Abstract
ABSTRACTIn Brucella abortus, two small RNAs (sRNAs), AbcR1 and AbcR2, are responsible for regulating transcripts encoding ABC-type transport systems. AbcR1 and AbcR2 are required for Brucella virulence, as a double chromosomal deletion of both sRNAs results in attenuation in mice. Although these sRNAs are responsible for targeting transcripts for degradation, the mechanism utilized by the AbcR sRNAs to regulate mRNA in Brucella has not been described. Here, two motifs (M1 and M2) were identified in AbcR1 and AbcR2, and complementary motif sequences were defined in AbcR-regulated transcripts. Site-directed mutagenesis of M1 or M2 or of both M1 and M2 in the sRNAs revealed transcripts to be targeted by one or both motifs. Electrophoretic mobility shift assays revealed direct, concentration-dependent binding of both AbcR sRNAs to a target mRNA sequence. These experiments genetically and biochemically characterized two indispensable motifs within the AbcR sRNAs that bind to and regulate transcripts. Additionally, cellular and animal models of infection demonstrated that only M2 in the AbcR sRNAs is required for Brucella virulence. Furthermore, one of the M2-regulated targets, BAB2_0612, was found to be critical for the virulence of B. abortus in a mouse model of infection. Although these sRNAs are highly conserved among Alphaproteobacteria, the present report displays how gene regulation mediated by the AbcR sRNAs has diverged to meet the intricate regulatory requirements of each particular organism and its unique biological niche.
Highlights
In Brucella abortus, two small RNAs, AbcR1 and AbcR2, are responsible for regulating transcripts encoding ABC-type transport systems
Caswell et al demonstrated that chromosomal isogenic deletion of either abcR1 or abcR2 in B. abortus resulted in a wild-type virulence phenotype but that a double abcR1 abcR2 mutant led to significant attenuation in a mouse model of infection [15]
Individual deletions of either abcR1 or abcR2 did not lead to altered target mRNA levels; a strain in which both abcR1 and abcR2 were deleted exhibited statistically significantly increased levels of the mRNA targets compared to wild-type strain 2308, clearly demonstrating that AbcR1 and AbcR2 possess redundant regulatory functions in B. abortus
Summary
In Brucella abortus, two small RNAs (sRNAs), AbcR1 and AbcR2, are responsible for regulating transcripts encoding ABC-type transport systems. One of the M2-regulated targets, BAB2_0612, was found to be critical for the virulence of B. abortus in a mouse model of infection These sRNAs are highly conserved among Alphaproteobacteria, the present report displays how gene regulation mediated by the AbcR sRNAs has diverged to meet the intricate regulatory requirements of each particular organism and its unique biological niche. In B. abortus, expression of abcR2, but not abcR1, is controlled by VtlR, the orthologous LysR-type transcriptional regulator found upstream of the genes encoding the A. tumefaciens and S. meliloti AbcR sRNAs [14] These differences in regulatory roles, genetic organization, and transcriptional regulation highlight the evolutionary differentiation of the AbcR regulatory system, which may have helped drive the host-bacterium relationships of Alphaproteobacteria
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