Acid Resistance Mechanisms Research Articles

Microbial removal of phenol and ammonium from acidic wastewater using recycled polyurethane: Performance and mechanism

Using solid waste as fortifying agent can improve the efficiency of biological treatment of wastewater and solve the problem of difficult treatment of solid waste. In this study, the waste polyurethane was used as the immobilized carrier of strain, and the repair effect of the immobilized system on acidic phenol-containing wastewater was investigated under different conditions and the mechanism of microbial degradation and acid resistance enhanced by the carrier was discussed. The results showed that polyurethane immobilization improved microorganisms' resistance to harsh environments, strain growth, and pollutant removal efficiency in wastewater. Optimizing the culture conditions of the immobilized system increased the average removal efficiency of phenol and NH4+ by 68.77 % and 135.12 %, respectively, compared to the free strain. Additionally, immobilization enhanced the strain's tolerance to phenol, achieving efficient removal of high-concentration phenol-containing wastewater (2200 mg L−1) that free bacteria could not handle. In the MBBR reactor, the immobilized polyurethane-strain Hly3 system operated stably for 20 cycles, removing 95 % of 2000 mg L−1 phenol and 65 % of 100 mg L−1 NH4+. The mechanism by which the carrier improved microbial degradation and acid resistance was studied. It was found that carrier protection, accelerated material transfer, enhanced strain resistance, and increased activity of the acid-resistant reaction system were the main reasons for the improved acid resistance and degradation ability of strain Hly3. From an environmental and economic perspective, recycling polyurethane buffer materials as carriers to immobilize microorganisms offers a cost-effective wastewater treatment solution, also addressing the challenge of difficult solid plastic waste treatment.

A self-isolated acid-tolerant Parachlorella kessleri with high efficiency in treating rare earth mining sewage

Rare earth mining sewage is a significant environmental concern due to its high acidity and ammonia nitrogen levels. Finding a sustainable and cost-effective treatment method is essential. Parachlorella kessleri FM2, a green algae strain isolated in-house, has demonstrated remarkable abilities to grow and remove ammonia nitrogen (NH4+-N) from highly acidic rare earth wastewater without the need for alkaline additives. After optimizing conditions, P. kessleri FM2 achieved an impressive NH4+-N removal rate of 7.94 mg/L/d and a removal efficiency of 98.71% in a 1.5-L photobioreactor. In semi-continuous culture, the average NH4+-N removal rate remained high at 6.67 mg/L/d. When scaled up to continuous culture in a 5-L photobioreactor, P. kessleri FM2 maintained stability with an NH4+-N removal rate of 6.79 mg/L/d. Additionally, we conducted a preliminary analysis of P. kessleri FM2's acid resistance mechanism, further highlighting its potential as a candidate for treating acidic wastewater.

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.

Genomic and proteomic analysis of Salmonella Enteritidis isolated from a patient with foodborne diarrhea.

Salmonella is a major cause of foodborne diseases and clinical infections worldwide. This study aimed to investigate the drug resistance, genomic characteristics, and protein expression of foodborne Salmonella in Shanxi Province. We isolated a strain of Salmonella Enteritidis from patient feces and designated it 31A. The drug resistance of 31A against 14 antibiotics was determined using an antimicrobial susceptibility test. Whole-genome sequencing and quantitative proteomic analysis were performed on the 31A strain. Functional annotation of drug resistance genes/proteins and virulence genes/proteins was conducted using various databases, such as VFDB, ARDB, CAZY, COG, KOG, CARD, GO, and KEGG. The focus of this study was understanding the mechanisms related to food poisoning, and the genetic evolution of 31A was analyzed through comparative genomics. The 31A strain belonged to ST11 Salmonella Enteritidis and showed resistance to β-lactam and quinolone antibiotics. The genome of 31A had 70 drug resistance genes, 321 virulence genes, 12 SPIs, and 3 plasmid replicons. Functional annotation of these drug resistance and virulence genes revealed that drug resistance genes were mainly involved in defense mechanisms to confer resistance to antibiotics, while virulence genes were mainly associated with cellular motility. There were extensive interactions among the virulence genes, which included SPI-1, SPI-2, flagella, fimbriae, capsules and so on. The 31A strain had a close relationship with ASM2413794v1 and ASM130523v1, which were also ST11 Salmonella Enteritidis strains from Asia and originated from clinical patients, animals, and food. These results suggested minimal genomic differences among strains from different sources and the potential for interhost transmission. Differential analysis of the virulence and drug resistance-related proteins revealed their involvement in pathways related to human diseases, indicating that these proteins mediated bacterial invasion and infection. The integration of genomic and proteomic information led to the discovery that Salmonella can survive in a strong acid environment through various acid resistance mechanisms after entering the intestine with food and then invade intestinal epithelial cells to exert its effects. In this study, we comprehensively analyzed the drug resistance and virulence characteristics of Salmonella Enteritidis 31A using a combination of genomic and proteomic approaches, focusing on the pathogenic mechanism of Salmonella Enteritidis in food poisoning. We found significant fluctuations in various virulence factors during the survival, invasion, and infection of Salmonella Enteritidis, which collectively contributed to its pathogenicity. These results provide important information for the source tracing, prevention, and treatment of clinical infections caused by Salmonella Enteritidis.

Combined genomic-proteomic approach in the identification of Campylobacter coli amoxicillin-clavulanic acid resistance mechanism in clinical isolates.

Aminopenicillins resistance among Campylobacter jejuni and Campylobacter coli strains is associated with a single mutation in the promoting region of a chromosomal beta-lactamase blaOXA61, allowing its expression. Clavulanic acid is used to restore aminopenicillins activity in case of blaOXA61 expression and has also an inherent antimicrobial activity over Campylobacter spp. Resistance to amoxicillin-clavulanic acid is therefore extremely rare among these species: only 0.1% of all Campylobacter spp. analyzed in the French National Reference Center these last years (2017-2022). Whole genome sequencing with bioinformatic resistance identification combined with mass spectrometry (MS) was used to identify amoxicillin-acid clavulanic resistance mechanism in Campylobacters. A G57T mutation in blaOXA61 promoting region was identified in all C. jejuni and C. coli ampicillin resistant isolates and no mutation in ampicillin susceptible isolates. Interestingly, three C. coli resistant to both ampicillin and amoxicillin-clavulanic acid displayed a supplemental deletion in the promoting region of blaOXA61 beta-lactamase, at position A69. Using MS, a significant difference in the expression of BlaOXA61 was observed between these three isolates and amoxicillin-clavulanic acid susceptible C. coli. A combined genomics/proteomics approach allowed here to identify a rare putative resistance mechanism associated with amoxicillin-clavulanic acid resistance for C. coli.

Acid-resistant enzymes: the acquisition strategies and applications.

Enzymes have promising applications in chemicals, food, pharmaceuticals, and other variety products because of their high efficiency, specificity, and environmentally friendly properties. However, due to the complexity of raw materials, pH, temperature, solvents, etc., the application range of enzymes is greatly limited in the industry. Protein engineering and enzyme immobilization are classical strategies to overcome the limitations of industrial applications. Although the pH tendency of enzymes has been extensively researched, the mechanism underlying enzyme acid resistance is unclear, and a less practical strategy for altering the pH propensity of enzymes has been suggested. This review proposes that the optimum pH of enzyme is determined by the pKa values of active center ionizable amino acid residues. Three levels of acquiring acid-resistant enzymes are summarized: mining from extreme environments and enzyme databases, modification with protein engineering and enzyme microenvironment engineering, and de novo synthesis. The industrial applications of acid-resistant enzymes in chemicals, food, and pharmaceuticals are also summarized. KEY POINTS: • The mechanism of enzyme acid resistance is fundamentally determined. • The three aspects of the method for acquiring acid-resistant enzymes are summarized. • Computer-aided strategies and artificial intelligence are used to obtain acid-resistant enzymes.

The role of PhoP/PhoQ system in regulating stress adaptation response in Escherichia coli O157:H7.

The development of acid tolerance response (ATR) as a result of low pH in Escherichia coli O157:H7 (E. coli O157:H7) contaminating beef during processing is considered a major food safety concern. Thus, in order to explore the formation and molecular mechanisms of the tolerance response of E. coli O157:H7 in a simulated beef processing environment, the resistance of a wild-type (WT) strain and its corresponding ΔphoP mutant to acid, heat, and osmotic pressure was evaluated. Strains were pre-adapted under different conditions of pH (5.4 and 7.0), temperature (37°C and 10°C), and culture medium (meat extract and Luria-Bertani broth media). In addition, the expression of genes related to stress response and virulence was also investigated among WT and ΔphoP strains under the tested conditions. Pre-acid adaptation increased the resistance of E. coli O157:H7 to acid and heat treatment while resistance to osmotic pressure decreased. Moreover, acid adaptation in meat extract medium simulating slaughter environment increased ATR, whereas pre-adaptation at 10°C reduced the ATR. Furthermore, it was shown that mildly acidic conditions (pH=5.4) and the PhoP/PhoQ two-component system (TCS) acted synergistically to enhance acid and heat tolerance in E. coli O157:H7. Additionally, the expression of genes related to arginine and lysine metabolism, heat shock, and invasiveness was up-regulated, which revealed that the mechanism of acid resistance and cross-protection under mildly acidic conditions was mediated by the PhoP/PhoQ TCS. Both acid adaptation and phoP gene knockout reduced the relative expression of stx1 and stx2 genes which were considered as critical pathogenic factors. Collectively, the current findings indicated that ATR could occur in E. coli O157:H7 during beef processing. Thus, there is an increased food safety risk due to the persistence of tolerance response in the following processing conditions. The present study provides a more comprehensive basis for the effective application of hurdle technology in beef processing.

Acid resistance and failure mechanism of zinc-rich primer/micaceous iron oxide/polyurethane composite coating immersed in 5 wt% sulfuric acid

In order to study the reliable resistance to sulfuric acid corrosion of epoxy zinc rich primer/micairon oxide intermediate paint/acrylic polyurethane topcoat composite coatings for power transmission and transformation equipment. In this paper, under the condition of immersion in 5 wt% sulfuric acid, the sulfuric acid resistance behaviour of zinc-rich epoxy/micaceous iron oxide/polyurethane composite coating was evaluated by performing soaking tests for 50 h, 100 h and 150 h, and the failure mechanism of the film was studied. The changes in the surface properties of the coating during sulfuric acid soaking were evaluated by testing the hardness, glossiness and adhesion, and the changes in the capacitance and resistance characteristics of the coating during acid soaking were studied by Electrochemical Impedance Spectroscopy (EIS). The micromorphology and chemical element distribution of the cross-section of the composite coatings were studied by Scanning electron microscope (SEM) and Energy-dispersive X-ray spectroscopy (EDS). The functional group changes were studied by Fourier transform infrared spectroscopy (FT-IR). The results show that the corrosive effect of sulfuric acid solution immersion on acrylic polyurethane is weak, while the hydrolysis of water molecules will lead to aging and failure of acrylic polyurethane. Sulfuric acid leads to the formation of zinc deficient areas in zinc-rich primers, which reduces the cathodic protection effect of zinc-rich primers. The service life of the zinc-rich primer/ Micaceous Iron Oxide/acrylic polyurethane composite coating is less than 150 h.

Molecular mechanism of engineered Zymomonas mobilis to furfural and acetic acid stress

Acetic acid and furfural (AF) are two major inhibitors of microorganisms during lignocellulosic ethanol production. In our previous study, we successfully engineered Zymomonas mobilis 532 (ZM532) strain by genome shuffling, but the molecular mechanisms of tolerance to inhibitors were still unknown. Therefore, this study investigated the responses of ZM532 and its wild-type Z. mobilis (ZM4) to AF using multi-omics approaches (transcriptomics, genomics, and label free quantitative proteomics). Based on RNA-Seq data, two differentially expressed genes, ZMO_RS02740 (up-regulated) and ZMO_RS06525 (down-regulated) were knocked out and over-expressed through CRISPR-Cas technology to investigate their roles in AF tolerance. Overall, we identified 1865 and 14 novel DEGs in ZM532 and wild-type ZM4. In contrast, 1532 proteins were identified in ZM532 and wild-type ZM4. Among these, we found 96 important genes in ZM532 involving acid resistance mechanisms and survival rates against stressors. Furthermore, our knockout results demonstrated that growth activity and glucose consumption of mutant strains ZM532∆ZMO_RS02740 and ZM4∆ZMO_RS02740 decreased with increased fermentation time from 42 to 55 h and ethanol production up to 58% in ZM532 than that in ZM532∆ZMO_RS02740. Hence, these findings suggest ZMO_RS02740 as a protective strategy for ZM ethanol production under stressful conditions.

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.

Trehalose transport occurs via TreB in Listeria monocytogenes and it influences biofilm development and acid resistance

Listeria monocytogenes is a pathogenic bacterium that can inhabit a diverse range of environmental niches. This is largely attributed to the high proportion of carbohydrate-specific phosphotransferase system (PTS) genes in its genome. Carbohydrates can be assimilated as sources of energy but additionally they can serve as niche-specific cues for L. monocytogenes to shape its global gene expression, in order to cope with anticipated stresses. To examine carbon source utilization among wild L. monocytogenes isolates and to understand underlying molecular mechanisms, a diverse collection of L. monocytogenes strains (n = 168) with whole genome sequence (WGS) data available was screened for the ability to grow in chemically defined media with different carbon sources. The majority of the strains grew in glucose, mannose, fructose, cellobiose, glycerol, trehalose, and sucrose. Maltose, lactose, and rhamnose supported slower growth while ribose did not support any growth. In contrast to other strains, strain1386, which belonged to clonal complex 5 (CC5), was unable to grow on trehalose as a sole carbon source. WGS data revealed that it carried a substitution (N352K) in a putative PTS EIIBC trehalose transporter, TreB, while this asparagine residue is conserved in other strains in this collection. Spontaneous mutants of strain 1386 that could grow in trehalose were found to harbour a reversion of the substitution in TreB. These results provide genetic evidence that TreB is responsible for trehalose uptake and that the N352 residue is essential for TreB activity. Moreover, reversion mutants also restored other unusual phenotypes that strain 1386 displayed, i.e. altered colony morphology, impaired biofilm development, and reduced acid resistance. Transcriptional analysis at stationary phase with buffered BHI media revealed that trehalose metabolism positively influences the transcription of genes encoding amino acid-based acid resistance mechanisms. In summary, our results demonstrated that N352 is key to the function of the sole trehalose transporter TreB in L. monocytogenes and suggest that trehalose metabolism alters physiology to favour biofilm development and acid stress resistance. Moreover, since strain 1386 is among the strains recommended by the European Union Reference Laboratory for conducting food challenge studies in order to determine whether or not L. monocytogenes can grow in food, these findings have important implications for food safety.

Probiotic Characteristics and Safety Assessment of Lacticaseibacillus casei KGC1201 Isolated from Panax ginseng.

Panax ginseng is one of the most important herbal medicinal plants consumed as health functional food and can be fermented to achieve better efficacy. Lacticaseibacillus, one of the representative genera among lactic acid bacteria (LAB), has also been used as a probiotic material for health functional foods due to its beneficial effects on the human body. To achieve a synergistic effect by using these excellent dietary supplement ingredients together, a novel LAB strain was isolated from the root of 6-year-old ginseng. Through similarity analysis of 16S rRNAs and whole-genome sequences, the strain was confirmed as belonging to the genus Lacticaseibacillus and was named L. casei KGC1201. KGC1201 not only met all safety standards as food, but also showed excellent probiotic properties such as acid resistance, bile salt resistance, and intestinal adhesion. In particular, KGC1201 exhibited superior acid resistance through morphological observation identifying that the cell surface damage of KGC1201 was less than that of the L. casei type strain KCTC3109. Gene expression studies were conducted to elucidate the molecular mechanisms of KGC1201's acid resistance, and the expression of the glycosyltransferase gene was found to be significantly elevated under acidic conditions. Exopolysaccharides (EPSs) biosynthesized by glycosyltransferase were also increased in KGC1201 compared to KCTC3109, which may contribute to better protection of KGC1201 cells from strong acidity. Therefore, KGC1201, with its increased acid resistance through molecular mechanisms and excellent probiotic properties, can be used in health functional foods to provide greater benefit to overall human health and well-being.

Molecular evolution and population genetics of glutamate decarboxylase acid resistance pathway in lactic acid bacteria.

Glutamate decarboxylase (GAD) pathway (GDP) is a major acid resistance mechanism enabling microorganisms' survival in low pH environments. We aimed to study the molecular evolution and population genetics of GDP in Lactic Acid Bacteria (LAB) to understand evolutionary processes shaping adaptation to acidic environments comparing species where the GDP genes are organized in an operon structure (Levilactobacillus brevis) versus lack of an operon structure (Lactiplantibacillus plantarum). Within species molecular population genetic analyses of GDP genes in L. brevis and L. plantarum sampled from diverse fermented food and other environments showed abundant synonymous and non-synonymous nucleotide diversity, mostly driven by low frequency changes, distributed throughout the coding regions for all genes in both species. GAD genes showed higher level of replacement polymorphism compared to transporter genes (gadC and YjeM) for both species, and GAD genes that are outside of an operon structure showed even higher level of replacement polymorphism. Population genetic tests suggest negative selection against replacement changes in all genes. Molecular structure and amino acid characteristics analyses showed that in none of the GDP genes replacement changes alter 3D structure or charge distribution supporting negative selection against non-conservative amino acid changes. Phylogenetic and between species divergence analyses suggested adaptive protein evolution on GDP genes comparing phylogenetically distant species, but conservative evolution comparing closely related species. GDP genes within an operon structure showed slower molecular evolution and higher conservation. All GAD and transporter genes showed high codon usage bias in examined LAB species suggesting high expression and utilization of acid resistance genes. Substantial discordances between species, GAD, and transporter gene tree topologies were observed suggesting molecular evolution of GDP genes do not follow speciation events. Distribution of operon structure on the species tree suggested multiple independent gain or loss of operon structure in LABs. In conclusion, GDP genes in LABs exhibit a dynamic molecular evolutionary history shaped by gene loss, gene transfer, negative and positive selection to maintain its active role in acid resistance mechanism, and enable organisms to thrive in acidic environments.

New Insights into the Lactic Acid Resistance Determinants of Listeria monocytogenes Based on Transposon Sequencing and Transcriptome Sequencing Analyses.

Listeria monocytogenes is a foodborne pathogen that can tolerate a variety of extreme environments. In particular, its acid resistance (AR) capability is considered one of the key factors threating food safety. Here, we employed a microbial functional genomic technology termed transposon sequencing (Tn-seq), leading to the identification of two genes involved in cell wall peptidoglycan biosynthesis (murF) and phosphate transport (lmo2248) that play key roles in lactic acid resistance (LAR) of L. monocytogenes. Deletion of lmo2248 significantly impaired the ability of LAR in L. monocytogenes, demonstrating the accuracy of the Tn-seq results. Transcriptome analysis revealed that 31.7% of the L. monocytogenes genes on the genome were differentially expressed under lactic acid (LA) treatment, in which genes involved in phosphate transport were influenced most significantly. These findings shed light on the LAR mechanisms of L. monocytogenes, which may contribute to the development of novel strategies against foodborne pathogens. IMPORTANCE Listeria monocytogenes is a Gram-positive foodborne pathogen with high lethality and strong stress resistance, and its strong acid tolerance leads to many foodborne illnesses occurring in low-pH foods. Lactic acid is a generally recognized as safe (GRAS) food additive approved for use by the FDA. However, the genetic determinants of lactic acid resistance in L. monocytogenes have not been fully identified. In this study, the lactic acid resistance determinants of L. monocytogenes were comprehensively identified by Tn-seq on a genome-wide scale. Two genes, murF (cell wall peptidoglycan biosynthesis) and lmo2248 (phosphate transport), were identified to play an important role in the lactic acid resistance. Moreover, genome-wide transcriptomic analysis showed that phosphotransferase system (PTS)-related genes play a key role at the transcriptional level. These findings contribute to a better understanding of the lactic acid resistance mechanism of L. monocytogenes and may provide unique targets for the development of other novel antimicrobial agents.

Heterogeneity and Evolutionary Tunability of Escherichia coli Resistance against Extreme Acid Stress.

Since acidic environments often serve as an important line of defense against bacterial pathogens, it is important to fully understand how the latter manage to mount and evolve acid resistance mechanisms. Escherichia coli, a species harboring many pathovars, is typically equipped with the acid fitness island (AFI), a genomic region encoding the GadE master regulator together with several GadE-controlled functions to counter acid stress. This study reveals that gadE and consequently AFI functions are heterogeneously expressed even in the absence of any prior acid stress, thereby preemptively creating acid-resistant subpopulations within a clonal E. coli population. Directed evolution efforts selecting for modulated gadE expression confirm that a gain-of-function mutation in the EvgS sensor kinase can constitutively upregulate gadE expression and concomitant acid resistance. However, we reveal that such upregulation of EvgS also causes cross-resistance to heat stress because of SafA-mediated cross-activation of the PhoPQ regulon. Surprisingly, loss of function of the serC gene (encoding phosphoserine/phosphohydroxythreonine aminotransferase) can also significantly upregulate gadE expression, acid resistance, and heat cross-resistance, although via a currently cryptic mechanism. As such, our data reveal a noisy expression of gadE in E. coli that is functional for the survival of sudden acid stress and that can readily be genetically tuned. IMPORTANCE Acidic environments constitute one of the most important stresses for enteric bacteria and can be encountered in both natural (e.g., host gastrointestinal tract) and manmade (e.g., food processing) environments. The enteric species Escherichia coli harbors many pathovars and is well known for its ability to cope with acid stress. In this study, we uncover that E. coli's acid fitness island (AFI), a genomic region that encodes important functions to deal with acid stress, is by default expressed in a heterogeneous manner. In fact, using microfluidics-based single-cell approaches, we further demonstrate that this heterogeneity preemptively creates a clonal subpopulation that is much better equipped to survive a sudden acid shock. In addition, we reveal that environments with recurring acid stress can readily select for mutants displaying a higher fraction of AFI-expressing cells. These new insights are important to properly understand and anticipate the survival characteristics of E. coli.

Methionine addition improves the acid tolerance of Lactiplantibacillus plantarum by altering cellular metabolic flux, energy distribution, lipids composition

This paper reported a wine-derived lactic acid bacterium, Lactiplantibacillus plantarum XJ25, which exhibited higher cell viability under acid stress upon methionine supplementation. Cellular morphology and the composition of the cytomembrane phospholipids revealed a more solid membrane architecture presented in the acid-stressed cells treated with methionine supplementation. Transcriptional analysis showed L. plantarum XJ25 reduced methionine transport and homocysteine biosynthesis under acid stress. Subsequent overexpression assays proved that methionine supplementation could alleviate the cell toxicity from homocysteine accumulation under acid stress. Finally, L. plantarum XJ25 employed energy allocation strategy to response environmental changes by balancing the uptake methionine and adjusting saturated fatty acids (SFAs) in membrane. These data support a novel mechanism of acid resistance involving methionine utilization and cellular energy distribution in LAB and provide crucial theoretical clues for the mechanisms of acid resistance in other bacteria.

More than Meets the Eye: Untargeted Metabolomics and Lipidomics Reveal Complex Pathways Spurred by Activation of Acid Resistance Mechanisms in Escherichia coli.

Escherichia coli is a ubiquitous group of bacteria that can be either commensal gut microbes or enterohemorrhagic food-borne pathogens. Regardless, both forms must survive acidic environments in the stomach and intestines to reach and colonize the gut, a process that partially relies on amino acid-dependent acid resistance (AR) mechanisms and modifications to membrane phospholipids. However, only the basic tenets of these mechanisms have been elucidated. In this paper, we aim to conduct a full-scale metabolic and lipidomic characterization of E. coli's adaptations to acid stress. We hypothesized that the use of untargeted metabolomics and lipidomics would reveal mechanisms downstream of AR processes that provide novel contributions to acid stress survival. We detected significant differences in the extracellular metabolome and the lipidome induced by amino acid supplementation (glutamine, arginine, or lysine) and contextualized these results using real-time quantitative polymerase chain reaction (RT-qPCR). We additionally identified several metabolic pathways as well as a significant alteration in phospholipid synthetic pathways induced by differential amino acid supplementation. These results demonstrate that AR may extend beyond canonical mechanisms to a coordinated metabolic phenotype. Future studies may benefit from our analysis to further elucidate distinct targets for prebiotic supplements to cultivate commensal strains or therapies to combat pathogenic ones.

Revealing novel synergistic defense and acid tolerant performance of Escherichia coli in response to organic acid stimulation.

Escherichia coli is an important producer of mono- and di-acids, such as D-lactic acid, itaconic acid, and succinic acid. However, E. coli has limited acid tolerance and requires neutralizers in large-scale fermentation, which leads to increased production costs. Mutagenesis breeding has been shown to be inefficient in improving the acid tolerance of strains. Therefore, it is crucial to analyze the acid resistance mechanism of E. coli. To this end, important regulatory genes and metabolic pathways in the highly evolved acid-resistant E. coli were identified based on transcriptome sequencing. By analyzing the overlap of the genes with significantly different expression levels in the four groups, a synergistic membrane-centric defense mechanism for E. coli against organic acid stress was identified. The mechanism includes four modules: signal perception, energy countermeasures, input conditioning, and envelope reinforcement. In addition, genes related to the ABC transporter pathway, polyketide metabolism, pyrimidine metabolism, and dual-arginine translocation system pathways were found for the first time to be potentially resistant to organic acid stress after overexpression. A new antacid ingredient, RffG, increases the survival rate of E. coli by 4509.6 times. This study provides new clues for improving the performance of acid-tolerant cells and reducing the production cost of industrial organic acid fermentation. KEY POINTS: • Systematic analysis of the mechanism of membrane protein partitioning in E. coli to resist organic acids • TAT system transports correctly folded hydrogenase accessory proteins to resistD-lactic acid stress • Enhanced PG synthesis and weakened hydrolysis to reduce acid penetration into cells • Overexpression of RffG in the polyketide synthesis pathway enhances acid tolerance.

RNA-Seq transcriptomic analysis reveals gene expression profiles of acetic acid bacteria under high-acidity submerged industrial fermentation process.

Acetic acid bacteria (AAB) are Gram-negative obligate aerobics in Acetobacteraceae family. Producing acetic acid and brewing vinegars are one of the most important industrial applications of AAB, attributed to their outstanding ability to tolerate the corresponding stresses. Several unique acid resistance (AR) mechanisms in AAB have been revealed previously. However, their overall AR strategies are still less-comprehensively clarified. Consequently, omics analysis was widely performed for a better understanding of this field. Among them, transcriptome has recently obtained more and more attention. However, most currently reported transcriptomic studies were conducted under lab conditions and even in low-acidity environment, which may be unable to completely reflect the conditions that AAB confront under industrialized vinegar-brewing processes. In this study, we performed an RNA-Seq transcriptomic analysis concerning AAB’s AR mechanisms during a continuous and periodical industrial submerged vinegar fermentation process, where a single AAB strain performed the fermentation and the acetic acid concentration fluctuated between ~8% and ~12%, the highest acidity as far we know for transcriptomic studies. Samples were directly taken from the initial (CK), mid, and final stages of the same period of the on-going fermentation. 16S rRNA sequence analysis indicated the participation of Komagataeibacter europaeus in the fermentation. Transcriptomic results demonstrated that more genes were downregulated than upregulated at both mid and final stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrich analysis reflected that the upregulated genes mainly carried out tricarboxylic acid cycle and oxidative phosphorylation processes, probably implying a considerable role of acetic acid overoxidation in AR during fermentation. Besides, upregulation of riboflavin biosynthesis pathway and two NAD+-dependent succinate-semialdehyde dehydrogenase-coding genes suggested a critical role of succinate oxidation in AR. Meanwhile, downregulated genes were mainly ribosomal protein-coding ones, reflecting that the adverse impact on ribosomes initiates at the transcription level. However, it is ambiguous whether the downregulation is good for stress responding or it actually reflects the stress. Furthermore, we also assumed that the fermentation stages may have a greater effect on gene expression than acidity. Additionally, it is possible that some physiological alterations would affect the AR to a larger extent than changes in gene expression, which suggests the combination of molecular biology and physiology research will provide deeper insight into the AR mechanisms in AAB.

Genome-guided prediction of acid resistance mechanisms in acidophilic methanotrophs of phylogenetically deep-rooted Verrucomicrobia isolated from geothermal environments.

Verrucomicrobia are a group of microorganisms that have been proposed to be deeply rooted in the Tree of Life. Some are methanotrophs that oxidize the potent greenhouse gas methane and are thus important in decreasing atmospheric concentrations of the gas, potentially ameliorating climate change. They are widespread in various environments including soil and fresh or marine waters. Recently, a clade of extremely acidophilic Verrucomicrobia, flourishing at pH < 3, were described from high-temperature geothermal ecosystems. This novel group could be of interest for studies about the emergence of life on Earth and to astrobiologists as homologs for possible extraterrestrial life. In this paper, we describe predicted mechanisms for survival of this clade at low pH and suggest its possible evolutionary trajectory from an inferred neutrophilic ancestor. Extreme acidophiles are defined as organisms that thrive in extremely low pH environments (≤ pH 3). Many are polyextremophiles facing high temperatures and high salt as well as low pH. They are important to study for both providing fundamental insights into biological mechanisms of survival and evolution in such extreme environments and for understanding their roles in biotechnological applications such as industrial mineral recovery (bioleaching) and mitigation of acid mine drainage. They are also, potentially, a rich source of novel genes and pathways for the genetic engineering of microbial strains. Acidophiles of the Verrucomicrobia phylum are unique as they are the only known aerobic methanotrophs that can grow optimally under acidic (pH 2–3) and moderately thermophilic conditions (50–60°C). Three moderately thermophilic genera, namely Methylacidiphilum, Methylacidimicrobium, and Ca. Methylacidithermus, have been described in geothermal environments. Most of the investigations of these organisms have focused on their methane oxidizing capabilities (methanotrophy) and use of lanthanides as a protein cofactor, with no extensive study that sheds light on the mechanisms that they use to flourish at extremely low pH. In this paper, we extend the phylogenetic description of this group of acidophiles using whole genome information and we identify several mechanisms, potentially involved in acid resistance, including “first line of defense” mechanisms that impede the entry of protons into the cell. These include the presence of membrane-associated hopanoids, multiple copies of the outer membrane protein (Slp), and inner membrane potassium channels (kup, kdp) that generate a reversed membrane potential repelling the intrusion of protons. Acidophilic Verrucomicrobia also display a wide array of proteins potentially involved in the “second line of defense” where protons that evaded the first line of defense and entered the cell are expelled or neutralized, such as the glutamate decarboxylation (gadAB) and phosphate-uptake systems. An exclusive N-type ATPase F0-F1 was identified only in acidophiles of Verrucomicrobia and is predicted to be a specific adaptation in these organisms. Phylogenetic analyses suggest that many predicted mechanisms are evolutionarily conserved and most likely entered the acidophilic lineage of Verrucomicrobia by vertical descent from a common ancestor. However, it is likely that some defense mechanisms such as gadA and kup entered the acidophilic Verrucomicrobia lineage by horizontal gene transfer.