Appl Microbiol Biotechnol DOI 10.1007/s00253-015-7204-y APPLIED MICROBIAL AND CELL PHYSIOLOGY A small periplasmic protein essential for Cytophaga hutchinsonii cellulose digestion Tengteng Yang 1 & Xuliang Bu1 & Qingqing Han1 & Xia Wang1 & Hong Zhou1 & Guanjun Chen1 & Weixin Zhang 1 & Weifeng Liu 1 Received: 12 October 2015 /Revised: 23 November 2015 /Accepted: 25 November 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract Cytophaga hutchinsonii is a gliding cellulolytic Keywords Cytophaga hutchinsonii . Cellulose degradation . bacterium that is ubiquitously distributed in soil. The mecha- Transposon mutagenesis . Periplasmic protein . Colony nism by which C. hutchinsonii achieves cellulose digestion, spreading . Gliding motility however, is still largely unknown. In this study, we obtained a C. hutchinsonii mutant that was defective in utilizing filter paper or Avicel as the sole carbon source by transposon mu- Introduction tagenesis. The interrupted gene locus, CHU_2981, encodes a hypothetical protein with only 130 amino acids. Cell fraction- Cytophaga hutchinsonii belonging to the phylum ation and western blot detection of CHU_2981 fused with a C- Bacteroidetes (also known as the Cytophaga- terminal green fluorescence protein (GFP) indicated that Flavobacterium-Bacteroides group) is an aerobic cellulolytic CHU_2981 is located in the periplasm. The CHU_2981- bacterium that is ubiquitously distributed in soil (Reichenbach disrupted mutant cells exhibited a significant growth defect 1992; Xie et al. 2007). It exhibits an excellent capability of on Avicel but not on glucose and cellobiose. The absence of thriving on crystalline cellulose, with its cellulolytic substrates CHU_2981 also resulted in a significant defect in colony including Avicel, filter paper, and cotton wool (Stanier 1942). spreading and individual cell motility compared to wild-type Whereas it has been generally accepted that cellulose- cells. Further analysis demonstrated that the CHU_2981- degrading microorganisms usually adopt one of the two com- disrupted mutant cells exhibited a different profile of mon strategies for efficient digestion of cellulose, either by cellulose-absorbed outer membrane proteins from that of secreting a suit of free cellulases or by producing large multi- wild-type cells, in which protein varieties and amounts were enzyme complexes termed cellulosomes that are attached to markedly decreased. Our results showed that CHU_2981, the cell surface (Beguin and Lemaire 1996). C. hutchinsonii is periplasmic non-cellulolytic protein, plays an important role considered to use an unusual cellulose-utilizing strategy that in both cellulose utilization and cell motility probably by be- is distinct from those two described above (Wilson 2008;Xie ing involved in the appropriate production of outer membrane et al. 2007). In recent years, many attempts have been made to proteins. clarify the unique cellulose-degrading mechanism in C. hutchinsonii. Four cellulase enzymes including CHU_1336, CHU_1107, CHU_2103, and CHU_1280 have Electronic supplementary material The online version of this article been biochemically characterized (Louime et al. 2007; Zhang (doi:10.1007/s00253-015-7204-y) contains supplementary material, which is available to authorized users. et al. 2014, 2015; Zhu et al. 2013). among which CHU_2103 and CHU_1280 may act as processive endoglucanases and * Weixin Zhang canbindtocellulose,althoughnosequenceencodingrecog- [email protected] nizable carbohydrate-binding modules (CBMs) are present. However, none of them seems to play a key role in 1 State Key Laboratory of Microbial Technology, School of Life C. hutchinsonii cellulose utilization. On the other hand, quite Science, Shandong University, No. 27 Shanda South Road, a few genes involved in cellulose utilization in C. hutchinsonii Jinan 250100, Shandong, People’s Republic of China have been identified by using random transposon mutagenesis Appl Microbiol Biotechnol or targeted gene inactivation, which includes CHU_0170, Materials and methods CHU_3237, CHU_1277, CHU_1719,andCHU_1798 (Ji et al. 2014;Lietal.2015;Wangetal.2014;Zhouetal. Strains, media, and cultivations 2015; Zhu and McBride 2014). The precise function of these genes and the involved mechanisms are, however, largely un- The wild-type C. hutchinsonii ATCC 33406 and its mutant known. Since the efficient digestion of cellulose by strains were routinely cultured in PY2 or PY10 medium (0.2 C. hutchinsonii depends on the direct cell contact with cellu- or 1 % peptone, 0.05 % yeast extract, pH 7.2) at 28 °C, sup- lose (Stanier 1942; Xie et al. 2007). it has thus been speculated plemented with 0.4 % glucose as the carbon source. For cul- that proteins located in the outer membrane and in the peri- tivation of mutant strains, appropriate antibiotics were added plasm may play an assignable role in cellulose digestion. at the following final concentrations: chloramphenicol, 40 μg/ Whereas several outer membrane proteins have indeed been mL; erythromycin, 60 μg/mL; and kanamycin, 40 μg/mL. identified to be involved in cellulose utilization (Ji et al. 2012, Escherichia coli strains DH5α and BL21(DE3) were grown 2014; Zhou et al. 2015). few periplasmic proteins were at 37 °C in Luria-Bertani medium with addition of 100 μg/mL identified. ampicillin when required. HimarEm mutagenesis of In addition to the efficient cellulose digestion, another C. hutchinsonii and identification of the insertion locus distinct feature of C. hutchinsonii is its gliding motility. pHimarEm1 (Braun et al. 2005) were introduced into the C. hutchinsonii is capable of gliding rapidly along the sur- wild-type C. hutchinsonii cells by electroporation as previous- face of solid medium (Stanier 1942). Gliding is thus spec- ly described (Zhu et al. 2013). HimarEm mutants were select- ulated to facilitate cellulose depolymerization by allowing ed by plating cells on PY10 agar containing 0.4 % glucose cells to move along cellulose fibers during the digestion plus erythromycin and cultured at 28 °C for 6 days. The (Xie et al. 2007). Bacteroidete gliding motility has been transformant colonies were, respectively, inoculated to 1-mL extensively studied in the distantly related Bacteroidete liquid PY10 medium containing glucose plus erythromycin, Flavobacterium johnsoniae wherein 19 Gld and Spr pro- cultured for 2–3 days to reach the mid-logarithmic phase, teins involved in motility were identified (Sato et al. 2010). washed and resuspended with PY10 medium without carbon GldK, GldL, GldM, and GldN have even been suggested to source, and then spotted on PY10 agar with autoclaved filter be components of the recently established type IX secre- paper on top. The mutant transformants defective in growth tion system (T9SS, previously referred to as the Por secre- with filter paper were chosen and inoculated to solid PY10 tion system) (McBride and Zhu 2013; Shrivastava et al. medium containing one thin top layer of 0.1 % ball-milled 2013). implying a functional relationship between motility Avicel. One mutant that can utilize neither filter paper nor and protein secretion. Genomic analysis demonstrated that Avicel, termed M5, was chosen for further study. To identify whereas homologous genes encoding the typical motility the insertion locus of the mutant, its chromosomal DNA was organelles like flagella or type IV pili are absent, isolated and digested with NsiI. The resulting fragments were orthologues of all the gld and spr genes involved in the self-circularized by using T4 DNA ligase and then trans- gliding motility of F. johnsoniae have been identified in formed into E. coli DH5α/λpir. E. coli colonies carrying the C. hutchinsonii (Xie et al. 2007). Thus, C. hutchinsonii is HimarEm1 and the chromosomal DNA fragments adjacent to considered to use the similar gliding strategy as that in the insertion site were selected from Luria-Bertani agar con- F. johnsoniae though the specific mechanism involved is taining 40 μg/mL kanamycin, and the retrieved plasmids were still not clear (Xie et al. 2007). Although several genes sequenced using primer 615 (Braun et al. 2005). (CHU_0134, CHU_0170, CHU_3237, CHU_1277, CHU_1719,andCHU_1798)havebeenidentifiedtoaffect not only the ability in efficient cellulose utilization but also Southern hybridization and reverse transcription PCR in gliding or colony spreading (Ji et al. 2012, 2014;Lietal. 2015;Wangetal.2014;Zhouetal.2015;Zhuand Southern hybridization was performed by using DIG High McBride 2014). the relationship between C. hutchinsonii Prime DNA Labeling and Detection Starter Kit I (Roche, gliding and cellulose digestion has not been clearly under- Basel, Switzerland) according to the manufacturer’sinstruc- stood, and more genes involved await further exploration. tions. Briefly, chromosomal DNA of the wild-type and trans- In this study, we identified and characterized a new poson mutant cells was isolated, respectively, digested with gene locus, CHU_2981,inC. hutchinsonii that was PstI, separated by agarose gel electrophoresis, and transferred shown to be essential in cellulose utilization by using to positively charged nylon membranes. The probe specifical- transposon mutagenesis. CHU_2981 encodes a hypothet- ly against a 780-bp internal fragment of the erythromycin- ical protein located in the cell periplasm, and its disrup- resistant gene (ermF) present in pHimarEm1 was prepared tion also led to a significant defect in colony spreading using PCR amplification (using primers ermF-F and ermF-R and individual cell motility.