Abstract
The haloalkalitolerant bacterium Egicoccus halophilus EGI 80432T exhibits high adaptability to saline–alkaline environment. The salinity adaptation mechanism of E. halophilus EGI 80432T was fully understood based on transcriptome analyses and physiological responses; however, the alkaline response mechanism has not yet been investigated. Here, we investigated the alkaline response mechanism of E. halophilus EGI 80432T by a transcriptomic comparison. In this study, the genes involved in the glycolysis, TCA cycle, starch, and trehalose metabolism for energy production and storage, were up-regulated under highly alkaline condition. Furthermore, genes responsible for the production of acidic and neutral metabolites, i.e., acetate, pyruvate, formate, glutamate, threonine, and ectoine, showed increased expression under highly alkaline condition, compared with the control pH condition. In contrast, the opposite results were observed in proton capture or retention gene expression profiles, i.e., cation/proton antiporters and ATP synthases. The above results revealed that E. halophilus EGI 80432T likely tended to adopt an “acidic metabolites production” strategy in response to a highly alkaline condition. These findings would pave the way for further studies in the saline–alkaline adaptation mechanisms of E. halophilus EGI 80432T, and hopefully provide a new insight into the foundational theory and application in ecological restoration with saline–alkaline strains.
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Introduction
Typical saline–alkaline environments like saline–alkaline lakes and soil harbor a number of halo(alkali)philic and halo(alkali)tolerant microorganisms (Banciu and Sorokin 2013). To thrive in those habitats, microorganisms deploy some adaptive strategies to cope with highly saline and/or alkaline stress. Microorganisms adopt the “salt-in-cytoplasm” strategy and the “compatible solute” strategy to withstand the salt stress (Chen et al. 2020, 2021). Besides, to overcome the burden of alkaline pH, microorganisms rely on some mechanisms of cytoplasmic pH homeostasis, e.g., the capture or retention of proton by the primary proton pumps (e.g., ATP synthase) and secondary active transporters [e.g., monovalent cation/proton antiporters (CPA)]; the production of acidic metabolites (e.g., acetate, pyruvate, and glutamate) through carbohydrate and amino acid metabolism; the modification of cell membrane by the alteration in membrane fatty acids components; the changes of secondary cell wall polymer containing various negatively charged residues, which favor H+ accumulation and deter OH− penetration (Slonczewski et al. 2009; Guo et al. 2019; Mamo 2020).
The class Nitriliruptoria, a higher taxon of phylum Actinobacteria, has six culturable members, namely Nitriliruptor alkaliphilus ANL-iso2T, Euzebya tangerina F10T, Euzebya rosea DSW09T, Euzebya sp. DY32-46, Egicoccus halophilus EGI 80432T, and Egibacter rhizosphaerae EGI 80759T, which exhibit great adaptability to various high-salt environments (Sorokin et al. 2009; Kurahashi et al. 2010; Zhang et al. 2016a, b; Yin et al. 2018; Xu et al. 2019). The genomic features playing a role in the adaptation to high-salt environments in Nitriliruptoria were analyzed by a comparative genomics approach (Chen et al. 2020). The research revealed that a similar synthesis systems of solutes, namely trehalose, glutamine, glutamate, and proline, were present in Nitriliruptoria. On the other hand, the specific mechanisms likely contributing to withstand various salt environments were found in each member of Nitriliruptoria species, including K+ influx and efflux, betaine and ectoine synthesis, and compatible solutes transport. Chen et al. (2021) performed physiological and transcriptomic analysis to reveal the salinity adaptation strategy in E. halophilus EGI 80432T. They proposed that E. halophilus EGI 80432T adopted the “salt-in-cytoplasm” strategy and the “compatible solute” strategy in response to moderate salinity condition, while the “compatible solute” strategy acted as a dominant strategy to withstand high salt stress. It is noteworthy that E. halophilus EGI 80432T is a haloalkalitolerant bacterium isolated from saline–alkaline soil. The salt-tolerant mechanism of E. halophilus EGI 80432T was elucidated by Chen et al. (2021), but the alkaline response mechanism of E. halophilus EGI 80432T still remains unknown.
Here, we tried to elucidate the alkaline response mechanism of E. halophilus EGI 80432T by comparing the transcriptome profile under highly alkaline condition with a control condition. We are confident that our research would be helpful for deeply understanding the adaptation mechanism of E. halophilus EGI 80432T to the saline–alkaline environment, and provide a theoretical support for its application in environmental domination.
Materials and methods
Strains and culture conditions
E. halophilus EGI 80432T (= CGMCC 1.14988T = KCTC 33612T) grown in the pH between pH 8.0 and pH 10.0, and optimally at pH 8.0–9.0, was isolated from a saline–alkaline soil in Xinjiang Province, north-west China (Zhang et al. 2016a). The strain was maintained on modified marine 2216E agar (Difco, Sparks, MD, USA) supplemented with 2% NaCl (w/v) and pH adjusted to 8.0 at 30 °C (Zhang et al. 2016a). The cells from modified marine 2216E agar were transferred to 50 mL fresh modified marine 2216E liquid medium in 250 mL Erlenmeyer flasks and incubated for 3 days at 30 °C with 150 rpm shake. The pre-cultures were used as inocula for the study.
Alkaline pH stress experiment and sample preparation
The pre-cultures were transferred to fresh modified marine 2216E liquid medium with different pH, namely pH 8.0 (control check, CK) and pH 10.0 (high alkali, HA), and cultured at 30 °C with shake of 150 rpm. The cultures grown to the mid-exponential growth phase in different alkaline conditions were harvested by centrifuging at 5,000 g for 10 min and washed three times with ddH2O for subsequent transcriptomic analysis.
RNA-seq sample preparation and transcriptome sequencing
Six RNA samples obtained from cells grown under control (pH 8.0) and high alkali (pH 10.0) treatments with three biological replicates were used to generate sequencing libraries. Total RNA per sample was extracted with a modified RNeasy midi kit (Qiagen Science, CA, USA) and treated with RNase-free DNase I (TaKaRa, China) to remove genomic DNA. Subsequently, the extracts were monitored on 1% agarose gels and checked with the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). The quantity and quality of RNA were measured using Qubit® 2.0 fluorometer with Qubit® RNA assay kit (Life Technologies, CA, USA), and Agilent Bioanalyzer 2100 system with the RNA Nano 6000 assay kit (Agilent Technologies, CA, USA), respectively. Sequencing libraries were constructed using NEBNext® Ultra™ Directional RNA library prep kit (NEB, USA) and sequenced on an Illumina Hiseq 2500 platform at Novogene Bioinformatics Technology Co. Ltd. (Beijing, China).
RNA-seq data analysis
Raw reads were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database (Bioproject: PRJNA718721 and the accession numbers: SRR14116862-SRR14116867). The raw data of fastq format were firstly processed through in-house perl scripts to remove reads containing adapter, ploy-N, and low-quality reads. The quality of clean data was assessed with Q20 and Q30 (Table 1). The high-quality clean data were aligned to E. halophilus EGI 80432T genome using Bowtie2-2.2.3 (Langmead and Salzberg 2012). The identification of novel genes and prediction of gene structure were performed by Rockhopper (McClure et al. 2013). The single-nucleotide polymorphisms (SNP) calling was performed by GATK (McKenna et al. 2020). Subsequently, the Shine–Dalgarno (SD) sequence and terminator sequence were predicted by RBSfinder and TransTermHP, respectively (Suzek et al. 2001; Kingsford et al. 2007). Finally, we used IntaRNA and RNAfold to predict the sRNA targets and RNA secondary structures, respectively (Hofacker and Stadler 2006; Busch et al. 2008).
Differentially expressed genes (DEGs) analysis and annotation
To estimate the levels of gene expression, the read numbers mapped to each gene were counted by HTSeq v0.6.1, and the effect of sequencing depth and gene length for each gene was calculated based on the fragments per kilobase of transcript sequence per millions of base pairs sequenced (FPKM) (Trapnell et al. 2010). Subsequently, we used the DESeq R package (1.18.0) to analyze the DEGs between the control group and the highly alkaline group (Anders and Huber 2013). In this study, we performed three biological replicates per group, such that the DEGs were identified with an adjusted P value < 0.05 (Anders and Huber 2013). Finally, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs identified between two groups were implemented by the GOseq R package and KOBAS software, respectively (Mao et al. 2005; Young et al. 2010).
Validation of RNAseq data by quantitative real-time PCR
Quantitative real-time PCR (qPCR) was performed to validate the RNA-seq data. Six randomly selected genes, namely ELR47_RS03560, ELR47_RS05540, ELR47_RS09510, ELR47_RS09675, ELR47_RS11550, and ELR47_RS14440, were used as target genes, and the chaperonin Cpn60 gene as an internal control. The primers used in this study were generated with DNAMAN 6.0 software and listed in Supplementary Table S1. The treated RNA (1 ng/μL) per sample was used to synthesize complementary DNAs (cDNAs) by the Hifair™ II super mix plus gDNA digester with the Oligo(dT) (Yeasen, China). The qPCR reaction was performed in the mixture containing Hieff® qPCR SYBR® Green master mix (Yeasen, China), a forward/reverse primer, a cDNA template, and ddH2O, with the procedure: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min, on a QuanStudio3 real-time PCR system (Applied Biosystems, USA). The 2−ΔΔCT method was used to calculate the relative expression of randomly selected genes (Livak and Schmittgen 2001). Three biological and three technical replicates were performed for the control and highly alkaline groups.
Results
Transcriptome sequencing and assembly analysis
To investigate the response mechanism to high pH shock in E. halophilus EGI 80432T, six libraries were generated and sequenced from cells grown under pH 8.0 (control check, CK) and pH 10.0 (high alkali, HA) conditions. Table 1 shows the results obtained from RNA-sequencing and assembly. After removing reads containing adapter, ploy-N, and low-quality reads, average 99% and 98.32% clean reads were obtained from average 16,905,009 and 31,220,560 raw reads generated for control and highly alkaline conditions, respectively. Moreover, average 16,079,234 (95%) and 29,580,620 (94.57%) reads were uniquely mapped to E. halophilus EGI 80432T reference genome and assembled into 3,840 and 3,837 genes. To validate the RNA-seq data reliability, we randomly selected 6 genes, ELR47_RS03560, ELR47_RS05540, ELR47_RS09510, ELR47_RS09675, ELR47_RS11550, and ELR47_RS14440, and performed the qPCR analysis. The qPCR results were in agreement with RNA-seq data (Supplementary Fig. S1).
Differentially expressed genes (DEGs) analysis and annotation
The overall transcription levels of genes were quantified by the FPKM metrics (Trapnell et al. 2010), and DEGs were identified with the standard threshold of P value < 0.05 (Anders and Huber 2013). Compared with the control, 1,129 genes were identified as DEGs at highly alkaline treatment, including 536 genes up-regulated and 593 genes down-regulated (Fig. 1a, Table 2). Furthermore, 733 DEGs (355 genes up-regulated and 378 genes down-regulated) and 606 DEGs (303 genes up-regulated and 303 genes down-regulated) were functionally annotated with GO and KEGG database, respectively (Table 2). According to KEGG pathway enrichment analysis, DEGs were classified into seventeen functional categories and mainly involved in the categories including “carbohydrate metabolism”, “energy metabolism”, “nucleotide metabolism”, “amino acid metabolism”, “metabolism of cofactors and vitamins”, “membrane transport”, and “signal transduction” (Fig. 1b).
Response of carbohydrate metabolism to highly alkaline stress
The highly alkaline environment significantly affected the expression of genes involved in carbohydrate metabolism, i.e., glycolysis, tricarboxylic acid cycle (TCA cycle), starch, and trehalose metabolism (Fig. 2). Six unigenes encoding enzymes, including fructose-1,6-bisphosphatase II, fructose-bisphosphate aldolase II, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphopyruvate hydratase, and pyruvate dehydrogenase E1, involved in glycolysis, were up-regulated under pH 10.0 condition. As for TCA cycle, four genes encoding fumarate hydratase class II, aconitate hydratase, and succinate-CoA ligase subunit alpha/beta, were up-expressed under highly alkaline condition. Similarly, the genes responsible for trehalose synthesis, i.e., treS (trehalose synthase), treY (malto-oligosyltrehalose synthase), and treX (glycogen debranching enzyme), showed positive response under highly alkaline shock. Moreover, genes required to synthesize starch from amylose (ELR47_RS05355) and the degradation of starch to dextrin (ELR47_RS11515) were highly expressed in highly alkaline stress.
Response of proton transport to highly alkaline stress
Proton capture or retention, performed by the primary proton pumps (e.g., respiratory chain complexes) and secondary active transporters [e.g., monovalent cation/proton antiporters (CPA)], is one major microbial strategy of maintaining intracellular pH homeostasis under a high pH environment (Slonczewski et al. 2009; Mamo 2020). The present work demonstrated up-regulated genes involved in the respiratory chain complexes (complex I, complex III, and complex IV) such as NADH–quinone oxidoreductase subunit I, cytochrome b subunit, cytochrome c1 subunit, and cytochrome c oxidase subunit I/II/III (Fig. 3). The main function of these genes is the proton production and translocation (Papa et al. 1994). In contrast, gene encoding ATP synthase for proton influx was down-regulated under high pH condition (Fig. 3).
The monovalent cation/proton antiporters regulate the influx of proton and the efflux of cations (Krulwich et al. 2011). Those antiporters were categorized into two superfamilies, the CPA families [CPA1, CPA2, and CPA3 (also known as Mrp-type)] and the Nha (Na+/H+ antiporter) families (NhaA, NhaB, NhaC, and NhaD) (Krulwich et al. 2009; Ito et al. 2017). Here, one Na+/H+ antiporter gene (ELR47_RS08060) and two cation/proton antiporter genes (ELR47_RS01215 and ELR47_RS08070) were down-expressed under higher alkaline condition (pH 10.0) (Fig. 3).
These findings suggested that E. halophilus EGI 80432T likely decreased the proton capture or retention under highly alkaline condition (pH 10.0), compared with the control pH condition (pH 8.0).
Response of organic acid metabolism to highly alkaline stress
The production of acidic metabolites that lower internal pH is another microbial strategy of maintaining intracellular pH homeostasis under a high pH environment (Slonczewski et al. 2009; Mamo 2020). We analyzed and compared the expression of genes involved in the production of common organic acids in E. halophilus EGI 80432T under highly alkaline treatment (pH 10.0) with those in the control treatment (pH 8.0) (Fig. 4a, c). Figure 4a shows that the highly alkaline stress showed positive effect on the production of acetate, which was performed by two pathways. One was performed by three up-regulated genes encoding pyruvate dehydrogenase, dihydrolipoyl dehydrogenase, and acetyl–CoA synthetase. The other one was performed by aldehyde dehydrogenase and alcohol dehydrogenase. Under highly alkaline condition, aldehyde dehydrogenase was encoded by two up-regulated genes (ELR47_RS06225 and ELR47_RS13150) and one down-regulated gene (ELR47_RS16010), and alcohol dehydrogenase was encoded by two up-regulated genes (ELR47_RS12650 and ELR47_RS17805) and two down-regulated genes (ELR47_RS08185 and ELR47_RS13890). In addition, genes encoding formate C-acetyltransferase and D-lactate dehydrogenase responsible for the pyruvate production from formate and lactate, respectively, were up-regulated under the pH 10.0 condition. The enhanced expression of a gene that encodes formyltetrahydrofolate deformylase involved in the formate synthesis and the decreased expression of a gene that encodes enoyl-CoA hydratase responsible for butanoate production were found under highly alkaline treatment.
We further analyzed the expression of genes involved in the acidic amino acid metabolism in E. halophilus EGI 80432T under highly alkaline condition (Fig. 4b, d). No significant difference was found in the expression of genes involved in the aspartate synthesis between highly alkaline treatment and control treatment. However, the highly alkaline stress led to the positive regulation in glutamate synthesized by genes encoding glutamate dehydrogenase, l-glutamate gamma-semialdehyde, carbamoyl-phosphate synthase, and aminotransferase. Surprisingly, the highly alkaline treatment also played a positive role in the gene encoding threonine synthase required for threonine synthesis. Furthermore, three ectoine and 5-hydroxyectoine synthesis genes encoding diaminobutyrate acetyltransferase, ectoine synthase, and ectoine hydroxylase were increased expression under high pH condition.
The above results revealed that E. halophilus EGI 80432T likely increased the production of acidic and neutral metabolites, i.e., acetate, pyruvate, formate, glutamate, threonine, ectoine, and 5-hydroxyectoine, in response to highly alkaline condition (pH 10.0).
Discussion
The development and popularization of next-generation sequencing provided more possibility to investigate the adaptation mechanism of microorganisms at various levels, e.g., gene, genome, and transcriptome (Cheng et al. 2016; Chen et al. 2020; Shu et al. 2020). Recently, RNA-sequencing, an effective method to evaluate the gene expression of organisms at various stage and state, was used to reveal the molecular mechanism of environmental stress response in microorganisms, e.g., temperature, salt, and pH (Raymond-Bouchard and Whyte 2017; Liang et al. 2020; Songserm et al. 2020; Chen et al. 2021). In this study, the alkaline response mechanism of haloalkalitolerant bacterium E. halophilus EGI 80432T was investigated by comparing the transcriptome profile under highly alkaline condition (pH 10.0) with control condition (pH 8.0). The clean reads were efficiently mapped to E. halophilus EGI 80432T reference genome (control condition for 95% and highly alkaline condition for 94.57%). Furthermore, 1129 DEGs (536 genes up-regulated and 593 genes down-regulated) were identified with the standard threshold of P value < 0.05 (Anders and Huber 2013).
It is a well-known fact that energy is required when microorganism adapt the environmental stress. The expression of genes responsible for energy production was significantly affected by environmental stress (Raymond-Bouchard and Whyte 2017; Wang et al. 2019; Songserm et al. 2020). The up-regulated genes involved in glycolysis and TCA cycle, which are the major pathways for ATP generation, were reported in E. halophilus EGI 80432T withstanding salt stress (Chen et al. 2021). Our transcriptomic analyses showed that the high alkali treatment also showed a positive effect on glycolysis and TCA cycle in E. halophilus EGI 80432T. Furthermore, we found that the expression of genes involved in the synthesis of trehalose and starch, which are used as the main source of carbon and energy, was increased under highly alkaline condition. These findings suggested that E. halophilus EGI 80432T likely increased energy production and reserve compounds synthesis to survive and grow in a highly alkaline environment. Previous researches proposed that trehalose was used not only as a source of carbon and energy but also as a stress protectant to cope with various environmental stresses, such as the compatible solute for coping with high salt environments (Wang et al. 2019; Chen et al. 2021) and the thermoprotectant for withstanding to high temperature environments (Reina-Bueno et al. 2012; Liu et al. 2019). The elevated expression of genes in trehalose synthesis pathways under highly alkaline condition suggested that trehalose may act as stress protectant in the highly alkaline response of E. halophilus EGI 80432T.
Even in a high pH environment, most microbes tend to maintain their cytoplasmic pH close to neutral (Slonczewski et al. 2009). For this, maintaining a relatively higher intracellular concentration of H+ is necessary. One important component of the cell membrane that contributes to cytoplasmic pH homeostasis is the primary proton pump, such as the respiratory system (Hicks and Krulwich 1995). Lewis et al. (1983) proposed that the proton translocation concomitant with respiration, and the H+ was extruded with a high H+/O ratio in the alkaliphiles. In the respiratory system of alkaliphiles, the NADH dehydrogenase was believed to be a proton--translocating complex (Hicks and Krulwich 1995), and the cytochrome c was thought to have a function in the transfer of electron-coupled H+ and the storage of electron and H+ for ATP production (Matsuno and Yumoto 2015). In E. halophilus EGI 80432T, the expression of genes encoding NADH–quinone oxidoreductase subunit I and cytochrome c1 subunit under pH 10.0 condition were higher than those under pH 8.0 condition, suggesting that E. halophilus EGI 80432T likely transferred and accumulated a large number of electron and H+ on the outer surface of membrane under higher alkaline condition. Researches proposed that ATP synthase utilize the electrochemical gradients of H+ on the surface of membrane to synthesize ATP and transfer H+ into the cytoplasm (Hicks et al. 2010). Here, the expression of ATP synthase gene was decreased under pH 10.0 condition, suggesting that the transfer of H+ to the cytoplasm by ATP synthase was lowered when E. halophilus EGI 80432T under higher alkaline condition.
The monovalent cation/proton antiporters that perform the exchange of the intracellular cations (e.g., Na+, Li+, and K+) and the extracellular H+ are thought to be a very crucial mechanism for proton capture (Mamo 2020). The monovalent cation/proton antiporters were encoded by diverse genes (Krulwich et al. 2009). However, not all the antiporter genes show differential expression during an alkaline pH environment. Desulfovibrio vulgaris possesses several putative Na+/H+ antiporter genes, but only one (DVU3108) was up-regulated in response to high alkali stress (Stolyar et al. 2007). Furthermore, the study of Cheng et al. (2016) revealed that the antiporter genes exhibited different transcriptional profiles under alkaline conditions. Compared with the pH 8.0 condition, antiporter genes, mrpA, mrpD, mrpE, nhaD2, and nhaP, were down-regulated in Halomonas sp. Y2 in response to higher alkaline condition (pH 10.17), whereas opposite results were detected in genes, mrpB, mrpC, mrpF, mrpG, and nhaD1. Fifteen putative monovalent cation/proton antiporter genes were detected in E. halophilus EGI 80432T (Chen et al. 2020). However, only three antiporter genes exhibited decreased expression in response to higher alkaline condition (pH 10.0), suggesting that the proton capture by monovalent cation/proton antiporters was low in E. halophilus EGI 80432T in response to a higher alkaline condition.
It is known that metabolic processes are significantly affected by extracellular pH. Under an alkaline environment, cells produce acidic compounds to maintain intracellular pH homeostasis (Mamo 2020). The increased production in lactate, formate, acetate, and butanoate were observed in Fusobacterium nucleatum cultured at pH 8.2 compared to pH 7.4 (Chew et al. 2012). Here, the transcription levels of genes involved in the synthesis of acidic end products from carbohydrate metabolism, i.e., acetate, pyruvate, and formate, exhibited increasing trends in E. halophilus EGI 80432T in response to higher alkaline condition. High pH favors the production of acid substances from carbohydrate and amino acid metabolism (Mamo 2020). The intracellular glutamate content of Streptomyces hygroscopicus in alkaline pH treatment was higher than the control (Jiang et al. 2018). The study of Chew et al. (2012) reported that the expression of glutamate dehydrogenase responsible for glutamate biosynthesis was significantly increased in F. nucleatum under alkaline condition. The positive effects in the four pathways for glutamate biosynthesis, namely glutamate dehydrogenase, l-glutamate gamma-semialdehyde, carbamoyl-phosphate synthase, and aminotransferase, were detected in E. halophilus EGI 80432T under higher alkaline condition. Moreover, the elevated expression of genes responsible for the synthesis of threonine, ectoine, and 5-hydroxyectoine, which were used for E. halophilus EGI 80432T withstanding higher salinity stress (Chen et al. 2021), was observed under highly alkaline stress, indicating that these compounds were responsible for E. halophilus EGI 80432T in response to a higher alkaline environment.
In summary, we compared the transcriptome of E. halophilus EGI 80432T under highly alkaline condition (pH 10.0) with control condition (pH 8.0), and proposed the putative mechanism of E. halophilus EGI 80432T in response to higher alkaline shock (Fig. 5). The energy production pathways, i.e., glycolysis and TCA cycle, and storage pathways, i.e., starch and trehalose metabolism, were extremely active, when E. halophilus EGI 80432T was cultured under higher alkaline condition, indicating that E. halophilus EGI 80432T may produce and store a large amount of energy to cope with higher alkaline environment. In some alkaliphilic and alkalitolerant microorganisms, the monovalent cation/proton antiporters and ATP synthase are the effective channels for trapping proton (Slonczewski et al. 2009; Mamo 2020). However, they likely were not the major strategy for E. halophilus EGI 80432T in response to higher alkaline condition, since the gene expression of cation/proton antiporters and ATP synthases was decreased under pH 10.0 condition. On the other hand, the acidic metabolites are thought to increase the cytoplasmic H+ concentration, which would relieve the burden of capturing proton to the cytoplasm (Mamo 2020). The higher gene expression was observed in the production of acidic and neutral metabolites, i.e., acetate, pyruvate, formate, glutamate, threonine, and ectoine, when E. halophilus was incubated under highly alkaline condition (pH 10.0), revealing that under higher alkaline condition, E. halophilus EGI 80432T likely tended to produce acidic metabolites to adjust the intracellular pH change.
Abbreviations
- CPA:
-
Cation/proton antiporter
- NCBI:
-
National Center for Biotechnology Information
- SRA:
-
Sequence Read Archive
- SNP:
-
Single-nucleotide polymorphisms
- SD:
-
Shine–Dalgarno
- DEG:
-
Differentially expressed gene
- FPKM:
-
Fragments per kilobase of transcript sequence per millions of base pairs
- GO:
-
Gene Ontology
- KEGG:
-
Kyoto Encyclopedia of Genes and Genomes
- qPCR:
-
Quantitative real-time PCR
- cDNA:
-
Complementary DNA
- TCA:
-
Tricarboxylic acid
References
Anders S, Huber W (2013) Differential expression of RNA-Seq data at the gene level-the DESeq package. European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.359.7464. Accessed 24 Feb 2013
Banciu HL, Sorokin DY (2013) Adaptation in haloalkaliphiles and natronophilic bacteria. In: Seckbach J, Oren A, Stanlotter H (eds) Polyextremophiles: life under multiple forms of stress. Cellular origin, life in extreme habitats and astrobiology 27. Springer, Dordrecht, pp 121–178. https://doi.org/10.1007/978-94-007-6488-0_5
Busch A, Richter AS, Backofen R (2008) IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions. Bioinformatics 24(24):2849–2856. https://doi.org/10.1093/bioinformatics/btn544
Chen DD, Tian Y, Jiao JY, Zhang XT, Zhang YG, Dong ZY, Xiong MJ, Xiao M, Shu WS, Li WJ (2020) Comparative genomics analysis of Nitriliruptoria reveals the genomic differences and salt adaptation strategies. Extremophiles 24:249–264. https://doi.org/10.1007/s00792-019-01150-3
Chen DD, Fang BZ, Manzoor A, Liu YH, Li L, Mohamad OAA, Shu WS, Li WJ (2021) Revealing the salinity adaptation mechanism in halotolerant bacterium Egicoccus halophilus EGI 80432T by physiological analysis and comparative transcriptomics. Appl Microbiol Biotechnol 105:2497–2511. https://doi.org/10.1007/s00253-021-11190-5
Cheng B, Meng YW, Cui TB, Li CF, Tao F, Yin HJ, Yang CY, Xu P (2016) Alkaline response of a halotolerant alkaliphilic Halomonas strain and functional diversity of its Na+(K+)/H+ antiporters. J Biol Chem 291(50):26056–26065. https://doi.org/10.1074/jbc.M116.751016
Chew J, Zilm PS, Fuss JM, Gully NJ (2012) A proteomic investigation of Fusobacterium nucleatum alkaline-induced biofilms. BMC Microbiol 12:189. https://doi.org/10.1186/1471-2180/12/189
Guo J, Ma ZP, Gao JS, Zhao JH, Wei L, Liu J, Xu N (2019) Recent advances of pH homeostasis mechanisms in Corynebacterium glutamicum. World J Microbiol Biotechnol 35:192. https://doi.org/10.1007/s11274-019-2770-2
Hicks DB, Krulwich TA (1995) The respiratory chain of alkaliphilic bacteria. Biochim Biophy Acta 1229:303–314. https://doi.org/10.1016/0005-2728(95)00024-D
Hicks DB, Liu J, Fujisawa M, Krulwich TA (2010) F1F0-ATP synthases of alkaliphilic bacteria: Lessons from their adaptations. Biochim Biophys Acta, Bioenerg 1797:1362–1377. https://doi.org/10.1016/j.bbabio.2010.02.028
Hofacker IL, Stadler PF (2006) Memory efficient folding algorithms for circular RNA secondary structures. Bioinformatics 22(10):1172–1176. https://doi.org/10.1093/bioinformatics/btl023
Ito M, Morino M, Krulwich TA (2017) Mrp antiporters have important roles in diverse bacteria and archaea. Front Microbiol 8:2325. https://doi.org/10.3389/fmicb.2017.02325
Jiang J, Sun YF, Tang X, He CN, Shao YL, Tang YJ, Zhou WW (2018) Alkaline pH shock enhanced production of validamycin A in fermentation of Streptomyces hygroscopicus. Bioresour Technol 249:234–240. https://doi.org/10.1016/j.biortech.2017.10.012
Kingsford CL, Ayanbule K, Salzberg SL (2007) Rapid, accurate, computational discovery of Rho-independent transcription terminators illuminates their relationship to DNA uptake. Genome Biol 8:R22. https://doi.org/10.1186/gb-2007-8-2-r22
Krulwich TA, Hicks DB, Ito M (2009) Cation/proton antiporter complements of bacteria: why so large and diverse? Mol Microbiol 74(2):257–260. https://doi.org/10.1111/j.1365-2958.2009.06842.x
Krulwich TA, Sachs G, Padan E (2011) Molecular aspects of bacterial pH sensing and homeostasis. Nat Rev Microbiol 9(5):330–343. https://doi.org/10.1038/nrmicro2549
Kurahashi M, Fukunaga Y, Sakiyama Y, Harayama S, Yokota A (2010) Euzebya tangerina gen. nov., sp. nov., a deeply branching marine actinobacterium isolated from the sea cucumber Holothuria edulis, and proposal of Euzebyaceae fam. nov., Euzebyales ord. nov. and Nitriliruptoridae subclassis nov. Int J Syst Evol Microbiol 60:2314–2319. https://doi.org/10.1099/ijs.0.016543-0
Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4):357–359. https://doi.org/10.1038/nmeth.1923
Lewis RJ, Krulwich TA, Reynafarje B, Lehningere AL (1983) Respiration-dependent proton translocation in alkalophilic Bacillus firmus RAB and its non-alkalophilic mutant Derivative*. J Biol Chem 258:2109–2111. https://doi.org/10.1016/S0021-9258(18)32891-6
Liang MH, Jiang JG, Wang L, Zhu JH (2020) Transcriptomic insights into the heat stress response of Dunaliella bardawil. Enzyme Microb Technol 132:109436. https://doi.org/10.1016/j.enzmictec.2019.109436
Liu XM, Wu XL, Gao W, Qu JB, Chen Q, Huang CY, Zhang JX (2019) Protective roles of trehalose in Pleurotus pulmonarius during heat stress response. J Integr Agric 18(2):428–437. https://doi.org/10.1016/S2095-3119(18)62010-6
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real time quantitative PCR and the 2-ΔΔCT method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Mamo G (2020) Challenges and adaptations of life in alkaline habitats. Adv Biochem Eng Biotechnol 172:85–134. https://doi.org/10.1007/10_2019_97
Mao XZ, Cai T, Olyarchuk JG, Wei LP (2005) Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics 21(19):3787–3793. https://doi.org/10.1093/bioinformatics/bti430
Matsuno T, Yumoto I (2015) Bioenergetics and the role of soluble cytochromes c for alkaline adaptation in gram-negative alkaliphilic Pseudomonas. BioMed Res Int. https://doi.org/10.1155/2015/847945
McClure R, Balasubramanian D, Sun Y, Bobrovskyy M, Sumby P, Genco CA, Vanderpool CK, Tjaden B (2013) Computational analysis of bacterial RNA-Seq data. Nucleic Acids Res 41:e140. https://doi.org/10.1093/nar/gkt444
McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA (2020) The genome analysis toolkit: a mapreduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303. https://doi.org/10.1101/gr.107524.110
Papa S, Lorusso M, Capitanio N (1994) Mechanistic and phenomenological features of proton pumps in the respiratory chain of mitochondria. J Bioenerg Biomembr 26:609–618. https://doi.org/10.1007/BF00831535
Raymond-Bouchard I, Whyte LG (2017) From transcriptomes to metatranscriptomes: cold adaptation and active metabolisms of psychrophiles from cold environments. In: Margesin R (ed) Psychrophiles: from biodiversity to biotechnology. Springer, New York, pp 437–445. https://doi.org/10.1007/978-3-319-57057-0_18
Reina-Bueno M, Argandoña M, Nieto JJ, Hidalgo-García A, Iglesias-Guerra F, Delgado MJ, Vargas C (2012) Role of trehalose in heat and desiccation tolerance in the soil bacterium Rhizobium etli. BMC Microbiol 12:207. https://doi.org/10.1186/1471-2180-12-207
Shu BS, Wu YX, Qu MQ, Pu XH, Wu ZZ, Lin JT (2020) Comparative transcriptomic analyses revealed genes and pathways responsive to heat stress in Diaphorina citri. Gene 727:144246. https://doi.org/10.1016/j.gene.2019.144246
Slonczewski JL, Fujisawa M, Dopson M, Krulwich TA (2009) Cytoplasmic pH measurement and homeostasis in bacteria and archaea. Adv Microb Physiol 55:1–317. https://doi.org/10.1016/S0065-2911(09)05501-5
Songserm P, Srimongkol P, Thitiprasert S, Tanasupawat S, Cheirsilp B, Assabumrungrat S, Karnchanatat A, Thongchul N (2020) Differential gene expression analysis of Aspergillus terreus reveals metabolic response and transcription suppression under dissolved oxygen and pH stress. J Evol Biochem Physiol 56(6):577–586. https://doi.org/10.1134/S0022093020060101
Sorokin DY, Pelt SV, Tourova TP, Evtushenko LI (2009) Nitriliruptor alkaliphilus gen. nov., sp. nov., a deeplineage haloalkaliphilic actinobacterium from soda lakes capable of growth on aliphatic nitriles, and proposal of Nitriliruptoraceae fam. nov. and Nitriliruptorales ord. nov. Int J Syst Evol Microbiol 59:248–253. https://doi.org/10.1099/ijs.0.002204-0
Stolyar S, He Q, He Z, Yang Z, Borglin SE, Joyner D, Huang K, Alm E, Hazen TC, Zhou J, Wall J, Arkin AP, Stahl DA (2007) Response of Desulfovibrio vulgaris to alkaline stress. J Bacteriol 189(24):8944–8952. https://doi.org/10.1128/JB.00284-07
Suzek BE, Ermolaeva MD, Schreiber M, Salzberg SL (2001) A probabilistic method for identifying start codons in bacterial genomes. Bioinformatics 17:1123–1130. https://doi.org/10.1093/bioinformatics/17.12.1123
Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515. https://doi.org/10.1038/nbt.1621
Wang DK, Hao ZQ, Zhao JS, Jin Y, Huang J, Zhou RQ, Wu CD (2019) Comparative physiological and transcriptomic analyses reveal salt tolerance mechanisms of Zygosaccharomyces rouxii. Process Biochem 82:59–67. https://doi.org/10.1016/j.procbio.2019.04.009
Xu L, Sun C, Huang MM, Wu YH, Yuan CQ, Dai WH, Ye KX, Han BN, Xu XW (2019) Complete genome sequence of Euzebya sp. DY32-46, a marine Actinobacteria isolated from the Pacific Ocean. Mar Genomics 44:65–69. https://doi.org/10.1016/j.margen.2018.09.008
Yin Q, Zhang L, Song ZM, Wu YH, Hu ZL, Zhang XH, Zhang Y, Yu M, Xu Y (2018) Euzebya rosea sp. nov., a rare actinobacterium isolated from the East China Sea and analysis of two genome sequences in the genus Euzebya. Int J Syst Evol Microbiol 68:2900–2905. https://doi.org/10.1099/ijsem.0.002917
Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol 11:R14. https://doi.org/10.1186/gb-2010-11-2-r14
Zhang YG, Chen JY, Wang HF, Xiao M, Yang LL, Guo JW, Zhou EM, Zhang YM, Li WJ (2016a) Egicoccus halophilus gen. nov., sp. nov., a halophilic, alkalitolerant actinobacterium and proposal of Egicoccaceae fam. nov. and Egicoccales ord. nov. Int J Syst Evol Microbiol 66:530–535. https://doi.org/10.1099/ijsem.0.000749
Zhang YG, Wang HF, Yang LL, Zhou XK, Zhi XY, Duan YQ, Xiao M, Zhang YM, Li WJ (2016b) Egibacter rhizosphaerae gen. nov., sp. nov., an obligately halophilic, facultatively alkaliphilic actinobacterium and proposal of Egibaceraceae fam. nov. and Egibacterales ord. nov. Int J Syst Evol Microbiol 66:283–289. https://doi.org/10.1099/ijsem.0.000713
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This work was supported by the National Natural Science Foundation of China (nos. 91751206, 32000084 and 32061143043) and China Postdoctoral Science Foundation (No. 2019M662952).
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DDC, WSS, and WJL conceived and designed the research. DDC conducted the experiments, analyzed the data, and wrote the original draft. YHL, SW, and BBL analyzed the data and modified the first draft of this manuscript. AM and SXG conducted the review and editing. DDC, YHL, HCJ, and WJL provided funding. All authors read and approved the manuscript.
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Chen, DD., Ahmad, M., Liu, YH. et al. Transcriptomic responses of haloalkalitolerant bacterium Egicoccus halophilus EGI 80432T to highly alkaline stress. Extremophiles 25, 459–470 (2021). https://doi.org/10.1007/s00792-021-01239-8
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DOI: https://doi.org/10.1007/s00792-021-01239-8