A high-throughput screening for phosphatases using specific substrates

A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.     

Fluorogenic probes for imaging cellular phosphatase activity

The ability to visualize enzyme activity in a cell, tissue, or living organism can greatly enhance our understanding of the biological roles of that enzyme. While many aspects of cellular signaling are controlled by reversible protein phosphorylation, our understanding of the biological roles of the protein phosphatases involved is limited. Here, we provide an overview of progress toward the development of fluorescent probes that can be used to visualize the activity of protein phosphatases. Significant advances include the development of probes with visible and near-infrared (near-IR) excitation and emission profiles, which provides greater tissue and whole-animal imaging capabilities. In addition, the development of peptide-based probes has provided some selectivity for a phosphatase of interest. Key challenges involve the difficulty of achieving sufficient selectivity for an individual member of a phosphatase enzyme family and the necessity of fully validating the best probes before they can be adopted widely.     

Spatial Patterns of Alkaline Phosphatase Expression within Bacterial Colonies and Biofilms in Response to Phosphate Starvation

The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in both Klebsiella pneumoniae and Pseudomonas aeruginosa biofilms. Some of the expression patterns observed revealed alkaline phosphatase activity at the boundary of the biofilm opposite the place where the staining substrate was delivered, indicating that the enzyme substrate penetrated the biofilm fully. Alkaline phosphatase accumulated linearly with time in K. pneumoniae colonies transferred from high-phosphate medium to low-phosphate medium up to specific activities of 50 μmol per min per mg of protein after 24 h. In K. pneumoniae biofilms and colonies, alkaline phosphatase was initially expressed in the region of the biofilm immediately adjacent to the carbon and energy source (glucose). In time, the region of alkaline phosphatase expression expanded inward until it spanned most, but not all, of the biofilm or colony depth. In contrast, expression of alkaline phosphatase in P. aeruginosa biofilms occurred in a thin, sharply delineated band at the biofilm-bulk fluid interface. In this case, the band of activity never occupied more than approximately one-sixth of the biofilm. These results are consistent with the working hypothesis that alkaline phosphatase expression patterns are primarily controlled by the local availability of either the carbon and energy source or the electron acceptor.     

Structural versatility that serves the function of the HRD motif in the catalytic loop of protein tyrosine kinase,Src

Site‐directed mutagenesis is a traditional approach for structure–function analysis of protein tyrosine kinases, and it requires the generation, expression, purification, and analysis of each mutant enzyme. In this study, we report a versatile high throughput bacterial screening system that can identify functional kinase mutants by immunological detection of tyrosine phosphorylation. Two key features of this screening system are noteworthy. First, instead of blotting bacterial colonies directly from Agar plates to nitrocellulose membrane, the colonies were cultured in 96‐well plates, and then spotted in duplicate onto the membrane with appropriate controls. This made the screening much more reliable compared with direct colony blotting transfer. A second feature is the parallel use of a protein tyrosine phosphatase (PTP)‐expressing host and a non‐PTP‐expressing host. Because high activity Src mutants are toxic to the host, the PTP system allowed the identification of Src mutants with high activity, while the non‐PTP system identified Src mutants with low activity. This approach was applied to Src mutant libraries randomized in the highly conserved HRD motif in the catalytic loop, and revealed that structurally diverse residues can replace the His and Arg residues, while the Asp residue is irreplaceable for catalytic activity.     

Bacterial Cell Surface Display of an Enzyme Library for Selective Screening of Improved Cellulase Variants

The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.     

Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants

The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.     

β-lactamase as a probe of membrane protein assembly and protein export

The enzyme TEM beta-lactamase constitutes a versatile gene-fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Cells producing translocated forms of beta-lactamase can be directly selected as ampicillin-resistant colonies, and consequently a beta-lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic beta-lactamase derivative depends on its level of expression, and therefore a beta-lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane-bound gene products expressed in E. coli.     

A Rapid Colony Test for Thiaminase Activity

A rapid method for the detection of very small amounts of thiaminase activity produced by bacterial colonies is described. It can be used routinely in screening for thiaminase-producing bacteria and other colony-forming micro-organisms and offers considerable advantages over other methods.     

Lysozyme, alkaline phosphatase and neutrophils in uterine secretions of mares with differing resistance to endometritis

A study was conducted to 1) determine differences in the inflammatory response following bacterial challenge between normal mares and mares with chronic endometritis and 2) to determine if enzyme activity in uterine fluid can be used to evaluate degree of inflammation in the equine uterus. Six normal mares (Group 1) and four mares with chronic endometritis (Group 2) received an intrauterine infusion of beta-hemolytic streptococci on the second day of estrus. Neutrophil concentration as well as lysozyme and alkaline phosphatase activity were determined in uterine secretions obtained by placing tampons in the uterus of mares. All mares had a similar inflammatory response following bacterial challenge of the uterus, as indicated by a neutrophil response of the same magnitude. Neutrophil numbers, lysozyme and alkaline phosphatase concentrations were all increased 12 h postinoculation and declined rapidly to normal preinoculation values by 48 h after inoculation. In spite of the similarity of the clinical signs, neutrophil concentrations and enzyme activity, mares in group 1 demonstrated a markedly higher ability to eliminate the infection than mares in group 2. It is concluded that factors other than neutrophil numbers, lysozyme and alkaline phosphatase activity account for the inability of the mare to eliminate uterine infections.     

Assay of aminoglycoside phosphotransferase in situ

A method for the evaluation of the aminoglycoside phosphotransferase activity in bacterial colonies directly is described. The method is based on the ability of the enzyme to modify the substrate immobilized on carboxymethylcellulose paper. The sensitivity and accuracy of the method were tested by comparing the results of the present assay to those obtained with conventional procedures. The method seems to be particularly useful for the detection within a bacterial population producing aminoglycoside phosphotransferase of those cells which do not make the enzyme and for rapid determination of the relative levels of enzyme produced by different clones.     

Molecular cloning of cDNA for rat glycine methyltransferase

Using a highly purified preparation of glycine methyltransferase mRNA, double-stranded cDNA was synthesized and inserted into the PstI site of pBR322. The resulting recombinant DNA was used to transform E. coli X 1776 by conventional methods. Among tetracycline-resistant transformants, a number of colonies were found to contain cDNA sequence for glycine methyltransferase as examined by hybrid-selected translation. A restriction endonuclease cleavage map was constructed covering about 720 base pairs. With the cDNA as the probe, the content of the glycine methyltransferase mRNA was quantitated in various rat tissues and was found to be proportional to the specific enzyme activity.     

Optical processing of bacterial libraries for directed evolution

Selection of phenotypically distinct bacterial colonies on a Petri dish is typically performed by one of two methods: chemical or mechanical. Chemical methods (e.g., antibiotic selection) rely on inherent growth advantages of the unique phenotypes desired and thus have limited applicability. Mechanical methods are generally slow and require relatively large colonies (typically hundreds of colonies per plate). Here the use of imaged light to select bacterial colonies is explored, employing either photodynamic therapy agents or a ferrochelatase mutation in combination with porphyrin precursors to sensitize the bacteria to light and a computer-controlled light projection system to illuminate some bacterial colonies while leaving others in the dark. A CCD camera was used to distinguish between bacteria expressing green fluorescent protein (GFP) from nonfluorescent colonies. The fluorescence image from the camera was then used to create a virtual masking image for photoselection. Using a simple commercial projector it was possible to confer a 56-fold selective advantage to colonies expressing GFP. This represents a potentially powerful tool in directed evolution experiments using large libraries.     

Synthesis of 2-arylbenzothiazole derivatives and their application in bacterial detection

A series of 2-arylbenzothiazole derivatives have been prepared as fluorogenic enzyme substrates in order to detect aminopeptidase, esterase, phosphatase and β-galactosidase activity in clinically important Gram-negative and Gram-positive bacteria. Substrates were incorporated into an agar-based culture medium and this allowed growth of intensely fluorescent bacterial colonies based on hydrolysis by specific enzymes. Substrate 20 targeted l-alanine aminopeptidase activity and was hydrolysed exclusively by a range of Gram-negative bacteria and inhibited the growth of a range of Gram-positive bacteria. Substrate 19a targeted β-alanyl aminopeptidase activity and generated fluorescent colonies of selected Gram-negative species including Pseudomonas aeruginosa. Substrate 21b targeted C8-esterase activity and resulted in strongly fluorescent colonies of selected species known to harbour such enzyme activity (e.g., Salmonella and Pseudomonas). Most Gram-negative species produced colonies with an intense blue fluorescence due to hydrolysis of phosphatase substrates 24a–c and substrate 24c was also hydrolysed by strains of Staphylococcus aureus. Compounds 26b and 26c targeted β-galactosidase activity and generated strongly fluorescent colonies with coliform bacteria that produced this enzyme (e.g., Escherichia coli).     

一株产高酶活性碱性磷酸酶解淀粉芽孢杆菌的分离及其phoD碱性磷酸酶基因的克隆与表达

[背景]碱性磷酸酶作为工具酶被广泛应用于各个领域,在免疫学检测方面应用较多的是PhoA家族的碱性磷酸酶,尚无关于PhoD家族的碱性磷酸酶在免疫学检测方面的研究。[目的]筛选出一株产高酶活性PhoD家族碱性磷酸酶的细菌,并将其phoD基因进行克隆表达,研究PhoD的酶学性质,为PhoD家族的碱性磷酸酶在免疫学检测方面的应用奠定一定的基础。[方法]采取有机质丰富的土样在有机磷平板中进行细菌分离,以4-硝基苯磷酸二钠盐（4-nitrophenyl phosphate disodium salt hexahydrate,p-NPP）为底物测定有机磷平板中单菌落的酶活性,选取酶活性高的菌株作为目的菌株,克隆其phoD基因。[结果]筛选到一株产碱性磷酸酶酶活性高的菌株S2-4,通过16S rRNA基因序列同源性比较分析,鉴定该菌株为解淀粉芽孢杆菌,克隆了其phoD基因并进行诱导表达。研究了纯化后PhoD的酶学性质,PhoD的最适反应温度为70℃;最适反应pH为9.8;PhoD最适Ca²⁺浓度为3 mmol/L,Mg²⁺对PhoD的酶活性有抑制作用,K     

Biochemical and genetic analysis of a mutant with altered alkaline phosphatase activity in Dictyostelium discoideum

Alkaline phosphatase is one of several enzymes that accumulate in a temporally regulated sequence during the development of Dictyostelium discoideum. These enzymes can be used to monitor specific gene expression; moreover, isolation and analysis of mutations in the structural gene(s) can serve to indicate some of the essential steps in programmed synthesis and morphogenesis. A mutation (alpA) which affects the activity and substrate affinity of alkaline phosphatase was isolated in D discoideum using a procedure for screening large numbers of clones. Alkaline phosphatase activity at all stages of vegetative growth and development was altered by the mutation. Several physical properties of the enzyme from growing cells and developed cells were compared and found to be indistinguishable. It is likely that a single enzyme is responsible for the majority of alkaline phosphatase activity in growth and development. The mutation is coexpressed in diploids heterozygous for alpA and maps to linkage group III. One of the haploid segregants isolated from these diploids carries convenient markers on each of the six defined linkage groups and can be used for linkage analysis of other genetic loci.     

Micromethods for the spectrophotometric determination of bacterial protease activities

Microassays for the spectrophotometric determination of bacterial proteases were developed using congo red elastin, a substrate specific for elastolytic activity, and hide powder azure, a substrate sensitive to more general proteolytic activity. The small reaction volume (0.1 ml) allows incubation, filtration and quantitation to be carried out in 96 well microassay plates. Using a simple spin filtration device constructed from microassay plates a large number (768) of microassays can be filtered simultaneously. The microassays are particularly useful for screening large numbers of bacterial colonies for proteolytic mutants since they allow the rapid and efficient handling of multiple samples. These assays also permit the qualitative estimation of enzyme levels.     

Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae.

Gene fusions between the cholera toxin structural genes and phoA, which encodes bacterial alkaline phosphatase, were identified after TnphoA mutagenesis of the cloned genes in Escherichia coli and were then mobilized into Vibrio cholerae. The activities of the hybrid proteins were detectable in V. cholerae and suggested that, like cholera toxin, they were secreted beyond the cytoplasm. To extend the utility of TnphoA to identify additional genetic export signals in V. cholerae and other gram-negative bacteria, TnphoA delivery vectors utilizing broad-host-range plasmids were developed. By using V. cholerae as a model system, insertion mutants carrying active phoA gene fusions were identified as colonies expressing alkaline phosphatase, which appeared blue on agar containing the indicator 5-bromo-4-chloro-3-indolyl phosphate. Since alkaline phosphatase is active only upon export from the cytoplasm, PhoA+ colonies resulting from the mutagenesis procedure were enriched for insertions in genes that encode secreted proteins. Insertion mutations were identified in the gene encoding a major outer membrane protein, OmpV, and in tcpA, which encodes a pilus (fimbrial) subunit. Mutant strains harboring chromosomal insertions isolated in this manner can be used to assess the role of the corresponding inactivated gene products on survival of V. cholerae in vivo. The expression of the hybrid proteins as determined by measuring alkaline phosphatase activity also allowed the convenient study of virulence gene expression.     

Induction of enhanced alkaline phosphatase activity in the melano-macrophage centres of Oreochromis aureus (Steindachner) through starvation and vaccination

Employing histochemical methods, alkaline phosphatase activity was studied in the melano-macrophage centres of the spleen of the cichlid fish Oreochromis aureus (Steindachner). Enzyme activity was observed to be very low in normal fish. Prolonged starvation induced an enhanced enzyme response. Starvation followed by antigenic stimulation through an intraperitoneal injection of a bacterial vaccine further elevated the levels of alkaline phosphatase activity. The marked response of the pigment-bearing macrophages to bacterial antigen provides further evidence of the lymphoreticular nature of these pigmented cell aggregates. The association of alkaline phosphatase activity and lipofuscin (the most common pigment in fish melano-macrophage centres) with phagocytic cells has been documented in higher animals including     

Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells

The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2–5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.     

Bacterial IMPDH gene used for the selection of mammalian cell transfectants

Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of the drug mycophenolic acid, which is an IMPDH inhibitor. We have demonstrated that transfection of human, monkey or Chinese hamster cell lines with an expression vector containing bacterial IMPDH and mycophenolic acid treatment resulted in the selection of colonies with a strikingly increased resistance to mycophenolic acid toxicity. Analysis of cells derived from these colonies indicated that the acquisition of this resistance was associated with bacterial IMPDH protein expression. As a proof of principle, we showed that mammalian cell transfection with a bicistronic IMPDH/GFP expression vector and mycophenolic acid treatment can be used to successfully select transfectants that express the fluorescent protein. These results indicate that bacterial IMPDH is a practical dominant selectable marker that can be used for the selection of transfectants that express exogenous genes or cDNAs in mammalian cells.