Genetic Examination and Mass Balance Analysis of Pyruvate/Amino Acid Oxidation Pathways in the Hyperthermophilic Archaeon Thermococcus kodakarensis

The present study investigated the simultaneous oxidation of pyruvate and amino acids during H₂-evolving growth of the hyperthermophilic archaeon Thermococcus kodakarensis. The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the ΔhyhBGSL strain) and the parent strain indicated that NADPH generated via H₂ uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in T. kodakarensis. No significant change was observed by deletion of pflDA, suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of ΔporDAB and ΔvorDAB strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H₂ yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of hyhBGSL and double deletion of hyhBGSL and vorDAB, respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH₃, suggesting the presence of unknown metabolisms in T. kodakarensis grown in the rich medium containing pyruvate.     

Identification of the main glutamine and glutamate transporters in Staphylococcus aureus and their impact on c-di-AMP production

A Staphylococcus aureus strain deleted for the c-di-AMP cyclase gene dacA is unable to survive in rich medium unless it acquires compensatory mutations. Previously identified mutations were in opuD, encoding the main glycine-betaine transporter, and alsT, encoding a predicted amino acid transporter. Here, we show that inactivation of OpuD restores the cell size of a dacA mutant to near wild-type (WT) size, while inactivation of AlsT does not. AlsT was identified as an efficient glutamine transporter, indicating that preventing glutamine uptake in rich medium rescues the growth of the S. aureus dacA mutant. In addition, GltS was identified as a glutamate transporter. By performing growth curves with WT, alsT and gltS mutant strains in defined medium supplemented with ammonium, glutamine or glutamate, we revealed that ammonium and glutamine, but not glutamate promote the growth of S. aureus. This suggests that besides ammonium also glutamine can serve as a nitrogen source under these conditions. Ammonium and uptake of glutamine via AlsT and hence likely a higher intracellular glutamine concentration inhibited c-di-AMP production, while glutamate uptake had no effect. These findings provide, besides the previously reported link between potassium and osmolyte uptake, a connection between nitrogen metabolism and c-di-AMP signalling in S. aureus.     

A novel KU70-mutant human leukemic cell line generated using CRISPR-Cas9 shows increased sensitivity to DSB inducing agents and reduced NHEJ activity

KU70 (XRCC6 gene in humans) is one of the proteins in the KU70-KU80 heterodimer which is the first component recruited to broken DNA ends during DNA double-strand break repair through nonhomologous end joining (NHEJ). Previous studies have shown that Ku70 deficient mouse cells are defective in NHEJ and V(D)J recombination. In contrast, heterozygous KU70 mutant human cell lines did not show any significant change in cell viability and sensitivity towards ionizing radiation. In this study, we used CRISPR-Cas9 technique to generate a KU70 mutant (heterozygous) human pre-B leukemic cell line (N6-KU70–2-DG). We observed that the N6-KU70–2-DG cells showed a prominent reduction in the expression of both KU70 mRNA and protein. The mutant cells showed reduced cell viability, increased sensitivity to DSB inducing agents such as ionizing radiation (IR) and etoposide, and increased number of unrepaired DSBs after exposure to IR. In addition, the mutant cells showed a reduction in the NHEJ activity and increased rate of microhomology mediated joining (MMEJ) activity. KU70 mutant cells also revealed enhanced level of senescence markers following irradiation. Thus, we report a novel KU70-mutant leukemic cell line (heterozygous) with reduced NHEJ, which is sensitive to DNA damaging agents, unlike the previously reported other KU heterozygous mutant cell lines.     

Exogenous Glutamate Inhibits the Root Growth and Increases the Glutamine Content in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Previously, our work with ginseng hairy root shows that the tissue of low-branching and slow-growing phenotype contains high level of glutamine. In order to check if the high glutamine concentration inhibits the root growth, we applied exogenous glutamine or glutamate into growth medium and check the root growth of Arabidopsis. While glutamine did not affect root growth, over 0.1 mM glutamate inhibited severe root growth. However, when the amino acid solution was adjusted to pH 5.7 and added into medium, Arabidopsis seedlings show normal growth pattern on medium containing glutamate or aspartate. These results demonstrated that inhibition of the root growth by high concentration of exogenous glutamate was a result of the low pH toxicity caused by acidic amino acid, although low concentration (0.05 mM) of glutamate has an inhibitory effect on the primary root growth. The application of exogenous glutamine or glutamate increases glutamine concentration within root tissue about 3- to 4-fold. However, concentration of glutamate is not significantly increased. The KO mutant on each of the Gln1_1, Gln1_2, or Glu2 gene was little effective on the root growth. These results indicate that high concentration of endogenous glutamine observed in root tissue does not affect root growth.     

Regulation of nitrogen fixation. Nitrogenase-derepressed mutants of Klebsiella pneumoniae

1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J. E., Prival, M. J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122–6128) for isolating histidase-constitutive mutants of a non-N₂-fixing bacterium.2. Nitrogenase levels of the new mutants in the presence of NH₄⁺ were as high as 100% of the nitrogenase activity detected in the absence of NH₄⁺.3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH₄⁺). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein.4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.     

Metabolic engineering of a diazotrophic bacterium improves ammonium release and biofertilization of plants and microalgae

The biological nitrogen fixation carried out by some Bacteria and Archaea is one of the most attractive alternatives to synthetic nitrogen fertilizers. However, with the exception of the symbiotic rhizobia-legumes system, progress towards a more extensive realization of this goal has been slow. In this study we manipulated the endogenous regulation of both nitrogen fixation and assimilation in the aerobic bacterium Azotobacter vinelandii. Substituting an exogenously inducible promoter for the native promoter of glutamine synthetase produced conditional lethal mutant strains unable to grow diazotrophically in the absence of the inducer. This mutant phenotype could be reverted in a double mutant strain bearing a deletion in the nifL gene that resulted in constitutive expression of nif genes and increased production of ammonium. Under GS non-inducing conditions both the single and the double mutant strains consistently released very high levels of ammonium (>20 mM) into the growth medium. The double mutant strain grew and excreted high levels of ammonium under a wider range of concentrations of the inducer than the single mutant strain. Induced mutant cells could be loaded with glutamine synthetase at different levels, which resulted in different patterns of extracellular ammonium accumulation afterwards. Inoculation of the engineered bacteria into a microalgal culture in the absence of sources of C and N other than N₂ and CO₂ from the air, resulted in a strong proliferation of microalgae that was suppressed upon addition of the inducer. Both single and double mutant strains also promoted growth of cucumber plants in the absence of added N-fertilizer, while this property was only marginal in the parental strain. This study provides a simple synthetic genetic circuit that might inspire engineering of optimized inoculants that efficiently channel N₂ from the air into crops.     

Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates

To study the relation between the number of hyphal tips and protein secretion during growth on a solid substrate, we have constructed two mutant strains of Aspergillus oryzae with increased hyphal branching. We have analysed hydrolytic enzyme activities during growth on wheat kernels (WK) of A. oryzae strains carrying the disrupted allele of the pclA gene encoding a secretion pathway specific (KEX2-like) endo-protease and the disrupted allele of the pg/pi-tp gene encoding a phosphatidylglycerol/phosphatidylinositol transfer protein. The biomass levels produced by the pclA and pg/pi-tp disrupted strains on wheat-based solid media were similar as found for the wild-type strain. However, the pclA disrupted strain showed much more compact colony morphology than the other two strains. Sporulation of the pclA and pg/pi-tp disrupted strains occurred, respectively, 2 days and 1 day later, compared to the wild type during fermentation on ground WK. During surface growth, microscopic analysis revealed that the hyphal growth unit length (L _hgu) of the pclA and pg/pi-tp disrupted strains was, on average, 50 and 74% of that of the wild-type strain. This implies that in both mutant strains, a higher branching frequency occurs than in the wild-type strain. Compared to the wild-type strain, the pclA and pg/pi-tp disrupted strains produced at least 50% more amylase, at least 100% more glucoamylase and at least 90% more protease activity levels after growth on WK. These results support the hypothesis that branching mutants with an increased branching frequency can improve the solid state fermentation process.     

Comparison of Assimilatory Organic Nitrogen, Sulfur, and Carbon Sources for Growth of Methanobacterium Species

Experiments document the ability of two species of autotrophic methanogens to assimilate and utilize organic substrates as the nutrient sulfur or nitrogen source and as a carbon source during growth on H₂-CO₂. Methanobacterium thermoautotrophicum strain ΔH and the mesophilic species Methanobacterium sp. strain Ivanov grew with glutamine as the nitrogen source or cysteine as the sulfur source. M. thermoautotrophicum also utilized urea as the nitrogen source and as a carbon precursor for methane and cell synthesis. Methanobacterium sp. strain Ivanov grew with methionine as the sulfur source. The growth rate of two different Methanobacterium species was lower on an organic N or S source than on ammonium or sulfide. ³⁵S and ¹⁴C tracer studies demonstrated that amino acid or urea assimilation correlated with time and amount of growth. The rate of [³⁵S]cysteine incorporation was similar in strain ΔH (34 nmol h⁻¹ mg of cells⁻¹) and strain Ivanov (23 nmol h⁻¹ mg of cells⁻¹). However, the rate of [¹⁴C]acetate incorporation was dramatically different (17 versus 208 nmol h⁻¹ mg of cells⁻¹ in strains ΔH and Ivanov, respectively). [¹⁴C]acetate accounted for 1.3 and 21.2% of the total cell carbon synthesized by strains ΔH and Ivanov, respectively. Amino acids and urea were mainly assimilated into the cell protein fraction, but accounted for less than 2.0% of the total cell carbon synthesized. The data suggest that a biochemical-genetic approach to understanding cell carbon synthesis in methanogens is feasible; mutants that are auxotrophic for either acetate, glutamine, cysteine, or methionine are suggested as future targets for genetic studies.     

Cloning,Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.     

Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium

Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections.     

Antioxidant defense system responses and role of nitrate reductase in the redox balance maintenance in Bradyrhizobium japonicum strains exposed to cadmium

In this work, we evaluated the effects of cadmium (Cd) on the antioxidant defense system responses and the role of nitrate reductase (NR) in the redox balance maintenance in Bradyrhizobium japonicum strains. For that, B. japonicum USDA110 and its NR defective mutant strain (GRPA1) were used. Results showed that the addition of 10 μM Cd did not modify the aerobic growth of the wild type strain while the mutant strain was strongly affected. Anaerobic growth revealed that only the parental strain was able to grow under this condition. Cd reduced drastically the NR activity in B. japonicum USDA110 and increased lipid peroxide content in both strains. Cd decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in B. japonicum USDA110 although, a significant increased was observed in the mutant GRPA1. GSH-related enzymes were induced by Cd, being more evident the increase in the mutant strain. This different behavior observed between strains suggests that NR enzyme plays an important role in the redox balance maintenance in B. japonicum USDA 110 exposed to Cd.     

Glycolate metabolism is under nitrogen control in chlorella

The utilization of nitrate and ammonia as nitrogen sources had different effects on the metabolism of glycolate in Cholorella sorokiniana. During photolithotrophic growth with nitrate as nitrogen source, glycolate was metabolized via the glycine-serine pathway. Ammonia, produced as a result of glycolate metabolism, was reassimilated by glutamine synthetase. Two isoforms of this enzyme were present at different relative abundance in C. sorokiniana wild type and in a mutant with an increased capacity for the metabolism of glycolate (strain OR).

During photolithotrophic growth in the presence of ammonia as sole nitrogen source, several lines of evidence indicated that glycolate was metabolized to malate, pyruvate, tricarboxylic acid cycle intermediates and related amino acids in C. sorokiniana wild-type cells. Malate synthase was induced and glycine decarboxylase and serine-glyoxylate aminotransferase were repressed in cells grown with ammonia. An inverse correlation was observed between aminating NADPH-glutamate dehydrogenase and the in vivo glycine decarboxylation rate.

     

phlF− mutant of Pseudomonas fluorescens J2 improved 2,4-DAPG biosynthesis and biocontrol efficacy against tomato bacterial wilt

Pseudomonas fluorescens J2 can produce 2,4-diacetylphloroglucinol (2,4-DAPG) as the main antibiotic compound and effectively inhibits the wilt pathogens Ralstonia solanacearum and Fusarium oxysporum. The phlF which negatively regulates the 2,4-DAPG synthesis in strain J2 was disrupted by homologous recombination to construct a mutant strain J2-phlF⁻. The mutant J2-phlF⁻ produced much more 2,4-DAPG and showed higher inhibitory effect on R. solanacearum than the wild type strain J2 in vitro. The mutant J2-phlF⁻ also showed more colonization of tomato roots and higher inhibition to R. solanacearum in soil than wild type strain J2. The biocontrol efficiency of mutant J2-phlF⁻ was higher against tomato bacterial wilt than wild type strain J2, but the differences were not significant. However, the application of both strains with organic fertilizer improved the colonization and biocontrol efficiency against tomato bacterial wilt and mutant strain J2-phlF⁻ showed higher biocontrol efficiency against tomato bacterial wilt than wild type strain J2. Both strains, J2 and J2-phlF⁻, could also promote the growth of tomato plants.     

Elevated Growth of Saccharomyces cerevisiae ATH1 Null Mutants on Glucose Is an Artifact of Nonmatching Auxotrophies of Mutant and Reference Strains

Yeast strains disrupted for ATH1, which encodes vacuolar acid trehalase, have been reported to grow to higher cell densities than reference strains. We showed that the increase in cell density is due to the URA3 gene introduced as a part of the disruption and concluded that the misinterpretation is a result of not using a control strain with matching auxotrophic markers.