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PMID:26471254

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Citation

Kamilla, S and Jain, V (2016) Mycobacteriophage D29 holin C-terminal region functionally assists in holin aggregation and bacterial cell death. FEBS J. 283:173-90

Abstract

Holins are phage-encoded small transmembrane proteins that perforate the bacterial cytoplasmic membrane. In most cases, this process allows the phage-encoded peptidoglycan hydrolases to act on the cell wall, resulting in host cell lysis and phage release. We report a detailed functional characterization of Mycobacterium phage D29 gp11 coding for a putative holin that, upon expression, rapidly kills both Escherichia coli and Mycobacterium smegmatis. We dissected Gp11 by making several deletions and expressing them in E. coli. The shortening of Gp11 from its C-terminus results in diminished cytotoxicity and smaller holes. Evidently, the two transmembrane domains (TMDs) present at the N-terminus of Gp11 are incapable of integrating into the cytoplasmic membrane and do not show toxicity. Interestingly, the fusion of two TMDs and a small C-terminal region that bears the coiled-coil motif resulted in restoration of the cell killing ability of the protein. We further show that the second TMD is dispensable in protein toxicity because its deletion does not abolish Gp11-mediated cell death. We conclude that Gp11 C-terminal region is necessary but not sufficient for toxicity. These results shed light on a yet undiscovered role of Gp11 C-terminal region that will help clarify the mechanism of holin-mediated membrane perforation. Finally, we abolish the toxicity of Gp11 using a specific Gly to Asp substitution in the putative loop region of the protein; the mutant protein may help to clarify how holin functions in mycobacteriophage D29.

Links

PubMed Online version:10.1111/febs.13565

Keywords


Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

BPMD2:HOLIN

involved_in

GO:0044660: viral release by cytolysis via pore formation in host cell membrane

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

BPMD2:VG11

NOT

GO:0051715: cytolysis in other organism

IDA: Inferred from Direct Assay:

P

Figure 2:

- (A) The growth curve was recorded after initiating HolFL expression in E. coli BL21(DE3) (in the case of pET and pTrc vectors) and TOP10 cells (in the case of pBAD vector), at time 0, by adding IPTG (+IPTG) and arabinose (+ara), respectively.
  * Measurement of E. coli when exposed to full-length Gp11. Host bacteria count decreased when exposed to gp11.
- (C) The growth curve of M. smegmatis expressing full-length Gp11 is recorded after induction with 0.2% acetamide at time 0.
  * Host (M. smeg) bacterial count decreased with addition of inducer acetamide.
- (E) Growth kinetics was recorded 1 min for 60 min after induction at time 0, in a 96-well plate, for E. coli carrying specified vectors. 
  * Gp11 Plasmid standalone and gp11 plasmid combination (with gp10) cause lysis in host bacteria of both M.smeg and E. coli

Quotes found in the results section give overview of cytolysis function of gp11 in phage D29:

- "we find that the expression of D29 gp11 is sufficient to kill [via gp11 mediated lysis] E. coli or M. smeg."
- "Gp10 and Gp11 in E. coli results in enhanced killing of bacterial cells."

complete


BPMD2:HOLIN

involved_in

GO:0044660: cytolysis by virus via pore formation in host cell membrane

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

BPMD2:HOLIN

GO:0020002: host cell plasma membrane

ECO:0000314:

C

Figure 2F shows a western blot performed after IPTG induction to detect the presence of HolFL by using anti-6xHis antibodies and Gp10 with anti-gp10 antibodies. pSKHol+gp10 shows higher expression between 42kDa and above, it also shows higher expression between 22kDa and 14kDa.

complete
CACAO 12894

BPMD2:HOLIN

GO:0044660: cytolysis by virus via pore formation in host cell membrane

ECO:0000315:

P

Mycobacterium phage D29 gene gp11 codes for the protein holin. The protein allows phage D29 to kill both Mycobacterium smegmatis and Escherichia coli by creating pores in the host membrane. The expression of the entire gp11 gene (using vectors) decreased the growth of E. coli significantly (Fig 2A and presence of gp11 confirmed by Western Blot in 2B). The expression of whole gp11 also decreased the growth of M. smegmatis (Fig 2C). Shortened versions Hol68 and Hol93, with amino acids cut off the C-terminal end, did not affect E. coli bacterial growth but Hol116 had a similar reduction in bacterial growth to full length holin protein (Fig. 3B). Figure 3C confirms the presence of holin in all shortened versions and compares them to full length holin. Spot assays confirm the data found in figure 3B because Hol116 showed little to no growth, whereas Hol68 and Hol93 show a significantly larger amount of growth compared to full length holin and Hol116 (Fig. 3D). The spot test of Hol116 also looked very similar to full length holin (Fig. 3D). This indicates that the N-terminal, up through amino acid 116, is important in the function of holin to cause bacterial cell death. The protein must be greater than 93 amino acids long to still contain the function of holin (Fig. 3, all).

complete
CACAO 13102

Notes

See also

References

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