Not Again!

Minimal Genome That Encodes Proteins
with Constrained Amino Acid Repertoire,
or
Quoth That Black Bird: Not Again!
Arcady Mushegian
Stowers Institute
Kansas City
Image: http://sarahforreal.files.wordpress.com/2010/12/2686429.jpg
MINIMAL GENE SET BUILDER GUIDE
Mycoplasma genitalium 468
240 orthologs
Haemophilus influenzae, 1703
- 2 “parasitism”
- 4 redundant
+ 22 missing links
Minimal set 256
MINIMAL GENE SET : STEPS OF REDUCTION
Mushegian AR, Koonin EV. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10268-73.
MINIMAL GENE SET : KNOCKING GENES OUT
Glass JI et al. Proc natl Acad Sci USA. 2006 103(2):425-30.
Cristen B et al. Molecular Systems Biol. 2011 7: 528.
MINIMAL GENE SET : ESTIMATED ORF COUNTS
Saccharomyces cerevisiae
Haemophilus influenzae
Acinetobacter baylyi
Mycoplasma pulmonis
Mycoplasma genitalium
Salmonella enterica
Typhi
Pseudomonas aeruginosa
Helicobacter pylori
Escherichia coli
Staphylococcus aureus
Bacillus subtilis
878
642
499
461
381
356
Single-gene-specific mutagenesis
Saturation transposon mutagenesis
Single-gene-specific mutagenesis
Saturation transposon mutagenesis
Saturation transposon mutagenesis
Saturation transposon mutagenesis
335
323
303
302
271
Saturation transposon mutagenesis
Saturation transposon mutagenesis
Single-gene-specific mutagenesis
Antisense RNA inhibition
Single-gene-specific mutagenesis
2
5
7
100
147
256
180
156
63
35
Comparative
Comparative
Comparative
Comparative
Comparative
genomics
genomics
genomics
genomics
genomics
Juhas M, Eberl L, Glass JI.. Trends Cell Biol. 2011 Oct;21(10):562-8.
Once upon a midnight dreary, while I pondered, weak and weary,
Over many a quaint and curious volume of forgotten lore,
While I nodded, nearly napping, suddenly there came a tapping,
As of someone gently rapping, rapping at my chamber door.
" 'Tis some visitor," I muttered, "tapping at my chamber door;
Only this, and nothing more."
The Raven, by Edgar Allan Poe
'Twas upon a midnight tristful I sat poring, wan and wistful,
Through many a quaint and curious list full of my consorts slain I sat nodding, almost napping, till I caught a sound of tapping,
As of spirits softly rapping, rapping at my door in vain.
"'Tis a visitor," I murmur'd, "tapping at my door in vain Tapping soft as falling rain."
That Black Bird, by Arthur Gordon Pym*
*In: A Void by George Perec, English by Gilbert Adair
Once upon a midnight dreary, while I pondered, weak and weary,
Over many a quaint and curious volume of forgotten lore,
While I nodded, nearly napping, suddenly there came a tapping,
As of someone gently rapping, rapping at my chamber door.
" 'Tis some visitor," I muttered, "tapping at my chamber door;
Only this, and nothing more."
The Raven, by Edgar Allan Poe
'Twas upon a midnight tristful I sat poring, wan and wistful,
Through many a quaint and curious list full of my consorts slain I sat nodding, almost napping, till I caught a sound of tapping,
As of spirits softly rapping, rapping at my door in vain.
"'Tis a visitor," I murmur'd, "tapping at my door in vain Tapping soft as falling rain."
That Black Bird, by Arthur Gordon Pym*
*In: A Void by George Perec, English by Gilbert Adair
MAKE DO WITH FEWER AMINO ACIDS :
GET RID OF THE RAREST ONE FIRST ?
CLASS I : NO CYSTEINES ,
OR THEY ARE MISSING IN HOMOLOGS
Keep in genome 437 genes from the minimal genome Is Cys present?
No
437 genes Is Cys present? Yes
Keep in genome
Orthologs search NO
Substitutions
?
Yes
No
Manual analysis YES
Is Cys essential? YES
ThyA, Tdk,
TopA, LigA,
RpoC, DnaJ,
RpmJ,
PtsI, PtsG, AtpA Analogs? (Cys function is unknown) NO
Genome 1
TpiA, Gpk
YES
Mutants? Orthologs 20 gene prediction mistakes 8 uncharacterized genes YES
NO
NO
Functional analysis Genome 3 CysS, NifS,
NifU, ThiI,
Spx, PdhD
YES
Cys metabolism? NO
Zn coordination? NO
Substitution? Genome 2 Manual analysis NO
YES
tRNA modification: GidA, MnmA, MnmE
Redox regulation: NrdE, TrxA, TrxB
Non-essential without Met: MsrA, Map
YES
Cdd,
YjeQ/EngC,
ThrS, UvrA,
MutM, Def
CLASS II : ALL HOMOLOGS WITH CYSTEINES ,
BUT ISOFUNCTIONAL ANALOGS WITHOUT
Keep in genome 437 genes from the minimal genome Is Cys present?
No
Yes
61 genes Orthologs search Is Cys essential? NO
Substitutions
?
Yes
Manual analysis No
Is Cys essential? NO
YES
20 gene prediction mistakes ThyA, Tdk,
TopA, LigA,
RpoC, DnaJ,
RpmJ,
8 uncharacterized genes YES
ThyA, Tdk,
TopA, LigA,
RpoC, DnaJ,
RpmJ,
YES
TpiA, Gpk
YES
NO
Analogs? PtsI, PtsG, AtpA Mutants? NO
Functional analysis (Cys function is unknown) CysS, NifS,
NifU, ThiI,
Spx, PdhD
YES
Cys metabolism? NO
NO
Zn coordination? NO
tRNA modification: GidA, MnmA, MnmE
Redox regulation: NrdE, TrxA, TrxB
Non-essential without Met: MsrA, Map
TpiA, Gpk
YES Mutants? NO
PtsI, PtsG, AtpA (Cys function is unknown) YES 20 gene prediction mistakes 8 uncharacterized genes Analogs? Functional analysis YES
Cdd,
YjeQ/EngC,
ThrS, UvrA,
MutM, Def
ISOFUNCTIONAL BUT NOT ORTHOLOGOUS :
DISPLACEMENT OF ORTHOLOGOUS GENES (DOG)
a.k.a. NON-ORTHOLOGOUS GENE DISPLACEMENT (NOD)
Differential loss
Independent recruitment
FUNCTIONAL ANALOGS MAY USE
DIFFERENT CHEMISTRY
Flavin-independent
thymidylate synthase ThyA :
conserved Cys nucleophile
Flavin-dependent
thymidylate synthase ThyX :
no conserved Cys
(none at all in Marinithermus)
CLASS III : NO Cys-SUBSTITUTED
HOMOLOGS NOR ANALOGS ?
20 genes
Keep in genome 437 genes from the minimal genome Is Cys present?
No
CysS, NifS,
NifU, ThiI,
Spx, PdhD
Yes
Orthologs search YES
Cys metabolism? Substitutions
?
Yes
Manual analysis No
NO
Is Cys essential? NO
Zn coordination? YES
Cdd,
YjeQ/EngC,
ThrS, UvrA,
MutM, Def
YES
ThyA, Tdk,
TopA, LigA,
RpoC, DnaJ,
RpmJ,
20 gene prediction mistakes 8 uncharacterized genes YES
Analogs? PtsI, PtsG, AtpA (Cys function is unknown) NO
TpiA, Gpk
YES
Mutants? NO
Functional analysis NO
CysS, NifS,
NifU, ThiI,
Spx, PdhD
YES
Cys metabolism? NO
Zn coordination? NO
tRNA modification: GidA, MnmA, MnmE
tRNA modification: GidA, MnmA, MnmE
Redox regulation: NrdE, TrxA, TrxB
Non-essential without Met: MsrA, Map
Redox regulation: TrxA, TrxB
Nucleotide synthesis: NrdE
Non-essential without Met: MsrA, Map
YES
Cdd,
YjeQ/EngC,
ThrS, UvrA,
MutM, Def
WHAT COORDINATES ZINC ?
THYIL RADICAL : IRREPLACEABLE Cys
IN RIBONUCLEOSIDE REDUCTASE ?
C439
N437
E441
C225
C462
Kolberg M, Strand KR, Graff P, Andersson KK.
Biochim Biophys. Acta. 2004 1699: 1-34.
And the Raven, never flitting, still is sitting, still is sitting
On the pallid bust of Pallas just above my chamber door;
And his eyes have all the seeming of a demon's that is dreaming,
And the lamplight o'er him streaming throws his shadow on the floor;
And my soul from out that shadow that lies floating on the floor
Shall be lifted- nevermore!
The Raven, by Edgar Allan Poe
And my Black Bird, still not quitting, still is sitting, still is sitting
On that pallid bust—still flitting through my dolorous domain;
But it cannot stop from gazing for it truly finds amazing
That, by artful paraphrasing, I such rhyming can sustain—
Notwithstanding my lost symbol I such rhyming can sustain—
Though I shan't try it again!
That Black Bird, by Arthur Gordon Pym*
*In: A Void by George Perec, English by Gilbert Adair
CONCLUSIONS : BY ARTFUL PARAPHRASING
•
Engineering Cys out requires dealing with 46 proteins
•
Most have cysteine-free homologs or analogs
•
~10-12 do not
• Redox regulators and redox damage repair factors
• Remove (non-initiatory) methionine
• Supply redox buffer - ironically, glutathione ?
• Some are not needed if Cys is not there (aaRSase-Cys)
• Nucleotide modification - U in tRNA, sugar in ribonucleosides
• ?????
•
In other news : ~50 proteins have invariant W
8 of them are aaRSases……..?????
WHO DID IT
Andrei Kucharavy
École Polytechnique
Marina Yurieva
Moscow State U
Open University
Olga Tsoy
Moscow State U
Kharkevich Institute
of R.A.S.