Chapter 15 : Chlamydial Genetics: Decades of Effort, Very Recent Successes

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This chapter addresses the challenging aspects of the chlamydial system and points out some avenues that can be exploited, perhaps opening the door to technologies for routine genetic modification of chlamydiae. It discusses the challenges in transforming chlamydiae. The rigid, extensively disulfide cross-linked outer membrane complex of the metabolically inactive elementary bodies (EBs) may hinder the introduction of exogenous DNA into the developmental form. The chapter then focuses on genomics and our understanding of natural lateral gene transfer in chlamydiae. The relative abundance of candidate gene transfer systems in members of the suggests that contemporary spp. evolved into their genetically intractable niche by reductive evolution from a genetically amenable ancestor. The chapter addresses the surprising finding that chlamydiae have the tools to naturally acquire and integrate homologous DNA into their genome as long as the DNA is donated by a related chlamydial strain. It reviews exciting new approaches that are being used to genetically manipulate the chlamydial genome and discusses how these efforts have helped one to understand different aspects of chlamydial biology. The chemical mutagenesis could be used to generate mutant chlamydial strains. A report was published describing the first stable transformation of chlamydiae by an exogenous plasmid, leading to replication of antibiotic-resistant and green fluorescent protein-expressing . The experimental studies demonstrate that chlamydiae can share DNA, can be transformed, and can incorporate introduced DNA into the genome via homologous recombination.

Citation: Jeffrey B, Rockey D, Maurelli A. 2012. Chlamydial Genetics: Decades of Effort, Very Recent Successes, p 334-351. In Tan M, Bavoil P (ed), Intracellular Pathogens I: . ASM Press, Washington, DC. doi: 10.1128/9781555817329.ch15

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Gene Expression and Regulation
Type IV Secretion Systems
Genetic Elements
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Image of FIGURE 1

Model for the integration of the tetracycline resistance gene into the -like gene in the genome. (A) The Tet(C) island of contains sequences that are very similar to sequences from other fish bacteria. R and cs605, which is composed of 200 and 1341 insertion sequences, are similar to sequences in HLHK5. Adjacent genes in the Tet(C) island are highly similar to plasmid maintenance genes from pRAS3.2, which is a plasmid from (B) The presence of this naturally occurring island in was then exploited as a tool for laboratory-based recombination experiments similar to those conducted by Demars and colleagues (Binet and Maurelli, 2009). rRNA operons in panel B are indicated by half-width open reading frame boxes. doi:10.1128/9781555817329.ch15.f1

Citation: Jeffrey B, Rockey D, Maurelli A. 2012. Chlamydial Genetics: Decades of Effort, Very Recent Successes, p 334-351. In Tan M, Bavoil P (ed), Intracellular Pathogens I: . ASM Press, Washington, DC. doi: 10.1128/9781555817329.ch15
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Image of FIGURE 2

The reverse-genetic approach used to generate targeted mutations in the operon. (A) Flowchart for generating and screening libraries. (B) Summary of the mutations identified in the analysis of the library of subpopulations. CEL I digestion of the 24 subpopulations harboring mutations are shown on the gels in the order of their genomic locations. The and open reading frames are illustrated by the horizontal arrows. The complete sets of SNPs identified by capillary sequencing are indicated below each sample. Locations of SNPs are indicated in the operon above the gel image. Genomic scale and the region corresponding to the PCR amplicon are also shown. The nonsense mutation in at position 991 (R331*) truncates the open reading frame by 186 bp. Used by permission of and the author. doi:10.1128/9781555817329.ch15.f2

Citation: Jeffrey B, Rockey D, Maurelli A. 2012. Chlamydial Genetics: Decades of Effort, Very Recent Successes, p 334-351. In Tan M, Bavoil P (ed), Intracellular Pathogens I: . ASM Press, Washington, DC. doi: 10.1128/9781555817329.ch15
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Image of FIGURE 3

Gene organization and plasmid constructs for the transformation vectors used by Binet and Maurelli. Shown are the regions of the ribosomal operons that were present in each of the transformation vectors used in the experiments and the base pair changes in the 16S and 23S rRNA genes that were used as markers in candidate transformants. Kasugamycin (Ksm) resistance was generated by a base pair change at position 794, and spectinomycin (Spc) resistance was associated with a change at position 1192. Two silent mutations were also present in some of the transformation vectors used by these authors. Note that one of the silent mutations (position 1071) allowed the screening of candidate transformants by the removal of an Hpa1 site in PCR products. The primers used to screen for positives are also indicated. Used by permission of . doi:10.1128/9781555817329.ch15.f3

Citation: Jeffrey B, Rockey D, Maurelli A. 2012. Chlamydial Genetics: Decades of Effort, Very Recent Successes, p 334-351. In Tan M, Bavoil P (ed), Intracellular Pathogens I: . ASM Press, Washington, DC. doi: 10.1128/9781555817329.ch15
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Image of Figure 4
Figure 4

Expression of the green fluorescent protein in cells transformed with a plasmid carrying . Untransformed L2/434/Bu (control) and L2/434/Bu transformed by pGFP::SW2 were grown on coverslips for two days prior to paraformaldehyde fixation and visualization by fluorescence microscopy. Panel A shows untransformed L2/434/Bu under white light (arrows indicate inclusions). Panel B is the same image, viewed in a fluorescence channel for visualization of GFP. Panel C shows L2/434/Bu transformed with plasmid pGFP::SW2 under white light, and panel D is the same field viewed by fluorescence. The scale bar in panel D represents 20 µm.

Citation: Jeffrey B, Rockey D, Maurelli A. 2012. Chlamydial Genetics: Decades of Effort, Very Recent Successes, p 334-351. In Tan M, Bavoil P (ed), Intracellular Pathogens I: . ASM Press, Washington, DC. doi: 10.1128/9781555817329.ch15
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Generic image for table

Antibiotics previously used to generate stable or transient resistance in spp.

Citation: Jeffrey B, Rockey D, Maurelli A. 2012. Chlamydial Genetics: Decades of Effort, Very Recent Successes, p 334-351. In Tan M, Bavoil P (ed), Intracellular Pathogens I: . ASM Press, Washington, DC. doi: 10.1128/9781555817329.ch15
Generic image for table

Antibiotic resistance markers that cannot be used for selection of transformants in

Citation: Jeffrey B, Rockey D, Maurelli A. 2012. Chlamydial Genetics: Decades of Effort, Very Recent Successes, p 334-351. In Tan M, Bavoil P (ed), Intracellular Pathogens I: . ASM Press, Washington, DC. doi: 10.1128/9781555817329.ch15

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