Acid-tolerant Mutant Research Articles

A mutant GH3 family β-glucosidase from Oenococcus oeni exhibits superior adaptation to wine stresses and potential for improving wine aroma and phenolic profiles

In this study, we conducted a comprehensive investigation into a GH3 family β-glucosidase (BGL) from the wild-type strain of Oenococcus oeni and its mutated counterpart from the acid-tolerant mutant strain. Our analysis revealed the mutant BGL's remarkable capacity to adapt to wine-related stress conditions, including heightened tolerance to low pH, elevated ethanol concentrations, and metal ions. Additionally, the mutant BGL exhibited superior hydrolytic activity towards various substrates. Through de novo modeling, we identified specific amino acid mutations responsible for its resilience to low pH and high ethanol environments. In simulated wine conditions, the mutant BGL outperformed both wild-type and commercial BGLs, efficiently releasing terpene and phenolic aglycones from glycosides in wine grapes. These findings not only expand our understanding of O. oeni BGLs but also highlight their potential in enhancing wine production. The mutant BGL's enhanced adaptation to wine stress conditions opens promising avenue for improving wine quality and flavor.

Malolactic fermentation performance of indigenous Oenococcus oeni strains from Shaanxi wine region (China) and their mutants on Pinot Noir and Chardonnay wines

This study investigated the malolactic fermentation (MLF) performance of three indigenous Oenococcus oeni strains isolated from the Shaanxi wine region (China) and mutants of those strains with different acid-tolerant phenotypes on Pinot Noir and Chardonnay wines. The MLF kinetics of these strains, as well as the basic chemical parameters and volatile compounds of wines after MLF, were determined. All strains successfully completed MLF in Pinot Noir wines with a pH of 3.45; however, only the acid-tolerant mutants were able to complete MLF in Chardonnay wines with a pH of 3.2. Compared with other strains, inoculation with the acid-tolerant mutants reduced the MLF duration up to 4.5 days in Pinot Noir wines, and significantly increased the concentrations of total volatile compounds in Pinot Noir wines and Chardonnay wines, with values rising from 324226.38 to 361450.91 μg/L and from 213322.96 to 279848.12 μg/L, respectively. These wines had higher levels of 2,3-butanediol, linalool, α-terpineol and β-damascenone, contributing to more pronounced butter and floral aroma profiles. MLF performance of these strains was positively correlated with their acid tolerance, and indigenous acid-tolerant mutants show great potential to be developed as MLF starters, contributing to the regional characteristics and high quality of wines.

Dynamic Responses of Streptomyces albulus QLU58 and Its Acid-Tolerant Derivatives to the Autoacidification in ε-Poly-l-Lysine Production

Streptomyces albulus is a kind of safety bacteria that is used to produce a natural food preservative named ε-poly-l-lysine (ε-PL). Environmental autoacidification (the pH declined from 6.8 to approximately 3.0) inevitably occurred in ε-PL biosynthesis by S. albulus. In this study, the dynamic responses of S. albulus QLU58 and its acid-tolerant mutants to autoacidification were investigated at the physiological and transcriptional levels. The results showed that cell growth, ε-PL production, cell respiratory activity, and intracellular pH (pHi) homeostasis were disturbed by autoacidification. In the initial autoacidification stage (before 24 h), the acid tolerance of S. albulus was effectively improved by increasing the intracellular ATP and related amino acids contents and the H+-ATPase activity, regulating the membrane fatty acids composition, and maintaining the pHi at about 7.7. However, as the autoacidification degree deepened (after 24 h), the metabolic activities decreased and negative cell growth appeared, which weakened the acid tolerance and caused the pHi to decline to about 6.5. Additionally, the acid-tolerant mutants exhibited better performances during autoacidification, which was also confirmed by the related genes’ improved transcription levels. These results provide references for the analysis of progressive environmental modification in ε-PL production.

Morphological Characteristics Selection of Acid-tolerant Leucaena leucocephala Mutant to Addition of IBA Hormone (Indole butyric acid) in Tissue Culture

Leucaena leucocephala is a legume forage plant that has a high protein content. Tissue culture is a technique that can be used to select mutants for forage plants in vitro. IBA (Indole butyric acid) is one type of auxin that can induce rooting and growth in plants. This study aimed to select the morphological characteristics of the acid-tolerant Leucaena leucocephala mutant to the addition of the hormone IBA in tissue culture. The design used in this study was a completely randomized design (CRD) with lamtoro plant explants as many as 11 acid-tolerant mutant lines pH 3.4 resulting from 400 gy irradiation which had been treated on 300 ppm Al3+ acid media, namely the K1-K11 strain (mutant+MS+1ppm IBA) and 2 parent trees Leucaena leucocephala without gamma irradiation, namely P0 as Leucaena leucocephala parent+MS+0ppm IBA, P1 as Leucaena leucocephala parent + MS + 1 ppm IBA with 15 replicates. The treatment which had a significant effect was continued with the test Tukey. Variables observed were an increase in root length, plant vertical height, number of shoots, and percentage of rooted plants. The increase in root length and increase in plant vertical height showed the best results on the K10 mutant strain, the number of shoots showed the best results on the K9 and K11 mutant strains, and the highest percentage of rooted plants on the K3 and K11 mutant strains. The addition of IBA can increase the morphological characteristics of the Leucaena leucocephala mutant.

EVALUATION AND SELECTION OF ALFALFA MUTANT RESULTS OF GAMMA RAY IRRADIATION ON ABIOTIC STRESS


 
 
 Alfalfa is a leguminous protein source with high nutritional value and economic value. Abiotic stress inhibits growth and reduces alfalfa production in many aquaculture systems. New varieties were developed by gamma-ray mutation induction. The selection was carried out to obtain superior abiotic stress-tolerant plants. This study aims to evaluate and select alfalfa mutants under abiotic stress conditions. The design used in this study was a comple- tely randomized design (CRD) with a factorial pattern. The first factor is the type of acid-tolerant mutants, namely A44, A54, A35, and A45. The second factor is the PEG level which is 10%, 15%, and 20%. The variables observed included viability, plant height, number of leaves, leaf wilting, leaf color, media shrinkage, and wet weight. Alfalfa mutants resulting from acid-tolerant gamma irradiation were evaluated under dry stress conditions. The results showed that mutation induction resulted in various responses to growth. Gamma-ray irradiation produces plants that are tolerant to abiotic stress conditions. From the selection results obtained superior mutant candidates that are tolerant of acid stress and dry conditions. The A44 mutant had better tolerance to dry stress than others and became an acid- and dry-tolerant alfalfa mutant.
 
 

Lipid profiles of acid-tolerant mutants of the green microalga Chlorella saccharophila reveal hydrocarbons and high-value lipids with potential industrial applications

This study analyzed the lipid profiles of two acid-tolerant mutants (M1 and M5) and a wild-type (WT) strain of Chlorella saccharophila. Six main types of compounds were identified in the lipid fractions: hydrocarbons, FAMEs (after transesterification of TAGs), free fatty acids, terpenes, sterols and fatty alcohols. Compared with the M1 and M5 mutant lines, the WT strain presented the highest relative abundance of hydrocarbons (73.63%), while the relative abundance of fatty acid methyl esters (FAMEs) obtained were higher in the M1 (28.39%) and M5 (29.17%) mutants than in the WT strain. High-value lipids, including terpenes and fatty alcohols, were also detected in higher proportions in the M1 and M5 mutants than in the WT strain, while sterols were only detected in the M1 and M5 mutants. These results show that C. saccharophila and its acid-tolerant mutants are a rich source of lipids with potential industrial applications.

Microbial fuel cell-assisted utilization of glycerol for succinate production by mutant of Actinobacillus succinogenes

BackgroundThe global production of glycerol is increasing year by year since the demands of biodiesel is rising. It is benefit for high-yield succinate synthesis due to its high reducing property. A. succinogenes, a succinate-producing candidate, cannot grow on glycerol anaerobically, as it needs a terminal electron acceptor to maintain the balance of intracellular NADH and NAD+. Microbial fuel cell (MFC) has been widely used to release extra intracellular electrons. However, A. succinogenes is a non-electroactive strain which need the support of electron shuttle in MFC, and pervious research showed that acid-tolerant A. succinogenes has higher content of unsaturated fatty acids, which may be beneficial for the transmembrane transport of lipophilic electron shuttle.ResultsMFC-assisted succinate production was evaluated using neutral red as an electron shuttle to recover the glycerol utilization. First, an acid-tolerant mutant JF1315 was selected by atmospheric and room temperature plasma (ARTP) mutagenesis aiming to improve transmembrane transport of neutral red (NR). Additionally, MFC was established to increase the ratio of oxidized NR to reduced NR. By combining these two strategies, ability of JF1315 for glycerol utilization was significantly enhanced, and 23.92 g/L succinate was accumulated with a yield of 0.88 g/g from around 30 g/L initial glycerol, along with an output voltage above 300 mV.ConclusionsA novel MFC-assisted system was established to improve glycerol utilization by A. succinogenes for succinate and electricity production, making this system as a platform for chemicals production and electrical supply simultaneously.

Enhanced production of triacylglycerols and polyunsaturated fatty acids in novel acid-tolerant mutants of the green microalga Chlorella saccharophila.

In this study, the microalga Chlorella saccharophila was subjected to ultraviolet (UV) mutagenesis, and mutant screening was conducted based on acidity tolerance to generate mutants with increased triacylglycerol (TAG) and polyunsaturated fatty acid (PUFA) contents. Two improved mutant strains (M1 and M5) were generated. M1 and M5 accumulated 27.2% and 27.4% more TAG, respectively, and showed stronger fluorescence intensity than the wild-type (WT) strain when the cells of these mutants were stained with the lipophilic Nile Red stain. In the M1 mutant, 50.5% of the fatty acid methyl esters (FAMEs) were saturated (C16:0 and C18:0) and 25.27% were monounsaturated (C18:1) fatty acids which are suitable for biofuels production. In the M5 mutant, 65.19% of the total FAMEs were nutritional PUFAs (C16:2, C18:2, and C18:3), while these FAMEs were not detected in the WT. These results demonstrated that UV mutagenesis coupled to an acid pH screening strategy represents a valuable and fast platform to generate mutants of C. saccharophila with improved TAG and PUFA contents for biofuels and nutraceutical applications, respectively.

Acid stress response ofStaphylococcus xylosuselicits changes in the proteome and cellular membrane

Coagulase-negative Staphylococcus xylosus strains are used as starter organisms for sausage fermentation. As those strains have to cope with low pH-values during fermentation, the aim of this study was to identify the acid adaptation mechanisms of S. xylosus TMW 2.1523 previously isolated from salami. A comparative proteomic study between two different acid tolerant mutants was performed. Therefore, both S. xylosus mutants were grown pH-static under acid stress (pH 5·1) and reference conditions (pH 7·0). Proteomic data were supported by metabolite and cell membrane lipid analysis. Staphylococcus xylosus acid stress adaptation is mainly characterized by a metabolic change towards neutral metabolites, enhanced urease activity, reduced ATP consumption, an increase in membrane fluidity and changes in the membrane thickness. This study corroborates mechanisms as previously described for other Gram-positive bacteria. Additionally, the adjustment of membrane structure and composition in S. xylosus TMW 2.1523 play a prominent role in its acid adaptation. This study demonstrates for the first time changes in the membrane lipid composition due to acid stress adaptation in staphylococci.

A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations

BackgroundAcetic acid, released during hydrolysis of lignocellulosic feedstocks for second generation bioethanol production, inhibits yeast growth and alcoholic fermentation. Yeast biomass generated in a propagation step that precedes ethanol production should therefore express a high and constitutive level of acetic acid tolerance before introduction into lignocellulosic hydrolysates. However, earlier laboratory evolution strategies for increasing acetic acid tolerance of Saccharomyces cerevisiae, based on prolonged cultivation in the presence of acetic acid, selected for inducible rather than constitutive tolerance to this inhibitor.ResultsPreadaptation in the presence of acetic acid was shown to strongly increase the fraction of yeast cells that could initiate growth in the presence of this inhibitor. Serial microaerobic batch cultivation, with alternating transfers to fresh medium with and without acetic acid, yielded evolved S. cerevisiae cultures with constitutive acetic acid tolerance. Single-cell lines isolated from five such evolution experiments after 50–55 transfers were selected for further study. An additional constitutively acetic acid tolerant mutant was selected after UV-mutagenesis. All six mutants showed an increased fraction of growing cells upon a transfer from a non-stressed condition to a medium containing acetic acid. Whole-genome sequencing identified six genes that contained (different) mutations in multiple acetic acid-tolerant mutants. Haploid segregation studies and expression of the mutant alleles in the unevolved ancestor strain identified causal mutations for the acquired acetic acid tolerance in four genes (ASG1, ADH3, SKS1 and GIS4). Effects of the mutations in ASG1, ADH3 and SKS1 on acetic acid tolerance were additive.ConclusionsA novel laboratory evolution strategy based on alternating cultivation cycles in the presence and absence of acetic acid conferred a selective advantage to constitutively acetic acid-tolerant mutants and may be applicable for selection of constitutive tolerance to other stressors. Mutations in four genes (ASG1, ADH3, SKS1 and GIS4) were identified as causative for acetic acid tolerance. The laboratory evolution strategy as well as the identified mutations can contribute to improving acetic acid tolerance in industrial yeast strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0583-1) contains supplementary material, which is available to authorized users.

Open fermentative production of fuel ethanol from food waste by an acid-tolerant mutant strain of Zymomonas mobilis

The aim of present study was to develop a process for open ethanol fermentation from food waste using an acid-tolerant mutant of Zymomonas mobilis (ZMA7-2). The mutant showed strong tolerance to acid condition of food waste hydrolysate and high ethanol production performance. By optimizing fermentation parameters, ethanol fermentation with initial glucose concentration of 200g/L, pH value around 4.0, inoculum size of 10% and without nutrient addition was considered as best conditions. Moreover, the potential of bench scales fermentation and cell reusability was also examined. The fermentation in bench scales (44h) was faster than flask scale (48h), and the maximum ethanol concentration and ethanol yield (99.78g/L, 0.50g/g) higher than that of flask scale (98.31g/L, 0.49g/g). In addition, the stable cell growth and ethanol production profile in five cycles successive fermentation was observed, indicating the mutant was suitable for industrial ethanol production.

Improvement of FK506 production by selection of 4-Aminobutyric acid-tolerant mutant and optimization of its fermentation using response surface methodology

Tacrolimus (FK506), a 23-membered polyketide macrolide with immunosuppressant activity, can be produced by Streptomyces tsukubaensis. We studied a variety of mutant strains of S. tsukubaensis for the microbial production of FK506. The best strain (CZ-19) was obtained from the parent strain LLZ-1 by nitrosoguanidine mutation and 4-Aminobutyric acid (FK506 precursor structure analogs) adaption. In the shake-flask experiments, titer of FK506 by CZ-19 was 532.44 mg/L, increased by 65.13% compared to that of the parent strain. Through single factor experiments and response surface methodology, we further optimized the medium for improved FK506 production by CZ-19 in shake flask culture. The optimal medium for enhanced FK506 production was as follows: 17.19 g/L corn starch, 21.78 g/L glucose, 8.06 g/L peptone, and 18.98 mg/L 4-aminobutyric acid. The predicted FK506 titer was 906.49 mg/L, and the experimental data confirmed the validity of the model. The present study demonstrates that S. tsukubaensis CZ-19 is a promising strain for industrial production of FK506.

Understanding of how Propionibacterium acidipropionici respond to propionic acid stress at the level of proteomics.

Propionic acid (PA) is an important platform chemical in the food, agriculture, and pharmaceutical industries and is mainly biosynthesized by propionibacteria. Acid tolerance in PA-producing strains is crucial. In previous work, we investigated the acid tolerance mechanism of Propionibacterium acidipropionici at microenvironmental levels by analyzing physiological changes in the parental strain and three PA-tolerant mutants obtained by genome shuffling. However, the molecular mechanism of PA tolerance in P. acidipropionici remained unclear. Here, we performed a comparative proteomics study of P. acidipropionici CGMCC 1.2230 and the acid-tolerant mutant P. acidipropionici WSH1105; MALDI-TOF/MS identified 24 proteins that significantly differed between the parental and shuffled strains. The differentially expressed proteins were mainly categorized as key components of crucial biological processes and the acid stress response. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to confirm differential expression of nine key proteins. Overexpression of the secretory protein glyceraldehyde-3-phosphate dehydrogenase and ATP synthase subunit α in Escherichia coli BL21 improved PA and acetic acid tolerance; overexpression of NADH dehydrogenase and methylmalonyl-CoA epimerase improved PA tolerance. These results provide new insights into the acid tolerance of P. acidipropionici and will facilitate the development of PA production through fermentation by propionibacteria.

Photo-biological hydrogen production by an acid tolerant mutant of Rhodovulum sulfidophilum P5 generated by transposon mutagenesis

Most of the photosynthetic bacterial strains exhibit optimum hydrogen production at neutral initial pH, and lower initial pH resulted in a sharp decrease in hydrogen yield. Thus, screening of acid-tolerant hydrogen-producing photosynthetic bacteria is very important. To obtain acid tolerant mutants, a Tn7-based transposon was randomly inserted into the genomic DNA of Rhodovulum sulfidophilum P5. An acid tolerant mutant strain TH-102 exhibited increased hydrogen production in acidic environment (pH 4.5-6.5) and at higher temperatures (35 and 37°C) than the wild-type strain. At pH 5.5 and 35°C, the mutant strain TH-102 continuously produced hydrogen. The hydrogen yield and average rate were 2.16 ± 0.10 mol/mol acetate and 10.06 ± 0.47 mL/Lh, which was about 17.32 and 15.37-fold higher than that of the wild-type strain, respectively. This acid- and temperature-tolerant mutant strain TH-102 could be used in a cost-effective hydrogen production process employing both dark fermentative and photosynthetic bacteria.

Acid tolerant mutants of Bifidobacterium animalis subsp. lactis with improved stability in fruit juice

The potential of UV mutagenesis combined with a specific selection step to generate more acid-resistant Bifidobacterium animalis subsp. lactis Bb-12 strains with improved viability in low pH food matrices was investigated in this study. A total of 144 Bb-12 UV-mutants were initially characterised. After prolonged storage in apple juice (pH 3.5) and various pheno- and genotypic tests (acid and bile tolerance, substrate utilisation, antibiotic susceptibility, aerotolerance, RAPD) two mutants (2.20 and 2.56) were chosen for further studies including cell surface morphology, stability of various traits after repeated inoculations, and performance in a fermenter and during down-stream processing. 2.20 and 2.56 showed over two Log-values better viability in pH 3.5 juice compared to Bb-12. Alterations in cell surface structures of 2.20 and 2.56 were detected with AFM, whereas other studied traits remained unchanged. In conclusion, UV mutagenesis and subsequent incubation in acidic medium enabled improving the stability of B. animalis subsp. lactis in low pH juice. Acid tolerance testing (HCl, pH 2.5, 2 h) results did not predict long-term stability of the strains in acidic food matrix (apple juice pH 3.5).

Ethanol fermentation by an acid-tolerant Zymomonas mobilis under non-sterilized condition

Ethanol fermentation under non-sterilized condition was developed in this paper. An acid-tolerant mutant of Zymomonas mobilis, which can begin fermentation at the low pH of 4.5, was selected. At this low pH, potential contamination of ethanol production was controlled at the normal level without autoclaving or filter sterilization. Compared with the filter sterilization process, the same ethanol yield of 73.1 g/l indicated that no contamination occurred in the non-sterilized process. Compared with the autoclaved process, ethanol yield increased from 70.3 to 73.2 g/l and residual sugar concentration decreased from 5.3 to 1.3 g/l with 40 h fermentation. The theoretical yield of ethanol from glucose was 0.488 g/g in this new ethanol process.

The acid-stress response in Lactobacillus sanfranciscensis CB1.

Lactobacillus sanfranciscensis CB1, an important sourdough lactic acid bacterium, can withstand low pH after initial exposure to sublethal acidic conditions. The sensitivity to low pH varied according to the type of acid used. Treatment of LB: sanfranciscensis CB1 with chloramphenicol during acid adaptation almost completely eliminated the protective effect, suggesting that induction of protein synthesis was required for the acid-tolerance response. Two constitutively acid-tolerant mutants, CB1-5R and CB1-7R, were isolated using natural selection techniques after sequential exposure to lactic acid (pH 3.2). Two-dimensional gel electrophoresis analysis of protein expression by non-adapted, acid-adapted and acid-tolerant mutant cells of LB: sanfranciscensis showed changes in the levels of 63 proteins. While some of the modifications were common to the acid-adapted and acid-tolerant mutant cells, several differences, especially regarding the induced proteins, were determined. The two mutants showed a very similar level of protein expression. Antibodies were used to identify heat-shock proteins DnaJ, DnaK, GroES and GrpE. Only GrpE showed an increased level of expression in the acid-adapted and acid-tolerant mutants as compared with non-adapted cells. The N-terminal sequence was determined for two proteins, one induced in both the acid-adapted and mutant cells and the other showing the highest induction factor of those proteins specifically induced in the acid-adapted cells. This second protein has 60% identity with the N-terminal portion of YhaH, a transmembrane protein of Bacillus subtilis, which has 54 and 47% homology with stress proteins identified in Listeria monocytogenes and Bacillus halodurans. The constitutively acid-tolerant mutants showed other different phenotypic features compared to the parental strain: (i) the aminopeptidase activity of CB1-5R decreased and that of CB1-7R markedly increased, especially in acid conditions; (ii) the growth in culture medium at 10 degrees C and in the presence of 5% NaCl was greater (the same was found for acid-adapted cells); and (iii) the acidification rate during sourdough fermentation in acid conditions was faster and greater.

Acid tolerance in Listeria monocytogenes influences invasiveness of enterocyte-like cells and macrophage-like cells

Clinical and food Listeria monocytogenes isolates, pre-exposed to mild acidic conditions, were able to readily develop acid tolerance, irrespective of their origin. We attempted to investigate the influence of acid tolerance mechanisms, either constitutive or induced, on the invasive behaviour of this facultative food-borne pathogen. Entry efficiency and intracellular growth of acid-tolerant strains were evaluated in in vitro cell models capable to mimic in vivo target cells, such as enterocytes and macrophages. An acid-adapted L. monocytogenes wild-type strain and a constitutively acid-tolerant mutant were able to enter enterocyte-like (Caco-2) cells as well as to survive and proliferate intracellularly in lipopolysaccharide-treated macrophage-like (J774.A1) cells, at a significant increased extent by respect of the non acid-adapted wild-type strain. These findings add new information about the influence of the acid tolerance response on L. monocytogenes virulence, suggesting that in acid-adapted bacteria the early events of pathogenesis which allow the colonization and the spread of bacteria in the host may be highly promoted.

Two-Dimensional Polyacrylamide Gel Electrophoresis Analysis of the Acid Tolerance Response in Listeria monocytogenes LO28

Listeria monocytogenes is capable of withstanding low pH after initial exposure to sublethal acidic conditions, a phenomenon termed the acid tolerance response (B. O'Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ. Microbiol. 62:1693-1698, 1996). Treatment of L. monocytogenes LO28 with chloramphenicol during acid adaptation abrogated the protective effect, suggesting that de novo protein synthesis is required for the acid tolerance response. Analysis of protein expression during acid adaptation by two-dimensional gel electrophoresis revealed changes in the levels of 53 proteins. Significant protein differences were also evident between nonadapted L. monocytogenes LO28 and a constitutively acid-tolerant mutant, ATM56. In addition, the analysis[S_TABC] revealed differences in protein expression between cells induced with a weak acid (lactic acid) and those induced with a strong acid (HCl). Comparison of both acid-adapted LO28 and ATM56 revealed that both are capable of maintaining their internal pH (pH(infi)) at higher levels than nonadapted control cells during severe acid stress. Collectively, the data demonstrate the profound alterations in protein synthesis which take place during acid adaptation in L. monocytogenes and ultimately lead to an increased ability to survive severe stress conditions.

Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence.

The ability of Listeria monocytogenes to tolerate low-pH environments is of particular importance because the pathogen encounters such environments in vivo, both during passage through the stomach and within the macrophage phagosome. In our study, L. monocytogenes was shown to exhibit a significant adaptive acid tolerance response following a 1-h exposure to mild acid (pH 5.5), which is capable of protecting cells from severe acid stress (pH 3.5). Susceptibility to pH 3.5 acid is growth phase dependent. Stationary-phase Listeria cultures are naturally resistant to the challenge pH (pH 3.5), while exponential-phase cultures require adaptation at pH 5.5 to induce acid tolerance. Adaptation requires protein synthesis, since treatment with chloramphenicol prevents the development of acid tolerance. Induction of the acid tolerance response also protects L. monocytogenes against the effect of other environmental stresses. Acid-adapted cells demonstrate increased tolerance toward thermal stress, osmotic stress, crystal violet, and ethanol. Following prolonged exposure of L. monocytogenes to pH 3.5, we isolated mutants which constitutively demonstrate increased acid tolerance at all stages of the growth cycle. These mutants do not display full acid tolerance, but their resistance to low pH can be further increased following adaptation to mild-acid conditions. The mutants demonstrated increased lethality for mice relative to that of the wild type when inoculated by the intraperitoneal route. When administered as lower inocula, the mutants reached higher levels in the spleens of infected mice than did the wild type. The data suggest that low-pH conditions may have the potential to select for L. monocytogenes mutants with increased natural acid tolerance and increased virulence.