A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli
- Mengyong Xiao†1, 2, 3,
- Xinna Zhu†1, 2,
- Hongtao Xu1, 2,
- Jinlei Tang1, 2,
- Ru Liu1, 2,
- Changhao Bi1, 2,
- Feiyu Fan1, 2Email author and
- Xueli Zhang1, 2Email author
© The Author(s). 2017
Received: 11 August 2016
Accepted: 10 February 2017
Published: 13 February 2017
Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory.
Here, we report that a point mutation leading to a single amino acid change (D654Y) within the β-subunit of DNA-dependent RNA polymerase (RpoB) significantly improved the osmotolerance of E. coli. Importation of the D654Y mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA production by more than 40% compared to that of the control under high glucose osmolality. The transcriptome profile, determined by RNA-sequencing, showed two distinct stress responses elicited by the mutated RpoB that counterbalanced the osmotic stress. Under non-stressed conditions, genes involved in the synthesis and transport of compatible solutes such as glycine-betaine, glutamate or proline were upregulated even without osmotic stimulation, suggesting a “pre-defense” mechanism maybe formed in the rpoB mutant. Under osmotic stressed conditions, genes encoding diverse sugar transporters, which should be down-regulated in the presence of high osmotic pressure, were derepressed in the rpoB mutant. Additional genetic experiments showed that enhancing the expression of the mal regulon, especially for genes that encode the glycoporin LamB and maltose transporter, contributed to the osmotolerance phenotype.
The D654Y single amino acid substitution in RpoB rendered E. coli cells resistant to osmotic stress, probably due to improved cell growth and viability via enhanced sugar uptake under stressed conditions, and activated a potential “pre-defense” mechanism under non-stressed conditions. The findings of this work will be useful for bacterial host improvement to enhance its resistance to osmotic stress and facilitate bio-based organic acids production.
KeywordsOsmotolerance RpoB Succinic acid Sugar transporter RNA-seq
Escherichia coli has been extensively developed for bio-based production of a wide variety of organic acids, including succinic acid (SA) [1, 2]. Although high yields of SA have been successfully achieved using E. coli as hosts on both laboratory and commercial scales [2–4], cells suffering from osmotic stress during fermentation remains a major barrier for hyper SA production. One of the main causes of osmotic stress is a high initial sugar concentration in the medium, which is beneficial for simplifying the carbon source feeding process. However, induced osmotic pressure also negatively impacts robustness and propagating fecundity of the bacterial cells. Alkali is usually added during SA fermentation to maintain the medium at a neutral pH [4, 5]. SA accumulates as the dissociated form, disodium succinate, which further aggravate the osmotic stress.
The molecular mechanisms underlying the inhibitory effect due to osmotic stress can be summarized in two aspects. First, since sugar molecules cannot freely travel across semi-permeable cell membranes by diffusion, the high concentrations of such external solvents lead to a strong tendency of cytoplasmic water efflux. This dehydration results in shrinkage of the cell volume and malfunction of cell membranes and embedded proteins, leading to osmotic stress . To counterbalance the deleterious effect of osmotic stress, compatible solutes (also called osmoprotectants), such as potassium ions , glycine-betaine , trehalose , glutamate , and proline  can spontaneously accumulate in cells via de novo synthesis or transport from the medium. Compatible solutes are usually impermeable to the cell membrane, less toxic at high internal concentrations, and not easily catabolized [6, 8], which greatly facilitates water remaining within the cytoplasm. In terms of SA production, it was reported that medium supplemented with glycine-betaine or proline improved cell osmotolerance and succinate production in E. coli  and Actinobacillus succinogenes . However, it is worth noting that the osmoprotective effects of these compatible solutes are conditional. For example, it was reported that internal glycine-betaine lost its protective effect in the presence of NaCl concentrations greater than 1 M . Second, inhibition of nutrition uptake might account for the attenuation of cell growth upon external osmolality. Previous studies using an isotopic labeling experiment demonstrated that in the presence of increased osmolality, the activity of nearly all known sugar transport systems in E. coli were inhibited, including the glucose phosphotransferase system (PTS), the binding protein mediated maltose transport system, lactose-proton symport system, and melibiose-sodium co-transport system . Sugar transportation defects leading to energy insufficiency could be partially explained by inhibition of DNA replication , protein synthesis and respiration  under an osmotic stress. It is noteworthy that such inhibitory effects on growth did not lead to cell death because cell growth and metabolic activity were still maintained at a low level . In addition to attenuation of sugar transport, transcriptional repression of genes encoding sugar transporters might also lead to inhibition of sugar uptake. It was experimentally shown that the transcripts abundance of galactitol and maltose transporter genes were drastically downregulated upon NaCl-induced osmotic stress , although transcriptional information for other sugar transporters has not been reported.
Our laboratory previously generated an E. coli strain, Suc-T110, for SA production that is highly susceptible to osmotic stress. After maintaining Suc-T110 for more than 1400 generations in a medium containing a high sugar concentration (12% w/v glucose), an osmotolerant strain, HX024, was obtained. Genome re-sequencing of HX024 showed that only seven genes had non-synonymous point mutations, including rpoB and agaR, which encode transcriptional regulators . In this work, we aimed to discover how these two mutations lead to phenotypic changes in osmotolerance.
Strain, medium and growth conditions
Strains used in this study
E.coli ATCC#8739, ΔldhA, ΔpflB, ΔptsI, Ppck*-galP, Ppck*-pck
Suc-T110, ΔackA-pta, Ppck*-aceBA, Ppck*-dcuC, ΔmgsA,adaptively evolved for 1440 generations
Suc-T110:: rpoB (D654Y)
In this study
Suc-T110:: agaR (R109W)
In this study
Suc-T110, Ppck*- malK-lamB-malM
In this study
In this study
RpoBD645Y, ∆malE-malF-malG ∆malK-lamB-malM
In this study
Gene knock-outs or overexpression mutants were constructed using a previously described two-step recombination method . Red recombinase was used to facilitate chromosomal gene deletion and modulation . All primers used to construction of mutants are listed in Additional file 1: Table S1. For importation of the mutated rpoB into Suc-T110, a cat-sacB cassette was amplified from plasmid pXZ-CS with the primer set rpoB-QC-cat-up/rpoB-QC-sacB-down, which was used to replace the native rpoB gene in the Suc-T110 chromosome via homologous recombination. Then the mutated rpoB gene was amplified from strain HX024 with primer set rpoB-up/rpoB-down, which was used to replace the cat-sacB cassette via a second recombination event. Since cells containing the sacB gene die when grown on sucrose due to the accumulation of levan, transformants containing the mutated rpoB gene were selected for resistance to sucrose . The mutated agaR gene was cloned into Suc-T110 by a similar method, and a single deletion of the malEFG operon as well as a double deletion of the malEFG and malK-lamB-malM operons in Suc-T110 were generated using the the same method. For over-expression of lamB in Suc-T110, the promoter for the malK-lamB-malM operon was replaced with a constitutive strong promoter Ppck* using the two-step recombination method as mentioned above.
Growth under normal or osmotic conditions
Fresh colonies were inoculated into 250-mL flasks containing 30 mL of modified NBS mineral salts medium  [per liter: 3.5 g of KH2PO4, 5.0 g of K2HPO4, 3.5 g of (NH4)2HPO4, 0.25 g of MgSO4 · 7 H2O, 15 mg CaCl2 · 2H2O, 0.5 mg of thiamine, 10.0 g of KHCO3 and 0.15 g of betaine HCl and 1 ml of trace metal stock]. The trace metal stock was prepared in 0.1 M HCl and contained the following (per liter):1.6 g of FeCl3, 0.2 g of CoCl2 · 6H2O, 0.1 g of CuCl2, 0.2 g of ZnCl2 · 4 H2O, 0.2 g of NaMoO4, 0.05 g of H3BO3. The medium contained 20 gL-1 glucose, and cultures were grown at 37 °C and 250 rpm for 12 h. Cultures were then transferred to 500-mL fermentation vessels, which contained 250 mL of NBS mineral salts medium supplemented with 50 gL-1 or 120 gL-1 glucose to represent the non-stressed or osmotic stressed conditions, respectively. Then, the cultures were incubated anaerobically for 96 h at 37 °C and 150 rpm. Cell mass was estimated by measuring the optical density at 550 nm (OD550) as described previously . The succinate and glucose concentration in the medium were measured by high-performance liquid chromatography according to a previously reported protocol .
After 48 h of fermentation under normal (5% w/v glucose) or osmotic-stressed (12% w/v glucose) conditions, samples of Suc-T110 and RpoBD645Y were harvested for total RNA isolation. Extraction and additional on-column DNase I treatment was performed with the RNeasy mini kit (Qiagen) according to the manufacturer’s protocol. The purified RNA was assessed with an Agilent 2100 Bioanalyser (Agilent) and quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies).
RNA sequencing and data analysis
Four 90-nt paired-end RNA-seq libraries were generated at Beijing Genomics Institute (BGI, Shenzhen, China) with the HiSeq™ 2000 platform. Quality control of sequencing reads was performed with the NGS QC Toolkit (version 2.3.3) , and the obtained high-quality reads were aligned to the E. coli ATCC#8739 genome (GenBank CP000946.1) with Bowtie 2 (version 2.2.5) . The aligned reads stored in SAM format file and the raw counts for reads mapping to unique gene were then tallied with HTSeq-count scripts (0.6.0) with the intersection-nonempty resolution mode . Abundance for each transcript was calculated using the Reads Per Kilobase per Million (RPKM) measure as described previously . Differential expression gene calling was performed with R package NOISeq (version 2.6.0)  with “probability of differential expression (q value)” ≥0.9 and |log2 Ratio| ≥ 1 as the final cut-off. MIPS FunCat online tools  were used to annotate genes with altered expression, and enriched categories (P value < 0.001) were marked out.
Statistical significance tests
Unless otherwise noted, all experiments were performed in triplicate, and statistical tests for significance were determined via a one-way ANOVA using R (version 3.1.1).
Results and Discussion
A point mutation in rpoB confers resistance to osmotic stress
In E. coli ATCC#8739, the gene rpoB (EcolC_4038) encodes the β-subunit of DNA-dependent RNA polymerase (RNAp). RNAp, which is the primary enzyme responsible for gene transcription in prokaryotes, consists of five subunits (α2ββ’ω) and is associated with one of several alternative sigma (σ) factors. Sigma factors, such as σS in E. coli , σB in Listeria monocytogenes , and RpoN in Campylobacter jejuni , have been reported to be involved in the regulation of osmotic stress responses. However, direct evidence for the involvement of the RNAp β-subunit in osmotolerance has not yet been reported. An analysis of the protein domain architecture of the RNAp β-subunit using SMART online tools  showed that the D654Y mutation was located in a Pfam domain (PF10385) called the external one region of the polymerase. However, the function of this domain has not been characterized. Given that the β-subunit, along with the β’ subunits, constitute the active center of RNAp , we speculated that the D654Y mutation might lead to a conformational change in the RNAp active site, and therefore, affect the overall gene transcription pattern. It has been shown that point mutations in the β subunit of Enterococci RNAp conferred the capability of adaption to drug stress induced by cephalosporin , which probably occurs via a similar mechanism.
Transcriptomic profiling revealed mutated RpoB caused osmotic response genes upregulated under non-osmotic stressed conditions
Of the 4200 annotated genes, 128 genes showed altered expression in the strain containing the RpoB D654Y mutation under normal osmolality; with 38 upregulated and 90 downregulated genes. Functional enrichment of the 38 up-regulated genes by MIPS FunCat tools showed that 11 genes were engaged in biological functions of amino acid metabolism (P-value = 4.60E-05; Additional file 3: Table S3, P1 and P3, Fig. 2b). As mentioned above, bacterial cells commonly accumulate glutamate and proline to high levels under osmotic stress. The transcriptional abundance of genes involved in the metabolism of glutamate and aspartate (which can be converted into glutamate through transamination ), such as EcolC_2869, EcolC_3652, EcolC_3653, EcolC_1833, EcolC_4075, and EcolC_3651, were markedly increased in RpoBD645Y under normal osmolality. Genes involved in proline metabolism were not detected in the up-regulated gene set. However, the expression of genes encoding the major proline transporter system ProU [6, 7] was markedly enhanced. Transcription levels of genes located in the proU operon, including proX (EcolC_1027, encoding substrate-binding protein), proW (EcolC_1028, encoding permease), and proV (EcolC_1029, encoding ATP-binding protein), increased 3- to 4- fold in RpoBD645Y. These results were consistent with previous observations that proline accumulation in E. coli was due to enhanced transportation and not by synthesis [6, 35]. In addition, the ProU system also functions as the major system for glycine betaine uptake in E. coli . A previous study showed that ProU-mediated glycine betaine transport was osmotically stimulated at the level of gene expression . However, to our knowledge, the observation that the proU operon highly expressed under non-osmotic stress conditions has not yet been reported. Given that our modified NBS mineral salts medium contains a small amount of betaine (1 mM), we speculated that this compatible solute could be taken up into RpoBD645Y via ProU under non-osmotic stressed conditions, although further study is needed to obtain more direct evidence. In summary, the rpoB mutation probably conferred E. coli with the ability to mount a “pre-defense” mechanism, such that the osmotic response genes were activated under non-osmotic stressed conditions, which helped the cells better adapt to subsequent osmotic shock.
Transcriptomic profiling showed that mutated RpoB conferred osmotolerance via derepression of sugar transporters
Under a high osmolality, 244 differentially transcribed genes were identified in RpoBD645Y when compared with Suc-T110; 132 were upregulated and 112 were downregulated (Additional file 3: Table S3, P2). Since genes involved in glutamate synthesis and proline/glycine betaine transportation showed similar transcription levels in RpoBD645Y and Suc-T110 (Additional file 2: Table S2 and Additional file 3: Table S3), strategies other than “pre-defense” mechanisms might be adopted by RpoBD645Y to survive under high osmolality. Functional analysis of 132 upregulated genes indicated that the most significantly enriched physiological function of RpoBD645Y upon osmotic stress was associated with carbon source transportation (FunCat term: 20.01.03 C-compound and carbohydrate transport, P-value =9.51E-05; Additional file 3: Table S3, P5, Fig. 2b). This group included genes encoding diverse sugar transporters. The expression levels of these genes have been reported to be drastically reduced under osmotic stress conditions [17, 37, 38]. We found that these sugar transporter genes were drastically repressed in Suc-T110 under osmotic stress, whereas in RpoBD645Y, their expression maintained stable under osmotic stress. For example, transcript levels of malE (EcolC_3995), malF (EcolC_3996), malG (EcolC_3997) malK (EcolC_3994), and lamB (EcolC_3993), which encode individual subunits of the maltose ABC (ATP-binding cassette) transporter, decreased 10- to 14- fold in Suc-T110 compared to the corresponding levels in RpoBD645Y under osmotic stress. Meanwhile, genes encoding galactose PTS (EcolC_1645, EcolC_1646, and EcolC_1647), mannose PTS (ManY: EcolC_1814 and ManZ: EcolC_1813), D-xylose proton symporter XylE (EcolC_3998), and the melibiose-sodium co-transport system (EcolC_3907) were decreased 2- to 5- fold in Suc-T110 when compared to the corresponding levels in RpoBD645Y. These data demonstrated that repression effects on sugar transport genes were alleviated in RpoBD645Y due to the rpoB mutation. Based on FunCat analysis, no other processes with potential osmotic responsive functions were markedly enriched (cut-off, P value ≤ 0.001) in RpoBD645Y (Additional file 3: Table S3, P5, Fig. 2b). We assumed that maintenance of sugar transporter gene expression may be helpful for maintaining growth under osmotic stress, which might be the primary reason for the osmotic stress resistance of RpoBD645Y.
The porin LamB contributes to osmotolerance in RpoBD645Y
Derepression of the inner membrane-associated maltose ABC transporter confers osmotolerance
In addition to porin proteins, a number of sugar transporters located on the inner membrane were also derepressed in RpoBD645Y under high osmolarity. Thus, additional experiments were carried out to test whether modifying the expression of these transporter genes would alter osmotolerance.
A novel point mutation (D654Y) within RpoB was identified in this work, which improved the osmotolerance of E. coli. This mutation affected the transcriptional activity of RpoB, leading to upregulation of several osmotic response genes which involved in the biosynthesis or transportation of compatible solutes under non-osmotic stressed conditions, probably contributes to compatible solute accumulation. This mutation also enhanced glucose uptake under high sugar osmolality via derepression of the mal regulon. Thus, this mutation can be used to improve cell growth under osmotic stress and increase the production of succinate and other organic acids.
Embden-Meyerhof-Parnas (EMP) glycolytic pathway
Phosphoenolpyruvate, carbohydrate phosphotransferase system
DNA-dependent RNA polymerase
Reads Per Kilobase per Million
Reductive tricarboxylic acid cycle
The authors wish to thank Ms. Ying Qin for the help in the experiments.
This research was supported by grants from the Key deployment project of the Chinese Academy of Sciences (ZDRW-ZS-2016-3), National High Technology Research and Development Program of China (2014AA021205), National Natural Science Foundation of China (31522002 and 31300089), and Tianjin Key Technology R&D program of Tianjin Municipal Science and Technology Commission (13ZCZDSY05300).
Availability of data and materials
The RNA-seq data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus  and are accessible through GEO Series accession number GSE84769 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84769).
MX, XiZ, FF, and XuZ designed the study and advised on protocols. MX and XiZ carried out the experimental procedures. HX, JT, RL, and CB helped with experimental procedures and manuscript preparation. The manuscript was read and approved by all the authors.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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