Cell Chromatography: Biocompatible Chromatographic Separation and Interrogation of Microbial Cells.

The isolation of pure, single colonies lies at the heart of experimental microbiology. However, a microbial colony typically contains around 1 million cells at all stages of the life cycle. Here, we describe a novel cell chromatography method that facilitates the capture, purification, and interrogation of microbial cell populations from both single and mixed cultures. The method described relies on, but is not limited to, differences in surface charge to separate bacterial strains. The method is fully biocompatible, leading to no significant loss of cell viability. The chromatographic capture of cells, combined with selective elution methods, facilitates a greater level of experimental control over the sample inputs required for downstream high-throughput and high-sensitivity analytical methods. The application of the method for interrogating the antibiotic resistance of bacterial strains and for the separation of bacteria from environmental samples is illustrated. IMPORTANCE This is the first report of a method for separating microbial cells using chromatography, with full retention of cell viability. Differences in the surface chemistry of microbial cells provides a means of attracting cells to immobilized microbeads. Some cells are attracted, and some are repelled. The differences in, for example, surface charge can be harnessed to capture, interrogate, and separate environmental samples, thus circumventing the need to use conventional bacterial plating methods. This method will greatly facilitate drug discovery and bioprospecting for novel microbial compounds.

Understanding the controls on deposited fine sediment in the streams of agricultural catchments.

Excessive sediment pressure on aquatic habitats is of global concern. A unique dataset, comprising instantaneous measurements of deposited fine sediment in 230 agricultural streams across England and Wales, was analysed in relation to 20 potential explanatory catchment and channel variables. The most effective explanatory variable for the amount of deposited sediment was found to be stream power, calculated for bankfull flow and used to index the capacity of the stream to transport sediment. Both stream power and velocity category were highly significant (p ≪ 0.001), explaining some 57% variation in total fine sediment mass. Modelled sediment pressure, predominantly from agriculture, was marginally significant (p<0.05) and explained a further 1% variation. The relationship was slightly stronger for erosional zones, providing 62% explanation overall. In the case of the deposited surface drape, stream power was again found to be the most effective explanatory variable (p<0.001) but velocity category, baseflow index and modelled sediment pressure were all significant (p<0.01); each provided an additional 2% explanation to an overall 50%. It is suggested that, in general, the study sites were transport-limited and the majority of stream beds were saturated by fine sediment. For sites below saturation, the upper envelope of measured fine sediment mass increased with modelled sediment pressure. The practical implications of these findings are that (i) targets for fine sediment loads need to take into account the ability of streams to transport/retain fine sediment, and (ii) where agricultural mitigation measures are implemented to reduce delivery of sediment, river management to mobilise/remove fines may also be needed in order to effect an improvement in ecological status in cases where streams are already saturated with fines and unlikely to self-cleanse.

Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases.

Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the interaction between the C5 MTase from Haemophilus haemolyticus (M.HhaI) and an oligodeoxynucleotide duplex containing 2-H pyrimidinone, an analogue often referred to as zebularine and known to give rise to high-affinity complexes with MTases. X-ray crystallography has demonstrated the formation of a covalent bond between M.HhaI and the 2-H pyrimidinone-containing oligodeoxynucleotide. This observation enables a comparison between the mechanisms of action of 2-H pyrimidinone with other mechanism-based inhibitors such as FdC. This novel complex provides a molecular explanation for the mechanism of action of the anti-cancer drug zebularine.

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity.

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with TAQ polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XBAI and by mass spectrometry of the PCR products.

Tracing the origin of the RACTK1 K(+) channel.

Potassium secretion by the kidney is vital for the maintenance of K(+) homeostasis. RACTK1, a putative inwardly rectifying potassium channel cloned from cultured rabbit collecting duct cells, has been proposed to play a role in this process. However, the lack of homology with any other cloned potassium channel and the inability to reproduce the results across different laboratories has brought into question the existence of RACTK1. Recently, it has been suggested that RACTK1 is a contamination from Escherichia coli. In this work we add conclusive evidence supporting the bacterial origin of RACTK1. Using both genomic PCR and RT-PCR we were unable to detect RACTK1 in a number of mammalian species. In addition sequencing of RACTK1 cDNA confirmed a complete homology between RACTK1 and a region of E. coli genomic DNA. Finally, a hypothesis on how RACTK1 could have been generated from a contamination by E. coli genomic DNA is presented.

A positive selection vector combining tetracycline resistance that eliminates the need for bacterial plating.

We describe here the construction of a plasmid that combines positive selection with tetracycline resistance. The vector comprises a modified version of the gene encoding the cytosine-specific DNA methyltransferase MspI and a modified form of the pBR322 tetA(C) gene. The combination of these two genes facilitates the selection of recombinant plasmids in broth cultures, thereby eliminating the need for bacterial plating.

The mechanism of DNA repair by uracil-DNA glycosylase: studies using nucleotide analogues.

2',4'-Dideoxy-4'-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by Tm and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4'-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4'-oxygen in recognition and cleavage of dU residues in DNA.

Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone.

DNA duplexes in which the target cytosine base is replaced by 2-H pyrimidinone have previously been shown to bind with a significantly greater affinity to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is shown that 2-H pyrimidinone, when incorporated into DNA duplexes containing the recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhibitory covalent nucleoprotein complexes. We have found that although covalent complexes are formed between 2-H pyrimidinone-modified DNA and both M.HgaI-2 and M.MspI, the kinetics of complex formation are quite distinct in each case. Moreover, the formation of a covalent complex is still observed between 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine residue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic attack by the residue at the catalytic position, which is enhanced by the absence of the 4-amino function in the base. The substitution of the catalytic cysteine residue by tyrosine or chemical modification of the wild-type enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2-H pyrimidinone-substituted duplex still binds to M.MspI with a greater affinity than a standard cognate duplex, since the 2-H pyrimidinone base is mis-paired with guanine.

Substitution of the conserved phenylalanine in the S-adenosyl-L-methionine binding site of M.MspI with tyrosine modifies the kinetic properties of the enzyme.

Cytosine (C-5)-specific DNA methyltransferases share a set of ten conserved motifs distributed evenly throughout the entire polypeptide chain. The first conserved motif contains a Phe, which is intimately associated with cofactor recognition. In the pseudo-DNA methyltransferase M.SpoI, encoded by the pmt1 gene in Schizosaccharomyces pombe, a Tyr replaces this Phe residue. We describe the properties of a mutant form of M.MspI, a typical cytosine (C-5)-specific DNA methyltransferase, in which Tyr replaces the conserved Phe. This mutant shows differences in ternary complex formation and in the pattern of covalent complex formation with an inhibitory, fluorinated DNA duplex which may be due to anomalous hydrogen bonding between the mutant Tyr hydroxyl group and the catalytic loop of the enzyme or through interference with cofactor binding.

Regulation of distal nephron K+ channels (ROMK) mRNA expression by aldosterone in rat kidney.

1. The expression of ROMK mRNA isoforms in rat kidney was measured using competitive polymerase chain reaction. Under basal conditions the expression of ROMK2 and 3 mRNA was significantly higher than that of ROMK1 or 6. 2. Administration of aldosterone for a period of 1 week significantly increased the mRNA expression not only of the alpha1-subunit of the Na+-K+-ATPase, but also of ROMK2, 3 and 6. 3. These data not only provide evidence that ROMK K+ channels may be involved with mineralocorticoid-sensitive K+ secretion in the distal nephron, but also demonstrate for the first time that ROMK6 may be involved in this process.

The polypeptide backbone of recombinant human zona pellucida glycoprotein-3 initiates acrosomal exocytosis in human spermatozoa in vitro.

Human gamete interaction is of fundamental biological importance, yet the molecular interactions between spermatozoa and the zona pellucida are poorly understood. Surprisingly, the role of the polypeptide backbone of zona pellucida glycoprotein 3 (ZP3), the putative ligand for spermatozoa activation, has been largely overlooked. Purified recombinant human ZP3 was expressed in Escherichia coli as a C-terminal fusion to the dimeric glutathione S-transferase (GST) from Schistosoma japonicum and was shown to induce acrosomal exocytosis in live, capacitated human spermatozoa. The level of exocytosis is comparable with that obtained using purified, glycosylated, recombinant human ZP3 [van Duin, M., Polman, J.E.M., DeBreet, I.T.M., Van Ginneken, K., Bunschoten, H., Grootenhuis, A., Brindle, J. and Aitken, R.J. (1994). Biol Reprod. 51, 607-617]. These data imply that the polypeptide chain of human ZP3 contributes to recognition of spermatozoa during acrosomal exocytosis in vitro.

Tyrosine phosphorylation of a 95 kDa protein and induction of the acrosome reaction in human spermatozoa by recombinant human zona pellucida glycoprotein 3.

Protein tyrosine phosphorylation and induction of the acrosome reaction (AR) in non-capacitated and capacitated human spermatozoa was investigated in response to recombinant human zona pellucida glycoprotein (rhZP3) produced by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA. rhZP3-containing medium promoted the AR in a high proportion of capacitated spermatozoa (48.6 +/- 3.2%; P < 0.01) compared with control (no rhZP3) samples (14.8 +/- 2.1%). However, rhZP3-containing medium did not cause increased acrosomal exocytosis in non-capacitated spermatozoa (16.8 +/- 3.0%). Induction of the AR was associated with increased tyrosine phosphorylation of a 95 +/- 5 kDa epitope only in capacitated spermatozoa. A dose-dependent increase in the protein phosphorylation of a 95 kDa epitope in response to rhZP3 was detected by [gamma-32P]-ATP labelling of detergent-solubilized sperm proteins. When spermatozoa were co-incubated with monoclonal antibody 97.25 (mAb 97.25) recognizing a 95 kDa tyrosine kinase epitope, there was no rhZP3 induction of tyrosine phosphorylation of the 95 kDa protein. Such co-incubation also markedly inhibited the AR (23.9 +/- 3.1%). These results support the model that initial interaction of the fertilizing spermatozoon with ZP3 involves the tyrosine phosphorylation of a 95 kDa tyrosine kinase protein and that this requires capacitation.

Biological activity of recombinant human ZP3 produced in vitro: potential for a sperm function test.

The human zona binding test is the most predictive test of sperm function yet the availability of human zona severely restricts its clinical use. The primary aim of this study was to use a commercially available in-vitro transcription and translation system to produce immobilized recombinant human ZP3 (rhuZP3) on agarose beads. The biological activity of this preparation was examined using sperm binding and the acrosome reaction. Significantly higher levels of sperm binding to rhuZP3 beads (n = 12, P < 0.05) compared with controls were observed and there was a significant induction (n = 12, P < 0.01) in the acrosome reaction after overnight incubation at 37 degrees C in 5% CO2 in air. In conclusion, the in-vitro transcription and translation system can produce sufficient quantities of purified immobilized biologically active rhuZP3. These preliminary experiments will enable further refinements to be made so that a solid-phase sperm function test based on rhuZP3 coated beads is likely to be developed in the near future.

Imaging ROMK1 inwardly rectifying ATP-sensitive K+ channel protein using atomic force microscopy.

The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been shown by immunocytochemistry to be expressed in relevant nephron segments. The development of the atomic force microscope has made possible the production of high resolution images of small particles, including a variety of biological macromolecules. Recently, a fusion protein of glutathione S-transferase (GST) and ROMK1 (ROMK1-GST) has been used to produce a polyclonal antibody for immunolocalization of ROMK1. We have used atomic force microscopy to examine ROMK1-GST and the native ROMK1 polypeptide cleaved from GST. Imaging was conducted with the proteins in physiological solutions attached to mica. ROMK1-GST appears in images as a particle composed of two units of similar size. Analyses of images indicate that the two units have volumes of approximately 118 nm3, which is close to the theoretical volume of a globular protein of approximately 65 kDa (the molecular mass of ROMK1-GST). Native GST exists as a dimer, and the images obtained here are consistent with the ROMK1-GST fusion protein's existence as a heterodimer. In experiments on ROMK1 in aqueous solution, single molecules appear to aggregate, but contact to the mica was maintained. Addition of ATP to the solution produced a change in height of the aggregates. This change (which was reversible) suggests that ATP induces a structural change in the ROMK1 protein. The data show that atomic force microscopy is a useful tool for examination of purified protein molecules under near-physiological conditions, and furthermore, that structural alterations in the proteins may be continuously investigated.

Recombinant human zona pellucida glycoprotein 3 induces calcium influx and acrosome reaction in human spermatozoa.

Recombinant human ZP3 (rhuZP3) generated by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA was used to study the acrosome reaction (AR) and intracellular calcium fluxes in capacitated human spermatozoa. Conditioned medium containing rhuZP3 significantly induced the AR (P < or = 0.005) in 59.4 +/- 4.7% of spermatozoa (control = 8.5 +/- 3.1%) and caused complete acrosomal loss in a further 17.2 +/- 3.8% of cells (control = 3.7 +/- 0.7%; mean +/- SEM, n = 5). Sperm motility was not affected and acrosomal exocytosis in response to rhuZP3 was also shown to be time-dependent. Basal concentrations of sperm intracellular calcium were measured (82 +/- 7 nM; mean +/- SEM, n = 9). A transient increase in intracellular calcium (typically up to 400-450 nM) occurred within 1 min of rhuZP3 addition and was followed by sustained lower values of calcium (200-400 nM). These responses were dependent on the amount of rhuZP3. This is the first report of zona protein-induced changes in intracellular calcium levels in human spermatozoa. The results support the premise that ZP3 is an agonist of the human sperm AR and that rhuZP3 generated in a eukaryotic cell is effective in this respect.

Activation of a yeast pseudo DNA methyltransferase by deletion of a single amino acid.

The biological methylation cytosine bases in DNA is central to such diverse phenomena as restriction and modification in bacteria, repeat induced point-mutation (RIPing) in fungi and for programming gene expression patterns in vertebrates. Structural studies on HhaI DNA methyltransferase, together with the sequence comparisons of around 40 cytosine-specific DNA methyltransferases, have recently provided a molecular framework for understanding the mechanism of action of the related group of enzymes that catalyse this base modification. There are, however, a number of organisms, including Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila melanogaster, which have no detectable DNA methylation. Here we report that the product of the pmt1 gene recently identified in S. pombe, which contains most of the primary structure elements of a typical cytosine-specific DNA methyltransferase, is catalytically inert owing to the insertion of a Ser residue between the Pro-Cys motif found at the active site of all such DNA methyltransferases. Following deletion of this Ser residue, catalytic activity is restored and, using a range of DNA binding experiments, it is shown that the enzyme recognises and methylates the sequence CC(A/T)GG, the same sequence that is modified by the product of the Escherichia coli dcm gene. The pmt gene of S. pombe therefore encodes a pseudo DNA methyltranferase, which we have called psiM.SpoI.

Identification of direct-repeat-binding protein 1 (DRP-1), a DNA-binding protein that binds specifically to the 'malic' enzyme gene promoter direct repeat element.

The 'malic' enzyme (ME) gene promoter contains three main regulatory regions. One of these, the direct repeat element (DRE), contains tandem degenerate Sp1-binding sites separated by a 3 bp intervening sequence. We now show that a previously unreported 95 kDa protein, which we have designated DRP-1, binds strongly to the DRE region in a highly specific manner. Western-blot analysis confirms that this protein is not Sp1, which has been shown to bind to similar degenerate sites. Competitive binding assays using purified DRP-1 further reveal that neither non-specific nor Sp1-consensus-site-containing oligonucleotides can displace those complexes formed between DRP-1 and the DRE sequence, thus confirming sequence-specific binding by this protein. SDS/PAGE analysis of DRE-protein complexes isolated by direct excision and transplantation from retardation gels confirms the presence of the 95 kDa protein and, in addition, suggests that more than one binding site exists for this protein within the DRE. This is in accord with the repeated nature of the DRE DNA sequence which contains two CACC box motifs.