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Functional and structural diversification of incomplete phosphotransferase system in cellulose-degrading clostridia

The ISME Journal volume 17, pages 823–835 (2023)Cite this article

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Abstract

Carbohydrate utilization is critical to microbial survival. The phosphotransferase system (PTS) is a well-documented microbial system with a prominent role in carbohydrate metabolism, which can transport carbohydrates through forming a phosphorylation cascade and regulate metabolism by protein phosphorylation or interactions in model strains. However, those PTS-mediated regulated mechanisms have been underexplored in non-model prokaryotes. Here, we performed massive genome mining for PTS components in nearly 15,000 prokaryotic genomes from 4,293 species and revealed a high prevalence of incomplete PTSs in prokaryotes with no association to microbial phylogeny. Among these incomplete PTS carriers, a group of lignocellulose degrading clostridia was identified to have lost PTS sugar transporters and carry a substitution of the conserved histidine residue in the core PTS component, HPr (histidine-phosphorylatable phosphocarrier). Ruminiclostridium cellulolyticum was then selected as a representative to interrogate the function of incomplete PTS components in carbohydrate metabolism. Inactivation of the HPr homolog reduced rather than increased carbohydrate utilization as previously indicated. In addition to regulating distinct transcriptional profiles, PTS associated CcpA (Catabolite Control Protein A) homologs diverged from previously described CcpA with varied metabolic relevance and distinct DNA binding motifs. Furthermore, the DNA binding of CcpA homologs is independent of HPr homolog, which is determined by structural changes at the interface of CcpA homologs, rather than in HPr homolog. These data concordantly support functional and structural diversification of PTS components in metabolic regulation and bring novel understanding of regulatory mechanisms of incomplete PTSs in cellulose-degrading clostridia.

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Fig. 1: Architectural and compositional diversification of phosphotransferase systems (PTS) in prokaryotes.
Fig. 2: The mini “PTS” of R. cellulolyticum is essential for microbial metabolism.
Fig. 3: Distinct roles of the mini “PTS” components in carbohydrate dependent transcriptional regulation.
Fig. 4: Distinct DNA binding profiles of R. cellulolyticum CcpA homologs that are independent of Cph and its phosphorylation status.
Fig. 5: Structural basis of Cph-independent regulation by CcpA and CcpB in R. cellulolyticum.

Data availability

Raw sequences of the ChIP-seq experiments are available in the NCBI SRA database (http://www.ncbi.nlm.nih.gov/sra) under accession number PRJNA748961. The MS proteomic data of IP-MS or phosphorylation determination are deposited in the ProteomeXchange consortium (http://www.proteomexchange.org/) via the iProX partner repository with the accession number PXD027554. Microarray data are deposited in the ArrayExpress under accession number E-MTAB-11755 and E-MTAB-11774. The structures of CcpB and Cph have been deposited to the Protein Data Bank (PDB) with accession numbers 7FF4 and 7FF5, respectively. Our in-house protein database can be accessed through http://www.ou.edu/ieg/publications/datasets. All other relevant data are available in Supplementary Information. Source data are provided with this paper.

Code availability

The genome mining and other analysis codes have been deposited in GitHub (https://github.com/adarobot/PTS).

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Acknowledgements

Genome mining, construction of mutant strains, phenotypic and omics experiments were supported by the Office of the Vice President for Research at the University of Oklahoma to JZ. Biochemical assays, crystallization, and structure determination were supported by the National Natural Science Foundation of China (Grant No. 31971422) to YH and (Grant No.31970103) to HG, and Key Research and Development Plan Project of Anhui Province (grant No. 202004a06020035) to NZ. We thank SSRF beamline BL18U1 for synchrotron data collection. We also thank the staff for providing technical support by using the facility of the Institute of Health Sciences and Technology of Anhui University.

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  1. These authors contributed equally: Tao Xu, Xuanyu Tao.

Authors and Affiliations

  1. Section on Pathophysiology and Molecular Pharmacology, Joslin Diabetes Center, Boston, MA, USA

    Tao Xu

  2. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA

    Tao Xu

  3. Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, USA

    Tao Xu, Xuanyu Tao, Megan L. Kempher, Daliang Ning & Jizhong Zhou

  4. School of Life Sciences, Anhui University, Hefei, 230601, PR China

    Hongxi He, Xiaochun Liu, Jun Wang, Honghua Ge & Nannan Zhang

  5. Institutes of Material Science and Information Technology, Anhui University, Hefei, 230601, PR China

    Hongxi He, Xiaochun Liu, Jun Wang, Honghua Ge & Nannan Zhang

  6. Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou, 730000, PR China

    Siping Zhang & Yong-Xing He

  7. Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, USA

    Dongyu Wang & Chongle Pan

  8. School of computer science, University of Oklahoma, Norman, OK, USA

    Chongle Pan

  9. School of Civil Engineering and Environmental Sciences, University of Oklahoma, Norman, OK, USA

    Jizhong Zhou

  10. Earth and Environmental Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Jizhong Zhou

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Contributions

TX, XT, and JZ developed the original concepts. TX, XT, YH, and NZ designed the experiments. TX conducted the genome mining analyses. XT and TX carried out the experiments for mutant construction, phenotype determination, microarray and ChIP-seq. XT, DW, and CP carried out the IP-MS experiment. HH, SZ, XL, JW, NZ, and YH carried out all biochemical experiments including protein purification, EMSA, ITC, and phosphorylation site determination. HH, HG, and NZ carried out the crystallization and collected the data. NZ and YH solved the structures. DN assisted with the statistical analysis. TX, XT, YH, and NZ wrote the paper. MLK and JZ edited the manuscript. All authors were given the opportunity to review the results and comment on the manuscript.

Corresponding authors

Correspondence to Nannan Zhang, Yong-Xing He or Jizhong Zhou.

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Xu, T., Tao, X., He, H. et al. Functional and structural diversification of incomplete phosphotransferase system in cellulose-degrading clostridia. ISME J 17, 823–835 (2023). https://doi.org/10.1038/s41396-023-01392-2

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  • DOI: https://doi.org/10.1038/s41396-023-01392-2

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