A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis

Garvin, Lotisha E.; Voorde, Rebecca Vande; Dickinson, Mary S; Carrell, Steven; Hybiske, Kevin; Rockey, Daniel D.

doi:10.1371/journal.pone.0261088

Cited by 6 publications

(8 citation statements)

References 28 publications

(37 reference statements)

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“…Since the first demonstration of reproducible Chlamydia transformation using a shuttle vector 12 years ago ( 35 ), the research community has leveraged this reverse genetic tool to investigate gene function via ectopic overexpression, insertional mutagenesis, deletion, and other methods ( 11 , 24 – 30 , 50 – 54 ). Nonetheless, the lack of effective strategies to disrupt truly essential genes, particularly those whose overexpression is toxic, has hampered research in Chlamydia and other biological systems.…”

Section: Discussionmentioning

confidence: 99%

Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance

Lu,

Wang,

Wurihan

et al. 2024

mBio

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Chlamydia , an obligate intracellular bacterial pathogen, has a unique developmental cycle involving the differentiation of invading elementary bodies (EBs) to noninfectious reticulate bodies (RBs), replication of RBs, and redifferentiation of RBs into progeny EBs. Progression of this cycle is regulated by three sigma factors, which direct the RNA polymerase to their respective target gene promoters. We hypothesized that the Chlamydia- specific transcriptional regulator GrgA, previously shown to activate σ66 and σ28, plays an essential role in chlamydial development and growth. To test this hypothesis, we applied a novel genetic tool known as dependence on plasmid-mediated expression to create Chlamydia trachomatis with conditional GrgA deficiency. We show that GrgA-deficient C. trachomatis RBs have a growth rate that is approximately half of the normal rate and fail to transition into progeny EBs. In addition, GrgA-deficient C. trachomatis fails to maintain its virulence plasmid. Results of RNA-Seq analysis indicate that GrgA promotes RB growth by optimizing tRNA synthesis and expression of nutrient-acquisition genes, while it enables RB-to-EB conversion by facilitating the expression of a histone and outer membrane proteins required for EB morphogenesis. GrgA also regulates numerous other late genes required for host cell exit and subsequent EB invasion into host cells. Importantly, GrgA stimulates the expression of σ54, the third and last sigma factor, and its activator, AtoC, and thereby indirectly upregulating the expression of σ54-dependent genes. In conclusion, our work demonstrates that GrgA is a master transcriptional regulator in Chlamydia and plays multiple essential roles in chlamydial pathogenicity. IMPORTANCE Hallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed dependence on plasmid-mediated expression (DOPE) that allows for the knockdown of essential genes in Chlamydia . We demonstrate that GrgA, a Chlamydia -specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.

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Section: Discussionmentioning

confidence: 99%

Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance

Lu,

Wang,

Wurihan

et al. 2024

mBio

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“…Current transformation protocols for Chlamydia focus on C. trachomatis or C. muridarum ( 14 ) and either require the use of cloning vectors with a broad host range ( 35 ) or the implementation of chlamydial plasmid sequences into the vector of choice, requiring at least the chlamydial ori ( 23 ).…”

Section: Discussionmentioning

confidence: 99%

“…Most routinely used vectors to date lead to allelic exchange with loss of the free plasmid vector over time ( 16 , 23 ). Full integration of a vector into the chromosome has so far been observed for a broad-spectrum vector isolated from Bordetella bronchiseptica that was modified for transformation into C. trachomatis L2 ( 35 ).…”

Section: Discussionmentioning

confidence: 99%

Chlamydia suis displays high transformation capacity with complete cloning vector integration into the chromosomal rrn-nqrF plasticity zone

Marti,

Biggel,

Shima

et al. 2023

Microbiol Spectr

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Chlamydia , comprising several human and zoonotic pathogens, is a genus of the conserved bacterial phylum Chlamydiota. Their obligate intracellular niche serves as a barrier for natural genetic exchange via horizontal gene transfer (HGT), and further limits the development and application of genetic tools. To date, the only example for recent inter-phylum HGT among the Chlamydiota is tetracycline resistance in the potentially zoonotic species Chlamydia suis , a close phylogenetic relative of human C. trachomatis , which causes bacterial sexually transmitted infections and ocular trachoma. Tetracycline resistance in porcine C. suis strains has been described worldwide and is always part of a genomic island dividing invasin ( inv ), located within a chromosomal region between the rRNA operon ( rrn ) and the nqrF reductase. Here, we aimed to expand the still modest number of available genetic manipulation systems for Chlamydia by generating allele-replacement and integration vectors for C. suis . These vectors comprised homologous C. suis sequences of the chromosomal region of interest, an E. coli origin of replication ( ori ) and selection markers but lacked the native chlamydial plasmids or its ori . We first recovered allele-replacement mutants using a vector that targets the tryptophan ( trp ) operon of C. suis . The vector was further successfully maintained as a free plasmid in C. trachomatis without allele replacement, suggesting complex plasmid dynamics in the absence of a chlamydial ori . Moreover, we showed that the hypervariable rrn-nqrF intergenic region of C. suis is highly susceptible to transformation, resulting in complete vector integration upstream of nqrF without interruption of the targeted inv gene. IMPORTANCE The obligate intracellular Chlamydia genus contains many pathogens with a negative impact on global health and economy. Despite recent progress, there is still a lack of genetic tools limiting our understanding of these complex bacteria. This study provides new insights into genetic manipulation of Chlamydia with the opportunistic porcine pathogen Chlamydia suis , the only chlamydial species naturally harboring an antibiotic resistance gene, originally obtained by horizontal gene transfer. C. suis is transmissible to humans, posing a potential public health concern. We report that C. suis can take up vectors that lack the native plasmid, a requirement for most chlamydial transformation systems described to date. Additionally, we show that C. trachomatis , the most common cause for bacterial sexually transmitted infections and infectious blindness worldwide, can be transformed with C. suis vectors. Finally, the chromosomal region that harbors the resistance gene of C. suis is highly susceptible to complete vector integration.

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“…This construct was maintained as both an episome and an integrated element in transformed strains. It is expected that further work with the pBBR1 vector system will perhaps allow the maintaining of non-chlamydial-plasmid-based genetic elements in transformed strains ( Garvin et al., 2021 ).…”

Section: Homologous Recombination and Genetic Engineeringmentioning

confidence: 99%

The Impact of Lateral Gene Transfer in Chlamydia

Marti

Suchland

Rockey

2022

Front. Cell. Infect. Microbiol.

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Lateral gene transfer (LGT) facilitates many processes in bacterial ecology and pathogenesis, especially regarding pathogen evolution and the spread of antibiotic resistance across species. The obligate intracellular chlamydiae, which cause a range of diseases in humans and animals, were historically thought to be highly deficient in this process. However, research over the past few decades has demonstrated that this was not the case. The first reports of homologous recombination in the Chlamydiaceae family were published in the early 1990s. Later, the advent of whole-genome sequencing uncovered clear evidence for LGT in the evolution of the Chlamydiaceae, although the acquisition of tetracycline resistance in Chlamydia (C.) suis is the only recent instance of interphylum LGT. In contrast, genome and in vitro studies have shown that intraspecies DNA exchange occurs frequently and can even cross species barriers between closely related chlamydiae, such as between C. trachomatis, C. muridarum, and C. suis. Additionally, whole-genome analysis led to the identification of various DNA repair and recombination systems in C. trachomatis, but the exact machinery of DNA uptake and homologous recombination in the chlamydiae has yet to be fully elucidated. Here, we reviewed the current state of knowledge concerning LGT in Chlamydia by focusing on the effect of homologous recombination on the chlamydial genome, the recombination machinery, and its potential as a genetic tool for Chlamydia.

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A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis

Cited by 6 publications

References 28 publications

Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance

Requirement of GrgA for Chlamydia infectious progeny production, optimal growth, and efficient plasmid maintenance

Chlamydia suis displays high transformation capacity with complete cloning vector integration into the chromosomal rrn-nqrF plasticity zone

The Impact of Lateral Gene Transfer in Chlamydia

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