A bioinformatic analysis of Pmp sequence and structure demonstrates that four of these encoded Pmps (PmpEFGH) vary consistently in relationship to the described phenotype, and these changes
include alterations of the net negative charge of the Pmp protein (Additional file 2: Tables S1-S2 and Additional file 3: Table S2). It is, however, preliminary to assess any property of a single protein or small set of proteins with the attachment efficiency distinction among strains. The recently developed genetic transcheck details formation system will be a critical technology selleck inhibitor in directly assessing such relationships in this species . The second phenotype investigated in our study was the formation of secondary inclusions within infected cells. This property of C. trachomatis strains varies not only between C. trachomatis serovars, but also between strains Epigenetics inhibitor within serovars . An intriguing result was the identification of high secondary inclusion formers in crosses between parents that exhibited very low secondary inclusion formation phenotypes (Table 1, Figure 7). While interpretations of this result are preliminary,
it appears that the phenotype is associated with two or more regions of the genome, and that a specific combination of genotypes at these positions is required for the high secondary inclusion formation phenotype to be manifested. Continued examination of novel recombinants, including backcrosses to integrate more parental genome into recombinant strains will add clarity to the phenotypes Resminostat we have discussed. We also continue to use the recombinants as tools to understand the basic processes associated with genetic exchange in the chlamydiae. Conclusion The described experiments characterize in detail the products of genetic exchange by C. trachomatis in vitro. Sequences representing over 1/3 of the chlamydial chromosome can be incorporated during these crosses. Selected phenotypes can be segregated in these crosses. This approach can be combined with the novel DNA transformation technologies being developed in these bacteria, leading
to novel approaches for determining the relationship between genetic makeup and chlamydial phenotype, both in vitro and in vivo. Methods Chlamydial strains and selection for resistance Antibiotic resistant C. trachomatis strains J/6276rif, RC-J/6276tet-rif, F(s)/70rif, F(s)/70tet-rif L2-434ofl,DUW/3Cx ofl, L1/440/LNrif or L3/404/LNrif were generated as previously described . Briefly, strains were grown in McCoy cells at a multiplicity of infection (MOI) of 1 in media containing sub-inhibitory concentrations, equivalent to half the minimum inhibitory concentration (MIC) of the appropriate drug. Serial passages of these strains were cultured in the media containing desired antibiotics until resistant mutants emerged or until passage was completely negative. Some strains required several attempts until resistant mutants were isolated.