A mechanistic understanding of DNA uptake in Vibrio cholerae.

Despite being defined as the etiological agent of cholera in 1854, Vibrio cholerae still causes vast loss of life, with around 130,000 people killed in 2010. V. cholerae inhabits the small intestine of humans, and upon ingestion enters a hyper-virulent state triggered by the rapid change in temperature and pH. During this state, the cholera toxin which comprised of 2 subunits, a and b is produced. The a subunit penetrates the host membrane, and is an ADP ribosyl transferase allowing transfer of NAD to a regulatory G protein. This G protein then activates adenyl cyclase, which converts ATP to cAMP. cAMP stimulates excretion of chloride ions, owing to osmotic and electrochemical gradients potassium and sodium ions are also secreted, thus causing extreme water loss. When not infecting humans, V. cholerae can be found in close association with the chitin-rich exoskeleton of zooplankton; chitin enables external DNA to be taken up by V. cholerae. The transfer of external DNA is an integral part of the V. cholerae life cycle; the cholera toxin in encoded for by the CTX is a bacteriophage derived gene thus V. cholerae needs to acquire this somehow. The ability to uptake genetic material is termed competence. Generally, external DNA can be taken up by bacteria via conjugation, transduction and transformation; these mechanisms are generally well understood.  

Previously, it was thought that DNA acquisition in V. cholerae was achieved through the cyclic extension and retraction action of a pilus, structurally similar to the type IV pili (Tfp). Uncertainty as to how these proteins functioned still remained however. It has been suggested that other competence enabling proteins may be at work, such as ComEA, in spite of this the experimental evidence to support this was lacking until the publication of work by Seitz et al. (2014).

In order to elucidate the importance of ComEA in DNA acquisition, it was necessary to identify its location and activity within the cell. This was achieved through use of translational fusion between ComEA and mCherry (a monomeric fluorophore). The lack of stop codon between the fluorescent protein gene and the gene of interest enables the gene of interest to be tagged. Researchers found that ComEA was located at the cell periphery (see Fig 1)

                                   Fig 1. Location of ComEA protein in V. cholerae cells. Column 1

                                   represents ComEA-mCherry translational fusion, here localization
                                   around the cell periphery can be observed. Column 2 represents DAPI
                                  stained individuals. Column 3 displayed a merge between column 1 and 2,                                     and the localisation of ComEA at the cell periphery is further exemplified.                                     Column 4 depicts phase contrast images. 

Validation of these results was also undertaken by replacing the comEA allele with a beta-lactamase (bla)-comEA translational fusion; these individuals displayed a lesser transformation ability (2.5 x10^-5 ± 3.0 x10^-5 compared with 7.9 x10^-5 ± 2.5 x10^-5). Furthermore, individuals displayed resistance against ampicillin; beta-lactamase can only function in the periplasm of gram-negative bacteria further supporting the evidence of the periplasmic localisation of ComEA-bla. Using fluorescence loss in photobleaching (FLIP), the protein dynamics of cells were examined (see Fig 2). One pole of a cell was subject to bleaching, this prevents fluorescence recovery. Mobile proteins will move to this degraded area and a subsequent decline in fluorescence will be observed. It appears that ComEA is highly motile as observed within the periplasm as indicated by the rapidly declining fluorescence. No net changes in fluorescence were observed in control individuals.

                                     Fig 2. On the left, depletion of relative fluorescence can be observed                                                owing to bleaching, and the subsequent movement of proteins. No change
                                     in fluorescence can be observed for non-bleached individuals.                                                          On the right, the corresponding sites of bleaching, and measurement                                               location of florescence measurement can be observed.
                                     

To determine the necessity of ComEA presence for DNA uptake, all comEA genes were substituted with comEA-mCherry alleles. A transformation assay and localisation of the comEA-mCherry allele encoded protein confirmed the functionality of the chromosomally encoded ComEA. External transforming DNA  (tDNA) was supplemented, and ComEA-mCherry centred, protein clusters were formed. The size of these structures varied in accordance with the length of supplemented DNA. Again, control conditions were set up; periplasmic mCherry alone did not aggregate. Thus, it can be inferred that ComEA binds to transforming DNA within the periplasm, and may be the active agent in the uptake of environmental DNA.

To confirm the presence of external DNA in the peri- or cytoplasm of a cell, a whole cell duplex PCR-based DNA uptake assay was employed. No tDNA was observed in V. cholerae deficient in comEA, those with comEA displayed significant uptake of tDNA however.  That said ComEA has a secondary function, to protect tDNA from degradation by nucleases. Detailed analysis of transformants and translocated tDNA confirmed that protection against nucelases is not the main function of ComEA. Despite this, other nucleases may be at work (other than the few examined), thus it is better to conclude that translocation of tDNA should not be solely attributed to Tfp-like structures.

Predictions of the structural architecture of ComEA were made using in silico techniques; unlike the many other proteins which interact with DNA, ComEA did not possess a helix-loop-helix, or helix-turn-helix motifs. Instead a helix-hairpin-helix motif is thought to be present. This hypothesis was achieved through comparative analysis of protein sequences of helix-hairpin-helix motifs, from a range of bacterial species from which ComEA/ComE homologs were characterised; a large number of conserved regions were present.

A number of other analyses to confirm the role of ComEA were carried out, including examination of the co-operativity for DNA binding, site of tDNA entry and ComEA function in other competent bacteria. However, these will not be reported here.

To conclude, Seitz et al. (2014) presented detailed and convincing examination of ComEA and its role in tDNA uptake. Whilst further work is required to fully elucidate its role, ComEA seems to play an important role in the life-style of V. cholerae. As aforementioned, the gene encoding for the cholera toxin is derived from a bacteriophage, and inability to acquire this genetic information would likely be deleterious for V. cholerae. However, this type of research may have application in the field of medicine; not just to treat cholera but also other bacterial infections which may show similar life-histories and DNA uptake mechanisms.

 Jack 

References

Seitz, P., Modarres, H.P., Borgeaud, S., Bulushev, R.D., Steinbock, L.J., Radenovic, A., Peraro, M.D. and Blokesch, M. (2014) ComEA is essential for the transfer of external DNA into the periplasm in naturally transformable Vibrio cholerae cells. PLOS Genetics. 10; (1).