Aehsas Sujaan, Sahoo Jyoti Prakash, Choudhury Neelanjana, Hossain Shaikh Nausad, Sahoo Anjali Lata, Sunani Sunil Kumar
Department of Biotechnology, Faculty of Agriculture and Allied Sciences, C.V. Raman Global University, Bhubaneswar, 752054, India.
Regional Institute of Biotechnology, Bhubaneswar, 751016, India.
Mol Biol Rep. 2025 Jul 12;52(1):706. doi: 10.1007/s11033-025-10796-6.
The study aimed to investigate the microbial diversity of soils from distinct environments, an urban park (SJ1) and an agricultural research field (SJ2) using serial dilution and spread plate techniques on nutrient agar, to identify region-specific bacterial strains with potential agricultural or biotechnological applications.
Bacterial isolates were purified through selective sub-culturing, characterized via Gram staining and biochemical tests (MR-VP, catalase, oxidase), and preserved at 4 °C. Genomic DNA was extracted, PCR-amplified targeting the 16S rRNA gene, and sequenced for BLAST-based identification. Phylogenetic analysis (MEGA 12, neighbor-joining, 1000 bootstraps) and evolutionary rate estimation (maximum likelihood, gamma-distributed site variation) were performed to assess genetic relationships and divergence. Morphological and biochemical analyses revealed Gram-positive rods in both isolates, exhibiting catalase positive, oxidase negative, methyl red negetive, and Voges-Proskauer positive activity. Genomic DNA was extracted, and the 16S rRNA gene was amplified (~ 1500 bp), sequenced, and analyzed via BLASTN. SJ1 showed 99.84-99.92% identity with Bacillus spp., while SJ2 matched Priestia megaterium (99.92%). Phylogenetic analysis placed SJ1 within Bacillus subtilis strain SBIS1 (GenBank: PV523294.1), and SJ2 within P. megaterium strain PIIS1 (GenBank: PV523528.1), indicating distinct evolutionary lineages. Maximum likelihood estimation revealed differing evolutionary rates, i.e., SJ1 exhibited high rate heterogeneity, while SJ2 showed uniform rates. Mean relative evolutionary rates across five discrete gamma categories for SJ1 ranged from 0.08 to 2.75, while for SJ2 they ranged from 0.90 to 1.10. SJ1 showed 25.23% (A), 20.09% (T), 23.43% (C), and 31.26% (G); while SJ2 had 24.87% (A), 21.26% (T), 24.20% (C), and 29.68% (G). Maximum log-likelihood values were -3315.740 for SJ1 (1268 aligned sites) and -1034.491 for SJ2 (750 aligned sites), confirming robust phylogenetic inference.
The study successfully identified distinct bacterial strains with unique evolutionary rates, genetic compositions, and robust divergence between the isolates, supporting their suitability for further agricultural or biotechnological exploration.
本研究旨在利用营养琼脂上的系列稀释和平板涂布技术，调查来自不同环境的土壤微生物多样性，即城市公园（SJ1）和农业研究田（SJ2）的土壤，以鉴定具有潜在农业或生物技术应用的区域特异性细菌菌株。
通过选择性继代培养纯化细菌分离株，通过革兰氏染色和生化试验（MR-VP、过氧化氢酶、氧化酶）进行表征，并保存在4°C。提取基因组DNA，针对16S rRNA基因进行PCR扩增，并进行测序以基于BLAST进行鉴定。进行系统发育分析（MEGA 12，邻接法，1000次重复）和进化速率估计（最大似然法，伽马分布的位点变异）以评估遗传关系和差异。形态学和生化分析显示，两种分离株均为革兰氏阳性杆菌，表现出过氧化氢酶阳性、氧化酶阴性、甲基红阴性和Voges-Proskauer阳性活性。提取基因组DNA，扩增16S rRNA基因（约1500 bp），测序并通过BLASTN分析。SJ1与芽孢杆菌属的同一性为99.84-99.92%，而SJ2与巨大Priestia菌匹配（99.92%）。系统发育分析将SJ1置于枯草芽孢杆菌菌株SBIS1（GenBank：PV523294.1）内，将SJ2置于巨大Priestia菌菌株PIIS1（GenBank：PV523528.1）内，表明它们具有不同的进化谱系。最大似然估计显示进化速率不同，即SJ1表现出高速率异质性，而SJ2表现出均匀速率。SJ1在五个离散伽马类别中的平均相对进化速率范围为0.08至2.75，而SJ2的范围为0.90至1.10。SJ1显示25.23%（A）、20.09%（T）、23.43%（C）和31.26%（G）；而SJ2分别为24.87%（A）、21.26%（T）、24.20%（C）和29.68%（G）。SJ1（1268个比对位点）的最大对数似然值为-3315.740，SJ2（750个比对位点）的最大对数似然值为-1034.491，证实了可靠的系统发育推断。
本研究成功鉴定出具有独特进化速率、遗传组成且分离株之间存在明显差异的不同细菌菌株，支持它们适合进一步的农业或生物技术探索。
