From DNA to lytic proteins: transcription and translation of the bacteriophage T5 holin/endolysin operon.

The analysis of transcriptional activity of the bacteriophage T5 hol/endo operon conducted in the paper revealed a strong constitutive promoter recognized by E. coli RNA polymerase and a transcription initiation point of the operon. It was also shown that the only translational start codon for holin was a non-canonical TTG. Translation initiation regions (TIRs) of both genes of the operon (hol and endo) were further analyzed using chimeric constructs, in which parts of the hol/endo regulatory regions were fused with the gene of a reporter protein (EGFP). It was found that TIR of hol was 20 times less effective than that of endo. As it turned out, the level of EGFP production was influenced by the composition of the constructs and the type of the hol start codon. Apparently, the translational suppression of holin's accumulation and posttranslational activation of endolysin by Ca2+ are the main factors ensuring the proper timing of the host cell lysis by bacteriophage T5. The approach based on the use of chimeric constructs proposed in the paper can be recommended for studying other native or artificial operons of any complexity: analyzing the impacts of separate DNA regions, as well as their coupled effect, on the processes of transcription and translation of recombinant protein(s).

Conservative Tryptophan Residue in the Vicinity of an Active Site of the M15 Family l,d-Peptidases: A Key Element in the Catalysis.

Bioinformatics analysis of the sequences of orthologous zinc-containing peptidases of the M15_C subfamily revealed the presence of a conserved tryptophan residue near the active site, which is not involved in the formation of the protein core. Site-directed mutagenesis of this Trp114/109 residue using two representatives of the family, l-alanoyl-d-glutamate peptidases of bacteriophages T5 (calcium-activated EndoT5) and RB49 (EndoRB49, without ion regulation) as examples, and further analysis of the 1H NMR spectra of the mutants showed that a decrease in the volume of the W → F → A residue leads to changes in the hydrophobic core and active center of the protein, and also decreases the affinity for regulatory Ca2+ in the EndoT5 mutants. The inactive T5W114A mutant lacks the ability to bind the substrate. In general, the conserved Trp114/109 residue, due to the spatial restrictions of its side chain, significantly affects the formation of the catalytically active form of the enzyme and is critical for catalysis.

A rapid and efficient technique for the isolation of Bacillus genomic DNA using a cocktail of peptidoglycan hydrolases of different type.

The paper suggests a rapid and efficient technique for isolation of genomic DNA from the bacteria of the genus Bacillus, which is based on the hydrolysis of cell wall peptidoglycan by a cocktail of peptidoglycan hydrolases of different type (L,D-peptidase and N-acetylmuramidase). The comparing of conventional techniques for the isolation of genomic DNA using: a microwave treatment; a treatment with ionic detergents (SDS, CTAB) or a chaotropic agent (GuSCN); and enzymatic hydrolysis (nonspecific, with proteinase K, or specific, with peptidoglycan hydrolases) conducted on Bacillus megaterium, B. subtilis, B. licheniformis, B. cereus showed that the most effective ones were techniques based on the specific hydrolysis of cell wall peptidoglycan. The highest efficiency of hydrolysis was obtained with an enzyme cocktail consisted of hen egg muramidase (HEWL) and highly active phage-specific L,D-peptidase EndoRB49 revealed a pronounced synergism between the peptidase and the muramidase. The cocktail treatment of Bacillus cells could be reduced to 10 min without affecting the yield of nucleic acids. The quality of DNA preparations was assessed using the restriction and PCR assays, as well as agarose gel electrophoresis. Using peptidoglycan hydrolases of different type, which have a good synergy, makes the technique very efficient and perspective for the application when rapid and effective disintegration of cell wall is crucial to avoid adverse effects of macromolecular denaturation.

Comparative analysis of the active sites of orthologous endolysins of the Escherichia lytic bacteriophages T5, RB43, and RB49.

The methods of solution NMR, circular dichroism (CD), and differential scanning calorimetry (DSC) were used to study two zinc-containing L-alanyl-D-glutamate peptidases - endolysins of the pseudo T-even myoviruses RB43 and RB49 (EndoRB43 and EndoRB49, respectively), which are orthologous to the EndoT5, which is a zinc-containing L-alanyl-D-glutamate peptidase of the T5 siphovirus. The spatial conservation of the Zn2+-binding sites for the enzymes EndoT5, EndoRB43, and EndoRB49 was established, and the key role of Zn2+ ions in the stabilization of the spatial structures of these three peptidases was confirmed. We are showing here that the binding of the Zn2+ ion in the active center of EndoRB49 peptidase causes conformational rearrangements similar to those observed in the EndoT5 peptidase upon binding of Zn2+ and Ca2+ ions and lead to the formation of a catalytically active form of the enzyme. Therefore, the binding of the Zn2+ ion to the active site of EndoRB49 peptidase is a necessary and sufficient condition for functioning of this protein.

On the roles of calcium and zinc ions in the formation of a catalytically active form of the metalloenzyme, l-alanyl-d-glutamate peptidase of the bacteriophage T5 (EndoT5).

Structural consequences of the binding of metal ions (regulatory Ca2+ and catalytic Zn2+) to the metalloenzyme l-alanyl-d-glutamate peptidase of the bacteriophage T5 (Endo T5) and some of its analogues containing single amino acid substitutions in the active center were analyzed by nuclear magnetic resonance (NMR), circular dichroism (CD) and calorimetry. Analyses revealed that the native EndoT5 undergoes strong structural rearrangements as a result of Zn2+ binding. This structural rearrangement resulting in the formation of an active enzyme is completed by the Ca2+ binding. In this case, the NMR spectra uncover the tautomerism of the NH protons of histidine imidazoles responsible for the Zn2+ coordination. For the EndoT5 analogues with point substitutions in the Ca2+-binding site, similar conformational rearrangements are observed upon Zn2+ binding. However, no characteristic changes in the NMR spectra associated with the Ca2+ binding were detected. The roles of the proton exchange in the process of Ca2+-induced activation of the enzymatic activity of EndoT5 is discussed.

Investigation of the calcium-induced activation of the bacteriophage T5 peptidoglycan hydrolase promoting host cell lysis.

Peptidoglycan hydrolase of bacteriophage T5 (EndoT5) is a Ca2+-dependent l-alanyl-d-glutamate peptidase, although the mode of Ca2+ binding and its physiological significance remain obscure. Site-directed mutagenesis was used to elucidate the role of the polar amino acids of the mobile loop of EndoT5 (111-130) in Ca2+ binding. The mutant proteins were purified to electrophoretic homogeneity, the overall structures were characterized by circular dichroism, and the calcium dissociation constants were determined via NMR spectroscopy. The data suggest that polar amino acids D113, N115, and S117 of EndoT5 are involved in the coordination of calcium ions by forming the core of the EF-like Ca2+-binding loop while the charged residues D122 and E123 of EndoT5 contribute to maintaining the loop net charge density. The results suggest that Ca2+ binding to the EndoT5 molecule could be essential for the stabilization of the long mobile loop in the catalytically active "open" conformation. The possible mechanism of Ca2+ regulation of EndoT5 activity during bacteriophage T5's life cycle through the Ca2+ concentration difference between the cytoplasm and the periplasm of the host bacteria cell has been discussed. The study reveals valuable insight into the role of calcium in the regulation of phage-induced bacterial lysis.

Effect of C-terminal His-tag and purification routine on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of the bacteriophage T5.

In this work, we studied the effect of the C-terminally attached poly-histidine tag (His-tag), as well as the peculiarities of the protein purification procedure by the immobilized metal affinity chromatography (IMAC) on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of bacteriophage T5 (EndoT5), whose zinc binding site and catalytic aspartate are located near the C-terminus. By itself, His-tag did not have a significant effect on either activity or folding of the polypeptide chain, nor on the binding of zinc and calcium ions to the protein. However, the His-tagged EndoT5 samples had low shelf-life, with storage of these samples resulting in an increased propensity for protein self-association and decreased enzymatic activity of EndoT5. Furthermore, disastrous effects on the activity of the enzyme were exerted by the presence of imidazole and nickel ions accompanying metal chelate chromatography. The activity of the protein can be restored by thorough washing off of these low molecular impurities via the prolonged dialysis of the His-tagged EndoT5 samples at the specifically elaborated conditions.

Two novel thermally resistant endolysins encoded by pseudo T-even bacteriophages RB43 and RB49.

Identification and cloning of genes as well as biochemical characterization of the gene products were carried out for two novel endolysins of pseudo T-even lytic bacteriophages RB43 and RB49, which represent different myovirus groups of the subfamily Tevenvirinae. Genes RB43ORF159c and RB49р102 were cloned in E. coli cells, and their products were purified to electrophoretic homogeneity with an up to 80 % yield of total activity. In respect to substrate specificity, both enzymes were found to be lytic l-alanoyl-d-glutamate peptidases belonging to the M15 family. The pH optimum functioning of both endolysins was within the range 7.0-9.0, whereas the optimal values of ionic strength were different for the two proteins (25 mM vs 100 mM for the RB43 and RB49 endolysins respectively). Both peptidases were thermally resistant, with the RB43 endolysin being more stable (it restored 81 % of enzyme activity and 96 % of secondary structure after a 10 min heating at 90 °C) than its RB49 counterpart (27 and 77% respectively). The possible origin of genes of lytic l-alanoyl-d-glutamate peptidases of myoviruses as a result of horizontal transfer in the variable parts of genomes between unrelated phages having a common host is discussed.

Evidence for the residual tertiary structure in the urea-unfolded form of bacteriophage T5 endolysin.

Using high-resolution NMR spectroscopy, we studied peculiarities of the unfolding process of the bacteriophage T5 endolysin (EndoT5) by strong denaturants. It was shown that in the absence of zinc ions this protein is mostly unfolded in the solution of 8 M urea or 6 M guanidine hydrochloride. However, in the presence of zinc ions EndoT5 unfolding can be achieved only in acidic solutions (at pH < 4.0), whereas at pH > 4.0 NMR spectra of the metal-bound protein (Zn2+-Ca2+-EndoT5 or Zn2+-EndoT5 complexes) exhibit a few chemical shifts characteristic of the native or native-like proteins. Our data, including the pH-titration curve with the pK of ~5, suggested involvement of the zinc-binding histidines in the stabilization of this protein. Up-field signals that appear in the NMR spectra of apo-EndoT5 in the presence of high concentrations of strong denaturants are probably derived from the amino acid residues included in the formation of structured hydrophobic cluster, which likely corresponds to the 81-93 region of EndoT5 and contains some residual tertiary structure. It is possible also that this hydrophobic fragment serves as a foundation for the formation of structured cluster in the unfolded state.

Structure and dynamics of the retro-form of the bacteriophage T5 endolysin.

Using high-resolution NMR spectroscopy we conducted a comparative analysis of the structural and dynamic properties of the bacteriophage T5 endolysin (EndoT5) and its retro-form; i.e., a protein with the reversed direction of the polypeptide chain (R-EndoT5). We show that structurally, retro-form can be described as the molten globule-like polypeptide that is easily able to form large oligomers and aggregates. To avoid complications associated with this high aggregation propensity of the retro protein, we compared EndoT5 and R-EndoT5 in the presence of strong denaturants. This analysis revealed that these two proteins possess different internal dynamics in solutions containing 8M urea, with the retro-form being characterized by larger dimensions and slower internal dynamics. We also show that in the absence of denaturant, both forms of the bacteriophage T5 endolysin are able to interact with micelles formed by the zwitterionic detergent dodecylphosphocholine (DPC), and that the formation of the protein-micelle complexes leads to the significant structural rearrangement of polypeptide chain and to the formation of stable hydrophobic core in the R-Endo T5.

Recent Origin of the Methacrylate Redox System in Geobacter sulfurreducens AM-1 through Horizontal Gene Transfer.

The origin and evolution of novel biochemical functions remains one of the key questions in molecular evolution. We study recently emerged methacrylate reductase function that is thought to have emerged in the last century and reported in Geobacter sulfurreducens strain AM-1. We report the sequence and study the evolution of the operon coding for the flavin-containing methacrylate reductase (Mrd) and tetraheme cytochrome с (Mcc) in the genome of G. sulfurreducens AM-1. Different types of signal peptides in functionally interlinked proteins Mrd and Mcc suggest a possible complex mechanism of biogenesis for chromoproteids of the methacrylate redox system. The homologs of the Mrd and Mcc sequence found in δ-Proteobacteria and Deferribacteres are also organized into an operon and their phylogenetic distribution suggested that these two genes tend to be horizontally transferred together. Specifically, the mrd and mcc genes from G. sulfurreducens AM-1 are not monophyletic with any of the homologs found in other Geobacter genomes. The acquisition of methacrylate reductase function by G. sulfurreducens AM-1 appears linked to a horizontal gene transfer event. However, the new function of the products of mrd and mcc may have evolved either prior or subsequent to their acquisition by G. sulfurreducens AM-1.

Identification and characterization of the metal ion-dependent L-alanoyl-D-glutamate peptidase encoded by bacteriophage T5.

Although bacteriophage T5 is known to have lytic proteins for cell wall hydrolysis and phage progeny escape, their activities are still unknown. This is the first report on the cloning, expression and biochemical characterization of a bacteriophage T5 lytic hydrolase. The endolysin-encoding lys gene of virulent coliphage T5 was cloned in Escherichia coli cells, and an electrophoretically homogeneous product of this gene was obtained with a high yield (78% of total activity). The protein purified was shown to be an L-alanoyl-D-glutamate peptidase. The enzyme demonstrated maximal activity in diluted buffers (25-50 mM) at pH 8.5. The enzyme was strongly inhibited by EDTA and BAPTA, and fully reactivated by calcium/manganese chlorides. It was found that, along with E. coli peptidoglycan, peptidase of bacteriophage T5 can lyse peptidoglycans of other Gram-negative microorganisms (Pectobacterium carotovorum, Pseudomonas putida, Proteus vulgaris, and Proteus mirabilis). This endolysin is the first example of an L-alanoyl-D-glutamate peptidase in a virulent phage infecting Gram-negative bacteria. There are, however, a great many sequences in databases that are highly similar to that of bacteriophage T5 hydrolase, indicating a wide distribution of endolytic L-alanoyl-D-glutamate peptidases. The article discusses how an enzyme with such substrate specificity could be fixed in the process of evolution.

Identification, cloning, and expression of bacteriophage T5 dnk gene encoding a broad specificity deoxyribonucleoside monophosphate kinase (EC 2.7.4.13).

The nucleotide sequence corresponding to 13-19.5% of the bacteriophage T5 genome in early region C was determined (GenBank AY 140897). One of the five major single-stranded interruptions (nicks) of bacteriophage T5 DNA was identified at 18.5%. The sequenced region was annotated and the putative functions of some open reading frames were proposed by comparison with databases. The dnk gene, encoding a deoxyribonucleoside monophosphate kinase, was identified using a previously defined N-terminal amino acid sequence. The gene was cloned and expressed in Escherichia coli, the enzyme was purified to homogeneity with high yield using two alternative methods, and the recombinant deoxyribonucleoside monophosphate kinase was found to have the same activity and specificity as the native enzyme.

Purification and characterization of the deoxynucleoside monophosphate kinase of bacteriophage T5.

Deoxynucleoside monophosphate kinase (dNMP kinase) of bacteriophage T5 (EC 2.7.4.13) was purified to apparent homogeneity from phage-infected Escherichia coli cells. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gel showed that the enzyme has a molecular mass of about 29 kDa. The molecular mass of dNMP kinase estimated by analytical equilibrium ultracentrifugation turned out to be 29.14 +/- 3.03 kDa. These data suggest that the enzyme exists in solution as a monomer. The isoelectric point of dNMP kinase was found to be 4.2. The N-terminal amino acid sequence, comprising 21 amino acids, was determined to be VLVGLHGEAGSGKDGVAKLII. A comparison of this amino acid sequence and those of known enzymes with a similar function suggests the presence of a nucleotide-binding site in the sequenced region.