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

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.

Keywords: Low temperature; Staphylococcus epidermidis; Transcriptional profile; Viable but nonculturable; gdpS.

Fig. 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 °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 °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

Fig. 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

Fig. 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 °C. The cultures were then placed at 4 °C, and the turbidity was measured every day at 600 nm. The experiment was repeated at least three times, and representative curves are shown

Fig. 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

Fig. 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)

Fig. 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

Fig. 7

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

Fig. 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