An antisense RNA regulates production of DnaA and affects sporulation in Bacillus subtilis

Abstract

DnaA is the replication initiator and a transcription factor in virtually all bacteria. Although the synthesis and activity of DnaA are highly regulated, the mechanisms of regulation vary between organisms. We found that production of DnaA in Bacillus subtilis is regulated by an antisense RNA that overlaps with the 5 untranslated region upstream of the dnaA open reading frame. We initially observed this RNA in in vitro transcription experiments and found that its production was inhibited by DnaA. This RNA, now called ArrA for antisense RNA repressor of dnaA, is made in vivo. We identified the arrA promoter and made a mutation that greatly reduced (or eliminated) production of ArrA RNA in vitro and in vivo. In vivo, this arrA promoter mutation caused an increase in the amount of mRNA and protein from dnaA and dnaN, indicating that arrA expression normally inhibits expression of the dnaA-dnaN operon. The arrA mutation also caused a delay in sporulation that was alleviated by loss of sda, a sporulation-inhibitory gene that is directly activated by DnaA. arrA appears to be conserved in some members of the Bacillus genus, indicating that arrA has evolved in at least some endospore-forming bacteria to modulate production of DnaA and enable timely and robust sporulation. Author summaryDnaA is the highly conserved replication initiator and transcription factor found in virtually all bacteria. The synthesis and activity of DnaA are highly regulated, and different types of bacteria use different mechanisms to control this key protein. We found that DnaA production in Bacillus subtilis is inhibited by an antisense RNA that overlaps with the 5 untranslated region of the dnaA mRNA. In the absence of this antisense RNA, there was an increase in the amount of dnaA mRNA and protein. There was also a delay in sporulation that depends on sda, a sporulation-inhibitory gene that is directly activated by DnaA. arrA appears to be conserved in several members of the Bacillus genus, indicating that it has evolved to temper production DnaA and enable robust sporulation in these species.