ABSTRACT
In
order to identify transcriptional regulators involved in virulence gene
control in Brucella melitensis, we generated a collection of
88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR,
and TetR families of regulators. This collection was named LiMuR
(library of mutants for regulators). We
developed a method to test several mutants simultaneously in one animal
in order to identify those unable to survive. This method, called the
plasmid-tagged mutagenesis method, was used to test the residual
virulence of mutants after 1 week in a mouse model of infection. Ten
attenuated mutants, of which six and three belong to the GntR and LysR
families, respectively, were identified and individually confirmed to
replicate at lower rates in mice. Among these 10 mutants, only
gntR10 and arsR6 are attenuated in cellular models.
The LiMuR also allows simple screenings to identify regulators of a
particular gene or operon. As a first example, we analyzed the
expression of the virB operon in the LiMuR mutants. We carried
out Western blottings of whole-cell extracts to analyze the production
of VirB proteins using polyclonal antisera against VirB proteins. Four
mutants produced small amounts of VirB proteins, and one mutant
overexpressed VirB proteins compared to the wild-type strain. In these
five mutants, reporter analysis using the virB promoter fused
to lacZ showed that three mutants control virB at the
transcriptional level. The LiMuR is a resource that will provide
straightforward identification of regulators involved in the control of
genes of
interest.