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Transcriptome Analysis to Understand Salt Stress Regulation Mechanism of Chromohalobacter salexigens ANJ207

ABSTRACT: Soil salinity is one of the major global issues affecting soil quality and agricultural productivity. The plant growth-promoting halophilic bacteria that can thrive in regions of high salt (NaCl) concentration have the ability to promote the growth of plants in salty environments. In this study, attempts have been made to understand the salinity adaptation of plant growth-promoting moderately halophilic bacteria Chromohalobacter salexigens ANJ207 at the genetic level through transcriptome analysis. In order to identify the stress-responsive genes, the transcriptome sequencing of C. salexigens ANJ207 under different salt concentrations was carried out. Among the 8,936 transcripts obtained, 93 were upregulated while 1,149 were downregulated when the NaCl concentration was increased from 5 to 10%. At 10% NaCl concentration, genes coding for lactate dehydrogenase, catalase, and OsmC-like protein were upregulated. On the other hand, when salinity was increased from 10 to 25%, 1,954 genes were upregulated, while 1,287 were downregulated. At 25% NaCl, genes coding for PNPase, potassium transporter, aconitase, excinuclease subunit ABC, and transposase were found to be upregulated. The quantitative real-time PCR analysis showed an increase in the transcript of genes related to the biosynthesis of glycine betaine coline genes (gbcA, gbcB, and L-pro) and in the transcript of genes related to the uptake of glycine betaine (OpuAC, OpuAA, and OpuAB). The transcription of the genes involved in the biosynthesis of L-hydroxyproline (proD and proS) and one stress response proteolysis gene for periplasmic membrane stress sensing (serP) were also found to be increased. The presence of genes for various compatible solutes and their increase in expression at the high salt concentration indicated that a coordinated contribution by various compatible solutes might be responsible for salinity adaptation in ANJ207. The investigation provides new insights into the functional roles of various genes involved in salt stress tolerance and oxidative stress tolerance produced by high salt concentration in ANJ207 and further support the notion regarding the utilization of bacterium and their gene(s) in ameliorating salinity problem in agriculture.

SUBMITTER: Srivastava A

PROVIDER: S-EPMC9279137 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Elucidating the mechanisms controlling the synthesis of hydroxyectoine is important to design novel genetic engineering strategies for optimizing the production of this biotechnologically relevant compatible solute. The genome of the halophilic bacterium Chromohalobacter salexigens carries two ectoine hydroxylase genes, namely ectD and ectE, whose encoded proteins share the characteristic consensus motif of ectoine hydroxylases but showed only a 51.9% identity between them. In this work, we have shown that ectE encodes a secondary functional ectoine hydroxylase and that the hydroxyectoine synthesis mediated by this enzyme contributes to C.␣salexigens thermoprotection. The evolutionary pattern of EctD and EctE and related proteins suggests that they may have arisen from duplication of an ancestral gene preceding the directional divergence that gave origin to the orders Oceanospirillales and Alteromonadales. Osmoregulated expression of ectD at exponential phase, as well as the thermoregulated expression of ectD at the stationary phase, seemed to be dependent on the general stress factor RpoS. In contrast, expression of ectE was always RpoS-dependent regardless of the growth phase and osmotic or heat stress conditions tested. The data presented here suggest that the AraC-GlxA-like EctZ transcriptional regulator, whose encoding gene lies upstream of ectD, plays a dual function under exponential growth as both a transcriptional activator of osmoregulated ectD expression and a repressor of ectE transcription, privileging the synthesis of the main ectoine hydroxylase EctD. Inactivation of ectZ resulted in a higher amount of the total ectoines pool at the expenses of a higher accumulation of ectoine, with maintenance of the hydroxyectoine levels. In addition to the transcriptional control, our results suggest a strong post-transcriptional regulation of hydroxyectoine synthesis. Data on the accumulation of ectoine and hydroxyectoine in rpoS and ectZ strains pave the way for using these genetic backgrounds for metabolic engineering for hydroxyectoine production.

Project description:Quantitative RNA sequencing (RNA-seq) and the complementary phenotypic assays were implemented to investigate the transcriptional responses of Chromohalobacter salexigens to osmotic and heat stress. These conditions trigger the synthesis of ectoine and hydroxyectoine, two compatible solutes of biotechnological interest. Our findings revealed that both stresses make a significant impact on C. salexigens global physiology. Apart from compatible solute metabolism, the most relevant adaptation mechanisms were related to "oxidative- and protein-folding- stress responses," "modulation of respiratory chain and related components," and "ion homeostasis." A general salt-dependent induction of genes related to the metabolism of ectoines, as well as repression of ectoine degradation genes by temperature, was observed. Different oxidative stress response mechanisms, secondary or primary, were induced at low and high salinity, respectively, and repressed by temperature. A higher sensitivity to H2O2 was observed at high salinity, regardless of temperature. Low salinity induced genes involved in "protein-folding-stress response," suggesting disturbance of protein homeostasis. Transcriptional shift of genes encoding three types of respiratory NADH dehydrogenases, ATP synthase, quinone pool, Na+/H+ antiporters, and sodium-solute symporters, was observed depending on salinity and temperature, suggesting modulation of the components of the respiratory chain and additional systems involved in the generation of H+ and/or Na+ gradients. Remarkably, the Na+ intracellular content remained constant regardless of salinity and temperature. Disturbance of Na+- and H+-gradients with specific ionophores suggested that both gradients influence ectoine production, but with differences depending on the solute, salinity, and temperature conditions. Flagellum genes were strongly induced by salinity, and further induced by temperature. However, salt-induced cell motility was reduced at high temperature, possibly caused by an alteration of Na+ permeability by temperature, as dependence of motility on Na+-gradient was observed. The transcriptional induction of genes related to the synthesis and transport of siderophores correlated with a higher siderophore production and intracellular iron content only at low salinity. An excess of iron increased hydroxyectoine accumulation by 20% at high salinity. Conversely, it reduced the intracellular content of ectoines by 50% at high salinity plus high temperature. These findings support the relevance of iron homeostasis for osmoadaptation, thermoadaptation and accumulation of ectoines, in C. salexigens.

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Transcriptome Analysis to Understand Salt Stress Regulation Mechanism of Chromohalobacter salexigens ANJ207

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