SUMMARY Replication-transcription collisions shape genomes, influence evolution, and promote genetic diseases. Although unclear why, head-on transcription (lagging strand genes) is especially disruptive to replication and promotes genomic instability. Here, we find that head-on collisions promote R-loop formation in Bacillus subtilis. We show that pervasive R-loop formation at head-on collision regions completely blocks replication, elevates mutagenesis, and inhibits gene expression. Accordingly, the activity of the R-loop processing enzyme RNase HIII at collision regions is crucial for stress survival in B. subtilis, as many stress response genes are head-on to replication. Remarkably, without RNase HIII, the ability of the intracellular pathogen Listeria monocytogenes to infect and replicate in hosts is weakened significantly, most likely because many virulence genes are head-on to replication. We conclude that the detrimental effects of head-on collisions stem primarily from excessive R-loop formation and that the resolution of these structures is critical for bacterial stress survival and pathogenesis.

Eukaryotes possess numerous quality control systems that monitor both the synthesis of RNA and the integrity of the finished products. We previously demonstrated that Saccharomyces cerevisiae possesses a quality control mechanism, nonfunctional rRNA decay (NRD), capable of detecting and eliminating translationally defective rRNAs. Here we show that NRD can be divided into two mechanistically distinct pathways: one that eliminates rRNAs with deleterious mutations in the decoding site (18S NRD) and one that eliminates rRNAs containing deleterious mutations in the peptidyl transferase center (25S NRD). 18S NRD is dependent on translation elongation and utilizes the same proteins as those participating in no-go mRNA decay (NGD). In cells that accumulate 18S NRD and NGD decay intermediates, both RNA types can be seen in P-bodies. We propose that 18S NRD and NGD are different observable outcomes of the same initiating event: a ribosome stalled inappropriately at a sense codon during translation elongation.

Summary Efforts to battle anti-microbial resistance (AMR) are generally focused on developing novel antibiotics. However, history shows that resistance arises regardless of the nature or potency of new drugs. Here, we propose and provide evidence for an alternate strategy to resolve this problem: inhibiting evolution. We determined that the DNA translocase Mfd is an “evolvability factor” that promotes mutagenesis and is required for rapid resistance development to all antibiotics tested, across highly divergent bacterial species. Importantly, hypermutator alleles that accelerate AMR development did not arise without Mfd, at least during evolution of trimethoprim resistance. We also show that Mfd’s role in AMR development depends on its interactions with the RNA polymerase subunit RpoB and the nucleotide excision repair protein UvrA. Our findings suggest that AMR development can be inhibited through inactivation of evolvability factors (potentially with “anti-evolution” drugs), and in particular Mfd, providing an unexplored route towards battling the AMR crisis.

Membrane-bound O -acyltransferases (MBOATs) represent a superfamily of integral transmembrane enzymes found in all kingdoms of life 1 . In bacteria, MBOATs modify protective cell surface polymers. In vertebrates, some MBOAT enzymes such as acyl-CoA:cholesterol acyltransferase (ACAT) and diacylglycerol acyltransferase 1 (DGAT1) are responsible for lipid biosynthesis and phospholipid remodeling 2 , 3 . Some other MBOATs, including porcupine (PORCN), hedgehog acyltransferase (HHAT) and ghrelin acyltransferase (GOAT), catalyze essential lipid modifications of secreted proteins such as Wnt, hedgehog and ghrelin, respectively 4 – 10 . Although many MBOAT proteins are important drug targets, little is known about their molecular architecture and functional mechanisms. Here we present crystal structures of DltB, a MBOAT responsible for D-alanylation of cell wall teichoic acid (TA) of Gram-positive bacteria 11 – 16 , by itself and in complex with the D-alanyl donor protein, DltC. DltB contains a ring of 11 peripheral transmembrane helices, which shield a highly conserved extracellular structural “funnel” extending into the middle of lipid bilayer. The conserved catalytic histidine residue is located at the bottom of this funnel and connected to the intracellular DltC through a narrow tunnel. Mutation of either the catalytic histidine or the DltC binding site of DltB abolishes LTA D-alanylation, and sensitizes the Gram-positive bacterium Bacillus subtilis to cell wall stress, suggesting cross-membrane catalysis involving the tunnel. Structure-guided sequence comparison among DltB and vertebrate MBOATs reveals a conserved structural core and suggests similar catalytic mechanisms. Our structures provide a template for understanding MBOAT structure-function relationships and for developing therapeutic MBOAT inhibitors.