Article Figures & Data
Figures
- Fig. 1.
Southern analysis of C. thermocellum genomic DNA. Genomic DNA was digested with EcoRI (lane 1),DraI (lane 2), ApaI (lane 3), NotI (lane 4), AscI (lane 5), and BamHI (lane 6) and fractionated on an agarose gel (1%). DNA fragments in the gel were transferred to a nylon membrane. The hybridization probe was the 0.9-kb PCR fragment amplified by using the CelK1F and CelK1R (Table 1). Digoxigenin was incorporated into the fragment during PCR amplification in the presence of digoxigenin-conjugated dUTP (Boehringer Mannheim). Stringency washing was done twice for 15 min at 42°C (A), 55°C (B), and 65°C (C). DNA standards labeled with digoxigenin were run under identical conditions, and their positions of migration are shown on the left.
- Fig. 2.
(A) Restriction map of celK. D, E, H, and X, restriction sites for DraI, EcoRI,HindIII, and XhoI, respectively. (B and C) Comparisons between partial sequences of celK andcbhA. Sequences compared include 5′ (B) and 3′ (C) ends of the two ORFs, together with some untranslated regions. Numbers on the left correspond to those in the databases. The deduced amino acid sequence for celK is shown as boldface; putative ribosome binding and transcription terminator sites are underlined.
- Fig. 3.
Analysis of PCR products amplified from C. thermocellum genomic DNA, using CelK4F and Celk2R (Table 1) as primers. The gel was loaded with PCR products amplified in the presence (lane 1) and absence (lane 2) of template. Lane 3 was loaded with 1-kb DNA standards (Promega).
- Fig. 4.
Comparison between the deduced amino acid sequences ofcelK and cbhA. The sequence in CbhA containing a family III CBD is underlined. The signal peptide in CelK is in boldface; amino acids in CelK determined by protein sequencing are italicized.
- Fig. 5.
Domain organizations of C. thermocellum CelK and CbhA. Assignments of domains are based on sequence similarities to domains of known functions. Symbols: SP, signal peptide; CBD IV, CBD of family IV; CD, catalytic domain; DD, dockerin domain; CBD III, CBD of family III; Fn3, fibronectin type 3-like domain.
- Fig. 6.
Alignment of the CBD of CelK with CBDs of family IV found in microbial cellulases. Abbreviations: Celk_Clotm, C. thermocellum CelK; Cbha_Clotm, C. thermocellum CbhA; Cenc_Celfi, Cellulomonas fimi CenC; Lica_Clotm, C. thermocellum LicA; Cel1_Strre, S. reticuli Cel1; E1_Thfu, T. fusca E1; Lama_Thne, Thermotoga neapolitana LamA.
- Fig. 7.
Alignment of the catalytic domain of CelK with those of other cellulases. Amino acid sequences that showed over 30% identity to the entire catalytic domain of CelK include CbhA of the same organism (CbhA_Clotm), CenC of Cellulomonas fimi(CenC_Celfi), CelA of S. reticuli (Cela_Strre), and CelA ofB. fibrosolvens (Cela_Butfi).
- Fig. 8.
SDS-PAGE of purified CelK. Lane 1, molecular mass standards; lane 2, CelK.
Tables
- Table 1.
Oligonucleotide primers used for amplification of thecelK gene by PCR
Name Peptide Sequencea Orientation Positionb CelK1F KLPDYKND 5′-AARYTICCIGAYTAYAARAAYGA-3′ Forward 1242–1265 CelK1R IPIEMPYA 5′-GCRTAIGGCATYTCDATIGGDAT-3′ Reverse 2119–2142 CelK2F 5′-CAGTGCTGATATTTACTCCA-3′ Forward 2051–2071 CelK2R GDVNDDG 5′-CCRTCRTCRTTRCARTCICC-3′ Reverse 3639–3658 CelK3F 5′-GCAGGCGGCATTAAAGCATG-3′ Forward 2732–2751 CelK3R 5′-ATGTGATTTCGCTGTTGTTGAT-3′ Reverse 1337–1358 CelK4F 5′-AGACTCATGGTCAACCAACGA-3′ Forward 3506–3526 CelK5R 5′-CATTATATGGCAGTTTTTTAT-3′ Reverse 3827–3847
