Part:BBa_K1378032

Endolysin from lambda phage

Endolysin is a kind of peptidoglycan hydrolase that are secreted by double-stranded DNA lambda phage to comprise the bacterial cell wall at the end of infection cycle.However, itself can not digest the peptidoglycan on its own because endolysin can not pass through the inner membrane unless there are other molecules' assistance.

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

The λ phage endolysin is an 18-kDa soluble protein with murein transglycosylase activity^[1]. In λ lysis system, enzymatically active endolysin accumulate in cytoplasm without harm to host bacteria before 'lysis time' because the holin accumulate in CM without disturbing its integrity during this time. However, at an allele-specific time, the holin oligomerizes to form a small number of large holes, allowing the endolysin to cross the CM and attack the PG ^[2][3] (Fig. 2).

Figure 2. Model for export and activation of λ phage endolysin. In λ phage, the holin is inserted in cell membrane without forming holes and endolysin is restricted within cytoplasm (Cyt) before 'lysis time'. However, at an allele-specific time, the holin oligomerizes to form holes in CM, allowing endolysin to reach and hydrolyze PG, leading to cell lysis.

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]Bieʼnkowska-Szewczyk, K., Lipiʼnska, B., & Taylor, A. (1981). The R gene product of bacteriophage &#955 is the murein transglycosylase. Molecular and General Genetics MGG, 184(1), 111-114.

[2]Wang, I. N., Smith, D. L., & Young, R. (2000). Holins: the protein clocks of bacteriophage infections. Annual Reviews in Microbiology, 54(1), 799-825.

[3]Dewey, J. S., Savva, C. G., White, R. L., Vitha, S., Holzenburg, A., & Young, R. (2010). Micron-scale holes terminate the phage infection cycle. Proceedings of the National Academy of Sciences, 107(5), 2219-2223.