Morpho-functional analysis of isogenic strains of Escherichia coli-- producers of penicillinacylases

A population of Escherichia coli strains producing penicillinacylase (PA) and differing in the level of their enzyme activity was studied. Their structural-functional analysis showed that selection according to the property of PA production yielded strains with aberrations in the processes of cell division. The population of microbial cells producing PA had a correlation between its morphological composition and enzyme activity, and the two characteristics depended on the conditions under which the strains were cultivated. The highest enzyme activity was exerted by normally dividing cells. A type of colonies optimal for stabilising the level of PA production was determined for the enzyme-producing strain. The structure of cell envelopes, their composition and permeability changed considerably as the ability to synthesize PA increased. The results allow one to specify further rational selection of strains superproducing the enzyme on the basis of changes in the membrane permeability.     

A novel protein kinase target for the lipid second messenger phosphatidic acid.

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.     

Palmitate increases superoxide production through mitochondrial electron transport chain and NADPH oxidase activity in skeletal muscle cells

The effect of unbound palmitic acid (PA) at plasma physiological concentration range on reactive oxygen species (ROS) production by cultured rat skeletal muscle cells was investigated. The participation of the main sites of ROS production was also examined. Production of ROS was evaluated by cytochrome c reduction and dihydroethidium oxidation assays. PA increased ROS production after 1 h incubation. A xanthine oxidase inhibitor did not change PA-induced ROS production. However, the treatment with a mitochondrial uncoupler and mitochondrial complex III inhibitor decreased superoxide production induced by PA. The importance of mitochondria was also evaluated in 1 h incubated rat soleus and extensor digitorum longus (EDL) muscles. Soleus muscle, which has a greater number of mitochondria than EDL, showed a higher superoxide production induced by PA. These results indicate that mitochondrial electron transport chain is an important contributor for superoxide formation induced by PA in skeletal muscle. Results obtained with etomoxir and bromopalmitate treatment indicate that PA has to be oxidized to raise ROS production. A partial inhibition of superoxide formation induced by PA was observed by treatment with diphenylene iodonium, an inhibitor of NADPH oxidase. The participation of this enzyme complex was confirmed through an increase of p47(phox) phosphorylation after treatment with PA.     

Improvement of posttranslational bottlenecks in the production of penicillin amidase in recombinant Escherichia coli strains

Using periplasmic penicillin amidase (PA) from Escherichia coli ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant E. coli expression hosts. Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production. Rate constants for both intracellular proteolytic breakdown (k(d)) and transport (k(t)) were used as quantitative tools for selection of the appropriate host system and cultivation medium. The production of mature active PA was increased up to 10-fold when the protease-deficient strain E. coli BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants k(d) and k(t). The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different E. coli host strains. Furthermore, we established that simultaneous coexpression of the OmpT pac gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production. In parallel, we made efforts to increase PA flux via coexpression with the kil gene (killing protein). The primary effects of the kil gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture.     

Differences between fruit-bearing and non-bearing apple spurs in activity of an enzyme system decomposing phloridzin

The activity of an enzyme system decomposing phloridzin was investigated in fruitbearing and nonbearing spurs of apple trees, Landsberger Reinette cv., throughout vegetation. Acetone powder obtained from xylem sap of apple spurs was incubated with phloridzinsubstratum in citric buffer at pH 5.5 for 12, 18 and 24 h at 30 °C. A paper and thin-layer chromatography as well aa a spectrophotometric assay were employed for tentative identification of enzymic degradation products. Phloretic acid (PA), co-factor of IAA-oxidase, as well as phloretin (Pin), and phloroglucinol (PI) were found after the digestion of phloridzin. The chromatographed enzyme reaction products were measured densitometrically. The activity of the enzyme system was estimated by its efficiency in PA production and phloridzin disappearance. Obtained values, expressed in percentages, showed that the enzyme activity in fruitbearing spurs was much higher than in nonbearing ones; 30 and 10% of released PA in July, respectively. Because fruitbearing spurs of the apple tree are possibly additionally supplied with auxin translocated from developing seeds, an adaptive character of the enzyme system producing PA, a known auxin repressor, is suggested.     

A novel protein kinase target for the lipid second messenger phosphatidic acid

Activation of phospholipase D occurs in response to a wide variety of hormones, growth factors, and other extracellular signals. The initial product of phospholipase D, phosphatidic acid (PA), is thought to serve a signaling function, but the intracellular targets for this lipid second messenger are not clearly identified. The production of PA in human neutrophils is closely correlated with the activation of NADPH oxidase, the enzyme responsible for the respiratory burst. We have developed a cell-free system, in which the activation of NADPH oxidase is induced by the addition of PA. Characterization of this system revealed that a multi-functional cytosolic protein kinase was a target for PA, and that two NADPH oxidase components were substrates for the enzyme. Partial purification of the PA-activated protein kinase separated the enzyme from known protein kinase targets of PA. The partially purified enzyme was selectively activated by PA, compared to other phospholipids, and phosphorylated the oxidase component p47-phox on both serine and tyrosine residues. PA-activated protein kinase activity was present in a variety of hematopoietic cells and cell lines and in rat brain, suggesting it has widespread distribution. We conclude that this protein kinase may be a novel target for the second messenger function of PA.     

Mutations in the translation initiation region of the <Emphasis Type="Italic">pac</Emphasis> gene resulting in increased levels of activity of penicillin G acylase

Penicillin G acylase (PA) is an important enzyme used in the industrial production of b-lactam antibiotics. In this study, the effects of mutations in the translation initiation region of the Escherichia coli pac gene, encoding periplasmic PA, were examined. Several mutations led to increased amounts of PA activity, including those that lengthened the spacer region between the ribosome binding site and the ATG start codon, and those with altered codons on positions +2 and +4 relative to the start codon. These results indicated that the wild-type sequence of the pac gene does not provide maximum expression levels and that the strategies applied in this study can be used to improve production of PA in E. coli. Unexpectedly, our study also suggested that translocation of PA was, in contrast to earlier reports, shown not to require the Twin-arginine translocation pathway for transport into the periplasm.     

Mammary plasminogen activator: correlation with involution, hormonal modulation and comparison between normal and neoplastic tissue.

We have analyzed the plasminogen activator (PA) content of normal rodent mammary glands at different stages of the mammary life cycle and after exposing the animals to various hormones; we have also assessed the PA response of mammary explants to a variety of hormonal environments. Similar studies were performed on a limited number of primary mammary tumors. Plasminogen activator production was clearly correlated with mammary involution. A large but transient increase in enzyme content followed the initiation of involution in all glands, and the enzyme was produced by mammary cells, not by macrophages or granulocytes. Oxytocin, prolactin and hydrocortisone, which slowed or blocked involution, produced parallel effects on gland regression and PA synthesis. PA synthesis by explants in organ culture was induced by hormonal environments that fostered involution and repressed by those that promoted lactation. Mammary tumors produced much more PA than normal tissue both in vivo and in vitro, and distinct differences were found in the response of enzyme synthesis to hormones. The results reinforce the association of PA with tissue remodeling; show that the enzyme can be used as an indicator of cellular response to a wide range of hormones in both normal and malignant tissue; and suggest that observations of this type in organ culture may be of some value in predicting physiological responses in vivo.     

A divergent Pseudomonas aeruginosa palmitoyltransferase essential for cystic fibrosis‐specific lipid A

Strains of Pseudomonas aeruginosa (PA) isolated from the airways of cystic fibrosis patients constitutively add palmitate to lipid A, the membrane anchor of lipopolysaccharide. The PhoPQ regulated enzyme PagP is responsible for the transfer of palmitate from outer membrane phospholipids to lipid A. This enzyme had previously been identified in many pathogenic Gram‐negative bacteria, but in PA had remained elusive, despite abundant evidence that its lipid A contains palmitate. Using a combined genetic and biochemical approach, we identified PA1343 as the PA gene encoding PagP. Although PA1343 lacks obvious primary structural similarity with known PagP enzymes, the β‐barrel tertiary structure with an interior hydrocarbon ruler appears to be conserved. PA PagP transfers palmitate to the 3′ position of lipid A, in contrast to the 2 position seen with the enterobacterial PagP. Palmitoylated PA lipid A alters host innate immune responses, including increased resistance to some antimicrobial peptides and an elevated pro‐inflammatory response, consistent with the synthesis of a hexa‐acylated structure preferentially recognized by the TLR4/MD2 complex. Palmitoylation commonly confers resistance to cationic antimicrobial peptides, however, increased cytokine production resulting in inflammation is not seen with other palmitoylated lipid A, indicating a unique role for this modification in PA pathogenesis.     

Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production.

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.     

Role of the intracellular proteolysis in the production of the periplasmic penicillin amidase in Escherichia coli

The yield of periplasmic enzyme, penicillin amidase (PA), from E. coli ATCC 11105 is regulated at the post-translational level by two competitive processes-intracellular proteolysis of newly synthesised pre-pro-PA (ppPA) in the cytoplasm and membrane transport and maturation of ppPA. Intracellular proteolysis results in a significant loss in the yield of active PA. Immunochemical analysis were used to study the influence of the cultivation temperature, phenylacetic acid, and glucose on the proteolysis of the ppPA in the cytoplasm. An increase in glucose concentrations or temperature during the cultivation resulted in a significant loss of PA activity due to the increased rate of intracellular proteolysis of ppPA. Addition of phenylacetic acid reduced the intracellular proteolysis of ppPA and, as a consequence, increased the PA production. Taken together, these data explain a new aspect of regulation of active PA production in E. coli.     

Improvement of Posttranslational Bottlenecks in the Production of Penicillin Amidase in Recombinant Escherichiacoli Strains

Using periplasmic penicillin amidase (PA) from Escherichia coli ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant E. coli expression hosts. Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production. Rate constants for both intracellular proteolytic breakdown (k_d) and transport (k_t) were used as quantitative tools for selection of the appropriate host system and cultivation medium. The production of mature active PA was increased up to 10-fold when the protease-deficient strain E. coli BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants k_d and k_t. The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different E. coli host strains. Furthermore, we established that simultaneous coexpression of the OmpT pac gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production. In parallel, we made efforts to increase PA flux via coexpression with the kil gene (killing protein). The primary effects of the kil gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture.     

Nitric oxide is critical for inducing phosphatidic acid accumulation in xylanase-elicited tomato cells

Nitric Oxide (NO) is a second messenger related to development and (a)biotic stress responses in plants. We have studied the role of NO in signaling during plant defense responses upon xylanase elicitation. Treatment of tomato cell cultures with the fungal elicitor xylanase resulted in a rapid and dose-dependent NO accumulation. We have demonstrated that NO is required for the production of the lipid second messenger phosphatidic acid (PA) via the activation of the phospholipase C (PLC) and diacylglycerol kinase (DGK) pathway. Defense-related responses downstream of PA were studied. PA and, correspondingly, xylanase were shown to induce reactive oxygen species production. Scavenging of NO or inhibition of either the PLC or the DGK enzyme diminished xylanase-induced reactive oxygen species production. Xylanase-induced PLDbeta1 and PR1 mRNA levels decreased when NO or PA production were compromised. Finally, we have shown that NO and PA are involved in the induction of cell death by xylanase. Treatment with NO scavenger cPTIO, PLC inhibitor U73122, or DGK inhibitor R59022 diminished xylanase-induced cell death. On the basis of biochemical and pharmacological experimental results, we have shown that PLC/DGK-derived PA represents a novel downstream component of NO signaling cascade during plant defense.     

Conjugation of penicillin acylase with the reactive copolymer of N-isopropylacrylamide: a step toward a thermosensitive industrial biocatalyst

Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 10(6) and, presumably, cross-linked protein-polymer aggregates of MW > 10(7). Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 microM) was found to be slightly higher than that of the free enzyme (12 microM), and evaluation of V(max) testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from d-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of beta-lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM).     

Urokinase-type and tissue-type plasminogen activators are essential for in vitro invasion of human melanoma cells

This study evaluates the contribution of two types of plasminogen activators (PAs; tissue-type PA (tPA) versus urokinase-type PA (uPA) toward the invasiveness of human melanoma cells in a novel in vitro assay. We identified two human melanoma cell lines, MelJuso and MeWo, expressing uPA or tPA as shown at mRNA, protein, and enzyme activity level. MelJuso cells produced uPA as well as plasminogen activator inhibitor-1 (PAI-1). The latter was, however, not sufficient to neutralize the cell-associated or secreted uPA activity. MeWo cells secreted tPA, but the enzyme was not found to be cell-associated. PAI-1 production by these cells was not detectable. Plasminogen activation and fibrinolytic capacity of both cell lines were reduced by anticatalytic monoclonal antibodies specific for the respective type of PA or by aprotinin. In a novel in vitro invasion assay, antibodies to PA as well as aprotinin decreased the invasiveness of both cell lines into a fibrin gel, Matrigel, or intact extracellular matrix. Our results confirm the importance of uPA-catalyzed plasminogen activation in tumor cell invasiveness. Furthermore, we provide evidence that tPA, beyond its key role in thrombolysis, can also be involved in in vitro invasion of human melanoma cells.     

Antioxidants successfully reduce ROS production in propionic acidemia fibroblasts

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. Most PA patients present in the neonatal period with metabolic acidosis and hyperammonemia, developing different neurological symptoms, movement disorders and cardiac complications. There is strong evidence indicating that oxidative damage could be a pathogenic factor in neurodegenerative, mitochondrial and metabolic diseases. Recently, we identified an increase in ROS levels in PA patients-derived fibroblasts. Here, we analyze the capability of seven antioxidants to scavenge ROS production in PA patients’ cells. Tiron, trolox, resveratrol and MitoQ significantly reduced ROS content in patients and controls’ fibroblasts. In addition, changes in the expression of two antioxidant enzymes, superoxide dismutase and glutathione peroxidase, were observed in PA patients-derived fibroblasts after tiron and resveratrol treatment. Our results in PA cellular models establish the proof of concept of the potential of antioxidants as an adjuvant therapy for PA and pave the way for future assessment of antioxidant strategies in the murine model of PA.     

Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles

Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca-interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps.     

Role of casein on induction and enhancement of production of a bacterial milk clotting protease from an indigenously isolated Bacillus subtilis

Aims: To isolate and enhance the yield of a bacterial milk clotting protease (MCP) through process optimization and scale up. Materials and Results: Bacillus subtilis was isolated as MCP producer with good milk clotting activity (MCA) per proteolytic activity (PA) index. The enzyme production was inducible with casein and enhanced with fructose and ammonium nitrate resulting in 571·43 U ml^?1 of enzyme. Conclusions: Medium containing 4% fructose, 0·75% casein, 0·3% NH₄NO₃ and 10 mmol l^–1 CaCl₂, pH 6·0, inoculated with 4% (v/v) inoculum, incubated at 37°C, 200 rev min^?1 for 72 h gave maximum production. A 6·67‐fold increase in MCP yield with very high MCA per PA index was observed after final optimization indicating similarity to rennets. Significance and Impact of the Study: Mostly fungal MCPs have been reported. The MCA and MCA per PA index of this bacterium is comparable to that of many fungal reports and better than quite a few bacterial MCPs. Thus, this enzyme by B. subtilis has good probability of successful use in cheese production.     

Study the influence of culture conditions on rennin production by Rhizomucor miehei using solid-state fermentations

Investigations were conducted on the production of Rennin enzyme from the fungi Rhizomucor miehei 3420 NRRL using Solid-State fermentation. Wheat bran was used as a substrate. The influence of moisture content, incubation temperature, and the initial pH of fermentation medium were studied. The protein content, milk clotting activity (MCA), specific activity, proteolytic activity (PA), and (MCA/PA) ratio of the extracted enzyme were calculated after 4 days of incubation to evaluate the quality of the enzyme. The results showed that the optimal conditions for production were as follows: incubation temperature of 40 °C, moisture content of 60%, and pH of (3). Under these conditions, a production process of Rennin enzyme was established, and the values of protein content, milk clotting activity, specific activity, proteolytic activity, and (MCA/PA) ratio reached to 4 mg/mL, 600 SU/mL, 150 SU/mg, 45 PU/mL, 13.3 respectively.