Mechanisms of Evolution in Rickettsia conorii and R. prowazekii Hiroyuki Ogata, Stéphane Audic, Patricia Renesto-Audiffren, Pierre-Edouard Fournier, Valérie Barbe, Delphine Samson, Véronique Roux, Pascale Cossart, Jean Weissenbach, Jean-Michel Claverie, and Didier Raoult

Figure 1

Supplemental Figure 2. (a) Dendrogram of ten Rickettsia species according to the PCR results presented in the Web Table 2. The classification was performed using the unweighted pair-group method with arithmetic mean using the Jaccard-Sneath distance. (b) Phylogenetic tree of citrate synthase genes (gltA) constructed by the neighbor-joining method. Bootstrap percentages are indicated in the tree. Branch lengths are arbitrarily scaled in both trees.

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Supplemental Table 1. Gene families in Rickettsia genomes and their distribution in the other 26 bacterial species.
Organism	ORFs	ATP/ADP translocase (Tlc)	Proline transporter (ProP)	(p)ppGpp/GTP pyrophospho-hydrolase (SpoT/RelA)	AmpG	Sca outer membrane protein
Rickettsia conorii	1374	5	11	4/0	4	5
Rickettsia prowazekii	834	5	7	4/0	3	4
Escherichia coli (K-12 MG1655)	4289	-	1	1/1	1	-
Haemophilus influenzae (Rd KW20)	1709	-	-	1/1	1	-
Xylella fastidiosa (9a5c)	2831	-	-	1/1	1	-
Vibrio cholerae (E1 Tor N16961)	3828	-	-	1/1	1	-
Pseudomonas aeruginosa (PA01)	5565	-	-	1/1	2	-
Buchnera sp. (APS)	575	-	-	-/-	-	-
Pasteurella multocida (PM70)	2014	-	-	1/1	1	-
Neisseria meningitides (MC58 serogroup B)	2025	-	-	1/1	1	-
Helicobacter pylori (26695)	1566	-	1	1/-	-	-
Campylobacter jejuni (NCTC11168)	1634	-	-	1/-	-	-
Mesorhizobium loti (MAFF303099)	6752	-	-	-/1	-	-
Caulobacter crescentus	3737	-	-	1/-	1	-
Bacillus subtilis (168)	4100	-	-	-/1	-	-
Mycoplasma genitalium (G-37)	480	-	-	1/-	-	-
Ureaplasma urealyticum (serovar 3)	611	-	-	1/-	-	-
Lactococcus lactis (subsp. Lactis IL1403)	2266	-	-	-/1	-	-
Streptococcus pyogenes (M1 class I)	1696	-	-	-/1	-	-
Staphylococcus aureus (N315 MRSA)	2612	-	1	-/1	-	-
Mycobacterium tuberculosis (H37Rv)	3918	-	-	-/1	-	-
Chlamydia trachomatis (serovar D)	894	2	-	-/-	-	-
Borrelia burgdorferi (B31)	1637	-	-	-/1	-	-
Treponema pallidum (Nichols)	1031	-	-	-/-	-	-
Synechocystis sp. (PCC6803)	3166	-	-	1/-	-	-
Deinococcus radiodurans (R1)	3102	-	-	-/1	-	-
Aquifex aeolicus (VF5)	1522	-	-	1/-	-	-
Thermotoga maritime (MSB8)	1846	-	-	-/1	-	-

Supplemental Table 2.R. conorii split genes and other genes of interest.
Function	ORF	Size (aa) / Strand	Primer sequence (5'-3'; F: forward; R: reverse)	Expected amplicon size (bp)	RT-PCR amplification *
Split genes found in R. conorii
Folate synthesis bifunctional protein	RC0035	101/-	F TCCTGGAATTGGTTTTGGAA R CTTTCCGATGCTTTGGAATTA	107	+
	RC0036	89/-	F TGCATTCATTAACCGTTCCA R AACGGATTTTCCAAAACCAA	130	+
	RC0037	140/-	F ACCTTTGCAGCAATAGCACA R CCCCTTCATTTATCAAGCGTA	134	+
	RC0038	79/-	F CGCTTAAGCAAGCTATTATTTTTG R GTACTTTTGCCCTGCACCAC	62	+
Ribose-phosphate pyrophosphokinase	RC0125	116/+	F TTTGGTTATGCAAGGCAAGA R GCTATAAGTGCTTAAAAACGGGATA	156	+
	RC0126	55/+	F AGAGGGCAAAAATTGCATGT R GGCAGAAAACACTGCATGA	94	-
Acetate kinase	RC0148	77/+	F TCTTTTTGAAGATTGGTGGAA R AAGGTTGATGCAAGGGACTTT	122	+
	RC0149	83/+	F TTTGCAGGTACTGGCTTTATTTC R CATTTCGTTAAAATGGCTGCT	155	-
	RC0150	122/+	F GGGCTTTAGCGTACTTGACG R TCAGGTGAGTTACTGGCAAGC	157	+
Prolyl endopeptidase	RC0215	88/-	F CATGAGGCGACTCAAGGAAT R AAGTATCGGCACTTCACATGC	160	+
	RC0216	121/-	F TGAATGGACAAAACCGGTAA R CATCCGAGCTTGTAGCTTCC	187	+
	RC0217	53/-	F TGCATCAAGAGGAAGTGACTG R CGGTAAAAGCTTACTGGCTGA	98	-
	RC0218	194/-	F GGATTATAAAAACCCGCAAGG R CATCCTTACCGCCAAATGAC	159	-
Multidrug resistance protein	RC0268	118/+	F GCCGTATTTATGGTCGAAGG R TTCCGCCATATGCAACAATA	142	+
	RC0269	66/+	F CAGGGGGCTTAAGTAGGTCA R CGGCAAACCAAATACTGTAAA	106	-
	RC0270	185/+	F CGTCGGCGATTTAATAATGG R TCGAAACTAACCGCACCTTT	161	+
	RC0271	75/+	F TTGATGAGGCAACAAGTTCG R CCTTTGTGCTGTAGCAAGGTT	159	-
D-alanyl-D-alanine dipeptidase	RC0317	66/+	F TGAGCTTTCTTCCAATATATGCT R TTAAAATTGCTTCCGGTGCT	110	+
	RC0318	53/+	F TTTGATGAAGCTTTACCTCATGATAGG R GCAAGTTGTATAATGCCACCA	88	-
Hypothetical 43.3 kD protein	RC0365	69/-	F TTGAATTAAAAACCGGCAAG R TTTTGGATGCCTTCTATAGCGTA	129	-
	RC0366	124/-	F CATGAAAGGTCAGCGAAAAA R GCCAACAAATGCAAAACCTT	174	-
	RC0367	159/-	F TTAGAGCCCATTTAGCAGCAA R GCCTGTTGGAGAATTTGGTC	188	-
ABC transporter ATP-binding protein	RC0413	65/+	F TGGTGTAGTAGGTGCCAACG R TTTTAAACAACCTATTTTGGAGTTTTT	74	-
	RC0414	69/+	F TTTTCTGCGTTGTCAGAAGG R CTACGGGAAAGAGCAATAATTC	160	-
RND efflux transporter	RC0564	64/-	F ACTATGCCTATTGCGCTTGG R AGGCAAAATTATCGGTGTGATG	88	+
	RC0565	239/-	F TTTCGGGCAATTACCTTCAG R GGCAAGTCCAAGCAATAAGC	153	+
	RC0566	154/-	F GACGCCGCTCAAACTATTTC R TAACGGCGTCAGCTCCTATT	156	+
	RC0567	170/-	F CATATGGGAAAAGGCGAGAG R CGAGATAACTCCCGAAAGCA	158	+
	RC0568	169/-	F AATTTACCGACCGGAGCTTT R CCCGGCTTATCCCTATACCA	160	+
	RC0569	72/-	F TTCAGTTGCATTACCGCTTG R TACAATATGAGGGCGGGGTA	146	-
Cell surface antigen	RC0630	50/+	F AAATTTTTAGCAACGGCATCA R CCTCCTACAAGACCGACACC	79	-
	RC0631	256/+	F GTGTTTTAGCTGCGGGAACT R CCTATATCTCCGGCACTTGG	162	+
Hypothetical protein	RC0641	144/+	F GGTGGGTAGGGGATAATAGCA R CAAAATCCCCCATATGAGCTT	174	-
	RC0642	73/+	F CTTATTACGTCCCGGGCTTT R TCCGCCGCTTGTTAAATAAT	105	-
	RC0643	73/+	F TGCAAAAAGGTAAACGCTCA R AAATCGGTCAATGAAATGCTT	137	-
	RC0644	86/+	F TGGGATGGGGATATAATGCT R ACCTCCGTTAATCGGTCTTG	160	-
	RC0645	78/+	F AATAGCCGGTGATACCGATG R ACCGCTGATTTTGTTCTTGG	116	-
Unknown function	RC0653	82/+	F AAGCATGCCGATGATTTACC R TGCAATTTCTTGAAGGCTTTT	115	+
	RC0654	70/+	F TGCCTGAAACAACAGAGATTTT R TTGCGTTATGCTTTGTATATTCG	146	-
	RC0655	52/+	F GTCAAAACCGGAAATTGCAC R AAATGAAGGTTCGTTAAAAGCA	61	-
Alkaline phosphatase synthesis sensor protein	RC0702	643/+	F ATCGATGGCAGAAGACACAAG R TTGCCATAATAACGCCGACT	162	+
	RC0703	132/+	F TTGGTGAAACCTTATGGGCTA R AATCCTTTCATGTAGCAATTCG	154	+
	RC0704	82/+	F TCGGTGTATTTGTCGTAAGCTC R TTTTCTGCCCAAATTTTTCC	70	+
LPS 1,2-glucosyltransferase	RC0720	60/+	F GCAAGATAAAAATATGCTTTTAGAAACT R TGTAAAACAGTTCCATCTCATCG	56	-
	RC0721	64/+	F TGAGACTCAAAGCCCTTATTTTTC R TTTGTCCTGATTTGTCTTCCA	58	-
Unknown function	RC0774	160/-	F GACCTTGGCAAAGAAGCAGA R TCGTTTAATGCTCCGGTTTT	190	+
	RC0775	206/-	F CGCTTGTTTTTAAGCCGGTA R CAGTTTCCATACCCGTACCAA	160	+
	RC0776	91/-	F GGAGGAGCTAAGGGAGCAAT R GGTGTCCCAAGAGCTACCAC	96	+
	RC0777	53/-	F TGAAGCAAGAGTCACAAACTCC R TCCTGCAATGTTTTTGTTTGA	71	-
3-hydroxyacyl-CoA dehydrogenase	RC0836	112/-	F GTGCAGCCGGATTACTTTACA R TCCTGCTCATAGGCAAGTAGC	233	+
	RC0837	53/-	F AGCTTGTGGTCTTGGTGTGG R AGCAAACATTTCGGTAACACC	110	-
	RC0838	98/-	F GCCGAAAATGACGGTAAAAA R CCACACCAAGACCACAAGC	161	+
	RC0839	70/-	F TTGGTTACAGCTGGAAATATGG R TGGCATAATATTGCGGAAGTG	77	-
	RC0840	52/-	F CGTAAAGGTGACGGTGGATT R CAAGAAGCGCATCAAGATCA	92	-
	RC0841	161/-	F GCCCGTAAAGCGATAAGTCA R TTGCAGGAAGAGAGGCAAGT	123	+
	RC0842	154/-	F TGTTATTGGTGCGGGAGTAA R AAAGGTGGAGGTGGAGGTTT	128	+
Competence protein F	RC0843	121/-	F TGGAAATTGCAAAAGTTACCG R AGCAGTGGTAATGACATCATCG	148	+
	RC0844	115/-	F GTTTAAGCGGTGCGGAAATA R AATAAGCTGCGAGCAAACTCA	148	+
Methylated-DNA--protein-cysteine methyltransferase	RC0867	58/-	F GCTGTTGCAAATGCTAATGG R CGATGTAATCCCCTGCCATA	67	-
	RC0868	193/-	F CGTTGCAAGCTTCTTTCACA R CAATACCCATACGCCTTGCT	164	+
	RC0869	77/-	F AGACCGACTTGTCCTGTACG R CACCAAGGATGTGAAAGAAGC	84	-
Proline/betaine transporter	RC0889	113/-	F TGGTAGCCCTTATGTCTCCTATC R TGCAAACATTAAAGCTCGTGA	153	-
	RC0890	170/-	F GTCGTAAGGCAACGGTCATT R ACCACCCAATAACGTTGAGG	178	+
Succinyl-CoA:3-ketoacid-coenzyme A transferase	RC0904	75/+	F TCGGTCTATGCGGTATTCCT R TCTCCGACATACGAAGAAATCA	121	-
	RC0905	120/+	F TGGGAATTCCGGCTTTTTAT R CACCTGCCATGACTGCATTA	167	+
Tryptophan repressor binding protein	RC0907	57/+	F TGGGTAGTTTAGCCAGATCGTT R AATGCTGCATAGCGAAGTGA	125	-
			(Alternative primer set) F CCTCAACTAGATGGGCACAA R AGCTGCTGAATACTGCAAACTT	60	-
	RC0908	75/+	F TGCAGCATTGTATGATTTGG R GCAGTTTTTAAATCGCCTTGA	134	-
			(Alternative primer set) F GCTGAAACTGCTCCGAATTT R TCAGGAGAAGATTTGTGGTTAGA	70	-
Bicyclomycin resistance protein	RC0923	122/-	F TGCAATCGCTATGATATGTGC R CTTATCGTTGGGCCGTGTAT	157	+
	RC0924	106/-	F TGCAAGCTGGCATTATGTTT R ATGCACCGATGATAAAAGCA	134	+
	RC0925	64/-	F TGAAAATTATTGCGAAGATTCC R TGCCGAGTGATTAATATACCAAGC	144	+
Mannose-1-phosphate guanylyltransferase	RC1039	209/+	F GGCCTTTGGCATCTAAGGAT R TAATGGCACAAACGGCAGTA	182	+
	RC1040	175/+	F AGCAATGAACCTTCAACCTGA R ACCAAGATCGTTCCAAGCAA	155	-
Unknown function	RC1042	67/-	F GGTAGGGCAAACAAAAGCTG R TGCCTTAAGTTTAGATTGCTTGAA	152	-
	RC1043	197/-	F GAATGGTTCTGCCAAATGGT R CCGGCATATTCATACCTGCT	162	+
Alpha-(1,3)-fucosyltransferase	RC1058	97/+	F TGGCCGGCTTTACAATAGAC R AGCTTTGGTCTAACCGATTCA	143	+
	RC1059	183/+	F ACGGCTGCATAGCAAGAAAT R TTGCAGTAACATACCCCTCAGA	144	+
Unknown function	RC1061	113/-	F AATGTCGGCAAGGCAATTAG R TTGCTGCAAAATAGCAAATCA	147	+
	RC1062	147/-	F TGTACCGGCTCAAGCAATTTA R TGTGGCATTTGCCGAACATA	182	-
	RC1063	190/-	F TTGGTTAACCGAAAAATCAGC R AAGACCGTACCGGGAAAGAT	156	+
ppGpp 3-pyrophosphohydrolase	RC1098	99/+	F TGGTTCACAAATGCGTCAGT R TAAAAAGCTTAGGCGCTTCG	64	+
	RC1099	57/+	F CTGAAAAGCTGATCATCGAAA R GCTTTCCTCGACACTGATTTG	59	-
Proline/betaine transporter	RC1100	58/-	F TGCTGTTTCTAGAGCCTTAATGTG R CCTGTTTCTTTTTCCAACTTTTG	112	-
	RC1101	77/-	F TGCAGCTCAGGTTATTCATCA R TTAAAAGGTATGGTGCAAATAGAA	115	-
	RC1102	75/-	F GGATTTAATTGGCGTCTTGC R TTTTTGTCGATATTTGCTTTTTCA	105	+
Acetyl-CoA acetyltransferase	RC1134	254/-	F TTACAGCGGGTAATGCTTCC R TTAACGCTCCAACCTGCTTT	162	+
	RC1135	106/-	F TTCGGCTCGTTTATGGGTAG R CGGCACTTCTTTCGGTATTC	161	+
Poly-betahydroxybutyrate polymerase	RC1136	151/-	F CCCGTGGGATTTTTCTCATT R TTCTTGCTCCTTATCGGAAGTT	148	+
	RC1137	87/-	F CCGTGGTTTGTCCAGAGATT R TCACCGTAGCTTCTCAAAGTTTC	167	+
Hypothetical protein	RC1190	63/-	F CACCGAGCATTACAAAATCAA R TCGCAAGACCTATTGCCTGT	85	-
	RC1191	100/-	F ATCCGTATTTTGCAGCCTTG R AAATCCTTCCCATGATGCAC	122	-
	RC1192	60/-	F CGGCGGATTTCCTTTATCAT R TCACTGCATAGACGGAATTG	90	-
	RC1193	84/-	F GACGTTATGTTTCTAATTTTCATATCG R AAAGCAAGCATCGGTTCATT	68	-
-lactamase	RC1243	100/-	F GATTGTTGGAAGCGTTACGG R AGAACGTTGCCCTGCATAAG	146	+
	RC1244	90/-	F CTCGTTATGCTCCATGCTCA R GGTGTTTGGGATTGTTTCCA	108	+
DNA repair protein	RC1277	103/+	F TTTTCGGCTATCCCTCGTAA R AGCTCACGTAGAGCAAAATTGA	113	+
	RC1278	122/+	F GCAATATGCGACTCGAACAA R TTGGCTTTTGAAGGTTCAGG	171	+
Proline/betaine transporter	RC1321	163/-	F GGGCGACGTCCTGTACTAAT R TGAAAGTGCAACACCGGTAA	160	+
	RC1322	152/-	F TGTTAGTATGTGCGGCGGTA R ACGCCTTGTTCCCTTAGCTT	159	-
	RC1323	62/-	F ATCGGCAACGCTTTAGAATG R CCGAGCGGTCGTACTACAAA	104	-
Na(+)/H(+) antiporter	RC1354	65/+	F TTTTTCAGCCGACATATATCTTTA R TGCCGGAGTATTGATTGTTTT	78	+
	RC1355	146/+	F AAGGCATTTGTTCCAACTCAA R GCAGCTGCTCTCAAACGTAA	191	+
Oxidoreductase	RC1363	66/+	F GAAAGAACTTGGGGCAAGG R TGCTCCCATTTATCTTCAGGA	140	-
	RC1364	86/+	F CGGTTACGTCAACACACCAT R GCCGCCATCTATCAAGAGTC	171	+
O-linked GlcNAc transferase	RC1366	99/-	F AAGGCAATCGAAGTAGATCCAA R TTCGTAATTTCCAAGTTCATCG	139	+
	RC1367	91/-	F AAAACAAAATCCGAATGATGC R GCTAAATCGTATTTTCCCAACTG	146	+
Genes absent in R. prowazekii
ppGpp 3-pyrophosphohydrolase	RC0374	177/+	F CTTGCGTTTACGCTCAAAAA R GCGTCTTCGGCTACAAACTC	165	+
ppGpp 3-pyrophosphohydrolase	RC0426	235/+	F AAGGCATGTAGCGGGTCTAA R TGAGCTTTTTCAGGCGATTT	128	-
Similar to lysozyme	RC0727	59/+	F CAAATCGCACCGCTAAAGTT R CATGCAAATCACCGGAAAAT	125	-
	RC0727		(Alternative primer set) F CAGTGAAATCAGGAGATGCTTT R CCCAGCCTACTGCGATATGA	72	-
Similar to N-terminal of biotin-protein ligase	RC0781	250/+	F GGACCATTGCTTGCTCCTTA R CCGTCGCCGTAAGTACATTT	178	+
	RC0781		(Alternative primer set) F ATTGCAAGGCAAAGGAAATG R CGGCTCTTGCTCCTTTATGT	184	+
Similar to type I restriction enzyme restriction subunit	RC0873	164/-	F TGCTTGCTCAAACAAAGGAA R GTCCCGGTCATCAGTAGCAG	160	+
	RC0873		(Alternative primer set) F AGATTAATTGCCGTTCGTTTT R TTTCAGGATCATCGGAACTTTT	171	+
Type I restriction enzyme	RC0873	164/-	F TGCTTGCTCAAATAGCAAAGGAA R GTCCCGGTCATCAGTAGCAG	160	+
ppGpp 3-pyrophosphohydrolase	RC0888	150/+	F ATTATTCGCACCCGATTGAG R GTTTTGCTACTTCCGGTCCA	140	+
Similar to WASP, N-WASP, MENA proteins	RC0909	520/+	F TCCACCACCGTCTCCTTAC R ACGTGGTTGATTTGCCAGTT	165	+
Similar to aminoglycoside 3'-phosphotransferase	RC0947	192/-	F TAACTTTTGGCGTGGAGGTG R CCTCCAAAATCGCTTACTGC	182	+
	RC0947		(Alternative primer set) F TGGGTATTGGTGATCCTGCT R CCAAACCATCTAGTGGAGCAA	124	+
Similar to lipopolysaccharide biosynthesis protein	RC1060	105/+	F AAGCATTTACCTACCGACTGG R CAAGAAGTTTTTGAGCAGCTTTT	128	-
	RC1060		(Alternative primer set) F TTGTCCTTGTCACATTCTAAAAACA R TGCATAAAAGCTTGGTACATTATC	143	+
Similar to lysozyme	RC1298	67/-	F GTGCCATTAACCGAAAACCA R CGTTTCACTAAGCCTTGCAG	145	-
	RC1298		(Alternative primer set) F AAGCCGCCTTAATCTCGTTT R AGACCGCCTTTAGCACACAT	99	-

* '+' for positive and '-' for negative RT-PCR results

Supplemental Table 3. PCR validation of R. conorii or R. prowazekii supplementary genes in other Rickettsia species.
Function	ORF	Primer sequence (5'-3'; F: forward; R: reverse)	Expected product size (bp)	Species exhibiting PCR products*
R. conorii
Acetylgultamate kinase (argB)	RC0139	F AAGCTAGTTTGGCAAAATTGC R ATACCTGTAATATCTCATATG	706	AU#, FE, HE, MA, RI
tRNA/rRNA methyltransferase	RC0160	F CTTAAATGAGCTTAGAATCG R AATGTTATGTATATCCTCTC	412	AK, AU, CA, FE, HE, MA, RI
Thermostable carboxypeptidase	RC0228	F GAATTTGCAACCATCTCAC R TTCTGCTCATATAAAGCATG	791	AK, AU, RI
Succinate semialdehyde dehydrogenase (gabD)	RC0621	F ACGGTGAATTTATAGAGTTC R GTAACAATTTTATGAGTGTTAC	373	FE, MA, RI
Methylated-DNA--protein-cystein methyltransferase (dat)	RC0870	F TTCTATGCTGGCAATTAGTG R GATAAGCCATTTTTTGTGATC	445	AU, CA, FE, HE, RI
Succinyl-CoA:3-ketoacid-coenzyme A transferase subunit B (scoB)	RC0906	F CAAATAACCGCAGAGGAAC R TATCAATATCAAAAATTCCAAG	517	AK#, AU, FE, HE#, MA#, RI
Magnesium and cobalt transport protein (corA)	RC0943	F GTTCTATAGTAAAAAATGCAG R GTAAATACTAGCTATTAAAGTG	810	AU, FE#, HE#, MA, RI
R. prowazekii
Unknown function	RP169	F TTCACAGGAGTTTAAATATG R TTCCTGGTTTACAAAATATCTTACC	235	FE
Unknown function	RP337	F TGCAGGGTTAGTATTGAATC R ATAAAACTGCCCTGCTATAC	609	TY
Unknown function	RP338	F TAATAGCCGTATAGTTTGGG R GATAATAGACCGGCACATTC	783	-
Similar to mannosyltransferase	RP340	F GGAAAAGTCGAAAATAGTCC R TGATTTTTATGAGCCTCACC	563	TY
Similar to ankyrin, brain variant 2 (ank2)	RP714	F TTAACACCACCTTTATCACC R GTGGCTAACTCAAAATTAGG	229	-
Unknown function	RP716	F GCTTGGATTCATCAAGATAG R CCTATACTATCAAGGTGTTC	602	-
Poly-beta-hydroxybutyrate polymerase (phbC2)	RP820	F TAAAGATGCTTTATGGGAGG R AAAGCCTTTCTTTGATAGGG	572	AK, AU, TY

* Species abbreviations are as follows. TY: R. typhi; CA: R. canadensis, HE: R. helvetica, FE: "R. felis", AU: R. australis, AK: R. akari, RI: R. rickettsii, MA: R. massiliae. Species whose PCR products exhibited internal stop codons are indicated by '#'.