SummaryMany microorganisms produce molecules having antibiotic activity and expel them into the environment, presumably enhancing their ability to compete with their neighbours. Given that these molecules are often toxic to the producer, mechanisms must exist to ensure that the assembly of the export apparatus accompanies or precedes biosynthesis. Streptomyces coelicolor produces the polyketide antibiotic actinorhodin in a multistep pathway involving enzymes encoded by genes that are clustered together. Embedded within the cluster are genes for actinorhodin export, two of which, actR and actA resemble the classic tetR and tetA repressor/efflux pump-encoding gene pairs that confer resistance to tetracycline. Like TetR, which represses tetA, ActR is a repressor of actA. We have identified several molecules that can relieve repression by ActR. Importantly (S)-DNPA (an intermediate in the actinorhodin biosynthetic pathway) and kalafungin (a molecule related to the intermediate dihydrokalafungin), are especially potent ActR ligands. This suggests that along with the mature antibiotic(s), intermediates in the biosynthetic pathway might activate expression of the export genes thereby coupling export to biosynthesis. We suggest that this could be a common feature in the production of many bioactive natural products.

TetR family transcriptional regulators (TFRs) are found in most bacteria and archea. Most of the family members that have been investigated to date are repressors of their target genes, and the majority of these, like the well-characterized protein TetR, regulate genes that encode transmembrane efflux pumps. In many cases repression by TFR proteins is reversed through the direct binding of a small-molecule ligand. The number of TFRs in the public database has grown rapidly as a result of genome sequencing and there are now thousands of family members; however virtually nothing is known about the biology and biochemistry they regulate. Generally applicable methods for predicting their regulatory targets would assist efforts to characterize the family. Here, we investigate chromosomal context of 372 TFRs from three Streptomyces species. We find that the majority (250 TFRs) are transcribed divergently from one neighboring gene, as is the case for TetR and its target tetA. We explore predicted target gene product identity and intergenic separation to see which either correlates with a direct regulatory relationship. While intergenic separation is a critical factor in regulatory prediction the identity of the putative target gene product is not. Our data suggest that those TFRs that are <200 bp from their divergently oriented neighbors are most likely to regulate them. These target genes include membrane proteins (26% of which 22% are probable membrane-associated pumps), enzymes (60%), other proteins such as transcriptional regulators (1%), and proteins having no predictive sequence motifs (13%). In addition to establishing a solid foundation for identifying targets for TFRs of unknown function, our analysis demonstrates a much greater diversity of TFR-regulated biochemical functions.

SummaryBecause most antibiotics are potentially lethal to the producing organism, there must be mechanisms to ensure that the machinery responsible for export of the mature antibiotic is in place at the time of biosynthesis. Simocyclinone D8 is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü 6040. Within the simocyclinone biosynthetic cluster are two divergently transcribed genes, simR and simX, encoding proteins that resemble the TetR/TetA repressor-efflux pump pair that cause widespread resistance to clinically important tetracyclines. Engineered expression of simX from a strong, heterologous promoter conferred high level simocyclinone D8 resistance on Streptomyces lividans, showing that simX encodes a simocyclinone efflux pump. Transcription of simX is controlled by SimR, which directly represses the simX and simR promoters by binding to two operator sites in the simX-simR intergenic region. Simocyclinone D8 abolishes DNA binding by SimR, providing a mechanism that couples the biosynthesis of simocyclinone to its export. In addition, an intermediate in the biosynthetic pathway, simocyclinone C4, which is essentially inactive as a DNA gyrase inhibitor, also induces simX expression in vivo and relieves simX repression by SimR in vitro.

Nutrient acquisition systems are often crucial for pathogen growth and survival during infection, and represent attractive therapeutic targets. Here, we study the protein machinery required for heme uptake in the opportunistic pathogen Acinetobacter baumannii. We show that the hemO locus, which includes a gene encoding the heme-degrading enzyme, is required for high-affinity heme acquisition from hemoglobin and serum albumin. The hemO locus includes a gene coding for a heme scavenger (HphA), which is secreted by a Slam protein. Furthermore, heme uptake is dependent on a TonB-dependent receptor (HphR), which is important for survival and/or dissemination into the vasculature in a mouse model of pulmonary infection. Our results indicate that A. baumannii uses a two-component receptor system for the acquisition of heme from host heme reservoirs.

Safe and effective vaccines are needed to end the COVID-19 pandemic. Here, we report the preclinical development of a lipid nanoparticle–formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern, including the Alpha, Beta, Gamma, and Delta lineages. No adverse effects were induced by PTX-COVID19-B in either mice or hamsters. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 2 clinical trial ongoing.