Temporal dynamics of stress response in Halomonas elongata to NaCl shock: physiological, metabolomic, and transcriptomic insights

Abstract

Background: The halophilic bacterium Halomonas elongata is an industrially important strain for ectoine production, with high value and intense research focus. While existing studies primarily delve into the adaptive mechanisms of this bacterium under fixed salt concentrations, there is a notable dearth of attention regarding its response to fluctuating saline environments. Consequently, the stress response of H. elongata to salt shock remains inadequately understood.

Results: This study investigated the stress response mechanism of H. elongata when exposed to NaCl shock at short- and long-time scales. Results showed that NaCl shock induced two major stresses, namely osmotic stress and oxidative stress. In response to the former, within the cell's tolerable range (1-8% NaCl shock), H. elongata urgently balanced the surging osmotic pressure by uptaking sodium and potassium ions and augmenting intracellular amino acid pools, particularly glutamate and glutamine. However, ectoine content started to increase until 20 min post-shock, rapidly becoming the dominant osmoprotectant, and reaching the maximum productivity (1450 ± 99 mg/L/h). Transcriptomic data also confirmed the delayed response in ectoine biosynthesis, and we speculate that this might be attributed to an intracellular energy crisis caused by NaCl shock. In response to oxidative stress, transcription factor cysB was significantly upregulated, positively regulating the sulfur metabolism and cysteine biosynthesis. Furthermore, the upregulation of the crucial peroxidase gene (HELO_RS18165) and the simultaneous enhancement of peroxidase (POD) and catalase (CAT) activities collectively constitute the antioxidant defense in H. elongata following shock. When exceeding the tolerance threshold of H. elongata (1-13% NaCl shock), the sustained compromised energy status, resulting from the pronounced inhibition of the respiratory chain and ATP synthase, may be a crucial factor leading to the stagnation of both cell growth and ectoine biosynthesis.

Conclusions: This study conducted a comprehensive analysis of H. elongata's stress response to NaCl shock at multiple scales. It extends the understanding of stress response of halophilic bacteria to NaCl shock and provides promising theoretical insights to guide future improvements in optimizing industrial ectoine production.

Keywords: Halomonas elongata; Ectoine; NaCl shock; Osmotic stress; Oxidative stress; Stress response.

Fig. 1

Changes in physiological parameters of H. elongata after NaCl shock. Biomass(a), the specific growth rate (µ) (b), oxygen uptake rate (c), the specific oxygen uptake rate (q_O2) (d), ectoine production(e), ectoine produced per biomass (p_ectoine) (f). The numbers inside the figure indicate the NaCl concentration in fermentation broth after shock

Fig. 2

Changes in intracellular sodium (a), potassium (b) ion and Na⁺/K⁺ ratio (c) levels after 8% NaCl shock. Where the “8% NaCl” represents the condition in fixed 8% NaCl. And the ROS levels (d), AEC ratio (e), and ATP contents (f) were also determined. The numbers (1, 2, 3, 4, and 5) on the x-axis in panels (e) and (f) represent different sampling time, which are 5 min before shock, 5, 10, 20, and 30 min after shock, respectively. Red lines indicate 8% NaCl shock group, green lines indicate 13% NaCl shock group. ** represents p < 0.01, *** represents p < 0.0005, **** represents p < 0.0001

Fig. 3

Heatmap for metabolite changes in H. elongata after 8% and 13% NaCl shock, and color-scaled with the contents of metabolites. The number (1, 2, 3, 4, and 5) in the heatmap indicates different sampling time, which are 5 min before shock, 5, 10, 20, and 30 min after shock, respectively

Fig. 4

Scatter plot of DEGs (a). Venn diagram of DEGs (b). And KEGG pathway enrichment analysis. (c), (d), (e), and (f) represent the KEGG enrichment analysis results for the Before vs 8 A, Before vs 8B, Before vs 13 A, and Before vs 13B comparison groups, respectively

Fig. 5

The expression level of four peroxiredoxin coding genes (a). The Log₂FC of key genes in sulfate metabolism, orange and blue rectangles with number represent gene expression changes in the Before vs Shock8B and Before vs Shock13B (b). Heatmap of the expression levels of upregulated DEGs in the amino acid biosynthesis pathways during the Before vs 8B and Before vs 13B comparisons (c). Schematic diagram of the ectoine metabolism (d). Heatmap of the expression levels of key genes involved in the ectoine metabolism in the Before, 8B, and 13B groups (e). * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.0005

Fig. 6

The DEGs in central carbon metabolism (a). Numbers represent the log₂FC of DEGs in Before vs 8B and Before vs 13B comparisons. The expression levels of genes in oxidative phosphorylation pathway (b). qRT-PCR analysis of eight genes in H. elongata at one hour after 8% NaCl shock (c). Where the abscissa (1 and 2) indicates different sampling time, which are 5 min before shock and 60 min after shock, respectively. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.0005. **** represents p < 0.0001

Fig. 7

Changes in physiological parameters of H. elongata after 8% NaCl shock in betaine and glutathione added groups. Biomass (a), ectoine production (b) and (d), ectoine produced per biomass (c). And the changes in ATP content (e) and AEC ratio (f). * represents p < 0.05

Fig. 8

The speculated stress response mechanism to NaCl shock in H. elongata within short time scale after 8% NaCl shock. Red upward arrows indicate the upregulated pathways or the increased metabolite contents. Green downward arrows indicate the downregulated pathways or the decreased metabolite contents. Green T-shaped line ends denote negative effect. Purple arrows indicate a promoting effect. The solid lines indicate direct actions; the dotted lines indicate unknown mechanisms