RESUMO
BACKGROUND: γ-aminobutyric acid (GABA) is a bioactive compound produced by lactic acid bacteria (LAB). The diversity of GABA production in the Lactococcus genus is poorly understood. Genotypic and phenotypic approaches were therefore combined in this study to shed light on this diversity. A comparative genomic study was performed on the GAD-system genes (gadR, gadC and gadB) involved in GABA production in 36 lactococci including L. lactis and L. cremoris species. In addition, 132 Lactococcus strains were screened for GABA production in culture medium supplemented with 34 mM L-glutamic acid with or without NaCl (0.3 M). RESULTS: Comparative analysis of the nucleotide sequence alignments revealed the same genetic organization of the GAD system in all strains except one, which has an insertion sequence element (IS981) into the PgadCB promoter. This analysis also highlighted several deletions including a 3-bp deletion specific to the cremoris species located in the PgadR promoter, and a second 39-bp deletion specific to L. cremoris strains with a cremoris phenotype. Phenotypic analysis revealed that GABA production varied widely, but it was higher in L. lactis species than in L. cremoris, with an exceptional GABA production of up to 14 and 24 mM in two L. lactis strains. Moreover, adding chloride increased GABA production in some L. cremoris and L. lactis strains by a factor of up to 16 and GAD activity correlated well with GABA production. CONCLUSIONS: This genomic analysis unambiguously characterized the cremoris phenotype of L. cremoris species and modified GadB and GadR proteins explain why the corresponding strains do not produce GABA. Finally, we found that glutamate decarboxylase activity revealing GadB protein amount, varied widely between the strains and correlated well with GABA production both with and without chloride. As this protein level is associated to gene expression, the regulation of GAD gene expression was identified as a major contributor to this diversity.
Assuntos
Cloretos , Lactococcus , Fenótipo , Meios de Cultura , Ácido gama-AminobutíricoRESUMO
BACKGROUND: The glutamate decarboxylase (GAD) system of Lactobacillus brevis involves two isoforms of GAD, GadA and GadB, which catalyze the conversion of L-glutamate to γ-aminobutyric acid (GABA) in a proton-consuming reaction contributing to intracellular pH homeostasis. However, direct experimental evidence for detailed contributions of gad genes to acid tolerance and GABA production is lacking. RESULTS: Molecular analysis revealed that gadB is cotranscribed in tandem with upstream gadC, and that expression of gadCB is greatly upregulated in response to low ambient pH when cells enter the late exponential growth phase. In contrast, gadA is located away from the other gad genes, and its expression was consistently lower and not induced by mild acid treatment. Analysis of deletion mutations in the gad genes of L. brevis demonstrated a decrease in the level of GAD activity and a concomitant decrease in acid resistance in the order of wild-type> ΔgadA> ΔgadB> ΔgadC> ΔgadAB, indicating that the GAD activity mainly endowed by GadB rather than GadA is an indispensable step in the GadCB mediated acid resistance of this organism. Moreover, engineered strains with higher GAD activities were constructed by overexpressing key GAD system genes. With the proposed two-stage pH and temperature control fed-batch fermentation strategy, GABA production by the engineered strain L. brevis 9530: pNZ8148-gadBC continuously increased reaching a high level of 104.38 ± 3.47 g/L at 72 h. CONCLUSIONS: This is the first report of the detailed contribution of gad genes to acid tolerance and GABA production in L. brevis. Enhanced production of GABA by engineered L. brevis was achieved, and the resulting GABA level was one of the highest among lactic acid bacterial species grown in batch or fed-batch culture.
Assuntos
Ácidos/farmacologia , Glutamato Descarboxilase/metabolismo , Levilactobacillus brevis/enzimologia , Ácido gama-Aminobutírico/biossíntese , Fermentação/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Loci Gênicos , Glutamato Descarboxilase/genética , Concentração de Íons de Hidrogênio , Isoenzimas/metabolismo , Levilactobacillus brevis/efeitos dos fármacos , Levilactobacillus brevis/genética , Levilactobacillus brevis/crescimento & desenvolvimento , Óperon/genética , Filogenia , Deleção de Sequência , Especificidade por Substrato/efeitos dos fármacos , Temperatura , Fatores de TempoRESUMO
Ultrasound creates cavitation phenomena, resulting in the formation of several free radicals, namely OHË and HË, due to the breakdown of the H2 O molecule. These radicals affect the cellular integrity of the bacteria, causing the inactivation of several processes, and thus it is important to unravel the mechanism of action of this technology. This research looks into the application and mechanism of action of ultrasound technology as a means of disinfection by acoustic cavitation. Sterile water and synthetic waste water were inoculated with different mutants of Escherichia coli K12 strains containing deletions in genes affecting specific functional properties of E. coli. These were: dnak soxR, soxS, oxyR, rpoS, gadA/gadB, gadC and yneL. Escherichia coli K-12 ΔoxyR appeared to be more resistant to the treatment together with gadW, gadX, gabT and gabD, whereas the mutant K-12 ΔdnaK was more sensitive with c. 2·5 log (CFU per ml) reduction in comparison to their isogenic wild-type E. coli K-12. This indicates that the dnaK gene participates in general stress response and more specifically to hyperosmotic stress. The other E. coli deleted genes tested (soxS, rpoS, gadB, gadC, yneL) did not appear to be involved in protection of microbial cells against ultrasound. SIGNIFICANCE AND IMPACT OF THE STUDY: This study looks at the mechanism of action of ultrasound technology for the disinfection of wastewater. Different mutants with deleted genes were used to study the respective sensitivity or resistance to this treatment. This is essential to characterize changes at the molecular level, which might be occurring during treatment, resulting in bacterial adaptation.
Assuntos
Desinfecção/métodos , Escherichia coli K12/genética , Escherichia coli K12/efeitos da radiação , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico HSP70/genética , Ondas Ultrassônicas , Antibacterianos/farmacologia , Escherichia coli K12/metabolismo , Radicais Livres/química , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/genética , Águas Residuárias/microbiologiaRESUMO
The adaptive response of bacterial cells to changing environmental conditions depends on the behavior of single cells within the population. Exposure of Listeria monocytogenes to sublethal acidic conditions in foods or in the gastrointestinal track of the host may induce injuries relevant to difficult physiological states within the dormancy continuum. In this study, exposure to acidic conditions (acetic-AA and hydrochloric acid-HCl adjusted to pH 3.0, 2.7, 2.5 at 20 °C for 5 h) was used to evaluate injury of L. monocytogenes, Scott A strain. To differentiate the resistant sub-population from the total, Tryptic Soy Agar with 0.6 % Yeast Extract (TSAYE) supplemented or not with 5 % NaCl were comparatively used. Sublethally injured cells were detected by comparing plate counts with fluorescence microscopy, using combinations of CFDA (viability) and Propidium-Iodide (death). Effect of acid stress on the relative transcription of clpP, mazE, mazF, relA, gadC, gadD, gadB, sigB, inlA and prfA upon transition of total population into different physiological stages was evaluated through RT-qPCR. AA treated cells showed measurable logarithmic reduction at pH 2.7 and 2.5, while there was a significant percentage of CFDA-/PI+ cells. Evaluation of the potentially culturable population on TSAYE, from the percentage of CFDA/PI-stained cells, revealed that unstained cells represented a non-culturable sub-population. Exposure to Ringer's solution pH 2.7, adjusted with AA, resulted in higher percentages of non-esterase active with membrane integrity cells (CFDA-/PI-) compared to the percentages of the enumerated culturable cells on TSAYE after 4 and 5 h. Under the same conditions, after 1 h of exposure macroscopic observation revealed size colony variations (SCVs) of the total population (CFU on TSAYE). L. monocytogenes retained its culturability after hydrochloric acid exposure, while cells remained metabolically active (CFDA+). However, a stochastic change in cell's shape, was detected after exposure to pH 3.0 and 2.5, adjusted with HCl, for 2 h at 20 °C. A pattern of gene up-regulation was observed during treatment with AA pH 2.7 and HCl pH 3.0 at the 3rd h of exposure. Deciphering L. monocytogenes sublethal injury sheds light into the physiological and molecular characteristics of this state and provides the food science community with quantitative data to improve risk assessment.
Assuntos
Listeria monocytogenes , Ácido Clorídrico/farmacologia , Cloreto de Sódio/farmacologia , Ácidos/farmacologia , Ágar/farmacologia , Microscopia de Fluorescência , Concentração de Íons de Hidrogênio , Contagem de Colônia MicrobianaRESUMO
Enterococcus avium (E. avium) is a common bacterium inhabiting the intestines of humans and other animals. Most strains of this species can produce gamma-aminobutyric acid (GABA) via the glutamate decarboxylase (GAD) system, but the presence and genetic organization of their GAD systems are poorly characterized. In this study, our bioinformatics analyses showed that the GAD system in E. avium strains was generally encoded by three gadB genes (gadB1, gadB2, and gadB3), together with an antiporter gene (gadC) and regulator gene (gadR), and these genes are organized in a cluster. This finding contrasts with that for other lactic acid bacteria. E. avium SDMCC050406, a GABA producer isolated from human feces, was employed to investigate the contribution of the three gadB genes to GABA biosynthesis. The results showed that the relative expression level of gadB3 was higher than those of gadB1 and gadB2 in the exponential growth and stationary phases, and this was accompanied by the synchronous transcription of gadC. After heterologous expression of the three gadB genes in Escherichia coli BL21 (DE3), the K m value of the purified GAD3 was 4.26 ± 0.48 mM, a value lower than those of the purified GAD1 and GAD2. Moreover, gadB3 gene inactivation caused decreased GABA production, accompanied by a reduction in resistance to acid stress. These results indicated that gadB3 plays a crucial role in GABA biosynthesis and this property endowed the strain with acid tolerance. Our findings provided insights into how E. avium strains survive the acidic environments of fermented foods and throughout transit through the stomach and gut while maintaining cell viability.
RESUMO
The extensive use of acids in a variety of manufacturing industries results in the increase of discharged acidic waste stream into the environment. Such co-pollution of acids and other organic pollutants limits the biodegradation capability of neutrophilic degraders. With high-throughput genetic techniques, we aim to improve the acid tolerance of a pollutant-degrading bacterium, Pseudomonas putida S16 by genetically engineering it with the glutamate decarboxylase (GAD)-dependent system and the global regulator (IrrE) of extreme radiation resistance. The engineered strains holding either GAD system or irrE regulator could grow under pH 4.5, compared to the wild type. They could also degrade over 90% of a selected pollutant (benzoate or nicotine) under pH 5.0 in 48 h, while no biodegradation was detected with the wild type under the same conditions. We conclude that acid stress tolerance by the possession of the GAD system or IrrE regulator in pollutant-degrading bacteria would be a promising approach to enhance their viability and biodegrading activities in bioremediation of acidic wastes.
RESUMO
Gamma-aminobutyrate (GABA) is an important chemical in the pharmaceutical field. GABA-producing lactic acid bacteria (LAB) offer the opportunity of developing this health-oriented product. In this study, the gadA, gadB, gadC, gadCB, and gadCA gene segments of Lactobacillus brevis were cloned into pMG36e, and strain Lb. brevis/pMG36e-gadA was selected for thorough characterization in terms of GABA production after analysis of GAD activities. Subsequently, a physiology-oriented engineering strategy was adopted to construct an FoF1-ATPase deficient strain NRA6 with higher GAD activity. As expected, strain NRA6 could produce GABA at a concentration of 43.65 g/L with a 98.42% GABA conversion rate in GYP fermentation medium, which is 1.22-fold higher than that obtained by the wild-type strain in the same condition. This work demonstrates how the acid stress response mechanisms of LAB can be employed to develop cell factories with improved production efficiency and contributes to research into the development of the physiology-oriented engineering.