Physical and genetic map of the obligate intracellular bacterium Coxiella burnetii

Abstract

Pulsed-field gel electrophoresis and PCR techniques have been used to construct a NotI macrorestriction map of the obligate intracellular bacterium Coxiella burnetii Nine Mile. The size of the chromosome has been determined to be 2,103 kb comprising 29 NotI restriction fragments. The average resolution is 72.5 kb, or about 3. 5% of the genome. Experimental data support the presence of a linear chromosome. Published genes were localized on the physical map by Southern hybridization. One gene, recognized as transposable element, was found to be present in at least nine sites evenly distributed over the whole chromosome. There is only one copy of a 16S rRNA gene. The putative oriC has been located on a 27.5-kb NotI fragment. Gene organization upstream the oriC is almost identical to that of Pseudomonas putida and Bacillus subtilis, whereas gene organization downstream the oriC seems to be unique among bacteria. The physical map will be helpful in investigations of the great heterogeneity in restriction fragment length polymorphism patterns of different isolates and the great variation in genome size. The genetic map will help to determine whether gene order in different isolates is conserved.

FIG. 1

CHEF-PFGE of NotI-digested total DNA. Different CHEF-PFGE parameters (see text) were applied to separate NotI fragments of 2 to 270 kb (B), 55 to 270 kb (A), and 6.7 to 55 kb (C). Lines indicate regions where resolution of NotI fragments has been increased. Panel A demonstrates fragments ranging in size from 210 to 268 kb to be clearly separated. Nevertheless, the two 227-kb NotI fragments are not distinguishable. Resolution enhancement is even more striking in panel C, where the 52.3-kb double fragment (B) divided into 53.8- and 50.8-kb NotI fragments. Panel C also demonstrates the 27.5- and 32.1-kb NotI fragments to be double fragments, as indicated by increased band intensities. In contrast, band intensity of the QpH1 plasmid suggests that there is only a single copy of the plasmid in C. burnetii Nine Mile.

FIG. 2

Physical and genetic map of the chromosome of C. burnetii Nine Mile. NotI fragments are indicated by numbers on the outer circle (sizes in kilobases): 1, 7.3; 2, 50.8; 3, 13.8; 4, 26.1; 5, 227; 6, 227; 7, 103; 8, 27.5; 9, 151; 10, 16.9; 11, 268; 12, 9.4; 13, 3.9; 14, 210; 15, 19.2; 16, 6.7; 17, 168; 18, 6.7; 19, 28; 20, 53.8; 21, 12.4; 22, 27.5; 23, 32.1; 24, 118; 25, 5.3; 26, 32.1; 27, 8.2; 28, 2.1; 29, 240. NotI recognition sites are indicated by lines connecting the inner and outer circles. ¹, contains the NotI recognition site. ², hybridization signal intensity suggests IS1111a to exist at least once on the 50.8- and 53.8-kb NotI fragments (in Fig. 1B, lane 1, indicated as the 52-kb fragment). ∗, Could not be related to one of the 227-kb NotI fragments; hybridization signal intensity suggests IS1111a to exist at least once on both 227-kb NotI fragments (Fig. 1B, lane 1). ∗∗, putative genes located around the oriC region: fmu, glysAB, ygi2, ygi1, omp, gidB, gidA, 50K, 60K, and 9K.

FIG. 3

Gene organization around the putative oriC of C. burnetii (B) compared to that of P. putida (A) and B. subtilis (C). The region upstream the oriC is almost identical to that of P. putida and B. subtilis (indicated by black boxes). Hypothetical proteins YGI1 and YGI2 of C. burnetii are homologous to YR55 and SpoJ of B. subtilis. Gene organization downstream the putative oriC of C. burnetii is quite different from that of P. putida and B. subtilis, as indicated by white boxes. Whereas in P. putida and B. subtilis genes around the oriC are transcribed in opposite direction, genes in C. burnetii are all transcribed in the same direction (indicated by arrows). Question marks above the white boxes indicate polypeptides without designation and with unknown function.