Genome Mining Enabled by Biosynthetic Characterization Uncovers a Class of Benzoxazolinate-Containing Natural Products in Diverse Bacteria.

Benzoxazolinate is a rare bis-heterocyclic moiety that interacts with proteins and DNA and confers extraordinary bioactivities on natural products, such as C-1027. However, the biosynthetic gene responsible for the key cyclization step of benzoxazolinate remains unclear. Herein, we show a putative acyl AMP-ligase responsible for the last cyclization step. We used the enzyme as a probe for genome mining and discovered that the orphan benzobactin gene cluster in entomopathogenic bacteria prevails across Proteobacteria and Firmicutes. It turns out that Pseudomonas chlororaphis produces various benzobactins, whose biosynthesis is highlighted by a synergistic effect of two unclustered genes encoding enzymes on boosting benzobactin production; the formation of non-proteinogenic 2-hydroxymethylserine by a serine hydroxymethyltransferase; and the types I and II NRPS architecture for structural diversity. Our findings reveal the biosynthetic potential of a widespread benzobactin gene cluster.

SREBP2-dependent lipid gene transcription enhances the infection of human dendritic cells by Zika virus.

The emergence of Zika virus (ZIKV) as a global health threat has highlighted the unmet need for ZIKV-specific vaccines and antiviral treatments. ZIKV infects dendritic cells (DC), which have pivotal functions in activating innate and adaptive antiviral responses; however, the mechanisms by which DC function is subverted to establish ZIKV infection are unclear. Here we develop a genomics profiling method that enables discrete analysis of ZIKV-infected versus neighboring, uninfected primary human DCs to increase the sensitivity and specificity with which ZIKV-modulated pathways can be identified. The results show that ZIKV infection specifically increases the expression of genes enriched for lipid metabolism-related functions. ZIKV infection also increases the recruitment of sterol regulatory element-binding protein (SREBP) transcription factors to lipid gene promoters, while pharmacologic inhibition or genetic silencing of SREBP2 suppresses ZIKV infection of DCs. Our data thus identify SREBP2-activated transcription as a mechanism for promoting ZIKV infection amenable to therapeutic targeting.

Global analysis of biosynthetic gene clusters reveals conserved and unique natural products in entomopathogenic nematode-symbiotic bacteria.

Microorganisms contribute to the biology and physiology of eukaryotic hosts and affect other organisms through natural products. Xenorhabdus and Photorhabdus (XP) living in mutualistic symbiosis with entomopathogenic nematodes generate natural products to mediate bacteria-nematode-insect interactions. However, a lack of systematic analysis of the XP biosynthetic gene clusters (BGCs) has limited the understanding of how natural products affect interactions between the organisms. Here we combine pangenome and sequence similarity networks to analyse BGCs from 45 XP strains that cover all sequenced strains in our collection and represent almost all XP taxonomy. The identified 1,000 BGCs belong to 176 families. The most conserved families are denoted by 11 BGC classes. We homologously (over)express the ubiquitous and unique BGCs and identify compounds featuring unusual architectures. The bioactivity evaluation demonstrates that the prevalent compounds are eukaryotic proteasome inhibitors, virulence factors against insects, metallophores and insect immunosuppressants. These findings explain the functional basis of bacterial natural products in this tripartite relationship.

The chloramphenicol/H+ antiporter CraA of Acinetobacter baumannii AYE reveals a broad substrate specificity.

OBJECTIVES: To identify major facilitator superfamily (MFS)-type chloramphenicol transporters of Acinetobacter baumannii AYE, to characterize its substrate specificity and identify CraA substrate and H+ binding sites. METHODS: Five ORFs predicted to encode chloramphenicol transporters were heterologously expressed in Escherichia coli and their substrate specificity was determined by drug susceptibility assays on solid agar medium. CraA transport properties were determined via whole cell fluorescence experiments using ethidium and dequalinium. ACMA quenching was used to characterize the H+/drug antiport process in everted membrane vesicles. The function of CraA in A. baumannii was determined by drug susceptibility assay using A. baumannii ATCC 19606 ΔcraA. RESULTS: CraA, ABAYE0913 and CmlA5 are functionally active when overproduced in E. coli. ABAYE0913 conferred resistance to florfenicol and benzalkonium, CmlA5 conferred resistance to chloramphenicol and thiamphenicol, and craA expression resulted in resistance to chloramphenicol, thiamphenicol, florfenicol, ethidium, dequalinium, chlorhexidine, benzalkonium, mitomycin C and TPP+. Cell expressing craA_E38A showed no resistance to all tested drugs, implying that Glu-38 is involved in the binding of drugs and/or protons. Functional assays indicated that substitution of Asp-46 to Ala resulted in severe susceptibility to cationic drugs, chloramphenicol and thiamphenicol. In contrast, Glu-338 is important for the recognition of chloramphenicol, florfenicol, chlorhexidine and dequalinium. CONCLUSIONS: This study suggests that CraA has a broad substrate specificity, similar to that of E. coli MdfA. However, due to the presence of three charged residues in the transmembrane region conferring different susceptibility profiles upon substitution to Ala, we postulate that CraA has a different substrate recognition mode compared with MdfA.