Abstract
The human pathogen Chlamydia trachomatis proceeds through a multi phenotypic developmental cycle with each cell form specialized for different roles in pathogenesis. Understanding the mechanisms regulating this complex cycle has historically been hampered by limited genetic tools. In an effort to address this issue, we developed a translational control system to regulate gene expression in Chlamydia using a synthetic riboswitch. Here we demonstrate that translational control via a riboswitch can be used in combination with a wide range of promoters in C. trachomatis. The synthetic riboswitch E, inducible with theophylline, was used to replace the ribosome binding site of the synthetic promoter T5-lac, the native chlamydial promoter of the pgp4 plasmid gene and an anhydrotetracycline responsive promoter. In all cases the riboswitch inhibited translation, and high levels of protein expression was induced with theophylline. Combining the Tet transcriptional inducible promoter with the translational control of the riboswitch resulted in strong repression and allowed for the cloning and expression of the potent chlamydial regulatory protein, HctB. The ability to control the timing and strength of gene expression independently from promoter specificity is a new and important tool for studying chlamydial regulatory and virulence genes.
Highlights
The bacterial species Chlamydia trachomatis (Ctr), are a group of human pathogens composed of over 15 distinct serovars causing trachoma, the leading cause of preventable blindness, and sexually acquired infections of the urogenital tract
Translational control of gene expression from a synthetic constitutive promoter Controlling the timing and level of gene expression is an important tool for uncovering the function of genes that are involved in chlamydial pathogenesis
A T5-lac promoter (T5)-E-clover3xFlag fragment was cloned into the chlamydial plasmid p2TK2-SW2 [15, 29] to make the p2TK2-SW2-T5-E-clover-3xflag plasmid (Fig 1A) and transformed into Ctr L2 resulting in the strain L2-E-clover-flag
Summary
The bacterial species Chlamydia trachomatis (Ctr), are a group of human pathogens composed of over 15 distinct serovars causing trachoma, the leading cause of preventable blindness, and sexually acquired infections of the urogenital tract. According to the CDC, Ctr is the most frequently reported sexually transmitted infection in the United States, costing the American healthcare system nearly $2.4 billion annually [1, 2]. These infections are widespread among all age groups and ethnic demographics, infecting ~3% of the human population worldwide [3]. Understanding the genetic factors that mediate infection and disease has historically been hindered by the lack of good genetic tools This has changed dramatically in the last few years with advances in chlamydial transformation.
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