The Course of Development
The Course of Development
Time
Events in time and space . . .
Complicated
The Course of Development
Time
Events in time and space . . .
ComplicatedReally
Free-living Nostoc
heterocysts
Matveyev and Elhai (unpublished)
N2
NH3
CyanobacteriaAnabaena grown without fixed nitrogen
Anabaena filament growing on nitrate
Anabaena filament growing on N2
removal of nitrate
18hours
Heterocysts only when needed
HetRGenes
needed for differentiation
Master regulator
Differentiation in cyanobacteriaIntegration of signals through HetR
Position in filament
Position in cell cycle
Nitrogen deprivation ??????
Promoter
GTA ..(8).. TAC
NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
hetR gene
How might hetR be controlled?
5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN
RNA Polymerase
Presence of fixed nitrogen
Regulatory protein
No HetR protein
Transcription
Absence of fixed nitrogen
GTA ..(8).. TAC
NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
hetR gene5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN
RNA Polymerase
Absence of fixed nitrogen
How might hetR be controlled?
GTA ..(8).. TAC
NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
hetR gene5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN
RNA Polymerase
Absence of fixed nitrogen
How might hetR be controlled?
HetR protein
Transcription
Promoter
DNA modification and differentiation
dmtB
DNA methyltransferase
No heterocysts!
DNA modification necessary
for differentiation?
Promoter
DNA modification and differentiation
dmtB
DNA methyltransferase
YES heterocysts!
DNA modification necessary
for differentiation?
trpD2
Promoter
DNA modification and differentiation
dmtB
Tryptophan biosynthesis
trpD2
Tryptophan biosynthesis necessary
for differentiation?
Promoter
DNA modification and differentiation
dmtB
Tryptophan biosynthesis
trpD2
Tryptophan biosynthesis necessary
for differentiation?
NO heterocysts!
DNA modification and differentiation
dmtB trpD2
Is there a gene between dmtB and trpD2 that may control heterocyst differentiation?
ORFs within intergenic region
dmtB trpD2ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC
Look for pattern:
(ATG | GTG | TTG)... ... (TAA | TAG | TGA)
start codon triplets stop codon
TATAAAGTTGGAGCGAAACTACTGTCAGCTAAAAAGCCATCCTATTGACGGTACGGAGAGTTTCATGTGTTTGAACTCAATACTGGTCAAGAGGATGTAG
LMTVDFSVG MPLKVHKLEL
LCTLRGMAVILPKNRLSSKRG
ORFs within intergenic region
dmtB trpD2ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC
Nothing compelling
What about RNA genes?
TATAAAGTTGGAGCGAAACTACTGTCAGCTAAAAAGCCATCCTATTGACGGTACGGAGAGTTTCATGTGTTTGAACTCAATACTGGTCAAGAGGATGTAG
LMTVDFSVG MPLKVHKLEL
LCTLRGMAVILPKNRLSSKRG
GTA ..(8).. TAC
NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
Critical gene5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN
RNA Polymerase
Absence of fixed nitrogen
How might a noncoding gene work?
dmtB trpD2ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC
NO heterocysts!
Find by Blast
How to find a noncoding gene? dmtB trpD2
ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC
Blast against Anabaena genome50 64 asl0749 alr07507 25 alr3353 alr335248 61 all47626 19 alr516453 66 alr450954 67 alr0279-9 4 alr4028 alr40299 25 alr2338 alr2339-15-3 alr13015 17 all1239 alr124052 64 all1663-7 5 alr5148-5 7 all392427 39 all0048 all0047-5 7 all228853 65 alr388551 63 all070655 67 alr5148 alr5149-1 11 all171616 28 all7645 alr7646
How to find a noncoding gene? dmtB trpD2
ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC
Blast against Anabaena genome50 64 asl0749 alr0750 7 25 alr3353 alr3352 -9 4 alr4028 alr40299 25 alr2338 alr2339 5 17 all1239 alr1240 27 39 all0048 all0047 55 67 alr5148 alr5149 16 28 all7645 alr7646
How to find a noncoding gene? dmtB trpD2
ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC
Blast against Anabaena genome50 64 asl0749 alr0750 7 25 alr3353
-9 4 alr40299 25 alr2339 5 17 all1239 alr1240 27 39 all0048 55 67 alr5149 16 28 all7645 alr7646
How to find a noncoding gene? dmtB trpD2
ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC
Blast against Anabaena genome50 64 asl0749 alr0750 7 25 alr3353
-9 4 alr40299 25 alr2339 5 17 all1239 alr1240 27 39 all0048 55 67 alr5149 16 28 all7645 alr7646
How to find a noncoding gene? dmtB trpD2
ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC
Blast against Anabaena genome
50 64 asl0749 alr0750 30S ribosomal protein hypothetical7 25 alr3353 putative peptididase-9 4 alr4029 ~Vitamin B12 transport9 25 alr2339 heterocyst differentiation5 17 all1239 alr1240 hypothetical Zam27 39 all0048 hypothetical55 67 alr5149 unknown16 28 all7645 alr7646 unknown MobB
How to find a noncoding gene? dmtB trpD2
ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC
Blast against Anabaena genome
7 25 alr3353 putative peptididase
9 25 alr2339 heterocyst differentiation
alr2339 is hetR
Characterization of matchbetween dmtB-trpD2 and pre-hetR dmtB trpD2
ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC
dmtB-trpD2 ATGACAGTCGATTTTTCGGTAGGATAACTGCCAT pre-hetR TTATTTTGTGATTTTTCGGTAAGATACAAGCACG
transcription
The match overlaps the promoter of hetR
Characterization of matchbetween dmtB-trpD2 and pre-hetR dmtB trpD2
ATATTTCAACCTCGCTTTGATGACAGTCGATTTTTCGGTAGGATAACTGCCATGCCTCTCAAAGTACACAAACTTGAGTTATGACCAGTTCTCCTACATC
dmtB-trpD2 ATGACAGTCGATTTTTCGGTAGGATAACTGCCAT pre-hetR TTATTTTGTGATTTTTCGGTAAGATACAAGCACG
transcription
The match is conserved in different cyanobacteria
dmtB-trpD2 ATAGCGGTCAATTTTCCGGTAGGATAGCTCCCAT pre-hetR GATATCTGTAATTTTCCAGTCAGATGCAGAAACA
Region from Nostoc punctiforme
Is there a gene between dmtB and trpD2 that may control heterocyst differentiation?
May be a noncoding gene
Striking match with hetR promoter
Conserved between Anabaena and Nostoc
Coding gene?
Unlikely
Novel mode of bacterial regulation
Andrey Matveyev
Virginia Commonwealth
University
Thanks to
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