Comparative genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica causing typhoid

Michael McClelland¹, Kenneth E. Sanderson², Sandra W. Clifton³, Phil Latreille³, Steffen Porwollik¹, Aniko Sabo³, Rekha Meyer³, Tamberlyn Bieri³, Phil Ozersky³, Michael McLellan³, C. Richard Harkins³, Chunyan Wang³, Christine Nguyen³, Amy Berghoff³, Glendoria Elliott³, Sara Kohlberg³, Cindy Strong³, Feiyu Du³, Jason Carter³, Colin Kremizki³, Dan Layman³, Shawn Leonard³, Hui Sun³, Lucinda Fulton ³, William Nash ³, Tracie Miner ³, Patrick Minx³, Kim Delehaunty³, Catrina Fronick³, Vincent Magrini³, Wesley Warren³, Liliana Florea⁴, John Spieth³, Michael Nhan³, & Richard K. Wilson³

1. Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121, USA
2. Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
3. Genome Sequencing Center, Washington University School of Medicine, 4444 Forest Park Boulevard, St Louis, Missouri 63108, USA
4. Informatics Research, Celera/Applied Biosystems, 45 W. Gude Drive, Rockville, MD 20850, USA.

* for correspondence: phone +1 858 450 5990 x 280, fax +1 858 550 3998, email: mmcclelland@sdibr.org


Supplementary Table 1: Genome annotation: Salmonella enterica serovar Paratyphi A ATCC9150.
This file contains an annotation and the corresponding DNA and amino acid sequences for this genome. It also contains the G+C content for each gene and the name of the most similar paralogues in the genome. A comparison of each ORF is performed with the Typhi CT18, Typhimurium LT2, and E. coli K12 genomes, using a sliding 100bp window and the percent identity. The identity in the best 100bp window is reported and whether this closest homologue is syntenic or not. The thresholds for DNA divergence are set at 90% for Salmonella and 70% for E. coli K12.
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Supplementary Figure 1: Genome annotation: Salmonella enterica serovar Paratyphi A ATCC9150.

Gene distribution:    Blue = Only in SPA Red =  SPA and Typhi, but not Typhimurium or E. coli. Green = SPA and Typhimurium, but not Typhi or E. coli. Yellow =SPA, Typhi, and Typhimurium, but not E. coli. Mauve = SPA, Typhi, Typhimurium, and E. coli. White = Structural RNAs. Grey =    Other distributions and paralogues. Crosshatching = Pseudogenes.

Supplementary Figure 2. Detection of phage SPA-3 DNA in supernatants of SPA liquid cultures after Mitomycin C treatment.
The contribution of each array feature to total signal is plotted in order of position of the genes on the SARB42 genome (10 gene moving average). A culture was grown in LB, and harvested 5 h after addition of Mitomycin C (final concentration 2 μg/ml). Cells were separated from the supernatants by centrifugation at 3,220 x g for 20 min at 4˚C. Phage present in the supernatants was precipitated using 5% PEG 8,000 and 0.5M NaCl on ice for 30 min and subsequent centrifugation at 20,000 x g for 20 min at 4˚C. The pellet was resuspended in 1xTE, and purified using the GenElute Bacterial Genomic DNA kit (Sigma, St. Louis, MO), according to the manufacturer’s recommendations. 100ng of purified DNA was labeled and hybridized to the Salmonella array as described in the materials and methods.


Supplementary Table 2: Comparison of gene content among twelve Paratyphi A strains. Strains investigated.

SGSC#	strain #	Source
3588	07255	R.G.E. Murray via Julian Davies, UBC
3526	WR01	W. Rabsch, Robert Koch Institute, Wernigerode, Germany
3023	RKS2907	R.K. Selander, Pennsylvania
2724	SPA3	Tikki Pang, Kuala Lumpur, Malaysia
2723	SPA2	Tikki Pang
2722	SPA1	Tikki Pang
2276	SA4249	Southern Alberta Provincial Lab (SAPL)
2686	R737	SAPL
2687	R120	SAPL
2688	R76	SAPL
2689	R291	SAPL
2679	SA4861	SAPL

 

Figure 2 in paper: Visualization of gene content differences among strains of Paratyphi A.
Blue, present. Red, absent. Predictions were established as described in Porwollik et al., J. Bacteriology (2004).

Table 3 in paper: Summary of gene content differences among strains of Paratyphi A. 

Region	# of genes missing	SPA gene numbers	# of strains missing	Start	End	Genes
I	3	1083-1085	1	1152663	1154381	hyaCDE
II	2	1629-1630	1	1691897	1693646	cobB, ycfX
III	45	2387-2431	1	2488061	2527582	Phage SPA-1: gtrC translocase, lysozymes (gp19, gp13), ninBDEXH
IVa	47	2554-2600	5	2662208	2695493	Phage SPA-2-SopE. lysozyme, nucD, adenine methylase
IVb	25	2601-2625	2	2696026	2724833	Phage SPA-3-P2

 

Supplementary Table 3. Pseudogenes in Paratyphi A.
A list of two deleted genes and 173 predicted pseudogenes and the nature of the lesion responsible.
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Supplementary Note. Further examples of potential functional consequences of pseudogenes.
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Supplementary Table 4. Putative orthologues of genes in Paratyphi A that are predicted to be pseudogenes in Typhi, Shigella flexneri, or Yersinia pestis.
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Supplementary Table 5. New predicted pseudogenes in Typhi.
A re-examination of the Typhi genomes in light of the SPA genome sequence reveals 16 previously unrecognized possible pseudogenes in the Typhi genome that were formerly annotated as 24 CDSs. This raises the total number of pseudogenes to 229 in Typhi CT18 and/or Ty2 (5% of a correspondingly adjusted number of 4,593 CDSs) of which 219 have a syntenic orthologous gene in SPA. Two of these putative pseudogenes are in regions where Typhi had no orthologues in previously sequenced genes so that the pseudogenes could not have been recognized. In addition, eutM and eutN are fused in Typhi, though they are separate in SPA, Typhimurium, and every other sequenced organism that has homologues. These genes for ethanolamine utilization may constitute a pseudogene in Typhi.

SPA orthologue	Gene name	STY gene fragment
SPA0019		STY0020+STY0019
SPA0080	yaaU	STY0089
SPA0085		STY0099+STY0098+STY0097
SPA0390	aegA	STY2717
SPA0928		STY2151+STY2152
SPA0930		STY2148+STY2147
SPA1187		STY1361
SPA1232		STY1413
SPA1349	rspB	STY1555
SPA1723		STY1164+STY1165
SPA2160		STY0616+STY0615
SPA2285		STY0476+STY0477+STY0478
SPA2503		STY0286+STY0287
SPA2778		STY3047+STY3046
SPA3861	yihR	STY3858
SPA4334		STY4870+STY4869