A membrane localized RTX-like protein mediates physiochemical properties of the Pantoea stewartii subsp. stewartii cell envelope that impact surface adhesion, cell surface hydrophobicity and plant colonization.

Pantoea stewartii subsp. stewartii (Pnss), is the bacterial causal agent of Stewart's wilt of sweet corn. Disease symptoms include systemic wilting and foliar, water-soaked lesions. A Repeat-in-toxin (RTX)-like protein, RTX2, causes cell leakage and collapse in the leaf apoplast of susceptible corn varieties and is a primary mediator of water-soaked lesion formation in the P. stewartii-sweet corn pathosystem. RTX toxins comprise a large family of proteins, which are widely distributed among Gram-negative bacteria. These proteins are generally categorized as cellulolysins, but the Biofilm-Associated Proteins (Bap) subfamily of RTX toxins are implicated in surface adhesion and other biofilm behaviors. The Pnss RTX2 is most phylogenetically related to other Bap proteins suggesting that Pnss RTX2 plays a dual role in adhesion to host surfaces in addition to mediating the host cell lysis that leads to water-soaked lesion formation. Here we demonstrated that RTX2 localizes to the bacterial cell envelope and influences physiochemical properties of the bacterial cell envelope that impact bacterial cell length, cell envelope integrity and overall cellular hydrophobicity. Interestingly, the role of RTX2 as an adhesin was only evident in absence of exopolysaccharide (EPS) production suggesting that RTX2 plays a role as an adhesin early in biofilm development before EPS production is fully induced. However, deletion of rtx2 severely impacted Pnss' colonization of the xylem suggesting that the dual role of RTX2 as a cytolysin and adhesin is a mechanism that links the apoplastic water-soaked lesion phase of infection to the wilting phase of the infection in the xylem.

Simultaneous removal of aliphatic and aromatic crude oil hydrocarbons by Pantoea agglomerans isolated from petroleum-contaminated soil in the west of Iran.

Hydrocarbons are considered as one of the most common and harmful environmental pollutants affecting human health and the environment. Bioremediation as an environmentally friendly, highly efficient, and cost-effective method in remediating oil-contaminated environments has been interesting in recent decades. In this study, hydrocarbon degrader bacterial strains were isolated from the highly petroleum-contaminated soils in the Dehloran oil field in the west of Iran. Out of 37 isolates, 15 can grow on M9 agar medium that contains 1.5 g L-1 of crude oil as the sole carbon source. The morphological, biochemical, and 16SrRNA sequencing analyses were performed for the isolates. The choosing of the isolates as the hydrocarbon degrader was examined by evaluating the efficacy of their crude oil removal at a concentration of 10 g L-1 in an aqueous medium. The results showed that five isolates belonging to Pseudomonas sp., Pseudomonas oryzihabitans, Roseomonas aestuarii, Pantoea agglomerans, and Arthrobacter sp. had a hyper hydrocarbon-degrading activity and they could remove more than 85% of the total petroleum hydrocarbon (TPH) after 96 h. The highest TPH removal of about 95.75% and biodegradation rate of 0.0997 g L-1 h-1 was observed for P. agglomerans. The gas chromatography-mass spectroscopy (GC-MS) analysis was performed during the biodegradation process by P. agglomerans to detect the degradation intermediates and final products. The results confirmed the presence of intermediates such as alcohols and fatty acids in the terminal oxidation pathway of alkanes in this biodegradation process. A promising P. agglomerans NB391 strain can remove aliphatic and aromatic hydrocarbons simultaneously.

Hitching a Ride in the Phyllosphere: Surfactant Production of Pseudomonas spp. Causes Co-swarming of Pantoea eucalypti 299R.

Here, we demonstrate the beneficial effect of surfactant-producing pseudomonads on Pantoea eucalypti 299R. We conducted a series of experiments in environments of increasing complexity. P. eucalypti 299R (Pe299R), and Pseudomonas sp. FF1 (Pff1) or Pe299R and surfactant-production deficient Pseudomonas sp. FF1::ΔviscB (Pff1ΔviscB) were co-inoculated in broth, on swarming agar plates, and on plants. In broth, there were no differences in the growth dynamics of Pe299R when growing in the presence of Pff1 or Pff1ΔviscB. By contrast, on swarming agar plates, Pe299R was able to co-swarm with Pff1 which led to a significant increase in Pe299R biomass compared to Pe299R growing with Pff1ΔviscB or in monoculture. Finally in planta, and using the single-cell bioreporter for reproductive success (CUSPER), we found a temporally distinct beneficial effect of Pff1 on co-inoculated Pe299R subpopulations that did not occur in the presence of Pff1ΔviscB. We tested three additional surfactant-producing pseudomonads and their respective surfactant knockout mutants on PE299R on swarming agar showing similar results. This led us to propose a model for the positive effect of surfactant production during leaf colonization. Our results indicate that co-motility might be common during leaf colonization and adds yet another facet to the already manyfold roles of surfactants.

Pantoea jilinensis D25 enhances tomato salt tolerance via altering antioxidant responses and soil microbial community structure.

As a major challenge to global food security, soil salinity is an important abiotic stress factor that seriously affects the crop growth and yield. In this study, the mechanism of salt resistance of Pantoea jilinensis D25 and its improving effect on salt tolerance of tomato were explored with salt resistance-related genes identified in strain D25 by genomic sequencing. The results showed that in comparison with the treatment of NaCl, strain D25 significantly increased the fresh weight, shoot length, root length, and chlorophyll content of tomato under salt stress by 46.7%, 20%, 42.4%, and 44.2%, respectively, with increased absorptions of various macronutrients and micronutrients and decreased accumulation of Na+. The activities of defense enzymes (peroxidase, catalase, superoxide dismutase, phenylalanine ammonia-lyase, and polyphenol oxidase) were enhanced, while the content of malondialdehyde was decreased. The results of quantitative real-time PCR analysis showed that the expressions of genes (SlSOS1, SlNHX1, SlHKT1.1, SlSOD1, SlAPX2, SlAOS, SlPin II, Solyc08g066270.1, Solyc03g083420.2 and SlGA20ox1) related to ion transporters, antioxidant machinery, key defense, serine/threonine protein kinase synthesis, and gibberellin (GA) signal protein were up-regulated and were the highest in the treatment of both NaCl and strain D25. The activities of enzymes (dehydrogenase, urease, invertase, and catalase activities) related to soil fertility were enhanced. The results of 16S rRNA sequencing showed that soil microbial diversity and the abundance of probiotics (e.g., Acidibacter, Limnobacter, and Romboutsia) were significantly increased. Our study provided strong experimental evidence to support the agricultural application of strain D25 in the promotion of growth in crops.

Efficient production of isomaltulose using engineered Yarrowia lipolytica strain facilitated by non-yeast signal peptide-mediated cell surface display.

BACKGROUND: Isomaltulose is a 'generally recognized as safe' ingredient and is widely used in the food, pharmaceutical and chemical industries. The exploration and development of efficient technologies is essential for enhancing isomaltulose yield. RESULTS: In the present study, a simple and efficient surface display platform mediated by a non-yeast signal peptide was developed in Yarrowia lipolytica and utilized to efficiently produce isomaltulose from sucrose. We discovered that the signal peptide SP1 of sucrose isomerase from Pantoea dispersa UQ68J (PdSI) could guide SIs anchoring to the cell surface of Y. lipolytica, demonstrating a novel and simple cell surface display strategy. Furthermore, the PdSI expression level was significantly increased through optimizing the promoters and multi-site integrating genes into chromosome. The final strain gained 451.7 g L-1 isomaltulose with a conversion rate of 90.3% and a space-time yield of 50.2 g L-1 h-1. CONCLUSION: The present study provides an efficient way for manufacturing isomaltulose with a high space-time yield. This heterogenous signal peptide-mediated cell surface display strategy featured with small fusion tag (approximately 2.2 kDa of SP1), absence of enzyme leakage in fermentation broth and ample room for optimization, providing a convenient way to construct whole-cell biocatalysts to synthesize other products and broadening the array of molecular toolboxes accessible for engineering Y. lipolytica. © 2024 Society of Chemical Industry.

Enhancement of herbicolin A production by integrated fermentation optimization and strain engineering in Pantoea agglomerans ZJU23.

BACKGROUND: The lipopeptide herbicolin A (HA) secreted by the biocontrol agent Pantoea agglomerans ZJU23 is a promising antifungal drug to combat fungal pathogens by targeting lipid rafts, both in agricultural and clinical settings. Improvement of HA production would be of great significance in promoting its commercialization. This study aims to enhance the HA production in ZJU23 by combining fermentation optimization and strain engineering. RESULTS: Based on the results in the single-factor experiments, corn steep liquor, temperature and initial pH were identified as the significant affecting factors by the Plackett-Burman design. The fermentation medium and conditions were further optimized using the Box-Behnken response surface method, and the HA production of the wild type strain ZJU23 was improved from ~ 87 mg/mL in King's B medium to ~ 211 mg/mL in HA induction (HAI) medium. A transposon library was constructed in ZJU23 to screen for mutants with higher HA production, and two transcriptional repressors for HA biosynthesis, LrhA and PurR, were identified. Disruption of the LrhA gene led to increased mRNA expression of HA biosynthetic genes, and subsequently improved about twofold HA production. Finally, the HA production reached ~ 471 mg/mL in the ΔLrhA mutant under optimized fermentation conditions, which is about 5.4 times higher than before (~ 87 mg/mL). The bacterial suspension of the ΔLrhA mutant fermented in HAI medium significantly enhanced its biocontrol efficacy against gray mold disease and Fusarium crown rot of wheat, showing equivalent control efficacies as the chemical fungicides used in this study. Furthermore, HA was effective against fungicide resistant Botrytis cinerea. Increased HA production substantially improved the control efficacy against gray mold disease caused by a pyrimethanil resistant strain. CONCLUSIONS: This study reveals that the transcriptional repressor LrhA negatively regulates HA biosynthesis and the defined HAI medium is suitable for HA production. These findings provide an extended basis for large-scale production of HA and promote biofungicide development based on ZJU23 and HA in the future.

Negatively Regulated Aerobactin and Desferrioxamine E by Fur in Pantoea ananatis Are Required for Full Siderophore Production and Antibacterial Activity, but Not for Virulence.

Pantoea ananatis is an emerging plant pathogen that causes disease in economically important crops such as rice, corn, onion, melon, and pineapple, and it also infects humans and insects. In this study, we identified biosynthetic gene clusters of aerobactin and desferrioxamine E (DFO-E) siderophores by using the complete genome of P. ananatis PA13 isolated from rice sheath rot. P. ananatis PA13 exhibited the strongest antibacterial activity against Erwinia amylovora and Yersinia enterocolitica (Enterobacterales). Mutants of aerobactin or DFO-E maintained antibacterial activity against E. amylovora and Y. enterocolitica, as well as in a siderophore activity assay. However, double aerobactin and DFO-E gene deletion mutants completely lost siderophore and antibacterial activity. These results reveal that both siderophore biosynthetic gene clusters are essential for siderophore production and antibacterial activity in P. ananatis PA13. A ferric uptake regulator protein (Fur) mutant exhibited a significant increase in siderophore production, and a Fur-overexpressing strain completely lost antibacterial activity. Expression of the iucA, dfoJ, and foxA genes was significantly increased in the Δfur mutant background, and expression of these genes returned to wild-type levels after fur compensation. These results indicate that Fur negatively regulates aerobactin and DFO-E siderophores. However, siderophore production was not required for P. ananatis virulence in plants, but it appears to be involved in the microbial ecology surrounding the plant environment. This study is the first to report the regulation and functional characteristics of siderophore biosynthetic genes in P. ananatis. IMPORTANCE Pantoea ananatis is a bacterium that causes diseases in several economically important crops, as well as in insects and humans. This bacterium has been studied extensively as a potentially dangerous pathogen due to its saprophytic ability. Recently, the types, biosynthetic gene clusters, and origin of the siderophores in the Pantoea genus were determined by using genome comparative analyses. However, few genetic studies have investigated the characteristics and functions of siderophores in P. ananatis. The results of this study revealed that the production of aerobactin and desferrioxamine E in the rice pathogen P. ananatis PA13 is negatively regulated by Fur and that these siderophores are essential for antibacterial activity against Erwinia amylovora and Yersinia enterocolitica (Enterobacterales). However, siderophore production was not required for P. ananatis virulence in plants, but it appears to be involved in the microbial ecology surrounding the plant environment.

Production of vitamin A and vitamin E: expression of vitreoscilla hemoglobin gene in Erwinia herbicola.

Vitamin A prevents eye problems, blindness and skin problems by strengthening the immune system. Vitamin E is a nutrient that has important roles in many areas such as skin health, eye health and hormonal order. Vitreoscilla hemoglobin (VHb) gives an advantage in later phases of grown conditions to cells. In this study, the intracellular and extracellular production of vitamin A and E in E. herbicola and its recombinant strains (vgb- and vgb+) in the three different M9 mediums with supplemented 0.1% glucose, 0.1% fructose and 0.1% sucrose was investigated. Additionally, the viable cell number and total cell mass (OD600) were measured by the host and the recombinant bacteria in these mediums. The VHb gene expression in E. herbicola enhanced vitamin A under different carbon conditionals. Especially, in the vgb + strain (carrying vgb gene) the production of total vitamin in 0.1% glucose medium was recorded as 0.14 µg/ml, while the production in fructose and sucrose media was recorded as 0.07 µg/ml. The production of intracellular vitamin E in the host strain (0.025 µg/ml) was about 13-fold (0.002 µg/ml) higher than vgb + recombinant strain in 0.1% fructose. The vgb + strain showed about 2-fold higher extracellular vitamin E production than the host strain.

Isolation and characterization of bacteria from activated sludge capable of degrading 17α-ethinylestradiol, a contaminant of high environmental concern.

The compound 17α-ethinylestradiol (EE2) is a synthetic oestrogen which is classified as a group 1 carcinogen by the World Health Organization. Together with other endocrine disruptor compounds, EE2 has been included in the surface water Watch List by the European Commission, since it causes severe adverse effects in ecosystems. Thus, it became a high priority to find or improve processes such as biodegradation of EE2 to completely remove this drug from the wastewater treatment plants (WWTPs). The present study aimed at the isolation of bacteria capable of degrading EE2 using environmental samples, namely a sludge from the Faro Northwest WWTP. Four isolates with ability to grow in the presence of 50 mg l-1 EE2 were obtained. The analysis of 16SrRNA gene sequences identified the isolated bacteria as Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans and Shinella zoogloeoides. The results of biodegradation assays showed that Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans and Shinella zoogloeoides were able to degrade 47±4 %, 55±3 %, 64±4% and 35±4 %, respectively of 13 mg l-1 EE2 after 168 h at 28 °C. To the best of our knowledge, these bacterial isolates were identified as EE2 degraders for the first time. In a preliminary experiment on the identification of metabolic products resulting from EE2 degradation products such as estrone (E1), γ-lactone compounds, 2-pentanedioic acid and 2-butenedioic acid an intermediate metabolite of the TCA cycle, were detected.

Fluorescent Protein Expression as a Proxy for Bacterial Fitness in a High-Throughput Assay.

Bacterial growth is classically assessed by measuring the increases in optical density of pure cultures in shaken liquid media. Measuring growth using optical density has severe limitations when studying multistrain interactions, as it is not possible to measure the growth of individual strains within mixed cultures. Here, we demonstrated that constitutively expressed fluorescent proteins can be used to track the growth of individual strains in different liquid media. Fluorescence measurements were highly correlated with optical density measurements and cell counts. This allowed us to assess bacterial growth not only in pure cultures but also in mixed bacterial cultures and determine the impact of a competitor on a focal strain, thereby assessing relative fitness. Furthermore, we were able to track the growth of two different strains simultaneously by using fluorescent proteins with differential excitation and emission wavelengths. Bacterial densities measured by fluorescence yielded more consistent data between technical replicates than optical density measurements. Our setup employs fluorescence microplate readers that allow high throughput and replication. IMPORTANCE We expand on an important limitation of the concept of measuring bacterial growth, which is classically limited to one strain at a time. By adopting our approach, it is possible to measure the growth of several bacterial strains simultaneously with high temporal resolution and in a high-throughput manner. This is important to investigate bacterial interactions, such as competition and facilitation.

Deciphering Microbial Metal Toxicity Responses via Random Bar Code Transposon Site Sequencing and Activity-Based Metabolomics.

To uncover metal toxicity targets and defense mechanisms of the facultative anaerobe Pantoea sp. strain MT58 (MT58), we used a multiomic strategy combining two global techniques, random bar code transposon site sequencing (RB-TnSeq) and activity-based metabolomics. MT58 is a metal-tolerant Oak Ridge Reservation (ORR) environmental isolate that was enriched in the presence of metals at concentrations measured in contaminated groundwater at an ORR nuclear waste site. The effects of three chemically different metals found at elevated concentrations in the ORR contaminated environment were investigated: the cation Al3+, the oxyanion CrO42-, and the oxycation UO22+. Both global techniques were applied using all three metals under both aerobic and anaerobic conditions to elucidate metal interactions mediated through the activity of metabolites and key genes/proteins. These revealed that Al3+ binds intracellular arginine, CrO42- enters the cell through sulfate transporters and oxidizes intracellular reduced thiols, and membrane-bound lipopolysaccharides protect the cell from UO22+ toxicity. In addition, the Tol outer membrane system contributed to the protection of cellular integrity from the toxic effects of all three metals. Likewise, we found evidence of regulation of lipid content in membranes under metal stress. Individually, RB-TnSeq and metabolomics are powerful tools to explore the impact various stresses have on biological systems. Here, we show that together they can be used synergistically to identify the molecular actors and mechanisms of these pertubations to an organism, furthering our understanding of how living systems interact with their environment. IMPORTANCE Studying microbial interactions with their environment can lead to a deeper understanding of biological molecular mechanisms. In this study, two global techniques, RB-TnSeq and activity metabolomics, were successfully used to probe the interactions between a metal-resistant microorganism, Pantoea sp. strain MT58, and metals contaminating a site where the organism can be located. A number of novel metal-microbe interactions were uncovered, including Al3+ toxicity targeting arginine synthesis, which could lead to a deeper understanding of the impact Al3+ contamination has on microbial communities as well as its impact on higher-level organisms, including plants for whom Al3+ contamination is an issue. Using multiomic approaches like the one described here is a way to further our understanding of microbial interactions and their impacts on the environment overall.

Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis.

BACKGROUND: Linalool, an acyclic monoterpene alcohol, is extensively used in the flavor and fragrance industries and exists as two enantiomers, (S)- and (R)-linalool, which have different odors and biological properties. Linalool extraction from natural plant tissues suffers from low product yield. Although linalool can also be chemically synthesized, its enantioselective production is difficult. Microbial production of terpenes has recently emerged as a novel, environmental-friendly alternative. Stereoselective production can also be achieved using this approach via enzymatic reactions. We previously succeeded in producing enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis, a member of the Enterobacteriaceae family of bacteria, via the heterologous mevalonate pathway with the highest linalool titer ever reported from engineered microbes. RESULTS: Here, we genetically modified a previously developed P. ananatis strain expressing the (S)-linalool synthase (AaLINS) from Actinidia arguta to further improve (S)-linalool production. AaLINS was mostly expressed as an insoluble form in P. ananatis; its soluble expression level was increased by N-terminal fusion of a halophilic ß-lactamase from Chromohalobacter sp. 560 with hexahistidine. Furthermore, in combination with elevation of the precursor supply via the mevalonate pathway, the (S)-linalool titer was increased approximately 1.4-fold (4.7 ± 0.3 g/L) in comparison with the original strain (3.4 ± 0.2 g/L) in test-tube cultivation with an aqueous-organic biphasic fermentation system using isopropyl myristate as the organic solvent for in situ extraction of cytotoxic and semi-volatile (S)-linalool. The most productive strain, IP04S/pBLAAaLINS-ispA*, produced 10.9 g/L of (S)-linalool in "dual-phase" fed-batch fermentation, which was divided into a growth-phase and a subsequent production-phase. Thus far, this is the highest reported titer in the production of not only linalool but also all monoterpenes using microbes. CONCLUSIONS: This study demonstrates the potential of our metabolically engineered P. ananatis strain as a platform for economically feasible (S)-linalool production and provides insights into the stereoselective production of terpenes with high efficiency. This system is an environmentally friendly and economically valuable (S)-linalool production alternative. Mass production of enantiopure (S)-linalool can also lead to accurate assessment of its biological properties by providing an enantiopure substrate for study.

Effects of feeding a Lactobacillus plantarum JL01 diet on caecal bacteria and metabolites of weaned piglets.

Currently, knowledge is limited concerning the impact of a Lactobacillus plantarum JL01 diet for weaned piglets on caecal bacteria and metabolite profiles. In our experiments, 24 weaned piglets were randomly divided into two groups; each piglet in the treatment groups (Cec-Lac) was fed a basic diet and administered 10 ml of L. plantarum JL01 (1·0 × 109  CFU per ml) every day. The control group (Cec-Con) was fed a basic diet. After feeding for 28 days, we analysed the parameters of the caecal digesta of weaned piglets. We used 16S rDNA gene sequencing and mass spectrometry (MS)-based metabolomics techniques to investigate the effect of a L. plantarum JL01 diet on intestinal microbial composition and its metabolite profiles in the caecum contents of weaned piglets. The results showed that the richness estimators (ACE and Chao indices) in the caecal bacteria increased in the Cec-Lac group. Prevotella_2 and Desulfovibrio decreased significantly, while Pantoea and Rectale_group increased in the caecum of weaned piglets in the Cec-Lac group. Furthermore, Pearson's correlation analysis revealed that the genus Rectale_group was positively correlated with indole-3-acetic acid (P < 0·05), and the genus Pantoea had the same correlation with 1-palmitoyl lysophosphatidic acid. The metabolomics analysis revealed that the L. plantarum JL01 diet supplementation had significant effects on tryptophan metabolism and fat digestion and absorption. The results indicated that the L. plantarum JL01 dietary supplementation not only altered the microbial composition but also mediated tryptophan metabolism and fat digestion and absorption in the caecum, factors that may further affect the health of the host.

Biocontrol activity and putative mechanism of Bacillus amyloliquefaciens (SF14 and SP10), Alcaligenes faecalis ACBC1, and Pantoea agglomerans ACBP1 against brown rot disease of fruit.

This study aimed at evaluating the antagonistic activity of 16 bacterial strains for the control of brown rot disease caused by Monilinia fructigena, and M. laxa under in vitro and a semi-commercial large-scale trial. These bacterial antagonists' belonging to the genera Alcaligenes, Bacillus, Brevibacterium, Pantoea, Pseudomonas, and Serratia were previously proven effective for control of fire blight of apple. The in vitro dual culture bioassay showed the highest inhibition rates of mycelial growth ranging from 55 to 95% and from 43 to 94% for M. fructigena and M. laxa, respectively. The in vivo bioassay showed moderate and strong inhibition for M. fructigena and M. laxa, respectively. The inhibition rates were dependent on incubation time as well as pathogen virulence. The free-cell bacterial filtrate revealed substantial mycelial growth inhibition ranging from 66 to 86%. The inhibition of conidial germination was from 32 to 78%, suggesting the involvement of metabolites in their biocontrol activity. The antifungal effect of the volatile compounds (VCOs) was observed for all bacteria with mycelial inhibition varying from 12 to 70%. Overall, their efficacy was substantially affected by the nature of the bacterial strains and the modes of action. Taken together, these results underscore that ACBC1 and SF14 for M. fructigena and SP10 and ACBP1 for M. laxa were the most effective bacterial strains. These strains were confirmed effective in a semi-commercial large-scale trial. Interestingly, their efficacies were found to be comparable to those of both commercial BCAs (B. subtilis Y1336 and P. agglomerans P10c), but slightly lower than thiophanate-methyl fungicide. The ability of most bacterial strains to produce lytic enzymes (Amylase, Protease or Cellulase) and lipopeptides (bacillomycin, fengycin, iturin and surfactin) was demonstrated by biochemical and molecular analyzes. Therefore, our findings suggest that the bacterial antagonists ACBC1, SF14, SP10 and ACBP1, have the potential to prevent brown rot disease.

Application of inorganic phosphate limitation to efficient isoprene production in Pantoea ananatis.

AIMS: Establishment of an efficient isoprene fermentation process by adopting inorganic phosphate limitation as the trigger to direct metabolic flux to the isoprene synthetic pathway. METHODS AND RESULTS: We constructed isoprene-producing strains of Pantoea ananatis (a member of the Enterobacteriaceae family) by integrating a heterologous mevalonate pathway and a metabolic switch that senses external inorganic phosphate (Pi) levels. This metabolic switch enabled dual-phase isoprene production, where the initial cell growth phase under Pi-saturating conditions was uncoupled from the subsequent isoprene production phase under Pi-limiting conditions. In fed-batch fermentation using our best strain (SWITCH-PphoC/pIspSM) in a 1-l bioreactor, isoprene concentration in the off-gas was maintained between 300 and 460 ppm during the production phase and at 20 ppm during the cell growth phase, respectively. The strain SWITCH-PphoC/pIspSM produced totally 2·5 g l-1 of isoprene from glucose with a 1·8% volumetric yield in 48 h. CONCLUSIONS: This proof-of-concept study demonstrated that our Pi-dependent dual-phase production system using a P. ananatis strain as a producer has potential for industrial-scale isoprene fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This Pi-dependent dual-phase fermentation process could be an attractive and economically viable option for the production of various commercially valuable isoprenoids.

Inoculation of pqqE gene inhabiting Pantoea and Pseudomonas strains improves the growth and grain yield of wheat with a reduced amount of chemical fertilizer.

OBJECTIVE: The present study was performed to examine the role of pqqE inhabiting rhizobacteria in organic acid production and relationship of the organic acids with phosphate solubilization by the bacteria in vitro as well as in vivo. METHODS AND RESULTS: The pqqE gene was PCR amplified and sequenced in genomic DNA of Pantoea sp. WP-5 and Pseudomonas sp. NN-4. Nucleotide sequence obtained from WP-5 and NN-4 showed maximum sequence similarity (88 and 89%, respectively) with the pqqE gene of Pseudomonas fluorescens strain CMR12a (KM251420). Deduced amino acid sequence from pqqE gene of Pseudomonas sp. NN-4 and Pantoea sp. WP-5 showed 75 and 93% similarity, respectively, with protein pyrroloquinoline quinone. Phosphate solubilization and acid production assay were quantified on spectrophotometer and high-profile liquid chromatograph, respectively, by each bacterial strain. Both strains produced organic acids such as acetic, citric, gluconic, succinic and malic acid and lowered the pH of Pikovskaya broth medium under laboratory conditions. Phosphate solubilization by Pantoea sp. WP-5 was 311 ± 4 and 204 ± 3 µg ml-1 in the culture medium supplemented with glucose and sucrose as carbon source, respectively. Pseudomonas sp. NN-4 solubilized 176 ± 3 and 298 ± 5 µg ml-1 phosphate in Pikovskaya broth medium under similar conditions. In field experiments conducted during two consecutive years, the concentration of acetic acid and gluconic acid was higher in root exudates of plants treated with Pantoea sp. WP-5 at 30% reduced doses of nitrogen (N)- and phosphorus (P)-based chemical fertilizers as compared to non-inoculated plants. Values of chlorophyll contents, crop growth rate, leaf area index, straw yield and P contents were recorded higher in plants inoculated with Pantoea sp. WP-5 and Pseudomonas sp. NN-4 as compared to non-inoculated control. Grain yield was increased by 10-12% due to inoculation with Pantoea sp. WP-5 and Pseudomonas sp. NN-4 over non-inoculated control in the field experiments. CONCLUSIONS: These results lead to the conclusions that the rhizobacteria inhabiting pqqE gene produced organic acids and solubilized the phosphate in vitro. On inoculation to wheat plants in field experiments, these strains produced the organic acids, solubilized the phosphate, and improved the P uptake and productivity of wheat. SIGNIFICANCE AND IMPACT OF THE STUDY: The Pantoea sp. WP-5 and Pseudomonas sp. NN-4 are the potential candidates for inoculation to wheat as phosphate solubilizer even with reduced chemical fertilizer dose. The inoculation of the strains may enhance grain yield and net income of the farmer even with less chemical fertilizer application. This practice will be helpfull inminimizing environmental pollution.

An roGFP2-Based Bacterial Bioreporter for Redox Sensing of Plant Surfaces.

The reduction-oxidation (redox) environment of the phytobiome (i.e., the plant-microbe interface) can strongly influence the outcome of the interaction between microbial pathogens, commensals, and their host. We describe a noninvasive method using a bacterial bioreporter that responds to reactive oxygen species and redox-active chemicals to compare microenvironments perceived by microbes during their initial encounter of the plant surface. A redox-sensitive variant of green fluorescent protein (roGFP2), responsive to changes in intracellular levels of reduced and oxidized glutathione, was expressed under the constitutive SP6 and fruR promoters in the epiphytic bacterium Pantoea eucalypti 299R (Pe299R/roGFP2). Analyses of Pe299R/roGFP2 cells by ratiometric fluorometry showed concentration-dependent responses to several redox active chemicals, including hydrogen peroxide (H2O2), dithiothreitol (DTT), and menadione. Changes in intracellular redox were detected within 5 min of addition of the chemical to Pe299R/roGFP2 cells, with approximate detection limits of 25 and 6 µM for oxidation by H2O2 and menadione, respectively, and 10 µM for reduction by DTT. Caffeic acid, chlorogenic acid, and ascorbic acid mitigated the H2O2-induced oxidation of the roGFP2 bioreporter. Aqueous washes of peach and rose flower petals from young blossoms created a lower redox state in the roGFP2 bioreporter than washes from fully mature blossoms. The bioreporter also detected differences in surface washes from peach fruit at different stages of maturity and between wounded and nonwounded sites. The Pe299R/roGFP2 reporter rapidly assesses differences in redox microenvironments and provides a noninvasive tool that may complement traditional redox-sensitive chromophores and chemical analyses of cell extracts.

Screening of tea saponin-degrading strain to degrade the residual tea saponin in tea seed cake.

Although tea seed cake (TSC) possesses high nutritional value, its high content of tea saponin (TS) limits its potential as feed. This study aimed to degrade TS in TSC by saponin-degrading strain and used a multistrains fermentation method to improve its nutritional value and palatability. Three saponin-degrading strains were isolated from Oleum Camelliae mill soil and identified as Citrobacter sp. FCTS301, Pantoea sp. FCTS302, and Enterobacter sp. FCTS303. Single-factor experiment showed that Citrobacter sp. FCTS301 had the highest degradation rate of TS. Response surface analysis for Citrobacter sp. FCTS301 indicated that the optimum culture conditions were as follows: initial pH of 7.2, culture temperature of 34.2 °C, inoculation amount of 7.3%, the agitation rate of 150 rpm, and the TS concentration of 10.0 g/L. Under these conditions, the maximum degradation rate was 82.6%. The fermentation process of TSC was obtained by a multistrains fermentation experiment. Considering the protein content, crude fiber degradation rate, and TS degradation rate of each group, the optimum inoculum amount of strains included Citrobacter sp. FCTS301, Aspergillus oryzae NCUF414, Saccharomyces cersvisiae NCUF306.5, and Lactobacillus plantarum NCUF201.1(5%, 0.5%, 1.0%, and 1.5%). After TS was degraded efficiently, fermented TSC can be presumed a potential feed raw material.

Identification and Characterization of pantocin wh-1, a Novel Cyclic Polypeptide Produced by Pantoea dispersa W18.

Pantoea dispersa W18, isolated from contaminated soil, was found to exert antimicrobial activity against Mycobacterium species, including Mycobacterium tuberculosis, an important human pathogen. Here, the anti-mycobacterial compound produced by Pantoea dispersa W18 was purified by a combination of hydrophobic interaction chromatography, cation exchange chromatography, and reverse phase HPLC. Active compounds from Pantoea dispersa W18 were identified as a natural peptide named pantocin wh-1 with a 1927 Da molecular weight. The primary structure of this compound was detected by N-terminal amino acid sequencing. The amino acid sequence of pantocin wh-1 consisted of 16 amino acid residues with a cyclic structure. The pantocin wh-1 could be inactivated by protease K, but was heat stable and unaffected by pH (2-12). However, the activity was not completely inactivated by trypsin and pepsin. This is the first report of a cyclic polypeptide purified from a strain of Pantoea dispersa.

Evaluation and improvement of phosphate solubilization by an isolated bacterium Pantoea agglomerans ZB.

A phosphate solubilizing bacterium ZB was isolated from the rhizosphere soil of Araucaria, which falls into the species Pantoea agglomerans. Optimization for phosphate solubilization by strain ZB was performed. At optimum culture conditions, the isolate showed great ability of solubilizing different insoluble inorganic phosphate sources viz. Ca3(PO4)2 (TCP), Hydroxyapatite (HP), CaHPO4, AlPO4, FePO4 along with rock phosphates (RPs). Inoculation with planktonic cells was found to enhance dissolved phosphorous as compared to that achieved by symplasma inoculation. Besides inoculation with different status of cells, pre-incubation could also exert a great effect on phosphate solubilization ability of P. agglomerans. When isolate ZB was cultured with glucose as carbon sources, phosphorous was more efficiently dissolved from HP and RP without pre-incubation in comparison to that obtained with pre-cultivation. Pre-cultivation, however, was more suitable for P solubilization than no pre-cultivation when bacteria were grown with xylose. A positive correlation was detected between the production of organic acids and phosphate solubilization. P. agglomerans ZB possessed many plant growth promotion traits such as N2 fixation and production of indole 3-acetic acid, phytase, alkaline phosphatase. Pot experiment showed inoculation with single isolate ZB or biofertilizer prepared from semi-solid fermentation of isolate ZB with spent mushroom substrate (SMS) compost could enhance plant growth with respect to number of leaves, plant leave area, stem diameter, root length, root dry mass, shoot dry mass and biomass when compared to the abiotic control, revealing strain ZB could be a promising environmental-friendly biofertilizer to apply for agricultural field.