Acid Resistance System Research Articles

New insight into acid-resistant enzymes from natural mutations of Escherichia coli Nissle 1917

The probiotic Escherichia coli Nissle 1917 (EcN), known for its superior acid resistance (AR), serves as a promising chassis for live therapeutics due to the effective colonization capabilities. However, the enzymatic activity regarding AR in EcN remains poorly understood. First, we investigated the AR systems of EcN by measuring cell growth under acidic stress and exploring the relationship of mutations to their corresponding enzymatic activities. As a result, the catalytic activity of inducible decarboxylases of GadB, AdiA and CadA, responsible for metabolizing glutamate, arginine, and lysine, exhibited an average 2-fold increase in EcN compared to the reference strain MG1655. Furthermore, we discovered that the glutamate-dependent AR2 system in EcN was meticulously regulated by specific regulons such as GadW. This study not only revealed the physiology of EcN under acidic conditions, but also highlighted that the mutated core enzymes in the AR system of EcN exhibit improved activities.

Characterization of Mild Acid Stress Response in an Engineered Acid-Tolerant Escherichia coli Strain.

Engineering acid-tolerant microbial strains is a cost-effective approach to overcoming acid stress during industrial fermentation. We previously constructed an acid-tolerant strain (Escherichia coli SC3124) with enhanced growth robustness and productivity under mildly acidic conditions by fine-tuning the expression of synthetic acid-tolerance module genes consisting of a proton-consuming acid resistance system (gadE), a periplasmic chaperone (hdeB), and ROS scavengers (sodB, katE). However, the precise acid-tolerance mechanism of E. coli SC3124 remained unclear. In this study, the growth of E. coli SC3124 under mild acid stress (pH 6.0) was determined. The final OD600 of E. coli SC3124 at pH 6.0 was 131% and 124% of that of the parent E. coli MG1655 at pH 6.8 and pH 6.0, respectively. Transcriptome analysis revealed the significant upregulation of the genes involved in oxidative phosphorylation, the tricarboxylic acid (TCA) cycle, and lysine-dependent acid-resistance system in E. coli SC3124 at pH 6.0. Subsequently, a weighted gene coexpression network analysis was performed to systematically determine the metabolic perturbations of E. coli SC3124 with mild acid treatment, and we extracted the gene modules highly associated with different acid traits. The results showed two biologically significant coexpression modules, and 263 hub genes were identified. Specifically, the genes involved in ATP-binding cassette (ABC) transporters, oxidative phosphorylation, the TCA cycle, amino acid metabolism, and purine metabolism were highly positively associated with mild acid stress responses. We propose that the overexpression of synthetic acid-tolerance genes leads to metabolic changes that confer mild acid stress resistance in E. coli. Integrated omics platforms provide valuable information for understanding the regulatory mechanisms of mild acid tolerance in E. coli and highlight the important roles of oxidative phosphorylation and ABC transporters in mild acid stress regulation. These findings offer novel insights to better the design of acid-tolerant chasses to synthesize value-added chemicals in a green and sustainable manner.

Molecular mechanism of acid stress response of Alicyclobacillus acidoterrestris DSM 3922T under sublethal pH

Alicyclobacillus acidoterrestris (A. acidoterrestris) causes the spoilage of pasteurized acidic fruit juice due to its unique thermoacidophilic properties, which caused economic losses to fruit juice industry. To reveal the molecular regulatory mechanism of how A. acidoterrestris responded to acid stress is the key to hazard control of this microbe. Herein, acid-responsive proteome expression profiles of A. acidoterrestris were analyzed using label-free quantitative mass spectrometry to investigate its acid resistance mechanism at sublethal pH. Totally, 325 differential expression proteins were identified during acid stress (pH 2.5, 15 min duration), of which the expressions of 83 proteins were up-regulated and the other 242 protein expressions were down-regulated. The identified differentially expressed proteins were mainly involved in small molecule metabolism, organic nitrogen compounds metabolism, organic acid metabolism, and signal transduction. Overall, they were mapped into 97 metabolic pathways. Combination of KEGG pathway analysis and protein functional analysis suggested that the pH homeostasis system variation and relevant changes in metabolic pathways, cell membrane permeability and DNA repair are the main acid resistance mechanisms of A. acidoterrestris. Our finding implied that A. acidoterrestris might sense and transmit pH signals from the external environment through NhaB protein which holds histidine-dependent acid resistance system, initiating a series of acid tolerance reactions. Taken together, our study demonstrated global physiological response of A. acidoterrestris to sublethal pH, which provided a better understanding of acid adaption mechanism of A. acidoterrestris.

Antibacterial mechanism of kojic acid and tea polyphenols against Escherichia coli O157:H7 through transcriptomic analysis

Escherichia coli O157:H7 is one of the major foodborne pathogenic bacterial that cause infectious diseases in humans. The previous found that a combination of kojic acid and tea polyphenols exhibited better activity against E. coli O157:H7 than using either alone. This study aimed to explore responses underlying the antibacterial mechanisms of kojic acid and tea polyphenols from the gene level. The functional enrichment analysis by comparing kojic acid and tea polyphenols individually or synergistically against E. coli O157:H7 found that acid resistance systems in kojic acid were activated, and the cell membrane and genomic DNA were destructed in the cells, resulting in "oxygen starvation". The oxidative stress response triggered by tea polyphenols inhibited both sulfur uptake and the synthesis of ATP, which affected the bacteria's life metabolic process. Interestingly, we found that kojic acid combined with tea polyphenols hindered the uptake of iron that played an essential role in the synthesis of DNA, respiration, tricarboxylic acid cycle. The results suggested that the iron uptake pathways may represent a novel approach for kojic acid and tea polyphenols synergistically against E. coli O157:H7 and provided a theoretical basis for bacterial pathogen control in the food industry.

Ahp deficiency-induced redox imbalance leads to metabolic alterations in E.coli

Alkyl hydroperoxide reductase (Ahp) is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli (E. coli). Ahp-deficient strains have been found to have high reactive oxygen species (ROS) levels, sufficient to cause cell damage. However, the exact role and underlying mechanisms of Ahp deficiency-induced cell damage remain largely unknown. Here, the E. coli MG1655 ΔAhp mutant strain was constructed as a model of deficiency to assess its role. The cells of the ΔAhp strain were found to be significantly longer than those of the wild strain, with elevated ROS and hydrogen peroxide (H2O2) levels. Proteome, redox proteome and metabolome analyses were performed to systematically present a global and quantitative profile and delineate the redox signaling and metabolic alterations at the proteome, metabolome, and cysteine oxidation site levels. The multiomics data revealed that Ahp deficiency disrupted the redox balance, activated the OxyR system, upregulated oxidative defense proteins and inhibited the TCA cycle to some extent. Surprisingly, the mutant strain shifted from aerobic respiration to anaerobic respiration and fermentation during the logarithmic phase in the presence of sufficient O2. The acid resistance system was activated to mitigate the effect of excessive acid produced by fermentation. Taken together, the results of this study demonstrated that Ahp deficiency triggered cellular redox imbalance and regulated metabolic pathways to confer resistance to submicromolar intracellular H2O2 levels in E. coli.

An underlying mechanism for MleR activating the malolactic enzyme pathway to enhance acid tolerance in Lacticaseibacillus paracasei L9.

Tolerance to acid stress is a crucial property of probiotics against gastric acids. The malolactic enzyme pathway is one of the most important acid resistance systems in lactic acid bacteria. It has been reported that the malolactic enzyme pathway was regulated by the transcriptional regulator, MleR. However, regulatory mechanisms underlying malolactic enzyme pathway to cope with acid stress remain unknown. In this study, the acid tolerance ability of the ΔmleR deletion strain was significantly lower than that of the wild-type strain, and the complementation of the mleR gene into the ΔmleR strain restored the acid tolerance of the ΔmleR strain, indicating that MleR was involved in acid tolerance response of Lacticaseibacillus paracasei L9. Real-time quantitative PCR and transcriptional fusion experiments confirmed MleR-activated transcription of the mleST gene cluster. Furthermore, MleR was confirmed to directly bind to the promoter region of the mleST operon using ChIP assays and EMSAs. The transcription start site G of the mleST operon was located at position -198 relative to the start codon of the mleS gene. The region from -80 to -61 upstream of the transcription start site was determined to be essential for MleR binding. Moreover, L-malic acid acted as an effector for MleR to activate the transcription of the mleST operon in a dose-dependent manner. These results revealed the regulatory mechanism behind MleR-mediated activation of the malolactic enzyme pathway to enhance acid tolerance in Lc. paracasei L9. IMPORTANCE Lacticaseibacillus paracasei is extensively used as probiotics in human health and fermented dairy production. Following consumption, Lc. paracasei is exposed to a variety of physico-chemical stresses, such as low pH in the stomach and bile salts in the intestines. The high acidity of the stomach severely inhibits bacterial metabolism and growth. Therefore, the acid tolerance response is critical for Lc. paracasei to survive. It has been reported that the malolactic enzyme (MLE) pathway plays an important role for LAB to resist acid stress. However, the regulatory mechanism has not yet been investigated. In this study, we determined that the LysR-type regulator MleR positively regulated the MLE pathway to enhance acid tolerance by binding -80 to -61 upstream of the transcription start site of the mleST operon. Further, L-malic acid acts as a co-inducer for MleR transcriptional regulation. Our study provides novel insights into acid tolerance mechanisms in LAB.

Acid tolerance of enterohemorrhagic Escherichia coli O157:H7 strain ATCC 43894 and its relationship with a large virulence plasmid pO157

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen that causes a severe intestinal infection including hemolytic uremic syndrome in humans. Various factors contribute to its pathogenesis, including a large virulence plasmid pO157. This F-like 92-kb plasmid is isolated in virtually all clinical EHEC isolates, and is considered a hallmark of EHEC virulence. A previous report stated that removal of pO157 from EHEC ATCC 43894 induced overexpression of GadAB that are essential in glutamate-dependent acid resistance (GDAR) system, yet the mechanism remains elusive. Based on this observation, we surmised that pO157 is involved in the regulation of GDAR system. We comparatively analyzed 43894 and its pO157-cured (ΔpO157) mutant 277 for i) their acid resistance, ii) changes in the transcriptional profiles and iii) expression of GDAR associated genes/proteins. Survivability of 43894 upon exposure to acidic conditions was significantly lower than the ΔpO157 mutant. In addition, RNA-sequencing revealed that genes involved in GDAR were significantly down-regulated in 43894 when compared to the ΔpO157 mutant. Exogenous expression of GadE in 43894 led to expression of GadAB, suggesting possible intervention of pO157 in GDAR regulation. Despite these findings, reintroduction of pO157 into 277 did not reverted Gad overexpression. Likewise, removing pO157 from 43894 using the plasmid incompatibility method did not induce Gad overexpression as shown in 277. Taken together, the results suggest that variation in acid resistance among EHEC isolates exists, and the large virulence plasmid pO157 has no effect on weak acid resistance phenotype displayed in 43894.

GadR4 mediates the acid resistance and pathogenicity of Listeria monocytogenes 10403S by negatively regulating the gadT2/gadD2 cluster.

Listeria monocytogenes is an important foodborne pathogen that can survive under acidic conditions. The glutamate decarboxylase (GAD) system is one of the acid resistance systems of L. monocytogenes. It usually comprises two glutamate transporters (GadT1/T2) and three glutamate decarboxylases (GadD1/D2/D3). Among them, gadT2/gadD2 contributes most significantly to the acid resistance of L. monocytogenes. However, the regulation mechanisms of gadT2/gadD2 still remain unclear. The results of this study indicated that gadT2/gadD2 deletion significantly decreases the survival rate of L. monocytogenes under different acidic conditions, including brain and heart infusion (BHI) broth, with a pH of 2.5, 2% citric acid, 2% acetic acid and 2% lactic acid. Further, gadT2/gadD2 cluster was expressed in the representative strains in response to alkaline stress rather than acid stress. To explore the regulation of gadT2/gadD2, we knocked out the five transcriptional factors belonging to the Rgg family in L. monocytogenes 10403S. We found that the deletion of gadR4, which exhibits the highest homology with the gadR of Lactococcus lactis, resulted in a significant increase in the survival rate of L. monocytogenes upon acid stress. Western blot analysis showed that gadR4 deletion significantly increased the gadD2 expression of L. monocytogenes under alkaline and neutral conditions. Furthermore, the gfp reporter gene showed that gadR4 deletion significantly increased the expression of the gadT2/gadD2 cluster. Adhesion and invasion assays indicated that gadR4 deletion significantly increased the rates of adhesion and invasion of L. monocytogenes to epithelial Caco-2cells. Virulence assays showed that gadR4 knockout significantly improved the colonization ability of L. monocytogenes in the livers and spleens of the infected mice. Taken together, our results showed that GadR4, a transcription factor belonging to the Rgg family, negatively regulates the gadT2/gadD2 cluster, thus, reducing the acid stress tolerance and pathogenicity of L. monocytogens 10403S. Our results provide a better understanding of the regulation of the GAD system of L. monocytogenes and a novel approach to potentially prevent and control listeriosis.

Acid-tolerant bacteria and prospects in industrial and environmental applications

Acid-tolerant bacteria such as Streptococcus mutans, Acidobacterium capsulatum, Escherichia coli, and Propionibacterium acidipropionici have developed several survival mechanisms to sustain themselves in various acid stress conditions. Some bacteria survive by minor changes in the environmental pH. In contrast, few others adapt different acid tolerance mechanisms, including amino acid decarboxylase acid resistance systems, mainly glutamate-dependent acid resistance (GDAR) and arginine-dependent acid resistance (ADAR) systems. The cellular mechanisms of acid tolerance include cell membrane alteration in Acidithiobacillus thioxidans, proton elimination by F1-F0-ATPase in Streptococcus pyogenes, biofilm formation in Pseudomonas aeruginosa, cytoplasmic urease activity in Streptococcus mutans, synthesis of the protective cloud of ammonia, and protection or repair of macromolecules in Bacillus caldontenax. Apart from cellular mechanisms, there are several acid-tolerant genes such as gadA, gadB, adiA, adiC, cadA, cadB, cadC, speF, and potE that help the bacteria to tolerate the acidic environment. This acid tolerance behavior provides new and broad prospects for different industrial applications and the bioremediation of environmental pollutants. The development of engineered strains with acid-tolerant genes may improve the efficiency of the transgenic bacteria in the treatment of acidic industrial effluents. KEY POINTS: • Bacteria tolerate the acidic stress by methylating unsaturated phospholipid tail • The activity of decarboxylase systems for acid tolerance depends on pH • Genetic manipulation of acid-tolerant genes improves acid tolerance by the bacteria.

ClC Chloride Channels in Gram-Negative Bacteria and Its Role in the Acid Resistance Systems.

Pathogenic bacteria that colonize the human intestinal tract have evolved strategies to overcome acidic conditions when they pass through the gastrointestinal tract. Amino acid-mediated acid resistance systems are effective survival strategies in a stomach that is full of amino acid substrate. The amino acid antiporter, amino acid decarboxylase, and ClC chloride antiporter are all engaged in these systems, and each one plays a role in protecting against or adapting to the acidic environment. The ClC chloride antiporter, a member of the ClC channel family, eliminates negatively charged intracellular chloride ions to avoid inner membrane hyperpolarization as an electrical shunt of the acid resistance system. In this review, we will discuss the structure and function of the prokaryotic ClC chloride antiporter of amino acid-mediated acid resistance system.

The mechanism of acid resistance by ornithine decarboxylase in Trichinella spiralis

Trichinella spiralis is a zoonotic parasite with worldwide distribution that can seriously harm human health and animal husbandry. Ornithine decarboxylase is a component of the acid resistance (AR) system in Escherichia coli. The aim of this study was to investigate the role that T. spiralis ornithine decarboxylase (TsODC) plays in the acid resistance mechanism of T. spiralis. This study involved assessing the transcription and expression of TsODC in worms under acidic conditions. According to mRNA sequences published by NCBI and the results of molecular biology experiments, the complete TsODC sequence was cloned and expressed. rTsODC had good immunogenicity, and immunofluorescence analysis revealed that TsODC was principally localized on the surface tissues of the nematode, especially at the head and tail. qRT‒PCR and Western blotting analysis indicated that the relative expression levels of TsODC mRNA and protein were highest when cultured at pH 2.5 for 2 h. The muscle larvae (ML) of T. spiralis were treated with curcumin and rapamycin, as well as arginine and TsODC polyantisera. The expression levels of TsODC mRNA and protein were significantly increased by arginine and suppressed by curcumin and rapamycin. After reducing the amount of TsODC, the relative expression of TsODC mRNA and the survival rate of T. spiralis ML were both reduced when compared to these values in the phosphate-buffered saline (PBS) group. The results indicated that TsODC is a member of the T. spiralis AR system and different treatments on TsODC have different effects; thus, these treatments might be a new way to prevent T. spiralis infection.

The Interplay of AphB and CadC to Activate Acid Resistance of Vibrio campbellii.

Bacteria have evolved different systems to sense and adapt to acid stress. For example, Vibrio campbellii, a marine pathogen for invertebrates, encounters acidic conditions in the digestive glands of shrimp. The main acid resistance system of V. campbellii is the Cad system, which is activated when cells are in a low-pH, amino acid-rich environment. The Cad system consists of the pH-responsive transcriptional activator CadC, the lysine decarboxylase CadA, and the lysine/cadaverine antiporter CadB. In many Vibrio species, the LysR-type transcriptional regulator AphB is involved in the regulation of the Cad system, but its precise role is unclear. Here, we examined AphB of V. campbellii in vivo and in vitro in the context of Cad activation. At low pH, an aphB deletion mutant was less able to grow and survive compared with the wild-type because it did not excrete sufficient alkaline cadaverine to increase the extracellular pH. AphB was found to upregulate the transcription of cadC, thereby increasing its protein copy number per cell. Moreover, AphB itself was shown to be a pH-sensor, and binding to the cadC promoter increased under low pH, as shown by surface plasmon resonance spectroscopy. By monitoring the activation of the Cad system over a wide range of pH values, we found that AphB-mediated upregulation of cadC not only adjusts CadC copy numbers depending on acid stress strength, but also affects the response of individual cells and thus the degree of heterogeneous Cad system activation in the V. campbellii population. IMPORTANCE Acid resistance is an important property not only for neutralophilic enteric bacteria such as Escherichia, Yersinia, and Salmonella, but also for Vibrio. To counteract acidic threats, the marine Vibrio campbellii, a pathogen for various invertebrates, activates the acid-resistance Cad system. The transcriptional activator of the Cad system is CadC, an extracellular pH-sensor. The expression of cadC is upregulated by the transcriptional regulator AphB to achieve maximum expression of the components of the Cad system. In vitro studies demonstrate that AphB binds more tightly to the DNA under low pH. The interplay of two pH-responsive transcriptional activators allows tight control of the activity of the Cad system.

Integrating Enzyme Evolution and Metabolic Engineering to Improve the Productivity of Γ-Aminobutyric Acid by Whole-Cell Biosynthesis in Escherichia Coli.

γ-Aminobutyric acid (GABA) is used widely in various fields, such as agriculture, food, pharmaceuticals, and biobased chemicals. Based on glutamate decarboxylase (GadBM4) derived from our previous work, three mutants, GadM4-2, GadM4-8, and GadM4-31, were obtained by integrating enzyme evolution and high-throughput screening methods. The GABA productivity obtained through whole-cell bioconversion using recombinant Escherichia coli cells harboring mutant GadBM4-2 was enhanced by 20.27% compared to that of the original GadBM4. Further introduction of the central regulator GadE of the acid resistance system and the enzymes from the deoxyxylulose-5-phosphate-independent pyridoxal 5'-phosphate biosynthesis pathway resulted in a 24.92% improvement in GABA productivity, reaching 76.70 g/L/h without any cofactor addition with a greater than 99% conversion ratio. Finally, when one-step bioconversion was applied for the whole-cell catalysis in a 5 L bioreactor, the titer of GABA reached 307.5 ± 5.94 g/L with a productivity of 61.49 g/L/h by using crude l-glutamic acid (l-Glu) as the substrate. Thus, the biocatalyst constructed above combined with the whole-cell bioconversion method represents an effective approach for industrial GABA production.

Effect of RNA interference with glutamate decarboxylase on acid resistance of Trichinella spiralis

Trichinella spiralis is a zoonotic parasite that infects most mammals, even humans. Glutamate decarboxylase (GAD) is an important enzyme in glutamate-dependent acid resistance system 2 (AR2), but the GAD of T. spiralis in AR2 is unclear. We aimed to investigate the role of T. spiralis glutamate decarboxylase (TsGAD) in AR2. We silenced the TsGAD gene to evaluate the AR of T. spiralis muscle larvae (ML) in vivo and in vitro via siRNA. The results showed that recombinant TsGAD was recognized by anti-rTsGAD polyclonal antibody (57 kDa), and qPCR indicated that TsGAD transcription peaked at pH 2.5 for 1 h compared to that with pH 6.6 phosphate-buffered saline. Indirect immunofluorescence assays revealed that TsGAD was expressed in the epidermis of ML. After TsGAD silencing in vitro, TsGAD transcription and the survival rate of ML decreased by 15.2% and 17%, respectively, compared with those of the PBS group. Both TsGAD enzymatic activity and the acid adjustment of siRNA1-silenced ML were weakened. In vivo, each mouse was orally infected with 300 siRNA1-silenced ML. On days 7 and 42 post-infection, the reduction rates of adult worms and ML were 31.5% and 49.05%, respectively. Additionally, the reproductive capacity index and larvae per gram of ML were 62.51±7.32 and 1250.22±146.48, respectively, lower than those of the PBS group. Haematoxylin-eosin staining revealed many inflammatory cells infiltrating the nurse cells in the diaphragm of mice infected with siRNA1-silenced ML. The survival rate of the F1 generation ML was 27% higher than that of the F0 generation ML, but there was no difference from the PBS group. These results first indicated that GAD plays a crucial role in AR2 of T. spiralis. TsGAD gene silencing reduced the worm burden in mice, providing data for the comprehensive study of the AR system of T. spiralis and a new idea for the prevention of trichinosis.

Acid shock protein Asr induces protein aggregation to promote E. coli O157:H7 entering viable but non-culturable state under high pressure carbon dioxide stress

Under stressful conditions, bacteria can enter viable but non-culturable (VBNC) state to survive. VBNC cells lost ability to grow on routine culture medium but are still alive and may revive in suitable conditions. The revived cells can consume nutrients or produce toxins, leading to food spoilage or human illness, posing great risk to food safety and public health. Previously, we have reported that high pressure carbon dioxide (HPCD), an environment-friendly sterilization technology, can induce VBNC formation. However, the underlying mechanism is unclear. By performing a comprehensive transcriptomic analysis, we revealed that HPCD initiated high expression of asr, encoding an acid shock protein, could promote VBNC formation of E. coli O157:H7. Quantitative reverse transcription PCR analysis suggested that high expression of asr (i) inhibited acid resistance (AR) systems, resulting in endogenous proton accumulation; (ii) inhibited hchA expression, a protein stabilizing factor. The two effects resulted in endogenous protein aggregation, which was highly correlated to VBNC formation. Accordingly, HPCD-stressed cells exhibited decreased efficiency of electron transfer chain and ATP production, which was also contributory to cytoplasmic protein aggregation. Taken together, HPCD-initiated high expression of Asr coupled with decreased ATP production led to protein aggregation, finally promoted the cells to enter VBNC state.

The mechanism of resistance in Escherichia coli to ridinilazole and other antibacterial head-to-head bis-benzimidazole compounds

The appY gene has been characterised as conferring resistance to a novel series of antimicrobial benzimidazole derivatives in E. coli MC1061 cells when expressed in high copy-number. A microarray approach was used to identify genes involved in the mechanism of appY-mediated antibacterial resistance, that were up- or down-regulated following induction of the gene in the appY knockout strain JW0553. In total, expression of 90 genes was induced and 48 repressed greater than 2.5-fold (P < 0.05), 45 min after appY induction. Over half the genes up-regulated following appY expression had confirmed or putative roles in acid resistance (AR) and response to oxidative and antibiotic stresses. These included the genes for MdtE and MdtF, which form a multi-drug transporter with TolC and have been implicated in resistance to several antibiotics including erythromycin. Amongst the acid resistance genes were gadAB and adiAC encoding the glutamate-dependant (AR2) and arginine-dependant (AR3) acid resistance systems respectively, in addition to the transcriptional activators of these systems gadE and gadX. In agreement with earlier studies, appA, appCB and hyaA-F were also up-regulated following induction of appY. This study has also confirmed that over-expression of mdtEF confers resistance to these antibacterial benzimidazoles, indicating that the observation of appY conferring resistance to these compounds, proceeds through an appY-mediated up-regulation of this efflux transporter. To assess the importance of the AppY enzyme to acid stress responses, the percentage survival of bacteria in acidified media (pH ≤ 2) was measured. From an initial input of 1 × 106 CFU/ml, the wild-type strain MG1655 showed 7.29% and 0.46% survival after 2 and 4 h, respectively. In contrast, strain JW0553 in which appY is deleted was completely killed by the treatment. Transformation of JW0553 with a plasmid carrying appY returned survival to wild-type levels (7.85% and 1.03% survival at 2 and 4 h). Further dissection of the response by prior induction of each of the three AR systems has revealed that AR1 and AR3 were most affected by the absence of appY. This work highlights an important and previously unidentified role for the AppY enzyme in mediating the responses to several stress conditions. It is likely that the appY gene fits into a complex transcriptional regulatory network involving σS and gadE and gadX. Further work to pinpoint its position in such a hierarchy and to assess the contribution of appY to oxidative stress responses should help determine its full significance. This work is also consistent with recent studies in C. difficile showing that the mechanism of action of ridinilazole involves AT-rich DNA minor groove binding.

TCS response regulator OmpR plays a major role in stress resistance, antibiotic resistance, motility, and virulence in Edwardsiella piscicida

Two-component regulatory systems (TCSs) are omnipresent in Gram-negative bacteria and play a major role in response to changes in environmental cues and pathogenicity. Edwardsiella piscicida is a serious pathogen of fresh and seawater aquaculture industries and has attracted increasing attention. However, extremely limited TCSs have been reported in E. piscicida. In this study, the role of response regulator OmpR which belongs to TCS EnvZ/OmpR was investigated in E. piscicida. By construction of a markerless ompR in-frame mutant strain, TX01ΔompR, we found that (i) in comparison to the wild type TX01, TX01ΔompR exhibited markedly compromised tolerance to acid stress, osmotic stress, and oxidative stress; (ii) the deletion of ompR significantly changed bacterial sensitivity to multiple antibiotics, (iii) the deficiency of ompR tremendously reduced bacterial motility, (iv) the deficiency of ompR abated bacterial colonization in host immune tissue and bacterial overall virulence. These results indicate OmpR is an important participant in E. piscicida's adversity resistance and pathogenicity. As a response regulator, OmpR was demonstrated to downregulate acid resistance system cadBA and to upregulate the porin ompC and flagellum mediator flhDC. Taken together, our results illustrate that OmpR is a vital regulator that coordinates the expressions of multiple genes during the response to an adverse environment and invasion to host.

Cooperative Interaction between Acid and Copper Resistance in Escherichia coli.

The persistence of pathogenic Escherichia coli under acidic conditions poses a serious risk to food safety, especially in acidic foods such as kimchi. To identify the bacterial factors required for acid resistance, transcriptomic analysis was conducted on an acid-resistant enterotoxigenic E. coli strain and the genes with significant changes in their expression under acidic pH were selected as putative resistance factors against acid stress. These genes included those associated with a glutamatedependent acid resistance (GDAR) system and copper resistance. E. coli strains lacking GadA, GadB, or YbaST, the components of the GDAR system, exhibited significantly attenuated growth and survival under acidic stress conditions. Accordantly, the inhibition of the GDAR system by 3-mercaptopropionic acid and aminooxyacetic acid abolished bacterial adaptation and survival under acidic conditions, indicating the indispensable role of a GDAR system in acid resistance. Intriguingly, the lack of cueR encoding a transcriptional regulator for copper resistance genes markedly impaired bacterial resistance to acid stress as well as copper. Conversely, the absence of YbaST severely compromised bacterial resistance against copper, suggesting an interplay between acid and copper resistance. These results suggest that a GDAR system can be a promising target for developing control measures to prevent E. coli resistance to acid and copper treatments.

Synthetic acid stress-tolerance modules improve growth robustness and lysine productivity of industrial Escherichia coli in fermentation at low pH

BackgroundDuring fermentation, industrial microorganisms encounter multiple stresses that inhibit cell growth and decrease fermentation yields, in particular acid stress, which is due to the accumulation of acidic metabolites in the fermentation medium. Although the addition of a base to the medium can counteract the effect of acid accumulation, the engineering of acid-tolerant strains is considered a more intelligent and cost-effective solution. While synthetic biology theoretically provides a novel approach for devising such tolerance modules, in practice it is difficult to assemble stress-tolerance modules from hundreds of stress-related genes.ResultsIn this study, we designed a set of synthetic acid-tolerance modules for fine-tuning the expression of multi-component gene blocks comprising a member of the proton-consuming acid resistance system (gadE), a periplasmic chaperone (hdeB), and reactive oxygen species (ROS) scavengers (sodB and katE). Directed evolution was used to construct an acid-responsive asr promoter library, from which four variants were selected and used in the synthetic modules. The module variants were screened in a stepwise manner under mild acidic conditions (pH 5–6), first by cell growth using the laboratory Escherichia coli strain MG1655 cultured in microplates, and then by lysine production performance using the industrial lysine-producing E. coli strain MG1655 SCEcL3 cultured first in multiple 10-mL micro-bioreactors, and then in 1.3-L parallel bioreactors. The procedure resulted in the identification of a best strain with lysine titer and yield at pH 6.0 comparable to the parent strain at pH 6.8.ConclusionOur results demonstrate a promising synthetic-biology strategy to enhance the growth robustness and productivity of E. coli upon the mildly acidic conditions, in both a general lab strain MG1655 and an industrial lysine-producing strain SCEcL3, by using the stress-responsive synthetic acid-tolerance modules comprising a limited number of genes. This study provides a reliable and efficient method for achieving synthetic modules of interest, particularly in improving the robustness and productivity of industrial strains.

Is energy excess the initial trigger of carbon overflow metabolism? Transcriptional network response of carbon-limited Escherichia coli to transient carbon excess

BackgroundEscherichia coli adapted to carbon-limiting conditions is generally geared for energy-efficient carbon utilization. This includes also the efficient utilization of glucose, which serves as a source for cellular building blocks as well as energy. Thus, catabolic and anabolic functions are balanced under these conditions to minimize wasteful carbon utilization. Exposure to glucose excess interferes with the fine-tuned coupling of anabolism and catabolism leading to the so-called carbon overflow metabolism noticeable through acetate formation and eventually growth inhibition.ResultsCellular adaptations towards sudden but timely limited carbon excess conditions were analyzed by exposing slow-growing cells in steady state glucose-limited continuous culture to a single glucose pulse. Concentrations of metabolites as well as time-dependent transcriptome alterations were analyzed and a transcriptional network analysis performed to determine the most relevant transcription and sigma factor combinations which govern these adaptations. Down-regulation of genes related to carbon catabolism is observed mainly at the level of substrate uptake and downstream of pyruvate and not in between in the glycolytic pathway. It is mainly accomplished through the reduced activity of CRP-cAMP and through an increased influence of phosphorylated ArcA. The initiated transcriptomic change is directed towards down-regulation of genes, which contribute to active movement, carbon uptake and catabolic carbon processing, in particular to down-regulation of genes which contribute to efficient energy generation. Long-term changes persisting after glucose depletion and consumption of acetete encompassed reduced expression of genes related to active cell movement and enhanced expression of genes related to acid resistance, in particular acid resistance system 2 (GABA shunt) which can be also considered as an inefficient bypass of the TCA cycle.ConclusionsOur analysis revealed that the major part of the trancriptomic response towards the glucose pulse is not directed towards enhanced cell proliferation but towards protection against excessive intracellular accumulation of potentially harmful concentration of metabolites including among others energy rich compounds such as ATP. Thus, resources are mainly utilized to cope with “overfeeding” and not for growth including long-lasting changes which may compromise the cells future ability to perform optimally under carbon-limiting conditions (reduced motility and ineffective substrate utilization).