Transformation and isolation of allelic exchange mutants of Chlamydia psittaci using recombinant DNA introduced by electroporation

Abstract

To facilitate genetic investigations in the obligate intracellular pathogens Chlamydia, the ability to construct variants by homologous recombination was investigated in C. psittaci 6BC. The single rRNA operon was targeted with a synthetic 16S rRNA allele, harboring three nucleotide substitutions over 398 bp, which imparts resistance to kasugamycin (Ksm) and spectinomycin (Spc) and causes loss of one HpaI restriction site. A fourth, silent mutation was introduced 654 bp downstream in the beginning of the 23S rRNA gene. C. psittaci 6BC infectious particles were electroporated with various concentrations of circular or linearized plasmids containing different lengths of the rRNA region homologous to the chromosomal copy except for the four nucleotide substitutions. Ksm and Spc were added 18 h after inoculation onto confluent cell monolayers in the plaque assay. Resistant plaques were picked and expanded with selection 10 days later before collecting DNA for analysis by PCR, restriction mapping, sequencing, or Southern. Spontaneous resistance to Ksm and Spc was never observed in mock electroporated bacteria (frequency <6.2 ؋ 10 ؊9 ). Conversely, double resistance and replacement of the 16S rRNA gene were observed when C. psittaci was electroporated with the recombination substrates. Highest efficiency was obtained with 10 g of circular vector prepared in a DNA methylase-deficient Escherichia coli (1.9 ؎ 1.1 ؋ 10 ؊6 , n ‫؍‬ 7). Coinheritance of the silent 23S rRNA mutation was seen in 46 of 67 recombinants analyzed, illustrating DNA exchange of up to 1,052 bp in length. These findings provide the first step toward genetic manipulation of Chlamydia.recombination ͉ selection ͉ plaque assay

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

“…Indeed, this barrier might explain the relatively low frequencies of insertion elements, phage remnants, and pathogenicity islands or the lack of genes coding for restriction enzymes in the genomes of chlamydial species. Nonetheless, DNA has successfully been introduced into Chlamydia following the electroporation and chemical transformation of EBs (14,18,19), although such transformation events require large amounts of DNA (5 to 10 g) and occur at a very low frequency.…”

“…Furthermore, the higher MICs required for the delivery of these antibiotics into the inclusion can lead to toxicity in the host cell, which imposes further limits on the use of antibiotics for selection. Several antibiotics have been used successfully in genetic selections, including chloramphenicol, kasugamycin, nalidixic acid, rifampin, spectinomycin, trimethoprim, tetracycline (only for naturally resistant veterinary strains), ␤-lactams (only for LGV serovars), and blasticidin S (12,14,18,19,27,31,(50)(51)(52)(53). However, the use of mutant versions of chlamydial factors, such as 16S rRNA, RpoB, and GyrA, that render them resistant to antibiotics as selectable markers is limited because the gene mutations that confer resistance to these antibiotics are often recessive.…”

“…Subsequent studies demonstrated that lateral gene transfer (LGT) events in Chlamydia are frequent and robust and occur both in nature and in the laboratory (4,(11)(12)(13). Transformation of exogenous DNA into Chlamydia has been achieved via several methods, including electroporation, dendrimer-based delivery, and a calcium chloride-based treatment (14)(15)(16)(17)(18)(19). Successful and stable transformation of C. trachomatis LGV L2 with an Escherichia coli-C. trachomatis L2 shuttle plasmid conferring ␤-lactam resistance led to the development of expression vectors for expressing Chlamydia open reading frames (ORFs), fluorescent proteins (green fluorescent protein [GFP], cyan fluorescent protein [CFP], mCherry, and mKate2), and reporter proteins (␤-galactosidase, adenylate cyclase, and glycogen synthase kinase [GSK]-tagged proteins) (19)(20)(21)(22)(23)(24)(25)(26).…”

Chlamydial Intracellular Survival Strategies

“…Early efforts achieved transient transformation of chimeric C. trachomatis ( pCT) plasmids (Tam et al 1994). In later experiments, Chlamydia psittaci was transformed with allelic exchange vectors that targeted the 16S rRNA gene, and the investigators were able to show successful replacement of 16S rRNA loci with the introduced DNA (Binet and Maurelli 2009). Very recently, Clarke and colleagues reported successful stable transformation of C. trachomatis with a pCTshuttle plasmid by calcium chloride-mediated transformation of EBs and selection for resistance to b lactams (Wang et al 2011).…”

Generation of targeted Chlamydia trachomatis null mutants

“…C. trachomatis genomes are amenable to manipulation as shown by naturally occurring recombination (5) and by the moderate frequency of in vitro lateral gene transfer (6,7). Chlamydiae have also been successfully transformed to antibiotic resistance by electroporation, but transformants either were unstable or successful transformation was limited to replacing the 16S rRNA with its antibiotic-resistant allele (8,9). Thus, these advancements have not led to the development of practical methods for genetic manipulation.…”