Indole-3-acetamide Research Articles

Elimination of ineffective inorganic salt component in medium for indole‐3‐acetic acid synthesis by Serratia plymuthica UBCF_13 and its effect on the growth of chili seedlings

Indole‐3‐acetic acid (IAA) is an essential phytohormone that controls a variety of plant growth mechanisms. Bacteria can produce IAA to stimulate plant growth, with its production influenced by the culture conditions. Serratia plymuthica UBCF_13 is recognized as an IAA‐producing bacterium, exhibiting maximum IAA production in a yeast medium comprising yeast extract, sucrose, K2HPO4, MgSO4, NaCl, and CaCO3. However, prior studies optimizing individual inorganic salt components indicated minimal impact on IAA synthesis within this medium. This study aimed to eliminate the unnecessary inorganic salt components and the medium was then applied to investigate the IAA biosynthesis pathway and the plant growth‐promoting assay. The elimination assay consisted of yeast sucrose medium devoid of K2HPO4, MgSO4, NaCl, or CaCO3, and yeast sucrose medium containing only MgSO4 and CaCO3. Various indole compounds were then added to the revised medium composition to investigate the IAA biosynthesis pathway of UBCF_13 using high‐performance liquid chromatography (HPLC). Furthermore, the effect of UBCF_13 culture supernatant, cultivated in the new medium, on chili plant growth was evaluated. The highest IAA production (138.8 µg/mL) was observed in the yeast sucrose with CaCO3 and MgSO4 (elimination of K2HPO4 and NaCl). The presence of indole‐3‐acetamide (IAM) compound from the medium extracts, supplemented with multiple indole compounds, revealed that UBCF_13 may use the IAM pathway. The application of UBCF_13 supernatant enhanced the shoot, root length, fresh weight, and germination time of chili seeds by 37.7%, 49.3%, 204.3%, and 38.6%, respectively. This study demonstrated that eliminating K2HPO4 and NaCl provided a new culture medium composition conducive to IAA production by UBCF_13. Moreover, the UBCF_13 extract has the potential to promote plant growth.

Intestinal Barrier Protection by Probiotic Lactobacillus-Associated Indoles in Caco-2 and Enteroids

Gut bacteria generate from dietary tryptophan (trp) indole compounds, which act as potent signaling molecules intricately involved in the regulation of intestinal homeostasis and barrier integrity. Lactobacillus rhamnosus GG (LGG), a widely used probiotic, has demonstrated ability to restore the gut barrier in animal models of colitis and sepsis, as well as in children with irritable bowel syndrome. To investigate the hypothesis that LGG-derived indoles are responsible for its gut barrier-enhancing properties, we fed gnotobiotic mice trp-suffcient and trp-deficient diets. We then monocolonized them with LGG, using PBS gavaged mice as a control, and analyzed the ileal transcriptome by bulk RNA sequencing and both fecal and serum metabolomes by LC-MS. Using our novel bioinformatic tool METRCA, which correlates metabolites and gene networks, we uncovered an unexpected and substantial influence of indole spatial distribution on genes associated with gut integrity: some were significantly positively linked to tight junction (TJ) genes exclusively from the luminal side, some exclusively from the serum, and others from both. Specifically, we identified nine indoles—indole-acetamide (IAM), indole-carboxaldehyde (IAL), indole-3-lactic acid (ILA), indole-3-propionic acid (IPA), indole-3-acrylic acid (IAR), indole-3-carbinol (ICB), indole-acetic acid (IAA), indole-3-acetonitrile (IAN), and indole-3-carboxylic acid (ICA)—that demonstrated the potential to maintain gut barrier function. To assess their impact, we used a high-throughput system that measured permeability through transepithelial electrical resistance (TEER) and FITC-dextran (4 kDa) in polarized Caco-2 cells. By luminally challenging cells with Clostridium diffcile toxins (TOX), we induced barrier disruption to model leaky gut. Intriguingly, when applied luminally, IAA, IAN, ICA, and ILA rescued the TEER of Caco-2 cells exposed to TOX, while IAM, IAL, and ILA were effective when applied basolaterally, suggesting a strong polar component to their barrier repair capacity. FITC transport revealed a significant basolateral rescue effect of IAM and luminal rescue effect of IAA and ICA, indicating that different sets of indoles are required to repair the pore and leak pathways. Furthermore, we confirmed that basolateral IAM rescued FITC permeability and induced increased expression of TJ genes Tjp1 and Ocln in enteroids challenged with lipopolysaccharides. In summary, our research highlights the potential beneficial therapeutic application of certain indoles in repairing disrupted gut barriers. NIHR01-AT010243 (NG, RF), R01DK119198 (NG), NSF 1754783 (RF). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bacterial indole-3-acetic acid: A key regulator for plant growth, plant-microbe interactions, and agricultural adaptive resilience

Indole-3-acetic acid (IAA), a fundamental phytohormone categorized under auxins, not only influences plant growth and development but also plays a critical role in plant-microbe interactions. This study reviews the role of IAA in bacteria-plant communication, with a focus on its biosynthesis, regulation, and the subsequent effects on host plants. Bacteria synthesize IAA through multiple pathways, which include the indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), and several other routes, whose full mechanisms remain to be fully elucidated. The production of bacterial IAA affects root architecture, nutrient uptake, and resistance to various abiotic stresses such as drought, salinity, and heavy metal toxicity, enhancing plant resilience and thus offering promising routes to sustainable agriculture. Bacterial IAA synthesis is regulated through complex gene networks responsive to environmental cues, impacting plant hormonal balances and symbiotic relationships. Pathogenic bacteria have adapted mechanisms to manipulate the host’s IAA dynamics, influencing disease outcomes. On the other hand, beneficial bacteria utilize IAA to promote plant growth and mitigate abiotic stresses, thereby enhancing nutrient use efficiency and reducing dependency on chemical fertilizers. Advancements in analytical methods, such as liquid chromatography–tandem mass spectrometry, have improved the quantification of bacterial IAA, enabling accurate measurement and analysis. Future research focusing on molecular interactions between IAA-producing bacteria and host plants could facilitate the development of biotechnological applications that integrate beneficial bacteria to improve crop performance, which is essential for addressing the challenges posed by climate change and ensuring global food security. This integration of bacterial IAA producers into agricultural practice promises to revolutionize crop management strategies by enhancing growth, fostering resilience, and reducing environmental impact.

Enhancement of tryptophan 2-monooxygenase thermostability by semi-rational enzyme engineering: a strategic design to minimize experimental investigation.

Tryptophan 2-monooxygenase (TMO) is an FAD-bound flavoenzyme which catalyzes the oxidative decarboxylation of l-tryptophan to produce indole-3-acetamide (IAM) and carbon dioxide. The reaction of TMO is the first step of indole-3-acetic acid (IAA) biosynthesis. Although TMO is of interest for mechanistic studies and synthetic biology applications, the enzyme has low thermostability and soluble expression yield. Herein, we employed a combined approach of rational design using computational tools with site-saturation mutagenesis to screen for TMO variants with significantly improved thermostability properties and soluble protein expression. The engineered TMO variants, TMO-PWS and TMO-PWSNR, possess melting temperatures (T m) of 65 °C, 17 °C higher than that of the wild-type enzyme (TMO-WT). At 50 °C, the stabilities (t 1/2) of TMO-PWS and TMO-PWSNR were 85-fold and 92.4-fold higher, while their soluble expression yields were 1.4-fold and 2.1-fold greater than TMO-WT, respectively. Remarkably, the kinetic parameters of these variants were similar to those of the wild-type enzymes, illustrating that they are promising candidates for future studies. Molecular dynamic simulations of the wild-type and thermostable TMO variants identified key interactions for enhancing these improvements in the biophysical properties of the TMO variants. The introduced mutations contributed to hydrogen bond formation and an increase in the regional hydrophobicity, thereby, strengthening the TMO structure.

Production of Acetic Indole Acid in Macrophomina phaseolina

The fungus Macrophomina phaseolina is a phytopathogen of great importance attacking various crops and causing severe losses especially in sorghum, beans, soybeans and others. Since acetic indole acid (AIA) is a hormone that some authors associate with the pathogenic power of other fungi such as Fusarium oxysporum, it was decided to perform this work to detect whether M phaseolina isolated from beans is layers of producing AIA and which synthesis pathways are involved in its production. The analyses performed using HPLC showed the production of indole acetamide (ACM) that occurs in the first 60 hours of incubation. Another compound produced is Indole (IND) which is released after 60 hours when ACM production has ceased. Similarly, the fungus can metabolize AIA in the culture medium and tryptophan, the main precursor of AIA, apparently has no effect on the amount of AIA synthesized.
 Conclusion: The phytopathogenic fungus Macrophomina phaseolina can produce indole acetic acid, a hormone that stimulates plant growth through the TRP-D pathway via indole acetamide, which occurs mainly in the first 60 hours of growth of the fungus in the culture medium. In addition, the presence of indole was detected, which allows us to estimate that the fungus uses another little explored route that is TRP-I and that from this compound the AIA can be synthesized directly or transformed into TRP. This means that when a metabolic pathway of IAA is turned off (ACM) it is replaced by another pathway (Indole). The convenient is to correlate the release of indole acetic acid with the pathogenicity of M phaseolina.

Growth Phase Influence the Gene Expression and Metabolite Production Related to Indole-3-Acetic Acid (IAA) Biosynthesis by Serratia plymuthica UBCF_13.

<b>Background and Objective:</b> The optimization of the indole-3-acetic acid (IAA) producing capability of <i>Serratia plymuthica</i> UBCF_13 has been intensively studied. This work tried to reveal the effect of growth phases on IAA production, gene expression and metabolite synthesis related to the IAA biosynthesis pathway. <b>Materials and Methods:</b> The growth curve and IAA production were measured every 3 hrs. The putative IAA biosynthesis pathway was investigated based on the UBCF_13 genome. To identify the possible pathway of IAA biosynthesis in UBCF_13, we applied the Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and High-Performance Liquid Chromatography (HPLC) analysis to measure the transcript levels of each gene and indole metabolite production based on tryptophan treatment at different times of incubation. <b>Results:</b> The optimal IAA production on colorimetric assay was at 9 hrs of incubation (initial stationary phase). The level expression of <i>puuC</i>, <i>DDC</i>, <i>oxdA</i>, <i>amiE</i>, <i>nthA</i> and <i>nthB</i> have been upregulated maximum in 3 hrs of culture time (lag phase), except <i>tyrB</i> and <i>ipdC</i>. The highest transcript level of the genes was found in nitrile hydratase genes (<i>nthA</i> and <i>nthB</i>) and indole-3- acetamide (IAM) has been detected as the only intermediate in the crude extract of UBCF_13 thus the IAM pathway may be used to produce IAA. The maximum IAA production on HPLC analysis was found at 21 hrs of incubation (late stationary phase). <b>Conclusion:</b> This study gives a new insight that the best time to measure gene expression and intermediates related to the IAA biosynthetic pathway in bacteria was found at a specific growth phase.

Comprehensive Phytohormone Profiling of Kohlrabi during In Vitro Growth and Regeneration: The Interplay with Cytokinin and Sucrose

The establishment of an efficient protocol for in vitro growth and regeneration of kohlrabi (Brassica oleracea var. gongylodes) allowed us to closely examine the phytohormone profiles of kohlrabi seedlings at four growth stages (T1–T4), additionally including the effects of cytokinins (CKs)—trans-zeatin (transZ) and thidiazuron (TDZ)—and high sucrose concentrations (6% and 9%). Resulting phytohormone profiles showed complex time-course patterns. At the T2 stage of control kohlrabi plantlets (with two emerged true leaves), levels of endogenous CK free bases and gibberellin GA20 increased, while increases in jasmonic acid (JA), JA-isoleucine (JA-Ile), indole-3-acetic acid (IAA) and indole-3-acetamide (IAM) peaked later, at T3. At the same time, the content of most of the analyzed IAA metabolites decreased. Supplementing growth media with CK induced de novo formation of shoots, while both CK and sucrose treatments caused important changes in most of the phytohormone groups at each developmental stage, compared to control. Principal component analysis (PCA) showed that sucrose treatment, especially at 9%, had a stronger effect on the content of endogenous hormones than CK treatments. Correlation analysis showed that the dynamic balance between the levels of certain bioactive phytohormone forms and some of their metabolites could be lost or reversed at particular growth stages and under certain CK or sucrose treatments, with correlation values changing between strongly positive and strongly negative. Our results indicate that the kohlrabi phytohormonome is a highly dynamic system that changes greatly along the developmental time scale and also during de novo shoot formation, depending on exogenous factors such as the presence of growth regulators and different sucrose concentrations in the growth media, and that it interacts intensively with these factors to facilitate certain responses.

A novel function of the key nitrogen-fixation activator NifA in beta-rhizobia: Repression of bacterial auxin synthesis during symbiosis.

Rhizobia fix nitrogen within root nodules of host plants where nitrogenase expression is strictly controlled by its key regulator NifA. We recently discovered that in nodules infected by the beta-rhizobial strain Paraburkholderia phymatum STM815, NifA controls expression of two bacterial auxin synthesis genes. Both the iaaM and iaaH transcripts, as well as the metabolites indole-acetamide (IAM) and indole-3-acetic acid (IAA) showed increased abundance in nodules occupied by a nifA mutant compared to wild-type nodules. Here, we document the structural changes that a P. phymatum nifA mutant induces in common bean (Phaseolus vulgaris) nodules, eventually leading to hypernodulation. To investigate the role of the P. phymatum iaaMH genes during symbiosis, we monitored their expression in presence and absence of NifA over different stages of the symbiosis. The iaaMH genes were found to be under negative control of NifA in all symbiotic stages. While a P. phymatum iaaMH mutant produced the same number of nodules and nitrogenase activity as the wild-type strain, the nifA mutant produced more nodules than the wild-type that clustered into regularly-patterned root zones. Mutation of the iaaMH genes in a nifA mutant background reduced the presence of these nodule clusters on the root. We further show that the P. phymatum iaaMH genes are located in a region of the symbiotic plasmid with a significantly lower GC content and exhibit high similarity to two genes of the IAM pathway often used by bacterial phytopathogens to deploy IAA as a virulence factor. Overall, our data suggest that the increased abundance of rhizobial auxin in the non-fixing nifA mutant strain enables greater root infection rates and a role for bacterial auxin production in the control of early stage symbiotic interactions.

The Indole-3-Acetamide-Induced Arabidopsis Transcription Factor MYB74 Decreases Plant Growth and Contributes to the Control of Osmotic Stress Responses.

The accumulation of the auxin precursor indole-3-acetamide (IAM) in the ami1 mutant has recently been reported to reduce plant growth and to trigger abiotic stress responses in Arabidopsis thaliana. The observed response includes the induction of abscisic acid (ABA) biosynthesis through the promotion of NCED3 expression. The mechanism by which plant growth is limited, however, remained largely unclear. Here, we investigated the transcriptional responses evoked by the exogenous application of IAM using comprehensive RNA-sequencing (RNA-seq) and reverse genetics approaches. The RNA-seq results highlighted the induction of a small number of genes, including the R2R3 MYB transcription factor genes MYB74 and MYB102. The two MYB factors are known to respond to various stress cues and to ABA. Consistent with a role as negative plant growth regulator, conditional MYB74 overexpressor lines showed a considerable growth reduction. RNA-seq analysis of MYB74 mutants indicated an association of MYB74 with responses to osmotic stress, water deprivation, and seed development, which further linked MYB74 with the observed ami1 osmotic stress and seed phenotype. Collectively, our findings point toward a role for MYB74 in plant growth control and in responses to abiotic stress stimuli.

Probing into the unique relationship between a soil bacterium, Pseudomonas putida AKMP7 and Arabidopsis thaliana: A case of “conditional pathogenesis”

Plant growth-promoting rhizobacteria (PGPR) are beneficial soil bacteria that colonise the rhizosphere and help plants in growth, development, and stress tolerance. While there is a significant body of research elucidating their benefits to plants, studies on the “abnormal” or “unexpected” behavior of these bacteria are almost non-existent. One such study from our laboratory has previously reported a unique situation in which a certain strain of drought and thermo-tolerant PGPR, namely, Pseudomonas putida AKMP7, becomes pathogenic towards Arabidopsis thaliana under drought conditions, but not under normal (well-watered) conditions. In this study, we have probed deeper into this phenomenon of “conditional pathogenesis”. We found that, AKMP7 imparts an enhancement in plant growth under well-watered conditions, while, causing a deterioration in plant health under drought conditions. In an attempt to understand the underlying reasons for this phenomenon, we analysed the phytohormones released by Pseudomonas putida AKMP7 using LC-ESI-MS/MS technique. We identified that AKMP7 releases zeatin (a cytokinin), the auxin derivative -indole acetamide and amino acid-conjugates of auxin (indole-3-acetyl-L-alanine, indole-3-acetyl-L-phenylalanine and indole-3-acetyl-L-aspartate) in the growth medium. By treating the plants with commercially obtained forms of these phytohormones, individually or in combination with AKMP7, we identified that zeatin and auxin derivative indole acetamide can play a crucial role in the conditional pathogenesis exhibited by this bacterium on A. thaliana under drought conditions. Our work lays a foundation for further understanding the precise molecular mechanisms involved in this unique phenomenon of conditional/opportunistic pathogenesis.

Metagenomic Approaches to Explore the Quorum Sensing-Mediated Interactions Between Algae and Bacteria in Sequence Membrane Photo-Bioreactors.

Algal–bacterial water treatment is more effective for better harvesting and promotes energy savings than other traditional treatments, while the relationships between them are multifarious. Among all the interactions, quorum sensing plays an essential ecological role. However, the relative contributions of signaling in the interaction between algae and bacteria are not clear. To elucidate the role of quorum sensing by indole-3-acetic acid (IAA) in terms of the algal–bacterial interaction during the nitrogen removal process, the bioreactors, respectively, inoculated with Chlorella, Phormidium, and both of them were started. We manifest the existence of multiple signaling-related proteins by alignment with the constructed database, and the signaling was analyzed using metagenomic sequence data obtained during bioreactor operation. We found that IAA was mainly synthetized depending on indole-3-acetamide (IAM) and indole-3-pyruvic acid (IPA) pathways by calculating the gene abundance of IAA synthetase. Both Chlorella and the co-culture reactor possessed higher nitrogen removal rate (NRR) than the Phormidium reactor, and the abundance profile of the signaling-related gene is similar with the NRR. The signaling-related gene abundance increased in Chlorella and co-culture reactors but decreased in the Phormidium reactor. Pseudomonas, Hydrogenophaga, and Zoogloea are the dominant signaled bacteria. Chlorella is the dominant signaled algae. The relative abundance of total signaled bacteria in the whole bacterial community increased during the start-up in Chlorella and co-culture reactors. According to the network analysis, phytoplankton prefers to positively correlate with signaled bacteria than non-signaled bacteria, which indicated that the signaling influences the algal–bacterial interaction. These findings hint at the significance of algal–bacterial signaling in this interkingdom interaction during nitrogen removal.

Versatile and Facile One-Pot Biosynthesis for Amides and Carboxylic Acids in E. coli by Engineering Auxin Pathways of Plant Microbiomes

The development of enzymatic routes toward amide and carboxylic acid bond formation in bioactive molecular scaffolds using aqueous conditions is a major challenge for biopharmaceutical and fine chemical industrial sectors. We report biocatalytic and kinetic characterization of two indole-3-acetamide (IAM) pathway enzymes, tryptophan-2-monooxygenase (iaaM) and indole-3-acetamide hydrolase (iaaH), present in plant microbiomes that produce indole-3-acetic acid (IAA). In this pathway, tryptophan is converted to indole-3-acetamide by the monooxygenase activity of iaaM, followed by its hydrolysis to form carboxylic acid by iaaH enzyme. Since IAA or auxin is an essential natural plant hormone and an important synthon for fine chemicals, the developed monooxygenase-based bioconversion route has a wider scope compared to currently available synthetic and biocatalytic methods to produce synthetic auxins and a range of amides and carboxylic acids for agrochemical and pharmaceutical applications. To display this, one-pot multienzyme biosynthetic cascades for preparative-scale production of IAA derivatives were performed by incorporating tryptophan synthase and tryptophan halogenase enzymes. We also report the creation of an efficient de novo biosynthesis for IAA and its derivatives from glucose or indoles via a reconstructed IAM pathway in Escherichia coli.

Metabolomics and Dual RNA-Sequencing on Root Nodules Revealed New Cellular Functions Controlled by Paraburkholderia phymatum NifA.

Paraburkholderia phymatum STM815 is a nitrogen-fixing endosymbiont that nodulate the agriculturally important Phaseolus vulgaris and several other host plants. We previously showed that the nodules induced by a STM815 mutant of the gene encoding the master regulator of nitrogen fixation NifA showed no nitrogenase activity (Fix−) and increased in number compared to P. vulgaris plants infected with the wild-type strain. To further investigate the role of NifA during symbiosis, nodules from P. phymatum wild-type and nifA mutants were collected and analyzed by metabolomics and dual RNA-Sequencing, allowing us to investigate both host and symbiont transcriptome. Using this approach, several metabolites’ changes could be assigned to bacterial or plant responses. While the amount of the C4-dicarboxylic acid succinate and of several amino acids was lower in Fix− nodules, the level of indole-acetamide (IAM) and brassinosteroids increased. Transcriptome analysis identified P. phymatum genes involved in transport of C4-dicarboxylic acids, carbon metabolism, auxin metabolism and stress response to be differentially expressed in absence of NifA. Furthermore, P. vulgaris genes involved in autoregulation of nodulation (AON) are repressed in nodules in absence of NifA potentially explaining the hypernodulation phenotype of the nifA mutant. These results and additional validation experiments suggest that P. phymatum STM815 NifA is not only important to control expression of nitrogenase and related enzymes but is also involved in regulating its own auxin production and stress response. Finally, our data indicate that P. vulgaris does sanction the nifA nodules by depleting the local carbon allocation rather than by mounting a strong systemic immune response to the Fix− rhizobia.

Indole Derivatives Obtained from Egyptian Enterobacter sp. Soil Isolates Exhibit Antivirulence Activities against Uropathogenic Proteus mirabilis.

Proteus mirabilis is a frequent cause of catheter associated urinary tract infections (CAUTIs). Several virulence factors contribute to its pathogenesis, but swarming motility, biofilm formation, and urease activity are considered the hallmarks. The increased prevalence in antibiotic resistance among uropathogens is alarming and requires searching for new treatment alternatives. With this in mind, our study aims to investigate antivirulence activity of indole derivatives against multidrug resistant P. mirabilis isolates. Ethyl acetate (EtOAc) extracts from Enterobacter sp. (rhizobacterium), isolated from Egyptian soil samples were tested for their ability to antagonize the virulence capacity and biofilm activity of P. mirabilis uropathogens. Extracts of two Enterobacter sp. isolates (coded Zch127 and Cbg70) showed the highest antivirulence activities against P. mirabilis. The two promising rhizobacteria Zch127 and Cbg70 were isolated from soil surrounding: Cucurbita pepo (Zucchini) and Brassica oleracea var. capitata L. (Cabbage), respectively. Sub-minimum inhibitory concentrations (Sub-MICs) of the two extracts showed potent antibiofilm activity with significant biofilm reduction of ten P. mirabilis clinical isolates (p-value < 0.05) in a dose-dependent manner. Interestingly, the Zch127 extract showed anti-urease, anti-swarming and anti-swimming activity against the tested strains. Indole derivatives identified represented key components of indole pyruvate, indole acetamide pathways; involved in the synthesis of indole acetic acid. Additional compounds for indole acetonitrile pathway were detected in the Zch127 extract which showed higher antivirulence activity. Accordingly, the findings of the current study model the feasibility of using these extracts as promising antivirulence agent against the P. mirabilis uropathogens and as potential therapy for treatment of urinary tract infections (UTIs).

Accumulation of the Auxin Precursor Indole-3-Acetamide Curtails Growth through the Repression of Ribosome-Biogenesis and Development-Related Transcriptional Networks.

The major auxin, indole-3-acetic acid (IAA), is associated with a plethora of growth and developmental processes including embryo development, expansion growth, cambial activity, and the induction of lateral root growth. Accumulation of the auxin precursor indole-3-acetamide (IAM) induces stress related processes by stimulating abscisic acid (ABA) biosynthesis. How IAM signaling is controlled is, at present, unclear. Here, we characterize the ami1 rooty double mutant, that we initially generated to study the metabolic and phenotypic consequences of a simultaneous genetic blockade of the indole glucosinolate and IAM pathways in Arabidopsis thaliana. Our mass spectrometric analyses of the mutant revealed that the combination of the two mutations is not sufficient to fully prevent the conversion of IAM to IAA. The detected strong accumulation of IAM was, however, recognized to substantially impair seed development. We further show by genome-wide expression studies that the double mutant is broadly affected in its translational capacity, and that a small number of plant growth regulating transcriptional circuits are repressed by the high IAM content in the seed. In accordance with the previously described growth reduction in response to elevated IAM levels, our data support the hypothesis that IAM is a growth repressing counterpart to IAA.

High-Level Production of Indole-3-acetic Acid in the Metabolically Engineered Escherichia coli.

Indole-3-acetic acid (IAA) is a critical plant hormone that regulates cell division, development, and metabolism. IAA synthesis in plants and plant-associated microorganisms cannot fulfill the requirement for large-scale agricultural production. Here, two novel IAA biosynthesis pathways, tryptamine (TAM) and indole-3-acetamide (IAM), were developed for IAA production by whole-cell catalysis and de novo biosynthesis in an engineered Escherichia coli MG1655. When 10 g/L l-tryptophan was used as a substrate, an MIA-6 strain containing a heterologous IAM pathway had the highest IAA titer of 7.10 g/L (1.34 × 103 mg/g DCW), which was 98.4 times more than MTAI-5 containing the TAM pathway by whole-cell catalysis. De novo IAA biosynthesis was optimized by improving NAD(P)H availability, resulting in an increased IAA titer of 906 mg/L obtained by the MGΔadhE::icd strain, which is 29.7% higher than the control. These strategies exhibit the potential for IAA production in engineered E. coli and possible industrial applications.

Design and synthesis of novel quinazolinones conjugated ibuprofen, indole acetamide, or thioacetohydrazide as selective COX-2 inhibitors: anti-inflammatory, analgesic and anticancer activities

Novel quinazolinones conjugated with indole acetamide (4a–c), ibuprofen (7a–e), or thioacetohydrazide (13a,b, and 14a-d) were designed to increase COX-2 selectivity. The three synthesised series exhibited superior COX-2 selectivity compared with the previously reported quinazolinones and their NSAID analogue and had equipotent COX-2 selectivity as celecoxib. Compared with celecoxib, 4 b, 7c, and 13 b showed similar anti-inflammatory activity in vivo, while 13 b and 14a showed superior inhibition of the inflammatory mediator nitric oxide, and 7 showed greater antioxidant potential in macrophages cells. Moreover, all selected compounds showed improved analgesic activity and 13 b completely abolished the pain response. Additionally, compound 4a showed anticancer activity in tested cell lines HCT116, HT29, and HCA7. Docking results were consistent with COX-1/2 enzyme assay results. In silico studies suggest their high oral bioavailability. The overall findings for compounds (4a,b, 7c, 13 b, and 14c) support their potential role as anti-inflammatory agents.

Auxin-producing fungal endophytes promote growth of sunchoke

Endophytic fungi were able to protect their host plants against pathogens and promote plant growth. No previous studies have been conducted on the growth promotion of sunchoke by endophytic fungi. This research was the first to characterize plant growth promoting properties of endophytic fungi including, Macrophomina phaseolina BUP2/3 and Diaporthe phaseolorum BUP3/1 isolated from sunchoke and Daldinia eschscholtzii 2NTYL11, Trichoderma koningii ST-KKU1, Trichoderma erinaceum ST-KKU2, Macrophomina phaseolina SS1L10 and Macrophomina phaseolina SS1R10 from medicinal plants. Also, their plant growth promoting efficiency in artichoke plants under greenhouse condition was evaluated. The highest phosphate solubility and production of extracellular enzymes including amylase, protease, cellulase and xylanase were found after 5 and 21 days of incubation, respectively. Moreover, Indole-3-Acetic Acid (IAA) in the crude extracts of fungi isolated from Jerusalem artichoke was higher than that from medicinal plants (p ≤ 0.05). HPLC analysis suggested the putative IAA biosynthesis pathways in our fungi were via Indole-3-lactic acid (ILA) and Indole-3-acetamide (IAM). In the pot experiment, the values of height, diameter, chlorophyll content and leaf dry weight in sunchoke plants inoculated with fungal endophytes were significantly higher than those of un-inoculated plants. Therefore, this study suggested that fungal endophytes could be used as a biofertilizer for promoting growth of sunchoke.

Indole microbial intestinal metabolites expand the repertoire of ligands and agonists of the human pregnane X receptor

The interplays between the metabolic products of intestinal microbiota and the host signaling through xenobiotic receptors, including pregnane X receptor (PXR), are of growing interest, in the context of intestinal health and disease. A distinct class of microbial catabolites is formed from dietary tryptophan, having the indole scaffold in their core structure, which is a biologically active entity. In the current study, we examined a series of ten tryptophan microbial catabolites for their interactions with PXR signaling. Utilizing a reporter gene assay, we identified indole (IND) and indole-3-acetamide (IAD) as PXR agonists. IND and IAD induced PXR-regulated genes CYP3A4 and MDR1 in human intestinal cancer cells. Using time-resolved fluorescence resonance energy transfer, we show that IND (IC50 292 μM) and IAD (IC50 10 μM) are orthosteric ligands of PXR. Binding of PXR in its DNA response elements was enhanced by IND and IAD, as revealed by chromatin immunoprecipitation assay. We demonstrate that tryptophan microbial intestinal metabolites IND and IAD are ligands and agonists of human PXR. These findings are of particular importance in understanding the roles of microbial catabolites in human physiology and pathophysiology. Furthermore, these results are seminal in expanding potential drug repertoire through microbial metabolic mimicry.

Indole-3-acetic acid biosynthesis and its regulation in plant-associated bacteria.

Numerous studies have reported the stimulation of plant growth following inoculation with an IAA-producing PGPB. However, the specific mode of IAA production by the PGPB is rarely elucidated. In part, this is due to the overwhelming complexity of IAA biosynthesis and regulation. The promiscuity of the enzymes implicated in IAA biosynthesis adds another element of complexity when attempting to decipher their role in IAA biosynthesis. To date, the majority of research on IAA biosynthesis describes three separate pathways classified in terms of their intermediates-indole acetonitrile (IAN), indole acetamide (IAM), and indole pyruvic acid (IPA). Each of these pathways is mediated by a set of enzymes, many of which are traditionally assumed to exist for that specific catalytic role. This lends the possibility of missing other, novel, enzymes that may also incidentally serve that function. Some of these pathways are constitutively expressed, while others are inducible. Some enzymes involved in IAA biosynthesis are known to be regulated by IAA or by IAA precursors, as well as by a multitude of environmental cues. This review aims to provide an update to our current understanding of the biosynthesis and regulation of IAA in bacteria. KEY POINTS: • IAA produced by PGPB improves bacterial stress tolerance and promotes plant growth. • Bacterial IAA biosynthesis is convoluted; multiple interdependent pathways. • Biosynthesis of IAA is regulated by IAA, IAA-precursors, and environmental factors.