SUPPLEMENTAL MATERIAL
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1 SUPPLEMENTAL MATERIAL SUPPLEMENTAL RESULTS Microarray analysis of F. tularensis LVS within hepatocytes: As described in Materials and Methods, the global gene expression of LVS organisms grown in FL83B hepatocytic cells for 48 hours was compared to that of bacteria cultured in broth for the same period using cdna microarrays. Data averaged from four independent hybridizations showed that expression of 53 genes was up-regulated and that of 20 genes was down-regulated more than two-fold in intracellular bacteria, as compared to bacteria cultured in MH+; these are listed in Table S2 below. The most highly upregulated genes belong to the fsl operon and are discussed in the text. Most of the other genes encode hypothetical s with unknown functions. Most also are present in the genomes of both the LVS and the highly virulent, type A Schu S4 strain. Twelve of the 17 genes that are located in the Francisella pathogenicity island were up-regulated from 2.0- to 5.0-fold, including pathogenicity island gene G (pigg), igld, pigb, pigi, piga, pigd, pdpc, pigc, pdpa, pigh, pdpd, and pdpb. Other pathogenicity island genes also were upregulated but to a lesser extent, including pige (1.8-fold), iglc (1.6-fold), iglb (1.8-fold), and igla (1.8-fold). Genes encoding MglA and MglB, which regulate expression of virulence genes and are required for growth in macrophages (1-4), were down-regulated approximately 2-fold. 1. Baron GS, Nano FE MglA and MglB are required for the intramacrophage growth of Francisella novicida. Mol. Microbiol. 29: Brotcke A, Weiss DS, Kim CC, Chain P, Malfatti S, Garcia E, Monack DM Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis. Infect. Immun. 74: Lauriano CM, Barker JR, Yoon SS, Nano FE, Arulanandam BP, Hassett DJ, Klose KE MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival. Proc. Natl. Acad. Sci. U. S. A 101: Santic M, Molmeret M, Klose KE, Jones S, Kwaik YA The Francisella tularensis pathogenicity island IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Cell Microbiol. 7:
2 Table S1: Primers used in this work Primer No. Sequence a 1 5'-GCAAGCTGAGCATATTCTCCCAG-3' 2 5'-CACTTGTAGCATGAATTGCCACTG-3' 3 5'-CGAGATTTACCGCAATGGC-3' 4 5'-GCTTAGAGTAGGGCTTATTCCTGC-3' 5 5'-TGGACCGATACTGACACTGAGG-3' 6 5'-AGAGCCTTTACACCGACTCC-3' 7 5'-AGACgtcgacTGCTTCGCCATGCTTTATCATC-3' 8 5'-AGACcatatgCCAATATGACAATGTAGCCCAGC-3' 9 5'-AGACcatatgTGAGCTAGGACGTAGTTTGGTCG-3' 10 5'-AGACgtcgacCTTGGCTTTAGATGGATGAACTCG-3' 11 5'-CCTATGGAAAAACGCCAGCAAC-3' AATACGCAAACCGCCTCTCC-3' 13 5'-TCGAGATTTACCGCAATGGC-3' 14 5'-TCGCTTAGAGTAGGGCTTATTCCTG-3' 15 5'-TGCTTTTCTCTCCTTTTTGGGG-3' 16 5'-GCTGCTATGATTATCGGGTCTGAG-3' 17 5'-GCCACTATCTGAAATCTATGGTCGC-3' 18 5'-CGAGTCCTTGAACAAAACGCC-3' 19 5'-ggatccGCTCTAGTTGGCAATCCAAACTGC-3' 20 5'-catatgACCAATACCTTTTGCTGCAACGACT-3' 21 5'-catatgACACTTGCTAAAGAGGTTGTAGTCGG-3' 22 5'-gtcgacGCAGCCAGTAAGATTTGCCACG-3' 23 5'-TGATATTGGTAGTCTGTGGCTTGC-3' 24 5'-CATTTGCGAGAATACCTGTGACAG-3' 25 5'-AGACcatatgGAAATCTCTGTGGCATTTTCCTGG-3' 26 5'-AGACacgcgtGGCAGCAAGAAATATACGCCC-3' 27 5'-AGACacgcgtGCAGATTGTTGAATCATTCGGTGC-3' 28 5'-AGACcatatgTTTACCAAGAAGCCCCTCACG-3' 29 5'-GGAGCCTATGGAAAAACGCC-3' 30 5'-GGAGAGCCTGAACCTTTGAAGC-3' 31 5'-AGTCgtcgacCAGGAACTCAAATCAAGGTATGTAAGG-3' 32 5'-AGTCggatccCGCCTGATTCGCTTTTATTACGACATTTC-3' 33 5'-AGTCgtcgacGAAATCTCTGTGGCATTTTCCTGG-3' 34 5'-AGTCggatccGGCAGCAAGAAATATACGCCC-3 a The following restriction sequences are indicated in lower-case letters: Sal I: gtcgac; Nde I: catatg; MluI: acgcgt; BamH I: ggatcc. 2
3 Table S2: Up- and down-regulated genes of the LVS grown in FL83B hepatocytic cells for 48 h in comparison with the LVS grown in MH+ broth ORF in LVS a ORF in Schu S4 a Product Symbol P-Value Fold Change FTL_1834 FTT0027c diaminopimelate decarboxylase fslc 4.4E FTL_1832 FTT0029c hypothetical fsla 3.8E FTL_1835 FTT0026c hypothetical fsld 5.4E FTL_1833 FTT0028c hypothetical fslb 5.1E FTL_1164 FTT1707 hypothetical pigg 3.1E FTL_0675 FTT1388 hypothetical 3.1E FTL_0953 FTT0677c hypothetical 5.4E FTL_1218 FTT0981 hypothetical 1.2E FTL_1160 FTT1711c intracellular growth locus, subunit D igld 2.6E FTL_0816 FTT1140 hypothetical 3.2E FTL_0867 FTT0602c hypothetical 1.4E FTL_0207 FTT0296 Pyrrolidone-carboxylate pcp 1.5E peptidase FTL_1169 FTT1702 hypothetical pigb 2.1E FTL_0473 FTT0403 peptide deformylase def1 1.5E FTL_1161 FTT1710 hypothetical pigi 7.4E FTL_1170 FTT1701 hypothetical piga 1.9E FTL_0026 NA b 3-hydroxyisobutyrate 6.1E dehydrogenase FTL_1217 NA b hypothetical 2.3E FTL_0815 NA b PRC-barrel 3.0E FTL_1231 FTT0970 hypothetical 5.3E FTL_0449 FTT0383 hypothetical 1.2E FTL_1678 FTT0101 conserved membrane 7.5E hypothetical FTL_1228 FTT0973 hypothetical 2.2E FTL_1167 FTT1704 hypothetical pidd 2.0E FTL_1162 FTT1709 hypothetical pdpc 2.5E FTL_1168 FTT1703 conserved hypothetical. pigc 2.6E NA b FTT1143 hypothetical 3.4E FTL_1219 FTT0980 hypothetical 5.0E FTL_1230 FTT0971 cysteine desulfurase activator sufb 1.2E complex subunit SufB FTL_1172 FTT1699 hypothetical pdpa 1.2E FTL_1163 FTT1708 hypothetical pigh 2.6E FTL_1154 NA b pseudogene 1.9E NA b FTT1143 hypothetical 4.7E FTL_1755 FTT0133 glycerol uptake facilitator glpf 1.3E FTL_1229 FTT0972 ABC transporter, ATP-binding sufc 2.2E
4 FTL_0924 NA b proton-dependent oligopeptide 1.0E transporter FTL_1016 FTT0558 short chain dehydrogenase 3.9E FTL_0663 FTT1400c hypothetical 1.8E FTL_1503 FTT0720c deoxyguanosinetriphosphate dgt 2.3E triphosphohydrolase FTL_1790 FTT0070c major facilitator superfamily (MFS) tranport ampg 2.2E FTL_0348 NA b pseudogene 1.2E FTL_0569 FTT1542c outer membrane 5.8E FTL_1709 FTT1639c hypothetical 5.4E FTL_0456 FTT0390c 30S ribosomal S21 rpsu 3.4E FTL_0217 FTT0306 fumarate hydratase, Class II fumc 2.1E FTL_0208 FTT0297 hypothetical membrane 4.5E NA b FTT0421 pseudogene 5.7E FTL_1509 NA b D-alanyl-D-alanine dacb2 1.4E carboxypeptidase/d-alanyl-d -alanine-endopeptidase FTL_1603 FTT0461 RNA-binding yhby 1.9E FTL_1601 FTT0463 trna/rrna methyltransferase yibk 2.6E FTL_1156 FTT1715c hypothetical pdpd 4.4E FTL_1171 FTT1700 hypothetical pdpb 1.4E NA b FTT0852 pseudogene 3.1E FTL_1184 FTT1276 macrophage growth locus, subunit B mglb 5.9E FTL_0046 FTT1647c diyroorotate dehydrogenase pyrd 7.1E FTL_0345 FTT0849 bile acid symporter family 1.1E FTL_0680 FTT0563 polyamine transporter, subunit poth 1.0E H, ABC transporter, membrane FTL_0075 FTT1674 riboflavin synthase beta subunit ribh 1.2E (6,7-dimethl-8-ribityllumazine synthase) FTL_0261 FTT0350 DNA-directed RNA polymerase, rpoa2 1.8E alpha subunit FTL_0245 FTT S ribosomal S17 rpsq 1.6E FTL_1185 FTT1275 macrophage growth locus, subunit A mgla 4.2E FTL_1025 FTT1061c 30S ribosomal S18 rpsr 1.2E FTL_0679 FTT0564 polyamine transporter, subunit I, ABC transporter, membrane poti 1.0E FTL_0045 FTT1648c Orotidine 5'-phosphate decarboxylase pyrf 1.6E FTL_1118 FTT1085 hypothetical 1.9E
5 FTL_1868 FTT1727c multidrug resistance, membrane located 2.4E FTL_0685 FTT1259 NH(3)-dependent NAD(+) synthetase nade 1.5E FTL_1190 FTT1270c Chaperone grpe (heat shock family 70 cofactor) grpe 3.6E FTL_0337 NA b pseudogene 2.0E FTL_0843 FTT1120c queuine trnaribosyltransferase. tgt 1.4E FTL_0863 FTT0598c Sodium-dicarboxylate symporter 5.3E family FTL_1478 FTT1317c Inosine-5-monophosphate guab 1.3E dehydrogenase FTL_0382 FTT0881c amino acid permease 1.9E a The open reading frame (ORF) designations are adopted from the annotations of the genomes of the LVS (GenBank Genome Accession No. NC_007880) and Schu S4 strain (GenBank Genome Accession No. NC_006570). b NA denotes the absence of an ortholog in the indicated strain. 5
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