Putrescine production via the ornithine decarboxylation pathway improves the acid stress survival of Lactobacillus brevis and is part of a horizontally transferred acid resistance locus

ABSTRACT Bacteria have evolved different signaling systems to sense and adapt to acid stress. One of these systems, the CadABC system, responds to a combination of low pH and lysine availability. In Escherichia coli, the two signals are sensed by the pH sensor and transcription activator CadC and the cosensor LysP, a lysine-specific transporter. Activated CadC promotes the transcription of the cadBA operon, which codes for the lysine decarboxylase CadA and the lysine/cadaverine antiporter CadB. The copy number of CadC is controlled translationally. Using a bioinformatics approach, we identified the presence of CadC with ribosomal stalling motifs together with LysP in species of the Enterobacteriaceae family. In contrast, we identified CadC without stalling motifs in species of the Vibrionaceae family, and the LysP cosensor is missing. Therefore, we compared the outputs of the Cad system in single cells of the distantly related organisms E. coli and Vibrio campbellii using fluorescently tagged CadB as the reporter. We observed a heterogeneous output in E. coli, and all the V. campbellii cells produced CadB. The copy number of the pH sensor CadC in E. coli was extremely low (≤4 molecules per cell), but it was 10-fold higher in V. campbellii. An increase in the CadC copy number in E. coli correlated with a decrease in heterogeneous behavior. This study demonstrated how small changes in the design of a signaling system allow a homogeneous output and, thus, adaptation of Vibrio species that rely on the CadABC system as the only acid resistance system. IMPORTANCE Acid resistance is an important property for bacteria, such as Escherichia coli, to survive acidic environments like the human gastrointestinal tract. E. coli possesses both passive and inducible acid resistance systems to counteract acidic environments. Thus, E. coli evolved sophisticated signaling systems to sense and appropriately respond to environmental acidic stress by regulating the activity of its three inducible acid resistance systems. One of these systems is the Cad system, which is induced only under moderate acidic stress in a lysine-rich environment by the pH-responsive transcriptional regulator CadC. The significance of our research lies in identifying the molecular design of the Cad systems in different proteobacteria and their target expression noise at the single-cell level during acid stress conditions.

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GlnR Negatively Regulates Glutamate-Dependent Acid Resistance in Lactobacillus brevis

Applied and Environmental Microbiology ◽

10.1128/aem.02615-19 ◽

2020 ◽

Vol 86 (7) ◽

Author(s):

Luchan Gong ◽

Cong Ren ◽

Yan Xu

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Glutamate Decarboxylase ◽

Wild Type Strain ◽

Acid Resistance ◽

Lactobacillus Brevis ◽

Deletion Strain ◽

Content Type ◽

Gaba Production ◽

Encoding Gene

ABSTRACT Lactic acid bacteria often encounter a variety of multiple stresses in their natural and industrial fermentation environments. The glutamate decarboxylase (GAD) system is one of the most important acid resistance systems in lactic acid bacteria. In this study, we demonstrated that GlnR, a nitrogen regulator in Gram-positive bacteria, directly modulated γ-aminobutyric acid (GABA) conversion from glutamate and was involved in glutamate-dependent acid resistance in Lactobacillus brevis. The glnR deletion strain (ΔglnR mutant) achieved a titer of 284.7 g/liter GABA, which is 9.8-fold higher than that of the wild-type strain. The cell survival of the glnR deletion strain was significantly higher than that of the wild-type strain under the condition of acid challenge and was positively correlated with initial glutamate concentration and GABA production. Quantitative reverse transcription-PCR assays demonstrated that GlnR inhibited the transcription of the glutamate decarboxylase-encoding gene (gadB), glutamate/GABA antiporter-encoding gene (gadC), glutamine synthetase-encoding gene (glnA), and specific transcriptional regulator-encoding gene (gadR) involved in gadCB operon regulation. Moreover, GABA production and glutamate-dependent acid resistance were absolutely abolished in the gadR glnR deletion strain. Electrophoretic mobility shift and DNase I footprinting assays revealed that GlnR directly bound to the 5′-untranslated regions of the gadR gene and gadCB operon, thus inhibiting their transcription. These results revealed a novel regulatory mechanism of GlnR on glutamate-dependent acid resistance in Lactobacillus. IMPORTANCE Free-living lactic acid bacteria often encounter acid stresses because of their organic acid-producing features. Several acid resistance mechanisms, such as the glutamate decarboxylase system, F1Fo-ATPase proton pump, and alkali production, are usually employed to relieve growth inhibition caused by acids. The glutamate decarboxylase system is vital for GAD-containing lactic acid bacteria to protect cells from DNA damage, enzyme inactivation, and product yield loss in acidic habitats. In this study, we found that a MerR-type regulator, GlnR, was involved in glutamate-dependent acid resistance by directly regulating the transcription of the gadR gene and gadCB operon, resulting in an inhibition of GABA conversion from glutamate in L. brevis. This study represents a novel mechanism for GlnR's regulation of glutamate-dependent acid resistance and also provides a simple and novel strategy to engineer Lactobacillus strains to elevate their acid resistance as well as GABA conversion from glutamate.

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Importance of Polyphosphate Kinase 1 for Campylobacter jejuni Viable-but-Nonculturable Cell Formation, Natural Transformation, and Antimicrobial Resistance

Applied and Environmental Microbiology ◽

10.1128/aem.01603-09 ◽

2009 ◽

Vol 75 (24) ◽

pp. 7838-7849 ◽

Cited By ~ 40

Author(s):

Dharanesh Gangaiah ◽

Issmat I. Kassem ◽

Zhe Liu ◽

Gireesh Rajashekara

Keyword(s):

Antimicrobial Resistance ◽

Campylobacter Jejuni ◽

Stress Responses ◽

Natural Transformation ◽

Cell Formation ◽

Acid Stress ◽

Therapeutic Interventions ◽

Wild Type ◽

Viable But Nonculturable ◽

Stress Survival

ABSTRACT Campylobacter jejuni, a gram-negative, microaerophilic bacterium, is a predominant cause of bacterial gastroenteritis in humans. Although considered fragile and fastidious and lacking many classical stress response mechanisms, C. jejuni exhibits a remarkable capacity for survival and adaptation, successfully infecting humans and persisting in the environment. Consequently, understanding the physiological and genetic properties that allow C. jejuni to survive and adapt to various stress conditions is crucial for therapeutic interventions. Of importance is polyphosphate (poly-P) kinase 1 (PPK1), which is a key enzyme mediating the synthesis of poly-P, an essential molecule for survival, mediating stress responses, host colonization, and virulence in many bacteria. Therefore, we investigated the role of PPK1 in C. jejuni pathogenesis, stress survival, and adaptation. Our findings demonstrate that a C. jejuni Δppk1 mutant was deficient in poly-P accumulation, which was associated with a decreased ability to form viable-but-nonculturable cells under acid stress. The Δppk1 mutant also showed a decreased frequency of natural transformation and an increased susceptibility to various antimicrobials. Furthermore, the Δppk1 mutant was characterized by a dose-dependent deficiency in chicken colonization. Complementation of the Δppk1 mutant with the wild-type copy of ppk1 restored the deficient phenotypes to levels similar to those of the wild type. Our results suggest that poly-P plays an important role in stress survival and adaptation and might contribute to genome plasticity and the spread and development of antimicrobial resistance in C. jejuni. These findings highlight the potential of PPK1 as a novel target for therapeutic interventions.

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Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of theWHI2Gene Identified through Inverse Metabolic Engineering

Applied and Environmental Microbiology ◽

10.1128/aem.03718-15 ◽

2016 ◽

Vol 82 (7) ◽

pp. 2156-2166 ◽

Cited By ~ 43

Author(s):

Yingying Chen ◽

Lisa Stabryla ◽

Na Wei

Keyword(s):

Saccharomyces Cerevisiae ◽

Acetic Acid ◽

Metabolic Engineering ◽

Acid Stress ◽

Acid Resistance ◽

Biofuel Production ◽

Gene Target ◽

Content Type ◽

Inverse Metabolic Engineering ◽

Acetic Acid Resistance

ABSTRACTDevelopment of acetic acid-resistantSaccharomyces cerevisiaeis important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target,WHI2(encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance inS. cerevisiae. Overexpression ofWHI2significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. TheWHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression ofWHI2gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 inS. cerevisiae. Meanwhile, thewhi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response inS. cerevisiae. Additionally, overexpression ofWHI2and of a cognate phosphatase gene,PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production.

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Comparative Genome-Wide Transcriptome Analysis of Brucella suis and Brucella microti Under Acid Stress at pH 4.5: Cold Shock Protein CspA and Dps Are Associated With Acid Resistance of B. microti

Frontiers in Microbiology ◽

10.3389/fmicb.2021.794535 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jorge A. de la Garza-García ◽

Safia Ouahrani-Bettache ◽

Sébastien Lyonnais ◽

Erika Ornelas-Eusebio ◽

Luca Freddi ◽

...

Keyword(s):

Transcriptome Analysis ◽

Protein Misfolding ◽

Cold Shock ◽

Stress Protein ◽

Acid Stress ◽

Acid Resistance ◽

Cold Shock Protein ◽

Physiological Adaptation ◽

Energy Conditions ◽

Brucella Suis

Brucellae are facultative intracellular coccobacilli causing brucellosis, one of the most widespread bacterial zoonosis affecting wildlife animals, livestock and humans. The genus Brucella comprises classical and atypical species, such as Brucella suis and Brucella microti, respectively. The latter is characterized by increased metabolic activity, fast growth rates, and extreme acid resistance at pH 2.5, suggesting an advantage for environmental survival. In addition, B. microti is more acid-tolerant than B. suis at the intermediate pH of 4.5. This acid-resistant phenotype of B. microti may have major implications for fitness in soil, food products and macrophages. Our study focused on the identification and characterization of acid resistance determinants of B. suis and B. microti in Gerhardt’s minimal medium at pH 4.5 and 7.0 for 20 min and 2 h by comparative RNA-Seq-based transcriptome analysis, validated by RT-qPCR. Results yielded a common core response in both species with a total of 150 differentially expressed genes, and acidic pH-dependent genes regulated specifically in each species. The identified core response mechanisms comprise proton neutralization or extrusion from the cytosol, participating in maintaining physiological intracellular pH values. Differential expression of 441 genes revealed species-specific mechanisms in B. microti with rapid physiological adaptation to acid stress, anticipating potential damage to cellular components and critical energy conditions. Acid stress-induced genes encoding cold shock protein CspA, pseudogene in B. suis, and stress protein Dps were associated with survival of B. microti at pH 4.5. B. suis response with 284 specifically regulated genes suggested increased acid stress-mediated protein misfolding or damaging, triggering the set-up of repair strategies countering the consequences rather than the origin of acid stress and leading to subsequent loss of viability. In conclusion, our work supports the hypothesis that increased acid stress resistance of B. microti is based on selective pressure for the maintenance of functionality of critical genes, and on specific differential gene expression, resulting in rapid adaptation.

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D-Methionine and D-Phenylalanine Improve Lactococcus lactis F44 Acid Resistance and Nisin Yield by Governing Cell Wall Remodeling

Applied and Environmental Microbiology ◽

10.1128/aem.02981-19 ◽

2020 ◽

Vol 86 (9) ◽

Author(s):

Hao Wu ◽

Ershu Xue ◽

Ning Zhi ◽

Qianqian Song ◽

Kairen Tian ◽

...

Keyword(s):

Amino Acids ◽

Cell Wall ◽

Lactococcus Lactis ◽

Acid Stress ◽

Acid Tolerance ◽

Acid Resistance ◽

Cell Wall Synthesis ◽

Nisin Production ◽

Content Type ◽

Nisin Yield

ABSTRACT Lactococcus lactis encounters various environmental challenges, especially acid stress, during its growth. The cell wall can maintain the integrity and shape of the cell under environmental stress, and d-amino acids play an important role in cell wall synthesis. Here, by analyzing the effects of 19 different d-amino acids on the physiology of L. lactis F44, we found that exogenously supplied d-methionine and d-phenylalanine increased the nisin yield by 93.22% and 101.29%, respectively, as well as significantly increasing the acid resistance of L. lactis F44. The composition of the cell wall in L. lactis F44 with exogenously supplied d-Met or d-Phe was further investigated via a vancomycin fluorescence experiment and a liquid chromatography-mass spectrometry assay, which demonstrated that d-Met could be incorporated into the fifth position of peptidoglycan (PG) muropeptides and d-Phe could be added to the fourth and fifth positions. Moreover, overexpression of the PG synthesis gene murF further enhanced the levels of d-Met and d-Phe involved in PG and increased the survival rate under acid stress and the nisin yield of the strain. This study reveals that the exogenous supply of d-Met or d-Phe can change the composition of the cell wall and influence acid tolerance as well as nisin yield in L. lactis. IMPORTANCE As d-amino acids play an important role in cell wall synthesis, we analyzed the effects of 19 different d-amino acids on L. lactis F44, demonstrating that d-Met and d-Phe can participate in peptidoglycan (PG) synthesis and improve the acid resistance and nisin yield of this strain. murF overexpression further increased the levels of d-Met and d-Phe incorporated into PG and contributed to the acid resistance of the strain. These findings suggest that d-Met and d-Phe can be incorporated into PG to improve the acid resistance and nisin yield of L. lactis, and this study provides new ideas for the enhancement of nisin production.

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Comparative Analysis of Acid Resistance between Susceptible and Multi-Antimicrobial-Resistant Salmonella Strains Cultured under Stationary-Phase Acid Tolerance–Inducing and Noninducing Conditions

Journal of Food Protection ◽

10.4315/0362-028x-66.5.732 ◽

2003 ◽

Vol 66 (5) ◽

pp. 732-740 ◽

Cited By ~ 28

Author(s):

R. T. BACON ◽

J. N. SOFOS ◽

P. A. KENDALL ◽

K. E. BELK ◽

G. C. SMITH

Keyword(s):

Stationary Phase ◽

Antimicrobial Agents ◽

Acid Stress ◽

Acid Tolerance ◽

Acid Resistance ◽

Low Ph ◽

Acid Exposure ◽

Acid Adaptation ◽

Acid Shock ◽

Resistant Strains

This study compared acid resistance levels among five antimicrobial-susceptible strains of Salmonella and five strains that were simultaneously resistant to a minimum of six antimicrobial agents. The induction of a stationary-phase acid tolerance response (ATR) was attempted by both transient low-pH acid shock and acid adaptation. For acid shock induction, strains were grown for 18 h in minimal E medium containing 0.4% glucose (EG medium) and exposed to sublethal acid stress (pH 4.3) for 2 h, and subsequently, both shocked and nonshocked cultures were acid challenged (pH 3.0) for 4 h. Acid adaptation was achieved by growing strains for 18 h in tryptic soy broth containing 1.0% glucose (TSB+G), while nonadapted cultures were grown for 18 h in glucose-free tryptic soy broth (TSB−G). Acid-adapted and nonadapted inocula were acid challenged (pH 2.3) for 4 h. Initial (0 h) mean populations of nonchallenged Salmonella were 8.5 to 8.7, 8.4 to 8.8, and 8.2 to 8.3 log CFU/ml for strains grown in EG medium, TSB−G, and TSB+G, respectively. After 4 h of acid challenge, mean populations were 3.0 to 4.8 and 2.5 to 3.7 log CFU/ml for previously acid-shocked susceptible and resistant strains, respectively, while corresponding counts for nonshocked strains were 4.3 to 5.5 log CFU/ml and 3.9 to 4.9 log CFU/ml. Following 4 h of acid exposure, acid-adapted cultures of susceptible and resistant strains had mean populations of 6.1 to 6.4 log CFU/ml and 6.4 to 6.6 log CFU/ml, respectively, while corresponding counts for nonadapted cultures were 1.9 to 2.1 log CFU/ml and 1.8 to 2.0 log CFU/ml, respectively. A low-pH–inducible ATR was not achieved through transient acid shock, while an ATR was evident following acid adaptation, as adapted populations were 4.2 to 4.8 log units larger than nonadapted populations following acid exposure. Although some strain-dependent variations in acid resistance were observed, results from this study suggest no association between susceptibility to antimicrobial agents and the ability of the Salmonella strains evaluated to survive low-pH stress.

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Chaperone Hsp31 Contributes to Acid Resistance in Stationary-Phase Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.02429-06 ◽

2006 ◽

Vol 73 (3) ◽

pp. 1014-1018 ◽

Cited By ~ 31

Author(s):

Mirna Mujacic ◽

Franï¿½ois Baneyx

Keyword(s):

Oxidative Stress ◽

Escherichia Coli ◽

Stationary Phase ◽

High Temperatures ◽

Protein Misfolding ◽

Acid Resistance ◽

Stress Survival

ABSTRACT Hsp31, the product of the σS- and σD-dependent hchA gene, is a heat-inducible chaperone implicated in the management of protein misfolding at high temperatures. We show here that Hsp31 plays an important role in the acid resistance of starved Escherichia coli but that it has little influence on oxidative-stress survival.

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