The  coding  poten-al  of  Pseudomonas  aeruginosa:  Compara-ve  genomics  and  ribosome  profiling  

Luciano  Brocchieri  

Department  of  Molecular  Gene-cs  &  Microbiology  and  Gene-cs  Ins-tute  University  of  Florida,  Gainesville,  FL  32610  

Gene finding and GC content

Coding  regions  are  characterized  by  typical  3-­‐base  periodicity  in  GC  content,  depending  on  the  overall  GC  content  of  the  sequence.  

(Bibb  et  al.  1984;  Borodovsky  and  McIninch  1993;  Besemer  et  al.  2001)  

GC  in  cod

on  posi-on

 

3  

2  

1   Pseudomonas  aeruginosa:  

67.7%  GC  

Frame analysis (Bibb et al 1984)

GC content is measured every third nucleotide in three phases. Compositional contrasts among S-profiles of GC content indicate presence

and frame of coding regions.

ggtgtccgcgtcccagacgtaggcctcgagcgtcgcgccgtagagcagggccgccgggtg...

!"#$%&' g..g..c..g..c..g..g..g..c..g..c..c..g..g..g..c..g..c..c..g.....

!"#$%&( .g..t..g..t..c..a..t..g..t..a..g..g..c..t..a..a..g..g..g..t....

!"#$%&) ..t..c..c..c..a..c..a..c..c..g..t..c..c..a..g..g..c..c..g..g...

!"#&$*%&'(*%)'(**(*%+'+

"&"""""""""""""""""""""&,""""""""""""""""""""""),"""""""""""""""""""""""-,""""""""""""""""""""""".,""""""""""""""""""""""/,"""""""""""""""""""""""0,

0.0

50.0

100.0%

GC

0.0

50.0

100.0

% G

C

0.0

50.0

100.0

% G

C

0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000

Annotation: dnaA dnaN recF gyrB lptA 0006 0007

0.0

50.0

100.0

% G

C

A

B

C

32 33 34 35 36 37 38 39 40

0

20

40

60

80

100

Annotation:

% G

C

PA0028

PA0029 PA0030 betC

PA0032

PA0033PA0034 trpA trpB

trpI

30 31

Genome position / Kbp

1,760

fabG ymfJ ymfM pgsA cinA recA pbpX ymdA ymdB spoVS

1,761 1,762 1,763 1,764 1,765 1,766 1,767 1,768 1,769 1,770

0

20

40

60

80

100

Annotation:

% G

CNC_002516 Pseudomonas aeruginosa PAO1 (%GC = 68.2)

NC_000964 Bacillus subtilis subtilis 168 (%GC = 43.1)

NC_000963 Rickettsia prowazekii Madrid E (%GC = 32.2)

214

sucA RP182 RP183 RP184 dnaK

215 216 217 218 219 220 221 222 223 224

0

20

40

60

80

100

Annotation:

% G

C

Frame analysis and sequence GC content

Relative representation of nucleotides at each codon position can be used as scores for nucleotide usage

Scores  that  depend  on  global  nucleo-de  composi-on    are  calculated  for  each  trinucleo-de  

-3

-2

-1

0

1

2

0.25 0.35 0.45 0.55 0.65GC

GWY

TGR

GCT

TGS

RWS

YSW

-1.0

-0.5

0

0.5

1.0

0.20 0.35 0.50 0.65 0.80

Codon base 1A1 C1 G1 T1

-1.0

-0.5

0

0.5

1.0

0.20 0.35 0.50 0.65 0.80

A2 C2 G2 T2

-1.0

-0.5

0

0.5

1.0

0.20 0.35 0.50 0.65 0.80

A3 C3 G3 T3

Codon base 2

Codon base 3Codons

GCGC

GC

Score

Score

Score(XYZ) = lnpX1

pY2pZ3

1− pstop(0)( )

pXpYpZ 1− pstop(1)( )

Cumulative scores and H-type hits

Cumula-ve  scores  can  be  used  to  translate  qualita-ve  visual  informa-on  on  composi-onal  contrasts  (leX)  into  sta-s-cally  characterized  (Karlin  and  

Altschul  1990,  Karlin  1994)  measures  of  coding  poten-al  for  precisely  defined  sequence  segments  (right).  

Posi-on  

Score  

0742  

Modified  0742  

p  =  10-­‐3  

Significant  contrasts  in  characterized  genes  

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!"! '()*+,"$-".-/0'%-1,+2-3-"(-&4+567-2,+%

25

30 35 40 45 50 55 60 65 70 75

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

5010015

020025

030035

040045

050055

060065

070075

080085

0900950

1000

% GC

Frac

tion

of c

hara

cter

ized

gen

es w

ith a

ny ty

pe h

its

p�����-3

Length/codons

p  ≤  0.01   p  ≤  0.001  

Power  of  the  tests  is  high  for  all  long  sequences  and  decreases  more  with  shorter  sequences  of  low  or  intermediate  GC  content  

ORF structure and local composition: The reading-frame prediction-unit

The  sequence  is  read  frame-­‐by-­‐frame  independently  considering  sequence   segments   included   between   consecu-ve   stop   codons  (i.e.,  analyzed  segments  do  not  contain  stop  codons).  

The   local   composi-on   of   the   each   sequence   segments   is  independently  calculated  from  each  selected  reading  frame  and  segment-­‐specific  scores  are  built  based  on  its  composi-on.  

Reading frame

Local composition

Only   hits   included  within  ORFs   are   considered   (i.e.,   a   poten-al  start  codon  must  be  iden-fied).  

ORF structure

NPACT  home  web  page  (hbp://genome.ufl.edu/npact/)  

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,--./0/1.-

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,--./0/1.-

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B,!!!(

?893 ?89C

/0? B,!!"" B,!!"# B,!!"$

B,!!"% B,!!"& /<D, B,!!"(

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,--./0/1.-

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56789:0--./0/6;

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,--./0/1.-

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,--./0/1.-

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NPACT  graphical  output  GC-profiles of Pseudomonas aeruginosa PAO1, complete genome.

0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 10,0000.0

20.040.060.080.0

100.0

Input file CDS

Hits

Newly identified ORFs

% G

C

dnaA dnaN recF gyrBlptA PA0006

PA0007

H-2*G

10,000 11,000 12,000 13,000 14,000 15,000 16,000 17,000 18,000 19,000 20,0000.0

20.040.060.080.0

100.0

Input file CDS

Hits

Newly identified ORFs

% G

C

PA0007glyS glyQ

tag PA0011 PA0013trkA PA0017

H-17*G

PA0012

PA0014 PA0015

H-15-A

20,000 21,000 22,000 23,000 24,000 25,000 26,000 27,000 28,000 29,000 30,0000.0

20.040.060.080.0

100.0

Input file CDS

Hits

Newly identified ORFs

% G

C

fmt defPA0020 PA0021 PA0022

qorhemF aroE plcB PA0027

H-25*a

PA0017

PA0028

30,000 31,000 32,000 33,000 34,000 35,000 36,000 37,000 38,000 39,000 40,0000.0

20.040.060.080.0

100.0

Input file CDS

Hits

Newly identified ORFs

% G

C

PA0028PA0029 PA0030 betC

PA0032PA0034 trpA trpB

trpI

H-32*GH-35*G

H-39*A

PA0033

40,000 41,000 42,000 43,000 44,000 45,000 46,000 47,000 48,000 49,000 50,0000.0

20.040.060.080.0

100.0

Input file CDS

Hits

Newly identified ORFs

% G

C

Sequence position / nt

trpI PA0040 PA0041PA0038

PA0039

Newly-­‐iden-fied  ORFs  in  P.  aeruginosa  strains  

Strain! ID! Genome lengh! GC%! Annotated

CDS!Newly-

identified ORFs!

PAO1! NC_002516! 6264404! 66.56! 5572! 179!UCBPP-PA14! NC_008463! 6537648! 66.29! 5892! 173!PA7! NC_009656! 6588339! 66.45! 6286! 189!LESB58! NC_011770! 6601757! 66.3! 5925! 258!M18! NC_017548! 6327754! 66.5! 5684! 161!NCGM2_S1! NC_017549! 6764661! 66.14! 6268! 250!DK2! NC_018080! 6402658! 66.27! 5883! 157!B136_33! NC_020912! 6421010! 66.42! 5828! 160!RP73! NC_021577! 6342034! 66.46! 5762! 187!

From  157  to  258  ORFs  with  significant  (p  <  0.001)  composi-onal  periodicity  and  not  corresponding  to  annotated  genes  are  iden-fied  in  different  strains  of  

P.  aeruginosa.  

Conserva-on  of  newly-­‐iden-fied      ORFs  between  Pseudomonas  aeruginosa  strains  

Strain! Cat! B136_33! DK2! LESB58! M18! NCGM2_S1! PA7! PAO1! RP73! PA14! Cons! Non Cons.!

B136_33! ORF!CDS!

 !27!

66!43!

47!47!

49!44!

78!45!

22!46!

48!44!

59!43!

66!48! 130! 30!

DK2! ORF!CDS!

66!48!

 !30!

60!54!

63!51!

57!49!

28!56!

61!52!

69!49!

55!59! 135! 22!

LESB58! ORF!CDS!

47!128!

60!130!

 !51!

66!131!

65!121!

22!130!

108!79!

62!127!

59!120! 212! 46!

M18! ORF!CDS!

49!53!

63!49!

66!56!

 !26!

56!48!

27!49!

62!44!

58!53!

62!54! 134! 27!

NCGM2_S1!

ORF!CDS!

78!79!

57!72!

65!57!

56!64!

 !31!

30!79!

58!51!

55!71!

66!65! 175! 75!

PA7! ORF!CDS!

22!47!

28!49!

22!39!

27!45!

30!45!

 !34!

27!45!

28!51!

32!49! 98! 91!

PAO1! ORF!CDS!

48!120!

61!121!

108!70!

62!115!

58!107!

27!113!

 !32!

58!125!

68!104! 187! 11!

RP73! ORF!CDS!

59!91!

69!98!

62!96!

58!99!

55!93!

28!93!

58!101!

 !49!

53!104! 171! 17!

PA14! ORF!CDS!

66!66!

55!64!

59!44!

40!55!

66!54!

32!60!

68!38!

53!65!

 !32! 146! 27!

Homologs  for  the  majority  of  newly  iden-fied  ORF  are  iden-fied  among  annotated  genes  or  newly-­‐iden-fied  ORFs  of  other  P.  aeruginosa  

strains.  

Name! Phyla! Genera! Species! Strains! Tot_con! Not_con! Tot! Tot Strain!

PAO1! 59! 26! 21! 13! 119! 60! 179! 109!UCBPP-PA14! 59! 10! 17! 11! 97! 76! 173! 89!PA7! 67! 11! 15! 17! 110! 79! 189! 95!LESB58! 128! 19! 25! 10! 182! 76! 258! 174!M18! 41! 17! 23! 18! 99! 62! 161! 88!NCGM2_S1! 105! 15! 27! 12! 159! 91! 250! 146!DK2! 42! 16! 18! 11! 87! 70! 157! 75!B136_33! 34! 11! 13! 13! 71! 89! 160! 62!RP73! 84! 17! 11! 7! 119! 68! 187! 110!Total! 619! 142! 170! 112! 1043! 671! 1714! 948!

Long-­‐range  onserva-on  of  ORFs  newly-­‐iden-fied  in  Pseudomonas  aeruginosa  strains  

Most  newly  iden-fied  ORF  conserved  among  P.  aeruginosa  strains  are  conserved  over  different  phyla  

Verifica-on  of  computa-onal  gene  predic-on  by  transcriptome  analysis  in  Pseudomonas  

aeruginosa  PAO1:  RNA-­‐seq  and  Ribosome  Footprin-ng  

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The  P.  aeruginosa  transcriptome:  RNA-­‐seq  

0

500

1000

1500

2000

2500

3000

3500

Long

Characterized

Long

Hypothetical

Short

Characterized

Short

Hypothetical

Tota

l nu

mb

er

Classes of annotated genes

0

10

20

30

40

50

60

Long

Characterized

Long

Hypothetical

Long Non-

conserved

Short

Characterized

Short

Hypothetical

Short Non-

conserved

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0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Long

Characterized

Long

Hypothetical

Short

Characterized

Short

Hypothetical

Fra

ction e

xpre

ssed

Classes of annotated genes

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Long

Characterized

Long

Hypothetical

Long Non-

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Short

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Short

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Short Non-

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Expression  of  predicted  genes  by  length  and  conserva-on  classes  Published  annota-on   Newly  iden-fied  ORFs  

ORFs  with  RNA-­‐seq  reads  

What  do  we  learn  about  gene  predic-ons  from  transcrip-on  in  bacteria?  

Unexpected  paberns  

33000 34000 35000 36000 37000 38000

0

2

4

2

4

Annotation

0.0

50.0

100.0

% C

+G

Log-count

Hits

New

betC

0032

0033 0034 trpA trpB

H-51*A

Contradictory  paberns  of  expression  of  well  defined  protein  coding  genes  

In  the  case  of  predic-on  of  H-­‐443*A  ,  sequence  features  appear  to  be  more  convincing  than  RNA  expression  data    

What  do  we  learn  about  gene  predic-ons  from  transcrip-on  in  bacteria?  

The  problem  of  an-sense  transcrip-on    

347000 348000 349000

0

2

4

2

4

Annotation

0.0

50.0

100.0

% C

+G

Lo

g-co

un

tHits

New

0306

0307

H-443*A

Ribosome  footprin-ng  (Ingolia  et  al,  Science  2009)  

Ribosome stalling with translation-elongation inhibitor tetracycline

Cell lysis and digestion of unprotected RNA

3XULÀFDWLRQ�RI�ULERVRPH�footprints

cDNA library preparation for deep-sequencing and genome mapping

Schema-c  representa-on  of  the  ribosome  footprin-ng  procedure  applied  to  P.  aeruginosa  

Ribosome  footprint  coverage  in  P.  aeruginosa  

3,110 3,111 3,112 3,113 3,114 3,115 3,116 3,117 3,118 3,119 3,120

0

2

4

2

4

Published

0.0

50.0

100.0

% C

+G

Log-

coun

t

New

2748 endA 2750 2751 2752 2753 2754 eco 2756 2757 2758 2759

H-3432-G H-3436*A H-3435*A H-3440*g H-3444*A

Genome position / Kbp

#  of  re

ads  

Example  of  ribosome  footprint  coverage  in  P.  aeruginosa  PAO1  showing  rela-on  with  S-­‐profiles,  annotated  genes  and  

newly  iden-fied  ORFs.  

Ribosome  footprints  of  ini-a-on  sites  

The  an-bio-c  tetracycline  inhibits  transla-on-­‐elonga-on  stalling  ac-vely-­‐transla-ng  ribosomes  

Ribosome  footprints  of  ini-a-on  sites  

However,  tetracycline  does  not  prevent  more  ribosomes  to  be  recruited  at  the  ini-a-on  site.  

Ribosome  footprints  of  ini-a-on  sites  

The  accumula-on  of  ribosomes  will  result  in  increased  numbers  of  profile-­‐reads  corresponding  to  the  ini-a-on  site.  

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

-30

-15 0 15 30 45 60 75 90 105

120

135

150

165

180

195

210

225

240

255

270

285

300

Rel

ativ

e co

vera

ge

Position from start of translation / nt

RNA-seq

Ribosome footprints

Ribosome  footprint  coverage  by  codon  posi-on  

Metagene  analysis  of  ribosome-­‐footprint  coverage    Coverage  is  averaged  over  all  genes,  rela-ve  to  the  start  of  transla-on  

Ribosome  footprint  coverage  by  codon  posi-on:  center  of  reads  

Metagene  analysis  of  coverage  by  read  center  +  2  nt    Coverage  is  averaged  over  all  genes,  rela-ve  to  the  start  of  transla-on  

0

10

20

30

40

50

60

70

80

-50 -40 -30 -20 -10 0 10 20 30 40 50 60 70 80 90 100

Rib

osom

e-fo

otpr

int c

over

age

Position relative to start of translation / nt

Transla-onal  evidence  by  ribosome  footprin-ng  in  P.  aeruginosa  

0

50

100

150

200

250

300

350

400

0 200 400 600 800 1000 1200 1400 1600

Cov

erag

e

Position from start of translation / nt

groEL (547 aa) c4917124..4915481 Characterized

Ribosome-­‐footprint  read-­‐count  paberns  iden-fy  mRNA  transla-on,  transla-on  ini-a-on  site,  and  transla-on  pausing.  

Transla-onal  evidence  by  ribosome  footprin-ng  in  P.  aeruginosa  

0

50

100

150

200

250

0 200 400 600 800 1000 1200 1400 1600

Cov

erag

e R

FP2

Position from start of translation / nt

RFP1

0

50

100

150

200

250

Cov

erag

e R

FP1

groEL (547 codons)

RFP2

Similar  coverage  paberns  are  observed  in  different  biological  replicates  

Scoring  RFP  expression  

“Strength”  of  evidence  decreases  for  poorly  translated  mRNA.  

0

1

2

3

4

5

6

0 100 200 300 400 500 600 700 800

Cov

erag

e

Position relative to predicted start of translation

2758 (295 aa) 3118296..3119183 Characterized

RFP control

Scoring  RFP  expression  

“Strength”  of  the  evidence  of  expression  is  measured  by  an  “Expression  Index”.  

=C0 lnC0C1

C0

C1

:  Count  of  RFP  reads  in  codon  posi-ons  [-­‐2,+3]  /  5;  

:  Count  of  RFP  reads  in  codon  posi-ons  [+9,  len/2]  /  (len/2  -­‐9);  

Expression  Index  

0.1

1

10

100

1000

0.001 0.01 0.1 1 10 100 1000 10000

Cov

erag

e St

art /

Cod

ing

regi

on

Coverage coding region

0.1

1

10

100

1000

0.001 0.01 0.1 1 10 100 1000 10000

Cov

erge

Sta

rt / C

odin

g re

gion

Coverage coding region

Long  published  genes   Short  published  genes  

Long  newly  iden-fied  ORFs   Short  newly  iden-fied  ORFs  

Expression  of  published  and  newly-­‐predicted  genes  in  Pseudomonas  aeruginosa  

*  Coverage  normalized  by  the  number  of  posi-ons  

0.1

1

10

100

1000

0.001 0.01 0.1 1 10 100 1000 10000

Cov

erag

e St

art /

Cod

ing

regi

on

Coverage coding region

0.1

1

10

100

1000

0.001 0.01 0.1 1 10 100 1000 10000

Cov

erag

e St

art /

Cod

ing

regi

on

Coverage coding region

0

500

1000

1500

2000

2500

3000

3500

Long

Characterized

Long

Hypothetical

Short

Characterized

Short

Hypothetical

Tota

l num

be

r

Classes of annotated genes

0

10

20

30

40

50

60

Long

Characterized

Long

Hypothetical

Long Non-

conserved

Short

Characterized

Short

Hypothetical

Short Non-

conserved

TRWDO�QXP

EHU�RI�QHZ

O\�LGHQWLÀHG�25)V

&ODVVHV�RI�QHZO\�LGHQWLÀHG�25)V

Expression  of  predicted  genes  by  length  and  conserva-on  classes  Published  annota-on   Newly  iden-fied  ORFs  

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Long

Characterized

Long

Hypothetical

Short

Characterized

Short

Hypothetical

Fra

ction e

xpre

ssed

Classes of annotated genes

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Long

Characterized

Long

Hypothetical

Long Non-

conserved

Short

Characterized

Short

Hypothetical

Short Non-

conserved

Fra

ctio

n e

xp

re

sse

d

&ODVVHV�RI�QHZO\�LGHQWLÀHG�25)V

ORFs  with  Expression  Index  ≥  12.0  

Iden-fica-on  of  transla-on-­‐ini-a-on  sites  by  ribosome-­‐footprin-ng  

0

20

40 60

80

100

120

140 160

180

0 100 200 300 400 500 600 700

Cov

erag

e

Position relative to predicted start of translation

fliA (247 aa) 1584795..1585538 Characterized

RFP control

0 50

100 150 200

250 300

350 400

450

-300 -200 -100 0 100 200 300

Cov

erag

e

Position relative to predicted start of translation

cheY (124 aa) 1585640..1586014 Characterized

RFP control

FliA,  sigma  factor  of  RNA  polymerase  for  flagellin  gene  transcrip-on.  CheY  is  involved  in  transmission  of  sensory  signal  to  the  flagellar  motor.  

Start  of  transla-on  iden-fica-on  by  RFP  read  accumula-on  

Annotated   Newly  iden.fied  

Same  start   0.850   0.778  

Different  start   0.150   0.222  

Ribosome  footprints  confirm  the  predicted  start  of  transla-on  for  85%  of  the  genes,  and  of  78%  of  the  newly-­‐iden-fied  ORFS,  among  those  with  evidence  of  transla-on.  

0

50

100

150

200

250

300

350

400

-100 0 100 200 300 400

Cov

erag

e

Position relative to predicted start of translation

eco (156 aa) 3116654..3117124 Characterized

RFP control

Iden-fica-on  of  new  genes  by  ribosome-­‐footprint  evidence  

A  new  gene  is  found  to  be  expressed  5’  of  the  gene  eco  for  Eco-n,  a  protease  inhibitor  localized  to  the  periplasmic  space.  

3,114,000 3,115,000 3,116,000 3,117,000 3,118,0000.0

20.040.060.080.0

100.0

Input file CDS:

Hits

Newly identified ORF:

% G

CPA2752

PA2753 ecoPA2756 PA2757

PA2754

H-3445*A

Sequence position / nt

Iden-fica-on  of  new  genes  by  ribosome-­‐footprint  evidence  

The  newly-­‐iden-fied  translated  ORF  (red  circle)  corresponds  to  a  region  of  weak  composi-onal  3-­‐base  periodicity.  

Ribosome  footprint  coverage  in  P.  aeruginosa  

Examples  of  RFP-­‐based  gene  discovery  in  P.  aeruginosa  PAO1  showing  rela-on  with  S-­‐profiles  and  annotated  genes.  

2,058 2,059 2,060 2,061

0

2

4

2

4

Annotated:

0.0

50.0

100.0

% G

C

Log-

coun

t

PAO1_1888

Sequence position / Kbp

PAO1_1889 PAO1_18890

0

100 200 300 400 500 600

-150 -100 -50 0 50 100 150 200 250

Cove

rage

Position relative to predicted start of translation

MHGP10 RFP1 control

MHGP10Identified by RFP:

Lab  members    

•  Steve  Oden  –  Postdoctoral  associate.  Development  of  gene  finding  methods  and  soXware,  gene  content  analysis  in  human  and  prokaryotes.  

•  Eric  Hernandez  –  Programmer.  

•  Dr.  Anna  Picca–  Postdoctoral  associate.  RNA-­‐seq  and  ribosome  profiling  

•  Dr.  Ying  Zhang  –  Postdoctoral  associate.  RNA-­‐seq  

•  Dr.  Shouguang  Jin  (Molecular  Gene-cs  and  Microbiology).  P.  aeruginosa  

•  Dr.  Silvia  Tornale|  (Medicine).  Transcrip-on  and  RNA.  

Collaborators  

•  Dr.  Rolf  Renne  and  MGM.  

•  Dr.  Jianhong  Hu  (Research  Scien-st)  

Sequencing  facility  and    support  

Thanks  to  

•  NIH  R01  GM087485-­‐01A2  

•  MGM,  Gene-cs  Ins-tute,  College  of  Medicine.  

Funding