Construction of Hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformable Neisseria mutants.

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformable Neisseria mutants. By random insertion of a selectable marker into the conjugative Neisseria plasmid ptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned in Escherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of an E. coli replicon that does not support autonomous replication in Neisseria, e.g. ColE1, p15A, or ori(fd), fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformable Neisseria strain involves a three-step process: (i) insertion of the desired gene into a +Hermes vector; (ii) transformation of Hermes into a Neisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the final Neisseria recipient. Several applications for the genetic manipulation of pathogenic Neisseriae are described.

Tetrapac (tpc), a novel genotype of Neisseria gonorrhoeae affecting epithelial cell invasion, natural transformation competence and cell separation.

We characterized a novel mutant phenotype (tetrapac, tpc) of Neisseria gonorrhoeae (Ngo) associated with a distinctive rough-colony morphology and bacterial growth in clusters of four. This phenotype, suggesting a defect in cell division, was isolated from a mutant library of Ngo MS11 generated with the phoA minitransposon TnMax4. The tpc mutant shows a 30% reduction in the overall murein hydrolase activity using Escherichia coli murein as substrate. Tetrapacs can be resolved by co-cultivation with wild-type Ngo, indicating that Tpc is a diffusible protein. Interestingly, Tpc is absolutely required for the natural transformation competence of piliated Ngo. Mutants in tpc grow normally, but show a approximately 10-fold reduction in their ability to invade human epithelial cells. The tpc sequence reveals an open reading frame of approximately 1 kb encoding a protein (Tpc) of 37 kDa. The primary gene product exhibits an N-terminal leader sequence typical of lipoproteins, but palmitoylation of Tpc could not be demonstrated. The ribosomal binding site of tpc is immediately downstream of the translational stop codon of the folC gene coding for an enzyme involved in folic acid biosynthesis and one-carbon metabolism. The tpc gene is probably co-transcribed from the folC promoter and a promoter located within the folC gene. The latter promoter sequence shares significant homology with E. coli gearbox consensus promoters. All three mutant phenotypes, i.e. the cell separation defect, the transformation deficiency and the defect in cell invasion can be restored by complementation of the mutant with an intact tpc gene. To some extent the tcp phenotype is reminiscent of iap in Listeria, lytA in Streptococcus pneumoniae and lyt in Bacillus subtilis, all of which are considered to represent murein hydrolase defects.

An improved TnMax mini-transposon system suitable for sequencing, shuttle mutagenesis and gene fusions.

A new collection of mini-transposons (mini-Tn) of the previously described TnMax series [Haas et al, Gene 130 (1993a) 23-31] has been constructed. The transposons (Tn) bear genes conferring resistance to either chloramphenicol (Cm) or kanamycin (Km). Each member of the new series (TnMax5-TnMax11) contains the general M13 forward (M13-FP) and reverse (M13-RP1) sequencing primers close to the inverted repeats (IR), facilitating the rapid and convenient determination of the DNA sequences flanking the transposon insertion site. Furthermore, the mini-Tn possess the infrequently occurring NotI sites, allowing the localization of genes on macro-restriction maps of bacterial species. Some derivatives contain promoterless trp-lacZ (TnMax11), xylE (TnMax10), phoA (TnMax6) or blaM (TnMax7, TnMax9) genes next to the IR, suitable for the generation of in vivo gene- and operon fusions to study gene regulation, protein export, or to determine the topology of proteins in bacterial membranes. A set of conjugative minimal plasmid vectors (pMin1, pMin2) are used to select for TnMax insertions into the cloned insert, rather than the vector sequences. Due to the small size of the mini-Tn, and a simple and efficient mutagenesis procedure, the TnMax system is a useful tool for targeting and sequencing of cloned genes in Escherichia coli, and especially for shuttle mutagenesis of bacterial species which cannot be targeted by direct transposition.

Generalized transposon shuttle mutagenesis in Neisseria gonorrhoeae: a method for isolating epithelial cell invasion-defective mutants.

One requirement for the invasion of, and tight adherence to, human epithelial cells by Neisseria gonorrhoeae is the synthesis of distinct opacity (Opa) outer membrane proteins, encoded by a family of phase-variable chromosomal genes. However, cloning and surface expression of invasion-promoting Opas in Escherichia coli is not sufficient for the efficient invasion of epithelial cells: additional factors besides Opa may be involved in this process. Using the phoA mini-transposon TnMax4, a library of gonococcal mutants affected in the expression of genes encoding exported proteins was generated through shuttle mutagenesis. Of a total of 608 PhoA+ plasmid clones identified in E. coli E145 approximately 40% were used successfully in transforming N. gonorrhoeae and in activating the corresponding chromosomal genes. Gonococci producing the invasion-promoting Opa50 served as the genetic background to identify 51 mutants unable to enter Chang human epithelial cells. We expect some of these mutations affect the interaction of N. gonorrhoeae with epithelial cells directly, while other mutants may carry defects in general house-keeping, secretory and/or regulatory determinants. In some mutants the loss of invasiveness appears to be due to a negative dominant effect of the PhoA+ fusions produced in these mutants. Some of the identified genes display a phase-variation phenomenon in E. coli and several genes are found in multiple copies in N. gonorrhoeae and/or present only in pathogenic Neisseria species.

TnMax--a versatile mini-transposon for the analysis of cloned genes and shuttle mutagenesis.

A series of Tn1721-based mini-transposons (TnMax) has been developed which are suitable for the targeting of cloned genes, shuttle mutagenesis, identification of protein export signals and the rescue of chromosomal markers. TnMax mini-Tn consist of two 38-bp inverted repeats (IR) flanking a resolution site (res), a suicide replication origin (orifd), and a gene conferring resistance to either chloramphenicol (TnMax1) or erythromycin (TnMax2). Other versions of TnMax (TnMax3 and TnMax4) carry a promoterless alkaline phosphatase-encoding gene (phoA') useful for the identification of protein export signals. The various mini-Tn cartridges are fused on the same plasmid with a common transposition control unit (TCU) comprising the tnpA (transposase) and tnpR (resolvase) genes under control of the inducible Ptrc promoter. This configuration causes a high frequency of transposition in Escherichia coli (approx. 10(-2) events/copy of target plasmid) and a minimum size of the mini-Tn. Like Tn1721, TnMax variants prefer random insertion into plasmids rather than into the E. coli chromosome, thus representing superb tools for the insertion mutagenesis of cloned genes. The TnMax-borne orifd simplifies the identification of targeted plasmids and facilitates shuttle mutagenesis, i.e., suicide delivery of a mutated gene with subsequent allelic replacement of a corresponding resident gene, in a variety of microorganisms. Rescue of such targeted chromosomal genes is easily accomplished by the excision of TnMax plus flanking segments using appropriate restriction endonucleases, ligation, and transformation of an E. coli host permissive for orifd-directed replication.