Applied Microbiology and Biotechnology

, Volume 99, Issue 10, pp 4321–4331 | Cite as

Identification and characterization of a novel locus in Cytophaga hutchinsonii involved in colony spreading and cellulose digestion

  • Hong Zhou
  • Xia Wang
  • Tengteng Yang
  • Weixin Zhang
  • Guanjun ChenEmail author
  • Weifeng LiuEmail author
Applied genetics and molecular biotechnology


Cytophaga hutchinsonii, an aerobic cellulolytic soil bacterium, is capable of degrading crystalline cellulose and gliding over surface rapidly. The involved mechanisms, however, are largely unknown. Here, we used the mariner-based transposon HimarEm1 to screen for C. hutchinsonii mutants deficient in utilizing filter paper as the sole carbon source. A novel gene locus, chu_1719, encoding a hypothetical protein was identified, whose inactivation resulted in a compromised growth of C. hutchinsonii on filter paper. Further analysis revealed that disruption of chu_1719 suppressed colony spreading but had no significant effect on Avicel degradation in liquid medium. Carboxymethylcellulase (CMCase) activity of the mutant membrane proteins was reduced by about 40 % as compared with the wild-type strain. Moreover, profiles of cellulose-adsorbed outer membrane proteins were significantly different between the mutant and wild-type (WT) strains. These results suggest that chu_1719 plays an important role in controlling the spreading motility and cellulose utilization probably by affecting the appropriate production of membrane proteins in C. hutchinsonii.


Cytophaga hutchinsonii Transposon insertion Colony spreading Membrane proteins Cellulose digestion 



We are grateful for Dr. MJ McBride for providing the plasmids and strains. This work is supported by grants from the National Basic Research Program (2011CB707402), the National Natural Science Foundation of China (31170762), New Century Excellent Talents in University (NCET-10-0546), Shandong Provincial Funds for Distinguished Young Scientists (JQ201108). We also thank the anonymous reviewers for their constructive comments.

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  1. Braun TF, McBride MJ (2005) Flavobacterium johnsoniae GldJ is a lipoprotein that is required for gliding motility. J Bacteriol 187(8):2628–2637. doi: 10.1128/JB.187.8.2628-2637.2005 CrossRefPubMedCentralPubMedGoogle Scholar
  2. Braun TF, Khubbar MK, Saffarini DA, McBride MJ (2005) Flavobacterium johnsoniae gliding motility genes identified by mariner mutagenesis. J Bacteriol 187(20):6943–6952. doi: 10.1128/JB.187.20.6943-6952.2005 CrossRefPubMedCentralPubMedGoogle Scholar
  3. Chang WT, Thayer DW (1977) The cellulase system of a Cytophaga species. Can J Microbiol 23(9):1285–1292CrossRefPubMedGoogle Scholar
  4. Chang LE, Pate JL, Betzig RJ (1984) Isolation and characterization of nonspreading mutants of the gliding bacterium Cytophaga johnsonae. J Bacteriol 159(1):26–35PubMedCentralPubMedGoogle Scholar
  5. Cooper AJ, Kalinowski AP, Shoemaker NB, Salyers AA (1997) Construction and characterization of a Bacteroides thetaiotaomicron recA mutant: transfer of Bacteroides integrated conjugative elements is RecA independent. J Bacteriol 179(20):6221–6227PubMedCentralPubMedGoogle Scholar
  6. Feldhaus MJ, Hwa V, Cheng Q, Salyers AA (1991) Use of an Escherichia coli β-glucuronidase gene as a reporter gene for investigation of Bacteroides promoters. J Bacteriol 173(14):4540–4543PubMedCentralPubMedGoogle Scholar
  7. Gong J, Forsberg CW (1989) Factors affecting adhesion of Fibrobacter succinogenes subsp. succinogenes S85 and adherence-defective mutants to cellulose. Appl Environ Microbiol 55(12):3039–3044PubMedCentralPubMedGoogle Scholar
  8. Gruss F, Zähringer F, Jakob RP, Burmann BM, Hiller S, Maier T (2013) The structural basis of autotransporter translocation by TamA. Nat Struct Mol Biol 20(11):1318–20. doi: 10.1038/ nsmb.2689 CrossRefPubMedGoogle Scholar
  9. Hu W, Hossain M, Lux R, Wang J, Yang Z, Li Y, Shi W (2011) Exopolysaccharide-independent social motility of Myxococcus xanthus. PLoS ONE 6(1):e16102. doi: 10.1371/journal.pone.0016102 CrossRefPubMedCentralPubMedGoogle Scholar
  10. 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. doi: 10.1007/s00253-012-4051-y CrossRefPubMedGoogle Scholar
  11. Ji X, Wang Y, Zhang C, Bai X, Zhang W, Lu X (2014) A novel outer membrane protein involved in cellulose and cellooligosaccharide degradation by Cytophaga hutchinsonii. Appl Environ Microbiol 80(15):4511–4518, AEM.00687-14/10.1128CrossRefPubMedCentralPubMedGoogle Scholar
  12. Jun HS, Qi M, Gong J, Egbosimba EE, Forsberg CW (2007) Outer membrane proteins of Fibrobacter succinogenes with potential roles in adhesion to cellulose and in cellulose digestion. J Bacteriol 189(19):6806–6815, JB.00560-07/10.1128CrossRefPubMedCentralPubMedGoogle Scholar
  13. Leyton DL, Rossiter AE, Henderson IR (2012) From self sufficiency to dependence: mechanisms and factors important for autotransporter biogenesis. Nat Rev Microbiol 10(3):213–225CrossRefPubMedGoogle Scholar
  14. Li LY, Shoemaker NB, Salyers AA (1995) Location and characteristics of the transfer region of a Bacteroides conjugative transposon and regulation of transfer genes. J Bacteriol 177(17):4992–4999PubMedCentralPubMedGoogle Scholar
  15. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66(3):506–577CrossRefPubMedCentralPubMedGoogle Scholar
  16. Lynd LR, van Zyl WH, McBride JE, Laser M (2005) Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol 16(5):577–583. doi: 10.1016/j.copbio. 2005.08.009 CrossRefPubMedGoogle Scholar
  17. McBride MJ (2001) Bacterial gliding motility: multiple mechanisms for cell movement over surfaces. Annu Rev Microbiol 55:49–75. doi: 10.1146/annurev.micro.55.1.49 CrossRefPubMedGoogle Scholar
  18. McBride MJ, Zhu Y (2013) Gliding motility and Por secretion system genes are widespread among members of the phylum Bacteroidetes. J Bacteriol 195(2):270–278. doi: 10.1128/JB.01962-12 CrossRefPubMedCentralPubMedGoogle Scholar
  19. Noinaj N, Kuszak AJ, Gumbart JC, Lukacik P, Chang H, Easley NC, Lithgow T, Buchanan SK (2013) Structural insight into the biogenesis of β-barrel membrane proteins. Nature 501(7467):385–390. doi: 10.1038/nature12521 CrossRefPubMedCentralPubMedGoogle Scholar
  20. Rhodes RG, Samarasam MN, Shrivastava A, van Baaren JM, Pochiraju S, Bollampalli S, McBride MJ (2010) Flavobacterium johnsoniae gldN and gldO are partially redundant genes required for gliding motility and surface localization of SprB. J Bacteriol 192(5):1201–1211. doi: 10.1128/ JB.01495-09 CrossRefPubMedCentralPubMedGoogle Scholar
  21. Rhodes RG, Nelson SS, Pochiraju S, McBride MJ (2011a) Flavobacterium johnsoniae sprB is part of an operon spanning the additional gliding motility genes sprC, sprD, and sprF. J Bacteriol 193(3):599–610. doi: 10.1128/JB.01203-10 CrossRefPubMedCentralPubMedGoogle Scholar
  22. Rhodes RG, Samarasam MN, Van Groll EJ, McBride MJ (2011b) Mutations in Flavobacterium johnsoniae sprE result in defects in gliding motility and protein secretion. J Bacteriol 193(19):5322–5327. doi: 10.1128/JB.05480-11 CrossRefPubMedCentralPubMedGoogle Scholar
  23. Rubin EJ, Akerley BJ, Novik VN, Lampe DJ, Husson RN, Mekalanos JJ (1999) In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria. Proc Natl Acad Sci U S A 96(4):1645–1650CrossRefPubMedCentralPubMedGoogle Scholar
  24. Sato K, Naito M, Yukitake H, Hirakawa H, Shoji M, McBride MJ, Rhodes RG, Nakayama K (2010) A protein secretion system linked to bacteroidete gliding motility and pathogenesis. Proc Natl Acad Sci U S A 107(1):276–281. doi: 10.1073/pnas.0912010107 CrossRefPubMedCentralPubMedGoogle Scholar
  25. Shrivastava A, Johnston JJ, van Baaren JM, McBride MJ (2013) Flavobacterium johnsoniae GldK, GldL, GldM, and SprA are required for secretion of the cell surface gliding motility adhesins SprB and RemA. J Bacteriol 195(14):3201–3212. doi: 10.1128/JB.00333-13 CrossRefPubMedCentralPubMedGoogle Scholar
  26. Stanier RY (1940) Studies on the Cytophagas. J Bacteriol 40(5):619–635PubMedCentralPubMedGoogle Scholar
  27. Stanier RY (1942) The Cytophaga group: A contribution to the biology of Myxobacteria. Bacteriol Rev 6(3):143–196PubMedCentralPubMedGoogle Scholar
  28. Walker E, Warren FL (1938) Decomposition of cellulose by Cytophaga. I Biochem J 32(1):31–43Google Scholar
  29. 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. doi: 10.1128/AEM. 01785-14 CrossRefPubMedCentralPubMedGoogle Scholar
  30. Wilson K (1990) Preparation of genomic DNA from bacteria. In: Ausubel FM, Brent R (eds) Current protocols in molecular biology. Greene Publ Assoc and Wiley Interscience, New York, pp 241–245Google Scholar
  31. Wilson DB (2008) Three microbial strategies for plant cell wall degradation. Ann N Y Acad Sci 1125:289–297. doi: 10.1196/annals.1419.026/1125/1/289 CrossRefPubMedGoogle Scholar
  32. 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, AEM.00225-07/10.1128CrossRefPubMedCentralPubMedGoogle Scholar
  33. Xu Y, Ji X, Chen N, Li P, Liu W, Lu X (2012) Development of replicative oriC plasmids and their versatile use in genetic manipulation of Cytophaga hutchinsonii. Appl Microbiol Biotechnol 93(2):697–705. doi: 10.1007/s00253-011-3572-0 CrossRefPubMedGoogle Scholar
  34. Zhu Y, McBride MJ (2014) Deletion of the Cytophaga hutchinsonii type IX secretion system gene sprP results in defects in gliding motility and cellulose utilization. Appl Microbiol Biotechnol 98(2):763–775. doi: 10.1007/s00253-013-5355-2 CrossRefPubMedGoogle Scholar
  35. Zhu Y, Li H, Zhou H, Chen G, Liu W (2010) Cellulose and cellodextrin utilization by the cellulolytic bacterium Cytophaga hutchinsonii. Bioresour Technol 101(16):6432–6437. doi: 10.1016/ j.biortech. 2010.03.041/S0960-8524(10)00518-3 CrossRefPubMedGoogle Scholar
  36. 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. doi: 10.1007/s00253-012-4259-x CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.The State Key Laboratory of Microbial Technology, School of Life ScienceShandong UniversityJinanPeople’s Republic of China

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