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.

A variable genetic island specific for Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates.

Neisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually transmitted disease gonorrhoea. Most gonococcal infections remain localized to the genital tract but, in a small proportion of untreated cases, the bacterium becomes systemic to produce the serious complication of disseminated gonococcal infection (DGI). We have identified a large region of chromosomal DNA in N. gonorrhoeae that is not found in a subset of gonococcal isolates (a genetic island), in the closely related pathogen, Neisseria meningitidis or in commensal Neisseria that do not usually cause disease. Certain versions of the island carry a serum resistance locus and a gene for the production of a cytotoxin; these versions of the island are found preferentially in DGI isolates. All versions of the genetic island encode homologues of F factor conjugation proteins, suggesting that, like some other pathogenicity islands, this region encodes a conjugation-like secretion system. Consistent with this hypothesis, a wild-type strain released large amounts of DNA into the medium during exponential growth without cell lysis, whereas an isogenic strain mutated in a peptidoglycan hydrolase gene (atlA) was drastically reduced in its ability to donate DNA for transformation during growth. This genetic island constitutes the first major discriminating factor between the gonococcus and the other Neisseria and carries genes for providing DNA for genetic transformation.

Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum.

Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum, the low efficiencies of transfection and the fact that integration of transfected DNA into chromosomes is observed only after long periods (typically 12 weeks or more) have made it difficult to genetically manipulate the blood stages of this major human pathogen. Here we show that co-transfection of a P. falciparum line with two plasmids, one expressing a green fluorescent protein (gfp) reporter and the other expressing a drug resistance marker (Tgdhfr-ts M23), allowed selection of a population in which about approximately 30% of the parasites produce GFP. In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 20 kb and could be maintained under drug pressure for at least 16 weeks. Our data suggest that chimera formation occurs early (detected by 7--14 days) and that it involves homologous recombination favored by presence of the same P. falciparum 5'hrp3 UTR promoting transcription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be used to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum.

Modulation of gonococcal piliation by regulatable transcription of pilE.

The gonococcal pilus, a member of the type IV family of pili, is composed of numerous monomers of the pilin protein and plays an important role in the initiation of disease by providing the primary attachment of the bacterial cell to human mucosal tissues. Piliation also correlates with efficient DNA transformation. To investigate the relationships between these pilus-related functions, the piliation state, and the availability of pilin, we constructed a derivative of MS11-C9 (DeltapilE1) in which the lacIOP regulatory sequences control pilE transcription. In this strain, MS11-C9.10, the steady-state levels of pilin mRNA and protein directly correlate with the concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) in the growth medium and can reach near-wild-type levels of expression. Transmission electron microscopy (TEM) demonstrated that the number of pili per cell correlated with the steady-state expression levels: at a low level of transcription, single long pili were observed; at a moderate expression level, many singular and bundled pili were expressed; and upon full gene expression, increased lateral association between pili was observed. Analysis of pilus assembly by TEM and epithelial cell adherence over a time course of induction demonstrated that pili were expressed as early as 1 h postinduction. Analysis at different steady-state levels of transcription demonstrated that DNA transformation efficiency and adherence of MS11-C9.10 to transformed and primary epithelial cells also correlated with the level of piliation. These data show that modulation of the level of pilE transcription, without a change in pilE sequence, can alter the number of pili expressed per cell, pilus bundling, DNA transformation competence, and epithelial cell adherence of the gonococcus.

Iron availability regulates DNA recombination in Neisseria gonorrhoeae.

The pilus of Neisseria gonorrhoeae (the gonococcus Gc), the causative agent of gonorrhoea, promotes attachment of the gonococcus to the host epithelium and is essential for the establishment of disease. The ability of N. gonorrhoeae to infect previously exposed individuals is partially due to pilus antigenic variation. In addition, variation of the pilus has been proposed to function in the adaptation of the gonococcus to host environments. Previously, we described the development of a competitive reverse transcriptase (RT)-PCR assay that quantifies the frequency of pilin antigenic variation within a gonococcal population. Using this assay, the effect of different biologically relevant environmental conditions on the frequency of pilin antigenic variation was tested. Of the environmental conditions examined in vitro, only limited iron affected a significant change in the frequency of antigenic variation. Further investigation revealed that an observed increase in pilin antigenic variation reflected an increase in other DNA recombination and DNA repair processes within iron-starved cultures. In addition, this low iron-induced increase was determined to be independent of changes in RecA expression and was observed in a Fur mutant strain. As gonococci encounter conditions of low iron during infection, these data suggest that iron-limitation signals for increased recombinational events that are important for gonococcal pathogenesis.

Molecular models accounting for the gene conversion reactions mediating gonococcal pilin antigenic variation.

The pilus antigenic variation (Av) system of Neisseria gonorrhoeae is one of several high-frequency variation systems that utilize gene conversion to switch between numerous forms of an antigen on the cell surface. We have tested three predictions of the first models that explain the movement of DNA during pilin Av: (i) Av requires two recombinations at short regions of identity, (ii) circular intermediates exist that carry pilE/pilS hybrid loci and (iii) these pilE/pilS hybrid loci target the pilS sequences to a recipient pilE gene. We confirm that normal pilin Av utilizes recombination at very short regions of DNA sequence identity and that these recombination events can occur independent of homologous recombination functions. We have isolated covalently closed circular DNA molecules carrying hybrid pilin loci, but propose that an alternative hybrid molecule is the intermediate of pilin Av. Our most striking finding is that transformation of isolated pilE/pilS hybrid loci targets the pilS sequences of the hybrid to a recipient pilE at frequencies much higher than normal recombination frequencies. These results show that the different steps of a model that explains pilin Av can be separately tested to support the validity of these novel models that account for the high-frequency gene conversions that mediate pilin Av.

A homologue of the recombination-dependent growth gene, rdgC, is involved in gonococcal pilin antigenic variation.

Neisseria gonorrhoeae pilin undergoes high-frequency changes in primary amino acid sequence that aid in the avoidance of the host immune response and alter pilus expression. The pilin amino acid changes reflect nucleotide changes in the expressed gene, pilE, which result from nonreciprocal recombination reactions with numerous silent loci, pilS. A series of mini-transposon insertions affecting pilin antigenic variation were localized to three genes in one region of the Gc chromosome. Mutational analysis with complementation showed that a Gc gene with sequence similarity to the Escherichia coli rdgC gene is involved in pilus-dependent colony phase variation and in pilin antigenic variation. Furthermore, we show that the Gc rdgC homologue is transcriptionally linked in an operon with a gene encoding a predicted GTPase. The inability to disrupt expression of this gene suggests it is an essential gene (engA, essential neisserial GTPase). While some of the transposon mutations in rdgC and insertions in the 5'-untranslated portion of engA showed a growth defect, all transposon insertions investigated conferred an aberrant cellular morphology. Complementation analysis showed that the growth deficiencies are due to the interruption of RdgC expression and not that of EngA. The requirement of RdgC for efficient pilin variation suggests a role for this protein in specialized DNA recombination reactions.

Isolation of Neisseria gonorrhoeae mutants that show enhanced trafficking across polarized T84 epithelial monolayers.

Initiation of a gonococcal infection involves attachment of Neisseria gonorrhoeae to the plasma membrane of an epithelial cell in the mucosal epithelium and its internalization, transepithelial trafficking, and exocytosis from the basal membrane. Piliation and expression of certain Opa proteins and the immunoglobulin A1 protease influence the transcytosis process. We are interested in identifying other genetic determinants of N. gonorrhoeae that play a role in transcellular trafficking. Using polarized T84 monolayers as a model epithelial barrier, we have assayed an N. gonorrhoeae FA1090 minitransposon (mTn) mutant bank for isolates that traverse the monolayer more quickly than the isogenic wild-type (WT) strain. From an initial screen, we isolated four mutants, defining three genetic loci, that traverse monolayers significantly more quickly than their WT parent strain. These mutants adhere to and invade cells normally and do not affect the integrity of the monolayer barrier. Backcrosses of the mutations into the WT FA1090 strain yielded mutants with a similar fast-trafficking phenotype. In two mutants, the mTns had inserted 370 bp apart into the same locus, which we have named fit, for fast intracellular trafficker. Backcrosses of one of these mutants into the MS11A genetic background also yielded a fast-trafficking mutant. The fit locus contains two overlapping open reading frames, fitA and fitB, whose deduced amino acid sequences have predicted molecular weights of 8.6 and 15.3, respectively. Neither protein contains a signal sequence. FitA has a potential helix-turn-helix motif, while the deduced sequence of FitB offers no clues to its function. fitA or fitB homologues are present in the genomes of Pseudomonas syringae and Rhizobium meliloti, but not Neisseria meningitidis. Replication of the MS11A fitA mutant in A431 and T84 cells is significantly accelerated compared to that of the isogenic WT strain. In contrast, growth of this mutant in liquid media is normal. Taken together, these results strongly suggest that traversal of N. gonorrhoeae across an epithelial barrier is linked to intracellular bacterial growth.

Active immunisation of black tiger prawn (Penaeus monodon) against vibriosis in Thailand.

Mechanisms of host defence in Crustaceae and vibriosis in shrimp and methods for its prevention are discussed as introduction. The own work deals with the development of a site-specific multivalent anti-vibrio vaccine and its application in Thailand. The vaccine was produced in a continuous bioreactor system from field strains of Vibrio alginolyticus, V. harveyi, V. parahaemolyticus and V. vulnificus, purified through ultrafiltration, and inactivated by formalin. It was applied to Penaeus monodon PL prior to release into the ponds via artemia larvae. As mean result from numerous trials carried out under commercial field conditions in Eastern Thailand it was shown that the PL tolerated the vaccine perfectly. Due to technical problems only few of the field trials could be monitored until harvest. The data available allow claiming an obvious protective effect of the vaccine.

Insertion mutations in pilE differentially alter gonococcal pilin antigenic variation.

Pilus antigenic variation in Neisseria gonorrhoeae occurs by the high-frequency, unidirectional transfer of DNA sequences from one of several silent pilin loci (pilS) into the expressed pilin gene (pilE), resulting in a change in the primary pilin protein sequence. Previously, we investigated the effects of large or small heterologous insertions in conserved and variable portions of a pilS copy on antigenic variation. We observed differential effects on pilin recombination by the various insertions, and the severity of the defect correlated with the disruption or displacement of a conserved pilin DNA sequence called cys2. In this study, we show that disruption or displacement of the pilE cys2 sequence by the same insertions or a deletion also affects pilin recombination. However, in contrast to the insertions in pilS, the analogous insertions in pilE impaired, but did not block, recombination of the flanking pilin sequences. These results, the change in the spectrum of donor silent copies used during variation, and our previous results with pilS mutations show that the donor pilS and recipient pilE play different roles in antigenic variation. We conclude that when high-frequency recombination mechanisms are blocked, alternative mechanisms are operative.

Comparisons between colony phase variation of Neisseria gonorrhoeae FA1090 and pilus, pilin, and S-pilin expression.

The gonococcal pilus is a primary virulence factor, providing the initial attachment of the bacterial cell to human mucosal tissues. Pilin, the major subunit of the pilus, can carry a wide spectrum of primary amino acid sequences which are generated by the action of a complex antigenic variation system. Changes in the pilin amino acid sequence can produce different pilus-dependent colony morphotypes, which have been previously shown to reflect phase variation of pili on the bacterial cell surface. In this study, we further examined the relationships between changes in pilus-dependent colony morphology, pilin sequence, pilus expression, and pilus function in Neisseria gonorrhoeae FA1090. A group of FA1090 colony variants expressed different pilin sequences and demonstrated different levels of pilin, S-pilin, and pilus expression. The analysis of these colony variants shows that they do not represent two distinct phases of pilus expression, but that changes in pilin protein sequence produce a spectrum of S-pilin production, pilus expression, and pilus aggregation levels. These different levels of pilus expression and aggregation influence not only colony morphology but also DNA transformation efficiency and epithelial cell adherence.

Frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Variation of the pilus of Neisseria gonorrhoeae occurs by the recombination of silent pilin DNA sequences into the pilin expression locus. We have developed a quantitative, competitive reverse transcription-PCR assay which measures the frequency of pilin antigenic variation independently of changes in gonococcal colony morphology and have determined this frequency within a gonococcal population. We have also studied the frequency of antigenic variation during growth and have concluded that growth does not dramatically influence the frequency of pilin antigenic variation, although a reproducible, twofold increase is observed upon the transition into late log/stationary phase.

The pathogenic neisseriae contain an inactive rpoN gene and do not utilize the pilE sigma54 promoter.

The sigma54 promoter (P3) upstream of the pilE gene in Neisseria gonorrhoeae was shown to be non-functional by transcriptional analysis of a PpilE::lacZ fusion containing only P3. A region on the chromosome of N. gonorrhoeae strain MS11-A was identified that potentially encodes a protein with a significant similarity to the Escherichia coli RpoN protein. However, this region (designated RLS for rpoN-like sequence) does not contain a single open reading frame (ORF) capable of encoding a functional RpoN protein. It appears that RLS may have arisen from an ancestral rpoN homologue that underwent a deletion removing the sequence encoding the essential helix-turn-helix (HTH) motif, and changing the subsequent reading frame. An RLS has been identified in several strains of N. gonorrhoeae and N. meningitidis. A 90-kDa gonococcal protein has previously been shown to react with a monoclonal antibody raised against the RpoN from Salmonella typhimurium. However, mutagenesis and Western blot analysis confirmed that the gene encoding this protein is not contained within RLS.

Differential roles of homologous recombination pathways in Neisseria gonorrhoeae pilin antigenic variation, DNA transformation and DNA repair.

Neisseria gonorrhoeae (Gc) pili undergo antigenic variation when the amino acid sequence of the pilin protein is changed, aiding in immune avoidance and altering pilus expression. Pilin antigenic variation occurs by RecA-dependent unidirectional transfer of DNA sequences from a silent pilin locus to the expressed pilin gene through high-frequency recombination events that occur at limited regions of homology. We show that the Gc recQ and recO genes are essential for pilin antigenic and phase variation and DNA repair but are not involved in natural DNA transformation. This suggests that a RecF-like pathway of recombination exists in Gc. In addition, mutations in the Gc recB, recC or recD genes revealed that a Gc RecBCD pathway also exists and is involved in DNA transformation and DNA repair but not in pilin antigenic variation.

A peptidoglycan hydrolase similar to bacteriophage endolysins acts as an autolysin in Neisseria gonorrhoeae.

We have identified a gene encoding an autolysin (atlA) from Neisseria gonorrhoeae. The deduced amino acid sequence of AtlA shows significant similarity to the peptidoglycan degrading transglycosylases (endolysins) of bacteriophages lambda and P2, suggesting that the encoded protein also functions in peptidoglycan hydrolysis. An atlA mutant was identical to the wild-type strain in exponential growth rate, but demonstrated reduced lysis and peptidoglycan turnover in the stationary phase of growth. When transferred into a buffer solution, at a pH non-permissive for other gonococcal autolysins, an autolytic activity was detectable in the wild-type strain that was not present in the mutant. The most dramatic phenotype of the mutant occurred after extended time in stationary phase. After approximately 16h in stationary phase, both strains underwent an apparent replication event, after which the wild-type strain died rapidly whereas the atlA mutant survived considerably longer. Even after both the wild-type and mutant cells were dead, many of the mutant cells maintained intact morphology, whereas the wild-type cells were lysed. These results suggest that AtlA is a peptidoglycan transglycosylase related to bacteriophage endolysins and acts as an autolysin in the stationary phase.

Insertionally inactivated and inducible recA alleles for use in Neisseria.

Two classes of recA mutations have been constructed for use in Neisseria gonorrhoeae: three insertionally inactivated ('knockout') mutations and three LacI-regulatable constructs that can be shifted between Rec- and Rec+ by the removal or addition of IPTG. The effects of regulating recA expression on the processes of DNA transformation, DNA repair and pilin-phase variation are described. These regulatable cassettes can also be used to control the expression of any chromosomal gene.

Analysis of protein binding to the Sma/Cla DNA repeat in pathogenic Neisseriae.

Antigenic variation of the pilus is an essential component of Neisseria gonorrhoeae pathogenesis. Unidirectional recombination of silent pilin DNA into an expressed pilin gene allows for substantial sequence variation of this highly immunogenic surface structure. While the RecA protein is required for pilin gene recombination, the factors which maintain the silent reservoir of pilin sequences and/or allow unidirectional recombination from silent to expression loci remain undefined. We have previously shown that a conserved sequence at the 3'end of all pilin loci (the Sma/Cla repeat) is required to be present at the expression locus for efficient recombination from the silent loci. In this study, the binding of gonococcal proteins to this DNA sequence was investigated. Gel mobility shift assays and competition experiments using deletion derivatives of the repeat, show that multiple activities bind to different regions of the Sma/Cla repeat and define the boundaries of the binding sequences. Moreover, only the pathogenic Neisseria harbor proteins which specifically bind to this repeat, suggesting a correlation between the expression of these DNA binding proteins and the potential to cause disease.

Random shuttle mutagenesis: gonococcal mutants deficient in pilin antigenic variation.

Shuttle mutagenesis has been adapted to randomly mutate the genome of Neisseria gonorrhoeae (gonococcus; Gc). A size-restricted plasmid library of Gc strain FA1090 was mutated with the mini-transposon mTnEGNS. Radomness was tested by checking for transposon insertion bias between vector and insert DNA, Gc transformation efficiency of individual mutated clones, and representation of unique clones before and after Gc transformation with a mutated pool of DNA. Mutants created by random shuttle mutagenesis were screened, using a colony-based polymerase chain reaction assay, for the ability to undergo pilin antigenic variation. Out of 8,064 mutants screened, 22 unique transposon insertion mutants were found to be antigenic variation deficient (Avd). The Avd mutants were separated into five types according to recombination defect-associated phenotypes, including colony growth, natural DNA transformation competence, and repair of DNA damage caused by ultraviolet radiation.

Environment, incidence, aetiology, epizootiology and immunoprophylaxis of soil-borne diseases in north-east Mexico.

Within the framework of an extensive research programme, the socio-economic and environmental conditions which influence the emergence of soil-borne diseases in north-eastern Mexico were analysed. Furthermore, specimens collected from carcasses in the field were bacteriologically examined and the causal organisms of soil-borne diseases differentiated by means of gas chromatographic analysis of their metabolic products and the long-chained fatty acids contained in the cell. With experimental clostridial vaccines prepared with the Goettingen Bioreactor Technique, trials to protect cattle and guinea-pigs against gas gangrene were carried out. It was found that the farm structure and the dry climate as well as the specific soil conditions and plant cover favour the emergence of soil-borne diseases. Causal organisms B. anthracis, C. perfringens, C. sordellii, C. haemolyticum, C. chauvoei/septicum, C. novyi A, C. botulinum and site-specific field strains of clostridia were detected. Experimental site-specific vaccines proved to be highly efficient in protecting cattle and guinea pigs.

The size and position of heterologous insertions in a silent locus differentially affect pilin recombination in Neisseria gonorrhoeae.

Gonococcal pilus antigenic and phase variation result from unidirectional, RecA-dependent recombination of DNA sequences from a silent pilin copy (pilS) into the expressed pilin gene (pilE). To develop a quantitative assay for pilin gene recombination that is independent of phase variation, a promoterless cat gene was inserted into pilS, and recombination of "cat into pilE was detected by selection of chloramphenicol-resistant (CmR) variants expressing "cat from the pilin promoter. Although RecA-dependent CmR variants occurred, none were generated by the simple transfer of "cat into pilE. Instead, each CmR variant contained a new pilin locus that was a hybrid of sequences from the pilE and the pilS1::cat loci in addition to the two starting loci. Therefore, this system could not be used to quantify antigenic variation. However, combined studies of these hybrid loci and of recombination products generated during additional pilS mutational analyses demonstrated that both the size and position of an insertion in pilS differentially affect pilin recombination. Also, the hybrid loci appear to be intermediates of antigenic variation. This enabled the creation of molecular models for the recombination reactions that result in pilin antigenic variation.