Development of acidic-pH tolerant mutants of Z. mobilis through adaptive laboratory evolution (ALE)
Z. mobilis was reported to be able to grow within a broad pH range (3.5-7.5) [22], the growth of Z. mobilis in pH range below pH 4.0 was further investigated in this study. The result showed that ZM4 can grow below pH 4.0 (Fig. 1), which is consistent with previously reported [22, 44]. However, when the pH value decreased from 4.0 to 3.5, the lower of the medium pH was, the longer of lag phase took and the lower of biomass produced (Fig. 1). Cells almost could not grow at pH below 3.5 (Fig. 1), which might be ascribed to the damage of cell membrane structure and protein configuration at acidic pH [45]. Therefore, the development of an acidic pH tolerant strain could benefit directly in commercial bioethanol production under acidic fermentation conditions using lignocellulosic feedstock hydrolyzed by acid as the substrate.
Subsequently, adaptive evolution was carried out with two parallel experiments in RMG2, which firstly adapted at pH 4.0 with 30 cycles and then transferred to pH 3.5 and pH 3.6 for acidic pH evolution (Fig. 2A). Finally, after 55 cycles of cultivation at pH 3.5 and 75 cycles at pH 3.6, four evolved cultures with enhanced acidic pH tolerance were obtained, and named as 3.5M-1, 3.5M-2, 3.6M-1, and 3.6M-2, respectively. The stability of these four adapted cultures was then analyzed at pH 3.6 with three colonies of each as replicates (Fig. 2B). The results showed that the growth of replicates from 3.5M-1 and 3.6M-1 was more uniformed than that of 3.5M-2 and 3.6M-2. The growth of these four mutants were then compared with wild-type ZM4 under different pH conditions, including pH 3.5, pH 3.6, pH 4.0, and pH 6.0 (Fig. 3).
Under pH 3.5 condition (Fig. 3A), all four mutants had higher growth rates and final OD values than ZM4, in which the mutant 3.6M-1 exhibited the maximum cell growth, followed by 3.6M-2, 3.5M-2 and 3.5M-1 successively. Under pH 3.6 condition, the mutants still had higher growth rates and shorter time reaching stationary phase than ZM4 (Fig. 3B). The mutants had no obvious advantage than ZM4 at either pH 4.0 or pH 6.0 (Fig. 3C, 3D). These results suggested that all mutants had enhanced tolerance at lower acidic pH conditions. Two mutants 3.5M-1 and 3.6M-1 with the most growth difference from ZM4 among all mutants were selected and renamed as 3.5M and 3.6M correspondingly for further studies.
Evaluation of cell growth, glucose consumption, and ethanol production of mutant strains 3.5M and 3.6M at acidic and neutral pH conditions
Since the acidic pH condition affects fermentation, ethanol production and glucose consumption, two mutant strains 3.5M and 3.6M were investigated at acidic and neutral pH conditions of pH 3.8 and pH 6.2, respectively. Both mutants 3.5M and 3.6M exhibited better cell growth and faster ethanol production than wild-type ZM4 at acidic pH 3.8 (Fig. 4A, 4B). The growth rate of 3.5M and 3.6M was 0.23 h-1 and 0.35 h-1 respectively, while that of ZM4 was only 0.14 h-1 (Table 1). Consistent with the fast cell growth, the fermentation time were reduced significantly from 22 h for ZM4 to 18 h and 13 h for 3.5M and 3.6M respectively, leading to the increase of ethanol productivity by 21.21% and 64.65% correspondingly (Table 1; Fig. 4A, 4B).
Under neutral pH condition of pH 6.2, cell growth and ethanol production of 3.6M were similar to those of ZM4, but performed better than those of 3.5M (Table 1; Fig. 4C, 4D). These results suggested that 3.5M and 3.6M possessed relatively fast glucose consumption and ethanol production at acidic condition, and 3.6M maintained similar capacities as ZM4 at neutral pH condition, which can be used to replace ZM4 as the biocatalyst for bioethanol production fermenting well in both acidic and neutral pH conditions (Table 1; Fig. 4C, 4D).
Table 1. Fermentation performance of time to consume all glucose (Time), growth rate, as well as ethanol titer, yield, and productivity of the wild-type Z. mobilis ZM4 and mutant strains 3.5M and 3.6M in RMG5 at pH 3.8 and pH 6.2.
Condition & Strain
|
Glucose used (g·L-1)
|
Time (h)
|
Growth rate (h-1)
|
Titer (g·L-1)
|
Yield (%)
|
Productivity
(g·L-1·h-1)
|
pH 3.8
|
|
|
|
|
|
|
ZM4
|
44.95±0.12
|
22
|
0.14±0.01
|
21.74±0.43
|
94.55±1.89
|
0.99±0.02
|
3.5M
|
44.91±0.01
|
18
|
0.23±0.01
|
21.62±0.14
|
94.13±0.64
|
1.20±0.01
|
3.6M
|
44.98±0.00
|
13
|
0.35±0.01
|
21.22±0.28
|
92.24±1.22
|
1.63±0.02
|
pH 6.2
|
|
|
|
|
|
|
ZM4
|
44.91±0.11
|
10
|
0.49±0.007
|
20.21±1.33
|
87.99±5.88
|
2.02±0.13
|
3.5M
|
44.97±0.00
|
12
|
0.39±0.005
|
20.05±0.82
|
87.19±3.56
|
1.67±0.07
|
3.6M
|
44.95±0.00
|
10
|
0.49±0.01
|
20.24±0.36
|
90.00±1.20
|
2.02±0.04
|
The underlying mechanism of acidic-pH tolerance through NGS-based genome resequencing and RNA-Seq
To illustrate the underlying genetic basis responsible for the enhanced acidic pH tolerance, samples of mutant and wild-type strains cultured at acidic pH 3.8 and neutral pH 6.2 were collected for WGR to determine the genetic changes in 3.5M and 3.6M using the genome of parental strain ZM4 as the reference (ATCC 31821) (GenBank accession No. of NZ_CP023715 for chromosome, and NZ_CP023716, NZ_CP023717, NZ_CP023718, NZ_CP023719 for four plasmids) [46]. RNA-Seq was also employed to explore the global transcriptional differences among these strains at acidic and neutral pH conditions.
The WGR result identified several single-nucleotide polymorphisms (SNPs) in the mutants. These SNPs included mutations located in chromosome, 7 SNPs in 3.5M and 5 SNPs in 3.6M, which were listed in Table 2. Among these mutations, 4 common SNPs were found in both mutants locating at the coding sequence (CDS) region of 4 genes: ZMO0421 (Ala67Thr), ZMO0712 (Gly539Asp), ZMO1432 (Pro480Leu), and ZMO1733 (Thr7Lys) respectively (Fig. 5A). These common mutations may contribute to the enhanced acidic-pH tolerance of mutant strains, while other unique mutations that are not shared by these strains may contribute to the unique phenotypic differences of these strains.
Table 2. Single-nucleotide polymorphisms (SNPs) in mutant strains 3.5M and 3.6M compared with wild-type ZM4. “+/−” indicates the presence/absence of variation, and the numbers in bracket after “+” represent the frequency (%) of the SNP. AA means amino acid.
Locus
|
Ref
|
SNP
|
AA change
|
3.5M
|
3.6M
|
ZM4
|
Gene
|
Product
|
424761
|
C
|
T
|
Ala67Thr
|
+ (99.76)
|
+ (98.86)
|
-
|
ZMO0421 (hisC2)
|
Histidinol-phosphate aminotransferase HisC
|
711194
|
G
|
A
|
Gly539Asp
|
+ (99.24)
|
+ (99.73)
|
-
|
ZMO0712 (ppk)
|
Polyphosphate kinase
|
1449594
|
G
|
A
|
Pro480Leu
|
+ (100.0)
|
+ (99.72)
|
-
|
ZMO1432
|
Membrane protein component of efflux system
|
1779278
|
C
|
A
|
Thr7Lys
|
+ (100.0)
|
+ (99.71)
|
-
|
ZMO1733 (oxyR)
|
Transcriptional regulator OxyR
|
1306151
|
C
|
T
|
Trp485*
|
+ (99.50)
|
-
|
-
|
ZMO1291
|
Peptidase S10 serine carboxypeptidase
|
1701191
|
G
|
A
|
Leu77Phe
|
+ (99.08)
|
-
|
-
|
ZMO1651 (ptsP)
|
Signal transduction protein
|
173653
|
T
|
C
|
|
+ (100.0)
|
-
|
-
|
Intergenic region
|
Between ZMO0183 and ZMO0184
|
1451222
|
A
|
G
|
|
-
|
+ (100.0)
|
-
|
Intergenic region
|
Between ZMO1432 and ZMO1433
|
Additionally, the differentially expressed genes (DEGs) were identified through analysis of variance (ANOVA) using strains and different conditions as variables. A total of 914 genes were identified by comparing any two conditions with p-value < 0.05 (Table S1). There has 267, 303, and 681 DEGs comparing acidic pH condition with neutral pH condition of 3.5M, 3.6M and wild-type ZM4 respectively (Fig. S1A). 304, 319 and 134 DEGs were also identified comparing 3.5M with ZM4, 3.6M with ZM4, and 3.5M with 3.6M at acidic pH respectively (Fig. S1B). The DEGs from comparison of same strain under different pH conditions or different strains under acidic pH condition were then further analyzed.
Association of genes with common changes in mutants with enhanced acidic-pH tolerance: A common mutation was found in gene ZMO0421, encoding histidinol-phosphate aminotransferase HisC2, which catalyzes the seventh step in the histidine biosynthesis pathway. Previous studies in Z. mobilis showed that HisC2 has broad substrate specificity and participates in transamination reactions for tyrosine and aromatic amino acid (phenylalanine) biosynthesis, which is essential in all studied organisms [47]. The mutation in ZMO0421 (Ala67Thr) was located in the amino transfer domain (PF00155, 32-357 aa) catalyzing the transamination reaction, which probably can enhance the enzymatic activity efficiency in acidic pH condition although detailed experiment is needed in the future.
Another common mutation was found in ppk gene (ZMO0712), which encodes polyphosphate kinase that transfers the γ-Pi of ATP to form a long chain polyphosphate (polyP) reversibly [48]. Several biological functions have been identified for cellular polyP including buffering capacities for pH homeostasis, DNA damage repair, cell cycle, motility, and biofilm formation [49-51]. Studies in other bacteria showed that polyP was rapidly accumulated by PPK under environmental stresses including acidic conditions [52-54]. Our transcriptomic data indicated that the expression of ppk in wild-type ZM4 was upregulated under acidic pH compared with neutral pH (Table S2), which is consistent with above reported conclusion. However, this gene was not significantly differentially expressed in mutant strains under acidic pH condition. Considering that the mutation in ppk (Gly539Asp) was located in the C2 domain (PF13090, 503-687), which is highly conserved in the PPK family and essential for the enzymatic activity [54], the mutation in this enzyme may help improve the activity of PPK resulting in the acceleration of polyP production to respond to the toxic acidic condition.
Additionally, mutation in gene ZMO1432, encoding the inner membrane component protein of a RND efflux system containing 12 transmembrane domains [55], was observed in both mutants. The mutation (Pro480Leu) was located at the eleventh transmembrane (TM11) domain, which may play an important role in the process of substrates extrusion from cytoplasm to periplasm by proton-motive force (PMF) with the conformational changes of RND system [55]. According to the prediction by TMHMM Server v. 2.0 [56], the transmembrane probability of TM11 domain in the mutant protein was improved from 0.7 to 1.0 (Fig. S2). Therefore, the mutation in ZMO1432 (Pro480Leu) may increase the stability and rigidity of TM11 and hence indirectly improve the efficiency to resist acidic stress by pumping toxic substances out, such as organic acids or anions [57].
Moreover, a mutation (A to G) was also found in the intergenic region between ZMO1432 and ZMO1433 in mutant 3.6M (Table 2), which is in the upstream of the promoter region of ZMO1432 predicted by BPROM [58]. As shown in the RNA-Seq result, the expression of the whole operon encoding a tripartite RND efflux system consisted of ZMO1432, ZMO1431, ZMO1430 and ZMO1429 was significantly upregulated at acidic pH in two mutant strains compared with ZM4, and 3.6M had the highest expression level among these strains (Fig. 5E). The mutation in the intergenic region in mutant 3.6M could help upregulate the expression of downstream genes, since the expression of these genes was also upregulated under pH 6.2 in 3.6M compared with ZM4 (Table S2). Combining these mutations and transcriptomic results, the RND efflux pump may play a crucial role on acidic-pH resistance in mutant strains.
The last common mutation shared in both mutant strains was within oxyR gene (ZMO1733). OxyR is a LysR family transcriptional regulator consisting of an N-terminal DNA-binding domain (DBD) and a C-terminal regulatory domain (RD), which controls the OxyR regulon consisting of almost 40 genes that can help protect cells from oxidative stress [59]. The mutation in OxyR (Thr7Lys) was in the N-terminal of LysR-type helix-turn-helix (HTH) DNA-binding domain (PS50931, 6-63 aa), which likely changes the binding affinity of HTH with its target DNA sequence due to the amino acid change from threonine with short side chain to lysine with long side chain (Table 2). Our RNA-Seq results showed that several genes involved in reactive oxygen species (ROS) detoxification possibly regulated by OxyR, such as ZMO0918 (catalase), ZMO1060 (superoxide dismutase), ZMO1732 (alkyl hydroperoxide reductase), and ZMO1211 (glutathione reductase), were significantly upregulated in all strains, especially in ZM4 at acidic pH compared with neutral pH condition (Table S2). Since acidic pH could induce a secondary oxidative stress and the acid tolerance response overlaps with the oxidative stress response [60, 61], the mutation in oxyR could contribute to the acidic pH tolerance in mutant strains.
Since mutant strains with these mutations exhibited advantages under acidic pH condition compared with wild-type ZM4, these mutations could be crucial for Z. mobilis to resist the acidic pH stress although further investigation is needed to help confirm whether they are necessary for the acidic-pH resistance phenotype and how.
Upregulation of genes associated with membrane components for enhanced acidic-pH tolerance: The lipid composition of cell membrane could be reconfigured at acidic pH condition, which will affect proton permeability directly or indirectly [62]. Hopanoids, one of the outer membrane component in Gram-negative bacteria such as Z. mobilis playing a role in regulating the fluidity and permeability of membrane [63, 64], are also contributed to the high ethanol tolerance of Z. mobilis [65]. It was reported that the deletion of hopanoid biosynthesis associated gene shc (ZMO0872 in ZM4), encoding the squalene-hopene cyclase protein, led the mutant more sensitive to acidic pH than the wild-type strains of Burkholderia cenocepacia or Rhodopseudomonas palustris TIE-1 [64, 66]. Our RNA-Seq data indicated that most hopanoid biosynthesis associated genes (ZMO0867, ZMO0872, ZMO0873, ZMO0874) were significantly downregulated in ZM4 at acidic pH compared with those at neutral pH, but not in mutant strains (Fig. 5B; Table S2), which suggested that mutant strains may have relative higher hopanoid expression than ZM4 at acidic pH. Notably, gene ZMO0867, encoding hopanoid-associated sugar epimerase HpnA protein, is a member of H+ and NADPH-consuming protein (EC1.1.1.219). Compared with ZM4, the upregulation of hopanoid biosynthesis gens such as hpnA (ZMO0876) in two mutant strains under acidic pH condition (Fig. 5B; Table S2) suggested that mutant strains may consume more cytoplasmic protons during hopanoid biosynthesis resulting in higher content of hopanoids and less cytoplasmic protons than ZM4 for enhanced acidic-pH tolerance.
The modification of the phospholipids in the inner membrane is also a strategy to reduce proton permeability. In many bacteria, the resistance to acidic pH is associated with the conversion of unsaturated fatty acids (UFAs) into cyclopropane fatty acids (CFAs) through the addition of a methyl group to the double bond of UFA, which is associated with cyclopropane fatty acid synthase (Cfa). The expression of cfa gene is usually upregulated under acidic conditions [67, 68], and a similar up-regulation was observed for gene ZMO1033 encoding Cfa in ZM4 at acidic pH, which suggested that cfa gene may be associated with outer membrane modification and acidic-pH tolerance (Fig. 5B; Table S2).
Energy generation through increased glycolysis and energy conservation through decreased cell growth and motility for acidic-pH resistance: It is reported that Streptococcus mutans altered its metabolism by increasing the glycolytic activity to produce ATP at acidic pH condition [69, 70], and ATP utilization was further derived from cell growth for acid tolerance [71]. Although ED pathway only produces one mole ATP per single mole glucose, it is reported that ED pathway in Z. mobilis is nearly twice as thermodynamically favorable as EMP pathway in E. coli or S. cerevisiae [72]. Our RNA-Seq result demonstrated that 6 genes involved in glycolysis pathway (ZMO1478 (pgl), ZMO0997 (eda), ZMO0177 (gap), ZMO1240 (gpmA), ZMO1608 (eno) and ZMO0152 (pyk)) were significantly upregulated at acidic pH compared with neutral pH in ZM4 and 3.6M, which could help produce more ATP for acidic pH tolerance (Fig. 5C; Table S2). Correspondingly, the final log2OD values of three strains at pH 3.8 were lower than those at pH 6.2, which was about 1.90 and 2.34, respectively (Fig. 4A, 4C), indicating more energy was used for acidic pH resistance instead of cell growth. This energy-demanding process might explain the uncoupling between glycolytic and biosynthetic reaction in Z. mobilis [73] with more energy consumed for acidic pH resistance. Additionally, gene ZMO1754 encoding SsdA that catalyzes the production of acetate from acetaldehyde was upregulated significantly at pH 3.8 compared with pH 6.2 for both mutants and especially wild-typeZM4, which was however significantly downregulated at pH 3.8 in mutant background compared with ZM4 (Fig. 5C; Table S2). These results indicated that more acetate might be produced at acidic pH than at neutral pH condition, and mutants produced less protonated acetate and consumed less NAD+ for a more efficient glycolysis and less acidified cytoplasmic environment than wild-type ZM4.
In addition, a number of genes encoding flagellar structure proteins and chemotaxis related proteins were significantly downregulated under acidic pH condition compared with neutral pH condition in both ZM4 and mutant strains (Fig. 5D; Table S2), which could also help save energy for survival in the acidic-pH condition [74]. Therefore, the conservation of energy from cell growth and cell motility may help re-distribute the cellular energy for inhibitory acidic-pH resistance in Z. mobilis.
Upregulation of transporter and efflux pump helped maintain pH homeostasis in acidic conditions: In acidic pH conditions (pH < 4.76), some protonated acids such as acetic acid enter the cell through the Fps1p channel or by diffusion in uncharged state, which will then be dissociated in cytoplasm with a neutral internal pH environment, and then affect cell metabolism and growth [75, 76]. HCl, which used in this study for pH adjustment, could enter the cytoplasm in undissociated state [75] at acidic pH, and then dissociate into H+ and Cl- in the cytoplasm. The accumulation of the H+ could activate cellular acid resistant system, while the accumulation of Cl- could change the membrane potential. It was reported that the CLC Cl-/H+ exchangers control the homeostasis of cellular compartments in most living organisms by catalyzing the exchange of two Cl- for one H+ in opposite direction [77, 78]. Our result indicated that the transcriptional level of CLC H+/Cl- antiporter, encoded by ZMO0841, was significantly downregulated in ZM4 under acidic pH, but upregulated at acidic pH in 3.6M compared with ZM4 (Fig. 5E; Table S2). These results suggested that CLC transporter may play an important role in homeostasis by controlling the membrane potential under acidic pH conditions, especially for the enhanced acidic-pH resistance capability of mutant strain 3.6M.
The increase of glycolysis pathway activity at acidic pH suggested that the accumulation of acidic end-products such as acetate and lactate could lead to an acidic intracellular condition [69]. Therefore, it is important for cell to export acidic products to maintain intracellular homeostasis. ATP-binding cassette (ABC) transporters transport a wide spectrum of substrates from small inorganic and organic molecules to larger organic compounds [79], and have been confirmed to contribute to acetic acid tolerance as an efflux pump of acetic acid [79]. Our results demonstrated that five genes encoding ABC transporters (ZMO0143, ZMO1017, ZMO0799-ZMO0801) were significantly upregulated in both mutant strains compared with ZM4 at acidic pH condition (Fig. 5E; Table S2). Moreover, RND efflux pump is well-known for transporting various compounds including cationic dyes, antibiotics, detergents and even simple organic solvents with the proton antiport [57, 80, 81]. Our result indicated that an RND efflux pump encoded by ZMO1429-ZMO1432 was also significantly upregulated at acidic pH in both mutant strains compared with ZM4. The upregulation of ABC transporter and efflux pump may suggest an enhanced capability of mutant strains to maintain cytoplasmic homeostasis in acidic pH conditions.
In addition, pumping H+ out of the cytoplasm is another efficient way in the maintenance of pH homeostasis [82]. F1Fo ATP synthase (F1Fo ATPase) can utilize the proton gradient for ATP synthesis, it can also reverse and hydrolyze ATP to pump H+ out to maintain the intracellular pH homeostasis [83, 84]. For example, genes encoding F1Fo ATPase in Streptococcus mutans was upregulated at acidic pH to help resist acid stress [85]. Another work indicated that when respiration was impeded, F1Fo ATPase hydrolyzed ATP to pump protons and contributed to intracellular neutral condition maintaining the essential mitochondrial membrane potential [86]. Our results demonstrated that 7 genes encoding F1Fo ATP synthase (ZMO0239, ZMO0240, ZMO0241, ZMO0667, ZMO0668, ZMO0669, ZMO0671) and another gene encoding F1Fo ATP synthase assembly protein (ZMO2005) were significantly upregulated at acidic pH compared with neutral pH for mutant strain 3.6M (Fig. 5F; Table S2). Since cellular respiration process was uncoupled with cell growth in Z. mobilis [87], and the ATP generation was majorly from glycolysis whose activity was increased as discussed above, the upregulation of F1Fo ATPase genes may possibly help pump H+ out from the cytoplasm through consuming ATP.
Furthermore, proton translocation was suggested to result in an alkalization of the intracellular medium in Z. mobilis at pH 6.5 during the respiration by transferring the H+ out of cytoplasm [88]. Nine genes related to respiration chain for transferring electrons to oxygen, ZMO0012, ZMO0568, ZMO0956-ZMO0958, ZMO1253-ZMO1255, and ZMO1258, were significantly downregulated in ZM4 at acidic pH compared with neutral pH (Fig. 5F; Table S2). In addition, six genes (ZMO1809-ZMO1814) encoding Rnf complex and an assembly gene (ZMO1808) were also downregulated at acidic pH compared with neutral pH in ZM4 but not in mutant strains (Fig. 5F; Table S2). The Rnf complex is required for electron transfer to nitrogenase during nitrogen fixation with proton excretion in Rhodobacter capsulatus [89]. Furthermore, gene ZMO0456 encoding the ferredoxin which is the electron acceptor from NADH and electron donor for nitrogenase was also downregulated at acidic pH compared with neutral pH in ZM4 (Fig. 5F; Table S2). The downregulation of genes associated with electron transfer chain at acidic pH condition in wild-type ZM4 could make the excretion of protons against proton gradient from cytoplasm difficult, leading to growth inhibition. In contrast, the expression of these genes in mutant background was not significantly downregulated in acidic pH compared with neutral pH condition. Instead, they were upregulated compared with ZM4 in acidic pH condition (Fig. 5F; Table S2). These results indicated that mutants could maintain relatively high proton transportation capacity against acidic pH condition.
Proton consumption and alkaline compound production for enhanced acidic-pH resistance: Biosynthesis of branched-chain amino acids (BCAAs) was reported to reduce H+ concentration in the cytoplasm by consuming proton or producing ammonia [69]. Two genes involved in the conversion of isoleucine from threonine in Z. mobilis (ZMO0687 and ZMO1275) were significantly up-regulated under acidic pH in mutant 3.6M compared with ZM4 (Fig. 5G; Table S2). ZMO0687, encoding acetolactate synthase large subunit, participates in the process with proton consumption in the first step. And ZMO1275, encoding threonine dehydratase, is involved in the process with ammonia production in the second step. Meanwhile, the fourth step of this conversion was catalyzed by dihydroxy-aid dehydratase encoded by ZMO1792, which had a reduced expression level at acidic pH compared with neutral pH only in wild type (Fig. 5G; Table S2). Moreover, genes ZMO0687 and ZMO1792 are also involved in the first step with proton consumption and the third step of valine biosynthesis from pyruvate. Interestingly, it was reported that the deviation of pyruvate away from lactic acid and acetate production toward other metabolic pathways was effective toward acidic end-product modulation in S. mutans [90]. The deviation of pyruvate from acidic production in mutants especially 3.6M was consistent with the result of less acetate production at acidic pH condition in mutants than ZM4 as described above. These changes indicated that 3.6M possesses higher activity on the conversion of BCAAs from threonine with more proton consumption and ammonia production than ZM4 and 3.5M.
In addition, gene ZMO0296 encoding adenosine deaminase (Ada) to convert adenosine into inosine with ammonia production was significantly upregulated at acidic pH in both mutant strains compared with ZM4 (Fig. 5G; Table S2). Furthermore, the expression of ZMO1207 gene encoding nitrilase (Nit, EC 3.5.5.1) that catalyzes the substrate containing cyanogroup to ammonia was also upregulated in mutant strain 3.6M only at acidic pH condition (Fig. 5G; Table S2). At acidic pH condition, ammonia could react with proton to produce the ammonium ion [91], which indicated that mutant strains possess greater capacity than ZM4 to neutralize the intracellular pH by proton-consuming and alkali-producing reactions resulting in enhanced acidic pH resistance.
However, the cytoplasmic pH homeostasis is connected with the proton motive force (PMF), which consists of two components of a transmembrane pH gradient (ΔpH) and a transmembrane electrical potential (Δψ) maintaining intercellular negative relative to outside [91]. The production of NH4+ from NH3 and proton would reduce the ΔpH, but would result in excess intracellular positive charges, which would lead to a positive internal Δψ at any time that could destroy the PMF and impair cellular functions. To balance the excess intracellular positive charges, exporting NH3 and NH4+ by ammonium transporter would avoid excessive positive charges hyperpolarizing the cell membrane [91]. our RNA-Seq result showed that the transcriptional level of ammonium transporter encoded by ZMO0346 was upregulated significantly at acidic pH compared with neutral pH in both mutant strains (Fig. 5G; Table S2), which ensures the normal PMF function on the membrane. Moreover, it was reported that the conversion of CO2 to HCO3- by carbonate anhydrase (CA) also contributed to acid-base equilibrium in H. pylori [22, 91]. It is interesting that the transcriptional level of ZMO1133 encoding carbonate anhydrase was significantly upregulated at acidic pH compared with neutral pH in all strains (Fig. 5G; Table S2). Since Z. mobilis can consume sugars and produce CO2 efficiently [92], CO2/HCO3- could also be involved in keeping acid-base equilibrium at acidic pH conditions.
Reduced energy consumption on macromolecular repair for enhanced acidic-pH tolerance of mutant strains: Cell membrane, proteins and DNA would be damaged when bacteria were cultured in acidic environments. To reduce the damage, the expression of repair and defense proteins such as DnaK, RecA, UvrA, IrrE and AP endonuclease would be increased to protect the macromolecules from the damage [69, 84]. Our result showed that the transcription level of ZMO0660 (dnaK) together with its co-chaperone ZMO1690 (dnaJ) and regulator ZMO0016 (grpE), and ZMO1588 (uvrA) with its subunit ZMO0362 (uvrB) were upregulated in ZM4 at acidic pH than neutral pH (Fig. 5H; Table S2), which demonstrated that it is necessary to enhance the expression of these proteins in order to protect DNA and protein from damage in acidic cytoplasm.
However, the expression level of these proteins was down-regulated at acidic pH in mutants compared with wild-type ZM4, except for gene recA that had no significant changes at different pH conditions in any strains. In addition, the transcriptional level of ZMO1929, encoding protein GroEL, which was important during adaptation to acid [69], was downregulated at acidic pH in mutant strains compared with wild type (Fig. 5H; Table S2). The deficient in HtrA, a surface protease involved in the degradation of aberrant proteins, reduced the ability of the mutant strain to endure acidic conditions [93], which demonstrated that this protein is important for cell to defense acid conditions. The transcriptional level of ZMO0015, encoding transcription repressor HrcA, was also upregulated significantly at acidic pH compared with neutral pH in wild type, and downregulated at acidic pH in mutant strains compared with wild type (Fig. 5H; Table S2). Moreover, the expression level of Clp protease complex, ZMO0948 (clpP), ZMO0949 (clpX), ZMO0405 (clpA) and ZMO1424 (clpB), which is involved in the remodeling and reactivation activities of proteins [69, 94], altered as same as ZMO1929 and ZMO0015. The phenomenon that the expression level of macromolecular repair proteins which are indispensable for acid resistance were upregulated at acidic pH only in wild type or downregulated significantly in both mutant strains compared with wild type at acidic pH, indicated that great demand on these proteins is needed for wild-type ZM4 to survive in acidic pH conditions, while mutants managed to avoid triggering defense responses and thus conserved energy used for macromolecule repair in ZM4.
Genetics confirmation of genes associated with acidic-pH resistance in Z. mobilis ZM4
To evaluate the impact of candidate genes associated with acidic-pH resistance identified through our genomics and transcriptomics study as discussed above, six plasmids containing candidate operons were constructed based on the shuttle vector pEZ15Asp with Ptet as the promoter [95]. These candidate operons including ZMO0142-ZMO0145 encoding ABC transporter, ZMO0798-ZMO0801 encoding multiple drug efflux, ZMO0956-ZMO0958 encoding cytochrome bc1 complex, ZMO0238-ZMO0242 encoding ATP synthesis F1 submits, ZMO1428-ZMO1432 encoding RND efflux system with a mutation in ZMO1432, and ZMO2005, ZMO0667-ZMO0671 encoding ATP synthesis F0 submits were cloned into pEZ15Asp shuttle vector, which were named pEZ-Tc1, pEZ-Tc2, pEZ-Tc3, pEZ-Tc4, pEZ-Tc5(M) and pEZ-Tc6, respectively. These plasmid constructs including the empty vector pEZ15Asp as the control were then introduced into ZM4 separately. These recombinant strains were then investigated under different conditions to examine their impact on cell growth (Fig. 6, Fig. S2).
With the increase of tetracycline inducer concentration from 0 to 0.8 μg/mL, the growth advantage of recombinant strain containing pEZ-Tc3 decreased in acidic pH condition (Fig. 6A, 6B, 6C). Our previous work demonstrated that Ptet promoter driven the operon expression used in this study is leaky even when tetracycline was not supplemented into the medium [95, 96]. Therefore, this result suggested that the cytochrome bc1 complex encoded by the operon ZMO0956-ZMO0958 in the recombinant strain containing pEZ-Tc3 could contribute to the acidic pH tolerance in Z. mobilis at a low expression level, which is consistent with our RNA-Seq result that the reduced expression of genes associated with electron transfer chain impacted the acid resistance of wild-type ZM4 (Fig. 5F; Table S2). On the contrary, with the increase of tetracycline inducer concentration from 0 to 0.8 μg/mL, the growth advantage of recombinant strain containing pEZ-Tc5(M) increased in acidic pH condition (Fig. 6D, 6E, 6F), which is again consistent with the significant upregulation of these genes in our RNA-Seq study (Fig. 5E; Table S2). Although further investigation is still needed to understand the association of acidic pH resistance with the different expression levels of these genes, our result suggested that the mutation in the intergenic region of upstream of ZMO1432 in mutant 3.6M may contribute to the upregulation of downstream gene, and a higher expression of RND efflux pump is more advantageous for strain to defend acidic pH condition.
Recombinant strains containing other four operons had no advantageous effect for cell growth in acidic pH condition with or without tetracycline induction (Fig. S3). Instead, the growth of recombinant strain containing pEZ-Tc2 dramatically impeded when induced with 0.4 μg/mL tetracycline (Fig. S3C, S3D), and the growth of recombinant strain containing pEZ-Tc6 was inhibited with or without the supplementation of tetracycline inducer (Fig. S3G, S2H). Since these operons encode ABC transporter, multiple drug efflux, and ATP synthase submits, which may function with other cellular component coordinately, a delicate balance of these operons with other genes may be needed for acidic pH resistance similar to previous report that the tailored expression of multiple genes simultaneously was essential for enhanced low-pH tolerance in E. coli [97].