Brucellosis, a zoonotic disease, has re-emerged in both humans and animals, causing significant economic losses globally. Recently, an increasing number of rifampicin-resistant
Brucella
strains have been isolated worldwide without detectable mutations in known antibiotic resistance genes. Here, this study identified the deletion of serine/threonine protein kinase (STPK) gene in
B. melitensis
as an efficient trigger for rifampicin resistance using bioinformatics predictions, a transposon mutant library, and gene mutation strains. Notably, the absence of the STPK could increase the expression of ribosomal proteins and genes involved in sulfur metabolism and reduced glutathione, and decrease NADPH oxidase activity and NADP
+
/NADPH ratio, which is associated with the antioxidant capacity of
B. melitensis
. Moreover, co-immunoprecipitation revealed that STPK could efficiently interact with the ribosomal protein RpsD, possibly altering protein translation and riboswitch expression. These findings demonstrate that the STPK gene mediates resistance by regulating sulfur metabolism to counteract the reactive oxygen species induced by rifampicin. Furthermore, the approaches developed in this study provide a platform for screening new resistance genes in
Brucella
spp., and the identified STPK or its pathway can serve as a potential target for new drug development against rifampicin-resistant
Brucella
spp.
IMPORTANCE
New rifampicin resistance gene in
Brucella melitensis
is identified via bioinformatics predictions and a whole-genome transposon mutant library, new mechanisms of rifampicin resistance in
B. melitensis
, and new function of serine/threonine protein kinase gene and its interaction proteins.