Bacillus subtilis stress-associated mutagenesis and developmental DNA repair.

SUMMARYThe metabolic conditions that prevail during bacterial growth have evolved with the faithful operation of repair systems that recognize and eliminate DNA lesions caused by intracellular and exogenous agents. This idea is supported by the low rate of spontaneous mutations (10-9) that occur in replicating cells, maintaining genome integrity. In contrast, when growth and/or replication cease, bacteria frequently process DNA lesions in an error-prone manner. DNA repairs provide cells with the tools needed for maintaining homeostasis during stressful conditions and depend on the developmental context in which repair events occur. Thus, different physiological scenarios can be anticipated. In nutritionally stressed bacteria, different components of the base excision repair pathway may process damaged DNA in an error-prone approach, promoting genetic variability. Interestingly, suppressing the mismatch repair machinery and activating specific DNA glycosylases promote stationary-phase mutations. Current evidence also suggests that in resting cells, coupling repair processes to actively transcribed genes may promote multiple genetic transactions that are advantageous for stressed cells. DNA repair during sporulation is of interest as a model to understand how transcriptional processes influence the formation of mutations in conditions where replication is halted. Current reports indicate that transcriptional coupling repair-dependent and -independent processes operate in differentiating cells to process spontaneous and induced DNA damage and that error-prone synthesis of DNA is involved in these events. These and other noncanonical ways of DNA repair that contribute to mutagenesis, survival, and evolution are reviewed in this manuscript.

Inorganic phosphate modifies stationary phase fitness and metabolic pathways in Lactiplantibacillus paraplantarum CRL 1905.

Inorganic phosphate (Pi) concentration modulates polyphosphate (polyP) levels in diverse bacteria, affecting their physiology and survival. Lactiplantibacillus paraplantarum CRL 1905 is a lactic acid bacterium isolated from quinoa sourdough with biotechnological potential as starter, for initiating fermentation processes in food, and as antimicrobial-producing organism. The aim of this work was to evaluate the influence of the environmental Pi concentration on different physiological and molecular aspects of the CRL 1905 strain. Cells grown in a chemically defined medium containing high Pi (CDM + P) maintained elevated polyP levels up to late stationary phase and showed an enhanced bacterial survival and tolerance to oxidative stress. In Pi sufficiency condition (CDM-P), cells were ~ 25% longer than those grown in CDM + P, presented membrane vesicles and a ~ 3-fold higher capacity to form biofilm. Proteomic analysis indicated that proteins involved in the "carbohydrate transport and metabolism" and "energy production and conversion" categories were up-regulated in high Pi stationary phase cells, implying an active metabolism in this condition. On the other hand, stress-related chaperones and enzymes involved in cell surface modification were up-regulated in the CDM-P medium. Our results provide new insights to understand the CRL 1905 adaptations in response to differential Pi conditions. The adjustment of environmental Pi concentration constitutes a simple strategy to improve the cellular fitness of L. paraplantarum CRL 1905, which would benefit its potential as a microbial cell factory.

A new type of phasin characterized by the presence of a helix-hairpin-helix domain is required for normal polyhydroxybutyrate accumulation and granule organization in Caulobacter crescentus.

Bacteria frequently store excess carbon in hydrophobic granules of polyhydroxybutyrate (PHB) that in some growth conditions can occupy most of the cytoplasmic space. Different types of proteins associate to the surface of the granules, mainly enzymes involved in the synthesis and utilization of the reserve polymer and a diverse group of proteins known as phasins. Phasins have different functions, among which are regulating the size and number of the granules, modulating the activity of the granule-associated enzymes and helping in the distribution of the granules inside the cell. Caulobacter crescentus is an oligotrophic bacterium that shows several morphological and regulatory traits that allow it to grow in very nutrient-diluted environments. Under these conditions, storage compounds should be particularly relevant for survival. In this work, we show an initial proteomic characterization of the PHB granules and describe a new type of phasin (PhaH) characterized by the presence of an N-terminal hydrophobic helix followed by a helix-hairpin-helix (HhH) domain. The hydrophobic helix is required for maximal PHB accumulation and maintenance during the stationary phase while the HhH domain is involved in determining the size of the PHB granules and their distribution in the cell.

Recent Trends in Graphene-Based Sorbents for LC Analysis of Food and Environmental Water Samples.

This review provides an overview of recent advancements in applying graphene-based materials as sorbents for liquid chromatography (LC) analysis. Graphene-based materials are promising for analytical chemistry, including applications as sorbents in liquid chromatography. These sorbents can be functionalized to produce unique extraction or stationary phases. Additionally, graphene-based sorbents can be supported in various materials and have consequently been applied to produce various devices for sample preparation. Graphene-based sorbents are employed in diverse applications, including food and environmental LC analysis. This review summarizes the application of graphene-based materials in food and environmental water analysis in the last five years (2019 to 2023). Offline and online sample preparation methods, such as dispersive solid phase microextraction, stir bar sorptive extraction, pipette tip solid phase extraction, in-tube solid-phase microextraction, and others, are reviewed. The review also summarizes the application of the columns produced with graphene-based materials in separating food and water components and contaminants. Graphene-based materials have been reported as stationary phases for LC columns. Graphene-based stationary phases have been reported in packed, monolithic, and open tubular columns and have been used in LC and capillary electrochromatography modes.

Application of an in-house packed octadecylsilica-functionalized graphene oxide column for capillary liquid chromatography analysis of hormones in urine samples.

Graphene oxide-based LC stationary phases were developed and applied for separating hormones from urine using capillaryLC-MS/MS. Using two analytical approaches - direct injection and column-switching arrangement - it was possible to evaluate the chromatographic parameters and perform tests on the raw biological fluid. Two stationary phases (SPs) were produced, varying the amino silica support particle diameter (Si, 5, and 10 µm). Graphene oxide was covalently bonded to the surface of Si particles, and this material was functionalized by the insertion of octadecylsilica groups, generating the SiGO-C18. Infra-red spectroscopy assays revealed that both steps were successful - supporting GO onto Si and further C18 customization. Scanning electron microscopy showed spherical geometries with minor irregularities and narrow particle size distribution for the produced SPs. The GO-coating rate was higher on the Si particles of 10 µm. As a result, the 10 µm produced column reported better resolution, efficiency, and peak capacity. Therefore, this SiGO-C18 capillary column (100 mm × 0.32 mm i.d., 10 µm dp) was applied successfully in a column-switching method to separate hormones in urine. Linearity (R2 above 0.99), quantification limits (between 1.0 and 5 µg/L), and other figures of merit of the method were determined. It is worth mentioning that the SiGO-C18 capillaryLC column performed adequately, separating the target compounds in less than 6 min. We hope this work could significantly contribute to shedding some light on graphene-based materials as a promising class of stationary phase for miniaturized liquid chromatography.

Ilex paraguariensis: the effect of genotypes and growth phase on biomass, secondary metabolism and antioxidant activity of in vitro cultivated calli / Ilex paraguariensis: el efecto de los genotipos y la fase de crecimiento sobre la biomasa, metabolismo secundario y la actividad antioxidante de los callos cultivados in vitro

Yerba mate ( Ilex paraguariensis ) produces several secondary metabolites of interest to the phar maceutical industry, such as chlorogenic acids and methylxanthines. These compounds have been produced in vitro by callus culture from different species. However, for I. paraguariensis , no studies upon the production of these compounds in vitro have been p erformed to date. In this work, we show that the concentration of secondary metabolites from I. paraguariensis callus is possible and highly dependent on the callus growth phase. We observed that the best phase for the production of secondary compounds in calli of yerba mate is the stationary growth phase on both genotypes tested. In this phase, higher levels of phenolic compounds, chlorogenic acid and 3,5 - dicaffeoylquinic acid and greater antioxidant activity were observed. Chlorogenic acid and 3,5 - dicaffe oylquinic acid presented positive correlation with antioxidant activity. For the first time, secondary compounds were reported in yerba mate calli cultivated in vitro .

La yerba mate ( Ilex paraguariensis ) produce varios metabolitos secundarios de interés para la industria farmacéutica, como los ácidos clorogénicos y las metilxantinas. Estos compuestos se han producido in vitro mediante cultivo de ca llos de diferentes especies. Sin embargo, para I. paraguariensis , hasta la fecha no se han realizado estudios sobre la producción de estos compuestos in vitro . En este trabajo, mostramos que la concentración de metabolitos secundarios desde callos de I. pa raguariensis es posible y altamente dependiente de la fase de crecimiento del callo. Observamos que la mejor fase para la producción de compuestos secundarios en callos de yerba mate es la fase de crecimiento estacionario en ambos genotipos probados. En es ta fase se observaron niveles más altos de compuestos fenólicos, ácido clorogénico y ácido 3,5 - dicafeoilquínico y una mayor actividad antioxidante. El ácido clorogénico y el ácido 3,5 - dicafeoilquínico presentaron correlación positiva con la actividad antio xidante. Por primera vez, se reportaron compuestos secundarios en callos de yerba mate cultivados in vitro .

Enantioselective metabolomics by liquid chromatography-mass spectrometry.

Metabolomics strives to capture the entirety of the metabolites in a biological system by comprehensive analysis, often by liquid chromatography hyphenated to mass spectrometry. A particular challenge thereby is the differentiation of structural isomers. Common achiral targeted and untargeted assays do not distinguish between enantiomers. This may lead to information loss. An increasing number of publications demonstrate that the enantiomeric ratio of certain metabolites can be meaningful biomarkers of certain diseases emphasizing the importance of introducing enantioselective analytical procedures in metabolomics. In this work, the state-of-the-art in the field of LC-MS based metabolomics is summarized with focus on developments in the recent decade. Methodologies, tagging strategies, workflows and general concepts are outlined. Selected biological applications in which enantioselective metabolomics has documented its usefulness are briefly discussed. In general, targeted enantioselective metabolomics assays are often based on a direct approach using chiral stationary phases (CSP) with polysaccharide derivatives, macrocyclic antibiotics, chiral crown ethers, chiral ion exchangers, donor-acceptor phases as chiral selectors. Rarely, these targeted assays focus on more than 20 analytes and usually are restricted to a certain metabolite class. In a variety of cases, pre-column derivatization of metabolites has been performed, especially for amino acids, to improve separation and detection sensitivity. Triple quadrupole instruments are the detection methods of first choice in targeted assays. Here, issues like matrix effect, absence of blank matrix impair accuracy of results. In selected applications, multiple heart cutting 2D-LC (RP followed by chiral separation) has been pursued to overcome this problem and alleviate bias due to interferences. Non-targeted assays, on the other hand, are based on indirect approach involving tagging with a chiral derivatizing agent (CDA). Besides classical CDAs numerous innovative reagents and workflows have been proposed and are discussed. Thereby, a critical issue for the accuracy is often neglected, viz. the validation of the enantiomeric impurity in the CDA. The majority of applications focus on amino acids, hydroxy acids, oxidized fatty acids and oxylipins. Some potential clinical applications are highlighted.

The Saccharomyces cerevisiae Ncw2 protein works on the chitin/ß-glucan organisation of the cell wall.

The NCW2 gene was recently described as encoding a GPI-bounded protein that assists in the re-modelling of the Saccharomyces cerevisiae cell wall (CW) and in the repair of damage caused by the polyhexamethylene biguanide (PHMB) polymer to the cell wall. Its absence produces a re-organization of the CW structure that result in resistance to lysis by glucanase. Hence, the present study aimed to extend the analysis of the Ncw2 protein (Ncw2p) to determine its physiological role in the yeast cell surface. The results showed that Ncw2p is transported to the cell surface upon O-mannosylation mediated by the Pmt1p-Pmt2p enzyme complex. It co-localises with the yeast bud scars, a region in cell surface formed by chitin deposition. Once there, Ncw2p enables correct chitin/ß-glucan structuring during the exponential growth. The increase in molecular mass by hyper-mannosylation coincides with the increasing in chitin deposition, and leads to glucanase resistance. Treatment of the yeast cells with PHMB produced the same biological effects observed for the passage from exponential to stationary growth phase. This might be a possible mechanism of yeast protection against cationic biocides. In conclusion, we propose that Ncw2p takes part in the mechanism involved in the control of cell surface rigidity by aiding on the linkage between chitin and glucan layers in the modelling of the cell wall during cell growth.

Stationary phase based on cellulose dodecanoate physically immobilized on silica particles for high-performance liquid chromatography.

The chemical agent free preparation of a stationary phase using a natural macromolecule was the focus of this paper. Thermal immobilization of cellulose dodecanoate on silica particles was used for the preparation of a stationary phase without the use of chemical reagents. Cellulose modification was performed to produce a hydrophobic macromolecule with solubility in common organic solvents. The new stationary phase was characterized morphologically and physico-chemically, presenting as spherical particles immobilized with a thin cellulose dodecanoate layer. The degree of substitution of cellulose dodecanoate was 1.7, which resulted in a separation mechanism in reversed phase mode, but with lower hydrophobicity and higher steric selectivity, which are properties from cellulose. These characteristics resulted in a stationary phase with intrinsic selectivity that was able to separate mixtures of polar drugs, homologs of an anionic surfactant and omeprazole isomers, which are not well resolved in typical C18 phases. Considering that cellulose is a natural polymer and the preparation method of stationary phase involves only physical processes of silica modification, the final material presents as a stationary phase with specific retention properties coming from both dodecanoate and cellulose.

Proteomic Study of the Exponential-Stationary Growth Phase Transition in the Haloarchaea Natrialba magadii and Haloferax volcanii.

The dynamic changes that take place along the phases of microbial growth (lag, exponential, stationary, and death) have been widely studied in bacteria at the molecular and cellular levels, but little is known for archaea. In this study, a high-throughput approach was used to analyze and compare the proteomes of two haloarchaea during exponential and stationary growth: the neutrophilic Haloferax volcanii and the alkaliphilic Natrialba magadii. Almost 2000 proteins were identified in each species (≈50% of the predicted proteome). Among them, 532 and 432 were found to be differential between growth phases in H. volcanii and N. magadii, respectively. Changes upon entrance into stationary phase included an overall increase in proteins involved in the transport of small molecules and ions, stress response, and fatty acid catabolism. Proteins related to genetic processes and cell division showed a notorious decrease in amount. The data reported in this study not only contributes to our understanding of the exponential-stationary growth phase transition in extremophilic archaea but also provides the first comprehensive analysis of the proteome composition of N. magadii. The MS proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier JPST000395.

Controlled crosslinking of trimethylolpropane trimethacrylate for preparation of organic monolithic columns for capillary liquid chromatography.

Organic monolithic columns based on single crosslinking of trimethylolpropane trimethacrylate (TRIM) monomer were prepared in a single step by living/controlled free-radical polymerization. Full optimization of the preparation, such as using different percentages of TRIM and different amounts of radical promoter as well as various porogen solvents were explored. The resulting monolithic columns were characterized by scanning electronic microscopy and nitrogen sorption for structure morphology studies and surface area measurements, respectively. Using capillary liquid chromatography, 150 µm i.d. columns were applied to separate a mixture of small hydrophobic molecules. The results indicated that column performance is highly sensitive to the type and the amount of porogen solvents used in the polymerization mixture composition. Good resolution factors and methylene selectivity were obtained, indicating the promising potential of this material for capillary liquid chromatography separations.

A plasmid-encoded DsbA homologue is a growth-phase regulated thioredoxin.

The Pseudomonas aeruginosa plasmid pUM505 contains in a pathogenicity island the dsbA2 gene, which encodes a product with similarity to DsbA protein disulfide isomerases, enzymes that catalyze formation and isomerization of disulfide bonds in protein cysteine residues. Using transcriptional fusions, it was found that dsbA2 gene promoter is activated during the stationary phase, suggesting that DsbA2 protein may be required for adaptive changes that occur during this stage of bacterial growth. Transfer of the pUM505 dsbA2 gene to a cadmium-sensitive P. aeruginosa PAO1-derivative affected in the chromosomal dsbA gene, restored cadmium resistance, suggesting a role of DsbA2 in protecting protein disulfide bonds. PAO1 dsbA2 transformants displayed increased sensitivity to intercalating agent mitomycin C, indicating that DsbA2 functions as a thioredoxin enzyme able to modify and activate toxicity of this compound. These results highlight the adaptive role of the pUM505 plasmid in its P. aeruginosa hosts.

Acyl homoserine lactone changes the abundance of proteins and the levels of organic acids associated with stationary phase in Salmonella Enteritidis.

Quorum sensing (QS) is cell-cell communication mechanism mediated by signaling molecules known as autoinducers (AIs) that lead to differential gene expression. Salmonella is unable to synthesize the AI-1 acyl homoserine lactone (AHL), but is able to recognize AHLs produced by other microorganisms through SdiA protein. Our study aimed to evaluate the influence of AI-1 on the abundance of proteins and the levels of organic acids of Salmonella Enteritidis. The presence of N-dodecyl-homoserine lactone (C12-HSL) did not interfere on the growth or the total amount of extracted proteins of Salmonella. However, the abundance of the proteins PheT, HtpG, PtsI, Adi, TalB, PmgI (or GpmI), Eno, and PykF enhanced while the abundance of the proteins RplB, RplE, RpsB, Tsf, OmpA, OmpC, OmpD, and GapA decreased when Salmonella Enteritidis was anaerobically cultivated in the presence of C12-HSL. Additionally, the bacterium produced less succinic, lactic, and acetic acids in the presence of C12-HSL. However, the concentration of extracellular formic acid reached 20.46 mM after 24 h and was not detected when the growth was in the absence of AI-1. Considering the cultivation period for protein extraction, their abundance, process and function, as well as the levels of organic acids, we observed in cells cultivated in presence of C12-HSL a correlation with what is described in the literature as entry into the stationary phase of growth, mainly related to nitrogen and amino acid starvation and acid stress. Further studies are needed in order to determine the specific role of the differentially abundant proteins and extracellular organic acids secreted by Salmonella in the presence of quorum sensing signaling molecules.

Adaptation of Propionibacterium freudenreichii to long-term survival under gradual nutritional shortage.

BACKGROUND: Propionibacterium freudenreichii is an Actinobacterium widely used in the dairy industry as a ripening culture for Swiss-type cheeses, for vitamin B12 production and some strains display probiotic properties. It is reportedly a hardy bacterium, able to survive the cheese-making process and digestive stresses. RESULTS: During this study, P. freudenreichii CIRM-BIA 138 (alias ITG P9), which has a generation time of five hours in Yeast Extract Lactate medium at 30 °C under microaerophilic conditions, was incubated for 11 days (9 days after entry into stationary phase) in a culture medium, without any adjunct during the incubation. The carbon and free amino acids sources available in the medium, and the organic acids produced by the strain, were monitored throughout growth and survival. Although lactate (the preferred carbon source for P. freudenreichii) was exhausted three days after inoculation, the strain sustained a high population level of 9.3 log10 CFU/mL. Its physiological adaptation was investigated by RNA-seq analysis and revealed a complete disruption of metabolism at the entry into stationary phase as compared to exponential phase. CONCLUSIONS: P. freudenreichii adapts its metabolism during entry into stationary phase by down-regulating oxidative phosphorylation, glycolysis, and the Wood-Werkman cycle by exploiting new nitrogen (glutamate, glycine, alanine) sources, by down-regulating the transcription, translation and secretion of protein. Utilization of polyphosphates was suggested.

Transcriptomic analysis of the stationary phase response regulator SpdR in Caulobacter crescentus.

BACKGROUND: As bacterial cells enter stationary phase, they adjust their growth rate to comply with nutrient restriction and acquire increased resistance to several stresses. These events are regulated by controlling gene expression at this phase, changing the mode of exponential growth into that of growth arrest, and increasing the expression of proteins involved in stress resistance. The two-component system SpdR/SpdS is required for the activation of transcription of the Caulobacter crescentus cspD gene at the onset of stationary phase. RESULTS: In this work, we showed that both SpdR and SpdS are also induced upon entry into stationary phase, and this induction is partly mediated by ppGpp and it is not auto-regulated. Global transcriptional analysis at early stationary phase of a spdR null mutant strain compared to the wild type strain was carried out by DNA microarray. Twenty-three genes showed at least twofold decreased expression in the spdR deletion mutant strain relative to its parental strain, including cspD, while five genes showed increased expression in the mutant. The expression of a set of nine genes was evaluated by quantitative real time PCR, validating the microarray data, and indicating an important role for SpdR at stationary phase. Several of the differentially expressed genes can be involved in modulating gene expression, including four transcriptional regulators, and the RNA regulatory protein Hfq. The ribosomal proteins NusE and NusG, which also have additional regulatory functions in transcription and translation, were also downregulated in the spdR mutant, as well as the ParE1 toxin. The purified SpdR protein was shown to bind to the regulatory region of CC0517 by Electrophoretic Mobility Shift Assay, and the SpdR-regulated gene CC0731 was shown to be expressed at a lower level in the null cspD mutant, suggesting that at least part of the effect of SpdR on the expression of this gene is indirect. CONCLUSIONS: The results indicate that SpdR regulates several genes encoding proteins of regulatory function, which in turn may be required for the expression of other genes important for the transition to stationary phase.

Dormancy models for Mycobacterium tuberculosis: A minireview.

Dormancy models for Mycobacterium tuberculosis play important roles in understanding various aspects of tuberculosis pathogenesis and in the testing of novel therapeutic regimens. By simulating the latent tuberculosis infection, in which the bacteria exist in a non-replicative state, the models demonstrate reduced susceptibility to antimycobacterial agents. This minireview outlines the models available for simulating latent tuberculosis both in vitro and in several animal species. Additionally, this minireview discusses the advantages and disadvantages of these models for investigating the bacterial subpopulations and susceptibilities to sterilization by various antituberculosis drugs.

Dormancy models for Mycobacterium tuberculosis: A minireview

Dormancy models for Mycobacterium tuberculosis play important roles in understanding various aspects of tuberculosis pathogenesis and in the testing of novel therapeutic regimens. By simulating the latent tuberculosis infection, in which the bacteria exist in a non-replicative state, the models demonstrate reduced susceptibility to antimycobacterial agents. This minireview outlines the models available for simulating latent tuberculosis both in vitro and in several animal species. Additionally, this minireview discusses the advantages and disadvantages of these models for investigating the bacterial subpopulations and susceptibilities to sterilization by various antituberculosis drugs..(AU)

Dormancy models for Mycobacterium tuberculosis: A minireview

Dormancy models for Mycobacterium tuberculosis play important roles in understanding various aspects of tuberculosis pathogenesis and in the testing of novel therapeutic regimens. By simulating the latent tuberculosis infection, in which the bacteria exist in a non-replicative state, the models demonstrate reduced susceptibility to antimycobacterial agents. This minireview outlines the models available for simulating latent tuberculosis both in vitro and in several animal species. Additionally, this minireview discusses the advantages and disadvantages of these models for investigating the bacterial subpopulations and susceptibilities to sterilization by various antituberculosis drugs.

Isolation and characterization of Caulobacter mutants impaired in adaptation to stationary phase

The entry into stationary phase causes a change in the pattern of gene expression of bacteria, when the cells must express a whole set of genes involved mainly with resistance to starvation and to environmental stresses. As an attempt to identify genes important for the survival of Caulobacter crescentus in stationary phase, we have screened a library of 5,000 clones generated by random transposon mutagenesis for mutants that showed reduced viability after prolonged growth. Four clones were selected, which displayed either lower viability or a longer time of recovery from stationary phase. The genes disrupted were identified, and the gene products were found to be mainly involved with amino acid metabolism (glutamate N-acetyltransferase, 4-hydroxyphenylpyruvate dioxygenase and L-aspartate oxidase) or with recombination (exonuclease RecJ). Each mutant was tested for resistance to stresses, such as oxidative, saline, acidic, heat and UV exposure, showing different responses. Although the mutations obtained were not in genes involved specifically in stationary phase, our results suggest that amino acids metabolism may play an important role in keeping viability during this growth phase.

A entrada em fase estacionária causa uma mudança no padrão de expressão gênica de bactérias, quando as células devem expressar um novo conjunto de genes envolvidos principalmente com resistência à carência alimentar e a estresses ambientais. Em uma tentativa de identificar genes importantes para a sobrevivência de Caulobacter crescentus em fase estacionária, nós varremos uma biblioteca de 5.000 clones gerados por transposição aleatória em busca de mutantes que mostrassem viabilidade reduzida após crescimento prolongado. Quatro clones foram selecionados, que mostraram menor viabilidade ou um maior tempo de recuperação da fase estacionária. Os genes interrompidos foram identificados, e os produtos gênicos mostraram-se estar envolvidos principalmente com o metabolismo de aminoácidos (glutamato N-acetiltransferase, 4-hidroxifenilpiruvato dioxigenase e L-aspartato oxidase) ou com recombinação (exonuclease RecJ). Cada mutante foi testado para resistência a estresses, como oxidativo, salino, ácido, calor e exposição à luz UV, mostrando respostas diferentes. Embora as mutações obtidas não tenham sido em genes envolvidos especificamente com fase estacionária, nossos resultados sugerem que o metabolismo de aminoácidos tem papel importante na manutenção da viabilidade durante esta fase do crescimento.