Oxydifficidin is a natural polyketide antibiotic that has long been recognized as a ribosome-targeting agent that inhibits protein synthesis. In this paper, we describe Bacillus velezensis strain EV17 and compare its complete genome sequence with that of the previously characterized B. velezensis strain K-3618 and the difficidin biosynthetic gene cluster (BGC) combined with mass spectrometry to elucidate the production of oxydifficidin by these strains. Toeprinting and small fluorescent peptide assays showed that isolated oxydifficidin induces a generalized inhibition of translation at every step of elongation in protein biosynthesis. In previous studies, it has been demonstrated that oxydifficidin targets bL12 protein. Although spontaneous mutations conferring resistance to oxydifficidin in ribosomal protein bL12 located relatively close to the thiostrepton binding site on uL11, our data show that oxydifficidin binding does not interfere with thiostrepton, thereby refining previous findings about its putative ribosomal target. We are the first to show that this compound does not affect eukaryotic translation and has two orders of magnitude lower effect on eukaryotic cells compared to bacteria. These facts are important to further investigate its potential as a bioprotectant against phytopathogens or even as a therapeutic agent.

In our large-scale search for antimicrobial-producing bacteria, we isolated an actinomycete strain from rhizospheric soil of Bambusa vulgaris. The strain designated BP-8 showed noticeable antibacterial activity. BP-8 was subjected to a whole-genome analysis via a polyphasic taxonomy approach, and its antibacterial metabolite was identified by HRLS-MS. The results of the physiological and morphological analyses indicated that BP-8 is an aerobic, neutrophilic, mesophilic organism that is tolerant to 8% NaCl and can use a wide range of carbohydrates. It forms curly sporophores with a warty surface. The results of the phylogenetic and average nucleotide identity analyses and in silico DNA–DNA hybridization calculation indicated that BP-8 represents the type strain of a novel Streptomyces species. A comparative in silico analysis of the genome sequences of BP-8 and its closest related strains revealed the presence of genes encoding chemotaxonomic markers characteristic of Streptomyces. The antibacterial compound was identified as amicetin. Genomic mining also revealed more than 10 biosynthetic gene clusters that have not been described previously and may lead to the discovery of new valuable compounds. On the basis of these results, strain BP-8^T (=VKM Ac-3066^T = CCTCC AA 2024094^T) is proposed as the type strain of the novel species Streptomyces sirii sp. nov.

Oxydifficidin is a natural polyketide antibiotic that is a long-known ribosome-targeting antibiotic that inhibits protein synthesis. In this paper, we describe Bacillus velezensis strain EV17 and compare its complete genome sequence with that of the previously characterized strain K-3618 and the difficidin biosynthetic gene cluster (BGC) combined with mass spectrometry, to elucidate the production of oxydifficidin by these strains. Isolated oxydifficidin was determined to increase generalized inhibition of translation at each step of protein biosynthesis using toeprinting and small fluorescent peptide assays. In previous studies, it has been demonstrated that oxydifficidin targets L7/L12 protein. Although spontaneous mutations in ribosomal protein L7/L12, located relatively close to the thiostrepton binding site on uL11, confer resistance to oxydifficidin, our data show that oxydifficidin binding does not interfere with thiostrepton, thereby refining previous findings about its putative ribosomal target. We show that this compound does not affect eukaryotic translation and has no toxic effect on eukaryotic cells. These facts are important to further investigate its potential as a bioprotectant against phytopathogens or even as a therapeutic activity.

Antimicrobial resistance is a major threat to modern society and healthcare, as it severely compromises the efficacy of standard antibiotic treatments. To meet the ever-increasing demand for novel antimicrobial drugs, it is crucial to develop new strategies for screening antimicrobial compounds and improve existing high-throughput techniques. Reporter systems that employ specific genetic markers are powerful tools not only for detecting antimicrobial activity of the substance being studied, but also for identifying the potential mechanism of its action. Among other metabolic pathways, RNA biosynthesis machinery is considered a promising molecular target as it remains underutilized in current antimicrobial therapy and therefore is rarely exposed to drug pressure. However, there is no suitable biomarker for identifying compounds that inhibit the transcription in Gram-negative bacteria. Combining bioinformatic search and RT-qPCR experimental validation, we have established the overexpression of the spermidine synthase gene (speE) as a biomarker associated with impaired transcription in Escherichia coli. Monitoring the expression level of speE in antibiotic-treated cells enables reliable detection of compounds that inhibit bacterial RNA-polymerase, such as rifampicin and fidaxomicin. Moreover, our screening system was successfully applied in practice to analyze chromatography fractions from fermentation broth of antibiotic producers, with compounds of the rifamycin family being identified as hits and isolated. The proposed method has the potential to be used in sequential screening procedures to reveal active antimicrobial compounds that inhibit bacterial transcription process, giving the world novel antimicrobials with minimal risk of resistance development.