Scadden Jacob, Ansorge Rebecca, Romano Stefano, Telatin Andrea, Baker Dave J, Evans Rhiannon, Gherghisan-Filip Cristina, Zhang Zhenrun J, Mayer Melinda J, Narbad Arjan
Quadram Institute Bioscience, Norwich Research Park, Norwich, UK.
Present address: School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia.
Microbiology (Reading). 2025 Feb;171(2). doi: 10.1099/mic.0.001531.
Nisin O is an antimicrobial peptide encoded by the human gut bacterium A2-162 which has antimicrobial activity against clinically relevant organisms. The nisin O biosynthetic gene cluster (BGC) varies from other nisin BGCs as it lacks a leader-peptide cleaving protease and contains two bacterial two-component response regulator-histidine kinase (RK) systems. The dissemination of the nisin O cluster, the final proteolytic biosynthesis step and the regulation of nisin O are currently unknown and are the foci of this study. We identified six nisin O-like BGCs across , and species using comparative genomics. These BGCs show evidence of genetic transfer between genera, with genes involved in transposition discovered up- and downstream of the BGCs. All nisin O-like BGCs contained two RK systems but no protease. Mining the A2-162 genome identified candidate proteases that were cloned and used in pre-nisin O leader peptide cleavage assays. None of the candidate proteases removed the leader; however, cleavage was achieved using trypsin. To maximize the expression of the peptides, the interactions of the two RK systems with predicted promoters in the nisin O cluster were assessed using a PepI reporter assay. We observed that the P promoter was constitutively expressed, with NsoR1K1 increasing its activity, and that there was increased expression when the nisin A RK system and nisin A were present. Long-read cDNA sequencing confirmed gene transcription in the heterologous expression system and identified a novel, highly expressed gene. This study provides evidence that the nisin O BGC has been transferred between different gut-associated genera, with all clusters lacking a protease and containing two RK systems. We hypothesize that this BGC has lost its protease due to negative selection as a result of high trypsin concentrations in the gut. Further work is required to maximize nisin O expression for it to be used as a potential antimicrobial therapy.
乳链菌肽O是一种由人类肠道细菌A2-162编码的抗菌肽，对临床相关微生物具有抗菌活性。乳链菌肽O生物合成基因簇（BGC）与其他乳链菌肽BGC不同，因为它缺乏前导肽切割蛋白酶，并且包含两个细菌双组分反应调节因子-组氨酸激酶（RK）系统。目前尚不清楚乳链菌肽O基因簇的传播、最终的蛋白水解生物合成步骤以及乳链菌肽O的调控，而这些正是本研究的重点。我们通过比较基因组学在多个物种中鉴定出了六个类乳链菌肽O的BGC。这些BGC显示出属间基因转移的证据，在BGC的上下游发现了与转座相关的基因。所有类乳链菌肽O的BGC都包含两个RK系统，但没有蛋白酶。对A2-162基因组进行挖掘，鉴定出了候选蛋白酶，将其克隆并用于前乳链菌肽O前导肽切割试验。没有一种候选蛋白酶能够切割前导肽；然而，使用胰蛋白酶可以实现切割。为了使肽的表达最大化，使用PepI报告基因试验评估了两个RK系统与乳链菌肽O基因簇中预测启动子的相互作用。我们观察到P启动子组成性表达，NsoR1K1可增加其活性，并且当存在乳链菌肽A的RK系统和乳链菌肽A时，表达会增加。长读长cDNA测序证实了异源表达系统中的基因转录，并鉴定出一个新的高表达基因。本研究提供了证据表明乳链菌肽O基因簇已在不同的肠道相关属之间转移，所有基因簇都缺乏蛋白酶且包含两个RK系统。我们推测，由于肠道中胰蛋白酶浓度较高，该基因簇因负选择而失去了蛋白酶。需要进一步开展工作以最大化乳链菌肽O的表达，使其能够用作潜在的抗菌疗法。
