Cloning and characterization of an autonomous replication sequence from Coxiella burnetii

Abstract

A Coxiella burnetii chromosomal fragment capable of functioning as an origin for the replication of a kanamycin resistance (Kanr) plasmid was isolated by use of origin search methods utilizing an Escherichia coli host. The 5.8-kb fragment was subcloned into phagemid vectors and was deleted progressively by an exonuclease III-S1 technique. Plasmids containing progressively shorter DNA fragments were then tested for their capability to support replication by transformation of an E. coli polA strain. A minimal autonomous replication sequence (ARS) was delimited to 403 bp. Sequencing of the entire 5.8-kb region revealed that the minimal ARS contained two consensus DnaA boxes, three A + T-rich 21-mers, a transcriptional promoter leading rightwards, and potential integration host factor and factor of inversion stimulation binding sites. Database comparisons of deduced amino acid sequences revealed that open reading frames located around the ARS were homologous to genes often, but not always, found near bacterial chromosomal origins; these included identities with rpmH and rnpA in E. coli and identities with the 9K protein and 60K membrane protein in E. coli and Pseudomonas species. These and direct hybridization data suggested that the ARS was chromosomal and not associated with the resident plasmid QpH1. Two-dimensional agarose gel electrophoresis did not reveal the presence of initiating intermediates, indicating that the ARS did not initiate chromosome replication during laboratory growth of C. burnetii.

Transformation of Coxiella burnetii to ampicillin resistance

“…Two-dimensional agarose gel electrophoresis of chromosomal DNA isolated from C. burnetii grown in tissue culture did not reveal DNA replication initiations within ars. It was concluded that, while the C. burnetii ars sequence was not a site for initiation of replication during growth in laboratory hosts, it might function as such in natural hosts (36). This was not the first report of a bacterial ars failing to demonstrate chromosomal origin function.…”

“…Specific probes were labeled by the incorporation of [␣-32 P]dCTP (Amersham) into DNA by using either oligomer random priming (11) (kit supplied by Boehringer Mannheim), nick translation (31) (kit supplied by Boehringer Mannheim), or PCR (33). PCR-generated probes were labeled by the PCR incorporation of either digoxigenin dUTP (Boehringer Mannheim; reaction conditions as specified by the manufacturer) or [␣- 32 P]dCTP (Amersham) (33,36). The unincorporated nucleotide triphosphates were removed by using Sephadex G-50 spin columns (22).…”

“…The total DNA of C. burnetii was isolated by either an alkaline phenol extraction (37) or a guanidine hydrochloride procedure (18,36). Plasmid DNA was extracted from E. coli by an alkaline lysis minipreparation or by alkaline lysis followed by purification with a polyethylene glycol procedure (32).…”

“…The use of E. coli as a host for C. burnetii genes was also implemented for the cloning of the C. burnetii chromosomal origin of DNA replication. These origin search techniques resulted in the isolation of a 5.8-kb C. burnetii chromosomal DNA fragment which initiates plasmid DNA replication within E. coli (36). The minimal ars sequence required for plasmid replication in an E. coli polA strain is 403 bp (6,36); it demonstrates limited similarity to origins of other bacteria (Fig.…”

Characterization of a Coxiella burnetii ftsZ Mutant Generated by Himar1 Transposon Mutagenesis

“…Using a plasmid containing a 5.8-kb C. burnetii autonomous replication sequence, they transformed C. burnetii to ampicillin resistance (4,35,36). Transformants exhibited both extrachromosomal replication and integration of the plasmid into the chromosome by homologous recombination.…”