Single-copy chromosomal integration systems for Francisella tularensis

Abstract

Francisella tularensis is a fastidious Gram-negative bacterium responsible for the zoonotic disease tularemia. Investigation of the biology and molecular pathogenesis of F. tularensis has been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of genetic tools. However, recent advances have substantially improved the ability of researchers to genetically manipulate this organism. To expand the molecular toolbox we have developed two systems to stably integrate genetic elements in single-copy into the F. tularensis genome. The first system is based upon the ability of transposon Tn7 to insert in both a site-and orientation-specific manner at high frequency into the attTn7 site located downstream of the highly conserved glmS gene. The second system consists of a sacB-based suicide plasmid used for allelic exchange of unmarked elements with the blaB gene, encoding a b-lactamase, resulting in the replacement of blaB with the element and the loss of ampicillin resistance. To test these new tools we used them to complement a novel D-glutamate auxotroph of F. tularensis LVS, created using an improved sacB-based allelic exchange plasmid. These new systems will be helpful for the genetic manipulation of F. tularensis in studies of tularemia biology, especially where the use of multi-copy plasmids or antibiotic markers may not be suitable.

The Francisella tularensis FabI Enoyl-Acyl Carrier Protein Reductase Gene Is Essential to Bacterial Viability and Is Expressed during Infection

“…Plasmids used in this study were recombinant derivatives of F. tularensis chromosomal integration vectors pMP812 and pMP815 (13), provided by H. P. Schweitzer, Colorado State University. Cloning and strain maintenance were carried out in Escherichia coli DH5␣ (Life Technologies) in Luria-Bertani broth (Becton, Dickinson).…”

“…All electroporations, transformations of sucrose-competent F. tularensis LVS and Schu4 with pMP815-or pMP812-based allelic exchange constructs, and subsequent generation of primary and secondary recombinants were performed as described by LoVullo et al (13). The FtfabI merodiploid strain was constructed as follows.…”

“…The ⌬FtfabI mutant was created using two nonsequential fragments with extensions providing overlapping ends amplified from the FtfabI genomic region of LVS DNA. The two fragments were combined in a second amplification and were cloned into a sacB suicide plasmid pMP812 (13) to form a 356-bp deletion of the fabI promoter and 5= CDS sequences flanked by 535 bp upstream and 759 bp downstream. The FtfabI conditional mutation allele created from a segment of the Schu4 FtfabI genomic region including 435 bp upstream and 437 bp downstream of sequence coding residue T241 was cloned into pMP812.…”

Working toward the Future: Insights intoFrancisella tularensisPathogenesis and Vaccine Development

“…Third-generation pMP-based vectors have also been developed, in which stability has been further enhanced, useful multiple cloning sites introduced, and heterologous promoters added for gene expression studies (120). Finally, a single-copy chromosomal integration system for Francisella has been developed by that group (119). Vectors designed for this system include plasmids allowing integration at the attachment site for the Tn7 transposon (located downstream of the glmS gene) or within the blaB gene, encoding resistance to the antibiotic ampicillin (119).…”

“…Finally, a single-copy chromosomal integration system for Francisella has been developed by that group (119). Vectors designed for this system include plasmids allowing integration at the attachment site for the Tn7 transposon (located downstream of the glmS gene) or within the blaB gene, encoding resistance to the antibiotic ampicillin (119). Development of an integration system for F. tularensis represents a major advancement for the field, as it alleviates some of the previous issues inherent with use of multicopy shuttle vectors, including lack of stability, use of heterologous antibiotic resistance determinants, and multicopy expression artifacts.…”