Primer design to assess bacterial degradation of glyphosate and other phosphonates.

Phosphonates are organic phosphorous (P) compounds frequently detected in the environment due to a very stable CP bond that render them relatively recalcitrant. Glyphosate [N-phosphonomethyl glycine] is the most widely used and best-known synthetic phosphonate, and one of the most concerning herbicides in the world today. Microbial degradation of glyphosate and organophosphonates in general, is the main dissipation mechanism operating in most environments. One microbial metabolic pathway in this process is the CP lyase pathway, entailing an enzymatic complex encoded by about 14 genes (the Phn operon). Our goal was to develop a quantitative polymerase chain reaction (qPCR) assay for a key enzyme, the CP lyase that breaks down the CP bond, via quantification of the codifying phnJ gene. The primers designed in this study fulfill the requirements for a successful qPCR assay, with high efficiency and sensitivity, as well as specific detection of the target sequence in a wide range of taxonomic groups. This is, to our knowledge, the first report of primers designed to target phnJ in both pure cultures and metagenomic DNA from different environmental sources. Direct quantification of phnJ may be a cost-effective proxy to determine glyphosate degradation potential in different matrixes.

Discovering radical-dependent enzymes in the human gut microbiota.

Human gut microbes have a tremendous impact on human health, in part due to their unique chemical capabilities. In the anoxic environment of the healthy human gut, many important microbial metabolic transformations are performed by radical-dependent enzymes. Although identifying and characterizing these enzymes has been challenging, recent advances in genome and metagenome sequencing have enabled studies of their chemistry and biology. Focusing on the glycyl radical enzyme family, one of the most enriched protein families in the human gut microbiota, we highlight different approaches for discovering radical-dependent enzymes that influence host health and disease.

Translation efficiency of heterologous proteins is significantly affected by the genetic context of RBS sequences in engineered cyanobacterium Synechocystis sp. PCC 6803.

BACKGROUND: Photosynthetic cyanobacteria have been studied as potential host organisms for direct solar-driven production of different carbon-based chemicals from CO2 and water, as part of the development of sustainable future biotechnological applications. The engineering approaches, however, are still limited by the lack of comprehensive information on most optimal expression strategies and validated species-specific genetic elements which are essential for increasing the intricacy, predictability and efficiency of the systems. This study focused on the systematic evaluation of the key translational control elements, ribosome binding sites (RBS), in the cyanobacterial host Synechocystis sp. PCC 6803, with the objective of expanding the palette of tools for more rigorous engineering approaches. RESULTS: An expression system was established for the comparison of 13 selected RBS sequences in Synechocystis, using several alternative reporter proteins (sYFP2, codon-optimized GFPmut3 and ethylene forming enzyme) as quantitative indicators of the relative translation efficiencies. The set-up was shown to yield highly reproducible expression patterns in independent analytical series with low variation between biological replicates, thus allowing statistical comparison of the activities of the different RBSs in vivo. While the RBSs covered a relatively broad overall expression level range, the downstream gene sequence was demonstrated in a rigorous manner to have a clear impact on the resulting translational profiles. This was expected to reflect interfering sequence-specific mRNA-level interaction between the RBS and the coding region, yet correlation between potential secondary structure formation and observed translation levels could not be resolved with existing in silico prediction tools. CONCLUSIONS: The study expands our current understanding on the potential and limitations associated with the regulation of protein expression at translational level in engineered cyanobacteria. The acquired information can be used for selecting appropriate RBSs for optimizing over-expression constructs or multicistronic pathways in Synechocystis, while underlining the complications in predicting the activity due to gene-specific interactions which may reduce the translational efficiency for a given RBS-gene combination. Ultimately, the findings emphasize the need for additional characterized insulator sequence elements to decouple the interaction between the RBS and the coding region for future engineering approaches.

iTRAQ-based quantitative proteomics analysis of rice leaves infected by Rice stripe virus reveals several proteins involved in symptom formation.

BACKGROUND: Rice plants infected by Rice stripe virus (RSV) usually leads to chlorosis and death of newly emerged leaves. However, the mechanism of RSV-induced these symptoms was not clear. METHODS: We used an iTRAQ approach for a quantitative proteomics comparison of non-infected and infected rice leaves. RT-qPCR and Northern blot analyses were performed for assessing the transcription of candidate genes. RESULTS: As a whole, 681 (65.8% downregulated, 34.2% upregulated infected vs. non-infected) differentially accumulated proteins were identified. A bioinformatics analysis indicated that ten of these regulated proteins are involved in chlorophyll biosynthesis and three in cell death processes. Subsequent RT-qPCR results showed that downregulation of magnesium chelatase was due to reduced expression levels of the genes encoding subunits CHLI and CHLD, which resulted in chlorophyll reduction involved in leaf chlorosis. Three aspartic proteases expressed higher in RSV-infected leaves than those in the control leaves, which were also implicated in RSV-induced cell death. Northern blot analyses of CHLI and p0026h03.19 confirmed the RT-qPCR results. CONCLUSIONS: The magnesium chelatase and aspartic proteases may be associated with RSV-induced leaf chlorosis and cell death, respectively. The findings may yield new insights into mechanisms underlying rice stripe disease symptom formation.

Correlation between development of female flower buds and expression of the CS-ACS2 gene in cucumber plants.

Ethylene plays a key role in sex determination of cucumber flowers. Gynoecious cucumber shoots produce more ethylene than monoecious shoots. Because monoecious cucumbers produce both male and female flower buds in the shoot apex and because the relative proportions of male and female flowers vary due to growing conditions, the question arises as to whether the regulation of ethylene biosynthesis in each flower bud determines the sex of the flower. Therefore, the expression of a 1-aminocyclopropane-1-carboxylic acid synthase gene, CS-ACS2, was examined in cucumber flower buds at different stages of development. The results revealed that CS-ACS2 mRNA began to accumulate just beneath the pistil primordia of flower buds at the bisexual stage, but was not detected prior to the formation of the pistil primordia. In buds determined to develop as female flowers, CS-ACS2 mRNA continued to accumulate in the central region of the developing ovary where ovules and placenta form. In gynoecious cucumber plants that produce only female flowers, accumulation of CS-ACS2 mRNA was detected in all flower buds at the bisexual stage and at later developmental stages. In monoecious cucumber, flower buds situated on some nodes accumulated CS-ACS2 mRNA, but others did not. The proportion of male and female flowers in monoecious cucumbers varied depending on the growth conditions, but was correlated with changes in accumulation of CS-ACS2 mRNA in flower buds. These results demonstrate that CS-ACS2-mediated biosynthesis of ethylene in individual flower buds is associated with the differentiation and development of female flowers.

ATPase activity of magnesium chelatase subunit I is required to maintain subunit D in vivo.

During biosynthesis of chlorophyll, Mg(2+) is inserted into protoporphyrin IX by magnesium chelatase. This enzyme consists of three different subunits of approximately 40, 70 and 140 kDa. Seven barley mutants deficient in the 40 kDa magnesium chelatase subunit were analysed and it was found that this subunit is essential for the maintenance of the 70 kDa subunit, but not the 140 kDa subunit. The 40 kDa subunit has been shown to belong to the family of proteins called "ATPases associated with various cellular activities", known to form ring-shaped oligomeric complexes working as molecular chaperones. Three of the seven barley mutants are semidominant mis-sense mutations leading to changes of conserved amino acid residues in the 40 kDa protein. Using the Rhodobacter capsulatus 40 and 70 kDa magnesium chelatase subunits we have analysed the effect of these mutations. Although having no ATPase activity, the deficient 40 kDa subunit could still associate with the 70 kDa protein. The binding was dependent on Mg(2+) and ATP or ADP. Our study demonstrates that the 40 kDa subunit functions as a chaperon that is essential for the survival of the 70 kDa subunit in vivo. We conclude that the ATPase activity of the 40 kDa subunit is essential for this function and that binding between the two subunits is not sufficient to maintain the 70 kDa subunit in the cell. The ATPase deficient 40 kDa proteins fail to participate in chelation in a step after the association of the 40 and 70 kDa subunits. This step presumably involves a conformational change of the complex in response to ATP hydrolysis.

Molecular cloning and functional analysis of a Schizosaccharomyces pombe homologue of Escherichia coli endonuclease III.

The Escherichia coli endonuclease III (Nth-Eco) protein is involved in the removal of damaged pyrimidine residues from DNA by base excision repair. It is an iron-sulphur enzyme possessing both DNA glycosylase and apurinic/apyrimidinic lyase activities. A database homology search identified an open reading frame in genomic sequences of Schizosaccharomyces pombe which encodes a protein highly similar to Nth-Eco. The gene has been subcloned in an expression vector and the protein purified to apparent homogeneity. The S.pombe Nth homologue (Nth-Spo) is a 40.2 kDa protein of 355 amino acids. Nth-Spo possesses glycosylase activity on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded polymers. The eukaryotic protein removes urea more efficiently than the prokaryotic enzyme, whereas its efficiency in excising thymine glycol is lower. A nicking assay was used to show that the enzyme also exhibits an AP lyase activity on UV- and gamma-irradiated DNA substrates. These findings show that Nth protein is structurally and functionally conserved from bacteria to fission yeast.

Microbial succession and intestinal enzyme activities in the developing rat.

The succession of gut bacteria and selected intestinal enzyme activities in developing 7-35-d-old rats was studied. Aerobes and anaerobes were identified as members of four broad major bacterial groups, i.e. Gram-positive rods, Gram-positive cocci, Gram-negative rods and obligate anaerobes. The enzyme activities of nitro and azo reductases, beta-glucuronidase, dechlorinase and dehydrochlorinase were determined by anaerobic incubation of intestinal homogenates with 3,4-dichloronitrobenzene, methyl orange, p-nitrophenyl-beta-D-glucuronide, and p,p'-DDT respectively. Nitroreductase and azo reductase activities increased significantly with the appearance of anaerobes in the large intestine. No increase in either nitroreductase or azo reductase activities in the small intestine was found. The early and high level of beta-glucuronidase activity in the small and large intestines coincided with high numbers of coliforms recovered in 7 and 14 d animals. Dehydrochlorinase activity appeared early but was undetectable at both 21 and 28 d. Its activity increased at 35 d. Dechlorinase activity was variable in development. The rapid changes in the microbial flora and intestinal enzyme activities may influence the susceptibility of pre-pubescent rats to a variety of toxicants. Therefore, age-dependent toxicity may be important in the risk assessment of some environmental chemicals.

A continuous spectrophotometric assay for ureidoglycolase activity with lactate dehydrogenase or glyoxylate reductase as coupling enzyme.

A spectrophotometric assay for ureidoglycolase activity (both ureidoglycolate lyase and hydrolase), based on the reduction of glyoxylate to glycolate catalyzed by glyoxylate reductase or lactate dehydrogenase with the stoichiometric and continuous NADH oxidation, is described. The assay has been optimized for the amount of coupling enzyme, reagent concentrations, buffers, and the nonenzymatic degradation of ureidoglycolate. Under optimal assay conditions, ureidoglycolase activity can be followed with either lactate dehydrogenase or glyoxylate reductase as coupling enzyme and reaction can be started by addition of substrate or enzyme extract. Once the reaction was started, NADH oxidation was linear with time after a lag phase of 1-2 min. This linear NADH oxidation was directly proportional to enzyme concentration in the assay mixture until changes in absorbance of 0.12 per minute. This method is easy and reliable for the accurate determination of ureidoglycolase activity in crude and purified extracts from Chlamydomonas reinhardtii cells and no notable interferences have been detected. Since lactate dehydrogenase is much cheaper and has a lower Km for its substrate than glyoxylate reductase and can be used as supplied, its use as the coupling enzyme of choice is recommended.

Expression of proteins encoded by the Escherichia coli cyn operon: carbon dioxide-enhanced degradation of carbonic anhydrase.

Cyanase catalyzes the reaction of cyanate with bicarbonate to give 2CO2. The cynS gene encoding cyanase, together with the cynT gene for carbonic anhydrase, is part of the cyn operon, the expression of which is induced in Escherichia coli by cyanate. The physiological role of carbonic anhydrase is to prevent depletion of cellular bicarbonate during cyanate decomposition due to loss of CO2 (M.B. Guilloton, A.F. Lamblin, E. I. Kozliak, M. Gerami-Nejad, C. Tu, D. Silverman, P.M. Anderson, and J.A. Fuchs, J. Bacteriol. 175:1443-1451, 1993). A delta cynT mutant strain was extremely sensitive to inhibition of growth by cyanate and did not catalyze decomposition of cyanate (even though an active cyanase was expressed) when grown at a low pCO2 (in air) but had a Cyn+ phenotype at a high pCO2. Here the expression of these two enzymes in this unusual system for cyanate degradation was characterized in more detail. Both enzymes were found to be located in the cytosol and to be present at approximately equal levels in the presence of cyanate. A delta cynT mutant strain could be complemented with high levels of expressed human carbonic anhydrase II; however, the mutant defect was not completely abolished, perhaps because the E. coli carbonic anhydrase is significantly less susceptible to inhibition by cyanate than mammalian carbonic anhydrases. The induced E. coli carbonic anhydrase appears to be particularly adapted to its function in cyanate degradation. Active cyanase remained in cells grown in the presence of either low or high pCO2 after the inducer cyanate was depleted; in contrast, carbonic anhydrase protein was degraded very rapidly (minutes) at a high pCO2 but much more slowly (hours) at a low pCO2. A physiological significance of these observations is suggested by the observation that expression of carbonic anhydrase at a high pCO2 decreased the growth rate.

Steroidogenic activity of atretic follicles in the cycling hamster ovary and relation to ultrastructural observations.

To evaluate the relation between the steroidogenic activity and cell proliferation of individual follicles in mature hamster ovaries during the estrous cycle, the localization of enzymes involved in estrogen biosynthesis and bromodeoxyuridine (BrdU) incorporation were examined immunohistochemically. Moreover, granulosa cells from the early atretic follicle were examined by scanning and transmission electron microscopy. Immunoreactivity for aromatase was localized in the granulosa cells of healthy developing follicles and Graafian follicles, as well as in newly formed granulosa lutein cells. In the healthy follicles of an ovulation cycle, intensity of aromatase immunoreactivity was suddenly decreased on day 3. The theca interna cells of healthy developing follicles were immunopositive for 17 alpha-hydroxylase/C17-C20 lyase (17 alpha-lyase) from day 2 to the morning of day 4, but on the evening of day 4 most theca interna cells were immunonegative except for only a few cells of the large Graafian follicles. BrdU incorporation was observed in the granulosa cells of healthy developing follicles, in the endothelial cells of capillaries around the developing follicles, and of newly formed corpora lutea. Very early morphological signs of atresia was the pyknotic change of a few granulosa cells lining the antral cavity. In that follicle, the number of BrdU-incorporating granulosa cells was suddenly decreased whilst immunoreactivity of aromatase and 17 alpha-lyase were gradually decreased. These data suggest that the mechanism of the loss of aromatase activity from the granulosa cells of atretic follicles appears to differ from that in cycling follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

A continuous, anaerobic spectrophotometric assay for chorismate synthase activity that utilizes photoreduced flavin mononucleotide.

A sensitive, continuous, spectrophotometric assay for chorismate synthase has been developed utilizing photoreduced flavin mononucleotide (FMNH2) as a cofactor under anaerobic conditions. The assay monitors directly the formation of chorismate from 5-enolpyruvylshikimate-3-phosphate (EPSP) at 275 nm with a precision of +/- 2 microM product. The assay conditions have been optimized with respect to FMNH2 (cofactor), EPSP (substrate) and enzyme concentrations, buffer type, and pH. The potential of the assay for detailed steady-state kinetic studies to elucidate the mechanism of action of this commercially important enzyme is also demonstrated.

Cytosolic C-S lyase activity in human kidney samples-relevance for the nephrotoxicity of halogenated alkenes in man.

Human renal cortex cytosolic samples were screened for C-S lyase (EC 4.4.1.13) activity using cysteine conjugates of halogenated aliphatic and aromatic hydrocarbons as substrates. Cystosolic activity was greatest with S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) (72.0 +/- 26.8 and 74.4 +/- 38.3 nmol pyruvate formed/mg protein/120 min. respectively). Less than five fold inter-individual variation was observed. In contrast to the low C-S lyase activity detectable in rat cytosol, no cleavage of the aromatic conjugates S-(2-benzothiazolyl)-L-cysteine (BTC), S-(2,3,5,6-tetrachlorophenyl)-L-cysteine (TCPC) and S-(4-bromophenyl)-L-cysteine (4-BPC) was detectable in human cytosol. Structure-activity relationships showed that increasing the fluorinated carbon chain length of the halogenated hydrocarbon species decreased conjugate cleavage by C-S lyase. The position and number of fluorine and chlorine atoms on the parent hydrocarbon determined the extent of cysteine conjugate C-S cleavage. Activity increased with an increase in fluorine and chlorine substitution and shortening of carbon chain length in the rat, although in human cytosol an increase in chlorine substitution resulted in decreased activity.

High activities of glutamine transaminase K (dichlorovinylcysteine beta-lyase) and omega-amidase in the choroid plexus of rat brain.

Certain halogenated hydrocarbons, e.g., dichloroacetylene, are nephrotoxic to experimental animals and neurotoxic to humans; cysteine-S-conjugate beta-lyases may play a role in the nephrotoxicity. We now show that with dichlorovinylcysteine as substrate the only detectable cysteine-S-conjugate beta-lyase in rat brain homogenates is identical to glutamine transaminase K. The predominant (mitochondrial) form of glutamine transaminase K in rat brain was shown to be immunologically distinct from the predominant (cytosolic) form of the enzyme in rat kidney. Glutamine transaminase K and omega-amidase (constituents of the glutaminase II pathway) activities were shown to be widespread throughout the rat brain. However, the highest specific activities of these enzymes were found in the choroid plexus. The high activity of glutamine transaminase K in choroid plexus was also demonstrated by means of an immunohistochemical staining procedure. Glutamine transaminase K has a broad specificity toward amino acid and alpha-keto acid substrates. The omega-amidase also has a broad specificity; presumably, however, the natural substrates are alpha-ketoglutaramate and alpha-ketosuccinamate, the alpha-keto acid analogues of glutamine and asparagine, respectively. The high activities of both glutamine transaminase K and omega-amidase in the choroid plexus suggest that the two enzymes are linked metabolically and perhaps are coordinately expressed in that organ. The data suggest that the natural substrate of glutamine transaminase K in rat brain is indeed glutamine and that the metabolism of glutamine through the glutaminase II pathway (i.e., L-glutamine and alpha-keto acid-->alpha-ketoglutarate and L-amino acid + ammonia) is an important function of the choroid plexus. Moreover, the present findings also suggest that any explanation of the neurotoxicity of halogenated xenobiotics must take into account the role of glutamine transminase K and its presence in the choroid plexus.

Tissue-specific molecular diversity of amidating enzymes (peptidylglycine alpha-hydroxylating monooxygenase and peptidylhydroxyglycine N-C lyase) in Xenopus laevis.

We investigated the molecular diversity of the paired enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N-C lyase (PHL), involved in peptide C-terminal amidation. Three kinds of amidating enzyme (AE) cDNAs (AE-I, AE-II and AE-III) have previously been isolated from Xenopus laevis skin. While AE-I cDNA encodes only PHM, AE-III cDNA encodes a protein containing both PHM and PHL sequences and a transmembrane domain. On the other hand, the translated product of AE-II has not been detected yet. Endoproteolytic cleavage of the AE-III protein generates separated forms of PHM and PHL that are purified from X. laevis skin. Expression of AE-III in insect cells using a baculovirus expression vector system indicated that PHM and PHL exist as a membrane-associated, bifunctional enzyme without endoproteolysis in insect cells. Both PHM and PHL activities were detected in all the X. laevis tissues examined. Particularly, the highest levels of both activities were found in skin, brain and heart. We identified basically three types of enzymes in X. laevis; soluble PHM, soluble PHL and a membrane-associated, bifunctional enzyme that has both PHM and PHL domains. While the skin contained soluble types of PHM and PHL, the brain and heart predominantly contained the membrane-associated, bifunctional type. Analysis of mRNA levels by the reverse-transcript polymerase chain reaction method and Western blot analysis using PHM-specific antibody revealed that such molecular diversity of PHM and PHL among the tissues are produced by changing the ratio of AE-I mRNA/AE-III mRNA, and by endoproteolytic processing of the membrane-associated precursor protein.

Immunocytochemical localization of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in human endocrine pancreas.

We studied the distribution of the enzymes that are involved in the post-translational alpha-amidation of regulatory peptides in human endocrine pancreas, using immunocytochemical methods for light and electron microscopy. Immunoreactivity for the two enzymes involved, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), was located in the periphery of the islets of Langerhans and in ductal endocrine cells. Staining of reverse-face serial sections demonstrated that these immunoreactivities co-localize with glucagon but not with pancreatic polypeptide (PP), insulin, or somatostatin. Double immunogold staining for electron microscopy confirmed the previous results and revealed a different localization for each enzyme inside the secretory granule: PHM is present in the central core of the glucagon-containing granules, whereas PAL is predominantly located near the granule membrane. The existence of an amidated peptide, GLP1, in the A-cells explains the presence of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in these cells. The absence of the enzymes in the PR-cells raises the possibility that a different form of amidating enzyme may be involved in the post-translational processing of this peptide.

Dose-dependent induction or depression of cysteine conjugate beta-lyase in rat kidney by N-acetyl-S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L-cysteine.

The influence of N-acetyl-S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L-cysteine (NAc-PCBD) on cysteine conjugate beta-lyase in female rat kidney has been examined. After a single, non-nephrotoxic dose of NAc-PCBD (3 mg/kg), cytosolic beta-lyase enzyme activity was increased 1.5 to 3-fold commensurate with a corresponding increase in enzyme protein levels as assessed by both Western blot and ELISA analyses. Using a cDNA probe for beta-lyase, this induction was found to be accompanied by an increase in the cognate mRNA. In contrast, a higher, nephrotoxic dose of NAc-PCBD (10 mg/kg) decreased all the above parameters. These effects appeared to be specific to the cytosolic form of the enzyme as no changes in kidney mitochondrial beta-lyase or enzyme protein levels were observed. Repeated dosing with the lower dose level (3 mg/kg) resulted in either no change, or in some instances, a reduction in the above parameters, suggesting an accumulation of the xenobiotic and a masking of the induction phenomenon. The molecular mechanisms underlying these observations are discussed in terms of the nephrotoxicity of halogenated xenobiotics.

A localized differentiation-inducing-factor sink in the front of the Dictyostelium slug.

Differentiation-inducing factor 1 [DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-hexan-1-one] induces stalk cell differentiation during Dictyostelium development. It is present as a gradient in the multicellular slug, its lowest concentration being in the anterior. Here we demonstrate the existence of a localized sink for DIF-1, also in the anterior of the slug, which could be responsible for generating the DIF-1 gradient. DIF-1 is metabolized extensively by developing cells, initially by a mono-dechlorination. We used an enzyme assay for DIF-1 dechlorinase to examine its distribution in the slug. DIF-1 dechlorinase activity is 30-fold higher in prestalk cells (largely anterior) compared with prespore cells (posterior) when these are separated from each other on Percoll density gradients. Dissection experiments showed that DIF-1 dechlorinase is 25-fold enriched in the anterior 13% of the slug compared with the rest. These experiments also showed that DIF-1 dechlorinase is more anterior-enriched than the standard prestalk markers, the ecmA and ecmB mRNAs. When cut from a slug, both prestalk and prespore fragments regulate to restore the missing cell type. Prespore fragments rapidly regain (by 30 min) a DIF-1 sink in their anteriors, and prestalk fragments restore a posterior zone with low DIF-1 dechlorinase by 4 hr after cutting. The reappearance of the DIF-1 sink in the anterior of prespore fragments is accomplished without detectable cell sorting and may, therefore, be in response to positional signals. Finally, a localized sink may provide a general way of producing a gradient of a signal substance in a developing embryo.