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The International Committee on Systematics of Prokaryotes (ICSP) discussed and rejected in 2020 a proposal to modify the International Code of Nomenclature of Prokaryotes to allow the use of gene sequences as type for naming prokaryotes. An alternative nomenclatural code, the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode), which considers genome sequences as type material for naming species, was published in 2022. Members of the ICSP subcommittee for the taxonomy of the phylum Chlamydiae (Chlamydiota) consider that the use of gene sequences as type would benefit the taxonomy of microorganisms that are difficult to culture such as the chlamydiae and other strictly intracellular bacteria. We recommend the registration of new names of uncultured prokaryotes in the SeqCode registry.

Chlamydia trachomatisis an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infections. Changes in DNA topology in this pathogen have been linked to its pathogenicity-associated developmental cycle. Here, evidence is provided that the balanced activity of DNA topoisomerases (Topos) contributes toChlamydiadevelopmental processes. Utilizing catalytically inactivated Cas12 (dCas12) based-clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology, we demonstrate targeted knockdown of chromosomaltopAtranscription inC. trachomatiswithout detected toxicity of dCas12. Repression oftopAimpaired the growth ofC. trachomatismostly through disruption of its differentiation from a replicative form to an infectious form. Consistent with this, expression of late developmental genes ofC. trachomatiswas downregulated while early genes maintained their expression. Importantly, the growth defect associated withtopAknockdown was rescued by overexpressingtopAat an appropriate degree and time, directly linking the growth patterns to the levels oftopAexpression. Interestingly,topAknockdown had pleiotropic effects on DNA gyrase expression, indicating a potential compensatory mechanism for survival to offset TopA deficiency.C. trachomatiswithtopAknocked down displayed hypersensitivity to moxifloxacin that targets DNA gyrase in comparison with the wild type. These data underscore the requirement of integrated topoisomerase actions to support the essential development and transcriptional processes ofC. trachomatis.ImportanceWe used genetic and chemical tools to demonstrate the relationship of topoisomerase activities and their obligatory role for the chlamydial developmental cycle. Successfully targeting the essential genetopAwith a CRISPRi approach, using dCas12, inC. trachomatisindicates that this method will facilitate the characterization of the essential genome. These findings have an important impact on our understanding of the mechanism(s) by which well-balanced topoisomerase activities enableC. trachomatisto adapt to unfavorable growth conditions imposed by antibiotics.

Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium that undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms, each of which expresses its own specialized repertoire of proteins. The transitions between the infectious, non-dividing elementary body (EB) and the non-infectious, replicative reticulate body (RB) are not mediated by division events that re-distribute intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation require protein turnover. The Clp protease system is well conserved in bacteria and, minimally, relies on a serine protease subunit, ClpP, and a AAA+ ATPase, such as ClpX, that recognizes and unfolds substrates for ClpP degradation. In Chlamydia , clpX is encoded within an operon adjacent to clpP2 . We present evidence that the chlamydial ClpX ortholog, and the co-transcribed ClpP2, play a key role in organism viability and development. We demonstrate here that chlamydial ClpX is a functional ATPase and forms the expected homohexamer in vitro . Overexpression of a ClpX mutant lacking ATPase activity had a limited impact on DNA replication or secondary differentiation but, nonetheless, reduced EB viability. Conversely, the overexpression of an inactive ClpP2 mutant significantly impacted later developmental cycle progression by reducing the overall number of organisms. Blocking clpP2X transcription using CRISPR interference led to a decrease in bacterial growth, which did not occur when the non-essential gene incA was targeted. Taken together, our data indicate that ClpX and the associated ClpP2 play a critical role in developmental cycle progression and differentiation. Importance Chlamydia trachomatis is the leading cause of infectious blindness globally and the most reported bacterial sexually transmitted infection both domestically and internationally. Given the economic burden, the lack of an approved vaccine, and the use of broad-spectrum antibiotics for treatment of infections, a further understanding of chlamydial growth and development is critical for the advancement of novel, targeted antibiotics. The Clp proteins comprise an important and conserved protease system in bacteria. Our work highlights the importance of the chlamydial Clp proteins to this clinically important bacterium. Additionally, our study implicates the Clp system playing an integral role in chlamydial developmental cycle progression, which may help establish models of how Chlamydia spp. and other bacteria progress through their respective developmental cycles. Our work also contributes to a growing body of Clp-specific research that underscores the importance and versatility of this system throughout bacterial evolution and further validates Clp-proteins as drug targets.