Affiliation:
1. Division of Microbiology, National Center for Toxicological Research , Jefferson, Arkansas, USA
Abstract
ABSTRACT
The ability to better understand the function of proteins expressed by bacteria has typically relied upon the development of genetic mutant strains. This approach has been especially challenging for plasmid-encoded genes, as most of the previously described allelic replacement vectors are inefficient for plasmid genome mutation as they either rely on plasmid-derived counterselection toxins or depend on other strategies such
sacB
,
tetA
, and
rpsL
which have been proven to be less efficient for mutant selection. Integrative vectors lack chromophore indicators, thus requiring laborious screening or excision of the vector’s backbone relies on the introduction of a flippase (FLP)-expressing plasmid. The allelic replacement vector, designated here as pDG1, expresses an X-Gal hydrolyzing enzyme (BgaB) that can be used for blue/white screening allowing identification of colonies that integrated and successfully removed the mutagenesis plasmid without a bias for those still carrying it. pDG1 was further improved by including the rhamnose-inducible Tse2 toxin as a potent counterselection system. The efficacy of pDG1 was validated by deleting portions of the plasmid-encoded VirB4/D4 type IV secretion system and aerobactin-synthesizing operons in
Salmonella enterica
. The integrative vectors, which contain an ΦC31
attP
site and genes encoding ΦC31 integrase (
int
), can seamlessly integrate to target ΦC31
attB
on
Salmonella
plasmids or chromosome. These vectors were improved by inserting
bgaB
and FLP-encoding genes so that, following integration, most of the vector’s backbone encompassing
int
,
bgaB
, and FLP genes can be excised by FLP, without the need for another FLP-expressing vector, creating white colonies carrying a stably integrated target gene. As such, we were able to integrate a 9.3-kb DNA fragment to
Salmonella
chromosome and flipped out most of the integrated vector in one step, leaving the target fragment in the chromosome.
IMPORTANCE
In spite of the dissemination of multidrug-resistant plasmids among Gram-negative pathogens, including those carrying virulence genes, vector tools for studying plasmid-born genes are lacking. The allelic replacement vectors can be used to generate plasmid or chromosomal mutations including markless point mutations. This is the first report describing a self-excising integrative vector that can be used as a stable single-copy complementing tool to study medically important pathogens including
in vivo
studies without the need for antibiotic selection. Overall, our newly developed vectors can be applied for the assessment of the function of plasmid-encoded genes by specifically creating mutations, moving large operons between plasmids and to/from the chromosome, and complementing phenotypes associated with gene mutation. Furthermore, the vectors express chromophores for the detection of target gene modification or colony isolation, avoiding time-consuming screening procedures.
Funder
HHS | U.S. Food and Drug Administration
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Cell Biology,Microbiology (medical),Genetics,General Immunology and Microbiology,Ecology,Physiology