Part:BBa_K1378031

Holin from lambda phage

Holin is a 105-amino-acid-residue cytoplasmic membrane protein with three transmembrane domains, naturally expressed by double-stranded lambada phage. Holin will oligomerize and form a hole on the inner membrane of host bacteria at a certain time at an allele-specific time. And then the formation of hole will help Endolysin, a kind of lysozyme, come out from cytoplasm to periplasm to degrade peptidoglycan and inhibit the respiration by eliminating proton gradient.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Holin is a generic term to describe a group of small proteins produced by double-stranded DNA bacteriophage to trigger holes formation at the end of lytic cycle. In our project, we design our suicide switch based on the λ lysis model. The S holin, also called S₁₀₅, encoded by S gene, a dual-start motif of λ phage, is an 105-amino-acid-residue CM protein with three transmembrane domains (TMD)^[1]. S₁₀₇, also called antiholin, is the other protein encoded by S gene, differing from the S holin only by the Met-Lys N-terminal extension. However, this difference confers to S₁₀₇ an extra positive charge, which prevents its TMD1 from inserting into the CM^[2]. Additionally, as its name suggests, S₁₀₇ can bind to S105 and inhibit its function specifically^[3]. In λ lysis system, S₁₀₇ and S₁₀₅ are encoded by S gene at ratio of approximately 1:2, which is defined by the two RNA structure, and if the amount of S₁₀₇ is increased relative to S₁₀₅, the 'lysis time' will be delayed^[4]. The inhibition function of S₁₀₇ can be subverted by collapsing proton motive force, which also allow insertion of TMD1 of S₁₀₇ into CM, instantly increasing the amount of active holin by making previously inactive S₁₀₇ - S₁₀₅ complexes functional (Fig. 1).

Figure 1. The model for the membrane topology of S₁₀₇ and S₁₀₅. S₁₀₅ consist of three transmembrane domains (TMD) with an N-out, C-in topology while S₁₀₇ only has two TMDs, caused by an extra positive charge conferred by Lys2. The S₁₀₇ can inhibit the function of S₁₀₅, preventing it from forming holes in cell membrane. However, this inhibition can be subverted by the dissipation of proton motive force and in this case, S₁₀₇ will become active holin, accelerating the rate of pore formation.

Characterization

We choose λ lysis system to construct suicide switch due to its high efficiency and natural occurrence, and we introduce both endolysin and holin because of their cooperativity in cell lysis, which improves the performance of our suicide switch. In our design, endolysin is controlled by a constitutive promoter while holin by inducible promoter, P_lac, because high concentration of holin can cause cell death alone (Fig. 4).

Figure 4. The final construct of killing switch. The transcription unit that expresses endolysin is inserted into the plasmid pSB1A2 and that for holin is inserted to pSB1C3. Endolysin is expressed under a constitutive promoter and holin is expressed under an inducible promoter, P_lac.

<p>We transformed the two plasmids into E.coli. Then, 1mM of inducer was applied empirically and the growth rate was measured. Compared with the bacteria carrying blank plasmid, the efficiency of our suicide switch can be evaluated.

<figure><img src=""><figcaption>Figure 5. The growth curves of the E. coli carrying suicide switch and blank plasmid. X axis is the culture time and we get OD_595nm value every five minutes. Y axis is the OD_595nm of E. coli. 1mM IPTG was added in experimental group (blue line) while none of IPTG was added in control group (red line). (a) The growth curves of E. coli carrying suicide switch. We have repeated this experiment for three times and the surrounding light-colored lines are error bars. (b) The growth curves of E. coli carrying blank plasmid of the first-time experiment. (c) The growth curves of E. coli carrying blank plasmid of the second-time experiment.</figcaption></figure>

The Fig. 5(a) shows that the difference between the OD_595nm of experimental group and control group is obvious in the late logarithmic phase, indicating that our suicide switch can inhibit the growth of E. coli. A possible explanation for the decrease in OD_595nm is that the E. coil have entered decline phase. The Fig. 5(b) and Fig. 5(c) show that the growth curves of the E.coli carrying blank plasmid after the addition of 1mM IPTG are nearnly coincident with that without addition of IPTG, excluding the possibility that the toxicity of IPTG leads to the noticeable OD_595nm’s difference. The difference between the pattern in Fig. 5(b) and Fig. 5(c) may be caused by the different culture environment. However, both the curves in Fig. 5(b) and Fig. 5(c) have consistency, and thus our experiment should be repeatable. Hence, the OD_595nm’s difference should be caused by the slowed growth rate or cell death, and combining the working mechanism of holin and endolysin, we believe the cell death may be the main cause and our suicide switch may have some bactericidal effect.

Reference

[1]Gründling, A., Bläsi, U., & Young, R. (2000). Biochemical and genetic evidence for three transmembrane domains in the class I holin, lambda S. Journal of Biological Chemistry, 275(2), 769-776.

[2]Young, R., Wang, I. N., & Roof, W. D. (2000). Phages will out: strategies of host cell lysis. Trends in microbiology, 8(3), 120-128.

[3]Bläsi, U., Chang, C. Y., Zagotta, M. T., Nam, K. B., & Young, R. (1990). The lethal lambda S gene encodes its own inhibitor. The EMBO journal, 9(4), 981.

[4]Bläsi, U., Nam, K., Hartz, D., Gold, L., & Young, R. (1989). Dual translational initiation sites control function of the lambda S gene. The EMBO journal, 8(11), 3501.