Interstrain gene transfer in Chlamydia trachomatis in vitro: mechanism and significance

Abstract

The high frequency of between-strain genetic recombinants of Chlamydia trachomatis among isolates obtained from human sexually transmitted infections suggests that lateral gene transfer (LGT) is an important means by which C. trachomatis generates variants that have enhanced relative fitness. A mechanism for LGT in C. trachomatis has not been described, and investigation of this phenomenon by experimentation has been hampered by the obligate intracellular development of this pathogen. We describe here experiments that readily detected LGT between strains of C. trachomatis in vitro. Host cells were simultaneously infected with an ofloxacin-resistant (Ofx(r)) mutant of a serovar L1 strain (L1:Ofx(r)-1) and a rifampin-resistant (Rif(r)) mutant of a serovar D strain (D:Rif(r)-1). Development occurred in the absence of antibiotics, and the progeny were subjected to selection for Ofx(r) Rif(r) recombinants. The parental strains differed at many polymorphic nucleotide sites, and DNA sequencing was used to map genetic crossovers and to determine the parental sources of DNA segments in 14 recombinants. Depending on the assumed DNA donor, the estimated minimal length of the transferred DNA was > or = 123 kb in one recombinant but was > or = 336 to > or = 790 kb in all other recombinants. Such trans-DNA lengths have been associated only with conjugation in known microbial LGT systems, but natural DNA transformation remains a conceivable mechanism. LGT studies can now be performed with diverse combinations of C. trachomatis strains, and they could have evolutionary interest and yield useful recombinants for functional analysis of allelic differences between strains.

FIG. 1.

Distribution of genetic crossovers in 14 independent recombinant clones resulting from in vitro LGT. The locations of the loci on the 1,043-kb circular C. trachomatis chromosome are indicated by their midpoints (in kilobases of DNA) from the chromosomal Ori. Sequencing of DNA segments within each of the loci shown (see Tables S1, S2, and S3 in the supplemental material) that included polymorphic nucleotide differences between the serovar D and L1 parental strains used in the cross was used to determine the allelic signatures of LGT recombinants (Table 1) and the parental sources of chromosomal segments shown; DNA between these polymorphism-containing segments was not sequenced. Multiple occurrences (n > 1) of the same recombinant type indicate independent origins of the same general type of recombinant in more than one mixed-infection culture. The placement of crossovers in all recombinants indicates that a crossover could have occurred anywhere in the interval between the nearest polymorphic nucleotides on both sides of the crossover. Asterisks identify recombinants that had an exchange within a short, completely sequenced segment of either the rpoB gene (types III, V, V-A, and V-B) or the gyrA gene (types VII and VIII) (Table 1). In the type V-B recombinant, a ≤512-bp segment of serovar L1-derived DNA was inserted into the rpoB gene of a serovar D strain, while in the type VIII recombinant, a ≤257-bp segment of serovar L1-derived DNA was inserted into the gyrA gene of a serovar D strain. Gray bars above the chromosome diagrams indicate the minimum lengths of trans-DNA that could account for the allelic signatures of the recombinant chromosomes. The derivation of these lengths is based on assumed DNA donors and is explained in the text. (A) The assumed DNA donor is L1:Ofx^r-1. (B) The assumed donor is D:Rif^r-1. Recombinant type VI is used to illustrate the two ways in which lengths are estimated for trans-DNA segments that extend either clockwise from ompA (type VI-C) or counterclockwise from ompA (type VI-CC). The estimated minimum serovar D-derived trans-DNA lengths for all recombinants were 386 to 794 kb.