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CACAO Phage Hunters 2018

My Annotations

StatusPageDate/TimeGO Term (Aspect)ReferenceEvidenceNotesLinks
acceptableBPMD2:HOLIN2018-03-04 20:34:59 CSTGO:0044660 cytolysis by virus via pore formation in host cell membrane (P)PMID:26471254ECO:0000315 mutant phenotype evidence used in manual assertion

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).

acceptableBPMT4:WHIB2018-03-16 16:03:52 CDTGO:0006355 regulation of transcription, DNA-templated (P)PMID:20545868ECO:0000314 direct assay evidence used in manual assertion

The protein WhiBTM4 from mycobacteriophage TM4 serves as a transcription regulator in bacteria such as M. smegmatis, in which it inhibits WhiB2. When WhiBTM4 was expressed in M. smegmatis, it caused the cells to lose acid-fast properties and created long, branched cells. WhiBTM4 caused these changes, in which the results were similar to a WhiB2 knockout protein (Fig. 2). When M. smegmatis contained the protein WhiBTM4, WhiB2 mRNA levels (measured using quantitative RT-PCR) greatly decreased compared to the wildtype, which only contained pSD24 (Fig. 3B). A decrease in mRNA levels suggests that the mycobacteriophage TM4 protein WhiBTM4 inhibits transcription of WhiB2 in M. smegmatis. WhiBTM4 binds only to a DNA WhiB2 promoter, which shows that transcription is regulated through DNA (Fig 6E). This is shown by the band created by the protein/DNA complex in figure 6 B-E.

acceptableBPMD2:VG662018-04-01 08:56:08 CDTGO:0008081 phosphoric diester hydrolase activity (F)PMID:25307893ECO:0000314 direct assay evidence used in manual assertion

The gene Gp66 in mycobacteriophage D29 acts as a protein phosphatase. In the time span of 1 hour, protein dephosphorylation was observed when D29Gp66 was present (Fig 2B). Phosphoesterase assay using substrates pNPP, bis-pNPP and TmPP show that D29Gp66 can use both phosophomonoester and diester as substrates, with the catalytic activity for bis-NPP (diester) being ten times higher than pNPP (monoester) (Fig 2C). D29Gp66 appears to function mainly as a phosphodiesterase (Fig 2C). Gp66 has very high 2', 3' cyclic phosphodiesterase activity and no 3', 5' activity (Fig 2D).

acceptableBPMD2:LYSB2018-04-15 22:17:24 CDTGO:0016787 hydrolase activity (F)PMID:19555454ECO:0000314 direct assay evidence used in manual assertion

The protein LysB from mycobacteriophage D29 was expressed (and also purified) using the plasmid pLAM3, with its molecular weight being around 30kDa (Fig. 2A). This purified LysB protein was used to measure LysB ability to perform hydrolysis of p-nitrophenyl butyrate (pNPB), in which it releases p-nitrophenol. A specific activity of 0.72 U/mg was measured for D29 LysB (Fig. 2B). This number was much higher than 0.12 U/mg, which was measured for a similar protein, Ms6 LysB. Lypolytic activity also decreased as D29 LysB had longer substrates (Fig. 2B).

acceptableBORBU:RECA2018-04-15 22:47:00 CDTGO:0008094 DNA-dependent ATPase activity (F)PMID:29088268ECO:0000315 mutant phenotype evidence used in manual assertion

The protein RecA in B. burgdorferi readily binds to ATP (Fig. 2A). RecA was shown to have DNA-dependent ATPase activity, with preference toward single stranded DNA (Fig. 2B and C). BbRecA had a 2.8 fold slower rate of ATP hydrolysis compared to E. coli RecA (Fig. 2C).



Annotations challenged by AHutchison

StatusAuthor,GroupPageGO Term (Aspect)ReferenceEvidenceLinksPage history
Team Blue B 2018
BPSPP:TUBEGO:0098005 - viral head-tail joining (P)PMID:24443902ECO:0000314 direct assay evidence used in manual assertionchallengeC: 1

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