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Invited presentation from the 2007 IS-MPMI plant pathology meeting in Sorrento, Italy. Presentation delivered 26th July 2007.
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Comparative genomics: What makes the enterobacterial plant pathogen Pectobacterium atrosepticum different to its animal pathogenic relatives? And other questions.
Leighton PritchardPaul BirchIan Toth
Pectobacterium atrosepticum (Pba, formerly Erwinia carotovora subsp. atroseptica):•potato pathogen: blackleg (stem rot), rotting of stored tubers•major rot symptoms due to plant cell wall-degrading enzymes (PCWDEs)•also has T3SS and effectors, and phytotoxins•stealth (host manipulation) and brute force
Pectobacterium atrosepticum (Pba, formerly Erwinia carotovora subsp. atroseptica):•plant, rather than animal-associated enterobacterium•soft-rot enterobacterium (with Dickeya spp., Pectobacterium carotovorum etc).•temperature/climate-related disease profiles•Pba-centric genomic and transcriptomic comparisons
Genome sequenced 2004:
•SCRI/Sanger
•Lab strain SCRI1043
•5 Mb
•4472 CDS
•51% (G + C)
•17 putative horizontally-
acquired islands
Bell et al. (2004) Proc. Natl. Acad. Sci. USA 101 11105-11110
Circular representation
Common sequence:similarity
Gaps :dissimilarity
Bell et al. (2004) Proc. Natl. Acad. Sci. USA 101 11105-11110
Reciprocal best hits(FASTA, 30% ID,
80% overlap)
Coloured by taxonomicgrouping
Extended comparison to allavailable genomes
Colours indicate similarity:•red: high similarity•blue: low similarity
Most similar organisms onouter rings
Extended comparison to allavailable genomes
Colours indicate similarity:•red: high similarity•blue: low similarity
Most similar organisms onouter rings
Radial gaps, with highly-similarsequences in less-similarorganisms indicate potential HGT or gene loss
Selection pressure in allenvironments
Loss of functions important only in a former niche
Gain of function on adaptation to a novel niche
Acquisition from organismsinhabiting novel niche
What does Pba have that plant-associated bacteria have, but animal-associated enterobacteria do not?
Marked features: more similar to PAB
than to AAE[>1.5X mean bit score](497, >10% of genome)
Plant-associated bacteria(PAB)
Animal-associated enterobacteria (AAE)
Toth et al. (2006) Ann. Rev. Phytopath. 44 305-336
Attachment
Nitrogen fixation
Coronafacic acid
Type III Secretion System
Toth et al. (2006) Ann. Rev. Phytopath. 44 305-336
AggA contributes to root adhesion in Pseudomonas putida
Pba ECA3266, aggA are similar to PAB, not AAE, and found in HAI
See Poster PS2-176 (Sonia HUMPHRIS)
cfa synthesis genes similar to PAB, not AAE, and found in HAI
A series of cfa synthesisgene knockouts wasconstructed
Lesion length much reduced in the knockouts compared to WT
WT cfa-
Bell et al. (2004) Proc. Natl. Acad. Sci. USA 101 11105-11110
See Poster PS13-642 (Michael RAVENSDALE)
11k Agilent arrays Pba, Dda
Challenge Pba array withDda and Pcc gDNA
(Complementary analysis to RBH of prepublication Dda genome)
Ravirala et al. (2007) Mol. Plant-Microbe Int. 20 313-320
See Poster PS13-637 (Hui LIU)
Pba Pcc Dda Pba Pcc Dda Pba Pcc Dda
Pba Pcc Dda
Whole-genome Southern hybridisation
Exact match of 20 probes to Dda genome
Over 900 probes hybridise strongly to Dda gDNA (3200 total)
PAB AAE
No RBH in Dda(1351)
No hyb to Pcc(1035)
30 selected islands of interest that don’t hyb to
Pcc, or make no RBH to Dda
cfa synthesis genes
Pcc BAC spanning SPI-7/cfa genessequenced and annotated (Sanger)
Blue bars indicatematches (BLAST)
cfa gene probes do not hyb to Pcc; have no counterpart insyntenous SPI-7region
Pba
Pcc
Region of Pba:•in HAI•no hyb to Pcc•RBH matches to Dda•RBHs to other PAB
nif: nitrogen fixation
•Prediction:•WT Pba fixes N•Pba nif knockouts do not fix N•WT Dda fixes N•WT Pcc does not fix N
Pba Pcc Dda
•three WT Pba strains fix N•two WT Dickeya spp. Fix N•one of six tested Pcc WT strains fixes N•Pba nifA- mutant does notfix N
•Prediction:•WT Pba fixes N•Pba nif knockouts do not fix N•WT Dda fixes N•WT Pcc does not fix N
What makes Pba different from animal-associated enterobacteria, andfrom other soft-rotting plant pathogens?
HGT activity
Putatively acquired functions:•coronafacic acid synthesis•root adhesion•nitrogen fixation
about 25% of genome also distinguishes Pba from close relatives…
What else to find out:
•What has Pba lost, in respect to animal-associated enterobacteria?
•(and closer relatives)?
•What do they all have in common?
SCRIPaul BirchIan Toth
Hui LiuSonia HumphrisLucy MolelekiMichael RavensdalePete Hedley
Eduard VenterGunnhild TakleBeth HymanJennifer White
SangerPathogen Sequencing Unit
FundingSEERAD, BBSRC
Comparisons against other bacterial genomes:
Reciprocal best hits(FASTA, 30% ID,
80% overlap)
linear representation
Coloured by taxonomicgrouping
Bell et al. (2004) Proc. Natl. Acad. Sci. USA 101 11105-11110
database .gbk .crunch …
Python Script GenomeDiagram
Reportlab
database .gbk .crunch …
Python Script GenomeDiagram
Reportlab
229 bacterial comparisons
185970 RBH
23Gb of data
24h on 50-node cluster
Visualisation issues
Pritchard et al. (2006) Bioinformatics 22 616-617
Failure to hybridise does notimply that the Pba gene isabsent
Dda: 2910 RBH to Pba; 949strongly hybridising probes(ca. 3200 total)
Weakly-hybridising probesare seen with > 90% aminoacid identity
E. coli CFT073 Pasteurella multocida
Salmonella entericasubsp. enterica
serovar Typhi LT2str. CT18
Plasmid hotspots
Toth et al. (2006) Ann. Rev. Phytopath. 44 305-336
