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Haemophilus ducreyi causes cutaneous ulcers in children and the genital ulcer disease chancroid in adults. In humans, H. ducreyi is found in the anaerobic environment of an abscess; previous studies comparing bacterial gene expression levels in pustules with the inocula (∼4-h aerobic mid-log-phase cultures) identified several upregulated differentially expressed genes (DEGs) that are associated with anaerobic metabolism. To determine how H. ducreyi alters its gene expression in response to anaerobiosis, we performed RNA sequencing (RNA-seq) on both aerobic and anaerobic broth cultures harvested after 4, 8, and 18 h of growth. Principal-coordinate analysis (PCoA) plots showed that anaerobic growth resulted in distinct transcriptional profiles compared to aerobic growth. During anaerobic growth, early-time-point comparisons (4 versus 8 h) identified few DEGs at a 2-fold change in expression and a false discovery rate (FDR) of

DNA replication is essential for the growth and development of Chlamydia trachomatis ; however, it is unclear how this process contributes to and is controlled by the pathogen’s biphasic life cycle. While inhibitors of transcription, translation, cell division, and glucose-6-phosphate transport all negatively affect chlamydial intracellular development, the effects of directly inhibiting DNA polymerase have never been examined. We isolated a temperature-sensitive dnaE mutant (the dnaE ts mutant) that exhibits an ∼100-fold reduction in genome copy number at the nonpermissive temperature (40°C) but replicates similarly to the parent at the permissive temperature of 37°C. We measured higher ratios of genomic DNA nearer the origin of replication than the terminus in the dnaE ts mutant at 40°C, indicating that this replication deficiency is due to a defect in DNA polymerase processivity. The dnaE ts mutant formed fewer and smaller pathogenic vacuoles (inclusions) at 40°C, and the bacteria appeared enlarged and exhibited defects in cell division. The bacteria also lacked both discernible peptidoglycan and polymerized MreB, the major cell division-organizing protein in Chlamydia responsible for nascent peptidoglycan biosynthesis. We also found that the absolute genome copy number, rather than active genome replication, was sufficient for infectious progeny production. Deficiencies in both genome replication and inclusion expansion were reversed when the dnaE ts mutant was shifted from 40°C to 37°C early in infection, and intragenic suppressor mutations in dnaE ts also restored genome replication and inclusion expansion in the dnaE ts mutant at 40°C. Overall, our results show that genome replication in C. trachomatis is required for inclusion expansion, septum formation, and the transition between the microbe’s replicative and infectious forms. IMPORTANCE Chlamydiae transition between infectious, extracellular elementary bodies (EBs) and noninfectious, intracellular reticulate bodies (RBs). Some checkpoints that govern transitions in chlamydial development have been identified, but the extent to which genome replication plays a role in regulating the pathogen’s infectious cycle has not been characterized. We show that genome replication is dispensable for EB-to-RB conversion but is necessary for RB proliferation, division septum formation, and inclusion expansion. We use new methods to investigate developmental checkpoints and dependencies in Chlamydia that facilitate the ordering of events in the microbe’s biphasic life cycle. Our findings suggest that Chlamydia utilizes feedback inhibition to regulate core metabolic processes during development, likely an adaptation to intracellular stress and a nutrient-limiting environment.

A critical step in intracellular Chlamydia infection is the production of infectious progeny through the expression of late genes. This differentiation step involves conversion from a reticulate body (RB), which is the replicating form of the bacterium, into an elementary body (EB), which is the developmental form that spreads the infection to a new host cell. EUO is an important chlamydial transcription factor that controls the expression of late genes, but the mechanisms that regulate EUO are not known. We report that a plasmid-encoded protein, Pgp4, enhanced the repressor activity of EUO. Pgp4 did not function as a transcription factor because it did not bind or directly modulate transcription of its target promoters. Instead, Pgp4 increased the ability of EUO to bind and repress EUO-regulated promoters in vitro and physically interacted with EUO in pulldown assays with recombinant proteins. We detected earlier onset of EUO-dependent late gene expression by immunofluorescence microscopy in Pgp4-deficient C. trachomatis and C. muridarum strains. In addition, the absence of Pgp4 led to earlier onset of RB-to-EB conversion in C. muridarum. These data support a role for Pgp4 as a negative regulator of chlamydial transcription that delays late gene expression. Our studies revealed that Pgp4 also has an EUO-independent function as a positive regulator of chlamydial transcription. IMPORTANCEChlamydia trachomatis is an important human pathogen that causes more than 150 million active cases of genital and eye infection in the world. This obligate intracellular bacterium produces infectious progeny within an infected human cell through the expression of late chlamydial genes. We showed that the ability of a key chlamydial transcription factor, EUO, to repress late genes was enhanced by a plasmid-encoded protein, Pgp4. In addition, studies with Chlamydia Pgp4-deficient strains provide evidence that Pgp4 delays late gene expression in infected cells. Thus, Pgp4 is a novel regulator of late gene expression in Chlamydia through its ability to enhance the repressor function of EUO.

Chlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l -tryptophan to N -formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694 , were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells.

Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia -specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperature-shift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle. IMPORTANCE Study of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms.