Download LysB of Phage D29

LysB of Phage D29
Subtitle
General Endolysin B Roles
• Works in conjuncture with LysA- generally downstream with 4 or less
intermediate holin (or holin-like- SAR) genes as intermediates.
• Unlike LysA it is not modular
• Works only on Gram positive bacteria by cleaving the mycobond mesh and
covalently between the host cell wall and the outer membrane’s terminal
arabionglycan to release many free mycolic acids.
• A high genetic sequence divergence of less than 20% identity among
members.
D29 LysB Function
• Works only on Gram positive bacteria
by cleaving the mycobond mesh and
covalently between the host cell wall
and the outer membrane’s terminal
arabinogalactan to release many free
mycolic acids.
• Occurs late in lysis stage
• Does not contain prediction of
peptidoglycan-binding domain at the
n-terminius like the rest of pham
01471
What Makes D29 LysB such a bad mothaaa….
• Belongs to pham 73
• 254 bp at gp12
• Purified to near homogeneity and high solubility(>10mgs/mL)
• Position 82- contains putative active site serine
• Position 117-119 -GXP (~40 bp downstream)
• Both postions -> 3 serine esterase (Ser, Asp, His) for catalytic triad
• Proposed aspartic acid at 166- highly conserved glutamic acid residue but
histadine trace leads another Gxp motif and unclear on role
Pham genetic relation to infer function• Compared pham 73 – and created a mutant by
deleting the catalytic serene with alanine at
position 82 and compared both to Ms6
• lyse B had a 40% amino identity with ms6
• Tested lipolytic activity by hydrolosis of p-nitrile
butyrate (pNPB) to yield p-nirophenol
• D29 was (.72 U/mg) compared to (Ms6 .12 U/mg)
• Mutant was inactive showing the triad is essential
to function
• When tested with longer chain substrates function
of D29 slowed
• Proves it is critical in catalytic function of lysis
Structure
• 253 residues- 170 residues make up alpha sheets
sandwiching a beta sheet (A)
• Fold is similar to fold of Cryptococcus Cutinase protein
• Low sequence similarity (c-alpha-2.45 Å), but high Z
score (20.3)
• 81 remaining residues (B) have 4 alpha helices
connecting c-terminal to the backface of the beta sheet.
This connective loop facilitates the c-terminal’s helical
linking and movement that allows bulky and hydrophobic
acyl chains from the mycolic acid to be accommodated.
• Catalytic triad at Ser82-Asp166-His240 on edge of beta
sheet between layers (proved by comparison of
superimposing Crypto and Lyse b)
Structure is good for you.
• Terrible TRIAD
• Ser 82 – invariant when compared to other
serine catalytic
• Asp-166 has a conserved alignment
• Hist-240 varies between plaques, poor
conservation of the residues. Also has loop
region positioning that further complicates
uniformity
• GXP is glycine and proline residues that are
adjacent to the triads
• Gly117-ASN118-PRO119 is at the end of the
beta 4 strand, allowing a 180 turn underneath
ASP 166 with 117 and 119 forming hydrogen
Bonds with met120, Arg121, and Asp160
D29 and Hydrolosis
• LysB is a mycolylara binoglactan
esterase (serine esterase)
• Cassette position in d29 infers an
esterlinked lipid cellular wall
component due to unusual linker
position.
•
(mAGP) Mycolyl
arabinogalactanopeptidoglycan with
tetrabutylammonium (TBAH) is a
proposed substrate candidate for the
hydrolysis
Spectrography of Hydrolysis
• Isolated a cell wall segment of M. smegmatis ( due to a high mAGP
count) and took the wild and mutant type D29s.
• To compare the concentration of release of lipids from the isolated
cell wall from the hydrolosis over a period of time to show
effectiveness of the D29.
• Products were proven to be of hydrolysis of M. smegmatis from
treating with idomethane to methyl-esterfy them to yield the lipids
that M. smegmatis would have produced in a TBAH treatment.
• Mass Spectrometry and NMR are consistent in proving d29 yields
free mycolic acids. Mutant omission of post 82 yielded no activity
proving catalytic serine essential to the function.
• FIRST EXPIRIMENT TO DESCRIBE mycolylarabinoglactan.
MEET GILES
• Giles is of the same pham and was chosen because we
know what lyseA does, and lyseB is different in identity (to
avoid recombination) yet has been proven to hydrolysis of
M. smegmatis similarly to d29.
• We got him ready for this dance by trimming the lyse b
and adding 100 bps to each side. Then we deleted 1146
bp’s and fused 15 codons to minimize any interference
from the other genes and to keep gene polarity from being
interfered with. And then we shoved him into the M. smeg
recombinated strain and watched the party begin.
• were done with M.smeg and a control lawn with Giles and
the Giles recombinant mutant.
• 22 were viable for DADA and PCR screening
• Only 2 resulted in a mix of wildtype and mutant strains, one
was chosen, resuspended, and plated ~600 plaques
• The mutant was equally present and non compenting with the
wildtype, verifying that lyse b does not effect lytic growth. (A)
• However the mutant produces smaller plaques on wild type
than on parent M. smeg so there was less bacterial doublings
and less rounds of phage infection.
• The mutant was only present in the small plaques
• 100 fold average decrease in in mutants wild lawn plaquing
• This shows lyse b required for efficient phage release
• Optical densities of wild vs mutant (B)
• Wild would peak infection at 3 hours after initiation and
decrease at 5 hours
• Mutant would decline at 3.5 and terminate at 5 hours
• Monitor ATP release (C)
• Wild atp production correlated with the optical density findings,
there was no atp until hour 3 and increased to 4.5- 5 hours
• Mutant type had a delayed release at 30 minutes and never
reached the levels of the wild type showing that there was a
lysis defect with the mutant type
• Findings of the ATP release correlated to the RZ or RZ1 findings
Of phage lamda.
• Phage particle release (D,E)
• Mutant shows no defect in particle production
• 4 hours after infection the wild type and mutant have a 90%
concentration of phage particles, but only 45% of mutant
particles are in unlysed cells
• When tested for mutant by PCR, shows that it is proportiaonally
equivalent and viable due to homogenous mutant verified
present in 30 plaques
FIN