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. (the gonococcus) is the etiologic agent of a sexually transmitted disease: gonorrhea.

Phase-variable Type I methyltransferase M.NgoAV from 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 FA1090.

A New Vaccination Method Based on Phage NgoΦ6 and Its Phagemid Derivatives.

Phagemid particles based on the filamentous phage NgoΦ6 were used as a vaccine delivery system. We demonstrate that the host proteins incorporated into/associated with these particles can be encoded by chromosomal genes of the host bacterium or from plasmids able to replicate as an autonomous entity in the phagemid host. Phagemid particles were prepared from three types of cells, namely, ser.

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

HP1 is a temperate bacteriophage, belonging to the family and infecting Rd. By in silico analysis and molecular cloning, we characterized and gene products, present in the previously proposed lytic module of HP1 phage. The amino acid sequence of the gene product revealed the presence of signal-arrest-release (SAR) and muraminidase domains, characteristic for some endolysins.

Phage proteins are expressed on the surface of Neisseria gonorrhoeae and are potential vaccine candidates.

All Neisseria gonorrhoeae strains whose DNA sequences have been determined possess filamentous phage sequences representing their full genomes. The presence of filamentous phage DNA sequences in all sequenced N. gonorrhoeae strains suggest that purified phage particles might be used as a gonococcal vaccine.

Oral Immunization of Rabbits with S. enterica Typhimurium Expressing Neisseria gonorrhoeae Filamentous Phage Φ6 Induces Bactericidal Antibodies Against N. gonorrhoeae.

All Neisseria gonorrhoeae strains whose DNA sequences have been determined possess filamentous phage DNA sequences. To ascertain if phage encoded proteins could form the basis of a gonococcal vaccine, rabbits were orally infected with S. enterica Typhimurium strain χ3987 harboring phagemid NgoΦ6 fm.

nagZ Triggers Gonococcal Biofilm Disassembly.

Bacterial-bacterial interactions play a critical role in promoting biofilm formation. Here we show that NagZ, a protein associated with peptidoglycan recycling, has moonlighting activity that allows it to modulate biofilm accumulation by Neisseria gonorrhoeae. We characterize the biochemical properties of NagZ and demonstrate its ability to function as a dispersing agent for biofilms formed on abiotic surfaces.

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.

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.

Novel non-specific DNA adenine methyltransferases.

The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N(6)-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene.

Deletion of one nucleotide within the homonucleotide tract present in the hsdS gene alters the DNA sequence specificity of type I restriction-modification system NgoAV.

As a result of a frameshift mutation, the hsdS locus of the NgoAV type IC restriction and modification (RM) system comprises two genes, hsdS(NgoAV1) and hsdS(NgoAV2). The specificity subunit, HsdS(NgoAV), the product of the hsdS(NgoAV1) gene, is a naturally truncated form of an archetypal specificity subunit (208 N-terminal amino acids instead of 410). The presence of a homonucleotide tract of seven guanines (poly[G]) at the 3' end of the hsdS(NgoAV1) gene makes the NgoAV system a strong candidate for phase variation, i.

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.

Characterization of the NgoAXP: phase-variable type III restriction-modification system in Neisseria gonorrhoeae.

Methyltransferases associated with type III restriction-modification (RM) systems are phase-variably expressed in a variety of pathogenic bacteria. NgoAXP, the type III RM system encoded by Neisseria gonorrhoeae, was characterized in this study. The cloned resngoAXP and ngoAXPmod genes were expressed in Escherichia coli strains.

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.

Characterization of the dsDNA prophage sequences in the genome of Neisseria gonorrhoeae and visualization of productive bacteriophage.

Background: Bioinformatic analysis of the genome sequence of Neisseria gonorrhoeae revealed the presence of nine probable prophage islands. The distribution, conservation and function of many of these sequences, and their ability to produce bacteriophage particles are unknown.

Results: Our analysis of the genomic sequence of FA1090 identified five genomic regions (NgoPhi1 - 5) that are related to dsDNA lysogenic phage.

Analysis of the filamentous bacteriophage genomes integrated into Neisseria gonorrhoeae FA1090 chromosome.

Bioinformatic analysis of the genome sequence of Neisseria gonorrhoeae revealed presence of four specific prophage islands. Based on the similarity with other DNA phage sequences they seem to belong to the filamentous ssDNA phages group. Phages belonging to this group are also present in the genome of Neisseria meningitidis.

Cloning and preliminary characterization of a GATC-specific beta2-class DNA:m6A methyltransferase encoded by transposon Tn1549 from Enterococcus spp.

A recent study revealed a subfamily of N6-adenine (m6A) methyltransferases that comprises a few functionally studied eukaryotic members acting on mRNA and prokaryotic members acting on DNA as well as numerous uncharacterized open reading frames. Here, we report cloning and functional characterization of a prokaryotic member of this family encoded by transposon Tn1549 from Enterococcus spp.

Biochemical analysis of Lpt3, a protein responsible for phosphoethanolamine addition to lipooligosaccharide of pathogenic Neisseria.

The inner core of neisserial lipooligosaccharide (LOS) contains heptose residues that can be decorated by phosphoethanolamine (PEA). PEA modification of heptose II (HepII) can occur at the 3, 6, or 7 position(s). We used a genomic DNA sequence of lpt3, derived from Neisseria meningitidis MC58, to search the genomic sequence of N.

The role of Dam methylation in phase variation of Haemophilus influenzae genes involved in defence against phage infection.

Haemophilus influenzae uses phase variation (PV) to modulate the activity of its defence systems against phage infection. The PV of the restriction-modification (R-M) system HindI, the main defence system against phage infection and incoming chromosomal and phage DNA in H. influenzae Rd, is driven by changes of the pentanucleotide repeat tract within the coding sequence of the hsdM gene and is influenced by lack of Dam methylation.

Identification of amino acids important for target recognition by the DNA:m5C methyltransferase M.NgoPII by alanine-scanning mutagenesis of residues at the protein-DNA interface.

DNA:m(5)C MTases comprise a catalytic domain with conserved residues of the active site and a strongly diverged TRD with variable residues involved in DNA recognition and binding. To date, crystal structures of 2 DNA:m(5)C MTases complexed with the substrate DNA have been obtained; however, for none of these enzymes has the importance of the whole set of DNA-binding residues been comprehensively studied. We built a comparative model of M.

Characterization of a dam mutant of Haemophilus influenzae Rd.

The gene encoding Dam methyltransferase of Haemophilus influenzae was mutagenized by the insertion of a chloramphenicol-resistance cassette into the middle of the Dam coding sequence. This mutant construct was introduced into the H. influenzae chromosome by transformation and selection for Cam(R) transformants.

DNA methyltransferases from Neisseria meningitidis and Neisseria gonorrhoeae FA1090 associated with mismatch nicking endonucleases.

The genes encoding the DNA methyltransferases M.NmeDI and M.NmeAI from Neisseria meningitidis associated with the genes encoding putative Vsr endonucleases were overexpressed in Escherichia coli.

Tetra-amino-acid tandem repeats are involved in HsdS complementation in type IC restriction-modification systems.

All known type I restriction and modification (R-M) systems of Escherichia coli and Salmonella enterica belong to one of four discrete families: type IA, IB, IC or ID. The classification of type I systems from a wide range of other genera is mainly based on complementation and molecular evidence derived from the comparison of the amino acid similarity of the corresponding subunits. This affiliation was seldom based on the strictest requirement for membership of a family, which depends on relatedness as demonstrated by complementation tests.

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes.

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

Biochemical properties of Neisseria gonorrhoeae LgtE.

A fragment of chromosomal DNA encoding the lgtE gene of Neisseria gonorrhoeae strain F62 was amplified by PCR and cloned into the expression vector pET15b. Functional LgtE was purified and its biochemical properties were determined. The purified enzyme was maximally active in buffer containing manganese; minimal activity was obtained in buffer containing other divalent cations.