Amino Acid-rich Medium Research Articles

New insight into the molecular etiopathogenesis of konzo: Cyanate could be a plausible neurotoxin contributing to konzo, contrary to thiocyanate

IntroductionChronic cassava-derived cyanide poisoning is associated with the appearance of konzo, a tropical spastic paraparesis due to selective upper motor neuron damage. Whether the disease is caused by a direct action of cyanide or its metabolites is still an open question. This preliminary study assessed the neurotoxic effects of thiocyanate (SCN) and cyanate (OCN), two cyanide metabolites hypothesized to be plausible toxic agents in konzo. MethodsCultured mouse neuroblastoma (Neuro-2A) and human neuroblastoma (SH-SY5Y) cell lines were incubated (24, 48, and 72hours) in sodium OCN or sodium SCN in a disease-relevant concentration range. Cell viability, caspase (3, 8, and 9) activities, and reactive oxygen species (ROS) generation were evaluated using appropriate assay kits. Additionally, electrophysiological responses induced by OCN and SCN in primary spinal cord neurons (from Sprague Dawley rats) were assessed by whole-cell patch-clamp techniques. ResultsBoth OCN and SCN were toxic in a dose-dependent way, even if SCN toxicity appeared at very high concentrations (30mM, corresponding to more than 100-fold higher than normal plasmatic levels), contrary to OCN (0.3-3mM). OCN was markedly more toxic in a poor culture medium (MEM; IC50 = 3.2mM) compared to a glucose- and amino acid-rich medium (DMEM; IC50=7.6mM). OCN treatment increased the ROS generation by 8.9 folds, as well as the Caspase-3, Caspase-8, and Caspase-9 activities by 3.2, 2.5, and 2.6 folds, respectively. Finally, OCN (and SCN to a lesser extent) induced depolarizing currents in primary spinal cord neurons, through an activation of ionotropic glutamate receptors. ConclusionOur results suggest OCN as the most plausible neurotoxic agent involved in konzo, while SCN toxicity could be questioned at such high concentrations. Also, they support apoptosis, oxidative stress, and excitotoxicity as probable mechanisms of OCN neurotoxicity.

Elevated energy costs of biomass production in mitochondrial respiration-deficient Saccharomyces cerevisia.

Microbial growth requires energy for maintaining the existing cells and producing components for the new ones. Microbes therefore invest a considerable amount of their resources into proteins needed for energy harvesting. Growth in different environments is associated with different energy demands for growth of yeast Saccharomyces cerevisiae, although the cross-condition differences remain poorly characterized. Furthermore, a direct comparison of the energy costs for the biosynthesis of the new biomass across conditions is not feasible experimentally; computational models, on the contrary, allow comparing the optimal metabolic strategies and quantify the respective costs of energy and nutrients. Thus in this study, we used a resource allocation model of S. cerevisiae to compare the optimal metabolic strategies between different conditions. We found that S. cerevisiae with respiratory-impaired mitochondria required additional energetic investments for growth, while growth on amino acid-rich media was not affected. Amino acid supplementation in anaerobic conditions also was predicted to rescue the growth reduction in mitochondrial respiratory shuttle-deficient mutants of S. cerevisiae. Collectively, these results point to elevated costs of resolving the redox imbalance caused by de novo biosynthesis of amino acids in mitochondria. To sum up, our study provides an example of how resource allocation modeling can be used to address and suggest explanations to open questions in microbial physiology.

Towards an advanced testing strategy for genotoxicity using image-based 2D and 3D HepG2 DNA damage response fluorescent protein reporters.

In vitro assessment of mutagenicity is an essential component in the chemical risk assessment. Given the diverse modes of action by which chemicals can induce DNA damage, it is essential that these in vitro assays are carefully evaluated for their possibilities and limitations. In this study, we used a fluorescent protein HepG2 reporter test system in combination with high content imaging. To measure induction of the DNA damage response (DDR), we used three different green fluorescent protein reporters for p53 pathway activation. These allowed for accurate quantification of p53, p21 and BTG2 (BTG anti-proliferation factor 2) protein expression and cell viability parameters at a single cell or spheroid resolution. The reporter lines were cultured as 2D monolayers and as 3D spheroids. Furthermore, liver maturity and cytochrome P450 enzyme expression were increased by culturing in an amino acid-rich (AAGLY) medium. We found that culture conditions that support a sustained proliferative state (2D culturing with normal DMEM medium) give superior sensitivity when genotoxic compounds are tested that do not require metabolisation and of which the mutagenic mode of action is dependent on replication. For compounds, which are metabolically converted to mutagenic metabolites, more differentiated HepG2 DDR reporters (e.g. 3D cultures) showed a higher sensitivity. This study stratifies how different culture methods of HepG2 DDR reporter cells can influence the sensitivity towards diverse genotoxicants and how this provides opportunities for a tiered genotoxicity testing strategy.

Distinct Amino Acid Availability-Dependent Regulatory Mechanisms of MepS and MepM Levels in Escherichia coli.

Peptidoglycan (PG) hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and antibiotic resistance. However, the regulatory mechanisms of their expression are poorly understood. In this study, we have uncovered novel regulatory mechanisms of the protein levels of the synthetically lethal PG endopeptidases MepS and MepM, which are involved in PG synthesis. A mutant defective for both MepS and MepM was lethal in an amino acid-rich medium, whereas it exhibited almost normal growth in a minimal medium, suggesting the expendability of MepS and MepM in a minimal medium. Protein levels of MepS and MepM dramatically decreased in the minimal medium. Although MepM was revealed as a substrate of Prc, a periplasmic protease involved in the proteolysis of MepS, only the decrease in the MepS level in the minimal medium was affected by the prc depletion. Phenotypic and biochemical analyses showed that the presence of aromatic amino acids in the medium induced the accumulation of MepS, but not MepM, while the presence of glutamate increased the level of MepM, but not MepS. Together, these results demonstrate that the protein levels of the two major PG endopeptidases are regulated in an amino acid availability-dependent manner, but their molecular mechanisms and signaling are significantly distinct.

Deletion of Gcw13 represses autophagy in Pichia pastoris cells grown in methanol medium with sufficient amino acids.

The purpose of this article is to study the underlying cause of the induction of autophagy in Pichia pastoris cells grown in amino acid-rich methanol medium during methanol adaptation. Autophagy was induced in P. pastoris GS115 when cells were grown in amino acid-rich methanol medium. Transcriptome analysis revealed that genes involved in amino acid biosynthesis were upregulated. The deletion of Gcw13, a GPI-anchored protein that plays a role in the endocytosis of the general amino acid permease Gap1, resulted in the inhibition of autophagy, the activation of TORC1 and an increase in the uptake of glutamine and asparagine in methanol-grown cells. Our results demonstrated that the autophagy induced in P. pastoris cells grown in amino acid-rich methanol medium was nitrogen source independent and may be due to a Gcw13-dependent decrease in amino acid uptake during methanol adaptation.

Identification of a High-Affinity Pyruvate Receptor in Escherichia coli

Two-component systems are crucial for signal perception and modulation of bacterial behavior. Nevertheless, to date, very few ligands have been identified that directly interact with histidine kinases. The histidine kinase/response regulator system YehU/YehT of Escherichia coli is part of a nutrient-sensing network. Here we demonstrate that this system senses the onset of nutrient limitation in amino acid rich media and responds to extracellular pyruvate. Binding of radiolabeled pyruvate was found for full-length YehU in right-side-out membrane vesicles as well as for a truncated, membrane-integrated variant, confirming that YehU is a high-affinity receptor for extracellular pyruvate. Therefore we propose to rename YehU/YehT as BtsS/BtsR, after “Brenztraubensäure”, the name given to pyruvic acid when it was first synthesized. The function of BtsS/BtsR was also assessed in a clinically relevant uropathogenic E. coli strain. Quantitative transcriptional analysis revealed BtsS/BtsR importance during acute and chronic urinary-tract infections.

Phagosomal Neutralization by the Fungal Pathogen Candida albicans Induces Macrophage Pyroptosis.

The interaction of Candida albicans with the innate immune system is the key determinant of the pathogen/commensal balance and has selected for adaptations that facilitate the utilization of nutrients commonly found within the host, including proteins and amino acids; many of the catabolic pathways needed to assimilate these compounds are required for persistence in the host. We have shown that C. albicans co-opts amino acid catabolism to generate and excrete ammonia, which raises the extracellular pH, both in vitro and in vivo and induces hyphal morphogenesis. Mutants defective in the uptake or utilization of amino acids, such as those lacking STP2, a transcription factor that regulates the expression of amino acid permeases, are impaired in multiple aspects of fungus-macrophage interactions resulting from an inability to neutralize the phagosome. Here we identified a novel role in amino acid utilization for Ahr1p, a transcription factor previously implicated in regulation of adherence and hyphal morphogenesis. Mutants lacking AHR1 were defective in growth, alkalinization, and ammonia release on amino acid-rich media, similar to stp2Δ and ahr1Δ stp2Δ cells, and occupied more acidic phagosomes. Notably, ahr1Δ and stp2Δ strains did not induce pyroptosis, as measured by caspase-1-dependent interleukin-1β release, though this phenotype could be suppressed by pharmacological neutralization of the phagosome. Altogether, we show that C. albicans-driven neutralization of the phagosome promotes hyphal morphogenesis, sufficient for induction of caspase-1-mediated macrophage lysis.

Robust Extracellular pH Modulation by Candida albicans during Growth in Carboxylic Acids.

ABSTRACTThe opportunistic fungal pathogen Candida albicans thrives within diverse niches in the mammalian host. Among the adaptations that underlie this fitness is an ability to utilize a wide array of nutrients, especially sources of carbon that are disfavored by many other fungi; this contributes to its ability to survive interactions with the phagocytes that serve as key barriers against disseminated infections. We have reported that C. albicans generates ammonia as a byproduct of amino acid catabolism to neutralize the acidic phagolysosome and promote hyphal morphogenesis in a manner dependent on the Stp2 transcription factor. Here, we report that this species rapidly neutralizes acidic environments when utilizing carboxylic acids like pyruvate, α-ketoglutarate (αKG), or lactate as the primary carbon source. Unlike in cells growing in amino acid-rich medium, this does not result in ammonia release, does not induce hyphal differentiation, and is genetically distinct. While transcript profiling revealed significant similarities in gene expression in cells grown on either carboxylic or amino acids, genetic screens for mutants that fail to neutralize αKG medium identified a nonoverlapping set of genes, including CWT1, encoding a transcription factor responsive to cell wall and nitrosative stresses. Strains lacking CWT1 exhibit retarded αKG-mediated neutralization in vitro, exist in a more acidic phagolysosome, and are more susceptible to macrophage killing, while double cwt1Δ stp2Δ mutants are more impaired than either single mutant. Together, our observations indicate that C. albicans has evolved multiple ways to modulate the pH of host-relevant environments to promote its fitness as a pathogen.

Sugar-based crosslinker forms a stable atelocollagen hydrogel that is a favorable microenvironment for 3D cell culture.

We created sugar-based crosslinkers for precise chemical crosslinking of atelocollagen to form a stable hydrogel microenvironment for three-dimensional (3D) cell culture. Crosslinkers were synthesized by partially oxidizing glucose, fructose, maltose, sucrose, or raffinose with periodate. The partial oxidization of sugar generated multiple aldehydes in the molecule, which acted as multifunctional crosslinkers, allowing atelocollagen to form chemical hydrogels. The crosslink reaction of atelocollagen was competitively suppressed by the addition of amino acid-rich medium, enabling flexible control of 3D molecular density in the acquired hydrogel. To evaluate structural stability, ultrasonic stress was loaded onto the acquired hydrogel and sequential measurement of water content showed that the crosslinking considerably stabilized the hydrogel structure. The effectiveness of the atelocollagen hydrogel as a 3D culture scaffold was analyzed by embedding and culturing endothelial cells or cardiomyocytes in it. Endothelial cells formed 3D capillary-like structures at 12 h, and cardiomyocytes formed a beating 3D netlike structure by 7 days. These findings suggest that crosslinked atelocollagen hydrogel effectively functions as a stable scaffold in 3D culture.

Genome-scale metabolic network reconstruction and in silico flux analysis of the thermophilic bacterium Thermus thermophilus HB27.

BackgroundThermus thermophilus, an extremely thermophilic bacterium, has been widely recognized as a model organism for studying how microbes can survive and adapt under high temperature environment. However, the thermotolerant mechanisms and cellular metabolism still remains mostly unravelled. Thus, it is highly required to consider systems biological approaches where T. thermophilus metabolic network model can be employed together with high throughput experimental data for elucidating its physiological characteristics under such harsh conditions.ResultsWe reconstructed a genome-scale metabolic model of T. thermophilus, iTT548, the first ever large-scale network of a thermophilic bacterium, accounting for 548 unique genes, 796 reactions and 635 unique metabolites. Our initial comparative analysis of the model with Escherichia coli has revealed several distinctive metabolic reactions, mainly in amino acid metabolism and carotenoid biosynthesis, producing relevant compounds to retain the cellular membrane for withstanding high temperature. Constraints-based flux analysis was, then, applied to simulate the metabolic state in glucose minimal and amino acid rich media. Remarkably, resulting growth predictions were highly consistent with the experimental observations. The subsequent comparative flux analysis under different environmental conditions highlighted that the cells consumed branched chain amino acids preferably and utilized them directly in the relevant anabolic pathways for the fatty acid synthesis. Finally, gene essentiality study was also conducted via single gene deletion analysis, to identify the conditional essential genes in glucose minimal and complex media.ConclusionsThe reconstructed genome-scale metabolic model elucidates the phenotypes of T. thermophilus, thus allowing us to gain valuable insights into its cellular metabolism through in silico simulations. The information obtained from such analysis would not only shed light on the understanding of physiology of thermophiles but also helps us to devise metabolic engineering strategies to develop T. thermophilus as a thermostable microbial cell factory.

Human embryonic stem cell differentiation into insulin secreting β‐cells for diabetes

hESC (human embryonic stem cells), when differentiated into pancreatic β ILC (islet-like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five-step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine β islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti-hyperglycaemic hormone-Glp1 (glucagon-like peptide 1) analogue Liraglutide with prolonged t(½) and Exendin 4. The differentiated islet-like 3D clusters expressed bonafide mature and functional β-cell markers-PDX1 (pancreatic and duodenal homoeobox-1), C-peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C-peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D β islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long-term in vivo functionally viable islets from hESC.

Amino acids downregulate the expression of several autophagy-related genes in rainbow trout myoblasts

Many fish species experience long periods of fasting often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as proteins of this tissue act as a major energy source. However, the molecular components involved in muscle protein degradation as well as the regulatory networks that control their function are still incompletely defined in fish. The present work aimed to characterize the response of the autophagy-lysosomal degradative pathway to nutrient and serum availability in primary culture of rainbow trout myoblasts. In this aim, 4-day-old cells were incubated in a serum and amino acid-rich medium (complete medium), a serum and amino acid-deprived medium (minimal medium) or a minimal medium plus amino acids, and both the transcription-independent short-term response and the transcription-dependent long-term response of the autophagy-lysosomal degradative pathway were analyzed. We report that serum and amino acids withdrawal is accompanied by a rapid increase of autophagosome formation but also by a slower induction of the expression of several autophagy-related genes (LC3B, gabarapl1, atg4b). We also showed that this latter response is controlled by amino acid (AA) availability and that both TOR-dependent and TOR-independent pathways are involved in this effect. Together these results suggest an important role for AA released by muscle proteolysis during the fasting period in regulating the subtle balance between using proteins as disposable furniture to provide energy, and conserving muscle through protein sparing.

Salmonella enterica Modulates Its Infectivity in Response to Intestinal Stimuli

En route to its intestinal target cells Salmonella enterica passes different host niches and encounters various environmental cues. These are expected to promote Salmonella in the decision of changing its extracellular life style to intracellular. We find that prior incubation of bacteria in the presence of signals which are characteristic for the small intestine affects invasion in a model system: Salmonella grown at high osmotic pressure in the presence of bile or in amino acid rich medium, infect host cells most efficiently. Hence, Salmonella enterica modulates its infectivity in response to these stimuli which consequently determines the success of infection. Our results close the current gap between signal and actual behavior and may serve as a basis for further investigations for example if Salmonella has an adaptive prediction of environmental changes.

A Dehydrogenase Activity Test for Monitoring the Growth of Streptomyces Venezuelae in a Nutrient Rich Medium

Jadomycin is a novel antibiotic that has shown activities against bacteria, yeasts and fungi as well as cytotoxic properties to cancer cells. Because of the wide range of its inhibitory actions, jadomycin shows promise as a novel antibiotic and cancer treatment drug. Streptomyces venezuelae are aerobic bacteria that are capable of producing jadomycin when shocked by alcohol in a nutrient deprived amino acid rich medium. The size of the bacterial population that is transferred from the growth medium to the production medium can significantly affect the jadomycin yield. Therefore, the number of transferred bacteria must be accurately measured. In this study, a dehydrogenase activity measurement test was developed for S. venezuelae using triphenyl tetrazolium chloride (TTC) to measure the cell growth and activity in maltose-yeast extract-malt extract (MYM) broth. The dehydrogenase activity was determined by measuring the visible color changes of the TTC to triphenyl formazan (TF). The test conditions which included extraction solvent, number of extractions, incubation time, incubation temperature and medium pH were evaluated. The results showed that the triphenyl formazan was related to the number of cells. Methanol was better able to permeate the cells and extract higher amount of TF than ethanol. The amount of TF increased with the number of extractions for both solvents. A lower medium pH and/or lower temperature produced the highest amount of TF. The best test conditions that produced the highest TF yield were three extractions using methanol after an incubation time of 1 hour at a temperature of 30oC and a medium pH of 6.

Phagocytosis by neutrophils induces an amino acid deprivation response in Saccharomyces cerevisiae and Candida albicans.

The transcriptional profiles of yeast cells that have been phagocytosed by either human neutrophils or monocytes were compared by using whole genome arrays. After phagocytosis by neutrophils, both Saccharomyces cerevisiae and Candida albicans respond by inducing genes of the methionine and arginine pathways. Neither of these pathways is induced upon phagocytosis by monocytes. Both fungi show a similar induction of these pathways when transferred from amino acid-rich medium to amino acid-deficient medium. These data suggest that the internal phagosome of the neutrophil is an amino acid-deficient environment.

The influence of sugars and amino acids on the blood-feeding behaviour, oviposition and longevity of laboratory colony of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae, Phlebotominae).

Schneider's Drosophila medium, a complex amino acid rich medium was tested alone and with seven different sugars for some aspects of the biology of Lutzomyia longipalpis. Statistically significant results were obtained when sucrose was used alone, indicating that among the sugars tested, this is still the most suitable and practical one for the maintenance of L. longipalpis colonies. However, the addition of Schneider's medium to a pool of different sugars, was suggested to be related with the acceptance of the first and second blood meals and to longevity, these being, obviously, quite relevant aspects when transmission experiments are contemplated.

Alpha-keto acids are novel siderophores in the genera Proteus, Providencia, and Morganella and are produced by amino acid deaminases.

Growth promotion and iron transport studies revealed that certain alpha-keto acids generated by amino acid deaminases, by enterobacteria of the Proteus-Providencia-Morganella group (of the tribe Proteeae), show significant siderophore activity. Their iron-binding properties were confirmed by the chrome azurol S assay and UV spectra. These compounds form ligand-to-metal charge transfer bands in the range of 400 to 500 nm. Additional absorption bands of the enolized ligands at 500 to 700 nm are responsible for color formation. Siderophore activity was most pronounced with alpha-keto acids possessing an aromatic or heteroaromatic side chain, like phenylpyruvic acid and indolylpyruvic acid, resulting from deamination of phenylalanine and tryptophan, respectively. In addition, alpha-keto acids possessing longer nonpolar side chains, like alpha-ketoisocaproic acid or alpha-ketoisovaleric acid and even alpha-ketoadipic acid, also showed siderophore activity which was absent or negligible with smaller alpha-keto acids or those possessing polar functional groups, like pyruvic acid, alpha-ketobutyric acid, or alpha-ketoglutaric acid. The fact that deaminase-negative enterobacteria, like Escherichia coli and Salmonella spp., could not utilize alpha-keto acids supports the view that specific iron-carboxylate transport systems have evolved in members of the tribe Proteeae and are designed to recognize ferric complexes of both alpha-hydroxy acids and alpha-keto acids, of which the latter can easily be generated by L-amino acid deaminases in an amino acid-rich medium. Exogenous siderophores, like ferric hydroxamates (ferrichromes) and ferric polycarboxylates (rhizoferrin and citrate), were also utilized by members of the tribe Proteeae.

Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum.

Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x 10(5) and 6.0 x 10(5) cells per 35-mm dish. Even at a density of 9.0 x 10(5) cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.

Overexpression of the relA gene in Escherichia coli

Intracellular levels of guanosine 3',5'-bispyrophosphate (ppGpp) governed by the relA gene are normally regulated by aminoacyl-tRNA availability for protein synthesis. An experimental system is described in which cellular levels of ppGpp are controlled instead by induction of plasmid pKK223-3 derivatives with the relA structural gene, or portions thereof, under control of the Ptac promoter. In amino acid-rich media, isopropyl-1-thio-beta-D-galactopyranoside induction of transcription of the wild type relA gene in pSM10 yields about a 100-fold overexpression of a metabolically stable, full length (743 amino acid) RelA protein to levels approximating the number of cellular ribosomes. This overexpression is accompanied by a roughly parallel and relC-dependent elevation of ppGpp levels. Induction of a relA gene deletion mutant in pSM11 containing 455 amino-terminal amino acids results in much lower levels of expression of a metabolically unstable 55-kDa protein and elevated ppGpp levels that are almost equivalent to induced pSM10 and are relC-independent. Induction of a larger deletion in pSM12 containing 331 amino-terminal amino acids does not provoke ppGpp accumulation. We are able to elicit high levels of ppGpp without changing nutritional abundance and without massive overexpression of the RelA protein by inducing the metabolically unstable, truncated RelA protein. We find the effects of elevated ppGpp levels to include a slowing of growth, an inhibition of stable RNA accumulation, an inhibition of cellular rrn P1 promoter activities as measured by primer extension, and changes in the pattern of gene expression viewed by two-dimensional electrophoresis of cellular proteins.

Catalase and Superoxide Dismutase of Root-Colonizing Saprophytic Fluorescent Pseudomonads

Root-colonizing, saprophytic fluorescent pseudomonads of the Pseudomonas putida-P. fluorescens group express similar levels of catalase and superoxide dismutase activities during growth on a sucrose- and amino acid-rich medium. Increased specific activities of catalase but not superoxide dismutase were observed during growth of these bacteria on components washed from root surfaces. The specific activities of both enzymes were also regulated during contact of these bacteria with intact bean roots. Increased superoxide dismutase and decreased catalase activities were observed rapidly, by 10 min upon inoculation of cells onto intact bean roots. Catalase specific activity increased with time to peak at 12 h before declining. By 48 h, the cells displayed this low catalase but maintained high superoxide dismutase specific activities. Catalase with a low specific activity and a high superoxide dismutase activity also were present in extracts of cells obtained from 7-day-old roots colonized from inoculum applied to seed. This specific activity of superoxide dismutase of root-contacted cells was about fourfold-higher in comparison to cells grown on rich medium, whereas the specific activity for catalase was reduced about fivefold. A single catalase isozyme, isozyme A, and one isozyme of superoxide dismutase, isozyme 1, were detected during growth of the bacteria on root surface components and during exposure of cells to intact bean roots for 1 h. An additional catalase, isozyme B, was detected from bacteria after exposure to the intact bean roots for 12 h. Catalase isozyme A and superoxide dismutase isozyme 1 were located in the cytoplasm and catalase band B was located in the membrane of P. putida.