Sugar-rich environments represent an important challenge for microorganisms. The osmotic and molecular imbalances resulting from this condition severely limit microbial metabolism and growth. Gluconacetobacter diazotrophicus is one of the most sugar-tolerant prokaryotes, able to grow in the presence of sucrose concentrations up to 30%. However, the mechanisms that control its tolerance to such conditions remain poorly exploited. The present work investigated the key mechanisms of tolerance to high sugar in G. diazotrophicus. Comparative proteomics was applied to investigate the main functional pathways regulated in G. diazotrophicus when cultivated in the presence of high sucrose. Among 191 proteins regulated by high sucrose, regulatory pathways related to sugar metabolism, nutrient uptake, compatible solute synthesis, amino acid metabolism, and proteolytic system were highlighted. The role of these pathways on high-sucrose tolerance was investigated by mutagenesis analysis, which revealed that the knockout mutants zwf:Tn5 (sugar metabolism), tbdr:Tn5 (nutrient uptake), mtlK:Tn5 (compatible solute synthesis), pepN:Tn5 (proteolytic system), metH:Tn5 (amino acid metabolism), and ilvD:Tn5 (amino acid metabolism) became more sensitive to high sucrose. Together, our results identified mechanisms involved in response to high sugar in G. diazotrophicus, shedding light on the combination of osmotolerance and sugar-tolerance mechanisms. Key points: • G. diazotrophicus intensifies glycolysis to metabolize the excess of sugar. • G. diazotrophicus turns down the uptake of nutrients in response to high sugar. • G. diazotrophicus requires amino acid availability to resist high sugar.
Keywords: Sugar-rich environment; Abiotic stress; PGPB; Proteomics
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00253-021-11590-7.
Sugar-rich environments are widespread in the microbial habitats. This condition challenges bacterial life through multiple aspects, such as the imbalance on macromolecular systems and decreased water activity (Oliveira et al. [
The decrease in water activity resulting from the exposure to solutes, including sugars, leads to an osmotic imbalance on microorganism cells, which can cause the efflux of water, turgor pressure loss, decrease in metabolic functions, and ultimately culminate in cell death (Esbelin et al. [
Sugars can alter the viscosity of both extracellular and intracellular compartments, and that viscosity per se is a stress parameter that inhibits the bacterial metabolic activities (Lievens et al. [
The plant growth-promoting bacterium (PGPB) Gluconacetobacter diazotrophicus is classified as one of the most sugar-tolerant prokaryotes (Cavalcante and Dobereiner [
G. diazotrophicus has also been isolated from pineapple (Ananas comosus), sweet potato (Ipomoea batatas), and coffee plants (Coffea arabica) (Luna et al. [
Boniolo et al. ([
The present work investigated mechanisms of G. diazotrophicus involved in the response and tolerance to high-sucrose concentrations. The effects of these conditions on cell morphology and viability were investigated by epifluorescence microscopy. The molecular mechanisms regulated in response to high-sucrose concentrations were investigated by comparative proteomics, and the proteins essential for tolerance to sugar stress were identified by using knockout mutants.
G. diazotrophicus wild-type (PAL5 standard strain, ATCC 49,037) used in this study was obtained in the culture collection of the Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF, Campos dos Goytacazes, Rio de Janeiro State, Brazil). The knockout mutants of G. diazotrophicus PAL5, defective in the synthesis of proteins glucose-6-phosphate 1-dehydrogenase (A9H326—zwf:Tn5), MtlK (A9HBL5—mtlK:Tn5), aminopeptidase N (A9HEL6—pepN:Tn5), methionine synthase (A9HFG3—metH:Tn5), dihydroxy-acid dehydratase (A9HA40—ilvD:Tn5), and a TonB-dependent receptor (A9HNM4—tbdr:Tn5), were obtained from the previously generated "G. diazotrophicus PAL5 Tn5 insertion mutant library" of the Laboratório de Biotecnologia—UENF (Intorne et al. [
High-sucrose exposure assays were performed as previously described (Oliveira et al. [
Morphological and cell viability analysis was performed as previously described (Leandro et al. [
Proteomic analyses were performed as previously described (Leandro et al. [
Spectral processing and database searching were performed using the software ProteinLynx Global Server (PLGS; version 3.0.2, provided by Waters Corporation) and ISOQuant (Distler et al. [
Comparative label-free quantification analysis was performed with ISOQuant software with previously described settings and algorithms (Distler et al. [
The data were analyzed using Student's t test (two-tailed), and differentially regulated proteins (DAPs) (p < 0.05) were considered up-regulated if the fold change (FC) was higher than 1.5 and down-regulated if the FC was lesser than 0.667.
Additionally, DAP subcellular localization (outer membrane, periplasm, cytoplasmic membrane, or cytoplasm) was predicted using the FUEL-mLoc subcellular localization prediction server at:
Reverse genetics analysis was performed as previously described (Leandro et al. [
Aiming to evaluate the effects of sucrose on G. diazotrophicus growth, morphology, and cell viability, bacterial cultures were grown under sucrose concentrations ranging from 0 to 800 mM.
G. diazotrophicus growth was inhibited by all sucrose concentrations tested, ranging from 14% inhibition, at 400 mM sucrose, to 40% inhibition, at 800 mM (Fig. 1). None of the sugar concentrations caused apparent morphological changes to bacterial cells (Supplemental Fig. S1). Additionally, epifluorescence microscopy analyses show that sucrose slightly affects the cell viability of G. diazotrophicus, even at the highest concentrations of sucrose tested (Supplemental Fig. S2).
Graph: Fig. 1 Sucrose inhibits G. diazotrophicus growth. G. diazotrophicus was cultivated for 12 h in liquid medium supplemented with different concentrations of sucrose, and its growth performance was analyzed through optical density. Three technical replications were used for each treatment, and means followed by different letters are significantly different from each other at 5% probability level by Tukey test. This analysis was repeated, at least, in three independent times, and similar results were observed
These results indicate that, despite the inhibitory effect of high sucrose on G. diazotrophicus growth, bacterial cells contain adaptative mechanisms that allow cell viability maintenance. So, the treatment with 600 mM sucrose was selected for further analyses of the molecular aspects of the bacterial response to high sucrose by proteomic analyses.
To investigate mechanisms regulated in G. diazotrophicus under a high-sucrose environment, total protein extracts from bacterial cells, cultivated in the presence and absence of 600 mM sucrose, were analyzed by comparative proteomics. A total of 601 proteins were identified, of which 191 (~ 30%) were differentially regulated (Supplemental Table S2, Fig. 2A). Among regulated proteins, 98 were up-regulated, while 93 were down-regulated in response to sucrose (Fig. 2B).
Graph: Fig. 2 High-sucrose concentration changes the proteome profile of G. diazotrophicus. Volcano plot of all identified proteins was performed (A). The spots represent differential abundance (log2 fold change) of identified proteins in the function of statistical significance (− log10 p value), and up-regulated, down-regulated, and non-regulated proteins are represented by blue, red, and gray spots, respectively. A graphical representation of the proportion of differentially accumulated proteins (DAPs) increased and decreased was also performed (B)
The 191 proteins differentially regulated in response to sucrose were categorized according to their predicted localization (see methods). As shown in Fig. 3, most of the regulated proteins are located in the cytoplasm and inner membrane, with a higher proportion of up-regulated proteins. Proteins from the outer membrane and periplasm were mainly down-regulated in response to sucrose.
Graph: Fig. 3 High-sucrose concentration changes the protein profile of the cellular compartments of G. diazotrophicus. Differentially accumulated proteins (DAPs) were classified by predicted subcellular localization (cytoplasm; inner membrane; periplasm; outer membrane) with fuel-mloc software
Five functional pathways regulated during the G. diazotrophicus response to high sucrose were identified through the functional categorization of DAPs: sugar metabolism, nutrient uptake, osmotic adjustment, amino acid metabolism, and proteolytic system. Such pathways are described below.
Among the proteins up-regulated in response to high sucrose, 15 participate in pathways involved with sugar metabolism (Table 1). Six proteins are components of glucose catabolism (glycolysis): glucose-6-phosphate 1-dehydrogenase (A9H0G0, Zwf), 6-phosphogluconate dehydrogenase, decarboxylating (A9H324, Gnd), glucokinase (A9HI04, Glk), bifunctional transaldolase/phosoglucose isomerase (A9H320, Tal/Pgi), 6-phosphogluconolactonase (A9HJ42, Pgl), and α-D-glucose phosphate-specific phosphoglucomutase (A9HSH5, Pgm); two belong to the pyruvate dehydrogenase complex: dihydrolipoyl acetyltransferase (A9HJB2) and pyruvate dehydrogenase E1 (A9HJA9, PdhB); and three participate in the two-step pathway of ethanol oxidation: NAD(P)-dependent alcohol dehydrogenase (A9HNN4, Adh), alcohol dehydrogenase (A9HNA5, AdhP) and aldehyde dehydrogenase (A9H4V7, AldA). Additionally, the proteins gluconolactonase (A9Hl52) and galactose mutarotase (A9HBF6, GalM), essential to gluconic acid production and galactose metabolism, respectively, were also up-regulated. These results indicate that, in response to high sucrose, G. diazotrophicus intensifies sugar metabolism, producing gluconic acid and oxidizing ethanol.
Table 1 Functional pathways regulated in G. diazotrophicus cells exposed to high sucrose
Accession Description Fold change a. Sugar metabolism A9H0G0 Glucose-6-phosphate 1-dehydrogenase (Zwf) 2.78 A9HI04 Glucokinase (Glk) 2.95 A9H320 Bifunctional transaldolase/phosoglucose isomerase (Tal/Pgi) 3.75 A9HSH5 α-D-Glucose phosphate-specific phosphoglucomutase (Pgm) 1.83 A9HJ42 6-Phosphogluconolactonase (Pgl) 1.64 A9H324 6-Phosphogluconate dehydrogenase, decarboxylating (Gnd) 1.64 A9H317 Transketolase (TktA) 3.05 A9HGX3 Phosphoketolase 4.79 A9HJB2 Dihydrolipoyl acetyltransferase 4.47 A9HJA9 Pyruvate dehydrogenase E1 (PdhB) 3.97 A9HNN4 NAD(P)-dependent alcohol dehydrogenase (Adh) 2.11 A9HNA5 Alcohol dehydrogenase (AdhP) 3.39 A9H4V7 Aldehyde dehydrogenase (AldA) 2.17 A9HL52 Gluconolactonase 1.70 A9HBF6 Galactose mutarotase (GalM) 2.64 b. Nutrient uptake A9HPE7 sn-Glycerol-3-phosphate ABC transporter ATP-binding protein (UgpC) 0.51 A9HPE1 Sugar ABC transporter substrate-binding protein 0.12 A9HPF6 Carbohydrate porin (OprB) 0.12 A9HNP0 D-Xylose ABC transporter, periplasmic substrate-binding (XylF) 0.28 A9HPB9 ABC transporter substrate-binding protein (RbsB) 0.25 A9HPK6 D-Ribose-binding periplasmic protein (RbsB) 0.11 A9H932 TonB-dependent receptor 1.53 A9H7M7 TonB-dependent receptor 0.13 A9HEU6 TonB-dependent receptor 0.27 A9HDZ9 TonB-dependent receptor 0.42 A9H7L3 TonB-dependent siderophore receptor 0.11 c. Osmotic adjustment A9HBU3 Trehalose 6-phosphate phosphatase (OtsB) 1.52 A9HBL5 Mannitol 2-dehydrogenase (MtlK) 6.8 A9HBX1 Pyrroline-5-carboxylate reductase (ProC) 1.64 d. Amino acid metabolism A9HNX4 5-Methyltetrahydropteroyltriglutamate–homocysteine methyltransferase (MetE) 7.94 A9HNY2 Methylenetetrahydrofolate reductase (MetF) 4.0 A9GZJ4 Ketol-acid reductoisomerase (NADP(+)) (IlvC) 3.13 A9HCQ4 Homoserine dehydrogenase 1 2.07 e. Proteolytic system A9HEL6 M1 family metallopeptidase—aminopeptidase N (PepN) 1.74 A9HFU5 M1 family metallopeptidase—aminopeptidase N (PepN) 1.75 A9HN12 M61 family metallopeptidase (aminopeptidase) 2.05 A9HP02 M3 family metallopeptidase (dipeptidyl carboxypeptidase—Dcp) 1.53 A9HRE6 M13 family metallopeptidase—endopeptidase (PepO) 1.92 A9HKD9 M32 family metallopeptidase (carboxypeptidase) 1.61 A9HS00 Serine peptidase S10 (carboxypeptidase) 1.74
The functional pathway with the second-highest number of proteins regulated in response to high sucrose is related to nutrient uptake. Among the down-regulated proteins, 11 are related to sugar, iron, and other nutrient uptake (Table 1). Among these, six are involved with sugar uptake (A9HPE7, A9HPE1, A9HPF6, A9HNP0, A9HPB9, and A9HPK6), four are TonB-dependent receptors—TBDRs (A9H932, A9H7M7, A9HEU6, and A9HDZ9), and one is a TonB-dependent siderophore receptor (A9H7L3), involved with iron and other nutrient uptake. These results indicate that G. diazotrophicus turns down the uptake of sugar and other nutrients in response to a high-sucrose environment.
Three proteins involved with compatible solutes synthesis were up-regulated in G. diazotrophicus in response to high sucrose: trehalose 6-phosphate phosphatase (A9HBU3, OtsB), mannitol 2-dehydrogenase (A9HBL5, MtlK), and pyrroline-5-carboxylate reductase (A9HBX1, ProC) (Table 1). The regulation of proteins related to the synthesis of three different classes of compatible solutes suggests that a variety of these compounds may be required for G. diazotrophicus tolerance to high sucrose.
Among the cytoplasmic proteins up-regulated in response to sucrose, four are directly involved in amino acid metabolism (Table 1). Among these, two participate in the methionine biosynthetic pathway: 5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase (A9HNX4, MetE) and methylenetetrahydrofolate reductase (A9HNY2, MetF); one participates in the homoserine synthesis: homoserine dehydrogenase 1 (A9HCQ4); and one is involved in the biosynthesis of branched-chain amino acids (BCAA): ketol-acid reductoisomerase (NADP(+)) (A9GZJ4, IlvC).
Seven proteins with proteolytic activity were up-regulated in G. diazotrophicus in response to sucrose (Table 1). Among these, six belongs to the family of metallopeptidases: M1 aminopeptidases N-PepN (A9HEL6, A9HFU5), M61 aminopeptidase (A9HN12), M3 dipeptidyl carboxypeptidase—Dcp (A9HP02), M13 endopeptidase—PepO (A9HRE6), M32 carboxypeptidase (A9HKD9), and one is a serine peptidase: carboxypeptidase S10 (A9HS00), indicating that the exposure to a high-sucrose environment leads to the activation of proteolytic systems in G. diazotrophicus.
The five abovementioned functional pathways revealed by the proteomic analyses as regulated by high-sucrose were tested about its role on G. diazotrophicus tolerance to such conditions. To this, a "G. diazotrophicus knockout mutants library" was accessed to identify strains defective for key proteins of such pathways. As a result, we select the knockout mutant strains related to sugar metabolism (zwf:Tn5), nutrient uptake (tbdr:Tn5), compatible solute synthesis (mtlK:Tn5), amino acid metabolism (metH:Tn5, ilvD:Tn5), and proteolytic system (pepN:Tn5). Thereby, a reverse genetic approach was performed comparing the growth of the wild-type strain of G. diazotrophicus with the knockout mutant strains under moderate (300 mM) and high (600 mM) sucrose concentrations.
As shown in Fig. 4, all the knockout mutants were sensitive to the high-sucrose concentration tested. This result confirms that the functional pathways revealed by the proteomic analyses have a key role on G. diazotrophicus tolerance to high sucrose.
Graph: Fig. 4 Reverse genetics analysis revealed essential genes to high-sucrose concentration tolerance in G. diazotrophicus. Knockout mutants of G. diazotrophicus defectives in the synthesis of proteins related to protein functional groups identified in our proteomic analyses were selected to perform the sucrose resistance assay. Results were registered after 5 days of sucrose exposure
The present work investigated the key mechanisms involved in the tolerance of G. diazotrophicus to high-sucrose concentrations. Our analyses showed that, although high-sucrose inhibit the growth of G. diazotrophicus, no evident effects on bacterial cell morphology or cell viability were observed. Proteomic analyses highlighted the regulation of several proteins involved with sugar metabolism, nutrient uptake, compatible solute synthesis, amino acid metabolism, and proteolytic system. The use of knockout mutants revealed the essential role of these pathways for G. diazotrophicus tolerance to high sucrose.
The proteomic analyses of G. diazotrophicus responses to high sucrose showed the up-regulation of several proteins related to sugar metabolism and the down-regulation of proteins related to sugar import. This result suggests that the bacteria activate both the sucrose conversion and sugar import restriction to avoid its excess into the cytoplasm. G. diazotrophicus is not able to import sucrose, so it utilizes the extracellular enzyme levansucrase to break sucrose into glucose and fructose, which are taken up by the cell (Alvarez and Martinez-Drets [
Proteomics also revealed the down-regulation of proteins involved with nutrient uptake. A similar response was observed in G. diazotrophicus cells as a tolerance mechanism against the osmotic stress caused by PEG-400 (about 0.95 water activity), probably to avoid the entry of the harmful compounds in bacterial cells (Leandro et al. [
Our proteomic analysis revealed the down-regulation of several TBDRs involved in nutrient uptake. A similar response was observed when G. diazotrophicus was exposed to the ionic osmotic stressor NaCl (Leandro et al. [
Three proteins involved with the de novo synthesis of the compatible solutes mannitol (MtlK), trehalose (OtsB), and proline (ProC) were up-regulated in our proteomic analyses. The protein MtlK is an essential component of G. diazotrophicus tolerance to osmotic stress caused by PEG-400 (Leandro et al. [
The proteomic analyses also revealed the up-regulation of proteins of the proteolytic system. This may represent a bacterial response mechanism to hyperosmotic environments, leading to the regulation of amino acids into the cytoplasm to act as compatible solutes (Le Marrec et al. [
MetE and MetF, two proteins involved in de novo methionine biosynthesis, were up-regulated in our proteomic analyses. The last step of de novo methionine biosynthesis is catalyzed by either cobalamin (B12)-independent methionine synthase (MetE) or B12-dependent methionine synthase (MetH) (Weissbach and Brot [
Lery et al. ([
Table 2 Proteins up-accumulated in G. diazotrophicus in response to both sucrose and sugarcane co-cultivation
Accession Description Functional pathway A9H320 Bifunctional transaldolase/phosoglucose isomerase (Tal/Pgi) Sugar metabolism A9H317 Transketolase (TktA) Sugar metabolism A9H324 6-Phosphogluconate dehydrogenase, decarboxylating (Gnd) Sugar metabolism A9HGX3 Phosphoketolase Sugar metabolism A9HL52 Gluconolactonase Sugar metabolism A9H932 TonB-dependent receptor Nutrient uptake A9GZJ4 Ketol-acid reductoisomerase (NADP(+)) (IlvC) Amino acid metabolism A9HNX4 5-Methyltetrahydropteroyltriglutamate–homocysteine methyltransferase (MetE) Amino acid metabolism A9HFU5 M1 family metallopeptidase—aminopeptidase N (PepN) Proteolytic system A9HHS4 Large-conductance mechanosensitive channel (MscL) Hypoosmotic shock response A9HED6 OmpW family protein (OmpW) Multiple functional pathways A9H5P1 Bacteriocin protein Antibiotic activity A9H9C0 Adenosine kinase Adenosine monophosphate (AMP) synthesis A9HAP4 Hypothetical protein Unknown function
The main G. diazotrophicus mechanisms of response to high-sucrose, revealed by proteomic and reverse genetic analyses, are summarized in a schematic illustration (Fig. 5, Supplemental Table S3). We propose that high sucrose strongly activates the glycolysis pathway of G. diazotrophicus to metabolize the excess of sugar that entered the cell, leading to the production of gluconic acid and the oxidation of ethanol. Moreover, G. diazotrophicus turns down the regulation of proteins related to nutrient uptake to avoid the excess of sugar entrance into bacterial cells. G. diazotrophicus activates the de novo synthesis of compatible solutes to adjust its osmotic balance. The activation of the proteolytic system may represent a strategy to release amino acids within bacterial cells. G. diazotrophicus also activates the de novo synthesis of methionine and BCAAs, from bacterial central metabolism, which involves protein synthesis and the regulation of DNA and RNA.
Graph: Fig. 5 Schematic illustration of the main responses of G. diazotrophicus to high sugar. Red and blue forms represent proteins classified as down-regulated and up-regulated, respectively, and gray forms represent non-regulated proteins (A). Cytoplasmic functional groups with more than three proteins were separately illustrated (B, C, D). Detailed information about each protein in the scheme is in the Supplemental Table S3
Taken together, our results identified molecular mechanisms of G. diazotrophicus tolerance to high sucrose, shedding light on two ways of metabolic response that are related to osmotolerance and the metabolism of the excess of sugar. These findings contribute to understanding the tolerance to high sugar in bacteria and open new perspectives for the understanding of its association with sugar-rich hosts.
MRL, LFA, and GASF conceived and designed research. MRL, LFA, LSV, and FSS conducted experiments. VS contributed new reagents. MRL, JRM, VRP, RRB, and MVVO analyzed data. MRL wrote the manuscript. All authors read and approved the final manuscript.
This study was funded by the Coordination for the Improvement of Higher Education Personnel (CAPES), the Brazilian National Council for Scientific and Technological Development (CNPq), the Rio de Janeiro Research Foundation (FAPERJ), the Funding Authority for Research and Projects (FINEP), and the State University of North Fluminense "Darcy Ribeiro" (UENF).
The raw data generated during the current study are openly available the repository Zenodo, at https://
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This article does not contain any studies with human participants or animals performed by any of the authors.
All authors declare no competing interests.
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By Mariana Ramos Leandro; Leandro Fernandes Andrade; Luciano de Souza Vespoli; Fabiano Silva Soares; Julia Rosa Moreira; Vivian Ribeiro Pimentel; Roberta Ribeiro Barbosa; Marcos Vinícius Viana de Oliveira; Vanildo Silveira and Gonçalo Apolinário de Souza Filho
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