Abstract
A large number of hypothetical genes potentially encoding small proteins of unknown function are annotated in the Brucella abortus genome. Individual deletion of 30 of these genes identified four mutants, in BAB1_0355, BAB2_0726, BAB2_0470, and BAB2_0450 that were highly attenuated for infection. BAB2_0726, an YbgT-family protein located at the 3′ end of the cydAB genes encoding cytochrome bd ubiquinal oxidase, was designated cydX. A B. abortus cydX mutant lacked cytochrome bd oxidase activity, as shown by increased sensitivity to H2O2, decreased acid tolerance and increased resistance to killing by respiratory inhibitors. The C terminus, but not the N terminus, of CydX was located in the periplasm, suggesting that CydX is an integral cytoplasmic membrane protein. Phenotypic analysis of the cydX mutant, therefore, suggested that CydX is required for full function of cytochrome bd oxidase, possibly via regulation of its assembly or activity.
Highlights
Genes encoding small proteins are not well-characterized in the genomes of bacteria
TARGETED MUTAGENESIS OF SMALL HYPOTHETICAL ORFs Using the published genome sequences of B. abortus 2308, B. suis 1330, and B. melitensis 16 M, we identified ORFs with the potential to encode proteins smaller than 15 kDa
While a faint product for cydA was observed in both WT and the cydX mutant and BA582 (cydB) mutant, neither cydB nor cydX amplification products were found in the cydB mutant, suggesting a polar effect of the cydB mutation on cydX expression. These results suggested that cydX is transcribed together with the cytochrome bd oxidase subunits encoded by cydA and cydB
Summary
Genes encoding small proteins are not well-characterized in the genomes of bacteria. An examination of the sequenced genomes shows that these small genes are not consistently annotated across related genomes, which raises the question of whether they encode functional genes. COMPLEMENTATION OF THE cydX MUTANT In order to restore a functional copy of cydX in the mutant, a previously described groEL promoter (Gor and Mayfield, 1992; Lin et al, 1992) corresponding to Brucella abortus 2308 chromosome II DNA coordinates 184149–184551 with engineered restriction enzyme sites was amplified using primers GroEL-P-F and GroEL-P-R and cloned into the pCR2.1 TOPO cloning vector.
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