Myo-inositol Utilization Research Articles

Hierarchic regulation of a metabolic pathway: H-NS, CRP, and SsrB control myo-inositol utilization by Salmonella enterica.

The capacity to utilize myo-inositol (MI) as sole carbon and energy source is widespread among bacteria, among them the intestinal pathogen S. Typhimurium. This study elucidates the complex and hierarchical regulation that underlies the utilization of MI by S. Typhimurium under substrate limitation. A total of seven regulatory factors have been identified so far, allowing the pathogen an environment-dependent, efficient, and fine-tuned regulation of a metabolic property that provides growth advantages in different environments.

Combined Application of Myo-Inositol and Corn Steep Liquor from Agricultural Waste Alleviate Salt Stress in Brassica rapa.

Salinity poses a significant threat to plant growth through induction of osmotic and ionic stress and disruption of nutrient absorption. Biostimulants derived from agricultural waste offer a sustainable solution to alleviate salt-induced damage to plants and contribute to a circular and sustainable economy. In this study, we applied a combination of myo-inositol and corn steep liquor from waste sources to seedling cabbage (Brassica rapa subsp. pekinensis) and investigated their effects on plant growth under NaCl-simulated salt stress. Different concentrations of myo-inositol and corn steep liquor were applied to the roots, revealing that 150 mM NaCl significantly inhibited the growth and physiological metabolism of cabbage seedlings. Substrate application of myo-inositol, corn steep liquor, and their combination materials increased biomass, photosynthetic pigments, soluble sugars, soluble proteins, and the contents of K+, Ca2+, and Mg2+ in cabbage under salt stress conditions, while reducing malondialdehyde, electrolyte leakage, Na+ content, and the ratios of Na+/K+, Na+/Ca2+, and Na+/Mg2+. Therefore, root application of myo-inositol, corn steep liquor, and myo-inositol-corn steep liquor combination materials enhanced photosynthesis and enhanced cabbage salt stress resistance by maintaining cell osmotic and ion balance. The most pronounced positive effects were observed in the treatment with 0.1 mL L-1 corn steep liquor +288 mg L-1 myo-inositol. This study provides a theoretical basis and technical guidance for the combined utilization of myo-inositol and corn steep liquor to boost early growth and salt resistance in crops.

A Global Survey of Hypervirulent Aeromonas hydrophila (vAh) Identified vAh Strains in the Lower Mekong River Basin and Diverse Opportunistic Pathogens from Farmed Fish and Other Environmental Sources.

Hypervirulent Aeromonas hydrophila (vAh) has emerged as the etiologic agent of epidemic outbreaks of motile Aeromonas septicemia (MAS) in high-density aquaculture of farmed carp in China and catfish in the United States, which has caused millions of tons of lost fish. We conducted a global survey to better understand the evolution, geographical distribution, and phylogeny of vAh. Aeromonas isolates were isolated from fish that showed clinical symptoms of MAS, and pure cultures were screened for the ability to utilize myo-inositol as the sole carbon source. A total of 113 myo-inositol-utilizing bacterial strains were included in this study, including additional strains obtained from previously published culture collections. Based on a gyrB phylogeny, this collection included 66 A. hydrophila isolates, 48 of which were vAh. This collection also included five new vAh isolates from diseased Pangas catfish (Pangasius pangasius) and striped catfish (Pangasianodon hypophthalmus) obtained in Cambodia and Vietnam, respectively. Genome sequences were generated from representative vAh and non-vAh isolates to evaluate the potential for lateral genetic transfer of the myo-inositol catabolism pathway. Phylogenetic analyses of each of the nine genes required for myo-inositol utilization revealed the close affiliation of vAh strains regardless of geographic origin and suggested lateral genetic transfer of this catabolic pathway from an Enterobacter species. Prediction of virulence factors was conducted to determine differences between vAh and non-vAh strains in terms of virulence and secretion systems. Core genome phylogenetic analyses on vAh isolates and Aeromonas spp. disease isolates (55 in total) were conducted to evaluate the evolutionary relationships among vAh and other Aeromonas sp. isolates, which supported the clonal nature of vAh isolates. IMPORTANCE This global survey of vAh brought together scientists that study fish disease to evaluate the evolution, geographical distribution, phylogeny, and hosts of vAh and other Aeromonas sp. isolates. In addition to vAh isolates from China and the United States, four new vAh isolates were isolated from the lower Mekong River basin in Cambodia and Vietnam, indicating the significant threat of vAh to modern aquaculture and the need for improved biosecurity to prevent vAh spread.

Comparative genomic analysis of hypervirulent Aeromonas hydrophila strains from striped catfish (Pangasianodon hypophthalmus) in Vietnam

Motile Aeromonad septicemia (MAS) outbreaks caused by hypervirulent Aeromonas hydrophila (vAh) strains belonging to sequence type (ST) 251, have been reported and investigated widely in China and United States (US). In this study, for the first time, two genomes of vAh strains isolated from striped catfish in Vietnam have been identified and characterized. These two vAh strains caused catfish mortalities of 51.7–86.7% post immersion challenge with 2 × 108 CFU mL−1 or intraperitoneal injection challenge with 1 × 103 CFU fish−1, while no mortalities were observed in catfish challenged with regular A. hydrophila (rAh) strains (p < 0.05). These challenge trials indicate that vAh virulence is impacted by different degrees of penetration into the fish host and suggest that disease outbreaks in production ponds are affected by other pathogens or factors that contribute to vAh invasiveness. Comparative genomic analyses of vAh and rAh strains from Vietnam, together with reference A. hydrophila strains from China and US, were performed to gain an insight into the pathogenicity of the epidemic A. hydrophila strains collected from various locations worldwide. Besides the myo-inositol utilization and mobile genetic elements identified by previous studies, this work found that attachment and motility mediated by pili /fimbriae are also key factors underlying the hypervirulent nature of vAh strains. The vAh heterogeneity was observed on the core genome phylogenetic tree and the O-antigen gene clusters, where Vietnamese vAh strains showed closer genetic relationship with vAh strains isolated from carp species in China compared to vAh strains isolated from catfish in the US. The persistent occurrence of ST251 strains has been responsible for MAS outbreaks occurring frequently in Vietnamese catfish farms since 2013. This study provides important information about the spread of vAh in farmed catfish in Vietnam, its virulence in fish and the highly clonal nature of vAh strains identified in multiple countries.

Effect of magnesium ions on glucaric acid production in the engineered Saccharomyces cerevisiae

Glucaric acid has been successfully produced in Escherichia coli and fungus. Here, we first analyzed the effects of different metal ions on glucaric acid production in the engineered Saccharomyces cerevisiae Bga-3 strain harboring the glucaric acid synthesis pathway. We found that magnesium ions could promote the growth rate of yeast cells, and thus, increase the glucaric acid production by elevating the glucose and myo-inositol utilization of Bga-3 strain. RNA-Seq transcriptome analysis results showed that the upregulation of genes involved in the gluconeogenesis pathway, as well as the downregulation of genes associated with the glycolysis pathway and pentose phosphate pathway in response to MgCl2 were all benefit for the enhancement of the glucose-6-phosphate flux, which was the precursor for myo-inositol and glucaric acid. In addition, we found that MgCl2 could also increase the activity of MIOX4, which was also crucial for glucaric acid synthesis. At last, a final glucaric acid titer of 10.6 g/L, the highest reported titer, was achieved in the fed-batch fermentation using a 5-L bioreactor by adding 100 mM MgCl2. Our findings will provide a new way of promoting the production of other chemicals in the engineered yeast cells.

Myo-inositol utilization by Citrobacter koseri promotes brain infection

Citrobacter species are opportunistic bacterial pathogens that are implicated in both nosocomial and community-acquired infections. Among the Citrobacter species, Citrobacter koseri is often isolated from clinical material, and it can cause meningitis and brain abscesses in neonates and immunocompromised individuals, thus posing a great threat to human health. However, the virulence determinants of C. koseri remain largely unknown. Myo-inositol is an abundant carbohydrate in the environment and in certain organs of the human body, especially the brain. The C. koseri genome harbors a cluster of genes, QCQ70420.1 to QCQ70429.1 (named the Ino-cluster in this study), which encode IolBCDE, MmsA, and an ATP-binding cassette transporter. The gene cluster may be involved in the utilization of myo-inositol. To investigate the functions of the Ino-cluster in C. koseri, we constructed a mutant strain by deleting the Ino-cluster and found that the mutant could not use myo-inositol as the sole carbon source, confirming that this cluster is responsible for myo-inositol utilization. Moreover, we investigated the function of the Ino-cluster and myo-inositol utilization in C. koseri pathogenicity. Deletion of the Ino-cluster significantly impaired C. koseri colonization of the brain of infected Sprague–Dawley (SD) rats and BALB/c mice, and this increased the survival rate of the infected animals, indicating that the Ino-cluster and the ability to use myo-inositol are essential for C. koseri pathogenicity. Taken together, our findings suggest that using the Ino-cluster products, C. koseri can exploit the abundant myo-inositol in the brain as a carbon source for growth, thus promoting colonization and virulence.

High binding affinity of repressor IolR avoids costs of untimely induction of myo-inositol utilization by Salmonella Typhimurium

Growth of Salmonella enterica serovar Typhimurium strain 14028 with myo-inositol (MI) is characterized by a bistable phenotype that manifests with an extraordinarily long (34 h) and variable lag phase. When cells were pre-grown in minimal medium with MI, however, the lag phase shortened drastically to eight hours, and to six hours in the absence of the regulator IolR. To unravel the molecular mechanism behind this phenomenon, we investigated this repressor in more detail. Flow cytometry analysis of the iolR promoter at a single cell level demonstrated bistability of its transcriptional activation. Electrophoretic mobility shift assays were used to narrow the potential binding region of IolR and identified at least two binding sites in most iol gene promoters. Surface plasmon resonance spectroscopy quantified IolR binding and indicated its putative oligomerization and high binding affinity towards specific iol gene promoters. In competitive assays, the iolR deletion mutant, in which iol gene repression is abolished, showed a severe growth disadvantage of ~15% relative to the parental strain in rich medium. We hypothesize that the strong repression of iol gene transcription is required to maintain a balance between metabolic flexibility and fitness costs, which follow the inopportune induction of an unusual metabolic pathway.

The orphan regulator ReiD of Salmonella enterica is essential for myo-inositol utilization.

In Salmonella enterica serovar Typhimurium (S. Typhimurium), the genomic island GEI4417/4436 is responsible for the utilization of myo-inositol (MI) as carbon and energy source. Here, we report the characterization of a novel, island-encoded positive autoregulator termed ReiD (STM4423) that is specific to certain S. enterica strains and Escherichia coli strain ED1a able to use MI. ReiD was essential for growth with this polyol and also contributed to S. Typhimurium proliferation in swine caecum content. Providing higher copy numbers of ReiD reduced the long lag phase of 2 days during growth of S. Typhimurium in MI medium by 50%. In a heterologous host, expression of ReiD activated the transcription from the promoter of iolE/iolG, whose products catalyse the initial two steps in MI degradation. Episomal expression of iolE/iolG1 rescued the otherwise zero growth phenotype of a reiD deletion mutant in MI medium. Gel mobility shift assays with purified ReiD demonstrated directed interaction of ReiD with its own promoter and that of iolE. The repressor IolR bound the reiD promoter, implying that reiD is part of the IolR regulon. Taken together, the regulator ReiD is a trigger to accelerate the switch from more easily accessible nutrients to MI utilization by S. Typhimurium.

Genotype to phenotype: identification of diagnostic vibrio phenotypes using whole genome sequences

Vibrios are ubiquitous in the aquatic environment and can be found in association with animal or plant hosts. The range of ecological relationships includes pathogenic and mutualistic associations. To gain a better understanding of the ecology of these microbes, it is important to determine their phenotypic features. However, the traditional phenotypic characterization of vibrios has been expensive, time-consuming and restricted in scope to a limited number of features. In addition, most of the commercial systems applied for phenotypic characterization cannot characterize the broad spectrum of environmental strains. A reliable and possible alternative is to obtain phenotypic information directly from whole genome sequences. The aim of the present study was to evaluate the usefulness of whole genome sequences as a source of phenotypic information. We performed a comparison of the vibrio phenotypes obtained from the literature with the phenotypes obtained from whole genome sequences. We observed a significant correlation between the previously published phenotypic data and the phenotypic data retrieved from whole genome sequences of vibrios. Analysis of 26 vibrio genomes revealed that all genes coding for the specific proteins involved in the metabolic pathways responsible for positive phenotypes of the 14 diagnostic features (Voges-Proskauer reaction, indole production, arginine dihydrolase, ornithine decarboxylase, utilization of myo-inositol, sucrose and L-leucine, and fermentation of D-mannitol, D-sorbitol, L-arabinose, trehalose, cellobiose, D-mannose and D-galactose) were found in the majority of the vibrios genomes. Vibrio species that were negative for a given phenotype revealed the absence of all or several genes involved in the respective biochemical pathways, indicating the utility of this approach to characterize the phenotypes of vibrios. The absence of the global regulation and regulatory proteins in the Vibrio parahaemolyticus genome indicated a non-vibrio phenotype. Whole genome sequences represent an important source for the phenotypic identification of vibrios.

Implication of Lateral Genetic Transfer in the Emergence of Aeromonas hydrophila Isolates of Epidemic Outbreaks in Channel Catfish

To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase, led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2, and IolR. Determination of the phosphoenolpyruvate carboxykinase activity of the ΔramA, ΔgntR1 ΔgntR2, and ΔiolR deletion mutants indicated that RamA represses pck during growth on glucose about 2-fold, whereas GntR1, GntR2, and IolR activate pck expression about 2-fold irrespective of whether glucose or acetate served as the carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in a divergent orientation with respect to a iol gene cluster, encoding proteins involved in myo-inositol uptake and degradation. Comparative DNA microarray analysis of the ΔiolR mutant and the parental wild-type strain revealed strongly (>100-fold) elevated mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT1, and iolR. IolR therefore is presumably subject to negative autoregulation. A consensus DNA binding motif (5'-KGWCHTRACA-3') which corresponds well to those of other GntR-type regulators of the HutC family was identified. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism.

Down-regulation of the myo-inositol oxygenase gene family has no effect on cell wall composition in Arabidopsis

The enzyme myo-inositol oxygenase (MIOX; E.C. 1.13.99.1) catalyzes the ring-opening four-electron oxidation of myo-inositol into glucuronic acid, which is subsequently activated to UDP-glucuronic acid (UDP-GlcA) and serves as a precursor for plant cell wall polysaccharides. Starting from single T-DNA insertion lines in different MIOX-genes a quadruple knockdown (miox1/2/4/5-mutant) was obtained by crossing, which exhibits greater than 90% down-regulation of all four functional MIOX genes. Miox1/2/4/5-mutant shows no visible phenotype and produces viable pollen. The alternative pathway to UDP-glucuronic acid via UDP-glucose is upregulated in the miox1/2/4/5-mutant as a compensatory mechanism. Miox1/2/4/5-mutant is impaired in the utilization of myo-inositol for seedling growth. The incorporation of myo-inositol derived sugars into cell walls is strongly (>90%) inhibited. Instead, myo-inositol and metabolites produced from myo-inositol such as galactinol accumulate in the miox1/2/4/5-mutant. The increase in galactinol and raffinose family oligosaccharides does not enhance stress tolerance. The ascorbic acid levels are the same in mutant and wild type plants.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-011-1394-z) contains supplementary material, which is available to authorized users.

PCR screening and sequence analysis of iol clusters in Lactobacillus casei strains isolated from koumiss

The iol cluster (consisting of genes involved in myo-inositol utilization) was investigated in Lactobacillus casei strains isolated from koumiss. Ten strains were tested for the presence of iol cluster by PCR screening; three strains encoded this cluster. Full-sequencing procedure was conducted; the iol cluster was identical to that of L. casei BL23 (GenBank access. no. FM177140) except for an upstream transposase. The iol cluster is not a common feature for L. casei strains isolated from koumiss.

Identification of a Functional 2-keto-myo-Inositol Dehydratase Gene ofSinorhizobium frediiUSDA191 Required formyo-Inositol Utilization

Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently iolE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An iolE ortholog was cloned from USDA191. USDA191 iolE was expressed in Escherichia coli as a His(6)-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 iolE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis iolE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myo-inositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and iolE.

Characterization of myo -Inositol Utilization by Corynebacterium glutamicum : the Stimulon, Identification of Transporters, and Influence on l -Lysine Formation

Although numerous bacteria possess genes annotated iol in their genomes, there have been very few studies on the possibly associated myo-inositol metabolism and its significance for the cell. We found that Corynebacterium glutamicum utilizes myo-inositol as a carbon and energy source, enabling proliferation with a high maximum rate of 0.35 h-1. Whole-genome DNA microarray analysis revealed that 31 genes respond to myo-inositol utilization, with 21 of them being localized in two clusters of >14 kb. A set of genomic mutations and functional studies yielded the result that some genes in the two clusters are redundant, and only cluster I is necessary for catabolizing the polyol. There are three genes which encode carriers belonging to the major facilitator superfamily and which exhibit a >12-fold increased mRNA level on myo-inositol. As revealed by mutant characterizations, one carrier is not involved in myo-inositol uptake whereas the other two are active and can completely replace each other with apparent Kms for myo-inositol as a substrate of 0.20 mM and 0.45 mM, respectively. Interestingly, upon utilization of myo-inositol, the L-lysine yield is 0.10 mol/mol, as opposed to 0.30 mol/mol, with glucose as the substrate. This is probably not only due to myo-inositol metabolism alone since a mixture of 187 mM glucose and 17 mM myo-inositol, where the polyol only contributes 8% of the total carbon, reduced the L-lysine yield by 29%. Moreover, genome comparisons with other bacteria highlight the core genes required for growth on myo-inositol, whose metabolism is still weakly defined.