MGH-PGA

1
MGH-PGA Genomic Analysis of Stress and
Inflammation Sequence Analysis of Pseudomonas
aeruginosa Strain PA14
Nicole T. Liberati, Dan G. Lee, Jacinto M.
Villanueva and Frederick M. Ausubel Department
of Molecular Biology Massachusetts General
Hospital
Kate Montgomery, Wade Brown, David Sarracino and
Raju Kucherlapati Harvard-Parners Center for
Genetics and Genomics
2
PA14 Genomic Sequence
PAO1
vs.
PA14

• (Less pathogenic)
• 6.26 MB
• 5,570 genes

• (More pathogenic)
• 6.54 MB
• 5,800 genes

Stover et al, 2000, Nature 406 959-964.
3
Comparison of PA14 and PAO1 Genomes
PA14

(6.54 Mb)
PAO1

(6.26 Mb)

• 96.3 of the PAO1 genome is present in PA14
  92.4 of PA14 is present in PAO1.
• regions of PA14 inverted relative to those in
  PAO1 are shown as black vertical lines.
• regions of either genome absent in the other are
  indicated by violet vertical lines highlighted by
  arrows.
• 313 PA14-specific ORFS 145 PA01-Specific ORFS.
• Several large blocks of PA14 genes
  (pathogenicity islands) not present in PA01.
• magnified view of the largest PA14 pathogenicity
  island (PAPI-1) shown at top. Arrows indicate
  positions of hypothetical ORFs for which a
  transposon insertion strain in the ORF has been
  tested in mice. The percent mortality is
  indicated above each arrow (wild-type PA14
  results in 100 mortality).

4
A Second Example of a PA14-Specific ORF Cluster

• PA14 has a 7-ORF insertion in between genes
  corresponding to PA2422 and PA2423 of PAO1.
• 1 ORF (5998) has no homology to any entry in the
  NCBI nr database.
• 6 ORFs (5999-6004) are similar to 6 contiguous
  hypothetical proteins from Yersinia pestis
  (strain KIM). The order an orientations of the
  genes is identical to that of Y. pestis. Gene
  identities range from 34 to 66, and
  similarities range from 49 to 79.

5
PA14 Proteomics for Validation of PA14 Annotations

• Bacteria grown to mid-log phase, washed and
  frozen.
• Sonicated in presence of Urea, SDS, etc.
• Sample was reduced and alkylated.
• Proteins were separated by SDS-PAGE.
• In-gel tryptic digests.
• Analyzed only peptides from low-molecular weight
  proteins by MS in the initial pilot experiment.
• A number of PA14-specific ORFs (i.e. - not
  present in PAO1) have been verified in this
  initial pilot proteomic analysis.
• A large scale analysis of the entire proteome
  (all molecular weight fractions) has been
  initiated.

6
PA14 Proteomics Example of a Validated
PA14-Specific ORF

• PAO1 gene PA3362 lies in between lecB and amiR.
• PA3362 is annotated as a hypothetical protein.

• PA14 ORF 6327 lies in between lecB and amiR.
• 6327 is 53 identical (76 similar) to a
  conserved hypothetical protein in Pseudomonas
  putita.
• 6327 does NOT have a homolog in PAO1.
• 6327 has been validated by proteomics.

7
Genomic Typing via Spotted Oligo Microarrays

• Microarray for transcriptional profiling AND
  genomic typing.
• Shear genomic DNA from a panel of P. aeruginosa
  strains
• 4-base cutters
• DNaseI
• Label genomic DNA
• End labeling
• Random hexamer labeling with aa-dUTP
• Hybridize to microarray
• Determine which genomic regions are
  present/absent in each test strain. Those
  PA14-specific regions that are conserved in
  other clinical isolates are of particular
  interest.

8
Hybridizations of PA14 and PAO1 Genomic DNA to
Pilot Microarray

• A pilot array was produced consisting of 95
  oligonucleotides (and one blank) spotted in
  quadruplicate on each slide
• 14 oligos were designed for P. aeruginosa
  sequences present in both PA14 and PAO1
• 36 oligos were designed for P. aeruginosa
  sequences present in PA14 (but absent in PAO1).
• 32 oligos were designed for P. aeruginosa
  sequences present in PAO1 (but absent in PA14).
• 13 oligos were designed for P. aeruginosa
  sequences absent in both PA14 and PAO1.
• Genomic PA14 and PAO1 DNA was digested with a
  4-base cutter (HaeIII), purified, labeled, and
  hybridized to the pilot array.

9
Hybridizations of PA14 and PAO1 Genomic DNA to
Pilot Microarray

• Labeled PAO1 DNA (left) or PA14 DNA (right) was
  hybridized to the microarray and hybridization
  intensities (corrected for background) are shown.
• Turquoise bars below each graph indicate probes
  corresponding to sequences present in the strain.
• An arbitrary cut-off to assign sequences as
  present or absent was not based on absolute
  intensities, but rather normalized values in the
  following manner each intensity was divided by
  the average intensity for the entire sample to
  adjust for overall intensity differences between
  samples, and any probe with an adjusted intensity
  greater than an 0.5 was defined as present in the
  genome.
• Using this criterion, 94 of the probes were
  correctly assigned for PAO1 (6 probes in regions
  absent in PAO1 were incorrectly assigned as
  present) and 94 of the probes were correctly
  assigned for PA14 (3 probes in regions present in
  PA14 were incorrectly assigned as absent, and 3
  probes in regions absent in PA14 were incorrectly
  assigned as present). Incorrect assignments
  based on this arbitrary cut-off are indicated by
  white circles.