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The Genetic Transformation of Chlamydia pneumoniae.

ABSTRACT: We demonstrate the genetic transformation of Chlamydia pneumoniae using a plasmid shuttle vector system which generates stable transformants. The equine C. pneumoniae N16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearing C. pneumoniae transformants. Using the pRSGFPCAT-Cpn plasmid construct, expression of RSGFP in koala isolate C. pneumoniae LPCoLN was demonstrated. Furthermore, we discovered that the human cardiovascular isolate C. pneumoniae CV-6 and the human community-acquired pneumonia-associated C. pneumoniae IOL-207 could also be transformed with pRSGFPCAT-Cpn. In previous studies, it was shown that Chlamydia spp. cannot be transformed when the plasmid shuttle vector is constructed from a different plasmid backbone to the homologous species. Accordingly, we confirmed that pRSGFPCAT-Cpn could not cross the species barrier in plasmid-bearing and plasmid-free C. trachomatis, C. muridarum, C. caviae, C. pecorum, and C. abortus However, contrary to our expectation, pRSGFPCAT-Cpn did transform C. felis Furthermore, pRSGFPCAT-Cpn did not recombine with the wild-type plasmid of C. felis Taken together, we provide for the first time an easy-to-handle transformation protocol for C. pneumoniae that results in stable transformants. In addition, the vector can cross the species barrier to C. felis, indicating the potential of horizontal pathogenic gene transfer via a plasmid.IMPORTANCE The absence of tools for the genetic manipulation of C. pneumoniae has hampered research into all aspects of its biology. In this study, we established a novel reproducible method for C. pneumoniae transformation based on a plasmid shuttle vector system. We constructed a C. pneumoniae plasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase in C. pneumoniae C. pneumoniae transformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation in C. pneumoniae using pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions in C. pneumoniae biology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species.

SUBMITTER: Shima K

PROVIDER: S-EPMC6180227 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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The Genetic Transformation of Chlamydia pneumoniae.

Shima Kensuke K Wanker Maximilian M Skilton Rachel J RJ Cutcliffe Lesley T LT Schnee Christiane C Kohl Thomas A TA Niemann Stefan S Geijo Javier J Klinger Matthias M Timms Peter P Rattei Thomas T Sachse Konrad K Clarke Ian N IN Rupp Jan J

mSphere 20181010 5

We demonstrate the genetic transformation of Chlamydia pneumoniae using a plasmid shuttle vector system which generates stable transformants. The equine C. pneumoniae N16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearing C. pneumoniae ...[more]

PMID: 30305318

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Project description:Partial duplication of genetic material is prevalent in eukaryotes and provides potential for evolution of new traits. Prokaryotes, which are generally haploid in nature, can evolve new genes by partial chromosome duplication, known as merodiploidy. Little is known about merodiploid formation during genetic exchange processes, although merodiploids have been serendipitously observed in early studies of bacterial transformation. Natural bacterial transformation involves internalization of exogenous donor DNA and its subsequent integration into the recipient genome by homology. It contributes to the remarkable plasticity of the human pathogen Streptococcus pneumoniae through intra and interspecies genetic exchange. We report that lethal cassette transformation produced merodiploids possessing both intact and cassette-inactivated copies of the essential target gene, bordered by repeats (R) corresponding to incomplete copies of IS861. We show that merodiploidy is transiently stimulated by transformation, and only requires uptake of a ~3-kb DNA fragment partly repeated in the chromosome. We propose and validate a model for merodiploid formation, providing evidence that tandem-duplication (TD) formation involves unequal crossing-over resulting from alternative pairing and interchromatid integration of R. This unequal crossing-over produces a chromosome dimer, resolution of which generates a chromosome with the TD and an abortive chromosome lacking the duplicated region. We document occurrence of TDs ranging from ~100 to ~900 kb in size at various chromosomal locations, including by self-transformation (transformation with recipient chromosomal DNA). We show that self-transformation produces a population containing many different merodiploid cells. Merodiploidy provides opportunities for evolution of new genetic traits via alteration of duplicated genes, unrestricted by functional selective pressure. Transient stimulation of a varied population of merodiploids by transformation, which can be triggered by stresses such as antibiotic treatment in S. pneumoniae, reinforces the plasticity potential of this bacterium and transformable species generally.

Project description:The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy. The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D. Differences between C.trachomatis strains were focused on an approximately 50 kb 'plasticity zone' near the termination origins. In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli O157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region. The C. pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes. AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C. pneumoniae. Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene. Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region. The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites. In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens.

Dataset Information

The Genetic Transformation of Chlamydia pneumoniae.

Publications

The Genetic Transformation of Chlamydia pneumoniae.

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