The bacterium Bacillus subtilis produces the DNA integrity scanning protein (DisA), a checkpoint protein that delays sporulation in response to DNA damage. DisA scans the chromosome and pauses at sites of DNA lesions. Structural analysis showed that DisA synthesizes the small molecule cyclic diadenosine monophosphate (c‐di‐AMP). Here, we demonstrate that the intracellular concentration of c‐di‐AMP rises markedly at the onset of sporulation in a DisA‐dependent manner. Furthermore, exposing sporulating cells to DNA‐damaging agents leads to a global decrease in the level of this molecule. This drop was associated with stalled DisA complexes that halt c‐di‐AMP production and with increased levels of the c‐di‐AMP‐degrading enzyme YybT. Reduced c‐di‐AMP levels cause a delay in sporulation that can be reversed by external supplementation of the molecule. Thus, c‐di‐AMP acts as a secondary messenger, coupling DNA integrity with progression of sporulation.

Finding bacterial cellular targets for developing novel antibiotics has become a major challenge in fighting resistant pathogenic bacteria. We present a novel compound, Relacin, designed to inhibit (p)ppGpp production by the ubiquitous bacterial enzyme RelA that triggers the Stringent Response. Relacin inhibits RelA in vitro and reduces (p)ppGpp production in vivo. Moreover, Relacin affects entry into stationary phase in Gram positive bacteria, leading to a dramatic reduction in cell viability. When Relacin is added to sporulating Bacillus subtilis cells, it strongly perturbs spore formation regardless of the time of addition. Spore formation is also impeded in the pathogenic bacterium Bacillus anthracis that causes the acute anthrax disease. Finally, the formation of multicellular biofilms is markedly disrupted by Relacin. Thus, we establish that Relacin, a novel ppGpp analogue, interferes with bacterial long term survival strategies, placing it as an attractive new antibacterial agent. The development of new antibacterial agents has become the major demand for fighting against pathogenic bacteria. The identification of new unexplored cellular targets in this combat is essential to prevent a possible return to the pre-antibiotic era. Traditional antibiotics target essential cellular components such as ribosomes and cell wall constituents, making them effective mostly during bacterial growth. However, the ability of bacteria to reside in nature at durable stages sets the need to cope with these alternative survival strategies. In this report, we present a novel antibiotic, termed Relacin, which targets the Stringent Response, a process required for the transition into stationary phase, crucial for bacterial survival. Relacin inhibits the abundant bacterial Rel enzymes that synthesize the signaling molecules required to activate the Stringent Response. We found that Relacin perturbs the switch into stationary phase in Gram positive bacteria and leads to cell death. Further, Relacin inhibits sporulation and biofilm formation, additional bacterial long term survival strategies. The ubiquity of Rel enzymes among bacteria, combined with the absence of known homologues in mammalian cells, strengthen the potential of Relacin to turn into a therapeutic antibiotic.

Significance: How cells specifically express only a single gene among numerous equivalent copies within their genomes is one of the unsolved mysteries in the field of eukaryotic gene expression. The molecular mechanisms that underlie mutually exclusive gene expression are the key for understanding the virulence of Plasmodium falciparum , the parasite responsible for the deadliest form of human malaria. P. falciparum expresses its primary virulence determinants in a mutually exclusive manner and evades human immune attack through switches in expression between different variants of a large gene family named var . We found that var -specific antisense long noncoding RNA molecules incorporate into chromatin and determine how parasites select a single gene for expression while the rest of the family is maintained silenced.