Part:BBa_K2387069
T4 Endolysin lytic mechanism controlled by inducible araC/pBAD promoter
Endolysins are phage-encoded peptidoglycan hydrolases (PGHs) which are used by bacteriophages to enzymatically degrade the peptidoglycan (PG) layer of the host bacterium from the inside at the end of their lytic cycle. By breaking the PG layer, an osmotic lysis can be favoured leading to cell death. However, host cell lysis is strictly regulated and controlled by the help and action of T4 Holin (BBa_K112000). T4 Holin constitutes a second component of a lysis cassette of phages. Holins oligomerize and create holes in the cytoplasmic membrane allowing the cytoplasm accumulated endolysins to degrade PG substrate [1].
The endolysin is a muralytic enzyme, usually accumulating in the cytoplasm. T4 Endolysin gene encodes for a phage lysin, which is a phage-encoded peptidoglycan hydrolases (PGHs) used by the most of bacteriophages to enzymatically degrade the peptidoglycan layer of the host bacterium leading to a lytic multiplication cycle. T4 Endolysin gene is expressed under the pBAD promoter which is an E.coli promoter that is induced by L-arabinose. In the absence of arabinose, the repressor protein AraC (BBa_I13458) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription [1]. However, when arabinose is addedd to the medium, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, permitting transcription [3].[4] pBAD promoter allowsthen for the control of the lysis process to take place.
Four different lytic mechanisms (Colicin E7 system, T4 Endolysin, T4 Holin and T4 Endolysin + T4 Holin cassette) were assessed in two backbones (pSB1C3 and pSB4K5). All the mechanisms are expressed under the pBAD promoter (Figure 2) and therefore all the mechanisms were induced at different arabinose concentrations. The figure (Figure 1) depicts the OD after 20 hours of arabinose induction. Based on the results, it was concluded that already low inductions of L-arabinose are able to induce a complete lysis of the bacterial cells (Figure 1).
From the assessment it was concluded that T4 Holin and T4 Endolysin + T4 Holin were successfully achieving bacterial cell lysis.T4 Endolysin alone was not able to trigger the lysis of bacterial cells. However, when combined with T4 Holin, the lytic effect achieved was even higher than the one achieved with T4 Holin alone (Figure 2). The observed lytic function of T4 Endolysin when acting together with T4 Holin in the double lysis gene cassette is understandable as it requires the action of T4 Holin to make the initial holes in the membrane [7]. T4 Holin starts the membrane degradation process and after holes are made in the membrane, T4 Endolysin is able to further increase bacterial membrane lysis (Figure 2).
References
[1] L. H. Burch, L. Zhang, F. G. Chao, H. Xu, and J. W. Drake, “The bacteriophage T4 rapid-lysis genes and their mutational proclivities.,” J. Bacteriol., vol. 193, no. 14, pp. 3537–45, Jul. 2011.
[3]
Schlief, R. (2000). Regulation of the L-arabinose operon of Escherichia coli. Trends in Genetics. 16(12):559-565.[4]
Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, and Keasling JD. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology. 147(12):3241-7. Sequence and Features| None |