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.

Yeast Rrp8p, a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA.

Ribosomal RNA undergoes various modifications to optimize ribosomal structure and expand the topological potential of RNA. The most common nucleotide modifications in ribosomal RNA (rRNA) are pseudouridylations and 2'-O methylations (Nm), performed by H/ACA box snoRNAs and C/D box snoRNAs, respectively. Furthermore, rRNAs of both ribosomal subunits also contain various base modifications, which are catalysed by specific enzymes. These modifications cluster in highly conserved areas of the ribosome. Although most enzymes catalysing 18S rRNA base modifications have been identified, little is known about the 25S rRNA base modifications. The m(1)A modification at position 645 in Helix 25.1 is highly conserved in eukaryotes. Helix formation in this region of the 25S rRNA might be a prerequisite for a correct topological framework for 5.8S rRNA to interact with 25S rRNA. Surprisingly, we have identified ribosomal RNA processing protein 8 (Rrp8), a nucleolar Rossman-fold like methyltransferase, to carry out the m(1)A base modification at position 645, although Rrp8 was previously shown to be involved in A2 cleavage and 40S biogenesis. In addition, we were able to identify specific point mutations in Rrp8, which show that a reduced S-adenosyl-methionine binding influences the quality of the 60S subunit. This highlights the dual functionality of Rrp8 in the biogenesis of both subunits.

Genetic interactions of yeast NEP1 (EMG1), encoding an essential factor in ribosome biogenesis.

Nep1 methylates the hypermodified ψ1191 base of 18S rRNA and has an additional essential function during ribosome biogenesis. It is strongly conserved in eukaryotes and a point mutation causes the human Bowen-Conradi syndrome. To identify Δnep1-specific genetic interactions, viable deletions were screened genome-wide (SGA). Due to its essential function, we used, for the first time, query strain (Δnep1) with two additive suppressor conditions (mcRPS19B, nop6-1). Nep1 interacting genes correspond to ribosome biogenesis (RPS18A, RPS18B, RRP8, EFG1, UTP30), to ribosome quality control (UBP3, BRE5, UBP6) and to ribosome functional control (DOM34, no-go decay). Deletions in ribosome quality and functional control genes were synthetically sick with Δnep1. They cope with malfunctions and the respective deletions strengthen the Δnep1 growth deficiency. Except for Δrps18b, deletions in the identified ribosome biogenesis genes were synthetically lethal with Δnep1. While the synthetic lethalities of Δrrp8 and Δefg1 may result from additive defects, the Δutp30 deletion seems to be in close functional relationship. The Δutp30 deletion itself has no phenotype but it enforced all nep1-1(ts) mutant phenotypes. Furthermore, its overexpression partially restored the nep1-1(ts) growth deficiency. Our genetic and biochemical data suggest that Utp30 and Nep1 act together during pre-ribosomal complex formation and, along with Rps18, provide the surface for the Rps19 assembly to the 90S pre-ribosome.