A novel thermostable TP-84 capsule depolymerase: a method for rapid polyethyleneimine processing of a bacteriophage-expressed proteins.

BACKGROUND: In spite of the fact that recombinant enzymes are preferably biotechnologically obtained using recombinant clones, the purification of proteins from native microorganisms, including those encoded by bacteriophages, continues. The native bacteriophage protein isolation is often troubled by large volumes of the infected bacterial cell lysates needed to be processed, which is highly undesired in scaled-up industrial processing. A well-known ammonium sulphate fractionation is often a method of choice during purification of the native bacteriophage protein. However, this method is time-consuming and cumbersome, and requires large amounts of the relatively expensive reagent. Thus, other effective and inexpensive methods of reversible protein precipitation are highly desirable. We have previously characterized thermophilic TP-84 bacteriophage, defined a new genus TP84virus within Siphoviridae family, conducted the TP-84 genome annotation and proteomic analysis. The longest Open Reading Frame (ORF) identified in the genome is TP84_26. We have previously annotated this ORF as a hydrolytic enzyme depolymerizing the thick polysaccharides host's capsule. RESULTS: The TP84_26 'capsule depolymerase' (depolymerase) is a large, 112 kDa protein, biosynthesized by the infected Geobacillus stearothermophilus 10 (G. stearothermophilus 10) cells. The TP84_26 protein biosynthesis was confirmed by three approaches: (i) purification of the protein of the expected size; (ii) mass spectrometry (LC-MS) analysis and (iii) detection of the enzymatic activity toward G. stearothermophilus polysaccharide capsules. Streptomycin-resistant mutant of the host was generated and microbiological aspects of both the TP-84 and G. stearothermophilus 10 were determined. A new variant of polyethyleneimine (PEI)-mediated purification method was developed, using the novel TP-84 depolymerase as a model. The enzyme was characterized. Three depolymerase forms were detected: soluble, unbound proteins in the bacteriophage/cells lysate and another integrated into the TP-84 virion. CONCLUSIONS: The novel TP-84 depolymerase was purified and characterized. The enzyme exists in three forms. The soluble, unbound forms are probably responsible for the weakening of the capsules of the uninfected bacterial cells. The form integrated into virion particles may generate a local passage for the invading TP-84. The developed PEI purification method appears well suited for the scaled-up or industrial production of bacteriophage proteins.

The third restriction-modification system from Thermus aquaticus YT-1: solving the riddle of two TaqII specificities.

Two restriction-modification systems have been previously discovered in Thermus aquaticus YT-1. TaqI is a 263-amino acid (aa) Type IIP restriction enzyme that recognizes and cleaves within the symmetric sequence 5'-TCGA-3'. TaqII, in contrast, is a 1105-aa Type IIC restriction-and-modification enzyme, one of a family of Thermus homologs. TaqII was originally reported to recognize two different asymmetric sequences: 5'-GACCGA-3' and 5'-CACCCA-3'. We previously cloned the taqIIRM gene, purified the recombinant protein from Escherichia coli, and showed that TaqII recognizes the 5'-GACCGA-3' sequence only. Here, we report the discovery, isolation, and characterization of TaqIII, the third R-M system from T. aquaticus YT-1. TaqIII is a 1101-aa Type IIC/IIL enzyme and recognizes the 5'-CACCCA-3' sequence previously attributed to TaqII. The cleavage site is 11/9 nucleotides downstream of the A residue. The enzyme exhibits striking biochemical similarity to TaqII. The 93% identity between their aa sequences suggests that they have a common evolutionary origin. The genes are located on two separate plasmids, and are probably paralogs or pseudoparalogs. Putative positions and aa that specify DNA recognition were identified and recognition motifs for 6 uncharacterized Thermus-family enzymes were predicted.

Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site.

The aim of this study was to improve a useful molecular tool-TaqII restriction endonuclease-methyltransferase-by rational protein engineering, as well as to show an application of our novel method of restriction endonuclease activity modulation through a single amino acid change in the NPPY motif of methyltransferase. An amino acid change was introduced using site-directed mutagenesis into the taqIIRM gene. The mutated gene was expressed in Escherichia coli. The protein variant was purified and characterized. Previously, we described a TspGWI variant with an amino acid change in the methyltransferase motif IV. Here, we investigate a complex, pleiotropic effect of an analogous amino acid change on its homologue-TaqII. The methyltransferase activity is reduced, but not abolished, while TaqII restriction endonuclease can be reactivated by sinefungin, with an increased DNA recognition fidelity. The general method for engineering of the IIS/IIC/IIG restriction endonuclease activity/fidelity is developed along with the generation of an improved TaqII enzyme for biotechnological applications. A successful application of our novel strategy for restriction endonuclease activity/fidelity alteration, based on bioinformatics analyses, mutagenesis and the use of cofactor-analogue activity modulation, is presented.

A Method for Rapid Polyethyleneimine-Based Purification of Bacteriophage-Expressed Proteins from Diluted Crude Lysates, Exemplified by Thermostable TP-84 Depolymerase.

Purification of bacteriophage-expressed proteins poses methodological difficulties associated with the need to process entire culture medium volume upon bacteriophage-induced bacterial cell lysis. We have used novel capsule glycosylase-depolymerase (TP84_26 GD) from bacteriophage TP-84, infecting thermophilic Geobacillus stearothermophilus bacteria, as a representative enzyme to develop a method for rapid concentration and purification of the enzyme present in diluted crude host cell lysate. A novel variant of the polyethyleneimine (PEI)-based purification method was devised that offers a fast and effective approach for handling PEI-facilitated purification of bacteriophage-expressed native proteins. Due to the very basic nature of PEI, the method is suitable for proteins interacting with nucleic acids or acidic proteins, where either mixed PEI-DNA or RNA-protein complexes or PEI-acidic protein complexes are reversibly precipitated. (i) The method is of general use, applicable with minor modifications to a variety of bacteriophage cell lysates and proteins. (ii) In the example application, TP84_26 GD was highly purified (over 50%) in a single PEI step; subsequent chromatography yielded a homogeneous enzyme. (iii) The enzyme's properties were examined, revealing the presence of three distinct forms of the TP84_26 GD. These forms included soluble, unbound proteins found in host cell lysate, as well as an integrated form within the TP-84 virion.

Identification of bacterial species in probiotic consortiums in selected commercial cleaning preparations.

The role of environmentally coexisting microflora that often comprises human commensal microbiome is still underestimated. Modern lifestyle changes include hygienic practices, food preparation and eradication of many contagious diseases. In this context, probiotic microorganisms are biocontrol remedies still under development, solving a number of gastrointestinal and immunological issues, while fighting hazardous microbiological biofilms on different surfaces. Probiotics are mainly associated with Lactic Acid Bacteria, however environmental, non-dairy sources are promising ecological niches of probiotic spore-forming Bacillus species. Industrial applications of these "unconventional" probiotics take an advantage of their sporulating activity which greatly enhances their compatibility with chemical formulations used in the household, cosmetic or pharmaceutical chemistry. We have analysed 14 commercially available chemical products, labelled or described to contain a probiotic or biologically active component. It was determined that in the most part they relay on consortiums of spore-forming, very closely related Bacillus species, exhibiting bimodal existence in the environment and the gastrointestinal tract (GIT). In addition, we have found a number of non-sporulating species. Overall, the microorganisms found included: Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, Citrobacter freundii, Klebsiella oxytoca, Stenotrophomonas malthophila, Serratia liquefaciens, Bacillus altitudinis, Lactobacillus gastricus, Bacillus megaterium, Lactobacillus nagelii, Aromatoleum buckelii, Trichosporon mucoides, Clostridium novyi, Bacteroides uniformis. As some of the listed species may become opportunistic pathogens, this raises an important question concerning general safety of probiotics, as apparently the manufacturing procedures do not always lead to microbiologically defined or sufficiently controlled microorganism consortiums.

Sequence, genome organization, annotation and proteomics of the thermophilic, 47.7-kb Geobacillus stearothermophilus bacteriophage TP-84 and its classification in the new Tp84virus genus.

Bacteriophage TP-84 is a well-characterized bacteriophage of historical interest. It is a member of the Siphoviridae, and infects a number of thermophilic Geobacillus (Bacillus) stearothermophilus strains. Its' 47.7-kbp double-stranded DNA genome revealed the presence of 81 coding sequences (CDSs) coding for polypeptides of 4 kDa or larger. Interestingly, all CDSs are oriented in the same direction, pointing to a dominant transcription direction of one DNA strand. Based on a homology search, a hypothetical function could be assigned to 31 CDSs. No RNA or DNA polymerase-coding genes were found on the bacteriophage genome indicating that TP-84 relies on the host's transcriptional and replication enzymes. The TP84 genome is tightly packed with CDSs, typically spaced by several-to-tens of bp or often overlapping. The genome contains five putative promoter-like sequences showing similarity to the host promoter consensus sequence and allowing for a 2-bp mismatch. In addition, ten putative rho-independent terminators were detected. Because the genome sequence shows essentially no similarity to any previously characterised bacteriophage, TP-84 should be considered a new species in an undefined genus within the Siphoviridae family. Thus a taxonomic proposal of a new Tp84virus genus has been accepted by the International Committee on Taxonomy of Viruses. The bioinformatics genome analysis was verified by confirmation of 33 TP-84 proteins, which included: a) cloning of a selected CDS in Escherichia coli, coding for a DNA single-stranded binding protein (SSB; gene TP84_63), b) purification and functional assays of the recombinant TP-84 SSB, which has been shown to improve PCR reactions, c) mass spectrometric (MS) analysis of TP-84 bacteriophage capsid proteins, d) purification of TP-84 endolysin activity, e) MS analysis of the host cells from infection time course.

Correction: Sequence, genome organization, annotation and proteomics of the thermophilic, 47.7-kb Geobacillus stearothermophilus bacteriophage TP-84 and its classification in the new Tp84virus genus.

[This corrects the article DOI: 10.1371/journal.pone.0195449.].