Enhanced extracellular ammonium release in the plant endophyte Gluconacetobacter diazotrophicus through genome editing.

IMPORTANCE: Our results demonstrate increased extracellular ammonium release in the endophyte plant growth-promoting bacterium Gluconacetobacter diazotrophicus. Strains were constructed in a manner that leaves no antibiotic markers behind, such that these strains contain no transgenes. Levels of ammonium achieved by cultures of modified G. diazotrophicus strains reached concentrations of approximately 18 mM ammonium, while wild-type G. diazotrophicus remained much lower (below 50 µM). These findings demonstrate a strong potential for further improving the biofertilizer potential of this important microbe.

Structures and roles of BcsD and partner scaffold proteins in proteobacterial cellulose secretion.

Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. Although bacterial cellulose (BC) was first described in the 19th century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing ß- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.

Improved production of bacterial cellulose using Gluconacetobacter sp. LYP25, a strain developed in UVC mutagenesis with limited viability conditions.

Bacterial cellulose (BC), a natural polymer synthesized by bacteria, has received considerable attention owing to its impressive physicomechanical properties. However, the low productivity of BC-producing strains poses a challenge to industrializing this material and making it economically viable. In the present study, UV-induced random mutagenesis of Gluconacetobacter xylinus ATCC 53524 was performed to improve BC production. Sixty mutants were obtained from the following mutagenesis procedure: the correlation between UVC fluence and cell death was investigated, and a limited viability condition was determined as a UVC dose to kill 99.99 %. Compared to the control strain, BC production by the mutant strains LYP25 and LYP23 improved 46.4 % and 44.9 %, respectively. Fermentation profiling using the selected strains showed that LYP25 was superior in glucose consumption and BC production, 13.8 % and 41.0 %, respectively, compared to the control strain. Finally, the physicochemical properties of LYP25-derived BC were similar to those of the control strain; thus, the mutant strain is expected to be a promising producer of BC in the bio-industry based on improved productivity.

Gluconacetobacter diazotrophicus Gene Fitness during Diazotrophic Growth.

Plant growth-promoting (PGP) bacteria are important to the development of sustainable agricultural systems. PGP microbes that fix atmospheric nitrogen (diazotrophs) could minimize the application of industrially derived fertilizers and function as a biofertilizer. The bacterium Gluconacetobacter diazotrophicus is a nitrogen-fixing PGP microbe originally discovered in association with sugarcane plants, where it functions as an endophyte. It also forms endophyte associations with a range of other agriculturally relevant crop plants. G. diazotrophicus requires microaerobic conditions for diazotrophic growth. We generated a transposon library for G. diazotrophicus and cultured the library under various growth conditions and culture medium compositions to measure fitness defects associated with individual transposon inserts (transposon insertion sequencing [Tn-seq]). Using this library, we probed more than 3,200 genes and ascertained the importance of various genes for diazotrophic growth of this microaerobic endophyte. We also identified a set of essential genes. IMPORTANCE Our results demonstrate a succinct set of genes involved in diazotrophic growth for G. diazotrophicus, with a lower degree of redundancy than what is found in other model diazotrophs. The results will serve as a valuable resource for those interested in biological nitrogen fixation and will establish a baseline data set for plant free growth, which could complement future studies related to the endophyte relationship.

Essential role of extracytoplasmic proteins in the resistance of Gluconacetobacter diazotrophicus to cadmium.

Cadmium (Cd) is a heavy metal used as raw material for several fertilizers and pesticides. The increase of Cd concentration in soils has been observed in cultivated areas, affecting animals, plants, and microorganisms. Gluconacetobacter diazotrophicus is a plant growth-promoting bacterium able to survive under adverse environmental conditions. Here, we investigated key mechanisms involved with the resistance of G. diazotrophicus to Cd. Proteomic analyses revealed that the main pathways regulated in response to Cd are nutrient uptake, multidrug efflux pumps, response to oxidative stress, and protein quality control system. Extracytoplasmic proteins related to multidrug efflux pumps were up-accumulated, while several proteins related to nutrients uptake were down-accumulated. The relevance of these pathways for bacterial resistance to Cd was investigated by reverse genetic analysis using mutants defective for nutrient uptake (tdbr, ompW, and oprB), multidrug efflux (czcC), response to oxidative stress (ggt), and protein quality control system (clpX). Our data demonstrated the essential role of the tdbr and czcC genes for resistance to Cd in G. diazotrophicus. These results contribute to a better understanding of the resistance mechanisms to Cd in G. diazotrophicus, shedding light on responses associated with extracytoplasmic compartments.

Differential Gene Expression of Brachypodium distachyon Roots Colonized by Gluconacetobacter diazotrophicus and the Role of BdCESA8 in the Colonization.

Alternatives to synthetic nitrogen fertilizer are needed to reduce the costs of crop production and offset environmental damage. Nitrogen-fixing bacterium Gluconacetobacter diazotrophicus has been proposed as a possible biofertilizer for monocot crop production. However, the colonization of G. diazotrophicus in most monocot crops is limited and deep understanding of the response of host plants to G. diazotrophicus colonization is still lacking. In this study, the molecular response of the monocot plant model Brachypodium distachyon was studied during G. diazotrophicus root colonization. The gene expression profiles of B. distachyon root tissues colonized by G. diazotrophicus were generated via next-generation RNA sequencing, and investigated through gene ontology and metabolic pathway analysis. The RNA sequencing results indicated that Brachypodium is actively involved in G. diazotrophicus colonization via cell wall synthesis. Jasmonic acid, ethylene, gibberellin biosynthesis. nitrogen assimilation, and primary and secondary metabolite pathways are also modulated to accommodate and control the extent of G. diazotrophicus colonization. Cellulose synthesis is significantly downregulated during colonization. The loss of function mutant for Brachypodium cellulose synthase 8 (BdCESA8) showed decreased cellulose content in xylem and increased resistance to G. diazotrophicus colonization. This result suggested that the cellulose synthesis of the secondary cell wall is involved in G. diazotrophicus colonization. The results of this study provide insights for future research in regard to gene manipulation for efficient colonization of nitrogen-fixing bacteria in Brachypodium and monocot crops.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Biofabrication of a Hyaluronan/Bacterial Cellulose Composite Nanofibril by Secretion from Engineered Gluconacetobacter.

Naturally occurring polysaccharides, such as cellulose, hemicellulose, and chitin, have roles in plant skeletons and/or related properties in living organisms. Their hierarchically regulated production systems show potential for designing nanocomposite fabrication using engineered microorganisms. This study has demonstrated that genetically engineered Gluconacetobacter hansenii (G. hansenii) individual cells can fabricate naturally composited nanofibrils by simultaneous production of hyaluronan (HA) and bacterial cellulose (BC). The cells were manipulated to contain hyaluronan synthase and UDP-glucose dehydrogenase genes, which are essential for HA biosynthesis. Fluorescence microscopic observations indicated the production of composited nanofibrils and suggested that HA secretion was associated with the cellulose secretory pathway in G. hansenii. The gel-like nanocomposite materials produced by the engineered G. hansenii exhibited superior properties compared with conventional in situ nanocomposites. This genetic engineering approach facilitates the use of G. hansenii for designing integrated cellulose-based nanomaterials.

CowN sustains nitrogenase turnover in the presence of the inhibitor carbon monoxide.

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N2) to ammonia (NH3). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN's protection mechanism involves decreasing the binding affinity of CO to nitrogenase's active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase.

Major aldehyde dehydrogenase AldFGH of Gluconacetobacter diazotrophicus is independent of pyrroloquinoline quinone but dependent on molybdopterin for acetic acid fermentation.

Acetic acid fermentation involves the oxidation of ethanol to acetic acid via acetaldehyde as the intermediate and is catalyzed by the membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) of acetic acid bacteria. Although ADH depends on pyrroloquinoline quinone (PQQ), the prosthetic group associated with ALDH remains a matter of debate. This study aimed to address the dependency of ALDH of Gluconacetobacter diazotrophicus strain PAL5 on PQQ and the physiological role of ALDH in acetic acid fermentation. We constructed deletion mutant strains for both the ALDH gene clusters of PAL5, aldFGH and aldSLC. In addition, the adhAB operon for ADH was eliminated, since it shows ALDH activity. The triple-deletion derivative ΔaldFGH ΔaldSLC ΔadhAB failed to show ALDH activity, which suggested that ALDH activity in PAL5 is derived from these three enzyme complexes. Since the single-gene cluster deletion derivative ΔaldFGH lost most ALDH activity, and accumulated much higher acetaldehyde than wild type under acetic acid fermentation conditions, we concluded that AldFGH functions as the major ALDH in PAL5. Furthermore, deletion of the PQQ biosynthesis gene cluster (pqqABCDE) abolished ADH activity completely, but did not affect ALDH activity. Instead, the molybdopterin biosynthesis gene deletion derivatives lost ALDH activity. Thus, we concluded that the AldFGH and AldSLC complexes of Ga. diazotrophicus PAL5 require a form of molybdopterin but not PQQ for ALDH activity. KEY POINTS: • AldFGH is the major aldehyde dehydrogenase in Gluconacetobacter diazotrophicus PAL5. • Acetaldehyde accumulated from ethanol in the absence of AldFGH. • Molybdopterin, rather than pyrroloquinoline quinone, is required for AldFGH.

Comparative proteomics reveals essential mechanisms for osmotolerance in Gluconacetobacter diazotrophicus.

Plant growth-promoting bacteria are a promising alternative to improve agricultural sustainability. Gluconacetobacter diazotrophicus is an osmotolerant bacterium able to colonize several plant species, including sugarcane, coffee, and rice. Despite its biotechnological potential, the mechanisms controlling such osmotolerance remain unclear. The present study investigated the key mechanisms of resistance to osmotic stress in G. diazotrophicus. The molecular pathways regulated by the stress were investigated by comparative proteomics, and proteins essential for resistance were identified by knock-out mutagenesis. Proteomics analysis led to identify regulatory pathways for osmotic adjustment, de novo saturated fatty acids biosynthesis, and uptake of nutrients. The mutagenesis analysis showed that the lack of AccC protein, an essential component of de novo fatty acid biosynthesis, severely affected G. diazotrophicus resistance to osmotic stress. Additionally, knock-out mutants for nutrients uptake (Δtbdr and ΔoprB) and compatible solutes synthesis (ΔmtlK and ΔotsA) became more sensitive to osmotic stress. Together, our results identified specific genes and mechanisms regulated by osmotic stress in an osmotolerant bacterium, shedding light on the essential role of cell envelope and extracytoplasmic proteins for osmotolerance.

The structure of a novel membrane-associated 6-phosphogluconate dehydrogenase from Gluconacetobacter diazotrophicus (Gd6PGD) reveals a subfamily of short-chain 6PGDs.

The enzyme 6-phosphogluconate dehydrogenase catalyzes the conversion of 6-phosphogluconate to ribulose-5-phosphate. It represents an important reaction in the oxidative pentose phosphate pathway, producing a ribose precursor essential for nucleotide and nucleic acid synthesis. We succeeded, for the first time, to determine the three-dimensional structure of this enzyme from an acetic acid bacterium, Gluconacetobacter diazotrophicus (Gd6PGD). Active Gd6PGD, a homodimer (70 kDa), was present in both the soluble and the membrane fractions of the nitrogen-fixing microorganism. The Gd6PGD belongs to the newly described subfamily of short-chain (333 AA) 6PGDs, compared to the long-chain subfamily (480 AA; e.g., Ovis aries, Homo sapiens). The shorter amino acid sequence in Gd6PGD induces the exposition of hydrophobic residues in the C-terminal domain. This distinct structural feature is key for the protein to associate with the membrane. Furthermore, in terms of function, the short-chain 6PGD seems to prefer NAD+ over NADP+ , delivering NADH to the membrane-bound NADH dehydrogenase of the microorganisms required by the terminal oxidases to reduce dioxygen to water for energy conservation. ENZYME: ECnonbreakingspace1.1.1.343. DATABASE: Structural data are available in PDB database under the accession number 6VPB.

Temporal assessment of root and shoot colonization of elephant grass (Pennisetum purpureum Schum.) host seedlings by Gluconacetobacter diazotrophicus strain LP343.

Gluconacetobacter diazotrophicus is a species of great agronomic potential due to its growth-promotion traits. Its colonization process in different plants has been reported. However, there have been no studies regarding its structural colonization in elephant grass. This is a fast-growing C4-Poaceae plant, and its application in Brazil is mainly aimed at feeding dairy cattle, due to its high nutritional value. Also, in the last decade, this grass has been applied in the production of biofuels. The present study aimed to monitor the colonization process of strain LP343 of G. diazotrophicus inoculated in elephant grass seedlings of PCEA genotype, by using a mCherry-tagged bacterium. Samples of roots and shoots collected at different periods were visualized by confocal laser-scanning microscopy. The colony-counting assay was used to compare the number of cells recovered in different niches and a qPCR was performed for the quantification of endophytic cells in root and shoot tissues. Results suggested that the strain LP343 quickly recognized the PCEA roots as host, attached to the elephant grass roots at 6 h, and 7 days after inoculation were able to colonize the xylem vessels of roots and shoots of elephant grass. This study advances our knowledge about the colonization process of G. diazotrophicus species in elephant grass, contributing to future studies involving the plant-bacteria interaction cultivated under gnotobiotic conditions.

DegP protease is essential for tolerance to salt stress in the plant growth-promoting bacterium Gluconacetobacter diazotrophicus PAL5.

The use of plant growth-promoting bacteria represents an alternative to the massive use of mineral fertilizers in agriculture. However, some abiotic stresses commonly found in the environment, like salinity, can affect the efficiency of this approach. Here, we investigated the key mechanisms involved in the response of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus to salt stress by using morphological and cell viability analyses, comparative proteomics, and reverse genetics. Our results revealed that the bacteria produce filamentous cells in response to salt at 100 mM and 150 mM NaCl. However, such a response was not observed at higher concentrations, where cell viability was severely affected. Proteomic analysis showed that salt stress modulates proteins involved in several pathways, including iron uptake, outer membrane efflux, osmotic adjustment, cell division and elongation, and protein transport and quality control. Proteomic data also revealed the repression of several extracytoplasmic proteins, especially those located at periplasm and outer membrane. The role of such pathways in the tolerance to salt stress was analyzed by the use of mutant defectives for Δtbdr (iron uptake), ΔmtlK and ΔotsA (compatible solutes synthesis), and ΔdegP (quality control of nascent extracytoplasmic proteins). ΔdegP presented the highest sensitivity to salt stress, Δtbdr, andΔmtlK also showed increased sensitivity, but ΔotsA was not affected. This is the first demonstration that DegP protein, a protease with minor chaperone activity, is essential for tolerance to salt stress in G. diazotrophicus. Our data contribute to a better understanding of the molecular bases that control the bacterial response/tolerance to salt stress, shedding light on quality control of nascent extracytoplasmic proteins.

Metaproteomics of microbiota involved in submerged culture production of alcohol wine vinegar: A first approach.

Acetic acid bacteria form a complex microbiota that plays a fundamental role in the industrial production of vinegar through the incomplete oxidation reaction from ethanol to acetic acid. The organoleptic properties and the quality of vinegar are influenced by many factors, especially by the raw material used as acetification substrate, the microbial diversity and the technical methods employed in its production. The metaproteomics has been considered, among the new methods employed for the investigation of microbial communities, since it may provide information about the microbial biodiversity and behaviour by means of a protein content analysis. In this work, alcohol wine vinegar was produced through a submerged culture of acetic acid bacteria using a pilot acetator, operated in a semi-continuous mode, where the main system variables were monitored and the cycle profile throughout the acetification was obtained. Through a first approach, at qualitative level, of a metaproteomic analysis performed at relevant moments of the acetification cycle (end of fast and discontinuous loading phases and just prior to unloading phase), it is aimed to investigate the microbiota existent in alcohol wine vinegar as well as its changes during the cycle; to our knowledge, this is the first metaproteomics report carried out in this way on this system. A total of 1723 proteins from 30 different genera were identified; 1615 out of 1723 proteins (93.73%) belonged to the four most frequent (%) genera: Acetobacter, Gluconacetobacter, Gluconobacter and Komagataeibacter. Around 80% of identified proteins belonged to the species Komagataeibacter europaeus. In addition, GO Term enrichment analysis highlighted the important role of catalytic activity, organic cyclic compound binding, metabolic and biosynthesis processes throughout acetic acid fermentation. These findings provide the first step to obtain an AAB profile at omics level related to the environmental changes produced during the typical semi-continuous cycles used in this process and it would contribute to the optimization of operating conditions and improving the industrial production of vinegar.

Scaled-up biotechnological production of individual betalains in a microbial system.

The recent interest in plant pigment betalains as bioactive compounds and chemopreventive agents has led to the search for a reliable and scalable process to obtain them. The cloning of the novel and efficient enzyme 4,5-DOPA-extradiol dioxygenase from Gluconacetobacter diazotrophicus in an expression vector, and the subsequent heterologous expression in Escherichia coli cultures has led to the start-up of a biotechnological production system of individual pigments. The aim of this study was to search for the optimal conditions for the production of betalamic acid in microbial factories and the scaled-up obtention of the derived pigments. Four different betaxanthins and two betacyanins were obtained after the addition of non-transformable amines and amino acids and their condensation with the betalamic acid produced by the dioxygenase. The scaled-up obtention and purification of betalains improved the yields of the previous methodologies reaching quantities by up to 150 mg of pure compounds.

First Betalain-Producing Bacteria Break the Exclusive Presence of the Pigments in the Plant Kingdom.

The biosynthesis of antioxidant pigments, namely, betalains, was believed to be restricted to Caryophyllales plants. This paper changes this paradigm, and enzyme mining from bacterial hosts promoted the discovery of bacterial cultures producing betalains. The spectrum of possible sources of betalain pigments in nature is broadened by our description of the first betalain-forming bacterium, Gluconacetobacter diazotrophicus The enzyme-specific step is the extradiol cleavage of the precursor amino acid l-dihydroxyphenylalanine (l-DOPA) to form the structural unit betalamic acid. Molecular and functional work conducted led to the characterization of a novel dioxygenase, a polypeptide of 17.8 kDa with a Km of 1.36 mM, with higher activity and affinity than those of its plant counterparts. Its superior activity allowed the first experimental characterization of the early steps in the biosynthesis of betalains by fully characterizing the presence and time evolution of 2,3- and 4,5-seco-DOPA intermediates. Furthermore, spontaneous chemical reactions are characterized and incorporated into a comprehensive enzymatic-chemical mechanism that yields the final pigments.IMPORTANCE Several studies have demonstrated the health-promoting effects of betalains due to their high antioxidant capacity and their positive effect on the dose-dependent inhibition of cancer cells and their proliferation. To date, betalains were restricted to plants of the order Caryophyllales and some species of fungi, but the present study reveals the first betalain-producing bacterium, as well as the first steps in the formation of pigments. This finding demonstrates that betalain biosynthesis can be expanded to prokaryotes.

Assessment of the cutaneous wound healing efficiency of acidic, neutral and alkaline bacterial cellulose membrane in rat.

Recent research was conducted to evaluate the healing efficiency of bacterial cellulose (BC) as a wound dressing in different pHs and its possibility of being a smart wound dressing that can indicate pHs. BC was produced by environmentally isolated bacterial strains. After washing the best achieved BC, it was floated in normal saline with different pHs with phenol red used as a pH indicator. Finally the wound healing effects of the acidic, neutral and alkaline BC membranes were evaluated in rat cutaneous wounds. Results showed that one of the isolates which its partial 16srRNA genome had 95% similarity with Gluconacetobacter intermedius, had the thickest layer. The microscopic and macroscopic evaluations showed that the acidic BC had the best healing activity. Although the color of the films remained unchanged during the experiments because they were transparent and thin, these changes could not be easily seen. This suggests the use of thicker films such as the ones which are cross linked with some materials (e.g., sterile gauze). In conclusion the pH can affect the healing ability of natural BC and acidic pH had the best wound healing efficiency. In future it is better to use the acidic BC instead of natural one for different wound healing purposes.

Recombinant biosynthesis of bacterial cellulose in genetically modified Escherichia coli.

Bacterial cellulose (BC) exhibits unique properties such as high purity compared to plant-based cellulose; however, commercial production of BC has remained a challenge, primarily due to the strain properties of cellulose-producing bacteria. Herein, we developed a functional and stable BC production system in genetically modified (GM) Escherichia coli by recombinant expression of both the BC synthase operon (bcsABCD) and the upstream operon (cmcax, ccp Ax). BC production was achieved in GM HMS174 (DE3) and in GM C41 (DE3) by optimization of the culture temperature (22 °C, 30 °C, and 37 °C) and IPTG concentration. BC biosynthesis was detected much earlier in GM C41 (DE3) cultures (3 h after IPTG induction) than those of Gluconacetobacter hansenii. GM HMS174 (DE3) produced dense fibres having a length of approximately 1000-3000 µm and a diameter of 10-20 µm, which were remarkably larger than the fibres of BC typically produced by G. hansenii.

Leucine responsive regulatory protein is involved in methionine metabolism and polyamine homeostasis in acetic acid bacterium Komagataeibacter europaeus.

The leucine responsive regulatory protein (Lrp) is a global transcription factor that regulates the expression of genes involved in amino acid metabolism. To identify metabolic pathways and related genes under the control of Lrp in the acetic acid bacterium Komagataeibacter europaeus, the Kelrp null mutant (KGMA7110), which requires supplementation of all 20 amino acids for normal growth, was cultivated in minimal media containing or lacking particular amino acids. The results confirmed that KGMA7110 was auxotrophic for methionine and its catabolites S-adenosylmethionine (SAM) and spermidine (SPD). Quantitative reverse-transcription PCR analysis revealed lower metK (SAM synthetase) and mdtI (SPD efflux pump) expression in KGMA7110 than in wild-type KGMA0119. By contrast, these genes were significantly up-regulated in the Kelrp mutant lacking the putative C-terminal ligand-sensing domain (KGMA7203), indicating abnormal regulation of target genes by the KeLrp variant in KGMA7203. KGMA7110 (0.69±0.27 µM) and KGMA7203 (4.90±0.61 µM) excreted lower and higher quantities of SPD, respectively, than KGMA0119 (2.28±0.26 µM). This was attributed to imbalanced carbon flow caused by Kelrp disruption that respectively attenuated and stimulated metK and mdtI expression. These findings indicate that KeLrp plays a key role in SAM biosynthesis and intracellular polyamine homeostasis in K. europaeus.

Essential role of K+ uptake permease (Kup) for resistance to sucrose-induced stress in Gluconacetobacter diazotrophicus PAl 5.

Microorganisms are constantly challenged by stressful conditions, such as sugar-rich environments. Such environments can cause an imbalance of biochemical activities and compromise cell multiplication. Gluconacetobacter diazotrophicus PAl 5 is among the most sugar-tolerant bacteria, capable of growing in the presence of up to 876 mM sucrose. However, the molecular mechanisms involved in its response to high sucrose remain unknown. The present work aimed to identify sucrose-induced stress resistance genes in G. diazotrophicus PAl 5. Screening of a Tn5 transposon insertion library identified a mutant that was severely compromised in its resistance to high sucrose concentrations. Molecular characterization revealed that the mutation affected the kupA gene, which encodes a K+ uptake transporter (KupA). Functional complementation of the mutant with the wild type kupA gene recovered the sucrose-induced stress resistance phenotype. High sucrose resistance assay, under different potassium concentrations, revealed that KupA acts as a high-affinity K+ transporter, which is essential for resistance to sucrose-induced stress, when extracellular potassium levels are low. This study is the first to show the essential role of the KupA protein for resistance to sucrose-induced stress in bacteria by acting as a high-affinity potassium transporter in G. diazotrophicus PAl 5.