Zareen Maryam, Baig Deeba Noreen, Imran Muhammad, Khaliq Zabish, Malik Kausar Abdulla, Bechthold Andreas, Mehnaz Samina
KAM School of Life Sciences, Forman Christian College (A Chartered University), Ferozepur Road, Lahore, 54600, Pakistan.
Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs- Universitat Freiburg, Freiburg, 79104, Germany.
Arch Microbiol. 2025 May 8;207(6):138. doi: 10.1007/s00203-025-04345-9.
Pyocins are bacteriocins which are explicitly associated with pseudomonads. In this study, the genome mining and in-depth sequence analysis identified three similar S9-like (a, b, and c), an S3-like (d) and one R-type pyocin systems from P. chlororaphis subsp aurantiaca PB-St2. The phenotypic screening of bacteriocin production by PB-St2 indicated narrow-spectrum bactericidal activity against closely related Pseudomonas species i.e., Pseudomonas aeruginosa PAi, PAc1, PAc3, PAc4; Pseudomonas fluorescens Psi-RS1 and Pseudomonas kilonensis OSRS3. Herein, the proposed pyocin S9c was further selected for molecular and functional characterization. The presumptive N-terminal receptor binding domain of candidate system lacks significant similarity with any characterized HNH-type pyocin S DNases from P. aeruginosa. In contrast, the cytotoxic domain showed 53% sequence similarity with pyocin S8 and 70% to pyocin S9. Thus, pyocin S9c was suggested as an isoform under the Class I DNase (H-N-H) family in pyocin S9 cluster, commonly found in P. chlororaphis subsp. aurantiaca and P. chlororaphis subsp aureofaciens strains. Molecular screening of the pyocin S9c system revealed its presence in 6 out of 7 tested strains of P. chlororaphis subsp. aurantiaca GS1, GS3, GS4, GS6, ARS38, FS2 and one P. chlororaphis RP4 relative strains, isolated from diverse plant hosts. The 1.59 kb fragment consisting of two structural genes of pyocin-immunity operon (S9c) in P. aurantiaca PB-St2 were cloned in pET28a(+) and expressed in Escherichia coli BL21 DE3 (pLysS) strain as a fusion protein with histidine tag. The recombinant cytotoxic protein of pyocin S9c operon was purified with N-term His-tag with a molecular weight of ≈ 50 kDa. The identity of target protein was affirmed by tandem mass spectrometry analysis. The purified cytotoxic protein was active against P. chlororaphis subsp. aurantiaca GS7, with a minimum inhibitory concentration of 12.5 µg/ml. The mechanism of cytotoxicity was affirmed as a metal-dependent endonuclease by evidence of non-specific hydrolysis of pTZ57R plasmid isoforms. These results indicate that pyocin S9c can contribute to the rhizo-competence of this strain in plant-associated natural habitats, occupied by related Pseudomonas strains.
绿脓菌素是与假单胞菌明确相关的细菌素。在本研究中，通过基因组挖掘和深入的序列分析，从绿针假单胞菌亚种金黄色变种PB-St2中鉴定出三种相似的S9样（a、b和c）、一种S3样（d）和一种R型绿脓菌素系统。PB-St2产生细菌素的表型筛选表明，其对密切相关的假单胞菌具有窄谱杀菌活性，即铜绿假单胞菌PAi、PAc1、PAc3、PAc4；荧光假单胞菌Psi-RS1和基洛假单胞菌OSRS3。在此，进一步选择了拟绿脓菌素S9c进行分子和功能表征。候选系统推定的N端受体结合结构域与来自铜绿假单胞菌的任何已表征的HNH型绿脓菌素S DNase均无显著相似性。相比之下，细胞毒性结构域与绿脓菌素S8的序列相似性为53%，与绿脓菌素S9的序列相似性为70%。因此，绿脓菌素S9c被认为是绿脓菌素S9簇中I类DNase（H-N-H）家族的一种亚型，常见于绿针假单胞菌亚种金黄色变种和绿针假单胞菌亚种金霉素菌株中。对绿脓菌素S9c系统的分子筛选显示，在从不同植物宿主中分离出的7株绿针假单胞菌亚种金黄色变种GS1、GS3、GS4、GS6、ARS38、FS2和1株绿针假单胞菌RP4相关菌株中，有6株存在该系统。将绿针假单胞菌亚种金黄色变种PB-St2中由绿脓菌素-免疫操纵子（S9c）的两个结构基因组成的1.59 kb片段克隆到pET28a(+)中，并在大肠杆菌BL21 DE3（pLysS）菌株中作为带有组氨酸标签的融合蛋白进行表达。用N端His标签纯化了绿脓菌素S9c操纵子的重组细胞毒性蛋白，其分子量约为50 kDa。通过串联质谱分析确认了目标蛋白的身份。纯化的细胞毒性蛋白对绿针假单胞菌亚种金黄色变种GS7具有活性，最低抑菌浓度为12.5 μg/ml。通过pTZ57R质粒异构体的非特异性水解证据，确认细胞毒性机制为金属依赖性核酸内切酶。这些结果表明，绿脓菌素S9c可有助于该菌株在由相关假单胞菌菌株占据的植物相关自然生境中的根际竞争力。
