The organic acid environmental capacity of mangrove ecosystem in Dongzhai harbor, Hainan, China.

To establish a method for studying the organic acid environmental capacity of mangrove ecosystems, high-performance liquid chromatography was used to measure the organic acid detoxification agent; Using different cultivation methods to determine the toxicity threshold of organic acids on mangrove plants; Calculate the environmental capacity of organic acids by combining the toxicity threshold of organic acids with the volume of water in the study area. The results showed the range of toxicity thresholds of organic acids to 25.29-30 mg/L would have an inhibitory effect on the development of mangrove plant hypocotyls; The organic acid environmental capacity of Dongzhai harbor Mangrove Wetland Protection Area is 7.76 × 10^4 kg/d ~ 8.73 × 10^4 kg/d, while the estimated organic acid emissions from shrimp ponds around Dongzhai harbor are 7.06 × 10^3 kg/d ~ 7.83 × 10^3 kg/d. Therefore, the organic acid emissions from shrimp ponds around Dongzhai harbor are within the carrying range of the mangrove wetland ecosystem in Dongzhai harbor.

Lytic transglycosylase repertoire diversity enables intrinsic antibiotic resistance and daughter cell separation in Escherichia coli under acidic stress.

Peptidoglycan (PG) is an important architectural element that imparts physical toughness and rigidity to the bacterial envelope. It is also a dynamic structure that undergoes continuous turnover or autolysis. Escherichia coli possesses redundant PG degradation enzymes responsible for PG turnover; however, the advantage afforded by the existence of numerous PG degradation enzymes remains incompletely understood. In this study, we elucidated the physiological roles of MltE and MltC, members of the lytic transglycosylase (LTG) family that catalyze the cleavage of glycosidic bonds between disaccharide subunits within PG strands. MltE and MltC are acidic LTGs that exhibit increased enzymatic activity and protein levels under acidic pH conditions, respectively, and deletion of these two LTGs results in a pronounced growth defect at acidic pH. Furthermore, inactivation of these two LTGs induces increased susceptibility at acidic pH against various antibiotics, particularly vancomycin, which seems to be partially caused by elevated membrane permeability. Intriguingly, inactivation of these LTGs induces a chaining morphology, indicative of daughter cell separation defects, only under acidic pH conditions. Simultaneous deletion of PG amidases, known contributors to daughter cell separation, exacerbates the chaining phenotype at acidic pH. This suggests that the two LTGs may participate in the cleavage of glycan strands between daughter cells under acidic pH conditions. Collectively, our findings highlight the role of LTG repertoire diversity in facilitating bacterial survival and antibiotic resistance under stressful conditions.

Preliminary investigation on the potential involvement of an ABC-like gene in Halomicronema metazoicum (Cyanobacteria) tolerance to low seawater pH in an ocean acidification scenario.

Decreasing ocean surface pH, called ocean acidification (OA), is among the major risks for marine ecosystems due to human-driven atmospheric pCO2 increase. Understanding the molecular mechanisms of adaptation enabling marine species to tolerate a lowered seawater pH could support predictions of consequences of future OA scenarios for marine life. This study examined whether the ATP-binding cassette (ABC)-like gene slr2019 confers tolerance to the marine cyanobacterium Halomicronema metazoicum to low seawater pH conditions (7.7, 7.2, 6.5) in short- and long-term exposures (7 and 30 d). Photosynthetic pigment content indicated that the species can tolerate all three lowered-pH conditions. At day 7, slr2019 was up-regulated at pH 7.7 while no changes were observed at lower pH. After 30-d exposure, a significant decrease in slr2019 transcript levels was observed in all low-pH treatments. These first results indicate an effect of low pH on the examined transporter expression in H. metazoicum.

Meta-analysis Driven Strain Design for Mitigating Oxidative Stresses Important in Biomanufacturing.

As the availability of data sets increases, meta-analysis leveraging aggregated and interoperable data types is proving valuable. This study leveraged a meta-analysis workflow to identify mutations that could improve robustness to reactive oxygen species (ROS) stresses using an industrially important melatonin production strain as an example. ROS stresses often occur during cultivation and negatively affect strain performance. Cellular response to ROS is also linked to the SOS response and resistance to pH fluctuations, which is important to strain robustness in large-scale biomanufacturing. This work integrated more than 7000 E. coli adaptive laboratory evolution (ALE) mutations across 59 experiments to statistically associate mutated genes to 2 ROS tolerance ALE conditions from 72 unique conditions. Mutant oxyR, fur, iscR, and ygfZ were significantly associated and hypothesized to contribute fitness in ROS stress. Across these genes, 259 total mutations were inspected in conjunction with transcriptomics from 46 iModulon experiments. Ten mutations were chosen for reintroduction based on mutation clustering and coinciding transcriptional changes as evidence of fitness impact. Strains with mutations reintroduced into oxyR, fur, iscR, and ygfZ exhibited increased tolerance to H2O2 and acid stress and reduced SOS response, all of which are related to ROS. Additionally, new evidence was generated toward understanding the function of ygfZ, an uncharacterized gene. This meta-analysis approach utilized aggregated and interoperable multiomics data sets to identify mutations conferring industrially relevant phenotypes with the least drawbacks, describing an approach for data-driven strain engineering to optimize microbial cell factories.

Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation.

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KEY POINTS: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage.

Nutrient consumption patterns of Lactobacillus acidophilus.

BACKGROUND: One of the greatest challenges in using Lactobacillus acidophilus as a probiotic is acid stress. The current research aimed to identify substances that help L. acidophilus resist acid stress; this was achieved through assessing its nutrient consumption patterns under various pH conditions. RESULTS: The consumption rates of alanine, uracil, adenine, guanine, niacin, and manganese were consistently higher than 60% for L. acidophilus LA-5 cultured at pH 5.8, 4.9, and 4.4. The consumption rates of glutamic acid + glutamine and thiamine increased with decreasing pH and were higher than 60% at pH 4.9 and 4.4. The viable counts of L. acidophilus LA-5 were significantly increased under the corresponding acidic stress conditions (pH 4.9 and 4.4) through the appropriate addition of either alanine (3.37 and 2.81 mmol L-1), glutamic acid + glutamine (4.77 mmol L-1), guanine (0.13 and 0.17 mmol L-1), niacin (0.02 mmol L-1), thiamine (0.009 mmol L-1), or manganese (0.73 and 0.64 mmol L-1) (P < 0.05). The viable counts of L. acidophilus LA-5 cultured in a medium supplemented with combined nutritional factors was 1.02-1.03-fold of the counts observed in control medium under all acid conditions (P < 0.05). CONCLUSION: Alanine, glutamic acid + glutamine, guanine, niacin, thiamine, and manganese can improve the growth of L. acidophilus LA-5 in an acidic environment in the present study. The results will contribute to optimizing strategies to enhance the acid resistance of L. acidophilus and expand its application in the fermentation industry. © 2024 Society of Chemical Industry.

Survival and Expression of rpoS and grxB of Cronobacter sakazakii in Powdered Infant Formula Under Simulated Gastric Conditions of Newborns.

Cronobacter sakazakii can cause severe illnesses in infants, predominantly in preterm newborns, with consumption of contaminated powdered infant formula (PIF) being the major vehicle of infection. Using a dynamic human gastrointestinal simulator called the SHIME, this study examined the effects of gastric acidity and gastric digestion time of newborns on the survival and expression of stress genes of C. sakazakii. Individual strains, inoculated at 7 log CFU/mL into reconstituted PIF, were exposed to gastric pH values of 4.00, 5.00 and 6.00 for 4 h with gradual acidification. The survival results showed that C. sakazakii grew in the stomach portion of the SHIME during a 4-h exposure to pH 4.00, 5.00 and 6.00 by 0.96-1.05, 1.02-1.28 and 1.11-1.73 log CFU/mL, respectively. The expression of two stress genes, rpoS and grxB, throughout gastric digestion was evaluated using reverse transcription qPCR. The upregulation of rpoS and grxB during the 4-h exposure to simulated gastric fluid at pH 4.00 showed that C. sakazakii strains may be experiencing the most stress in the pH 4.00 treatment. The gene expression results also suggest that C. sakazakii strains appeared to develop an acid adaptation response during the 4-h exposure that may facilitate their survival. Altogether, this study highlights that a combination of low gastric acidity, long digestion time in the presence of reconstituted PIF, created a favorable environment for the adaptation and survival of C. sakazakii in the simulation of a newborn's stomach. This study gives directions for future research to further advance our understanding of the behavior of C. sakazakii in the GI tract of newborns.

Peeling the onion: additional layers of regulation in the acid stress response.

Bacteria are capable of withstanding large changes in osmolality and cytoplasmic pH, unlike eukaryotes that tightly regulate their pH and cellular composition. Previous studies on the bacterial acid stress response described a rapid, brief acidification, followed by immediate recovery. More recent experiments with better pH probes have imaged single living cells, and we now appreciate that following acid stress, bacteria maintain an acidic cytoplasm for as long as the stress remains. This acidification enables pathogens to sense a host environment and turn on their virulence programs, for example, enabling survival and replication within acidic vacuoles. Single-cell analysis identified an intracellular pH threshold of ~6.5. Acid stress reduces the internal pH below this threshold, triggering the assembly of a type III secretion system in Salmonella and the secretion of virulence factors in the host. These pathways are significant because preventing intracellular acidification of Salmonella renders it avirulent, suggesting that acid stress pathways represent a potential therapeutic target. Although we refer to the acid stress response as singular, it is actually a complex response that involves numerous two-component signaling systems, several amino acid decarboxylation systems, as well as cellular buffering systems and electron transport chain components, among others. In a recent paper in the Journal of Bacteriology, M. G. Gorelik, H. Yakhnin, A. Pannuri, A. C. Walker, C. Pourciau, D. Czyz, T. Romeo, and P. Babitzke (J Bacteriol 206:e00354-23, 2024, https://doi.org/10.1128/jb.00354-23) describe a new connection linking the carbon storage regulator CsrA to the acid stress response, highlighting new additional layers of complexity.

Novel Roles of the Greatwall Kinase Rim15 in Yeast Oxidative Stress Tolerance through Mediating Antioxidant Systems and Transcriptional Regulation.

The Greatwall-family protein kinase Rim15 is associated with the nutrient starvation response, whereas its role in oxidative stress responses remains unclear. Here, acetic acid and peroxide were used as two oxidative stress elicitors. The antioxidant indicator assay under acetic acid stress revealed the impaired growth in rim15Δ related to the regulation of antioxidant systems. Comparative transcriptome analysis revealed that differentially expressed genes (DEGs) are predicted to be mostly regulated by oxidative stress-responsive transcriptional factor Yap1. Among the DEGs, acetic acid stress-induced genes were found, and YAP1 disruption also inhibited their induction. The deletion of Rim15 or the Rim15 kinase domain in yap1Δ did not further decrease the gene expression, suggesting that Rim15 functions together with Yap1 in regulating acetic acid stress-induced genes, which requires Rim15 kinase activity. Additionally, Rim15 regulated H2O2 stress tolerance through partially similar but special mechanisms in that Rim15 kinase activity impacted acetic acid and H2O2 stress tolerance in different degrees, indicating the different mechanisms underlying Rim15-mediated redox regulation against different stressors. These results benefit the better understanding of stress signaling pathways related to Rim15. Given that Rim15 and some of its target genes are conserved across eukaryotes, these results also provide a basis for studies of oxidative stress-related processes in other organisms.

Transcriptomic analysis using RNA sequencing and phenotypic analysis of Salmonella enterica after acid exposure for different time durations using adaptive laboratory evolution.

Introduction: This study is the final part of a two-part series that delves into the molecular mechanisms driving adaptive laboratory evolution (ALE) of Salmonella enterica in acid stress. The phenotypic and transcriptomic alterations in the acid-evolved lineages (EL) of Salmonella enterica serovar Enteritidis after 70 days of acid stress exposure were analyzed. Materials and methods: The stability of phenotypic changes observed after 70 days in acetic acid was explored after stress removal using a newly developed evolutionary lineage EL5. Additionally, the impact of short-term acid stress on the previously adapted lineage EL4 was also examined. Results: The results indicate that the elevated antibiotic minimum inhibitory concentration (MIC) observed after exposure to acetic acid for 70 days was lost when acid stress was removed. This phenomenon was observed against human antibiotics such as meropenem, ciprofloxacin, gentamicin, and streptomycin. The MIC of meropenem in EL4 on day 70 was 0.094 mM, which dropped to 0.032 mM when removed from acetic acid stress after day 70. However, after stress reintroduction, the MIC swiftly elevated, and within 4 days, it returned to 0.094 mM. After 20 more days of adaptation in acetic acid, the meropenem MIC increased to 0.125 mM. The other human antibiotics that were tested exhibited a similar trend. The MIC of acetic acid in EL4 on day 70 was observed to be 35 mM, which remained constant even after the removal of acetic acid stress. Readaptation of EL4 in acetic acid for 20 more days caused the acetic acid MIC to increase to 37 mM. Bacterial whole genome sequencing of EL5 revealed base substitutions in several genes involved in pathogenesis, such as the phoQ and wzc genes. Transcriptomic analysis of EL5 revealed upregulation of virulence, drug resistance, toxin-antitoxin, and iron metabolism genes. Unstable Salmonella small colony variants (SSCV) of S. Enteritidis were also observed in EL5 as compared to the wild-type unevolved S. Enteritidis. Discussion: This study presents a comprehensive understanding of the evolution of the phenotypic, genomic, and transcriptomic changes in S. Enteritidis due to prolonged acid exposure through ALE.

Effects of Alterations in Acid-Base Effects on Insulin Signaling.

Insulin tightly regulates glucose levels within a narrow range through its action on muscle, adipose tissue and the liver. The activation of insulin receptors activates multiple intracellular pathways with different functions. Another tightly regulated complex system in the body is acid-base balance. Metabolic acidosis, defined as a blood pH < 7.35 and serum bicarbonate < 22 mmol/L, has clear pathophysiologic consequences including an effect on insulin action. With the ongoing intake of typical acid-producing Western diets and the age-related decline in renal function, there is an increase in acid levels within the range considered to be normal. This modest increase in acidosis is referred to as "acid stress" and it may have some pathophysiological consequences. In this article, we discuss the effects of acid stress on insulin actions in different tissues.

Genome-wide analysis of acid tolerance genes of Enterococcus faecalis with RNA-seq and Tn-seq.

Enterococcus faecalis, a formidable nosocomial and community-acquired opportunistic pathogen, can persist a wide range of extreme environments, including low pH and nutrient deficiency. Clarifying the survival mechanism of E. faecalis in low-pH conditions is the key to combating the infectious diseases caused by E. faecalis. In this study, we combined transcriptome profiling (RNA-seq) and transposon insertion sequencing (TIS) to comprehensively understand the genes that confer these features on E. faecalis. The metadata showed that genes whose products are involved in cation transportation and amino acid biosynthesis were predominantly differentially expressed under acid conditions. The products of genes such as opp1C and copY reduced the hydrion concentration in the cell, whereas those of gldA2, gnd2, ubiD, and ubiD2 mainly participated in amino metabolism, increasing matters to neutralize excess acid. These, together with the folE and hexB genes, which are involved in mismatch repair, form a network of E. faecalis genes necessary for its survival under acid conditions.

IMPORTANCE: As a serious nosocomial pathogen, Enterococcus faecalis was considered responsible for large numbers of infections. Its ability to survive under stress conditions, such as acid condition and nutrient deficiency was indispensable for its growth and infection. Therefore, understanding how E. faecalis survives acid stress is necessary for the prevention and treatment of related diseases. RNA-seq and TIS provide us a way to analyze the changes in gene expression under such conditions.

Multitier regulation of the E. coli extreme acid stress response by CsrA.

CsrA is an RNA-binding protein that regulates processes critical for growth and survival, including central carbon metabolism, motility, biofilm formation, stress responses, and expression of virulence factors in pathogens. Transcriptomics studies in Escherichia coli suggested that CsrA repressed genes involved in surviving extremely acidic conditions. Here, we examine the effects of disrupting CsrA-dependent regulation on the expression of genes and circuitry for acid stress survival and demonstrate CsrA-mediated repression at multiple levels. We show that this repression is critical for managing the trade-off between growth and survival; overexpression of acid stress genes caused by csrA disruption enhances survival under extreme acidity but is detrimental for growth under mildly acidic conditions. In vitro studies confirmed that CsrA binds specifically to mRNAs of structural and regulatory genes for acid stress survival, causing translational repression. We also found that translation of the top-tier acid stress regulator, evgA, is coupled to that of a small leader peptide, evgL, which is repressed by CsrA. Unlike dedicated acid stress response genes, csrA and its sRNA antagonists, csrB and csrC, did not exhibit a substantial response to acid shock. Furthermore, disruption of CsrA regulation of acid stress genes impacted host-microbe interactions in Caenorhabditis elegans, alleviating GABA deficiencies. This study expands the known regulon of CsrA to genes of the extreme acid stress response of E. coli and highlights a new facet of the global role played by CsrA in balancing the opposing physiological demands of stress resistance with the capacity for growth and modulating host interactions.IMPORTANCETo colonize/infect the mammalian intestinal tract, bacteria must survive exposure to the extreme acidity of the stomach. E. coli does this by expressing proteins that neutralize cytoplasmic acidity and cope with molecular damage caused by low pH. Because of the metabolic cost of these processes, genes for surviving acid stress are tightly regulated. Here, we show that CsrA negatively regulates the cascade of expression responsible for the acid stress response. Increased expression of acid response genes due to csrA disruption improved survival at extremely low pH but inhibited growth under mildly acidic conditions. Our findings define a new layer of regulation in the acid stress response of E. coli and a novel physiological function for CsrA.

Unraveling the Genetic Adaptations in Cell Surface Composition and Transporters of Lactiplantibacillus plantarum for Enhanced Acid Tolerance.

This study employed adaptive laboratory evolution to improve the acid tolerance of Lactiplantibacillus plantarum, a vital strain in food fermentation and a potential probiotic. Phenotype and genomic analyses identified the overexpression of stress response proteins, ATP synthases, and transporters as pivotal in conferring acid tolerance to the evolved strains. These adaptations led to a shorter lag phase, improved survival rates, and higher intracellular pH values compared to the wild-type strain under acid stress conditions. Additionally, the evolved strains showed an increased expression of genes in the fatty acid synthesis pathway, resulting in a higher production of unsaturated fatty acids. The changes in cell membrane composition possibly prevented H+ influx, while mutant genes related to cell surface structure contributed to observed elongated cells and thicker cell surface. These alterations in cell wall and membrane composition, along with improved transporter efficiency, were key factors contributing to the enhanced acid tolerance in the evolved strains.

Effect of the initial pH of the culture medium on the nutrient consumption pattern of Bifidobacterium animalis subsp. lactis Bb12 and the improvement of acid resistance by purine and pyrimidine compounds.

AIMS: During fermentation, the accumulation of acidic products can induce media acidification, which restrains the growth of Bifidobacterium animalis subsp. lactis Bb12 (Bb12). This study investigated the nutrient consumption patterns of Bb12 under acid stress and effects of specific nutrients on the acid resistance of Bb12. METHODS AND RESULTS: Bb12 was cultured in chemically defined medium (CDM) at different initial pH values. Nutrient consumption patterns were analyzed in CDM at pH 5.3, 5.7, and 6.7. The patterns varied with pH: Asp + Asn had the highest consumption rate at pH 5.3 and 5.7, while Ala was predominant at pH 6.7. Regardless of the pH levels (5.3, 5.7, or 6.7), ascorbic acid, adenine, and Fe2+ were vitamins, nucleobases, and metal ions with the highest consumption rates, respectively. Nutrients whose consumption rates exceeded 50% were added individually in CDM at pH 5.3, 5.7, and 6.7. It was demonstrated that only some of them could promote the growth of Bb12. Mixed nutrients that could promote the growth of Bb12 were added to three different CDM. In CDM at pH 5.3, 5.7, and 6.7, it was found that the viable cell count of Bb12 was the highest after adding mixed nutrients, which were 8.87, 9.02, and 9.10 log CFU ml-1, respectively. CONCLUSIONS: The findings suggest that the initial pH of the culture medium affects the nutrient consumption patterns of Bb12. Specific nutrients can enhance the growth of Bb12 under acidic conditions and increase its acid resistance.

Streptococcal arginine deiminase system defences macrophage bactericidal effect mediated by XRE family protein XtrSs.

The arginine deiminase system (ADS) has been identified in various bacteria and functions to supplement energy production and enhance biological adaptability. The current understanding of the regulatory mechanism of ADS and its effect on bacterial pathogenesis is still limited. Here, we found that the XRE family transcriptional regulator XtrSs negatively affected Streptococcus suis virulence and significantly repressed ADS transcription when the bacteria were incubated in blood. Electrophoretic mobility shift (EMSA) and lacZ fusion assays further showed that XtrSs directly bind to the promoter of ArgR, an acknowledged positive regulator of bacterial ADS, to repress ArgR transcription. Moreover, we provided compelling evidence that S. suis could utilize arginine via ADS to adapt to acid stress, while ΔxtrSs enhanced this acid resistance by upregulating the ADS operon. Moreover, whole ADS-knockout S. suis increased arginine and antimicrobial NO in the infected macrophage cells, decreased intracellular survival, and even caused significant attenuation of bacterial virulence in a mouse infection model, while ΔxtrSs consistently presented the opposite results. Our experiments identified a novel ADS regulatory mechanism in S. suis, whereby XtrSs regulated ADS to modulate NO content in macrophages, promoting S. suis intracellular survival. Meanwhile, our findings provide a new perspective on how Streptococci evade the host's innate immune system.

An oxalate decarboxylase-like cupin domain containing protein is involved in imparting acid stress tolerance in Bacillus amyloliquefaciens MBNC.

We report here the structural and functional properties of an oxalate decarboxylase (OxDC)-like cupin domain-containing protein of Bacillus amyloliquefaciens MBNC and its role in imparting tolerance to acid stress conditions. Quantitative real-time PCR (qPCR) analysis revealed 32-fold and 20-fold upregulation of the target gene [(OxDC')cupin] under acetic acid stress and hydrochloric acid stress, respectively, indicating its association with the acid stress response. Bacterial cells with targeted inactivation of the (OxDC')cupin gene using the pMUTIN4 vector system showed decreased growth and survival rate in acidic pH, with drastically reduced exopolysaccharide production. In Silico protein-protein interaction studies revealed seven genes (viz. glmS, nagA, nagB, tuaF, tuaF, gcvT, and ykgA) related to cell wall biosynthesis and biofilm production to interact with OxDC-like cupin domain containing protein. While all these seven genes were upregulated in B. amyloliquefaciens MBNC after 6 h of exposure to pH 4.5, the mutant cells containing the inactivated (OxDC')cupin gene displayed significantly lower expression (RQ: 0.001-0.02) (compared to the wild-type cells) in both neutral and acidic pH. Our results indicate that the OxDC-like cupin domain containing protein is necessary for cell wall biosynthesis and biofilm production in Bacillus amyloliquefaciens MBNC for survival in acid-stress conditions.

Physiology of γ-aminobutyric acid production by Akkermansia muciniphila.

Gut bacteria hold the potential to produce a broad range of metabolites that can modulate human functions, including molecules with neuroactive potential. One such molecule is γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter of the central nervous system in animals. Metagenomic analyses suggest that the genomes of many gut bacteria encode glutamate decarboxylase (GAD), the enzyme that catalyzes GABA production. The genome of Akkermansia muciniphila, a mucin specialist and potential next-generation probiotic from the human gut, is predicted to encode GAD, suggesting a contributing role in GABA production in the human gut. In this study, A. muciniphila was grown in batch cultures with and without pH control. In both experiments, A. muciniphila was found to produce GABA as a response to acid (pH <5.5), although only when GABA precursors, either glutamate or glutamine, were present in the medium. Proteomic analysis comparing A. muciniphila grown with and without precursors at pH 4 did not show a difference in GAD expression, suggesting that it is expressed regardless of the presence of GABA precursors. To further investigate the function of A. muciniphila GAD, we heterologously expressed the gad gene (encoded by locus tag Amuc_0372) with a His tag in Escherichia coli and purified the GAD protein. Enzyme assays showed GAD activity in a pH range between 4 and 6, with the highest specific activity at pH 5 of 144 ± 16 µM GABA/min/mg. Overall, our results demonstrate the ability of A. muciniphila to produce GABA as an acid response and unravel the conditions under which GABA production in A. muciniphila occurs.IMPORTANCEAkkermansia muciniphila is considered to be a beneficial bacterium from the human gut, but the exact mechanisms by which A. muciniphila influences its host are not yet fully understood. To this end, it is important to identify which metabolites are produced and consumed by A. muciniphila that may contribute to a healthy gut. In the present study, we demonstrate the ability of A. muciniphila to produce γ-aminobutyric acid (GABA) when grown in an acidic environment, which often occurs in the gut. GABA is the major inhibitory neurotransmitter in the central nervous system and is present in the human gut. For this reason, it is considered an important bacterial metabolite. Our finding that A. muciniphila produces GABA in acidic environments adds to the growing body of understanding of its relationship with host health and provides an explanation on how it can survive acid stress in the human gut.

Improvement of the viability of Tetragenococcus halophilus under acidic stress by forming the biofilm cell structure based on RNA-Seq and iTRAQ analyses.

BACKGROUND: Tetragenococcus halophilus is a halophilic lactic acid bacterium (LAB) isolated from soya sauce moromi. During the production of these fermented foods, acid stress is an inevitable environmental stress. In our previous study, T. halophilus could form biofilms and the cells in the biofilms exhibited higher cell viability under multiple environmental stresses, including acid stress. RESULTS: In this study, the effect of preformed T. halophilus biofilms on cell survival, cellular structure, intracellular environment, and the expression of genes and proteins under acid stress was investigated. The result showed that acid stress with pH 4.30 for 1.5 h reduced the live T. halophilus cell count and caused cellular structure damage. However, T. halophilus biofilm cells exhibited greater cell survival under acid stress than the planktonic cells, and biofilm formation reduced the damage of acid stress to the cell membrane and cell wall. The biofilm cells maintained a higher level of H+ -ATPase activity and intracellular ammonia concentration after acid stress. The RNA-Seq and iTRAQ technologies revealed that the genes and proteins associated with ATP production, the uptake of trehalose and N-acetylmuramic acid, the assembly of H+ -ATPase, amino acid biosynthesis and metabolism, ammonia production, fatty acid biosynthesis, CoA biosynthesis, thiamine production, and acetoin biosynthesis might be responsible for the stronger acid tolerance of T. halophilus biofilm cells together. CONCLUSION: These findings further explained the mechanisms that allowed LAB biofilm cells to resist environmental stress. © 2023 Society of Chemical Industry.

Inter-strain variability on the cardinal parameters (pH and Aw) of clinical and food isolates of Listeria monocytogenes using turbidimetric measurements.

Listeria monocytogenes is a foodborne pathogen which, in 2021, was considered the fifth most commonly reported zoonosis in humans in the European Union (EU). Ready-to-eat (RTE) fishery products, deli meats or soft cheeses have been mostly involved in food safety alerts and outbreaks in the last years. Hurdle technology by food industries has been widely used to enhance the safety of foods. Among the barriers, the application of acid and osmotic stress during processing is extensively used worldwide. This study aims to gain knowledge about the inter-strain variability of twenty-six clinical and food L. monocytogenes isolates with the estimation of their cardinal parameters using turbidimetric measurements. To analyse the data and to obtain the estimated cardinal values, a common statistical procedure was set up. The estimation of cardinal parameters showed a high inter-strain variability of L. monocytogenes, and no correlation was observed between Aw min and pHmin values for the studied strains. By grouping the strains in clinical, meat and fish origin, it was observed that strains from the meat group presented the lowest average pHmin values (4.57), thus showing potential acid adaptation. This work contributes to gain knowledge of the inter-strain variability of L. monocytogenes in relation with pH and Aw cardinal values, as well as provide a starting point for future validation studies in fish and meat food matrices.