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Mutation of gdpS gene induces a viable but non-culturable state in Staphylococcus epidermidis and changes in the global transcriptional profile

BMC Microbiology volume 22, Article number: 288 (2022) Cite this article

Abstract

Background

In the genome of staphylococci, only the gdpS gene encodes the conserved GGDEF domain, which is the characteristic of diguanylate cyclases. In our previous study, we have demonstrated that the gdpS gene can modulate biofilm formation by positively regulating the expression of ica operon in Staphylococcus epidermidis. Moreover, this regulation seems to be independent of the c-di-GMP signaling pathway and the protein-coding function of this gene. Therefore, the biological function of the gdpS gene remains to be further investigated.

Results

In the present study, it was observed that mutation of the gdpS gene induced S. epidermidis to enter into a presumed viable but nonculturable state (VBNC) after cryopreservation with glycerol. Similarly, when moved from liquid to solid culture medium, the gdpS mutant strain also exhibited a VBNC state. Compared with the wild-type strain, the gdpS mutant strain autolyzed more quickly during storage at 4℃, indicating its increased susceptibility to low temperature. Transcriptional profiling analysis showed that the gdpS mutation affected the transcription of 188 genes (92 genes were upregulated and 96 genes were downregulated). Specifically, genes responsible for glycerol metabolism were most markedly upregulated and most of the altered genes in the mutant strain are those involved in nitrogen metabolism. In addition, the most significantly downregulated genes included the betB gene, whose product catalyzes the synthesis of glycine betaine and confers tolerance to cold.

Conclusion

The preliminary results suggest that the gdpS gene may participate in VBNC formation of S. epidermidis in face of adverse environmental factors, which is probably achieved by regulating expression of energy metabolism genes. Besides, the gdpS gene is critical for S. epidermidis to survive low temperature, and the underlying mechanism may be partly explained by its influence on expression of betB gene.

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Introduction

The GGDEF domain is characteristic of diguanylate cyclases (DGCs) that are responsible for synthesis of bis-(3′, 5′)-cyclic dimeric GMP (c-di-GMP). The domain is named after its conserved signature motif Gly-Gly-Asp-Glu-Phe. C-di-GMP is a ubiquitous and important second messenger in bacteria [1, 2]. It has been implicated in a growing number of physiological processes, including biofilm formation, motility, virulence, cell cycle and differentiation [2, 3]. Generally, low levels of the intracellular second messenger are related to planktonic growth, whereas increased concentrations favor surface attachment and biofilm formation [3, 4].

Intracellular pools of c-di-GMP fluctuate dynamically in response to internal or external stimuli. This is achieved through the antagonistic activities of DGCs and c-di-GMP-specific phosphodiesterases (PDEs). Two classes of structurally and mechanistically distinct PDEs, which typically contain EAL and HD-GYP domains respectively, have been described. The EAL domain catalyzes the cleavage of c-di-GMP to generate the linear molecule 5′-phosphoguanylyl-(3′-5′)-guanosine (pGpG). The HD-GYP domain is responsible for degradation of c-di-GMP into two molecules of GMP [3, 5, 6]. It has been demonstrated that c-di-GMP exerts its regulatory function by binding to a wide variety of effectors including kinases or phosphorylases, transcription factors, PilZ domain proteins, degenerate DGCs or PDEs, and riboswitches [7].

Biofilm formation is a key virulence determinant for many microorganisms that cause chronic and device-associated infections [8, 9]. The bacteria enclosed within the biofilm are recalcitrant to the hosts’ immune response and antimicrobial agent clearance, so that medical interventions such as surgery are often required to treat infected tissues and/or remove indwelling devices[10, 11]. In a number of Staphylococcus epidermidis and Staphylococcus aureus strains, the major component of biofilm matrix is the exopolysaccharide termed polysaccharide intercellular adhesin (PIA) or polymeric N-acetyl-glucosamine (PNAG). The synthesis of PIA/PNAG is encoded by the ica operon [12, 13].

Only one gene encoding the GGDEF domain protein, designated as gdpS, is present in sequenced staphylococcal genomes. Moreover, neither genes encoding EAL or HD-GYP domain proteins nor those encoding PilZ domain proteins are found in their genomes [14]. Holland et al. and we have investigated the role of the gdpS gene in biofilm formation in S. epidermidis [15]. It was found that gdpS can promote biofilm formation by elevating ica operon transcription. When exploring the mechanism by which gdpS regulates ica transcription, unexpectedly, the regulation was found to be independent of c-di-GMP synthesis. Mutagenesis of the GGDEF domain did not abolish the capacity of gdpS to restore the biofilm defect of the gdpS mutant. Furthermore, heterologous DGC expressed in trans failed to complement the gdpS mutant, and recombinant GdpS protein exhibited no DGC activity in vitro [15, 16]. These observations indicated that the gdpS gene might represent remnants of the c-di-GMP signaling pathway.

Indeed, this speculation is also supported by the mechanistic insight into the non-coding role of gdpS on spa gene expression in S. aureus, as reported by Shang [14]. They found that the RNA transcript of gdpS can directly bind to the 5’ UTR of sarS mRNA, leading to stabilization of the latter [17]. The global regulatory locus sarS is a sarA homolog and can activate transcription of spa [18]. Inspired by these findings, through site-mutagenesis of start codon and by complementation experiments, we have suggested that gdpS in S. epidermidis may also modulate biofilm formation at the post-transcriptional level [16]. However, the protein product of the gdpS gene has also been detected in S. aureus and S. epidermidis through Western blot analysis [16, 17]. Therefore, it is worthwhile to explore the biological function of the gdpS-encoding protein. In the present study, we sought to investigate the physiological role of gdpS by comparing phenotypic and transcriptional profiling variations between S. epidermidis strain 35984 M and its gdpS mutant derivative.

Results and discussion

Mutation of gdpS induces S. epidermidis to enter into a VBNC state upon low temperature and osmotic pressure

When we reactivated the gdpS mutant strain that has been preserved at -80 Â°C with glycerol as cryoprotectant, obvious growth retardation was observed in comparison with the wild-type strain. As shown in Fig. 1A, when frozen cultures were inoculated into TSB medium at a dilution of 1:100 and incubated at 37 Â°C with an agitation of 200 rpm for more than 16 h, subcultures of the wild-type and complementation strains exhibited significant turbidity, whereas those of the gdpS mutant and the empty vector control strains grew poorly. To determine whether this difference in reactivation is caused by the loss of cell viability after freezing and during frozen storage, both thawed cultures of the gdpS mutant strain and its parent strain were spotted onto the TSB agar and Columbia blood agar plates respectively, and cultivated for 24 h. As illustrated in Fig. 1B, when grown on the TSB agar plate, the gdpS mutant indeed formed significantly fewer colonies than the wild-type strain. However, when grown on the Columbia blood agar plate, there were no obvious differences in colony formation between the gdpS mutant strain and the wild-type strain. The same is true for the complementation strain and the empty vector control strain (Fig. 1C). Moreover, no differences in growth curves were detected between the gdpS mutant strain and the wild-type strain after they were fully reactivated.

Fig. 1
figure 1

Mutation of gdpS induces S. epidermidis to enter into a VBNC state after cryopreservation. The cryopreserved S. epidermidis strains were thawed, inoculated directly into liquid TSB medium (A) at a dilution of 1:100, and then incubated with agitation at 37 Â°C for over 16 h. Meanwhile, the thawed cultures and their corresponding dilutions were spotted onto TSB agar plate (B) and Columbia blood agar plate (C) respectively, and cultivated at 37 Â°C for 24 h. WT: the wild type strain, ΔgdpS: the gdpS mutant strain, C-gdpS: the gdpS mutant strain complemented with the native gdpS gene, C-pCN: the gdpS mutant strain complemented with the empty vector pCNcat. The experiment was repeated at least three times, and a representative figure is shown

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These results indicated that a sub-population of the gdpS mutant strain after frozen storage cannot be cultured in TSB medium although they are alive, which corresponds to the concept of viable but nonculturable (VBNC) cells. Since its first proposal in 1982, many bacterial species have been found to exist in a VBNC state. VBNC cells are characterized by the loss of culturability on routine agar, which hampers their detection by conventional plate count techniques. Although it is controversial that entering VNBC state may be a general strategy adopted by bacteria to survive unfavorable conditions, this leads to an underestimation of total viable cells in environmental and clinical samples, and thus poses a threat to public health [19]. Exposure to various stresses can induce the VBNC state. One of the most frequent inducing factors is low temperature [20, 21], the condition that triggered the presumed VBNC state of the gdpS mutant strain in the present study. In addition, previous studies have shown that bacteria in the VBNC state could be resuscitated by rich medium. Our study indeed observed that culturability of the gdpS mutant after cryopreservation could be recovered by the nutrient-rich Columbia blood agar. Therefore, we speculate that mutation of gdpS can induce S. epidermidis to enter into a presumed VBNC state upon low temperature challenge.

More intriguingly, we were surprised to find that when the gdpS mutant was cultured in liquid TSB medium until the mid-exponential phase, and then serially diluted and spotted onto TSB agar, its culturability was significantly reduced compared to the wild-type strain (Fig. 2). One of the key differences between the liquid and solid culture media used here is a change of osmotic pressure. It is thus rational to deduce that osmotic pressure may also trigger the VBNC state of the gdpS mutant.

Fig. 2
figure 2

Mutation of gdpS induces S. epidermidis to enter into a VBNC state under osmotic pressure. All the cryopreserved S. epidermidis strains were resuscitated on Columbia blood agar plate and single colonies were inoculated into liquid TSB medium for overnight culture. The overnight cultures were subcultured 1:100 and grown to the exponential phase with identical OD600 values in fresh TSB medium, and then serially diluted (1:10), spotted onto TSB agar plate (A) and Columbia blood agar plate (B) for cultivation, respectively. The experiment was repeated at least three times, and a representative figure is shown

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Mutation of gdpS increases the susceptibility of S. epidermidis to low temperature

Subsequently, to investigate whether the gdpS mutation affects the survival of S. epidermidis at low temperature, bacterial cultures in mid-exponential phase were kept at 4℃ for several days, and the optical density at 600 nm was monitored each day after thorough shaking. As illustrated in Fig. 3, compared with the wild-type strain, OD600 values of the gdpS mutant declined sharply during storage at 4℃, indicating that massive cell death and autolysis occurred in the bacterial cultures. The complementation strain had almost restored survival to the wild-type level at low temperature, while the empty vector control strain displayed a phenotype similar to that of the gdpS mutant. These findings suggest that gdpS mutation increased the susceptibility of S. epidermidis to low temperature.

Fig. 3
figure 3

Susceptibility of the ΔgdpS strain to low temperature. All the S. epidermidis strains were inoculated in fresh TSB medium and grown to logarithmic phase (4 h; OD600 = 2) at 37 Â°C. The cultures were then placed at 4 Â°C, and the turbidity was measured every day at 600 nm. The experiment was repeated at least three times, and representative curves are shown

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Transcriptome changes induced by loss of gdpS function

To assess the global impact of losing gdpS function on S. epidermidis physiology, RNA sequencing was performed to compare the gene expression profile between the gdpS mutant and the wild-type strain. As a result, a total of 188 DEGs (differentially expressed genes) with fold change >  = 1.5 was found between the gdpS mutant and its parent strain. Of these, 92 genes were significantly upregulated, and 96 genes were downregulated in the gdpS mutant relative to the wild-type strain (Fig. 4). To evaluate the reliability of the RNA-seq data, five randomly selected DEGs were analyzed by qRT-PCR, using the gyrB gene as reference. The expression level of five DEGs obtained by qRT-PCR were consistent with the results from RNA-seq (Fig. 5), indicating that the data generated by RNA-seq could be used to further investigate the expression of specific genes.

Fig. 4
figure 4

A volcano plot revealing the differences in gene expression between the ΔgdpS strain and the wild-type strain. Genes with |log2FC|> = 0.58496 and adjusted p-value < 0.05 were considered differentially expressed. Each point represents one gene: dark dots are non-DEGs, red and blue dots are upregulated and downregulated genes, respectively

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Fig. 5
figure 5

Validation results of RNA-seq profiles by qPCR. Data are means ± SEM of three independent experiments with three replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ΔgdpS, vs. wild-type (WT)

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In order to investigate the functions of DEGs, they were assigned by the GO (gene ontology) databases. A total of 38 DEGs were successfully annotated with three main GO terms, of which 32 DEGs were categorized into the ‘biological process’, 24 DEGs into the ‘cellular component’, and 27 DEGs into the ‘molecular function’ (Fig. 6). Among biological processes, the dominant GO terms were ‘cellular process’, ‘metabolic process’ and ‘single-organism process’. For cellular component, ‘cell’, ‘cell part’ and ‘membrane’ were major GO terms. Under the category of molecular function, ‘catalytic activity’ and ‘transporter activity’ were the dominant terms.

Fig. 6
figure 6

Gene ontology classification of differentially expressed genes (DEGs). The x-axis is the name of category and the y-axis is the number of genes. Red and green denote the upregulated and downregulated genes, respectively

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Metabolic pathways are differentially regulated in the gdpS mutant strain

The most highly upregulated genes in the gdpS mutant are involved in glycerol metabolism, including glpF, glpK and glpD, with fold changes of 9.9, 4.5 and 25, respectively (Table 1). The glpF gene encodes a glycerol facilitator belonging to the aquaporin family of passive transporters. The glpF gene usually forms an operon together with the glpK gene, which encodes a glycerol kinase phosphorylating glycerol to glycerol-3-phosphate (G3P). The glpD gene encodes a membrane-bound, aerobic G3P dehydrogenase. This enzyme is a flavoprotein that catalyzes the oxidation of G3P to dihydroxyacetone-phosphate (DHAP). The oxidation is coupled to the reduction of ubiquinone or menaquinone of the respiratory electron transport chain. DHAP is subsequently catabolized through the Embden-Meyerhof-Parnas (EMP) pathway. In the presence of the above three genes, glycerol can be aerobically utilized by bacteria as a carbon source. The uptake of glycerol into bacterial cells is achieved by facilitated diffusion mediated by GlpF. It is converted by GlpK to G-3-P, which is then dehydrogenated by GlpD to yield DHAP [22,23,24]. In addition to participating in glycerol metabolism, these genes have also been implicated in formation of persister cell in Escherichia coli, Pseudomonas aeruginosa and S. aureus [25,26,27]. The persister cells have been defined along with VBNC cells as two dormancy strategies that cope with adverse environments. Previous studies have shown that VBNC cells are not only present with persister cells at much higher numbers in exponential phase but also withstand antibiotic treatment alongside persister cells. Therefore, the two survival modes are viewed as a continuum between actively growing and dead cells, with VBNC cells being in a deeper dormancy depth than persister cells [28, 29]. It is plausible that gdpS mutation might cause S. epidermidis to enter a VBNC state by increasing expression of genes involved in glycerol metabolism. This warrants further investigation.

Table 1 Main genes affected by gdpS mutation in S. epidermidis (RNA-seq)
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In Borrelia burgdorferi, the causative agent of Lyme disease, expression of the glpFKD operon is essential for fitness of the spirochetes in ticks, which can produce glycerol as a cryoprotective molecule. It has been shown that c-di-GMP can positively regulate the glpFKD operon via the c-di-GMP effector PlzA. Moreover, in the absence of c-di-GMP, PlzA also can function as a negative regulator of glpFKD expression [30,31,32]. In the present study, transcriptomic data indicates gdpS negatively controls expression of the glpFKD gene cluster in S. epidermidis. In addition, inactivation of gdpS in S. aureus results in upregulation of glpT gene encoding a G3P transporter, as revealed by microarray data [14]. These findings imply that while gdpS involvement in c-di-GMP synthesis in Staphylococci is under debate, the regulatory role of the GGDEF-containing gene in glycerol metabolism may be highly conserved.

The KEGG pathway enrichment analysis revealed that the most affected genes in the gdpS mutant are those involved in nitrogen metabolism (Fig. 7, Supplementary File 1), including narT gene and narABC, narGHJI and nirBD operons, with almost 2- to threefold increased expression (Table 1). The narT gene encodes a transport protein that is required for nitrate uptake and nitrite export [33]. The narGHJI operon encodes a membrane-bound respiratory nitrate reductase that is responsible for generation of nitrite [34]. Under anaerobic conditions, nitrite can be further reduced to ammonium by the nirBD encoded cytosolic NADH-dependent nitrite reductase [35]. In contrast to nitrate reduction, NirBD-catalyzed nitrite dissimilation is not coupled to the formation of proton motive force and, hence, is not a respiratory pathway. It serves rather to detoxify the nitrite that accumulates in nitrate-consuming cells and as an electron sink to reproduce NAD+ [36, 37]. The three-cistron operon nreABC has been identified to encode an oxygen sensing two-component system NreB/NreC and a nitrate receptor NreA. When oxygen is depleted, autophosphorylation activity of the cytoplasmic histidine kinase NreB increases in the presence of nitrate and NreA. The response regulator NreC is subsequently phosphorylated by NreB and specifically binds to the promoters of the narT gene and of the narGHJI and nirBD operons to activate transcription [37,38,39]. Since previous studies have shown that both aerobic and anaerobic G3P dehydrogenase can transfer hydrogens from G3P to nitrate, leading to growth of E. coli and S. aureus on glycerol as the carbon source and nitrate as the hydrogen acceptor [40, 41]. We speculate the gdpS gene may negatively modulate the entire respiratory pathway consisting of glycerol catabolism and nitrate reduction in S. epidermidis.

Fig. 7
figure 7

Functional categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs using the Majorbio cloud platform (p < 0.01)

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It is noteworthy that the betB gene encoding betaine-aldehyde dehydrogenase was downregulated approximately twofold in the gdpS mutant. Under the catalysis of this gene product, betaine-aldehyde can be oxidized to glycine betaine (GB). GB has been widely studied as an excellent osmoprotectant in both the prokaryotic and eukaryotic world [42]. Recently, GB has also been reported to confer tolerance against low temperature in bacteria and plants by preventing cold-induced aggregation of cellular proteins and maintaining an optimum membrane fluidity [43,44,45]. In the present study, loss of gdpS function resulted in increased susceptibility of S. epidermidis to low temperature, which led us to speculate that gdpS may promote tolerance of S. epidermidis to low temperature through regulating the expression of betB gene responsible for glycine betaine biosynthesis.

Genes involved in pathogenesis are altered in the gdpS mutant strain

Apart from multi-organism process, pathogenesis is the significantly enriched GO term (Fig. 8, Supplementary File 2). Under this category, the ica operon involved in PIA biosynthesis and the psmβ operon encoding phenol-soluble modulins (PSMs) are included. It has been demonstrated by Holland et.al and our group that the gdpS gene promotes biofilm formation in an ica-dependent manner [15]. Transcriptional profiling analysis in this study discloses the ica operon as the most remarkably downregulated genes in the gdpS mutant. Also the beta subclass of PSMs has also been proposed to promote S. epidermidis biofilm mature and dissemination by mediating biofilm cluster detachment through its surfactant-like property [46]. In the present study, transcript levels of the three genes, psmβ1a, psmβ1b and psmβ3, which constitute part of the four-gene psmβ operon, increased approximately two-fold in the gdpS mutant. Therefore, the transcriptomic data imply that gdpS might enhance biofilm formation in S. epidermidis not only by elevating expression of ica operon but also repressing transcription of psmβ operon.

Fig. 8
figure 8

Functional categories and gene Ontology (GO) enrichment analysis of DEGs using the Majorbio cloud platform. Seventy-three terms were identified; the first 5 enrichment terms of upregulated and downregulated genes are shown based on the P values from low to high, respectively; p < 0.05

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Conclusion

This study explored more physiological roles of gdpS gene apart from enhancing biofilm formation in S. epidermidis. These include that it may participate in formation of presumed VBNC state under induction of cryopreservation and confer S. epidermidis tolerance against low temperature. Transcriptome experiments displayed that this may be attributed to its influence on expression of genes related to energy metabolism and synthesis of osmoprotectant.

Materials and methods

Bacterial strains and growth conditions

The gdpS deletion mutant strain (ΔgdpS) of S. epidermidis 35984 M (WT) and its complementation strain C-gdpS, vector control strain C-pCN (ΔgdpS complemented with the empty vector pCNcat) were constructed in our laboratory previously [16]. All the strains were maintained as glycerol (40%, v/v) stocks, which were prepared after growth to mid-exponential phase (approximately 4 h), and were frozen at − 80 Â°C. For liquid cultivation, overnight cultures of S. epidermidis from each single colony were inoculated respectively into TSB medium (BD Difco) at a dilution of 1:100 with a ratio of flask volume to medium volume of 5:1. The subcultures were grown to mid-exponential phase (approximately 4 h) under 200 rpm agitation at 37 Â°C. When necessary, chloramphenicol at a final concentration of 10 Î¼g/mL was added. For solid cultivation, an aliquot of 5 μL of glycerol stocks or serially diluted cultures was spotted onto the TSB agar plate or Columbia blood agar plate and incubated at 37 Â°C for about 24 h. The bacterial colonies on the agar plates were photographed.

Susceptibility to low temperature

To detect the susceptibility of the gdpS mutant strain to low temperature, overnight cultures of S. epidermidis strains were diluted into fresh TSB medium and grown to logarithmic phase (4 h, OD600 = 2) at 37 Â°C. Bacterial cultures were kept at 4 Â°C for four days and the turbidity was measured at 600 nm each day.

RNA extraction

For transcriptome sequencing, all the S. epidermidis strains were grown to mid-exponential phase in 6 mL TSB medium under the conditions described above. Prior to RNA isolation, two volumes of RNAprotect Bacteria Reagent (Qiagen, cat#76,506) were added to one volume of bacterial culture to provide immediate stabilization of RNA. Thereafter, bacterial cells were pelleted by centrifugation at 5000 g for 10 min and vortexed with 0.9 mL Buffer RLT plus 1 mL Ziconia-silica beads (0.1 mm diameter). The bacterial sediments were then mechanically disrupted on a Mini-Beadbeater (Biospec) at maximum speed four times for 40 s with intermittent cooling on ice. The subsequent RNA extraction using RNeasey Mini kit (Qiagen, cat#74,104) were performed according to the manufacturer’s instruction. To eliminate any genomic DNA contamination, DNase digestion in solution and on-column DNase digestion were both carried out using RNase-Free DNase Set (Qiagen, cat#79,254) as recommended by the manufacturer during RNA extraction. The total RNA was eventually eluted from the column with 60 μL of RNase-free water.

Transcriptome sequencing

For genome-wide RNA sequencing, total RNA of each sample was subsequently submitted to Majorbio Co., Ltd. (Majorbio, Shanghai, China). The quality of the total RNA was assessed using a 2100 Bioanalyzer (Agilent, USA) and its amount was quantified using an ND-2000 instrument (NanoDrop Technologies, USA). Afterward, ribosomal RNA was removed from the total RNA using the Ribo-Zero magnetic kit (Epicentre, USA) and the yielded mRNA was chemically fragmented to approximately 200-nt-long oligonucleotides using fragmentation buffer. The cDNA libraries were then generated from enriched mRNA samples using the Illumina TruSeq RNA sample prep kit as follows. Synthesis of double-stranded cDNA was performed using the SuperScript double-stranded cDNA synthesis kit (Invitrogen, USA) with random hexamer primers (Illumina, USA). Then, the synthesized cDNA was subjected to end repair, A-base addition and adapter ligation according to Illumina’s library construction protocol. The libraries were size-selected for cDNA target fragments of 200 bp on 2% low-range ultra-agarose followed by PCR amplification using Phusion DNA polymerase (NEB, USA) for 15 PCR cycles. After quantification by TBS-380, the paired-end RNA-seq library was sequenced with the Illumina HiSeq4000 platform (2 × 150-bp read length).

Bioinformatics analysis

The trimming and quality control of raw end-paired reads were performed using SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) with default parameters. Clean data from each sample were then aligned to the genome of S.epidermidis strain ATCC35984 (NCBI Reference Sequence: NC_002976.3) using Bowtie2 (http://www.bowtie-bio.sourceforge.net/bowtie2/manual). To estimate the expression level of each gene, FPKM (fragments per kilobase of transcript per million fragments) was calculated using RSME (http://deweylab.biostat.wisc.edu/rsem/). The differentially expressed genes (DEGs) were analyzed with EdgeR (http://www.bioconductor.org/packages/2.12/bioc/html/edgeR.html) by comparing the transcript abundance between the gdpS mutant and its parent strain. The DEGs were selected using the following filter criteria: an adjusted p value < 0.05 and fold change >  = 1.5 (|log2FC|> = 0.58496). The GO and KEGG pathway enrichment analysis for the DGEs were conducted by Goatools (https://github.com/tanghaibao/Goatools) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do) [47, 48].

Experimental validation of RNA-seq profiles by qPCR

To confirm the reliability of the transcriptome data, five DEGs namely icaA, glpA, sarZ, saeS and narT were chosen for qRT-PCR validation. The cDNA was synthesized from the same RNA samples used for RNA-seq by using the GoScriptâ„¢ Reverse Transcription Mix, Random Primers (Promega) according to the manufacturer’s instructions. The qPCR reactions were performed using the FastStart Essential DNA Green Master (Roche, USA) on a LightCycler® 96 instrument (Roche, USA). The PCR amplification program was set as follows: initial denaturation at 95 Â°C for 10 min, followed by 40 cycles of 95 Â°C for 15 s, 60 Â°C for 15 s. Melting curve analysis was monitored at the end of the PCR amplification by first heating to 95 Â°C for 10 s, cooling to 65 Â°C for 60 s, and then melting with continuous acquisition (5 readings/°C) of fluorescence signal until 97 Â°C. Each reaction was performed in triplicate, and the gyrB gene was employed as internal control for normalization in the assay. The relative expression levels of target genes were calculated using the 2−△△Ct method and compared with the results of RNA-seq analysis. All primer pairs for five selected genes and the gyrB gene were listed in Supplementary file 3.

Statistical analysis

All experiments were performed in triplicate or separately reproduced three times. Student’s t test on the VassarStats Web site was used to compare data between two groups. A p value of < 0.05 indicated that there were significant differences between groups.

Availability of data and materials

The raw data for the transcriptome sequencing of S. epidermidis 35984 M and the S. epidermidis ΔgdpS mutant have been deposited in the NCBI Sequence Read Archive (SRA) database under BioProject accession number PRJNA865168.

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Acknowledgements

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Funding

This work was supported by the Key Program of Educational Commission of Anhui Province (KJ2020A0602), the National Natural Science Foundation of China (No. 82072249, 81991532, 81871622), the Support Program for University Outstanding Youth Talent of Anhui Province (gxyq2019043), Open Research Fund Program of Key Laboratory of Medical Molecular Virology (MOE/NHC), and Fudan University (FDMV-2020005).

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Authors and Affiliations

  1. Department of Medical Microbiology and Immunology, Wannan Medical College, Wuhu, 241002, People’s Republic of China

    Tao Zhu

  2. Department of Pharmacy, Wannan Medical College, Wuhu, 241002, People’s Republic of China

    Wei Wang & Han Wang

  3. Department of Laboratory Medicine, the Second Affiliated Hospital of Nanjing Medical University, Nanjing, 210011, People’s Republic of China

    Yanfeng Zhao

  4. Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai, 200032, People’s Republic of China

    Di Qu & Yang Wu

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  1. Tao Zhu
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  2. Wei Wang
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  3. Han Wang
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  4. Yanfeng Zhao
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  5. Di Qu
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Contributions

T Zhu, Y Wu and D Qu were responsible for the conception and design of the study. W Wang and H Wang carried out the qRT-PCR experiments. T Zhu carried out the analysis and interpretation of data. T Zhu and Y Wu wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Di Qu or Yang Wu.

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Supplementary Information

Additional file 1: Table S1.

List of KEGG pathways that were enriched with differentially expressed genes (DEG) detected between the ΔgdpS strain and the wild-type strain.

Additional file 2: Table S2.

List of Gene Ontology (GO) terms that were enriched with differentially expressed genes (DEG) detected between the ΔgdpS strain and the wild-type strain.

Additional file 3: Table S3.

Primers used in this study.

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Zhu, T., Wang, W., Wang, H. et al. Mutation of gdpS gene induces a viable but non-culturable state in Staphylococcus epidermidis and changes in the global transcriptional profile. BMC Microbiol 22, 288 (2022). https://doi.org/10.1186/s12866-022-02708-6

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Keywords

  • Staphylococcus epidermidis
  • gdpS
  • Viable but nonculturable
  • Low temperature
  • Transcriptional profile

BMC Microbiology

ISSN: 1471-2180

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