An effective antibiofilm strategy based on bacteriophages armed with silver nanoparticles.

The emerging antibiotic resistance in pathogenic bacteria is a key problem in modern medicine that has led to a search for novel therapeutic strategies. A potential approach for managing such bacteria involves the use of their natural killers, namely lytic bacteriophages. Another effective method involves the use of metal nanoparticles with antimicrobial properties. However, the use of lytic phages armed with nanoparticles as an effective antimicrobial strategy, particularly with respect to biofilms, remains unexplored. Here, we show that T7 phages armed with silver nanoparticles exhibit greater efficacy in terms of controlling bacterial biofilm, compared with phages or nanoparticles alone. We initially identified a novel silver nanoparticle-binding peptide, then constructed T7 phages that successfully displayed the peptide on the outer surface of the viral head. These recombinant, AgNP-binding phages could effectively eradicate bacterial biofilm, even when used at low concentrations. Additionally, when used at concentrations that could eradicate bacterial biofilm, T7 phages armed with silver nanoparticles were not toxic to eukaryotic cells. Our results show that the novel combination of lytic phages with phage-bound silver nanoparticles is an effective, synergistic and safe strategy for the treatment of bacterial biofilms.

Availability of iron ions impacts physicochemical properties and proteome of outer membrane vesicles released by Neisseria gonorrhoeae.

Outer membrane vesicles (OMVs) are bilayer structures released by bacteria for various purposes, e.g., response to environmental factors, bacterial communication, and interactions with host cells. One of the environmental variables bacteria need to react is the amount and availability of iron, a crucial element for bacteria biology. We have investigated the impact of the iron amount and availability on OMV secretion by pathogenic Neisseria gonorrhoeae, which, depending on the infection site, challenges different iron availability. N. gonorrhoeae releases OMVs in iron starvation and repletion growth environments. However, OMVs differed in physicochemical features and proteome according to iron amount and availability during the bacteria growth, as was analyzed by Liquid Chromatography-Tandem Mass Spectrometry, Infrared spectroscopy with a Fourier transform infrared spectrometer, and Atomic Force Microscopy. OMVs from iron starvation and repletion conditions had a higher variation in size, different flexibility, and different membrane protein and lipid components than OMVs isolated from control growth conditions. These OMVs also varied qualitatively and quantitatively in their total proteome composition and contained proteins unique for iron starvation and repletion conditions. Thus, the modulation of OMVs' properties seems to be a part of N. gonorrhoeae adaptation to surroundings and indicates a new direction of antigonococcal proceeding.

The potential for bacteriophages and prophage elements in fighting and preventing the gonorrhea.

Bacteriophages are the most numerous entities on earth and are found everywhere their bacterial hosts live. As natural bacteria killers, phages are extensively investigated as a potential cure for bacterial infections. Neisseria gonorrhoeae (the gonococcus) is the etiologic agent of a sexually transmitted disease: gonorrhea. The rapid increase of resistance of N. gonorrhoeae to antibiotics urges scientists to look for alternative treatments to combat gonococcal infections. Phage therapy has not been tested as an anti-gonococcal therapy so far. To date, no lytic phage has been discovered against N. gonorrhoeae. Nevertheless, gonococcal genomes contain both dsDNA and ssDNA prophages, and viral particle induction has been documented. In this review, we consider literature data about the attempts of hunting for a bacteriophage specific for gonococci - the gonophage. We also discuss the potential application of prophage elements in the fight against N. gonorrhoeae. Temperate phages may be useful in preventing and treating gonorrhea as a scaffold for anti-gonococcal vaccine development and as a source of lytic enzymes with anti-gonococcal activity.

The Structural Changes in the Membranes of Staphylococcus aureus Caused by Hydrolysable Tannins Witness Their Antibacterial Activity.

Polyphenols, including tannins, are phytochemicals with pronounced antimicrobial properties. We studied the activity of two hydrolysable tannins, (i) gallotannin-1,2,3,4,5-penta-O-galloyl-ß-D-glucose (PGG) and (ii) ellagitannin-1,2-di-O-galloyl-4,6-valoneoyl-ß-D-glucose (dGVG), applied alone and in combination with antibiotics against Staphylococcus aureus strain 8324-4. We also evaluated the effect of these tannins on bacterial membrane integrity and fluidity and studied their interaction with membrane proteins and lipids. A correlation between the antimicrobial activity of the tannins and their membranotropic action depending on the tannin molecular structure has been demonstrated. We found that the antibacterial activity of PGG was stronger than dGVG, which can be associated with its larger flexibility, dipole moment, and hydrophobicity. In addition, we also noted the membrane effects of the tannins observed as an increase in the size of released bacterial membrane vesicles.

How Streptococcus mutans Affects the Surface Topography and Electrochemical Behavior of Nanostructured Bulk Ti.

The metabolization of carbohydrates by Streptococcus mutans leads to the formation of lactic acid in the oral cavity, which can consequently accelerate the degradation of dental implants fabricated from commercially available microcrystalline Ti. Microstructure influences surface topography and hence interaction between bacteria cells and Ti surfaces. This work offers the first description of the effect of S. mutans on the surface topography and properties of nanostructured bulk Ti, which is a promising candidate for modern narrow dental implants owing to its superior mechanical strength. It was found that S. mutans incubation resulted in the slight, unexpected decrease of surface nanoroughness, which was previously developed owing to privileged oxidation in areas of closely spaced boundaries. However, despite the changes in nanoscale surface topography, bacteria incubation did not reduce the high level of protection afforded by the oxide layer formed on the nanostructured Ti surface. The results highlight the need-hitherto ignored-to consider Ti microstructure when analyzing its behavior in the presence of carbohydrate-metabolizing bacteria.

Phase-variable Type I methyltransferase M.NgoAV from Neisseria gonorrhoeae FA1090 regulates phasevarion expression and gonococcal phenotype.

The restriction-modification (RM) systems are compared to a primitive, innate, prokaryotic immune system, controlling the invasion by foreign DNA, composed of methyltransferase (MTase) and restriction endonuclease. The biological significance of RM systems extends beyond their defensive function, but the data on the regulatory role of Type I MTases are limited. We have previously characterized molecularly a non-canonical Type I RM system, NgoAV, with phase-variable specificity, encoded by Neisseria gonorrhoeae FA1090. In the current work, we have investigated the impact of methyltransferase NgoAV (M.NgoAV) activity on gonococcal phenotype and on epigenetic control of gene expression. For this purpose, we have constructed and studied genetic variants (concerning activity and specificity) within M.NgoAV locus. Deletion of M.NgoAV or switch of its specificity had an impact on phenotype of N. gonorrhoeae. Biofilm formation and planktonic growth, the resistance to antibiotics, which target bacterial peptidoglycan or other antimicrobials, and invasion of human epithelial host cells were affected. The expression of genes was deregulated in gonococcal cells with knockout M.NgoAV gene and the variant with new specificity. For the first time, the existence of a phasevarion (phase-variable regulon), directed by phase-variable Type I MTase, is demonstrated.

Both Neisseria gonorrhoeae and Neisseria sicca Induce Cytokine Secretion by Infected Human Cells, but Only Neisseria gonorrhoeae Upregulates the Expression of Long Non-Coding RNAs.

Bacteria of the Neisseria genus are Gram-negative diplococci including both pathogenic and commensal species. We focused on pathogenic Neisseria gonorrhoeae and commensal Neisseria sicca. We have demonstrated that not only N. gonorrhoeae, but also N. sicca induce the secretion of pro-inflammatory cytokines IL-6, TNF-α, and chemokines CXCL8 and CCL20 by infected epithelial cells. However, N. sicca triggers a lesser effect than does N. gonorrhoeae. Furthermore, N. gonorrhoeae and N. sicca invoke distinct effects on the expression of genes (JUNB, FOSB, NFKB1, NFKBIA) encoding protein components of AP-1 and NF-κB transcription factors. We have also shown that the infection of epithelial cells by N. gonorrhoeae leads to significant overexpression of the long non-coding RNAs (lncRNAs), including MALAT1, ERICD, and RP11-510N19.5. This effect was not identified for N. sicca. In conclusion, data on the expression of lncRNAs and cytokine secretion in response to Neisseria spp. exposure indicate new directions for research on Neisseria-host interactions and can provide further insights into virulence of not only pathogenic, but also commensal Neisseria spp.

Haemophilus influenzae HP1 Bacteriophage Encodes a Lytic Cassette with a Pinholin and a Signal-Arrest-Release Endolysin.

HP1 is a temperate bacteriophage, belonging to the Myoviridae family and infecting Haemophilus influenzae Rd. By in silico analysis and molecular cloning, we characterized lys and hol gene products, present in the previously proposed lytic module of HP1 phage. The amino acid sequence of the lys gene product revealed the presence of signal-arrest-release (SAR) and muraminidase domains, characteristic for some endolysins. HP1 endolysin was able to induce lysis on its own when cloned and expressed in Escherichia coli, but the new phage release from infected H. influenzae cells was suppressed by inhibition of the secretion (sec) pathway. Protein encoded by hol gene is a transmembrane protein, with unusual C-out and N-in topology, when overexpressed/activated. Its overexpression in E. coli did not allow the formation of large pores (lack of leakage of ß-galactosidase), but caused cell death (decrease in viable cell count) without lysis (turbidity remained constant). These data suggest that lys gene encodes a SAR-endolysin and that the hol gene product is a pinholin. HP1 SAR-endolysin is responsible for cell lysis and HP1 pinholin seems to regulate the cell lysis and the phage progeny release from H. influenzae cells, as new phage release from the natural host was inhibited by deletion of the hol gene.

Lactobacillus crispatus and its enolase and glutamine synthetase influence interactions between Neisseria gonorrhoeae and human epithelial cells.

Neisseria gonorrhoeae, an obligatory human pathogen causes the sexually transmitted disease gonorrhea, which remains a global health problem. N. gonorrhoeae primarily infects the mucosa of the genitourinary tract, which in women, is colonized by natural microbiota, dominated by Lactobacillus spp., that protect human cells against pathogens. In this study, we demonstrated that precolonization of human epithelial cells with Lactobacillus crispatus, one of the most prevalent bacteria in the female urogenital tract, or preincubation with the L. crispatus enolase or glutamine synthetase impairs the adhesion and invasiveness of N. gonorrhoeae toward epithelial cells, two crucial steps in gonococcal pathogenesis. Furthermore, decreased expression of genes encoding the proinflam-matory cytokines, TNFα and CCL20, which are secreted as a consequence of N. gonorrhoeae infection, was observed in N. gonorrhoeae-infected epithelial cells that had been preco-lonized with L. crispatus or preincubated with enolase and glutamine synthetase. Thus, our results indicate that the protection of human cells against N. gonorrhoeae infection is a complex process and that L. crispatus and its proteins enolase and glutamine synthetase can have a potential role in protecting epithelial cells against gonococcal infection. Therefore, these results are important since disturbances of the micro-biota or of its proteins can result in dysbiosis, which is associated with increased susceptibility of epithelium to pathogens.

Inactivation of Genes Encoding MutL and MutS Proteins Influences Adhesion and Biofilm Formation by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an etiological agent of gonorrhea, which remains a global health problem. This bacterium possesses MutL and MutS DNA repair proteins encoded by mutL and mutS genes, whose inactivation causes a mutator phenotype. We have demonstrated the differential gene expression in N. gonorrhoeae mutL and mutS mutants using DNA microarrays. A subset of differentially expressed genes encodes proteins that can influence adhesion and biofilm formation. Compared to the wild-type strain, N. gonorrhoeae mutL and mutS mutants formed denser biofilms with increased biofilm-associated biomass on the abiotic surface. The N. gonorrhoeae mutS::km, but not the mutL mutant, was also more adherent and invasive to human epithelial cells. Further, during infection of epithelial cells with N. gonorrhoeae mutS::km, the expression of some bacterial genes encoding proteins that can influence gonococcal adhesion was changed compared with their expression in cells infected with the wild-type gonococcus, as well as of human genes' encoding receptors utilized by N. gonorrhoeae (CD46, CEACAM 1, HSPG 2). Thus, deficiency in the mutS gene resulting in increased mutation frequency in singular organisms can be beneficial in populations because these mutants can be a source of features linked to microbial fitness.

Activity of Vsr endonucleases encoded by Neisseria gonorrhoeae FA1090 is influenced by MutL and MutS proteins.

BACKGROUND: The functioning of DNA repair systems is based on correct interactions between proteins involved in DNA repair. Very Short Patch (VSP) repair is a DNA repair system that corrects mismatches resulting from the deamination of 5-methylcytosine. The key enzyme in the VSP system is Vsr endonuclease, which can cleave mismatched DNA independently of accessory proteins. Until now, in vivo activity has only been shown for V.EcoKDcm - the only Vsr endonuclease in Escherichia coli. Additionally, the VSP system of E. coli is the only one for which interactions between proteins of the system have been demonstrated. Neisseria gonorrhoeae FA1090 is the first bacterium that we previously demonstrated to encode two active in vitro Vsr endonucleases: V.NgoAXIII and V.NgoAXIV. RESULTS: We elucidate the mutator phenotype of N. gonorrhoeae mutants with disrupted genes encoding V.NgoAXIII or V.NgoAXIV endonuclease. Furthermore, we investigate the interactions between gonococcal Vsr endonucleases and MutL and MutS proteins. The Vsr endonucleases physically interact with gonococcal MutL protein but not with MutS protein. In the presence of the MutL protein, the efficiency of DNA cleavage by both V.NgoAXIII and V.NgoAXIV endonucleases increases, resulting in a decrease in the amount of Vsr enzyme required to complete digestion of mismatched DNA. Both Vsr endonucleases are also stimulated in vitro by the MutL protein of E. coli. In turn, the gonococcal MutS protein hinders DNA cleavage by the Vsr endonucleases. However, this effect is overridden in the presence of MutL, and furthermore, the simultaneous presence of MutL and MutS causes an increase in the efficiency of DNA cleavage by the Vsr endonucleases compared to the reaction catalyzed by V.NgoAXIII or V.NgoAXIV alone. CONCLUSIONS: For the first time, interactions between proteins of the DNA repair system encoded by N. gonorrhoeae that are responsible for the correction of mismatches resulting from the 5-methylcytosine deamination were identified. The increase in activity of Vsr endonucleases in the presence of MutL protein could allow for reduced synthesis of the Vsr endonucleases in cells, and the susceptibility of gonococcal Vsr endonucleases on MutL protein of E. coli implies a universal mechanism of Vsr stimulation by MutL protein.

Characterization of Vsr endonucleases from Neisseria meningitidis.

DNA methylation is a common modification occurring in all living organisms. 5-methylcytosine, which is produced in a reaction catalysed by C5-methyltransferases, can spontaneously undergo deamination to thymine, leading to the formation of T:G mismatches and C→T transitions. In Escherichia coli K-12, such mismatches are corrected by the Very Short Patch (VSP) repair system, with Vsr endonuclease as the key enzyme. Neisseria meningitidis possesses genes that encode DNA methyltransferases, including C5-methyltransferases. We report on the mutagenic potential of the meningococcal C5-methyltransferases M.NmeDI and M.NmeAI resulting from deamination of 5-methylcytosine. N. meningitidis strains also possess genes encoding potential Vsr endonucleases. Phylogenetic analysis of meningococcal Vsr endonucleases indicates that they belong to two phylogenetically distinct groups (type I or type II Vsr endonucleases). N. meningitidis serogroup C (FAM18) is a representative of meningococcal strains that carry two Vsr endonuclease genes (V.Nme18IIP and V.Nme18VIP). The V.Nme18VIP (type II) endonuclease cut DNA containing T:G mismatches in all tested nucleotide contexts. V.Nme18IIP (type I) is not active in vitro, but the change of Tyr69 to His69 in the amino acid sequence of the protein restores its endonucleolytic activity. The presence of tyrosine in position 69 is a characteristic feature of type I meningococcal Vsr proteins, while type II Vsr endonucleases possess His69. In addition to the T:G mismatches, V.Nme18VIP and V.Nme18IIPY69H recognize and digest DNA with T:T or U:G mispairs. Thus, for the first time, we demonstrate that the VSP repair system may have a wider significance and broader substrate specificity than DNA lesions that only result from 5-methylcytosine deamination.

Type III Methyltransferase M.NgoAX from Neisseria gonorrhoeae FA1090 Regulates Biofilm Formation and Interactions with Human Cells.

Neisseria gonorrhoeae is the etiological factor of the sexually transmitted gonorrhea disease that may lead, under specific conditions, to systemic infections. The gonococcal genome encodes many restriction modification (RM) systems, which main biological role is to defend the pathogen from potentially harmful foreign DNA. However, RM systems seem also to be involved in several other functions. In this study, we examined the effect of inactivation the N. gonorrhoeae FA1090 ngoAXmod gene encoding M.NgoAX methyltransferase on the global gene expression, biofilm formation, interactions with human epithelial host cells and overall bacterial growth. Expression microarrays showed at least a twofold deregulation of a total of 121 genes in the NgoAX knock-out mutant compared to the wild-type (wt) strain under standard grow conditions. Genes with changed expression levels encoded mostly proteins involved in cell metabolism, DNA replication and repair or regulating cellular processes and signaling (such as cell wall/envelop biogenesis). As determined by the assay with crystal violet, the NgoAX knock-out strain formed a slightly larger biofilm biomass per cell than the wt strain. Live biofilm observations showed that the biofilm formed by the gonococcal ngoAXmod gene mutant is more relaxed, dispersed and thicker than the one formed by the wt strain. This more relaxed feature of the biofilm, in respect to adhesion and bacterial interactions, can be involved in pathogenesis. Moreover, the overall adhesion of mutant bacterial cells to human cells was lower than adhesion of the wt gonococci [adhesion index = 0.672 (±0.2) and 2.15 (±1.53), respectively]; yet, a higher number of mutant than wt bacteria were found inside the Hec-1-B epithelial cells [invasion index = 3.38 (±0.93) × 10(5) for mutant and 4.67 (±3.09) × 10(4) for the wt strain]. These results indicate that NgoAX knock-out cells have lower ability to attach to human cells, but more easily penetrate inside the host cells. All these data suggest that the NgoAX methyltransferase, may be implicated in N. gonorrhoeae pathogenicity, involving regulation of biofilm formation, adhesion to host cells and epithelial cell invasion.

The Cj0588 protein is a Campylobacter jejuni RNA methyltransferase.

TlyA proteins belong to 2'-O-methyltransferases. Methylation is a common posttranscriptional RNA modification. The Campylobacter jejuni Cj0588 protein belongs to the TlyA(I) protein family and is a rRNA methyltransferase. Methylation of ribosomal RNA catalyzed by Cj0588 appears to have an impact on the biology of the cell. Presence of the cj0588 gene in bacteria appears to be important for ribosome stability and virulence properties. Absence of the Cj0588 protein causes accumulation of the 50S ribosomal subunits, reduction in the amount of functional 70S ribosomes and confers increase resistance to capreomycin.

Neisseria gonorrhoeae filamentous phage NgoΦ6 is capable of infecting a variety of Gram-negative bacteria.

We constructed a phagemid consisting of the whole genome of the Neisseria gonorrhoeae bacteriophage NgoΦ6 cloned into a pBluescript plasmid derivative lacking the f1 origin of replication (named pBS::Φ6). Escherichia coli cells harboring pBS::Φ6 were able to produce a biologically active phagemid, NgoΦ6fm, capable of infecting, integrating its DNA into the chromosome of, and producing progeny phagemids in, a variety of taxonomically distant Gram-negative bacteria, including E. coli, Haemophilus influenzae, Neisseria sicca, Pseudomonas sp., and Paracoccus methylutens. A derivative of pBS::Φ6 lacking the phage orf7 gene, a positional homolog of filamentous phage proteins that mediate the interaction between the phage and the bacterial pilus, was capable of producing phagemid particles that were able to infect E. coli, Haemophilus influenzae, N. sicca, Pseudomonas sp., and Paracoccus methylutens, indicating that NgoΦ6 infects cells of these species using a mechanism that does not involve the Orf7 gene product and that NgoΦ6 initiates infection through a novel process in these species. We further demonstrate that the establishment of the lysogenic state does not require an active phage integrase. Since phagemid particles were capable of infecting diverse hosts, this indicates that NgoΦ6 is the first broad-host-range filamentous bacteriophage described.

The dam replacing gene product enhances Neisseria gonorrhoeae FA1090 viability and biofilm formation.

Many Neisseriaceae do not exhibit Dam methyltransferase activity and, instead of the dam gene, possess drg (dam replacing gene) inserted in the leuS/dam locus. The drg locus in Neisseria gonorrhoeae FA1090 has a lower GC-pairs content (40.5%) compared to the whole genome of N. gonorrhoeae FA1090 (52%). The gonococcal drg gene encodes a DNA endonuclease Drg, with GmeATC specificity. Disruption of drg or insertion of the dam gene in gonococcal genome changes the level of expression of genes as shown by transcriptome analysis. For the drg-deficient N. gonorrhoeae mutant, a total of 195 (8.94% of the total gene pool) genes exhibited an altered expression compared to the wt strain by at least 1.5 fold. In dam-expressing N. gonorrhoeae mutant, the expression of 240 genes (11% of total genes) was deregulated. Most of these deregulated genes were involved in translation, DNA repair, membrane biogenesis and energy production as shown by cluster of orthologous group analysis. In vivo, the inactivation of drg gene causes the decrease of the number of live neisserial cells and long lag phase of growth. The insertion of dam gene instead of drg locus restores cell viability. We have also shown that presence of the drg gene product is important for N. gonorrhoeae FA1090 in adhesion, including human epithelial cells, and biofilm formation. Biofilm produced by drg-deficient strain is formed by more dispersed cells, compared to this one formed by parental strain as shown by scanning electron and confocal microscopy. Also adherence assays show a significantly smaller biomass of formed biofilm (OD570 = 0.242 ± 0.038) for drg-deficient strain, compared to wild-type strain (OD570 = 0.378 ± 0.057). Dam-expressing gonococcal cells produce slightly weaker biofilm with cells embedded in an extracellular matrix. This strain has also a five times reduced ability for adhesion to human epithelial cells. In this context, the presence of Drg is more advantageous for N. gonorrhoeae biology than Dam presence.

Neisseria gonorrhoeae FA1090 carries genes encoding two classes of Vsr endonucleases.

A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases V.NgoAXIII and V.NgoAXIV from Neisseria gonorrhoeae FA1090 based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli, the proteins were biochemically characterized and the endonucleolytic activities and specificities of V.NgoAXIII and V.NgoAXIV were determined. V.NgoAXIII was found to be multispecific and to recognize T:G mismatches in every nucleotide context tested, whereas V.NgoAXIV recognized T:G mismatches in the following sequences: GTGG, CTGG, GTGC, ATGC, and CTGC. Alanine mutagenesis of conserved residues showed that Asp50 and His68 of V.NgoAXIII and Asp51 and His69 of V.NgoAXIV are essential for hydrolytic activity. Glu25, His64, and Asp97 of V.NgoAXIV and Glu24, Asp63, and Asp97 of V.NgoAXIII are important but not crucial for the activity of V.NgoAXIII and V.NgoAXIV. However, Glu24 and Asp63 are also important for the specificity of V.NgoAXIII. On the basis of our results concerning features of Vsr endonucleases expressed by N. gonorrhoeae FA1090, we postulate that at least two types of Vsr endonucleases can be distinguished.

The restriction endonuclease R.NmeDI from Neisseria meningitidis that recognizes a palindromic sequence and cuts the DNA on both sides of the recognition sequence.

The restriction endonuclease Type II R.NmeDI from Neisseria meningitidis 2120 (serogroup C, ST-11 complex) was characterized. The cloned nmeDIR gene was expressed in Escherichia coli cells, and the endonucleolytic and restriction activities of R.NmeDI were then observed in vitro and in vivo. The nmeDIR gene consists of 1056 bp coding 351 aa protein with a calculated molecular weight of M((r)) = 39 000 +/- 1000 Da. The R.NmeDI enzyme was purified to apparent homogeneity following overexpression, using metal affinity chromatography. This enzyme recognizes a palindrome sequence and cleaves double-stranded DNA upstream and downstream of its recognition sequence (12/7) RCCGGY (7/12) (R = A/G, Y = C/T) cutting out a 25-bp fragment. R.NmeDI cleaves in two steps. The enzyme cleaves the first strand randomly on either side of the recognition sequence generating an intermediate, and the second cleavage occurs more slowly and results in the production of a final reaction product. The R.NmeDI endonuclease requires two recognition sequences for effective cleavage. The tetramer is an active form of the R.NmeDI enzyme.

Unveiling hidden catalytic contributions of the conserved His/Trp-III in tyrosine recombinases: assembly of a novel active site in Flp recombinase harboring alanine at this position.

The catalytic pentad of tyrosine recombinases, that assists the tyrosine nucleophile, includes a conserved histidine/tryptophan (His/Trp-III). Flp and Cre harbor tryptophan at this position; most of their kin recombinases display histidine. Contrary to the conservation rule, Flp(W330F) is a much stronger recombinase than Flp(W330H). The hydrophobicity of Trp330 or Phe330 is utilized in correctly positioning Tyr343 during the strand cleavage step of recombination. Why then is phenylalanine almost never encountered in the recombinase family at this conserved position? Using exogenous nucleophiles and synthetic methylphosphonate or 5'-thiolate substrates, we decipher that Trp330 also assists in the activation of the scissile phosphate and the departure of the 5'-hydroxyl leaving group. These two functions are consistent with the hydrogen bonding property of Trp330 as well as its location in structures of the Flp recombination complexes. However, van der Waals contact between Trp330 and Arg308 may also be important for the phosphate activation step. A structure based suppression strategy permits the inactive variant Flp(W330A) to be rescued by a second site mutation A339M. Modeling alanine and methionine at positions 330 and 339, respectively, in the Flp crystal structure suggests a plausible mechanism for active site restoration. Successful suppression suggests the possibility of evolving, by design, new active site configurations for tyrosine recombination.

Protein-induced local DNA bends regulate global topology of recombination products.

The tyrosine family of recombinases produces two smaller DNA circles when acting on circular DNA harboring two recombination sites in head-to-tail orientation. If the substrate is supercoiled, these circles can be unlinked or form multiply linked catenanes. The topological complexity of the products varies strongly even for similar recombination systems. This dependence has been solved here. Our computer simulation of the synapsis showed that the bend angles, phi, created in isolated recombination sites by protein binding before assembly of the full complex, determine the product topology. To verify the validity of this theoretical finding we measured the values of phi for Cre/loxP and Flp/FRT systems. The measurement was based on cyclization of the protein-bound short DNA fragments in solution. Despite the striking similarity of the synapses for these recombinases, action of Cre on head-to-tail target sites produces mainly unlinked circles, while that of Flp yields multiply linked catenanes. In full agreement with theoretical expectations we found that the values of phi for these systems are very different, close to 35 degrees and 80 degrees, respectively. Our findings have general implications in how small protein machines acting locally on large DNA molecules exploit statistical properties of their substrates to bring about directed global changes in topology.