Amino Acids as Nitrogen Sources for the Growth of Euglena gracilis and Astasia longa

SYNOPSIS. Euglena gracilis (bacillaris variety, strain SM-L1, streptomycin-bleached) used the following amino adds (10⁻³ M) as sole nitrogen source for growth on a defined medium: glycine, alanine, valine, leucine, isoleucine, serine, threonine, and glutamic acid. Aspartic acid was used at 10⁻² M. Glutamine and asparagine were used at 10⁻³ M and were better N sources than their parent dicarboxylic amino acids. Not used as sole N source for growth were phenylalanine, tyrosine, tryptophan, cysteine, cystine, methionine, proline, hydroxyproline, histidine, arginine, lysine, and taurine. Astasia longa (Jahn strain) was more restricted than Euglena and used only asparagine and glutamine as N sources for growth.     

Utilization of Amino Acids as a Sole Source of Nitrogen by Obligate Chemolithoautotroph Thiobacillus ferrooxidans

An obligate chemolithoautotroph, Thiobacillus ferrooxidans API 9–3, could utilize amino acids, other than glycine, methionine and phenylalanine, as a sole source of nitrogen. However, both the growth rate and growth yield were lower than those in Fe²⁺-NH4 -salts medium, suggesting that the ammonium ion was a superior nitrogen source for the strain compared to amino acids. Methionine and phenylalanine strongly inhibited the cell growth on Fe²⁺-NH4-salts medium at 10 mm. [¹⁴C]Glycine could not be taken up into the cells, and this meant the strain could not use glycine as a sole source of nitrogen. The uptake of [¹⁴C]leucine into the cells was dependent on the presence of Fe^{2 +}. When the strain was cultured on Fe^{2 +} - leucine (lOmm)-salts medium lacking an inorganic nitrogen source for 5 days at 30°C, 83.5% and 16.5% of the cellular carbon were derived from carbon dioxide and leucine, respectively, indicating that carbon dioxide was a superior carbon source for the bacterium compared to leucine. The ammonium ion did not inhibit the utilization of leucine for cellular carbon. Leucine uptake was markedly inhibited by inhibitors of protein synthesis, such as chloramphenicol (94.3% at 1 mm), streptomycin (57.2% at 5mm) and rifampin (77.2% at 0.1 mm), respectively. Carbon dioxide uptake was also completely inhibited by chloramphenicol at 4mm. These results suggest that the transport of both amino acids and carbon dioxide into the cells was dependent on protein synthesis.     

Nitrogen Metabolism in Plant Cell Suspension Cultures: II. Role of Organic Acids during Growth on Ammonia

Tobacco cells (Nicotiana tabacum) are capable of growth on ammonia as a sole nitrogen source only when succinate, malate, fumarate, citrate, α-ketoglutarate, glutamate, or pyruvate is added to the growth medium. A ratio between the molar concentrations of ammonia to succinate (as a complementary organic acid) in the growth medium of 1.5 was optimal. Succinate had no effect on the rate of uptake of ammonia from the medium into the cells although it did affect the intracellular concentration of ammonia. However, the changes were not sufficient to explain inhibition of growth as being due to ammonia toxicity. The radioactivity from ¹⁴C-succinate was incorporated into malate, glutamate, and aspartate within 2 minutes.     

Experimental Studies on the Formation of Volatile Nitrogen Compounds Induced by Pseudomonas Fragi in A Synthetic Medium with Amino Acids as Source of Nitrogen

The influence of the growth of Pseudomonas fragi strain F 111 was studied in a synthetic medium. It was shown that volatile nitrogen compounds were rapidly liberated as a result of the decomposition at 5°C of arginine and histidine. A later and slower increase of TVN was observed in media, which contained lysine or urea. From the other 7 amino acids which were included in the test, no increase of TVN was observed to be caused by the strain F 11. It was shown that within the range of 0.05 to 0.8 % histidine added to the basic salt solution the amount of TVN liberated was correlated to the amount of histidine included in the media. At the TVN maximum approx. 50 % of the amino acid nitrogen of the medium was recovered as TVN. It was further shown that the liberation of TVN was correlated to the added amount of arginine and histidine included in the growth medium. The presence of lysine also influenced on the TVN maximum which was reached. In the synthetic medium to which arginine or histidine was added the growth of strain F 111 was stimulated by the presence of glucose. The acids produced by the oxidative breakdown of glucose were neutralized partly by the volatile bases produced in the decomposition of amino acids. The ability of the Pseudomonas fragi to grow in anaerobic conditions and to produce enzymes which could decompose histidine was studied. It was concluded that the anaerobic breakdown of histidine was similar to the anaerobic breakdown of arginine reported by other authors.     

Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

By taking advantage of MazF, an ACA codon-specific mRNA interferase, Escherichia coli cells can be converted into a bioreactor producing only a single protein of interest by using an ACA-less mRNA for the protein. In this single-protein production (SPP) system, we engineered MazF by replacing two tryptophan residues in positions 14 and 83 with Phe (W14F) and Leu (W83L), respectively. Upon the addition of an inducer (IPTG [isopropyl-β-d-thiogalactopyranoside]), the mutated MazF [MazF(ΔW)] can still be produced even in the absence of tryptophan in the medium by using a Trp auxotroph, while a target protein having Trp residues cannot be produced. However, at 3 h after the addition of IPTG, the addition of tryptophan to the medium exclusively induces production of the target protein at a high level. A similar SPP system was also constructed with the use of a His-less protein [MazF(ΔH)] and a His auxotroph. Using these dual-induction systems, isotopic enrichments of ¹³C, ¹⁵N, and ²H were highly improved by almost complete suppression of the production of the unlabeled target protein. In both systems, isotopic incorporation reached more than 98% labeling efficiency, significantly reducing the background attributable to the unlabeled target protein.The condensed single-protein production (cSPP) system was developed based on the endoribonuclease activity of an Escherichia coli toxin called MazF, which selectively cleaves cellular mRNAs at the ACA codon sequence (14, 16). Upon induction of MazF, protein synthesis is completely inhibited, and as a result, cell growth is also completely arrested. However, MazF-induced cells are in a quasidormant state, as they are metabolically fully active, producing ATP, amino acids, and nucleotides. Most significantly, in the quasidormant cells, machineries for protein synthesis and mRNA production are also fully functional, and therefore the MazF-induced quasidormant cells are still capable of synthesizing a protein of interest without producing any other cellular proteins, if the mRNA for the protein is engineered to have no ACA sequences (14). This system is thus termed the single-protein production (SPP) system. One of the most remarkable advantages of the SPP system is that the cell culture can be highly condensed without affecting protein yields (7, 12, 13). Using this cSPP system, one can achieve a cost savings of as much as 97.5% by condensing a culture 40-fold. This is particularly valuable when highly expensive isotopes or isotope-labeled compounds, such as amino acids and glucose, are used for the preparation of protein samples for structural study by nuclear magnetic resonance (NMR) spectroscopy. Furthermore, by use of the cSPP system, amino acid analogues or D₂O, which is toxic in conventional protein production systems, reducing protein yields, is not toxic, hardly affecting the final protein yields. However, one drawback of the current cSPP system is the use of IPTG (isopropyl-β-d-thiogalactopyranoside) as an inducer for both MazF and a target protein, such that the target protein is also produced at the same time as MazF. Since isotopes or isotope-labeled compounds are added 2 to 3 h after the addition of IPTG to avoid their incorporation into cellular proteins, non-isotope-labeled target protein is also produced during this preincubation period, resulting in a higher background of unlabeled target protein, which may be as high as 20% of the final yield of the target protein produced (10).The combination of both tetracycline- and IPTG-inducible systems was employed to separate the inductions of target protein and MazF, respectively (10). The main disadvantage of this system is that the expression level of target protein critically depends upon tetracycline concentration. The amounts of tetracycline being added to the cells for induction of target protein significantly affect the level of target protein synthesis, especially in the condensed SPP system. Therefore, a highly precise and accurate optimization of the tetracycline level is required for consistency in the expression of target proteins. To circumvent this problem, we developed a novel dual-induction system using amino acid auxotrophs. It has been shown previously that E. coli cells from a histidine (His) auxotroph can still produce a protein containing no His residues in the absence of histidine in the medium without producing any other cellular proteins (5). Therefore, it is assumed that even if both MazF and a target protein containing His residues or tryptophan (Trp) residues are coinduced by IPTG, only His-less MazF or Trp-less MazF would be produced in the absence of histidine or tryptophan by using a His auxotroph or a Trp auxotroph, respectively. In this fashion, the target protein may be induced by the addition of histidine or tryptophan in the medium a few hours after His-less or Trp-less MazF induction so that background production of the target protein may be avoided. Furthermore, this new SPP system could also be useful in studies involving specific replacement of amino acids with their analogues.In the present study, we employed two amino acid auxotrophs, of Trp and His, to construct the dual-induction SPP system. For this purpose, both Trp-less proteins and His-less MazF proteins [MazF(ΔW) and MazF(ΔH), respectively] were generated to create the new SPP system. Using this new system, we tested a number of proteins, such as (i) E. coli EnvZB, which is the ATP-binding domain of the histidine kinase EnvZ (161 residues) (15), (ii) E. coli CspA, which is the major cold shock protein (70 residues) (3), (iii) E. coli YaiZ, which is a plasma membrane protein (80 residues) (7), (iv) the antiapoptotic adenoviral protein E1B19K150 (150 residues) (2), (v) human granulocyte colony-stimulating factor (GCSF; 175 residues) (4), and (vi) human calmodulin (CaM), which is a calcium binding protein (148 residues) (6). It was found that isotope incorporation into these proteins was very tightly regulated so that the background due to the unlabeled target protein was significantly reduced. We also developed a pCold(W) system in which a Trp tag can be added to the N-terminal part of a protein so that the dual-induction expression can still be applied for Trp-less proteins.     

Acidaminococcus gen. n., Acidaminococcus fermentans sp. n., Anaerobic Gram-negative Diplococci Using Amino Acids as the Sole Energy Source for Growth

Acidaminococcus gen. n. and the type species Acidaminococcus fermentans sp. n. were described. Amino acids, of which glutamic acid is the most important, could serve as the sole energy source for growth. Acetic and butyric acids and CO(2) were produced; propionic acid and hydrogen were not produced. Amino acid media supporting growth and the amino acid and vitamin requirements were described. Glucose was frequently not fermented or was weakly catabolized. Derivative products from glucose autoclaved in media, but not glucose itself, stimulated or were required for growth in amino acid media. A wide range of polyols and carbohydrates were not attacked. Lactate, fumarate, malate, succinate, citrate, and pyruvate were not used as energy sources for growth. Pyruvate completely suppressed growth. Cytochrome oxidase and benzidine reactions were negative; catalase, indole, acetyl methyl carbinol, and H(2)S were not produced; nitrate and sulfonthalein indicators were not reduced; ammonia was produced; gelatin liquefaction was negative or slow and partial; vancomycin (7.5 mug/ml) was resisted. Acidaminococcus was different from Veillonella in morphology, serology, nutrition, utilization of substrates, and accumulation of products in media supporting growth; Acidaminococcus resembled Peptococcus in utilization of glutamic acid and accumulation of similar products, but the two genera differed in morphology, gram reaction, serology, guanine plus cytosine content of deoxyribonucleic acid, and nutrition.     

Characterization of a Mutant of Chlamydomonas reinhardtii That Uses L-Methionine-S-Sulfoximine and Phosphinothricin as Nitrogen Sources for Growth

A strain of Chlamydomonas reinhardtii, named ARF-1, which grows with the glutamine synthetase (GS) inhibitor L-methionine-S-sulfoximine (MSX), has been isolated and characterized. Mutant ARF-1 is affected at a single and dominant gene, tentatively assigned to the allele msr-1-2. Neither the uptake of ammonia nor the two GS isoenzyme activities of the mutant were affected by MSX in vivo. GS activities, however, were fully abolished in vitro, thus suggesting that neither GS isoform was an altered enzyme resistant to the inhibitor. Resistance to MSX does not seem to be due to either a defect in a permease responsible for the transport of MSX or over-expression of GS activity, nor did we find an alternative enzymatic pathway for the assimilation of ammonium. Resistance was independent of the nitrogen source used and was strongly enhanced by the addition of acetate. Unlike the parental strain, mutant ARF-1 can degrade and utilize MSX as the sole nitrogen source for growth, which could account for the observed resistance. Thus, this mutant can be classified as a novel type of MSX-resistant mutant. This mutant can also use phosphinothricin, methionine sulfone, or methionine sulfoxide as the sole sources of nitrogen. This capability cosegregated in the genetic crosses and was also observed in all the diploids isolated. An MSX/[alpha]-ketoglutarate aminotransferase activity, not present in the parental strain 305, was detected in mutant ARF-1 cells. Therefore, we propose that the locus msr-1-2 either codes for this transaminase activity or its product gene is necessary to express this transaminase activity.     

Ammonia Production by Ruminal Microorganisms and Enumeration, Isolation, and Characterization of Bacteria Capable of Growth on Peptides and Amino Acids from the Sheep Rumen

Excessive NH₃ production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH₃ production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH₃ production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH₃ production from Trypticase varied from 1.8 to 19.7 nmol mg of protein⁻¹ min⁻¹ depending on the substrate, its concentration, and the method used. Monensin (5 μM) inhibited NH₃ production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (P = 0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 μM monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group III, similar to Atopobium minutum), produced NH₃ at >250 nmol min⁻¹ mg of protein⁻¹, depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp. Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra). Group VI was 95% similar to Acidaminococcus fermentans. Growth of the Atopobium- and Desulfomonas-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH₃ production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.     

Characterization of the Fungal Gibberellin Desaturase as a 2-Oxoglutarate-Dependent Dioxygenase and Its Utilization for Enhancing Plant Growth

The biosynthesis of gibberellic acid (GA₃) by the fungus Fusarium fujikuroi is catalyzed by seven enzymes encoded in a gene cluster. While four of these enzymes are characterized as cytochrome P450 monooxygenases, the nature of a fifth oxidase, GA₄ desaturase (DES), is unknown. DES converts GA₄ to GA₇ by the formation of a carbon-1,2 double bond in the penultimate step of the pathway. Here, we show by expression of the des complementary DNA in Escherichia coli that DES has the characteristics of a 2-oxoglutarate-dependent dioxygenase. Although it has low amino acid sequence homology with known 2-oxoglutarate-dependent dioxygenases, putative iron- and 2-oxoglutarate-binding residues, typical of such enzymes, are apparent in its primary sequence. A survey of sequence databases revealed that homologs of DES are widespread in the ascomycetes, although in most cases the homologs must participate in non-gibberellin (GA) pathways. Expression of des from the cauliflower mosaic virus 35S promoter in the plant species Solanum nigrum, Solanum dulcamara, and Nicotiana sylvestris resulted in substantial growth stimulation, with a 3-fold increase in height in S. dulcamara compared with controls. In S. nigrum, the height increase was accompanied by a 20-fold higher concentration of GA₃ in the growing shoots than in controls, although GA₁ content was reduced. Expression of des was also shown to partially restore growth in plants dwarfed by ectopic expression of a GA 2-oxidase (GA-deactivating) gene, consistent with GA₃ being protected from 2-oxidation. Thus, des has the potential to enable substantial growth increases, with practical implications, for example, in biomass production.The GAs are a class of diterpenoid hormones that regulate many aspects of growth and development in plants, including stem extension (Thomas and Hedden, 2006). Despite being ubiquitous in higher plants, they were first discovered as secondary metabolites of the plant pathogenic fungus Gibberella fujikuroi, the causative agent of the bakanae disease of rice (Oryza sativa; Phinney, 1983). This fungus is now known to comprise a group of reproductively isolated species or mating populations, the rice pathogen belonging to mating group C and assigned the name Fusarium fujikuroi (Leslie and Summerell, 2006; Kvas et al., 2009). Details of the GA biosynthetic pathways in both plants and the fungus are known in considerable detail and have revealed that, although they give rise to common metabolites, the pathways utilize different types of enzymes for several steps and appear to have evolved independently (Hedden et al., 2001; Bömke and Tudzynski, 2009).Higher plants differ from the GA-producing fungi by possessing the means for GA inactivation, which is necessary to allow precise regulation of their GA concentration. In contrast, the fungi are not dependent on GAs for their development but produce and secrete large quantities of the compounds to modify the behavior of their hosts. It has been shown that GAs interfere with plant defense by suppressing jasmonate signaling and may thus compromise the host’s ability to evade fungal infection (Navarro et al., 2008; Hou et al., 2010). An apparent ubiquitous inactivation mechanism involves 2β-hydroxylation (Thomas et al., 1999), the effect of which reduces binding of the GA within the active site of the GID1 receptor (Murase et al., 2008). However, GAs such as GA₃ and GA₅, which are unsaturated on C-2, are protected from 2β-hydroxylation and, as a consequence, would be expected to be turned over more slowly than their saturated analogs (King et al., 2008). In accordance with the requirement to regulate GA content, shoots of higher plants contain relatively little 1,2-unsaturated GAs, although developing seeds of some species contain substantial quantities. They are produced in a two-step reaction via a 2,3-dehydro intermediate, which is then hydroxylated on C-3β with rearrangement of the double bond from C-2,3 to C-1,2 (Albone et al., 1990). The reactions are catalyzed by GA 3-oxidase-type enzymes, with a single enzyme catalyzing both reactions in cereal shoots to produce GA₃ from GA₂₀ as a minor by-product of GA₁ biosynthesis (Itoh et al., 2001; Appleford et al., 2006; Fig. 1). In developing seeds of Marah macrocarpus, which contain high concentrations of the 1,2-unsaturated GA, GA₇, the formation of this GA from GA₉ requires the activities of two functionally different GA 3-oxidases acting sequentially (Ward et al., 2010). However, direct formation of GA₇ from GA₄, such as occurs in F. fujikuroi, is not usual in higher plants.Open in a separate windowFigure 1.The GA biosynthetic pathway in plants and F. fujikuroi. The fungal pathway to GA₃ is indicated by the thick gray arrow. DES catalyzes the conversion of GA₄ to GA₇.While the late stages of GA biosynthesis in higher plants, including desaturation when it occurs and 2β-hydroxylation, are catalyzed by 2-oxoglutarate-dependent dioxygenases (ODDs), these enzymes have not been shown to be involved in GA biosynthesis in fungi. F. fujikuroi contains a cluster of seven genes for GA biosynthesis, including a geranylgeranyl diphosphate synthase that is specific to the GA pathway and a bifunctional terpene cyclase that converts geranylgeranyl diphosphate to ent-kaurene in two steps via ent-copalyl diphosphate (for review, see Hedden et al. [2001]; Bömke and Tudzynski [2009]). The formation of GA₃ from ent-kaurene requires the activity of five oxidases (Fig. 1), four of which are cytochrome P450 monooxygenases: P450-4 (ent-kaurene oxidase) oxidizes ent-kaurene to ent-kaurenoic acid (Tudzynski et al., 2001), which is converted to GA₁₄ by P450-1 (GA₁₄ synthase; Rojas et al., 2001); P450-2 functions as a GA 20-oxidase, converting GA₁₄ to GA₄ (Tudzynski et al., 2002), while, in the final step of the pathway, P450-3 13-hydroxylates GA₇ to form GA₁₃ (Tudzynski et al., 2003). However, the nature of the desaturase (DES), which converts GA₄ to GA₇ (Fig. 1), is unknown. When first described, it was found to have closest, albeit weak, homology to a component of the 7α-cephem-methoxylase from Nocardia lactamdurans, giving little indication of its mechanism (Tudzynski et al., 2003). Besides F. fujikuroi, several other ascomycetes, including Sphaceloma manihoticola (Bömke et al., 2008), Phaeosphaeria spp. (Kawaide, 2006), and two other species of the G. fujikuroi species complex, Fusarium konzum (Malonek et al., 2005) and Fusarium sacchari (Troncoso et al., 2010), have been shown to synthesize GAs, although the first two species do not carry out the desaturation step and do not contain a desaturase gene.The promotion of vegetative growth offers potential benefits, for example, in biomass production (Demura and Ye, 2010). In order to test the hypothesis that growth could be stimulated by increasing the shoot concentrations of GAs that are unsaturated on C-2 and therefore resistant to 2β-hydroxylation, we introduced the fungal desaturase gene into plants. The feasibility of this approach was reinforced by the demonstration that DES has the characteristics of an ODD and, therefore, would be expected to function in higher plants.     

Geostrategic Supply Risk and Economic Importance as Drivers for Implementation of Industrial Ecology Measures in a Nitrogen Fertilizer Production Company

Among other concerns, safeguarding the supply chains of raw materials is an important task for industrial companies. Therefore, not surprisingly, the number of scientific publications concerning the evaluation of resource criticality has increased in recent years. However, it was noticed that currently published methodologies are too complex to be applied by industrial companies on a daily basis. For this reason, the need to develop a methodology that would allow not only assessing resource criticality, but could also be integrated into widely applied methodological frameworks as an additional driver to improve resource efficiency was identified. Geostrategic supply risk and economic importance were chosen as key indicators to analyze and assess relative resource criticality. The developed methodology was field tested by applying it to a resource‐intensive nitrogen fertilizer production company. Five scenarios for resource efficiency improvements, consisting of cleaner production and industrial symbiosis measures, were investigated. If all the proposed measures were implemented, consumption of natural gas would decrease by 3.552 million cubic meters per year (0.3% of the total consumption). However, not all identified measures contribute to a reduction of the overall criticality of resources for the production company. Nevertheless, the integration of criticality assessments into the widely applied methodologies for development and implementation of resource efficiency innovations is a valuable addition and should be included in the analysis for sustainable innovations and development.     

Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as “acidified”). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This “switch-like” relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell''s pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways.     

Synergistic Effects of Metabolically Related Amino Acids on the Growth of a Multicellular Plant: II. Studies of 14C-Amino Acid Incorporation 1,2

The synergistic inhibition of the growth of Marchantia polymorpha gemmalings by lysine and threonine and its prevention by methionine has been investigated utilizing (14)C-labeled amino acids. Experiments involving the uptake of (14)C-lysine or (14)C-threonine in the presence or absence of methionine indicated that the synergistic growth effects were not a result of altered amino acid uptake. These data, as well as direct chemical analysis, indicated that growth inhibition was correlated with an inhibition of protein synthesis. Experiments utilizing (14)C-aspartic acid revealed that the presence of lysine and threonine resulted in increased (14)CO(2) production and an accumulation of soluble (14)C-aspartic acid and labeled ninhydrin-positive compounds. These metabolic alterations were prevented when methionine was also included in the growth media. A model depicting a sequence of events which involve the interaction of regulatory mechanisms is suggested to account for the effects of specific amino acids on plant growth.     

Characterization of a Planctomycetal Organelle: a Novel Bacterial Microcompartment for the Aerobic Degradation of Plant Saccharides

Bacterial microcompartments (BMCs) are organelles that encapsulate functionally linked enzymes within a proteinaceous shell. The prototypical example is the carboxysome, which functions in carbon fixation in cyanobacteria and some chemoautotrophs. It is increasingly apparent that diverse heterotrophic bacteria contain BMCs that are involved in catabolic reactions, and many of the BMCs are predicted to have novel functions. However, most of these putative organelles have not been experimentally characterized. In this study, we sought to discover the function of a conserved BMC gene cluster encoded in the majority of the sequenced planctomycete genomes. This BMC is especially notable for its relatively simple genetic composition, its remote phylogenetic position relative to characterized BMCs, and its apparent exclusivity to the enigmatic Verrucomicrobia and Planctomycetes. Members of the phylum Planctomycetes are known for their morphological dissimilarity to the rest of the bacterial domain: internal membranes, reproduction by budding, and lack of peptidoglycan. As a result, they are ripe for many discoveries, but currently the tools for genetic studies are very limited. We expanded the genetic toolbox for the planctomycetes and generated directed gene knockouts of BMC-related genes in Planctomyces limnophilus. A metabolic activity screen revealed that BMC gene products are involved in the degradation of a number of plant and algal cell wall sugars. Among these sugars, we confirmed that BMCs are formed and required for growth on l-fucose and l-rhamnose. Our results shed light on the functional diversity of BMCs as well as their ecological role in the planctomycetes, which are commonly associated with algae.     

Gene Cloning and Characterization of a Novel Highly Organic Solvent Tolerant Lipase from Proteus sp. SW1 and its Application for Biodiesel Production

Proteus sp. SW1 was found to produce an extracellular solvent tolerant lipase. The gene, lipA, encoding a bacterial lipase, was cloned from total Proteus sp. SW1 DNA. lipA was predicted to encode a 287 amino acid protein of 31.2?kDa belonging to the Group I proteobacterial lipases. Purified His-tagged LipA exhibited optimal activity at pH 10.0 and 55??C. It was highly stable in organic solvents retaining 112% of its activity in 100% isopropanol after 24?h, and exhibited more than 200% of its initial activity upon exposure to 60% acetone, ethanol, and hexane for 18?h. Biodiesel synthesis reactions, using a single step addition of 13% an acyl acceptor ethanol, showed that LipA was highly effective at converting palm oil into biodiesel.     

Amino Acids, Iron, and Growth Rate as Key Factors Influencing Production of the Pseudomonas Putida BTP1 Benzylamine Derivative Involved in Systemic Resistance Induction in Different Plants

The biological control bacterium Pseudomonas putida BTP1 exerts its protective effect mostly by inducing an enhanced state of resistance in the host plant against pathogen attack [induced systemic resistance (ISR)]. We previously reported that a specific compound derived from benzylamine may be involved in the elicitation of the ISR phenomenon by this Pseudomonas strain. In this article, we provide further information about the N,N-dimethyl-N-tetradecyl-N-benzylammonium structure of this determinant for ISR and show that the benzylamine moiety may be important for perception of the molecule by root cells of different plant species. We also investigated some regulatory aspects of elicitor production with the global aim to better understand how in situ expression of these ISR elicitors can be modulated by physiological and environmental factors. The biosynthesis is clearly related to secondary metabolism, and chemostat experiments showed that the molecule is more efficiently produced at low cell growth rate. Interestingly, the presence of free amino acids in the environment is necessary for optimal production, and a specific positive effect of phenylalanine was evidenced in pulsed continuous cultures. The influence of other abiotic factors, such as mineral content, oxygen concentration, or pH, on elicitor production is also reported and discussed with respect to the specific conditions that the producing strain undergoes in the rhizosphere environment.     

Isolation and Characterization of a Mutant of Arthrobacter sp. Strain GLP-1 Which Utilizes the Herbicide Glyphosate as Its Sole Source of Phosphorus and Nitrogen

Arthrobacter sp. strain GLP-1, grown on glucose as a carbon source, utilizes the herbicide glyphosate [N-(phosphonomethyl)glycine] as its sole source of phosphorus as well as its sole source of nitrogen. The mutant strain GLP-1/Nit-1 utilizes glyphosate as its sole source of nitrogen as well. In strain GLP-1, P_i was a potent competitive inhibitor of glyphosate uptake (K_i, 24 μM), while the affinity of P_i for the uptake system of strain GLP-1/Nit-1 was reduced by 2 orders of magnitude (K_i, 2.3 mM). It is concluded that the inability of strain GLP-1 to utilize glyphosate as a source of nitrogen is due to the stringent control of glyphosate uptake by excess phosphate released during the degradation of the herbicide.