Determination of Coxiella burnetii rpoB sequence and its use for phylogenetic analysis

Gene 207 (1998) 97–103

Determination of Coxiella burnetii rpoB sequence and its use for phylogenetic analysis Christophe Mollet, Michel Drancourt, Didier Raoult * ´ ´ ´ ´ Unite des Rickettsies, CNRS UPRES-A 6020, 27 Boulevard Jean Moulin, Universite de la Mediterranee, 13385 Marseille, France Received 21 July 1997; received in revised form 7 October 1997; accepted 8 October 1997; Received by A.M. Campbell

Abstract The nucleotide sequence of the rpoB, encoding the b-subunit of RNA polymerase of the obligate intracellular bacterium Coxiella burnetii, was determined using a polymerase chain reaction amplification and direct sequencing methodology. Comparison between C. burnetii and other eubacterial rpoB sequences indicated sequence similarity ranging from 53.6% to 67.6%. Coxiella burnetii rpoB consists of 4128 base pairs with a 45.3% GC content encoding 1375 amino acids with a calculated molecular mass of 153.67 kDa. Comparison of 512 bases of the rpoB variable region I, from eight C. burnetii strains isolated from various sources, revealed fewer than four base differences, although the distribution of these did not correlate with previously determined genotypic groupings with the species. Phylogenetic analysis of C. burnetii based on comparison of its rpoB sequence with sequences available for other bacteria is consistent with those previously derived from 16S rRNA gene sequence, and indicate that C. burnetii belongs to the c-group of Proteobacteria. Furthermore, phylogeny inferred from comparison of RpoB, or homologous sequences including Archae, Bacteria and Eukarya, concurred with these results. © 1998 Elsevier Science B.V. Keywords: c-Proteobacteria; RNA polymerase b-subunit; Nucleotide sequence; Phylogeny; Rifampicin; Q fever

1. Introduction Coxiella burnetii, the aetiological agent of Q fever, is an obligate intracellular bacterium recovered world-wide from various sources, including acarians, birds and mammals (Raoult and Saltzman, 1994). Phenotypic differences between strains (Stein and Raoult, 1992a) and genotypic characterization using pulsed field gel electrophoresis have led to the C. burnetii strains being divided into six genotypes (Heinzen et al., 1990). A tentative correlation between genotype, plasmid content and acute or chronic clinical form of Q fever has been proposed, but inconsistent data were obtained when larger collections of human isolates were analysed (Stein and Raoult, 1993; Thiele and Willems, 1994). 16S rRNA sequence analysis of six C. burnetii strains indicated the species to be extremely homogeneous (Stein et al., 1993). * Corresponding author. Tel. +33 4 91324375; Fax: +33 4 91830390; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); kb, kilobase(s); ORF, open reading frame; PCR, polymerase chain reaction; RBS, ribosome binding site; RNAP, RNA polymerase; RpoB, b-subunit of RNA polymerase; rRNA, ribosomal RNA.. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 6 18 - 5

The rpoB encodes the b-subunit of the RNAP, an enzymatic complex conserved among Bacteria (Ovchinnikov et al., 1981) and Archae ( Klenk and Zillig, 1994). Comparison of rpoB sequences has previously been used for phylogenetic inferences among Archae ¨ (Puhler et al., 1989a,b; Klenk and Zillig, 1994) and some Bacteria (Rowland et al., 1992). In this study, PCR amplification and direct sequencing of C. burnetii rpoB in eight isolates, including representatives of different genotypic groups, were used to assess the evolutionary homogeneity of the species and its overall phylogenetic position within the Proteobacteria.

2. Materials and methods 2.1. Amplification and sequencing of the C. burnetii Q 212 strain rpoB The C. burnetii reference strain Q212 (human Q fever endocarditis isolate, Canada, 1981) was co-cultivated at 35°C for 5 days with L929 cells. The absence of contamination by Mycoplasma species was verified using the Mycoplasma Detection Kit (Boehringer Mannheim,

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Table 1 Codon usage in Coxiella burnetii 40 coding sequences and rpoB 40 C. burnetii coding sequence

rpoB coding sequence

Amino acid

Number of amino acid

Codon

Number of codon

Amino acid

Ala

1087

95

793

Arg

96

Asn

502

Asn

50

Asp

652

Asp

97

Cys

159

Cys

7

Gln

588

Gln

38

Glu

953

Glu

116

Gly

934

Gly

102

His

284

His

23

Ile

836

Ile

89

Leu

1278

Leu

133

Lys

918

Lys

97

Met Phe

334 482

Met Phe

29 40

Pro

576

Pro

55

Ser

655

Ser

70

Thr

627

Thr

81

Trp Tyr

133 409

Trp Tyr

5 41

Val

892

Val

111

Stop

40

305 287 181 314 204 230 173 111 52 23 346 156 462 190 93 66 453 135 749 204 291 327 192 124 181 103 518 227 91 506 112 167 102 82 109 760 158 334 350 132 146 165 104 161 141 49 108 148 92 117 135 184 107 201 133 253 156 303 222 144 223 23 12 5

Ala

Arg

GCT GCC GCA GCG CGT CGC CGA CGG AGA AGG AAT AAC GAT GAC TGT TGC CAA CAG GAA GAG GGT GGC GGA GGG CAT CAC ATT ATC ATA TTA TTG CTT CTC CTA CTG AAA AAG ATG TTT TTC CCT CCC CCA CCG TCT TCC TCA TCG AGT AGC ACT ACC ACA ACG TGG TAT TAC GTT GTC GTA GTG TAA TGA TAG

Stop

Number of amino acid

1

x2 test Codon

Number of codon

p-Value

GCT GCC GCA GCG CGT CGC CGA CGG AGA AGG AAT AAC GAT GAC TGT TGC CAA CAG GAA GAG GGT GGC GGA GGG CAT CAC ATT ATC ATA TTA TTG CTT CTC CTA CTG AAA AAG ATG TTT TTC CCT CCC CCA CCG TCT TCC TCA TCG AGT AGC ACT ACC ACA ACG TGG TAT TAC GTT GTC GTA GTG TAA TGA TAG

28 22 15 30 22 30 20 19 3 2 29 21 71 26 5 2 25 13 92 24 37 24 27 14 12 11 55 31 3 50 40 13 11 4 15 84 13 29 30 10 17 17 3 18 11 16 13 15 12 3 14 30 8 29 5 26 15 32 31 22 26 0 1 0

0.88

0.51

0.11 0.63 0.49 0.11 0.86 0.11

0.27 0.046

0.30

0.34

0.74 0.12

0.0001

0.20

0.85 0.56

C. Mollet et al. / Gene 207 (1998) 97–103

Meylan, France). Infected cells were lysed in SDS/proteinase K. The origin of DNA extracted using a phenol/chloroform standard procedure (Sambrook et al., 1989) was confirmed by PCR amplification of the C. burnetii superoxide dismutase gene (Stein and Raoult, 1992b) and sequence analysis of the 16S rRNA gene ( Weisburg et al., 1989). The GenBank/EMBL accession numbers of the rplL, rpoB and rpoC used in this study were: E. coli V00339 and V00340, Brucella melitensis L27819, Buchnera aphidicola Z11913, Neisseria meningitidis Z54353 and Pseudomonas putida X16538. Primer CM100b, 5∞-CTGGGTCTGAAAGAAGCTAAAGAC-3∞, was designed by reference to an alignment of the rplL sequences of E. coli and B. melitensis. Primer BEGA-R1, 5∞-ACCGGTIGAACGCGCGTGCAT-3∞, was designed by reference to an alignment of rpoB sequences of Escherichia coli, Salmonella enterica typhimurium, P. putida and N. meningitidis. Primer RPOC130-R, 5∞-GTTTCIGGITTTTTAACTTCACC-3∞, was designed by reference to an alignment of rpoC sequences of E. coli, B. aphidicola and P. putida. Inosine was introduced into primers to resolve ambiguous base positions ( Knoth et al., 1988). The sequence of amplification primers specific for C. burnetii rpoB was determined as new regions of sequence were obtained. Cbu2300-D, 5∞-ACCTGCATTAATCAGCATCC-3∞, was chosen to pair with the RPOC130-R. PCR mixes incorporated final concentrations of 10−2 U ml−1 Gold Star Taq polymerase, 1× Taq buffer, 1.5 mM MgCl , 200 mM of each 2 dNTP and 0.2 mM of each primer ( Eurogentec, Seraing, Belgium). The following thermal programmes were used: for CM100b/BEGA-R1, 30 cycles consisting of denaturation of 94°C for 10 s, primer-annealing and extension at 68°C for 3 min, in which the annealing-extension time was increased by 20 s per cycle for the final 20 cycles; for Cbu2300-D/RPOC130-R, 35 cycles consisting of denaturation of 94°C for 10 s, primer-annealing at 55°C for 20 s and extension at 72°C for 50 s. Both programmes included a pre-denaturation step of 94°C for 90 s and a final elongation step of 72°C for 5 min. Sterile distilled water was used as a negative control. Amplicons, visualized after electrophoresis through an ethidium bromidestained 0.8% agarose gel, were purified using a QIAquik Spin PCR Purification Kit (QIAGEN Gmbh, Hilden, Germany) following the protocol of the supplier. Initial sequencing of PCR products used 5∞-end fluoresceinlabelled primers (Eurogentec) identical to those used for gene amplification (Mollet et al., 1995). Subsequently, primers were chosen by reference to the newly obtained sequences. Sequencing reactions used the reagents of the AmpliCycle Sequencing Kit (Perkin Elmer Cetus, Norwalk, CT, USA) according to the manufacturer’s instructions and using the following programme: predenaturation of 94°C for 90 s followed by 30 cycles of denaturation at 94°C for 15 s, primer-annealing temper-

99

ature depending on primer for 30 s and extension at 72°C for 50 s. A final post-elongation step at 72°C for 5 min completed the cycle. Products of sequencing reactions were resolved by electrophoresis in a 0.35 mm 6% polyacrylamide denaturing gel and recorded using an LKB ALF–DNA Sequencer (Pharmacia Biotech, Uppsala, Sweden) following the standard protocol of the supplier. The results obtained were processed into sequence data by the ALF Manager software (Pharmacia Biotech). Each base position was established at least three times in both the forward and reverse directions. Partial sequences were combined into a single consensus sequence using PC Gene software (Intel ligenetics, CA, USA). 2.2. Coxiella burnetii rpoB intraspecies variability study The reference strains of C. burnetii Q212, Nine Mile (a tick isolate, USA, 1935), Henzerling (human acute Q fever isolate, Italy, 1945), Priscilla (goat isolate, USA, 1980), and clinical strains isolated in our laboratory: MAC 12 (human acute Q fever isolate, France, 1991), MEI 2, ME 5 and ME 6 (human Q fever endocarditis isolates, France, 1989–1990) (Stein et al., 1993), were grown in L929 cells and the DNA was prepared as described before. Cbu1400D 5∞-CCGCGTGGGTCTTGTGCGGG-3∞ and Cbu2200R 5∞-CGATTCGCGAAGCGTCAACGG-3∞ primers were chosen, from the C. burnetii Q212 rpoB sequence obtained, to amplify a 806 bp region of the rpoB from all seven strains. After purification, a 512 bp fraqment of this product was sequenced as described above. 2.3. Sequence analysis of rpoB and deduced amino acid sequence Codon usage, computational translations and pairwise sequence comparisons for nucleic or peptidic sequence homology were determined using the PC Gene program. Secondary structure predictions were made by the MFOLD program (Zuker, 1989). Sequence multiple alignments were made using CLUSTAL (Higgins and Sharp, 1988) supported within the Bisance workstation (Dessen et al., 1990). Phylogenetic inferences derived from multiple alignments of 16S rRNA gene, rpoB and RpoB sequences used programs within PHYLIP (Farris, 1989; Felsenstein, 1993), again supported within the Bisance workstation. Bootstrap samples (100) were generated by SEQBOOT. Distance matrices were derived from nucleic alignments using DNADIST under the assumptions of Jukes and Cantor or Kimura algorithms and from peptidic alignments using PROTDIST under the assumptions of Dayhoff or Kimura algorithms. Branching processes were inferred from these matrices by the neighbour-joining method. Parsimony analysis was achieved using DNAPARS and PROTPARS for

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entire C. burnetii rpoB in two fragments, F1 (4132 bp) and F2 (2169 bp), extending from the rplL beyond the 5∞-terminal of rpoB to the rpoC beyond the 3∞-terminal of rpoB in E. coli and resulting in a C. burnetii rpoB ORF of 4128 bp. No amplification was obtained from negative controls. A partial ORF, found at the 5∞-end of the total sequence, was 68.5% similar to the E. coli rplL. The rpoB start codon was preceded by the purinerich sequence TGAGGTG at position 270, similar to the typical consensus RBS ( TGAGGAG). Downstream of the rpoB termination codon, a 392 bp region was found, which comprised a 126 bp coding region sharing 58.1% similarity with the E. coli rpoC. The entire amplification product had a GC content of 45.3%, consistent with the 43% GC content reported for the entire genome of C. burnetii (Schramek, 1968). The rpoB ORF of 4128 bp was translated in a polypeptide of 1375 aa with a calculated mol. mass of 153.671 kDa and a theoretical isoelectric point of 5.7. Codon use in C. burnetii rpoB was compared with that of 40 other C. burnetii coding sequences ( Table 1) and the overall usage was found to be similar, with the exception of those coding isoleucine and serine. Within the rpoB sequence, Ile was coded for by ATC markedly more often than by ATA, and Ser was coded for by TCC markedly more often than by AGC. Codon use in C. burnetii is similar to that reported for Mycoplasma capricolum (Osawa et al., 1992) and Rickettsia prowazekii (Anderson and Sharp, 1996), and could be correlated to the low GC content or the specific metabolism of these epicellular or intracellular pathogens.

Fig. 1. Potential RNA secondary structures predicted by MFOLD program within C. burnetii rpL–rpoB intergenic space numbers referring to nucleotide positions in U86688, showing a 53-bp translational attenuator structure similar to that reported for Gram-negative bacteria downstream of the rpIL coding sequence.

nucleic and peptidic alignments, respectively. Maximum likelihood analysis was achieved using DNAMLK with a molecular clock from nucleic alignments. A consensus tree derived from bootstrap samples was obtained using CONSENSE. 3. Results and discussion 3.1. Amplification, sequencing and analysis of the C. burnetii Q 212 strain rpoB The primer pairs CM100b/BEGA-R1 and Cbu2300-D/RPOC130-R allowed amplification of the

Table 2 Bacterial and archaebacterial rpoB sequences pairwise similarity (upper triangular matrix) and their deduced RpoB sequences ( lower triangular matrix) x

Bap

Bap Bbu Bsu Cbu Cpa Eco Hin Mle Msm Mtu Nme Ppu Sac Sau Sty Tac Tce Tma

48.08 49.79 68.11 44.51 83.61 77.12 48.43 47.31 49.28 60.80 67.88 18.03 50.85 83.61 15.56 18.54 41.65

Bbu

Bsu

Cbu

Cpa

Eco

Hin

Mle

Msm

Mtu

Nme

Ppu

Sac

Sau

Sty

Tac

Tce

Tma

63.56

58.96 60.27

66.87 63.09 62.02

64.64 61.65 60.66 63.22

67.76 59.52 64.02 67.54 59.03

72.03 62.28 61.61 66.91 64.58 73.05

53.19 53.33 59.41 59.92 54.44 62.80 57.43

51.65 51.53 59.32 58.58 51.27 63.65 56.50 83.53

52.24 52.31 59.42 59.20 52.23 63.61 56.42 86.30 85.30

62.57 59.66 62.06 65.02 60.78 67.46 65.77 62.74 61.74 61.57

59.30 57.13 60.39 64.78 57.13 73.81 64.46 63.98 64.30 64.94 66.64

59.07 53.00 52.91 57.56 54.05 55.49 58.30 47.91 46.32 47.56 57.26 54.78

62.98 60.91 73.12 63.12 64.82 61.40 64.92 56.98 55.07 55.57 63.29 58.64 55.13

67.21 59.14 60.80 67.56 57.43 93.65 72.45 62.88 63.62 62.37 67.59 73.37 54.72 60.36

52.01 49.67 50.42 54.54 49.64 55.66 54.15 50.62 51.28 51.33 56.22 55.46 57.91 50.77 55.60

52.92 46.30 51.71 56.75 44.69 58.27 56.16 52.75 53.07 54.67 58.71 60.40 52.24 51.26 57.61 62.51

56.83 60.39 60.33 57.46 60.14 58.31 55.70 58.45 58.03 58.21 56.94 57.86 53.62 58.78 58.15 52.12 54.62

48.34 50.87 44.06 49.13 49.39 46.34 44.59 46.16 47.12 47.91 17.14 47.29 49.48 14.31 17.11 41.54

51.63 51.68 51.47 50.38 56.49 56.03 56.33 52.14 50.13 18.03 79.27 51.47 18.52 21.39 45.85

46.24 70.27 67.09 49.36 49.62 49.96 62.55 69.64 19.63 52.03 70.04 16.90 20.50 41.73

46.33 45.06 47.96 47.96 48.05 41.70 46.06 18.13 52.13 45.78 16.32 17.59 45.24

83.16 50.30 49.70 50.72 64.38 71.16 19.63 52.28 98.51 16.40 22.37 42.52

48.01 50.30 49.70 62.10 68.43 17.94 52.28 83.38 14.39 19.07 41.57

88.79 93.29 46.48 47.92 16.61 56.06 49.87 15.10 16.93 43.85

89.99 47.48 47.73 17.58 56.80 50.38 12.75 15.42 43.54

49.19 47.83 18.21 56.16 49.87 15.46 17.02 44.01

62.86 19.01 51.35 64.23 16.74 19.79 40.30

19.54 49.58 71.09 16.15 18.18 42.04

17.58 18.47 49.02 53.30 17.67

51.86 19.12 20.94 46.36

15.90 21.21 42.60

53.74 15.73

18.36

Bap, Buchnera aphidicola, Z11913; Bbu, Borrelia burgdorferi, X71024; Bsu, Bacillus subtilis, D83789; Cbu, Coxiella burnetii, U86688; Cpa, Cyanophora paradoxa, U30821; Eco, Escherichia coli, V00339; Hin, Haemophilus influenzae, U68759; Mle, Mycobacterium leprae, Z14314; Msm, Mycobacterium smegmatis, U24494; Mtu, Mycobacterium tuberculosis, L27989; Nme, Neisseria meningitidis, Z54353; Ppu, Pseudomonas putida, X15849; Sac, Sulfolobus acidocaldarius, U56904; Sau, Staphylococcus aureus, X64172; Sty, Salmonella enterica typhimurium, X13854; Tac, Thermoplasma acidophilum, U02635; Tce, Thermococcus celer, X73633; Tma, Thermotoga maritima, X61562.

C. Mollet et al. / Gene 207 (1998) 97–103

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Fig. 2. Phylogenetic tree representation inferred by parsimony method from 43 available RpoB sequences, or homologous, multiple alignment.

3.2. Organization of the C. burnetii b-gene cluster The order rplL–rpoB–rpoC observed for C. burnetii probably represents part of the rplJ–rplL–rpoB–rpoC organization which has been previously described for

the rpoBC operon in other Eubacteria ( Tittawella, 1984; Aboshkiwa et al., 1992; Engel et al., 1990; Alekshun et al., 1997). The rplL–rpoB intergenic spacer of C. burnetii was 152 bp long, a size intermediate to that of Borrelia burgdorferi (82 bp) and E. coli (321 bp). A

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C. Mollet et al. / Gene 207 (1998) 97–103

translational attenuation site may exist within this intergenic space. The MFOLD program predicted two hairpins of 11 and 8 bp, with the first being closed by a stable GNRA tetra-loop ( Woese et al., 1990) ( Fig. 1), creating a local secondary structure similar to those previously described for the E. coli and B. burgdorferi attenuators (Barry et al., 1980; Ralling and Linn, 1987; Alekshun et al., 1997). Furthermore, a highly structured region with four stems of ≥7 bp was predicted overlapping the 5∞ end of rpoB (positions 246–374) (data not shown), which could form a RNase III processing site. 3.3. Coxiella burnetii rpoB intraspecies variability study Comparison of a 512 bp rpoB region sequence in eight C. burnetii strains showed position 42 to be a G for the Q 212 strain and A for all others; position 319 to be an A for the Priscilla strain provoking LeuMet and T for all others; and position 377 to be an A for the Q 212, Nine Mile and MAC 12 strains, and G for the Henzerling, MEI 2, ME 5, ME 6 and Priscilla strains provoking GluGly. These mutations do not dramatically affect the secondary structure of RpoB. Four strains, MEI 2, M 5, M 6 and Henzerling, had an indistinguishable sequence, as did the MAC 12 and Nine Mile strains. The 0.6% divergence observed among the strains confirms their phylogenetic homogeneity, as previously demonstrated when studying the 16S rRNA (Stein et al., 1993). These data do not support the tentative division of C. burnetii species based on plasmid content or macrorestriction analysis (Heinzen et al., 1990). Variation in this 512 bp rpoB region has been reported to influence rifampicin resistance ( Telenti et al., ´ 1993; Honore and Cole, 1993). However, variation in susceptibility to rifampin between C. burnetii Priscilla strain and Nine Mile strain ( Yeaman et al., 1989) was not confirmed in the shell-vial system ( Raoult et al., 1991) and the present study did not indicate any molecular basis to differences in susceptibility to rifampicin among C. burnetii strains, although other mechanisms of resistance to rifampicin cannot be excluded ( Yazawa et al., 1993) 3.4. Comparison of the C. burnetii rpoB sequence and phylogeny When compared with rpoB sequences available for other Bacteria and Archae, that of C. burnetii was found to be most similar to those of other Proteobacteria (64.78 to 67.6%). Lower similarities were found with other groups ( Table 2). As nucleotide inferrences are of limited sensitivity due to the small number of rpoB sequences available, topographies were confirmed by peptidic sequences analysis. Indeed, broad-spectrum phylogenetic studies based on comparison of genes encoding highly conserved proteins (such as RpoB) are

subject to errors due to the GC% bias, the inherent ambiguity of nucleotide alignments, and the fact that the reading frame is not taken into account in the alignment process. Much more reliable alignments can be produced by using protein sequences. Similarity values of 62.55–70.27% were found with other Proteobacteria RpoB sequences, whereas markedly lower similarities were found with members of other taxa ( Table 2). a protein multiple alignment was derived as a basis for inferring protein-based phylogenies. Neighbour-joining methods, whatever the distance algorithm used, and parsimony analysis resulted in reconstructions similar to nucleic acid-based phylogenetic topologies with significant bootstrap values at all nodes, except those within the c-subdivision of the Proteobacteria (data not shown). Furthermore, a largescale protein-based phylogeny including 43 bacterial RpoB sequences and homologous sequences for chloroplasts, Archae and Eukarya RNAP II ( Fig. 2) also supported a phylogenetic position of C. burnetii closely related to N. meningitidis ( b-subdivision) on a branch derived from the node which also supports P. putida (csubdivision) in the Proteobacteria.

4. Conclusions (1) Coxiella burnetii rpoB has been amplified and sequenced using consensus inosine-containing primers. The gene encodes an RNAP b-subunit of 1375 aa. (2) The organization of partial b-gene cluster comprises rplL–rpoB–rpoC. (3) A putative translational attenuation site, similar to that found in other bacteria, exists in the rplL–rpoB intergenic spacer. This suggests that the synthesis of the C. burnetii b- and b∞-subunits may be regulated at the translational level. (4) Phylogenetic analysis indicates that C. burnetii is a highly homogeneous species and does not support the existence of previously defined subgroups of strains based either on genotypic heterogeneity or on susceptibility to rifampicin. (5) Phylogenetic analysis based on nucleotide and peptidic sequence analysis indicates that C. burnetii is a member of the Proteobacteria, although its precise position within either the b- or c-subgroup is not clearly demonstrated.

Acknowledgement This work was made possible thanks to the funding ´ of BioMerieux. The authors acknowledge R. Birtles for reading the manuscript, G. Vestris for technical assistance in cultivating bacterial strains, A. Dolla for expert

C. Mollet et al. / Gene 207 (1998) 97–103

help in protein sequence analysis and D. Gautheret for expert advice on RNA folding.

References Aboshkiwa, M., Al-Ani, B., Coleman, G., Rowland, G., 1992. Cloning and physical mapping of the Staphylococcus aureus rplL, rpoB and rpoC genes, encoding ribosomal protein L7/L12 and RNA polymerase subunits b and b∞. J. Gen. Microbiol. 138, 1875–1880. Alekshun, M., Kashlev, M., Schwartz, I., 1997. Molecular cloning and characterization of Borrelia burgdorferi rpoB. Gene 186, 227–235. Anderson, S.G.E., Sharp, P.M., 1996. Codon usage and base composition in Rickettsia prowazekii. J. Mol. Evol. 42, 525–536. Barry, G., Squires, C., Squires, C.L., 1980. Attenuation and processing of RNA from the rplJL–rpoBC transcription unit of Escherichia coli. Proc. Natl. Acad. Sci. USA 77, 3331–3335. Dessen, P., Fondrat, C., Valencien, C., Mugnier, C., 1990. Bisance: a French service for access to biomolecular sequence databases. Cabios 6, 355–356. Engel, J.N., Pollack, J., Malik, F., Ganem, D., 1990. Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction. J. Bacteriol. 172, 5732–5741. Farris, J.S., 1989. PHYLIP—Phylogeny Inference Package version 3.2. Cladistics 5, 164–166. Felsenstein, J., 1993. PHYLIP: Phylogeny Inference Package ( University of Washington, Seattle), Version 3.5c. Heinzen, R., Stiegler, G.L., Whiting, L.L., Schmitt, S.A., Mallavia, L.P., Frazier, M.E., 1990. Use of pulsed-field gel electrophoresis to differentiate Coxiella burnetii strains. Ann. N.Y. Acad. Sci. 590, 504–513. Higgins, D.G., Sharp, P.M., 1988. Clustal: a package for performing multiple sequence alignment on a microcomputer. Gene 73, 237–244. ´ Honore, N., Cole, S.T., 1993. Molecular basis of rifampin resistance in Mycobacterium leprae. Antimicrob. Agents Chemother. 37, 414–418. Klenk, H.P., Zillig, W., 1994. DNA-dependent RNA polymerase subunit B as a tool for phylogenetic reconstructions: branching topology of the archaeal domain. J. Mol. Evol. 38, 420–432. Knoth, K., Roberds, S., Poteet, C., Tamkun, M., 1988. Highly degenerate, inosine-containing primers specifically amplify rare cDNA using the polymerase chain reaction. Nucleic Acids Res. 16, 10932 Mollet, C., Drancourt, M., Raoult, D., 1995. Deoxyinosine containing primers for solid-phase, automated DNA sequencing. Meth. Mol. Cell. Biol. 5, 125–127. Osawa, S., Jukes, T.H., Watanabe, K., Muto, A., 1992. Recent evidence for evolution of the genetic code. Microbiol. Rev. 56, 229–264. Ovchinnikov, Y.A., Monastyrskaya, G.S., Gubanov, V.V., Gurey, S.O., Chertov, O.Y., Modyanov, T.V., Grinkervich, U.A., Makarova, I.A., Marchenko, T.V., Polovnikova, I.N., Lipkin, V.M., Sverdlov, E.D., 1981. The primary structure of Escherichia coli RNA polymerase. Nucleotide sequence of the rpoB gene and amino-acid sequence of the b-subunit. Eur. J. Biochem. 116, 621–629. ¨ Puhler, G., Lottspeich, F., Zillig, W., 1989a. Organization and nucleotide sequence of the genes encoding the large subunits A, B, and C of the DNA-dependent RNA polymerase of the archaebacterium Sulfolobus acidocaldarius. Nucleic Acids Res. 17, 4517–4534.

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¨ Puhler, G., Leffers, H., Gropp, F., Palm, P., Klenk, H.S., Lottspeich, F., Garrett, R.A., Zillig, W., 1989b. Archaebacterial DNA-dependent RNA polymerases testify to the evolution of the eukaryotic nuclear genome. Proc. Natl. Acad. Sci. USA 86, 4569–4573. Ralling, G., Linn, T., 1987. Evidence that Rho and NusA are involved in termination in the rplL–rpoB intercistronic region. J. Bacteriol. 169, 2277–2280. Raoult, D., Torres, H., Drancourt, M., 1991. Shell-vial assay: evaluation of a new technique for determining antibiotic susceptibility, tested in 13 isolates of Coxiella burnetii. Antimicrob. Agents Chemother. 35, 2070–2077. Raoult, D., Saltzman, R.L. 1994. Q fever. In: Hoeprich, P.D., Jordan, M.C., Ronald, A.R. ( Eds.), Infectious Diseases, 5th Edn. J.B. Lippincott Company, Philadelphia, PA, pp. 417–420. Rowland, G.C., Aboshkiwa, M., Coleman, G., 1992. Comparative sequence analysis and predicted phylogeny of the DNA-dependent RNA polymerase b subunits of Staphylococcus aureus and other eubacteria. Biochem. Soc. Trans. 21, 40S Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Purification of nucleic acids. In: Sambrook, J., Fritsch, E.F., Maniatis, T. ( Eds.), Molecular Cloning, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. E3–E4. Schramek, S., 1968. Isolation and characterization of deoxyribonucleic acid from Coxiella burnetii. Acta Virol. 12, 18–22. Stein, A., Raoult, D., 1992a. Phenotypic and genotypic heterogeneity of 8 new human Coxiella burnetii isolates. Acta Virol. 36, 7–12. Stein, A., Raoult, D., 1992b. Detection of Coxiella burnetii by DNA amplification using polymerase chain reaction. J. Clin. Microbiol. 30, 2462–2466. Stein, A., Raoult, D., 1993. Lack of pathotype specific gene in human Coxiella burnetii isolates. Microb. Pathogen. 15, 177–185. Stein, A., Saunders, N.A., Taylor, A.G., Raoult, D., 1993. Phylogenic homogeneity of Coxiella burnetii strains as determined by 16S ribosomal RNA sequencing. FEMS Microbiol. Lett. 113, 339–344. Telenti, A., Imboden, P., Marchesi, F., Lowrie, D., Cole, S., Colston, M.J., Matter, L., Schopfer, K., Bodmer, T., 1993. Detection of rifampin-resistance mutations in Mycobacterium tuberculosis. Lancet 341, 647–650. Thiele, D., Willems, H., 1994. Is plasmid based differentiation of Coxiella burnetii in ‘acute’ and ‘chronic’ isolates still valid? Eur. J. Epidemiol. 10, 427–434. Tittawella, I.P.B., 1984. Evidence for clustering of RNA polymerase and ribosomal protein genes in six species of enterobacteria. Mol. Gen. Genet. 195, 215–218. Weisburg, W.G., Dobson, M.E., Samuel, J.E., Dasch, G.A., Mallavia, L.P., Baca, O.G., Mandelco, L., Sechrest, J.E., Weiss, E., Woese, C.R., 1989. Phylogenetic diversity of the Rickettsiae. J. Bacteriol. 171, 4202–4206. Woese, C.R., Winker, S., Gutell, R.R., 1990. Architecture of ribosomal RNA: constraints on the sequence of ‘tetra-loops’. Proc. Natl. Acad. Sci. USA 87, 8467–8471. Yazawa, K., Mikami, Y., Maeda, A., Akao, M., Morisaki, N., Iwasaki, S., 1993. Inactivation of rifampin by Nocardia brasiliensis. Antimicrob. Agents Chemother. 37, 1313–1317. Yeaman, M.R., Roman, M.J., Baca, O.G., 1989. Antibiotic susceptibility of two Coxiella burnetii isolates implicated in distinct clinical syndromes. Antimicrob. Agents Chemother. 33, 1052–1057. Zuker, M., 1989. On finding all suboptimal foldings of an RNA molecule. Science 244, 48–52.