scholarly journals Initial Characterization of the Two ClpP Paralogs ofChlamydia trachomatisSuggests Unique Functionality for Each

2018 ◽  
Vol 201 (2) ◽  
Author(s):  
Nicholas A. Wood ◽  
Krystal Y. Chung ◽  
Amanda M. Blocker ◽  
Nathalia Rodrigues de Almeida ◽  
Martin Conda-Sheridan ◽  
...  
Keyword(s):  
Host Cells ◽  
Developmental Cycle ◽  
Intracellular Bacteria ◽  
Clp Protease ◽  
Content Type ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Developmental Form ◽  
Obligate Intracellular Bacteria

ABSTRACTMembers ofChlamydiaare obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other.Chlamydiaspp. have five uncharacterizedclpgenes,clpX,clpC, twoclpPparalogs, andclpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactiveclpPmutants inChlamydiaspp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detectedin vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.IMPORTANCEChlamydia trachomatisis the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression inChlamydiaspp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

scholarly journals Initial Characterization of the Two ClpP Paralogs ofChlamydia trachomatisSuggests Unique Functionality for Each

2018 ◽  
Author(s):  
Nicholas A. Wood ◽  
Krystal Chung ◽  
Amanda Blocker ◽  
Nathalia Rodrigues de Almeida ◽  
Martin Conda-Sheridan ◽  
...  
Keyword(s):  
Host Cells ◽  
Developmental Cycle ◽  
Clp Protease ◽  
Developmentally Regulated ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Clp Proteases ◽  
Integral Role ◽  
Developmental Form

AbstractChlamydiais an obligate intracellular bacterium that differentiates between two distinct functional and morphological forms during its developmental cycle: elementary bodies (EBs) and reticulate bodies (RBs). EBs are non-dividing, small electron dense forms that infect host cells. RBs are larger, non-infectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other.Chlamydiahas five uncharacterizedclpgenes:clpX,clpC, twoclpPparalogs, andclpB. In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined these genes are expressed mid-cycle. Bioinformatic analyses of these proteins identified key residues important for activity. Over-expression of inactiveclpPmutants inChlamydiasuggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypic interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, ClpP2, but not ClpP1, protease activity was detectedin vitro. This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen.ImportanceChlamydia trachomatisis the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated, obligate intracellular pathogens that alternate between two functional and morphologic forms with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression inChlamydia, their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.

scholarly journals A Coming of Age Story: Chlamydia in the Post-Genetic Era

2015 ◽  
Vol 84 (3) ◽  
pp. 612-621 ◽  
Author(s):  
Anna J. Hooppaw ◽  
Derek J. Fisher
Keyword(s):  
Global Economy ◽  
Financial Burden ◽  
Developmental Cycle ◽  
Intracellular Bacteria ◽  
Content Type ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Reticulate Body ◽  
Technological Advances ◽  
Obligate Intracellular Bacteria

Chlamydiaspp. are ubiquitous, obligate, intracellular Gram-negative bacterial pathogens that undergo a unique biphasic developmental cycle transitioning between the infectious, extracellular elementary body and the replicative, intracellular reticulate body. The primaryChlamydiaspecies associated with human disease areC. trachomatis, which is the leading cause of both reportable bacterial sexually transmitted infections and preventable blindness, andC. pneumoniae, which infects the respiratory tract and is associated with cardiovascular disease. Collectively, these pathogens are a significant source of morbidity and pose a substantial financial burden on the global economy. Past efforts to elucidate virulence mechanisms of these unique and important pathogens were largely hindered by an absence of genetic methods. Watershed studies in 2011 and 2012 demonstrated that forward and reverse genetic approaches were feasible withChlamydiaand that shuttle vectors could be selected and maintained within the bacterium. While these breakthroughs have led to a steady expansion of the chlamydial genetic tool kit, there are still roads left to be traveled. This minireview provides a synopsis of the currently available genetic methods forChlamydiaalong with a comparison to the methods used in other obligate intracellular bacteria. Limitations and advantages of these techniques will be discussed with an eye toward the methods still needed, and how the current state of the art for genetics in obligate intracellular bacteria could direct future technological advances forChlamydia.

scholarly journals A SpoIID Homolog Cleaves Glycan Strands at the Chlamydial Division Septum

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Nicolas Jacquier ◽  
Akhilesh K. Yadav ◽  
Trestan Pillonel ◽  
Patrick H. Viollier ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Intracellular Bacteria ◽  
Human Pathogens ◽  
Content Type ◽  
Obligate Intracellular ◽  
Peptidoglycan Biosynthesis ◽  
Peptidoglycan Synthesis ◽  
Lytic Transglycosylase ◽  
Obligate Intracellular Bacteria

ABSTRACT Chlamydiales species are obligate intracellular bacteria lacking a classical peptidoglycan sacculus but relying on peptidoglycan synthesis for cytokinesis. While septal peptidoglycan biosynthesis seems to be regulated by MreB actin and its membrane anchor RodZ rather than FtsZ tubulin in Chlamydiales, the mechanism of peptidoglycan remodeling is poorly understood. An amidase conserved in Chlamydiales is able to cleave peptide stems in peptidoglycan, but it is not clear how peptidoglycan glycan strands are cleaved since no classical lytic transglycosylase is encoded in chlamydial genomes. However, a protein containing a SpoIID domain, known to possess transglycosylase activity in Bacillus subtilis, is conserved in Chlamydiales. We show here that the SpoIID homologue of the Chlamydia-related pathogen Waddlia chondrophila is a septal peptidoglycan-binding protein. Moreover, we demonstrate that SpoIID acts as a lytic transglycosylase on peptidoglycan and as a muramidase on denuded glycan strands in vitro. As SpoIID-like proteins are widespread in nonsporulating bacteria, SpoIID might commonly be a septal peptidoglycan remodeling protein in bacteria, including obligate intracellular pathogens, and thus might represent a promising drug target. IMPORTANCE Chlamydiales species are obligate intracellular bacteria and important human pathogens that have a minimal division machinery lacking the proteins that are essential for bacterial division in other species, such as FtsZ. Chlamydial division requires synthesis of peptidoglycan, which forms a ring at the division septum and is rapidly turned over. However, little is known of peptidoglycan degradation, because many peptidoglycan-degrading enzymes are not encoded by chlamydial genomes. Here we show that an homologue of SpoIID, a peptidoglycan-degrading enzyme involved in sporulation of bacteria such as Bacillus subtilis, is expressed in Chlamydiales, localizes at the division septum, and degrades peptidoglycan in vitro, indicating that SpoIID is not only involved in sporulation but also likely implicated in division of some bacteria.

scholarly journals In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport

2015 ◽  
Vol 83 (8) ◽  
pp. 3268-3280 ◽  
Author(s):  
Stephanie Dille ◽  
Eva-Maria Kleinschnitz ◽  
Collins Waguia Kontchou ◽  
Thilo Nölke ◽  
Georg Häcker
Keyword(s):  
Chlamydia Trachomatis ◽  
Golgi Apparatus ◽  
Bacterial Species ◽  
Human Cells ◽  
Host Cells ◽  
Developmental Cycle ◽  
Important Species ◽  
Content Type ◽  
Obligate Intracellular

TheChlamydialesare an order of obligate intracellular bacteria sharing a developmental cycle inside a cytosolic vacuole, with very diverse natural hosts, from amoebae to mammals. The clinically most important species isChlamydia trachomatis. Many uncertainties remain as to howChlamydiaorganizes its intracellular development and replication. The discovery of newChlamydialesspecies from other families permits the comparative analysis of cell-biological events and may indicate events that are common to all or peculiar to some species and more or less tightly linked to “chlamydial” development. We used this approach in the infection of human cells withWaddlia chondrophila, a species from the familyWaddliaceaewhose natural host is uncertain. Compared toC. trachomatis,W. chondrophilahad slightly different growth characteristics, including faster cytotoxicity. The embedding in cytoskeletal structures was not as pronounced as for theC. trachomatisinclusion.C. trachomatisinfection generates proteolytic activity by the proteaseChlamydiaprotease-like activity factor (CPAF), which degrades host substrates upon extraction; these substrates were not cleaved in the case ofW. chondrophila. UnlikeChlamydia,W. chondrophiladid not protect against staurosporine-induced apoptosis.C. trachomatisinfection causes Golgi apparatus fragmentation and redirects post-Golgi sphingomyelin transport to the inclusion; both were absent fromW. chondrophila-infected cells. When host cells were infected with both species, growth of both species was reduced. This study highlights differences between bacterial species that both depend on obligate intracellular replication inside an inclusion. Some features seem principally dispensable for intracellular development ofChlamydialesin vitrobut may be linked to host adaptation ofChlamydiaand the higher virulence ofC. trachomatis.

scholarly journals Role for GrgA in Regulation of σ28-Dependent Transcription in the Obligate Intracellular Bacterial PathogenChlamydia trachomatis

2018 ◽  
Vol 200 (20) ◽  
Author(s):  
Malhar Desai ◽  
Wurihan Wurihan ◽  
Rong Di ◽  
Joseph D. Fondell ◽  
Bryce E. Nickels ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chlamydia Trachomatis ◽  
Sigma Factor ◽  
Bacterial Pathogen ◽  
Direct Interaction ◽  
Developmental Cycle ◽  
Content Type ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Specific Transcription Factor

ABSTRACTThe obligate intracellular bacterial pathogenChlamydia trachomatishas a unique developmental cycle consisting of two contrasting cellular forms. Whereas the primaryChlamydiasigma factor, σ66, is involved in the expression of the majority of chlamydial genes throughout the developmental cycle, expression of several late genes requires the alternative sigma factor, σ28. In prior work, we identified GrgA as aChlamydia-specific transcription factor that activates σ66-dependent transcription by binding DNA and interacting with a nonconserved region (NCR) of σ66. Here, we extend these findings by showing GrgA can also activate σ28-dependent transcription through direct interaction with σ28. We measure the binding affinity of GrgA for both σ66and σ28, and we identify regions of GrgA important for σ28-dependent transcription. Similar to results obtained with σ66, we find that GrgA's interaction with σ28involves an NCR located upstream of conserved region 2 of σ28. Our findings suggest that GrgA is an important regulator of both σ66- and σ28-dependent transcription inC. trachomatisand further highlight NCRs of bacterial RNA polymerase as targets for regulatory factors unique to particular organisms.IMPORTANCEChlamydia trachomatisis the number one sexually transmitted bacterial pathogen worldwide. A substantial proportion ofC. trachomatis-infected women develop infertility, pelvic inflammatory syndrome, and other serious complications.C. trachomatisis also a leading infectious cause of blindness in underdeveloped countries. The pathogen has a unique developmental cycle that is transcriptionally regulated. The discovery of an expanded role for theChlamydia-specific transcription factor GrgA helps us understand the progression of the chlamydial developmental cycle.

Effect of bovine lactoferrin onChlamydia trachomatisinfection and inflammation

2017 ◽  
Vol 95 (1) ◽  
pp. 34-40 ◽  
Author(s):  
Rosa Sessa ◽  
Marisa Di Pietro ◽  
Simone Filardo ◽  
Alessia Bressan ◽  
Luigi Rosa ◽  
...  
Keyword(s):  
Acute Infection ◽  
Host Cells ◽  
Developmental Cycle ◽  
Bovine Lactoferrin ◽  
Bacterial Disease ◽  
Chronic Infections ◽  
Sexually Transmitted ◽  
Obligate Intracellular Pathogen

Chlamydia trachomatis is an obligate, intracellular pathogen responsible for the most common sexually transmitted bacterial disease worldwide, causing acute and chronic infections. The acute infection is susceptible to antibiotics, whereas the chronic one needs prolonged therapies, thus increasing the risk of developing antibiotic resistance. Novel alternative therapies are needed. The intracellular development of C. trachomatis requires essential nutrients, including iron. Iron-chelating drugs inhibit C. trachomatis developmental cycle. Lactoferrin (Lf), a pleiotropic iron binding glycoprotein, could be a promising candidate against C. trachomatis infection. Similarly to the efficacy against other intracellular pathogens, bovine Lf (bLf) could both interfere with C. trachomatis entry into epithelial cells and exert an anti-inflammatory activity. In vitro and in vivo effects of bLf against C. trachomatis infectious and inflammatory process has been investigated. BLf inhibits C. trachomatis entry into host cells when incubated with cell monolayers before or at the moment of the infection and down-regulates IL-6/IL-8 synthesized by infected cells. Six out of 7 pregnant women asymptomatically infected by C. trachomatis, after 30 days of bLf intravaginal administration, were negative for C. trachomatis and showed a decrease of cervical IL-6 levels. This is the first time that the bLf protective effect against C. trachomatis infection has been demonstrated.

scholarly journals Mimicry of the Pathogenic Mycobacterium VacuoleIn VitroElicits the Bacterial Intracellular Phenotype, Including Early-Onset Macrophage Death

2011 ◽  
Vol 79 (6) ◽  
pp. 2412-2422 ◽  
Author(s):  
Julie Early ◽  
Luiz E. Bermudez
Keyword(s):  
Early Onset ◽  
Disease Process ◽  
Host Cells ◽  
Intracellular Bacteria ◽  
Content Type ◽  
Time Points ◽  
Stepwise Reduction ◽  
Trace Elemental ◽  
Laboratory Tool

ABSTRACTMycobacterium aviumcomplex (MAC) within macrophages undergoes a phenotype change that allows for more efficient entry into surrounding host cells. We hypothesized that, by developing anin vitrosystem resembling the intravacuolar environment, one could generate insights into the mycobacterial intracellular phenotype. MAC was incubated in “elemental mixtures” that reproduce metal concentrations and pH in the vacuoles at different time points and then used to infect fresh macrophages. Incubation of MAC with the mixture corresponding to the vacuole environment 24 h postinfection infected macrophages at a significantly higher rate than bacteria that were incubated in Middlebrook 7H9 broth. Uptake occurred by macropinocytosis, similar to the uptake of bacteria passed through macrophages. Genes reported to be upregulated in intracellular bacteria, such asMav1365,Mav2409,Mav4487, andMav0996, were upregulated in MAC incubated in the 24-h elemental mixture. Like MAC obtained from macrophages, the vacuoles of bacteria from the 24-h elemental mixture were more likely to contain lysosome-associated membrane protein 1 (LAMP-1). A stepwise reduction scheme of the 24-h elemental mixture indicated that incubation in physiologically relevant concentrations of potassium chloride, calcium chloride, and manganese chloride was sufficient to induce characteristics of the intracellular phenotype. It was demonstrated that bacteria harboring the intracellular phenotype induced early-onset macrophage death more efficiently than bacteria grown in broth. This new trace elemental mixture mimicking the condition of the vacuole at different time points has the potential to become an effective laboratory tool for the study of the MAC andMycobacterium tuberculosisdisease process, increasing the understanding of the interaction with macrophages.

scholarly journals Single-Inclusion Kinetics of Chlamydia trachomatis Development

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Travis J. Chiarelli ◽  
Nicole A. Grieshaber ◽  
Anders Omsland ◽  
Christopher H. Remien ◽  
Scott S. Grieshaber
Keyword(s):  
Chlamydia Trachomatis ◽  
Mathematical Models ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Host Cells ◽  
Developmental Cycle ◽  
Cell Type ◽  
Content Type ◽  
Obligate Intracellular

ABSTRACT The obligate intracellular bacterial pathogen Chlamydia trachomatis is reliant on a developmental cycle consisting of two cell forms, termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but it does not replicate. The RB replicates in the host cell but is noninfectious. This developmental cycle is central to chlamydial pathogenesis. In this study, we developed mathematical models of the developmental cycle that account for potential factors influencing RB-to-EB cell type switching during infection. Our models predicted that two categories of regulatory signals for RB-to-EB development could be differentiated experimentally, an “intrinsic” cell-autonomous program inherent to each RB and an “extrinsic” environmental signal to which RBs respond. To experimentally differentiate between mechanisms, we tracked the expression of C. trachomatis development-specific promoters in individual inclusions using fluorescent reporters and live-cell imaging. These experiments indicated that EB production was not influenced by increased multiplicity of infection or by superinfection, suggesting the cycle follows an intrinsic program that is not directly controlled by environmental factors. Additionally, live-cell imaging revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a progression with expression from the euo and ihtA promoters evident in RBs, while expression from the promoter for hctA was apparent in early EBs/IBs. Finally, expression from the promoters for the true late genes, hctB, scc2, and tarp, was evident in the maturing EB. IMPORTANCE Chlamydia trachomatis is an obligate intracellular bacterium that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells, Chlamydia must complete a multiple-cell-type developmental cycle. The developmental cycle consists of specialized cells, the EB cell, which mediates infection of new host cells, and the RB cell, which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB-to-EB cell type switching, we demonstrate that RB-to-EB development follows a cell-autonomous program that does not respond to environmental cues. Additionally, we show that RB-to-EB development is a function of chlamydial growth and division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacterium’s pathogenesis.

scholarly journals Apoptosis Functions in Defense against Infection of Mammalian Cells with Environmental Chlamydiae

2020 ◽  
Vol 88 (6) ◽  
Author(s):  
Dominik Brokatzky ◽  
Oliver Kretz ◽  
Georg Häcker
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Intracellular Bacteria ◽  
Mitochondrial Apoptosis ◽  
Content Type ◽  
Obligate Intracellular ◽  
Human Epithelial Cells ◽  
Antiapoptotic Activity ◽  
Obligate Intracellular Bacteria ◽  
Induced Apoptosis

ABSTRACT Apoptotic cell death can be an efficient defense reaction of mammalian cells infected with obligate intracellular pathogens; the host cell dies and the pathogen cannot replicate. While this is well established for viruses, there is little experimental support for such a concept in bacterial infections. All Chlamydiales are obligate intracellular bacteria, and different species infect vastly different hosts. Chlamydia trachomatis infects human epithelial cells; Parachlamydia acanthamoebae replicates in amoebae. We here report that apoptosis impedes growth of P. acanthamoebae in mammalian cells. In HeLa human epithelial cells, P. acanthamoebae infection induced apoptosis, which was inhibited when mitochondrial apoptosis was blocked by codeletion of the mediators of mitochondrial apoptosis, Bax and Bak, by overexpression of Bcl-XL or by deletion of the apoptosis initiator Noxa. Deletion of Bax and Bak in mouse macrophages also inhibited apoptosis. Blocking apoptosis permitted growth of P. acanthamoebae in HeLa cells, as measured by fluorescence in situ hybridization, assessment of genome replication and protein synthesis, and the generation of infectious progeny. Coinfection with C. trachomatis inhibited P. acanthamoebae-induced apoptosis, suggesting that the known antiapoptotic activity of C. trachomatis can also block P. acanthamoebae-induced apoptosis. C. trachomatis coinfection could not rescue P. acanthamoebae growth in HeLa; in coinfected cells, C. trachomatis even suppressed the growth of P. acanthamoebae independently of apoptosis, while P. acanthamoebae surprisingly enhanced the growth of C. trachomatis. Our results show that apoptosis can be used in the defense of mammalian cells against obligate intracellular bacteria and suggest that the known antiapoptotic activity of human pathogenic chlamydiae is indeed required to permit their growth in human cells.

scholarly journals Interstrain Gene Transfer in Chlamydia trachomatis In Vitro: Mechanism and Significance

2007 ◽  
Vol 190 (5) ◽  
pp. 1605-1614 ◽  
Author(s):  
Robert DeMars ◽  
Jason Weinfurter
Keyword(s):  
Gene Transfer ◽  
Chlamydia Trachomatis ◽  
Host Cells ◽  
Relative Fitness ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Allelic Differences ◽  
Important Means ◽  
Conceivable Mechanism

ABSTRACT The high frequency of between-strain genetic recombinants of Chlamydia trachomatis among isolates obtained from human sexually transmitted infections suggests that lateral gene transfer (LGT) is an important means by which C. trachomatis generates variants that have enhanced relative fitness. A mechanism for LGT in C. trachomatis has not been described, and investigation of this phenomenon by experimentation has been hampered by the obligate intracellular development of this pathogen. We describe here experiments that readily detected LGT between strains of C. trachomatis in vitro. Host cells were simultaneously infected with an ofloxacin-resistant (Ofxr) mutant of a serovar L1 strain (L1:Ofxr-1) and a rifampin-resistant (Rifr) mutant of a serovar D strain (D:Rifr-1). Development occurred in the absence of antibiotics, and the progeny were subjected to selection for Ofxr Rifr recombinants. The parental strains differed at many polymorphic nucleotide sites, and DNA sequencing was used to map genetic crossovers and to determine the parental sources of DNA segments in 14 recombinants. Depending on the assumed DNA donor, the estimated minimal length of the transferred DNA was ≥123 kb in one recombinant but was ≥336 to ≥790 kb in all other recombinants. Such trans-DNA lengths have been associated only with conjugation in known microbial LGT systems, but natural DNA transformation remains a conceivable mechanism. LGT studies can now be performed with diverse combinations of C. trachomatis strains, and they could have evolutionary interest and yield useful recombinants for functional analysis of allelic differences between strains.

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