Applied Microbiology and Biotechnology

, Volume 102, Issue 15, pp 6593–6611 | Cite as

Effects of the histone-like protein HU on cellulose degradation and biofilm formation of Cytophaga hutchinsonii

  • Zhiwei Guan
  • Ying Wang
  • Lijuan Gao
  • Weican Zhang
  • Xuemei LuEmail author
Applied genetics and molecular biotechnology


Cytophaga hutchinsonii, belonging to Bacteroidetes, is speculated to use a novel cell-contact mode to digest cellulose. In this study, we identified a histone-like protein HU, CHU_2750, in C. hutchinsonii, whose transcription could be induced by crystalline but not amorphous cellulose. We constructed a CHU_2750-deleted mutant and expressed CHU_2750 in Escherichia coli to study the gene’s functions. Our results showed that although the deletion of CHU_2750 was not lethal to C. hutchinsonii, the mutant displayed an abnormal filamentous morphology, loose nucleoid, and obvious defects in the degradation of crystalline cellulose and cell motility. Further study indicated that the mutant displayed significantly decreased cell surface and intracellular endoglucanase activities but with β-glucosidase activities similar to the wild-type strain. Analyses by real-time quantitative PCR revealed that the transcription levels of many genes involved in cellulose degradation and/or cell motility were significantly downregulated in the mutant. In addition, we found that CHU_2750 was important for biofilm formation of C. hutchinsonii. The main extracellular components of the biofilm were analyzed, and the results showed that the mutant yielded significantly less exopolysaccharide but more extracellular DNA and protein than the wild-type strain. Collectively, our findings demonstrated that CHU_2750 is important for cellulose degradation, cell motility, and biofilm formation of C. hutchinsonii by modulating transcription of certain related genes, and it is the first identified transcriptional regulator in these processes of C. hutchinsonii. Our study shed more light on the mechanisms of cellulose degradation, cell motility, and biofilm formation by C. hutchinsonii.


Cytophaga hutchinsonii Histone-like protein HU Cellulose degradation Biofilm 



We are grateful to Dr. Mark J. McBride (University of Wisconsin-Milwaukee, USA) for providing C. hutchinsonii ATCC 33406. We sincerely thank Dr. Haiyan Yu and Dr. Xiaomin Zhao (Analysis & Testing Center of State Key Laboratory of Microbial Technology, Shandong University) for assistance in the SEM test. We sincerely thank Dr. Taiyong Quan (Shandong University) and Mr. Long Ma (Qilu Normal University) for assistance in the CLSM test. Thanks to Dr. Edward C. Mignot and Dr. Junshu Wang (Shandong University) for linguistic advice.


This study was funded by the National Natural Science Foundation of China (grant numbers 31770080 and 31371262).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9071_MOESM1_ESM.pdf (804 kb)
ESM 1 (PDF 803 kb)
253_2018_9071_MOESM2_ESM.mp4 (18.4 mb)
ESM 2 (MP4 18,887 kb)
253_2018_9071_MOESM3_ESM.mp4 (13.5 mb)
ESM 3 (MP4 13,798 kb)


  1. Agarwal S, Hunnicutt DW, McBride MJ (1997) Cloning and characterization of the Flavobacterium johnsoniae (Cytophaga johnsonae) gliding motility gene, gldA. Proc Natl Acad Sci U S A 94(22):12139–12344CrossRefPubMedPubMedCentralGoogle Scholar
  2. Alberti-Segui C, Arndt A, Cugini C, Priyadarshini R, Davey ME (2010) HU protein affects transcription of surface polysaccharide synthesis genes in Porphyromonas gingivalis. J Bacteriol 192(23):6217–6229. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Almarza O, Nunez D, Toledo H (2015) The DNA-binding protein HU has a regulatory role in the acid stress response mechanism in Helicobacter pylori. Helicobacter 20(1):29–40. CrossRefPubMedGoogle Scholar
  4. Azam TA, Ishihama A (1999) Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity. J Biol Chem 274(46):33105–33113CrossRefPubMedGoogle Scholar
  5. Bai X, Wang X, Wang S, Ji X, Guan Z, Zhang W, Lu X (2017) Functional studies of β-glucosidases of Cytophaga hutchinsonii and their effects on cellulose degradation. Front Microbiol 8:140. PubMedPubMedCentralCrossRefGoogle Scholar
  6. Balandina A, Claret L, Hengge-Aronis R, Rouviere-Yaniv J (2001) The Escherichia coli histone-like protein HU regulates rpoS translation. Mol Microbiol 39(4):1069–1079CrossRefPubMedGoogle Scholar
  7. Balandina A, Kamashev D, Rouviere-Yaniv J (2002) The bacterial histone-like protein HU specifically recognizes similar structures in all nucleic acids. DNA, RNA, and their hybrids. J Biol Chem 277(31):27622–27628. CrossRefPubMedGoogle Scholar
  8. Berger M, Farcas A, Geertz M, Zhelyazkova P, Brix K, Travers A, Muskhelishvili G (2010) Coordination of genomic structure and transcription by the main bacterial nucleoid-associated protein HU. EMBO Rep 11(1):59–64. CrossRefPubMedGoogle Scholar
  9. Bi HK, Sun LL, Fukamachi T, Saito H, Kobayashi H (2009) HU participates in expression of a specific set of genes required for growth and survival at acidic pH in Escherichia coli. Curr Microbiol 58(5):443–448. CrossRefPubMedGoogle Scholar
  10. Black WP, Yang Z (2004) Myxococcus xanthus chemotaxis homologs DifD and DifG negatively regulate fibril polysaccharide production. J Bacteriol 186(4):1001–1008CrossRefPubMedPubMedCentralGoogle Scholar
  11. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  12. Browning DF, Grainger DC, Busby SJ (2010) Effects of nucleoid-associated proteins on bacterial chromosome structure and gene expression. Curr Opin Microbiol 13(6):773–780. CrossRefPubMedGoogle Scholar
  13. Chen C, Ghosh S, Grove A (2004) Substrate specificity of Helicobacter pylori histone-like HU protein is determined by insufficient stabilization of DNA flexure points. Biochem J 383(Pt 2):343–351. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Claret L, RouviereYaniv J (1997) Variation in HU composition during growth of Escherichia coli: the heterodimer is required for long term survival. J Mol Biol 273(1):93–104. CrossRefPubMedGoogle Scholar
  15. Corinaldesi C, Danovaro R, Dell'Anno A (2005) Simultaneous recovery of extracellular and intracellular DNA suitable for molecular studies from marine sediments. Appl Environ Microbiol 71(1):46–50. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Dalai B, Zhou R, Wan Y, Kang M, Li L, Li T, Zhang S, Chen H (2009) Histone-like protein H-NS regulates biofilm formation and virulence of Actinobacillus pleuropneumoniae. Microb Pathog 46(3):128–134. CrossRefPubMedGoogle Scholar
  17. Dame RT, Goosen N (2002) HU: promoting or counteracting DNA compaction? FEBS Lett 529(2–3):151–156CrossRefPubMedGoogle Scholar
  18. Dang H, Lovell CR (2016) Microbial surface colonization and biofilm development in marine environments. Microbiol Mol Biol Rev : MMBR 80(1):91–138. CrossRefPubMedGoogle Scholar
  19. Devaraj A, Justice SS, Bakaletz LO, Goodman SD (2015) DNABII proteins play a central role in UPEC biofilm structure. Mol Microbiol 96(6):1119–1135. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Dillon SC, Dorman CJ (2010) Bacterial nucleoid-associated proteins, nucleoid structure and gene expression. Nat Rev Microbiol 8(3):185–195. CrossRefPubMedGoogle Scholar
  21. Dri AM, Rouviereyaniv J, Moreau PL (1991) Inhibition of cell-division in hupA hupB mutant bacteria lacking HU protein. J Bacteriol 173(9):2852–2863CrossRefPubMedPubMedCentralGoogle Scholar
  22. Drlica K, Rouviere-Yaniv J (1987) Histone-like proteins of bacteria. Microbiol Rev 51(3):301–319PubMedPubMedCentralGoogle Scholar
  23. Flemming HC, Wingender J (2010) The biofilm matrix. Nat Rev Microbiol 8(9):623–633. CrossRefPubMedGoogle Scholar
  24. Goodman SD, Obergfell KP, Jurcisek JA, Novotny LA, Downey JS, Ayala EA, Tjokro N, Li B, Justice SS, Bakaletz LO (2011) Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins. Mucosal Immunol 4(6):625–637. CrossRefPubMedGoogle Scholar
  25. Grove A (2011) Functional evolution of bacterial histone-like HU proteins. Curr Issues Mol Biol 13(1):1–12PubMedGoogle Scholar
  26. Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2(2):95–108. CrossRefPubMedGoogle Scholar
  27. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain-reaction. Gene 77(1):51–59. CrossRefPubMedGoogle Scholar
  28. Hodges-Garcia Y, Hagerman PJ, Pettijohn DE (1989) DNA ring closure mediated by protein HU. J Biol Chem 264(25):14621–14623PubMedGoogle Scholar
  29. Ji X, Xu Y, Zhang C, Chen N, Lu X (2012) A new locus affects cell motility, cellulose binding, and degradation by Cytophaga hutchinsonii. Appl Microbiol Biotechnol 96(1):161–170. CrossRefPubMedGoogle Scholar
  30. Ji X, Bai X, Li Z, Wang S, Guan Z, Lu X (2013) A novel locus essential for spreading of Cytophaga hutchinsonii colonies on agar. Appl Microbiol Biotechnol 97(16):7317–7324. CrossRefPubMedGoogle Scholar
  31. Ji X, Wang Y, Zhang C, Bai X, Zhang W, Lu X (2014) Novel outer membrane protein involved in cellulose and cellooligosaccharide degradation by Cytophaga hutchinsonii. Appl Environ Microbiol 80(15):4511–4518. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Kar S, Edgar R, Adhya S (2005) Nucleoid remodeling by an altered HU protein: reorganization of the transcription program. Proc Natl Acad Sci U S A 102(45):16397–16402. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Li Z, Zhang C, Wang S, Cao J, Zhang W, Lu X (2015) A new locus in Cytophaga hutchinsonii involved in colony spreading on agar surfaces and individual cell gliding. FEMS Microbiol Lett 362(14):fnv095. CrossRefPubMedGoogle Scholar
  34. Link AJ, LaBaer J (2011) Trichloroacetic acid (TCA) precipitation of proteins. Cold Spring Harb Protoc 2011(8):993–994. CrossRefPubMedGoogle Scholar
  35. Liu D, Yumoto H, Murakami K, Hirota K, Ono T, Nagamune H, Kayama S, Matsuo T, Miyake Y (2008) The essentiality and involvement of Streptococcus intermedius histone-like DNA-binding protein in bacterial viability and normal growth. Mol Microbiol 68(5):1268–1282. CrossRefPubMedGoogle Scholar
  36. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(−δ δ C) method. Methods 25(4):402–408. CrossRefPubMedGoogle Scholar
  37. Mangan MW, Lucchini S, Croinin TO, Fitzgerald S, Hinton JCD, Dorman CJ (2011) Nucleoid-associated protein HU controls three regulons that coordinate virulence, response to stress and general physiology in Salmonella enterica serovar Typhimurium. Microbiol-SGM 157:1075–1087. CrossRefGoogle Scholar
  38. Nguyen HH, de la Tour CB, Toueille M, Vannier F, Sommer S, Servant P (2009) The essential histone-like protein HU plays a major role in Deinococcus radiodurans nucleoid compaction. Mol Microbiol 73(2):240–252. CrossRefPubMedGoogle Scholar
  39. Nur A, Hirota K, Yumoto H, Hirao K, Liu D, Takahashi K, Murakami K, Matsuo T, Shu R, Miyake Y (2013) Effects of extracellular DNA and DNA-binding protein on the development of a Streptococcus intermedius biofilm. J Appl Microbiol 115(1):260–270. CrossRefPubMedGoogle Scholar
  40. Oberto J, Nabti S, Jooste V, Mignot H, Rouviere-Yaniv J (2009) The HU regulon is composed of genes responding to anaerobiosis, acid stress, high osmolarity and SOS induction. PLoS One 4(2):e4367. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Pettijohn DE (1988) Histone-like proteins and bacterial chromosome structure. J Biol Chem 263(26):12793–12796PubMedGoogle Scholar
  42. Phan NQ, Uebanso T, Shimohata T, Nakahashi M, Mawatari K, Takahashi A (2015) DNA-binding protein HU coordinates pathogenicity in Vibrio parahaemolyticus. J Bacteriol 197(18):2958–2964. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Rollefson JB, Stephen CS, Tien M, Bond DR (2011) Identification of an extracellular polysaccharide network essential for cytochrome anchoring and biofilm formation in Geobacter sulfurreducens. J Bacteriol 193(5):1023–1033. CrossRefPubMedGoogle Scholar
  44. Rouviere-Yaniv J, Gros F (1975) Characterization of a novel, low-molecular-weight DNA-binding protein from Escherichia coli. Proc Natl Acad Sci U S A 72(9):3428–3432CrossRefPubMedPubMedCentralGoogle Scholar
  45. Sarkar T, Vitoc I, Mukerji I, Hud NV (2007) Bacterial protein HU dictates the morphology of DNA condensates produced by crowding agents and polyamines. Nucleic Acids Res 35(3):951–961. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Stanier RY (1942) The Cytophaga group: a contribution to the biology of Myxobacteria. Bacteriol Rev 6(3):143–196PubMedPubMedCentralGoogle Scholar
  47. Tjokro NO, Rocco CJ, Priyadarshini R, Davey ME, Goodman SD (2014) A biochemical analysis of the interaction of Porphyromonas gingivalis HU PG0121 protein with DNA. PLoS One 9(3):e93266. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Wang Y, Wang Z, Cao J, Guan Z, Lu X (2014) FLP-FRT-based method to obtain unmarked deletions of CHU_3237 (porU) and large genomic fragments of Cytophaga hutchinsonii. Appl Environ Microbiol 80(19):6037–6045. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Wang S, Zhao D, Bai X, Zhang W, Lu X (2017) Identification and characterization of a large protein essential for degradation of the crystalline region of cellulose by Cytophaga hutchinsonii. Appl Environ Microbiol 83(1):AEM.02270–AEM.02216. CrossRefGoogle Scholar
  50. Wilson DB (2008) Three microbial strategies for plant cell wall degradation. Ann N Y Acad Sci 1125:289–297. CrossRefPubMedGoogle Scholar
  51. Wojtuszewski K, Hawkins ME, Cole JL, Mukerji I (2001) HU binding to DNA: evidence for multiple complex formation and DNA bending. Biochemistry 40(8):2588–2598CrossRefPubMedGoogle Scholar
  52. Xie G, Bruce DC, Challacombe JF, Chertkov O, Detter JC, Gilna P, Han CS, Lucas S, Misra M, Myers GL, Richardson P, Tapia R, Thayer N, Thompson LS, Brettin TS, Henrissat B, Wilson DB, McBride MJ (2007) Genome sequence of the cellulolytic gliding bacterium Cytophaga hutchinsonii. Appl Environ Microbiol 73(11):3536–3546. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Xu YX, Ji XF, Chen N, Li PW, Liu WF, Lu XM (2012) Development of replicative oriC plasmids and their versatile use in genetic manipulation of Cytophaga hutchinsonii. Appl Microbiol Biotechnol 93(2):697–705. CrossRefPubMedGoogle Scholar
  54. Zhang YH, Cui J, Lynd LR, Kuang LR (2006) A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 7(2):644–648. CrossRefPubMedGoogle Scholar
  55. Zhang C, Wang Y, Li Z, Zhou X, Zhang W, Zhao Y, Lu X (2014) Characterization of a multi-function processive endoglucanase CHU_2103 from Cytophaga hutchinsonii. Appl Microbiol Biotechnol 98(15):6679–6687. CrossRefPubMedGoogle Scholar
  56. Zhang C, Zhang W, Lu X (2015) Expression and characteristics of a Ca(2)(+)-dependent endoglucanase from Cytophaga hutchinsonii. Appl Microbiol Biotechnol 99(22):9617–9623. CrossRefPubMedGoogle Scholar
  57. Zhang C, Wang X, Zhang W, Zhao Y, Lu X (2017) Expression and characterization of a glucose-tolerant β-1,4-glucosidase with wide substrate specificity from Cytophaga hutchinsonii. Appl Microbiol Biotechnol 101(5):1919–1926. CrossRefPubMedGoogle Scholar
  58. Zhou H, Wang X, Yang T, Zhang W, Chen G, Liu W (2015) Identification and characterization of a novel locus in Cytophaga hutchinsonii involved in colony spreading and cellulose digestion. Appl Microbiol Biotechnol 99(10):4321–4331. CrossRefPubMedGoogle Scholar
  59. Zhou H, Wang X, Yang T, Zhang W, Chen G, Liu W (2016) An outer membrane protein involved in the uptake of glucose is essential for Cytophaga hutchinsonii cellulose utilization. Appl Environ Microbiol 82(6):1933–1944. CrossRefPubMedPubMedCentralGoogle Scholar
  60. Zhu Y, McBride MJ (2017) The unusual cellulose utilization system of the aerobic soil bacterium Cytophaga hutchinsonii. Appl Microbiol Biotechnol 101:7113–7127. CrossRefPubMedGoogle Scholar
  61. Zhu Y, Zhou H, Bi Y, Zhang W, Chen G, Liu W (2013) Characterization of a family 5 glycoside hydrolase isolated from the outer membrane of cellulolytic Cytophaga hutchinsonii. Appl Microbiol Biotechnol 97(9):3925–3937. CrossRefPubMedGoogle Scholar
  62. Zhu Y, Han L, Hefferon KL, Silvaggi NR, Wilson DB, McBride MJ (2016) Periplasmic Cytophaga hutchinsonii endoglucanases are required for use of crystalline cellulose as the sole source of carbon and energy. Appl Environ Microbiol 82(15):4835–4845. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Zhiwei Guan
    • 1
    • 2
  • Ying Wang
    • 3
  • Lijuan Gao
    • 1
  • Weican Zhang
    • 1
  • Xuemei Lu
    • 1
    Email author
  1. 1.State Key Laboratory of Microbial Technology, School of Life ScienceShandong UniversityJinanChina
  2. 2.School of Life ScienceQilu Normal UniversityJinanChina
  3. 3.Central Laboratory, Huai’an First People’s HospitalNanjing Medical UniversityHuai’anChina

Personalised recommendations