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Matveyev and Elhai (unpublished)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

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