Characterization of hypothetical proteins reveals function modules and virulence factors in Pseudomonas aeruginosa.
9 affiliations
- 1State Key Laboratory of Agricultural and Forestry Biosecurity, The Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing, China.
- 2Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Science and Technology Research on Fruit Tree, Guangzhou, China.
- 3Department of Biomedical Sciences, City University of Hong Kong, Kowloon Tong, Hong Kong SAR, China.
- 4College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China.
- 5School of Biological Sciences, The University of Hong Kong, Hong Kong SAR, China.
- 6State Key Laboratory of Agricultural and Forestry Biosecurity, The Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing, China. [email protected].
- 7Department of Biomedical Sciences, City University of Hong Kong, Kowloon Tong, Hong Kong SAR, China. [email protected].
- 8Department of Biomedical Sciences, City University of Hong Kong, Kowloon Tong, Hong Kong SAR, China. [email protected].
- 9Shenzhen Research Institute, City University of Hong Kong, Shenzhen, Guangdong, China. [email protected].
Abstract
Hypothetical proteins (HPs), known as biological dark matter, are genetic sequences of which the biological functions and regulatory processes remain largely unclear. In Pseudomonas aeruginosa, HPs account for 36.4% of all coding genes, representing a significant gap in understanding their functions and contribution to the virulence network. To address these challenges, we present a deletion repertoire comprising 938 strains targeting 1165 HPs in P. aeruginosa. Starting with transcriptome-wide analysis, we inferred putative functional predictions for HP mutants through systematic profiling of differential expression and candidate transcriptional pathways. We integrated our expression profiles with public datasets (5707 genes × 2312 conditions). This allowed us to distinguish constitutively expressed genes from variable genes. Using independent component analysis (ICA), we identified 56 independent regulatory Modules, providing a comprehensive transcriptional-association framework. These contributed to the discovery and experimental validation of 20 virulence-related hypothetical proteins (VRHPs) and 3 antibiotic resistance-related hypothetical proteins (ARHPs). Evolutionary analysis across bacterial taxa provided additional insights into HP sequence conservation and evolutionary dynamics. In summary, our study presents a large-scale HP mutant library combined with integrated transcriptomic and machine learning approaches, establishing both a valuable resource and a systematic strategy for predicting and prioritizing candidate functions of HPs across bacterial species.
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BibTeX
@article{yao2026characterization,
title = {{Characterization of hypothetical proteins reveals function modules and virulence factors in Pseudomonas aeruginosa.}},
author = {Chunyan Yao and Beifang Lu and Yiqing Ding and Letong Xu and Jingui Liu and Yingchao Zhang and Jiadai Huang and Yue Sun and Na Liu and Fang Chen and Shumin Li and Tianmin Li and Tongtong Feng and Youyue Li and Guoliang Qian and Aixin Yan and Xiaolong Shao and Xin Deng},
journal = {Nature communications},
year = {2026},
month = sep,
doi = {10.1038/s41467-026-77512-1},
url = {https://europepmc.org/article/MED/42897133},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Characterization of hypothetical proteins reveals function modules and virulence factors in Pseudomonas aeruginosa.
AU - Chunyan Yao
AU - Beifang Lu
AU - Yiqing Ding
AU - Letong Xu
AU - Jingui Liu
AU - Yingchao Zhang
AU - Jiadai Huang
AU - Yue Sun
AU - Na Liu
AU - Fang Chen
AU - Shumin Li
AU - Tianmin Li
AU - Tongtong Feng
AU - Youyue Li
AU - Guoliang Qian
AU - Aixin Yan
AU - Xiaolong Shao
AU - Xin Deng
PY - 2026
DA - 2026/09/17
JO - Nature communications
DO - 10.1038/s41467-026-77512-1
UR - https://europepmc.org/article/MED/42897133
AB - Hypothetical proteins (HPs), known as biological dark matter, are genetic sequences of which the biological functions and regulatory processes remain largely unclear. In Pseudomonas aeruginosa, HPs account for 36.4% of all coding genes, representing a significant gap in understanding their functions and contribution to the virulence network. To address these challenges, we present a deletion repertoire comprising 938 strains targeting 1165 HPs in P. aeruginosa. Starting with transcriptome-wide analysis, we inferred putative functional predictions for HP mutants through systematic profiling of differential expression and candidate transcriptional pathways. We integrated our expression profiles with public datasets (5707 genes × 2312 conditions). This allowed us to distinguish constitutively expressed genes from variable genes. Using independent component analysis (ICA), we identified 56 independent regulatory Modules, providing a comprehensive transcriptional-association framework. These contributed to the discovery and experimental validation of 20 virulence-related hypothetical proteins (VRHPs) and 3 antibiotic resistance-related hypothetical proteins (ARHPs). Evolutionary analysis across bacterial taxa provided additional insights into HP sequence conservation and evolutionary dynamics. In summary, our study presents a large-scale HP mutant library combined with integrated transcriptomic and machine learning approaches, establishing both a valuable resource and a systematic strategy for predicting and prioritizing candidate functions of HPs across bacterial species.
SN - 2041-1723
ER -