Identification of the Chlamydia trachomatis RecA-encoding gene

Abstract

DNA sequencing of the major outer membrane protein (MOMP) gene (omp1) from Chlamydia trachomatis shows that some strains have a mosaic structure suggestive of homologous recombination between two distinct omp1 genes. On the basis of this conjecture, we attempted to clone by complementation and sequence the chlamydial recA homolog from C. trachomatis serovar L 2. Chlamydial genomic DNA was partially restricted with XbaI, and fragments of 2 to 4 kb were ligated into pUC19. The recombinant plasmid was electroporated into Escherichia coli HB101 (RecA ؊), and colonies were selected in the presence of methyl methanesulfonate (MMS). A 2.1-kb fragment of C. trachomatis DNA in pUC19 conferred relative MMS resistance to E. coli HB101. When this recombinant plasmid (pX203) was electroporated into E. coli JC14604 (RecA ؊ lacZ), lac ؉ recombinants were isolated. Rabbit polyclonal antibodies produced to purified E. coli RecA were immunoreactive in an immunoblot assay with a 35-kDa antigen in RecA ؊ strains of E. coli transformed with pX203. The 2.1-kb insert was cycle sequenced by the dideoxy chain termination method. An open reading frame of 1,056 bp encoding 352 amino acids that had 44% sequence identity with E. coli RecA was identified. The finding of a recA homolog in C. trachomatis suggests that homologous recombination may occur in this organism. The cloned C. trachomatis RecA-encoding gene will be useful for the construction of a recA mutant once a gene transfer system is developed for chlamydiae.

Characterization of Chlamydia MurC‐Ddl, a fusion protein exhibiting D‐alanyl‐D‐alanine ligase activity involved in peptidoglycan synthesis and D‐cycloserine sensitivity

“…However, these hindrances have been offset by the recently available genome sequences of seven strains representing four species of Chlamydia (Stephens et al ., 1998; Read et al ., 2000; 2003) . Despite the inability to perform genetic manipulations in Chlamydia , knowledge of the unique biology of these organisms has been gained by using established genetic systems of heterologous host systems such as E. coli to identify the function of both homologous and Chlamydia‐ specific gene products (Tipples and McClarty, 1995; Zhang et al ., 1995; Wylie et al ., 1997; Sweet et al ., 2001; Schwoppe et al ., 2002; Hesse et al ., 2003; Karunakaran et al ., 2003; McCoy et al ., 2003) . Phylogenetically, Chlamydia lie very distant to other eubacteria thus the ability of a chlamydial gene to complement a homologous mutation in another eubacteria provides a strong indication that the gene product expressed in Chlamydia is functional.…”

The immunobiology and immunopathology of chlamydial infections

“…The simplest explanation is that recombination occurred within fused inclusions resulting from mixed serovar L1 and L2 infection. Interestingly, a gene analogous to the RecA gene mediating recombination in E. coli has been recognised in C. truchomutis (193). As both N and C terminal halves of MOMP are reported essential for the binding of serovar L1-specific typing antibody (34) the result was a major antigenic shift which prevented antibody binding to the neutralising serovar-specific epitope (61,62).…”

Molecular Cloning, Sequence Analysis and Functional Characterization of the Gene Kdsa, Encoding 3‐Deoxy‐D‐Manno‐2‐Octulosonate‐8‐Phosphate Synthase of Chlamydia Psittaci 6BC

“…Therefore, taking into account that the amino acid sequences of distantly related bacteria were compared in this study, we conclude that bacterial Kdo-8-P synthases are homologous proteins. Identity values of chlamydial proteins exceeding 40 % compared to E. coli are generally documented for those involved in housekeeping cellular processes like transcription, translation, recombination or heat shock response (Koehler et al, 1990;Gu et al, 1995;Zhang et al, 1995;Morrison et al, 1989;Schmiel and Wyrick, 1994). In addition, the sequence of KdsB which was recently reported for C. tachomatis L2 shows a high degree of similarity to the corresponding protein of E. coli (Tripples and McClarty, 1995).…”