Interference of Neisseria gonorrhoeae growth by aerobic bacterial representatives of the urogenital flora

Aerobic bacterial isolates obtained from endocervical, vaginal and urethral swabbings were tested for interference of neisseria gonorrhoeae growth on solid medium. Simultaneous antagonism was studied using the lawn spotting method, and delayed antagonism by the basal spot/lawn method. From 58 swabbings we recuperated a total of 181 isolates, 71 of those were found interfering with at least one out of four gonococcal strains (G-1, G-2, G-3 and G-4). Similar percentages of interfering isolates were obtained from each of the isolation sites. The identification of the interfering isolates has revealed that similar numbers of coagulase negative staphylococci and identical numbers of group D streptococci were found for each of those sites. The majority of the interfering isolates and also of the inhibitory coagulase negative staphylococci showed only simultaneous antagonism. To complete the interference spectrum, we have tested all the active urogenital isolates against four other gonococcal strains (G-7, G-9, G-10 and G-11). This spectrum showed clearly that interference is not an all or none phenomenon. While the gonococcal interference spectrum of most of the Gram positive cocci and the Acinetobacter sp. strains is broad, that of all the other isolates is relatively narrow. Gonococcal strains G-7 and G-9 were the most susceptible to inhibition by the interfering urogenital isolates while strain G-3 was the most resistant one.     

New tandem repeat region in the non-transcribed spacer of human ribosomal RNA gene.

A new repetitive DNA region was identified in the non-transcribed spacer of human rDNA, namely a long (4.6 kb) sequence motif (Xbal element) was present in two copies. The repeating unit composed of two parts. One of them consisted of unique nucleotide sequences, interrupted by some simple sequences. The other, about 3.1 kb long one assembled only from highly repeated simple sequences. The unique sequence region contained two, inverted copies of the human AluI type repetitive DNA family. The authors suggest that the XbaI elements may flank the tandem arrays of human rRNA genes as terminal repeats and they might function both as the origin of rDNA replication and/or site of homologous recombination.     

Characterization of the mitogenic activity elicited by Neisseria gonorrhoeae ribosomal fractions.

Ribosomal preparations from Neisseria gonorrhoeae types 1 and 4 were examined for their in vitro stimulation of mouse splenocytes to determine the ribosomal moiety or contaminant responsible for the immunoproliferative activity. In immunodiffusion tests with homologous rabbit antiserum, crude 70S ribosomes formed four precipitin bands while the purified 30S and 50S subunits showed one major line. The same antiserum reacted with lysed N. gonorrhoeae and Neisseria meningitidis A cells but no precipitation occurred with Escherichia coli cells purified N. gonorrhoeae lipopolysaccharide (LPS). No membrane or LPS contaminant was detected in the purified 30S and 50S preparations. All the ribosomal preparations from virulent and non-virulent N. gonorrhoeae consistently stimulated the murine splenocytes. The mitogenic activity of the 30S and 50S ribosomal preparation was destroyed by treatment with trypsin but only slightly decreased by ribonuclease. It is suggested that the lymphoproliferative response elicited by gonococcal ribosomes is triggered by the protein moiety of the 30S or 50S subunits.     

Plasmid-mediated chromosomal gene transfer in Neisseria gonorrhoeae.

An indigenous Neisseria gonorrhoeae conjugative plasmid, pLE2450, was tested for its ability to mediate chromosomal gene transfer between gonococcal strains. Plasmid-mediated chromosomal transfer was detected at a low frequency and can be used to establish certain linkage relationships between amino acid and antibiotic resistance markers.     

Structural analysis of the pilE region of Neisseria gonorrhoeae P9

We have determined the nucleotide sequence of an expressed structural pilus gene (pilE) derived from Neisseria gonorrhoeae strain P9-2. Detailed analysis of nucleotide sequences upstream from pilE revealed a silent, truncated pilin gene segment that was linked to families of DNA elements (RS1 and RS3) that have previously been identified at the major silent pilin gene locus (pilS1) and at pilE of the independently isolated N. gonorrhoeae strain MS11ms. A nucleotide sequence downstream from pilE was reminiscent of the recognition sequences of several recombinases, including Tn3 tnpR product (resolvase), suggesting a possible role for site-specific events in the recombinational modulation of pilus expression.     

Molecular analysis of the heterogeneity region of the human ribosomal spacer

The human ribosomal non-transcribed spacers are 30 X 10(3) base-pairs (or 30 kb) in length with a limited length heterogeneity localized in a specific region downstream from the 3' end of the transcribed region. Total DNA digested with EcoRI and BamHI and hybridized with a probe containing the 3' end of the 28 S ribosomal RNA coding region shows four major bands of 3.9 kb, 4.6 kb, 5.4 kb and 6.2 kb. The 5.4 kb band is the most abundant in every individual, followed by the 4.6 kb band. The longest and the shortest size classes are less well-represented and may even be absent. Every individual shows his own pattern of relative abundance of non-transcribed spacer length classes that can be followed through generations. We decided to investigate the molecular structure of the heterogeneity region, in order to cast light onto the mechanisms underlying the origin and maintenance of this length heterogeneity. Pertinent spacer regions of eight ribosomal clones from two human genomic libraries were subcloned and analyzed by restriction mapping and nucleotide sequencing. In the minimal length class, there is a sequence of 700 base-pairs that appears to be tandemly duplicated once, twice or three times in the other length classes. This repeated DNA module contains a region consisting of repetitions of simple pyrimidine groups like C-T, C-T-T-T or C-C-C-T. DNA module repeats may differ by the length of this pyrimidine-rich region. However, these length variations are not continuous, as revealed by Southern transfer analysis of several individuals and different cloned gene units: instead, the repeated modules fall into two discrete length classes of about 700 base-pairs and 800 base-pairs. An imperfect duplication of a short sequence of 86/89 base-pairs is present at the boundary between the heterogeneity region and the upstream flanking region, representing a very ancient duplication event.     

Analysis of the Piv recombinase-related gene family of Neisseria gonorrhoeae

Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of the disease gonorrhea. The gonococcal pilus undergoes antigenic variation through high-frequency recombination events between unexpressed pilS silent copies and the pilin expression locus pilE. The machinery involved in pilin antigenic variation identified to date is composed primarily of genes involved in homologous recombination. However, a number of characteristics of antigenic variation suggest that one or more recombinases, in addition to the homologous recombination machinery, may be involved in mediating sequence changes at pilE. Previous work has identified several genes in the gonococcus with significant identity to the pilin inversion gene (piv) from Moraxella species and transposases of the IS110 family of insertion elements. These genes were candidates for a recombinase system involved in pilin antigenic variation. We have named these genes irg for invertase-related gene family. In this work, we characterize these genes and demonstrate that the irg genes do not complement for Moraxella lacunata Piv invertase or IS492 MooV transposase activities. Moreover, by inactivation of all eight gene copies and overexpression of one gene copy, we conclusively show that these recombinases are not involved in gonococcal pilin variation, DNA transformation, or DNA repair. We propose that the irg genes encode transposases for two different IS110-related elements given the names ISNgo2 and ISNgo3. ISNgo2 is located at multiple loci on the chromosome of N. gonorrhoeae, and ISNgo3 is found in single and duplicate copies in the N. gonorrhoeae and Neisseria meningitidis genomes, respectively.     

The recX gene potentiates homologous recombination in Neisseria gonorrhoeae

In the pathogen Neisseria gonorrhoeae (Gc), the RecA protein is necessary for DNA repair, DNA transformation and pilus antigenic variation. Many bacteria contain a gene, recX, which has been suggested to downregulate recA through an unknown mechanism. To investigate the possible role of recX in Gc, we cloned and insertionally inactivated the recX gene. The recX loss-of-function mutant showed decreases in pilus phase variation, DNA transformation and DNA repair ability compared with wild type. We were able to complement all these deficiencies by supplying a functional copy of recX elsewhere in the chromosome. The recX mutant still showed increases in pilus phase variation under conditions of iron starvation, and the recX mutant showed levels of RecA protein equivalent to wild type. Although the precise role of recX in recombination remains unclear, RecX aids all RecA-related processes in Gc, and this is the first demonstration of a role for recX in homologous recombination in any organism.     

Length heterogeneity in a region of the human ribosomal gene spacer is not accompanied by extensive population polymorphism

We carried out a restriction enzyme analysis of human ribosomal DNA structure on total placental DNA using the Southern (1975) method. Studies on a single individual using HindIII, PstI, HpaI and BglII revealed that a region of the non-transcribed spacer near the 3′ end of the 28 S gene was heterogeneous in size. Four fragment classes were detected in this individual. Adjacent classes differed in size from one another by about 0·8 × 10³ bases. Analysis of 19 additional placental samples and four cell lines revealed no fragment classes other than those detected in the original sample. Mixing experiments carried out with all 20 placental DNA samples provided further evidence for the discrete nature of the population polymorphism in the length of this region of the spacer. This finding contrasts sharply with the almost continuous nature of the population polymorphism in the length of a region of the non-transcribed spacer in Xenopus laevis.     

Pilin gene variation in Neisseria gonorrhoeae: reassessing the old paradigms

Neisseria gonorrhoeae displays considerable potential for antigenic variation as shown in human experimental studies. Various surface antigens can change either by antigenic variation using RecA-dependent recombination schemes (e.g. PilE antigenic variation) or, alternatively, through phase variation (on/off switching) in a RecA-independent fashion (e.g. Opa and lipooligosaccharide phase variation). PilE antigenic variation has been well documented over the years. However, with the availability of the N. gonorrhoeae FA1090 genome sequence, considerable genetic advances have recently been made regarding the mechanistic considerations of the gene conversion event, leading to an altered PilE protein. This review will compare the various models that have been presented and will highlight potential mechanistic problems that may constrain any genetic model for pilE gene variation.     

Role of the Rep helicase gene in homologous recombination in Neisseria gonorrhoeae

In Escherichia coli, the Rep helicase has been implicated in replication fork progression, replication restart, homologous recombination, and DNA repair. We show that a Neisseria gonorrhoeae rep mutant is deficient in the homologous-recombination-mediated processes of DNA transformation and pilus-based colony variation but not in DNA repair.