Bi-modal Distribution of the Second Messenger c-di-GMP Controls Cell Fate and Asymmetry during the Caulobacter Cell Cycle

Many bacteria mediate important life-style decisions by varying levels of the second messenger c-di-GMP. Behavioral transitions result from the coordination of complex cellular processes such as motility, surface adherence or the production of virulence factors and toxins. While the regulatory mechanisms responsible for these processes have been elucidated in some cases, the global pleiotropic effects of c-di-GMP are poorly understood, primarily because c-di-GMP networks are inherently complex in most bacteria. Moreover, the quantitative relationships between cellular c-di-GMP levels and c-di-GMP dependent phenotypes are largely unknown. Here, we dissect the c-di-GMP network of Caulobacter crescentus to establish a global and quantitative view of c-di-GMP dependent processes in this organism. A genetic approach that gradually reduced the number of diguanylate cyclases identified novel c-di-GMP dependent cellular processes and unraveled c-di-GMP as an essential component of C. crescentus cell polarity and its bimodal life cycle. By varying cellular c-di-GMP concentrations, we determined dose response curves for individual c-di-GMP-dependent processes. Relating these values to c-di-GMP levels modeled for single cells progressing through the cell cycle sets a quantitative frame for the successive activation of c-di-GMP dependent processes during the C. crescentus life cycle. By reconstructing a simplified c-di-GMP network in a strain devoid of c-di-GMP we defined the minimal requirements for the oscillation of c-di-GMP levels during the C. crescentus cell cycle. Finally, we show that although all c-di-GMP dependent cellular processes were qualitatively restored by artificially adjusting c-di-GMP levels with a heterologous diguanylate cyclase, much higher levels of the second messenger are required under these conditions as compared to the contribution of homologous c-di-GMP metabolizing enzymes. These experiments suggest that a common c-di-GMP pool cannot fully explain spatiotemporal regulation by c-di-GMP in C. crescentus and that individual enzymes preferentially regulate specific phenotypes during the cell cycle.     

Stability test of six enzymes for internal quality control

The daily quality control for the determination of the catalytic activity concentrations of enzymes is an important aspect in clinical chemistry. Instead of the expensive, commercially available control sera, we have looked for a simple, reliable and cheap method for the quality control of enzyme determinations. Commercially available enzymes were suspended in an albumin solution and ampoules were filled with 1.0 ml of these various solutions. The ampoules were stored at 4 degrees C or -20 degrees C. Once a week, during 10 months, catalytic activities of these enzyme-albumin solutions were determined together with the same activities in freshly reconstituted control sera. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine kinase and gamma-glutamyltransferase were determined at 30 degrees C according to well-described methods. alpha-Amylase was determined with the Phadebas method at 37 degrees C. Except for creatine kinase, the stability and reliability of these enzyme solutions are fully comparable with control sera during the experimental period. The catalytic activity concentration of creatine kinase decreased slowly during the 10 months. The enzyme solutions react in the same manner as commercial test sera on changes in the reaction conditions for the enzyme determinations. The conclusion seems justified that these enzyme solutions can be used for the daily quality control of the enzyme determinations instead of control sera.     

Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14579

To monitor the ability of the food-borne opportunistic pathogen Bacillus cereus to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42 degrees C, B. cereus ATCC 14579 adapts to heat exposure at the lethal temperature of 50 degrees C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42 degrees C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of B. subtilis. These observations indicate that heat adaptation of B. cereus involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic B. cereus, which may result in increased survival in foods.     

In vitro determination of virulence factors activity associated with several Cryptococcus neoformans clinical isolates

Different phenotypic characteristics associated with virulence of the Cryptococcus neoformans species complex have shown an important role in their pathogenicity. In this study we have determined the role of phenotypically and genotypically factors of some virulence factors from clinical isolates in the two species of the complex; 35 C. neoformans and 19 Cryptococcus gattii. Growth at 37 degrees C, macroscopic and microscopic morphology, switching phenomenon, activity of 23 extracellular enzymes, variability of the colonies in agar with phloxin B; phospholipase B gene, and the mating type were determined by PCR; the molecular pattern was determined by URA5 RFLP. All isolates grew at 37 degrees C, the capsular size was greater in C. gattii (1.87 microm -/+1.47 microm) than in C. neoformans (0.83 microm -/+0.15 microm). Switching was observed mainly in isolates of C. gattii. All isolates expressed the enzyme urease, a lower activity of the proteases (Pz= 0.54), but a higher activity of the phospholipase (Pz=0.43) and phenoloxidase (Pz=0.003) was determined for C. gattii.     

Radical SAM enzymes in the biosynthesis of sugar-containing natural products

Carbohydrates play a key role in the biological activity of numerous natural products. In many instances their biosynthesis requires radical mediated rearrangements, some of which are catalyzed by radical SAM enzymes. BtrN is one such enzyme responsible for the dehydrogenation of a secondary alcohol in the biosynthesis of 2-deoxystreptamine. DesII is another example that catalyzes a deamination reaction necessary for the net C4 deoxygenation of a glucose derivative en route to desosamine formation. BtrN and DesII represent the two most extensively characterized radical SAM enzymes involved in carbohydrate biosynthesis. In this review, we summarize the biosynthetic roles of these two enzymes, their mechanisms of catalysis, the questions that have arisen during these investigations and the insight they can offer for furthering our understanding of radical SAM enzymology. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Inositol phospholipid metabolism in Arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C

Phosphoinositides (PIs) constitute a minor fraction of total cellular lipids in all eukaryotic cells. They fulfill many important functions through interaction with a wide range of cellular proteins. Members of distinct inositol lipid kinase families catalyze the synthesis of these phospholipids from phosphatidylinositol. The hydrolysis of PIs involves phosphatases and isoforms of PI-specific phospholipase C. Although our knowledge of the roles played by plant PIs is clearly limited at present, there is no doubt that they are involved in many physiological processes during plant growth and development. In this review, we concentrate on inositol lipid-metabolizing enzymes from the model plant Arabidopsis for which biochemical characterization data are available, namely the inositol lipid kinases and PI-specific phospholipase Cs. The biochemical properties and structure of characterized and genome-predicted isoforms are presented and compared with those of the animal enzymes to show that the plant enzymes have some features clearly unique to this kingdom.     

Steroidogenic enzymes and stem cell markers are upregulated during androgen deprivation in prostate cancer

Considerable levels of testosterone and dihydrotestosterone (DHT) are found in prostate cancer (PCa) tissue after androgen deprivation therapy. Treatment of surviving cancer-initiating cells and the ability to metabolize steroids from precursors may be the keystones for the appearance of recurrent tumors. To study this hypothesis, we assessed the expression of several steroidogenic enzymes and stem cell markers in clinical PCa samples and cell cultures during androgen depletion. Gene expression profiles were determined by microarray or qRT-PCR. In addition, we measured cell viability and analyzed stem cell marker expression in DuCaP cells by immunocytochemistry. Seventy patient samples from different stages of PCa, and the PCa cell line DuCaP were included in this study. The androgen receptor (AR) and enzymes (AKR1C3, HSD17B2, HSD17B3, UGT2B15 and UGT2B17 ) that are involved in the metabolism of adrenal steroids were upregulated in castration resistant prostate cancer (CRPC). In vitro, some DuCaP cells survived androgen depletion, and eventually gave rise to a culture adapted to these conditions. During and after this transition, most of the steroidogenic enzymes were upregulated. These cells also are enriched with stem/progenitor cell markers cytokeratin 5 (CK5) and ATP-binding cassette sub-family G member 2 (ABCG2). Similarly, putative stem/progenitor cell markers CK5, c-Kit, nestin, CD44, c-met, ALDH1A1, α2-integrin, CD133, ABCG2, CXCR4 and POU5F1 were upregulated in clinical CRPC. The upregulation of steroidogenic enzymes and stem cell markers in recurrent tumors suggests that cancer initiating cells can expand by adaptation to their T/DHT deprived environment. Therapies targeting the metabolism of adrenal steroids by the tumor may prove effective in preventing tumor regrowth.     

Stimulation of cell wall biosynthesis and structural changes in response to cytokinin- and elicitor-treatments of suspension-cultured Phaseolus vulgaris cells

Qualitative sugar flux into cell wall polysaccharides has been determined for two model systems. The first, treatment of suspension-cultured French bean (Phaseolus vulgaris L.) cells with an increase in the cytokinin/auxin ratio and in the concentration of sucrose, models some aspects of differentiation. Wall changes are characterised by up to a five-fold increase in thickness due to the laying down of extra wall material. Sugar flux following labelling of cells with [¹⁴C]-sucrose was examined during the period of maximum extractable catalytic activities of the enzymes of sugar nucleotide conversion determined previously. Increased secretion was observed in all major groups of polysaccharides, particularly the cellulosic fraction. Analysis of the sugars in the hemicellulosic fraction indicated that the newly synthesised polysaccharide was most probably xylan. It was confirmed by immunolocalisation of xylan in these walls. This treatment thus increases incorporation into the wall of components characteristic of secondary wall. In the second system, which models the defence response, suspension cultures were treated with an elicitor from the walls of Colletotrichum lindemuthianum. Again, sugar flux was determined by labelling cells with [¹⁴C]-sucrose and examined during the period determined previously of maximum extractable catalytic activities of the enzymes of sugar nucleotide conversion. Increased secretion into unextractable polymers was the major change and was consistent with the occurrence of oxidative processes leading to immobilisation of some wall components. Callose, a polysaccharide characteristic of the defence response was immunolocalised in these walls but not in those of control cells.     

Urea cycle disorder in C3H-H-2 degree mice with juvenile steatosis of viscera

We determined the activities of urea cycle enzymes in the liver of C3H-H-2 degree-jsv mice. The activities of all urea cycle enzymes decreased in the latter period of lactation. The activities of carbamylphosphate synthetase and ornithine transcarbamylase in some affected mice were undetectable. On the other hand, the activities of enzymes other than urea cycle enzymes were normal. We consider that the decrease in the urea cycle enzymes is caused by an abnormality in the mechanism of gene expression.     

Optimized extraction by cetyl trimethyl ammonium bromide reversed micelles of xylose reductase and xylitol dehydrogenase from Candida guilliermondii homogenate

The intracellular enzymes xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugar cane bagasse hydrolysate, were separated by reversed micelles of cetyl trimethyl ammonium bromide (CTAB) cationic surfactant. An experimental design was employed to optimize the extraction conditions of both enzymes. Under these conditions (temperature = 5 degree C, hexanol: isooctane proportion = 5% (v/v), 22 %, surfactant concentration = 0.15M, pH = 7.0 and electrical conductivity = 14 mScm(-1)) recovery values of about 100 and 80% were achieved for the enzymes XR and XD, respectively. The purity of XR and XD increased 5.6- and 1.8-fold, respectively. The extraction process caused some structural modifications in the enzymes molecules, as evidenced by the alteration of K(M) values determined before and after extraction, either in regard to the substrate (up 35% for XR and down 48% for XD) or cofactor (down 29% for XR and up 11% for XD). However, the average variation of V(max) values for both enzymes was not higher than 7%, indicating that the modified affinity of enzymes for their respective substrates and cofactors, as consequence of structural modifications suffered by them during the extraction, are compensated in some extension. This study demonstrated that liquid-liquid extraction by CTAB reversed micelles is an efficient process to separate the enzymes XR and XD present in the cell extract, and simultaneously increase the enzymatic activity and the purity of both enzymes produced by C. guilliermondii.     

Differential effects of pectolytic enzymes on tomato polyuronides in vivo and in vitro

There are marked differences between polyuronide degradation in vivo during tomato ripening and in vitro during the autolysis of cell wall preparations. Experiments using purified enzymes and enzymically inactive wall preparations show that the combined action of polygalacturonase (E.C. 3.2.1.15) and pectinesterase(E.C. 3.1.1.11)can mimic this in vitro autolysis of cell walls. Assuming these two enzymes are also responsible for polyuronide degradation in vivo their combined action must be restricted in some way.     

Broad-specificity GH131 β-glucanases are a hallmark of fungi and oomycetes that colonize plants

Plant-tissue-colonizing fungi fine-tune the deconstruction of plant-cell walls (PCW) using different sets of enzymes according to their lifestyle. However, some of these enzymes are conserved among fungi with dissimilar lifestyles. We identified genes from Glycoside Hydrolase family GH131 as commonly expressed during plant-tissue colonization by saprobic, pathogenic and symbiotic fungi. By searching all the publicly available genomes, we found that GH131-coding genes were widely distributed in the Dikarya subkingdom, except in Taphrinomycotina and Saccharomycotina, and in phytopathogenic Oomycetes, but neither other eukaryotes nor prokaryotes. The presence of GH131 in a species was correlated with its association with plants as symbiont, pathogen or saprobe. We propose that GH131-family expansions and horizontal-gene transfers contributed to this adaptation. We analysed the biochemical activities of GH131 enzymes whose genes were upregulated during plant-tissue colonization in a saprobe (Pycnoporus sanguineus), a plant symbiont (Laccaria bicolor) and three hemibiotrophic-plant pathogens (Colletotrichum higginsianum, C. graminicola, Zymoseptoria tritici). These enzymes were all active on substrates with β-1,4, β-1,3 and mixed β-1,4/1,3 glucosidic linkages. Combined with a cellobiohydrolase, GH131 enzymes enhanced cellulose degradation. We propose that secreted GH131 enzymes unlock the PCW barrier and allow further deconstruction by other enzymes during plant tissue colonization by symbionts, pathogens and saprobes.     

The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>

Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO₂ in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.     

Structural and biochemical studies of GH family 12 cellulases: improved thermal stability, and ligand complexes

In this review we will describe how we have gathered structural and biochemical information from several homologous cellulases from one class of glycoside hydrolases (GH family 12), and used this information within the framework of a protein-engineering program for the design of new variants of these enzymes. These variants have been characterized to identify some of the positions and the types of mutations in the enzymes that are responsible for some of the biochemical differences in thermal stability and activity between the homologous enzymes. In this process we have solved the three-dimensional structure of four of these homologous GH 12 cellulases: Three fungal enzymes, Humicola grisea Cel12A, Hypocrea jecorina Cel12A and Hypocrea schweinitzii Cel12A, and one bacterial, Streptomyces sp. 11AG8 Cel12A. We have also determined the three-dimensional structures of the two most stable H. jecorina Cel12A variants. In addition, four ligand-complex structures of the wild-type H. grisea Cel12A enzyme have been solved and have made it possible to characterize some of the interactions between substrate and enzyme. The structural and biochemical studies of these related GH 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can serve as a structural toolbox for the design of Cel12A enzymes with specific properties and features suited to existing or new applications.     

Protein kinase C regulatory domains: the art of decoding many different signals in membranes

Protein kinase C (PKC) is a member of a family of Ser/Thr phosphotransferases that are involved in many cellular signaling pathways. These enzymes possess two regulatory domains, C1 and C2, that are the targets of different second messengers. The purpose of this review is to describe in molecular terms the diverse mechanisms of activation of PKCs in the light of very significant advances made in this field over recent years. The role of some critical amino acid residues concerning activation of the enzymes and their location within known structures of isolated domains will be presented. For example, the recently deduced 3D structures of the C2 domains show that these domains can additionally act as PtdIns(4,5)P(2)-binding or phosphotyrosine-binding modules depending on the isoenzyme. All these capacities to play different roles in the cell wide web of signals underline the notion that we are dealing with a multifunctional family of enzymes which, after 30 years of investigation, we are just beginning to understand.     

The roles of residues Tyr150, Glu272, and His314 in class C β-lactamases

Serine β-lactamases contribute widely to the β-lactam resistance phenomena. Unfortunately, the intimate details of their catalytic mechanism remain elusive and subject to some controversy even though many “natural” and “artificial” mutants of these different enzymes have been isolated. This paper is essentially focused on class C β-lactamases, which contain a Tyr (Tyr150) as the first residue of the second conserved element, in contrast to their class A counterparts, in which a Ser is found in the corresponding position. We have modified this Tyr residue by site-directed mutagenesis. On the basis of the three-dimensional structure of the Enterobacter cloacae P99 enzyme, it seemed that residues Glu272 and His314 might also be important. They were similarly substituted. The modified enzymes were isolated and their catalytic properties determined. Our results indicated that His314 was not required for catalysis and that Glu272 did not play an important role in acylation but was involved to a small extent in the deacylation process. Conversely, Tyr150 was confirmed to be central for catalysis, at least with the best substrates. On the basis of a comparison of data obtained for several class C enzyme mutants and in agreement with recent structural data, we propose that the phenolate anion of Tyr150, in conjunction with the alkyl ammonium of Lys315, acts as the general base responsible for the activation of the active-site Ser64 during the acylation step and for the subsequent activation of a water molecule in the deacylation process. The evolution of the important superfamily of penicillin-recognizing enzymes is further discussed in the light of this proposed mechanism. © 1996 Wiley-Liss, Inc.     

An overview on nucleases (DNase,RNase, and phosphodiesterase) in snake venoms

In this review, we have compiled the data on pharmacological activities associated with endogenous purine release related enzymes—nucleases (DNases, RNases, and phosphodiesterases). The results of studies on toxic effects of these enzymes, emphasizing the future directions in this field, are summarized. One of the major problems facing toxicologists is the identification and characterization of specific venom nucleases since they share similar substrate specificities and biochemical properties. In this review, we have attempted to clarify some of the discrepancies about these enzymes. Further, we have tried to correlate the existence of nuclease enzymes in relation to endogenous release of purines, a multitoxin, during snake envenomation, and we also discuss the possible actions of purines. We hope that this review will stimulate renewed interest among toxicologists to biologically characterize these enzymes and elucidate their role in envenomation.     

Crab digestive lipase acting at high temperature: purification and biochemical characterization

In recent years, recovery and characterization of enzymes from fish and aquatic invertebrates have taken place and this had led to the emergence of some interesting new applications of these enzymes. However, much less is known about lipases from crustaceans. A lipolytic activity was located in the crab digestive glands (hepatopancreas), from which a crab digestive lipase (CDL) was purified. Pure CDL has a molecular mass of 65kDa as determined by SDS/PAGE analysis. Unlike known digestive lipases, CDL displayed its maximal activity on long and short-chain triacylglycerols at a temperature of 60 degrees C. A specific activity of 500U/mg or 130U/mg was obtained with TC(4) or olive oil as substrate, respectively. Only 10% of the maximal activity was detected at 37 degrees C. The enzyme retained 80% of its maximal activity when incubated during 10 min at 60 degrees C, and was completely inactivated at a temperature higher than 65 degrees C. Interestingly, neither colipase, nor bile salts were detected in the crab hepatopancreas. Which suggests that colipase evolved in invertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produce bile salts. No similarity between the 13 N-terminal amino acid residues of CDL was found with those of known other digestive lipases.     

Interaction of enzymes involved in triosephosphate metabolism. Comparison of yeast and rabbit muscle cytoplasmic systems

The affinity of baker's yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphate aldolase towards the metabolically related enzymes phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase was tested by using a fluorescence-probe technique with fluorescein isothiocyanate attached covalently to the enzymes. The dissociation constants of the enzyme-enzyme complexes, as well as the rate constants of association and dissociation, were determined. Data were compared with the parameters derived from a mammalian (rabbit muscle) system, known from the literature and determined under the same conditions (pH 7.5 or 8.5 in 0.05 M Tris/HCl buffer at 20 degrees C). The comparison reveals similarities in the supramolecular organization of these cytoplasmic enzymes in phylogenetically distant species. Moreover, the fact that in vitro hybrid complexes are formed of stability comparable to that of non-hybrid complexes indicates that this ancient characteristic is probably conserved during evolution. A possible regulatory mechanism is presented, based on the dynamic competition, with each other, of the enzymes involved in triosephosphate metabolism.