “resistant” to “resistance”; moreover, we have cut “ability”. Thanks for the carefully review of our manuscript which is very helpful.

24. Line 366 and 371. Spelling of “membrane permeability” and “medium”

Response: We have corrected the spelling mistake of the word “membrane permeability” and “medium” and thanks for the corrections.

25. Line 373. Space after “colony.”

Response: Special thanks for your careful and patient review of the manuscript. We are really sorry that we made many spelling mistakes in this paper and this would cause the misunderstanding. We have revised the mistakes correspondingly and checked the whole paper carefully.

26. Line 388 and 392. Spelling of “production” and “species”

Response: Thanks for the correction. We have corrected the spelling of the words “production” and “species”.

Response: Special thanks for your careful and patient review. We have checked Lines 413-420 carefully and revived as recommended accordingly. We have added “be” after “might” and “may”. It should be RpoS and we have changed “rpoS” to “RpoS”. We have changed “researches are” to “research is”. We have correct the spelling of the word “strains”.

28. Line 443. Replace “an” with “the”

Response: We have changed “an” to “the” and thanks again for the correction.

29. Line 467-8. Cut “in our study”

Response: We have cut the words “in our study” and thanks for the corrections.

30. Figure 4B. Y-axis – should be “pigA” rather than PigA.

Response: Special thanks for your positive and constructive comments. It should be pigA, and we have changed “PigA” rather than pigA.

Replies to Reviewer#2:

1. The data regarding rpoS mutations and their effects on stress resistance, growth, and membrane permeability are not very surprising, as RpoS is a well-known stress-response regulator in the Enterobacteriaceae. The role of RpoS in the stationary-phase-inducible, general-stress-responses of Escherichia coli and Salmonella enterica serovar Typhimurium have been well documented in recent decades, and the authors are missing some important references in this research field.

Response: We thank reviewer#2 for their positive and constructive comments. The stationary phase sigma factor RpoS is the master regulator of the general stress response (GSR) in E. coli [6]. RpoS is known to play an important role in adaptation of bacteria to general stresses such as carbon starvation, high temperature and oxidative stress [7-9]. However, the regulatory role of RpoS in environmental stress is diverse in different strains [10-12]. In E. coli, RpoS is positively related to the oxidative resistance, while in Borrelia burgdorferi, RpoS is negatively related to oxidative resistance [10, 13, 14]. In Burkholderia pseudomallei and Pseudomonas aeruginosa, role of RpoS in regulating resistance of osmotic stress is also different [10, 15, 16]. In different E. coli strains, the capability of resistance to the same stress is different in different rpoS mutant strains [17]. Therefore, although RpoS is a well-known stress-response regulator, it seems that the regulatory role of RpoS in stress resistance is not all the same in different species. At present, the regulatory role of RpoS in stress resistance in S. marcescens has not been reported and the specific role of RpoS in S. marcescens is still unclear. So, it seems that the work is not without merit. Actually, we do have found some interesting points in this study.

The role of RpoS in the stationary-phase-inducible, general-stress-responses of E. coli and S. enterica serovar Typhimurium have been well documented in recent decades and we have added the corresponding references to illustrate the research progress of RpoS in GSR of Escherichia coli and Salmonella enterica serovar Typhimurium. We have revised the “Introduction” and “Results” of the paper accordingly. Thanks again for the positive and constructive comments.

2. However, the data does provide evidence that prodigiosin is RpoS-regulated in Serratia marcescens. The other data presented in this manuscript provide evidence of an extensive RpoS-regulon in S. marcescens, but how this is relevant to prodigiosin production is less clear.

Response: Special thanks to reviewers for their positive comments on the conclusions of the article. The regulatory genes of prodigiosin production seem to be pleiotropic. The LysR-type transcriptional regulator, MetR, is a positively regulator in regulation of prodigiosin biosynthesis and it is also pleiotropic in regulation of methionine biosynthesis, cell motility, H2O2 tolerance, heat tolerance, and exopolysaccharide synthesis in Serratia marcescens [18]; An IgaA/UmoB family protein from Serratia marcescens is a positive regulator in prodigiosin regulation and it also regulates motility, capsular polysaccharide biosynthesis, and serratamolide production [19]; The positive regulator of prodigiosin production, RbsR, is a pleiotropic regulator of motility, virulence, siderophore and antibiotic production, gas vesicle morphogenesis and flotation in Serratia sp. ATCC 39006. Therefore, it seems that regulation of prodigiosin production is relevant to other phenotypes. However, the relationship between prodigiosin production and other phenotypes has not been reported. In this study, we do not have the direct evidences to prove the specific relationship between prodigiosin production and other diverse phenotypes, but we have proved that prodigiosin production and other phenotypes are regulated by RpoS and it seems that the extensive RpoS-regulon is relevant to prodigiosin production. Thanks again for your positive and constructive comments and we will continue to explore their specific relationship in future researches.

3. Although the authors show that RpoS-regulated functions are involved in various types of stress resistance and in growth rate, evidence that prodigiosin production is somehow directly connected to the SPANC balance in Serratia marcescens is again unclear. Is a potential connection between prodigiosin and other RpoS-regulated functions being inferred? Does less prodigiosin biosynthesis result in less stress resistance and greater nutritional competence? Perhaps the manuscript should focus on the RpoS regulon of Serratia marcescens instead of just focusing on prodigiosin production. In other words, a different manuscript title might solve some of these issues. Overall, it seems that the study of prodigiosin-deficient mutants led to the discovery of an RpoS regulon in this organism.

Response：Thank reviewer#2 for the positive and constructive comments. The physiological role of bacterial prodigiosin in vivo remains undefined [20]. A number of reports have suggested potential functions that may provide an advantage in resistance to other strains (biotic stress) and challenging natural environment (abiotic stress) [21, 22]. In this study, the ΔrpoS strain was unable to produce prodigiosin and the resistance of ΔrpoS strain to different stresses decreased compared to the pigment WT strain at the same time; in addition, the ΔrpoS strain outcompeted the pigment WT strain. Therefore, this results indirectly proved that less prodigiosin biosynthesis results in less stress resistance and greater nutritional competence. Not only RpoS but also the prodigiosin is relevant to the SPANC balance. However, the poor English style and grammar of this manuscript makes some sections of the manuscript very difficult to read and the manuscript title is not appropriate. It is the study of prodigiosin-deficient mutants leading to the discovery of an RpoS regulon in this organism.

We have revised the whole manuscript carefully and changed the title with “RpoS is a pleiotropic regulator of motility, biofilm formation, exoenzymes, siderophore and prodigiosin production, and trade-off during prolonged stationary phase in Serratia marcescens”.

4. Furthermore, the authors begin to discuss possible differences in the RpoS regulation of pig loci (prodigiosin biosynthesis operons) in different S. marcescens strains. This is an interesting point of discussion, but is not explained enough in the manuscript. An additional figure illustrating the potential differences in pig regulation could be helpful. In fact, any potential differences between the RpoS-regulated functions of this organism (1912768R) and those of other S. marcescens strains, or closely related species, should be emphasized more in the body of the manuscript.

Response: Special thanks for your your positive and constructive comments. We have added additional figure to illustrate the potential difference in pig regulation (Fig 8). As the regulatory pathway of RpoS in prodigiosin production is unclear, it is hard to fully clarify the reason why the regulatory role of RpoS is different in Serratia marcescens 1912768R and strain Serratia sp. ATCC 39006. We have emphasized the potential differences between the RpoS-regulated functions of strain 1912768R and those of ATCC 39006 and revised the paper accordingly.

5. The experimental strategies and methods appear to be scientifically sound, and there was sufficient statistical analysis of the data. But again, the authors’ findings with S. marcescens were somewhat predictable based on what is already known about the RpoS regulons of closely related species.

Response: Special thanks to reviewers for their positive comments on the experimental strategies and methods of the article. RpoS is a master regulator of different phenotypes and the RpoS regulons have been studied in many species due to the important role in regulation of exoenzymes secretion, stress resistance and secondary metabolites biosynthesis.

In Vibrio anguillarum, RpoS positively regulates the secretion of extracellular lipase and proteinase, and hemolysis ability [14]; on the contrary, secretion of extracellular lipase and proteinase increased in rpoS mutant strain of Pseudomonas chlororaphis PA23 [23, 24]. In Borrelia burgdorferi and Vibrio cholerae, the secretion of chitinase is dependent on RpoS, while in S. marcescens 1912768R, secretion of chitinase increased in rpoS mutant strains. In addition, the regulatory role of RpoS in environmental stress is diverse in different strains (please see the answer to question 1). In Pseudomonas species, the regulatory effect of RpoS on secondary metabolites is diverse or even opposite in some species [25]; it was until we sequenced and deleted rpoS in S. marcescens 1912768R that we prove RpoS is a positive regulator in prodigiosin biosynthesis and its regulatory role in prodigiosin production is opposite to that in Serratia sp. ATCC 39006 [26]. Therefore, the regulatory role of RpoS is diverse in different strains. Although RpoS have been proved to be a regulator in exoenzymes secretion and stress resistance, the specific role of RpoS in regulation of exoenzymes secretion and stress resistance is still unknown in S. marcescens. Some results of this paper may be predictable, but we do have some new findings reported. We tried our best to improve the manuscript and make revisions to make the points clearer. Once again, thank you very much for your comments and suggestions.

6. There are extensive English language issues throughout the manuscript. One result of these language problems is that the actual experimental data is not always described sufficiently in a clear manner. Some important experimental results are only described in a couple of sentences, and their impact is lost in the overall body of the manuscript. There are also several typographic errors throughout the manuscript, a few examples of which are listed below:

Line 86: “theses” should be “these”

Line 366: “menbrane” should read “membrane”

Line 388: “produciton” should read “production”

Line 392: “spicies” should be “species”

Response: Special thanks for your careful and patient review. The results of the manuscript have been revised and we have used editing service of American Journal Experts (https://www.aje.cn/) to polish the English language of our manuscript. It should be “these” and we have replaced “theses” with “these”. We have replaced “menbrane” with “membrane”. The wrong word “produciton” have been replaced with “production”. It should be “species” and we have removed the wrong word “spicies”.

We tried our best to improve the manuscript and make revisions following the reviewers’ comments in the manuscript. These changes will not influence the content or framework of the paper. We earnestly appreciate the the editor’s and reviewers’ thorough work, and hope that the revision will meet with their approval.

Once again, thank you very much for your comments and suggestions.

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PLoS One. doi: 10.1371/journal.pone.0232549.r003

Decision Letter 1

Marie-Joelle Virolle

17 Apr 2020

RpoS is a pleiotropic regulator of motility, biofilm formation, exoenzymes, siderophore and prodigiosin production, and trade-off during prolonged stationary phase in Serratia marcescens

PONE-D-20-03043R1

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PLoS One. doi: 10.1371/journal.pone.0232549.r004

Acceptance letter

Marie-Joelle Virolle

8 May 2020

PONE-D-20-03043R1

RpoS is a pleiotropic regulator of motility, biofilm formation, exoenzymes, siderophore and prodigiosin production, and trade-off during prolonged stationary phase in Serratia marcescens

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Primers used in this study.

(PDF)

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S2 Table. General genome features of S. marcescens strains 1912768R, 1912768WR and 1912768W.

(PDF)

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S1 Fig. Circular diagrams of S. marcescens 1912768R genome.

(TIF)

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S2 Fig. Comparison of pig cluster from S. marcescens 1912768R and Serratia sp. ATCC 39006.

(TIF)

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S3 Fig. Relative transcription of gene rpoS.

Relative transcription of gene rpoS from WT strain, ΔrpoS and rpoS complemented strain at 6 h (a) and 16 h (b).

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Data Availability Statement

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PERMALINK

RpoS is a pleiotropic regulator of motility, biofilm formation, exoenzymes, siderophore and prodigiosin production, and trade-off during prolonged stationary phase in Serratia marcescens

Han Qin

Ying Liu

Xiyue Cao

Jia Jiang

Weishao Lian

Dairong Qiao

Hui Xu

Yi Cao

Roles

Abstract

Introduction

Materials and methods

Bacterial strains, plasmids and growth conditions

Table 1. Strains and plasmids used in this study.

Genome sequencing and annotation

Genetic manipulations and plasmid construction

Sequence alignment

Detection of extracellular enzymes, hemolysin and biofilm formation

Motility assays

Stress and competition assays

RNA purification and qRT-PCR

Statistical analysis

Results

Selective advantage for nonpigmented mutants during prolonged stationary phase

Fig 1. Prolonged incubation of S. marcescens 1912768R and isolation of mutants with prodigiosin biosynthesis defects.

RpoS was screened as a candidate regulator of prodigiosin production

Fig 2. Distribution of SNPs and InDels in S. marcescens strain 1912768WR and 1912768W compared to WT.

Fig 3. Sequence alignment of RpoS.

RpoS positively regulates the production of prodigiosin in S. marcescens 1912768R

Fig 4. RpoS is a positive regulator of prodigiosin production, and the function of RpoS is conserved in S. marcescens 1912768R.

The disruption of rpoS has pleiotropic effects on S. marcescens physiology

Fig 5. The disruption of rpoS has diverse effects on the physiology and motility of 1912768R.

The ΔrpoS mutant displayed impaired resistance to stresses but higher competence than its isogenic WT strain

Fig 6. Stress resistance assays of S.marcescens 1912768R and the ΔrpoS mutant.

Fig 7. Effect of rpoS on membrane permeability and relative fitness of S. marcescens 1912768R and the ΔrpoS mutant.

Discussion

Fig 8. Proposed model of prodigiosin production regulation in Serratia sp. ATCC 39006 and S. marcescens 1912768R by luxS and rpoS.

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Marie-Joelle Virolle

Roles

Author response to Decision Letter 0

Decision Letter 1

Marie-Joelle Virolle

Roles

Acceptance letter

Marie-Joelle Virolle

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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