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 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). PA14 PAO1 (6.54 Mb) (6.26 Mb) % killing in a murine sepsis model compared to 100% for PA14 503825100 75635667038 50 insertions from non-redundant library in unknown ORFS PAPI-1 (108 Kb)

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%. No hits 6 contiguous hypothetical proteins from Yersinia pestis strain KIM PAO1 PA14

5. PA14 Proteomics for Validation of PA14 Annotations 1. Bacteria grown to mid-log phase, washed and frozen. 2. Sonicated in presence of Urea, SDS, etc. 3. Sample was reduced and alkylated. 4. Proteins were separated by SDS-PAGE. 5. In-gel tryptic digests. 6. 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. 7. 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 1.Microarray for transcriptional profiling AND genomic typing. 2.Shear genomic DNA from a panel of P. aeruginosa strains: 4-base cutters DNaseI 3.Label genomic DNA: End labeling Random hexamer labeling with aa-dUTP 4.Hybridize to microarray 5.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. PAO1PA14 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.