7723 APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Feb. 1997. p. 355-363 Vol. 63. Nl). :: 0099-2240/97/S04.00 +0 Copyright £' 1997. American Society for Microbiology Cloning of Cellobiose Phosphoenolpyruvate-Dependent Phosphotransferase Genes: Functional Expression in Recombinant Escherichia coii "and Identification of a Putative Binding Region for Disaccharidest XIAOKUANG LAI,I F. C. DAVIS, I R. B. HESPELV AND L. O. INGRAM 1* Depanmellt ofMicrobiology and Cell Science, University ofFlorida, Gainesville, Florida 32611. I and Fennenration Biochemistry Unit. National Cellter for Agricultural Utilization Research USDA Agricultural Research Service. Peoria, Illinois 61604: Received 9 July 1996/Accepted 4 November 1996 Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Pos­ itive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phos­ photransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio jibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pL0I1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin. and methylum­ belliferyl derivatives ofglucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-~-glucosidase,which appear to form an operon (casRAB). Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated sequences from the EIIC domains of cellobiose, aryl-~-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation. Cellulose, a [3-1,4-linked polymer of glucose, represents ap­ phosphotransferase systems (PTS) have been reported in proximately half of the dry weight of plant cell walls. Each year Streptococcus bovis (34), Bacillus stearothemwphilus (27). and 4 X 1010 metric tons of this polymer is produced by photosyn­ B. subtilis (15a). Cryptic PTS genes for cellobiose utilization thesis and degraded by microbial cellulases (8). Recent interest are present in Escherichia coli (16). Although the mechanisms in cellulose hydrolysis has focused on animal nutrition (32) and of uptake are unknown, Microbispora bispora (58) contains two on the potential development of environmentally benign pro­ genes which encode cellobiase activity. R flavefaciens. R. albus, cesses for microbial conversion into fuel ethanol (57). Cellulomonas uda, and C. favigena (48) contain intracellular In nature cellulose is solubilized bv a combination of endo­ phosphorylases which cleave cellobiose into glucose and glu­ glucanase and cellobiohydrolase activities with cellobiose as cose-phosphate, conserving energy from the glycosidic bond. the primary product. Cellobiose is a potent inhibitor of these Four PTS operons which encode proteins (Ell permease enzymes and must be continually removed by microbial activity and phospho-[3-glucosidase) for cellobiose utilization have (8, 58). This disaccharide is among the most abundant soluble been sequenced: B. subtilis celRABCD (15a). B. stearothermo­ substrates in nature for microbial growth and is metabolized in philus celRABCD (27), and two cryptic operons from E. coli, preference to glucose by some rumen organisms (17, 53). celABCDF (37) and ascFG (arbutin, salicin, and cellobiose) The ability to utilize cellobiose is widespread among gram­ (16). The three permeases encoded by eel operons are quite negative. gram-positive, andArchaeal genera (8). Bacterial cel­ similar but share little sequence similarity with the protein lobiase ([3-glucosidase) activity is typically cell associated and encoded by ascF. These four operons also encode three dif­ may hydrolyze cellobiose to glucose prior to uptake in some ferent types of phospho-[3-glucosidases. Only the E. coli ascB cases. Multiple systems for cellobiose utilization within a single and B. subtilis celC enzymes appear similar (56% identity). organism do not appear uncommon (12, 16, 58). Cellobiose In this study, we have used 4-methylumbelliferyl-[3-D-glu­ uptake in Clostridium themwcellum is energized directly by coside (MUG) and cellobiose-MacConkey agar to screen nine ATP hydrolysis (35). Inhibitor studies have established the different genomic libraries for cellobiose utilization genes presence of active transport systems for cellobiose in Rumino­ which are functionally expressed in E. coli. Subsequent se­ coccus flavefaciens (17), R albus (53), Fibrobacter succinogenes quencing of the most active clone and sequence analysis al­ (32), and Streptomyces granaticolor (23) which may be coupled lowed the identification of a highly conserved region within the to ion gradients. Cellobiose phosphoenolpyruvate-dependent ElIC domain which may be involved in disaccharide binding. MATERIALS AND METHODS * Corresponding author. Mailing address: Department of Microbi­ Bacterial strains, growth conditions, and plasmids. Bacteria and plasmids used in this study are described in Table 1. B. coagulans was grown at 55°C in ology and Cell Science, Museum Road, Bldg. 981. P.O. Box 110700, Difco Tryptic Soy medium. K oxylOca and E. coli were grown in Luria-Bertani University of Florida, Gainesville, FL 32611. Phone: (352) 392-8176. (LB) medium (2) at 30 and 37°C. respectively. PrevOielia (Bacteroides) rumini­ Fax: (352) 392-5922. E-mail address: [email protected]. cola. Butyrivibrio jibrisolvens. Selenomonas ruminamium. and Streptococcus bovis t Florida Agricultural Experiment Station publication no. R-05241. were grown anaerobically at ~oC in RGM medium (20). 355 356 LAI ET AL. APPL. El"VIRO;-';. MICROBIOL. TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid Genetic characteristic( s) Source or reference Bacterial strains .,.., Bacillus coagulalls XL-55-60 Prototroph _I B. subli/is YB886 trpC2 xill-1 61 PrevOIella ntnlillicola 23 20 BUlVrivibrio fibrisoll'ells H17c 20 Klebsiella Q.\"-y·toca P2 57 SelellOl1l0llaS nmlillanrium HD4 20 Slreptococcus bOl'is 26 20 Escherichia coli DHSe> F- lacZ~M15 recAl endAl hsdR17 (rl-;- ml-; -) supE44 BRL" E. coli JLT2 F- mcrB mrr hsdS20 (rB- mB-) recAl3 supE44 pIS! 54 E. coli JLT3 F- merB mrr hsdS20 (rB- mB-) recAl3 supE44 plsH 54 E. coli JL630 bglR67 bgIB::A1acZ bgL4.7 ~acX74 30 Plasmids pUC18 bla amp lacI' Z' BRL" pL0I902 pUC1S with B. slearolhemlOphilus cel operon 27 pL0I1901 pUCl8 with B. coagulans l3-glucoside PTS genes This stud\' pL0I1902 pUC18 with B. subli/is l3-glucoside PTS genes This stud,­ pL0I1903 pUCl8 with B. subti/is l3-glucoside PTS genes This stud~ pL0I1904 pUCl8 with S. bOI-is l3-glucoside PTS genes This stud\' pL0I1905 pUC1S with B. jibrisolvells l3-glucoside PTS genes This stud" pL0I1906 pUC18 with K oxytoca P2 l3-glucoside PTS genes This stud\' pL0I1907 pUClS with K planricola l3-glucoside PTS genes This stud\' pL0I1998 EagIJXbaI deletion of pL0I1906 retaining casR' AB This stud)' pL0I1992 SjilXbaI deletion of pL0I1906 (3' end of casA and all of casB) pL0I1997 San deletion of pL0I1998 (3' end of casB) This studY pL0I1974 Frameshift mutation in casA of pL0I1998 This stud)' pL0I1975 NcoIlBslXI deletion of pL0I1998 (internal deletion in casA) This study a Life Technologies. Inc.. Gaithersburg. Md. Recombinant E. coli was evaluated for carbohydrate utilization using LB agar harvested by centrifugation (5.000 x g. 5 min. 4'C). washed twice. and resus­ containing 10 mg of4-methylumbelliferyl-glucoside liter-I. M9 agar (2) contain­ pended in 50 mM NaKHP04 buffer (pH 7.2) to a density of approximately 50 ing arginine (50 mg liter-I) and 2 g of cellobiose liter-I. and MacConkey agar optical density at 550 nm ml- 1• Cells were disrupted by two passages through a base containing 10 g of I'-glucoside liter-I (cellobiose. arbutin. or salicin). Am­ French pressure cell at 20.000 Ib in -2. Lysates were assayed at 37'C in 50 mM I picillin (50 ....g ml- ) was added when appropriate for the selection. Chromogenic NaKHP04 buffer (pH 7.2) containing 5 mM MgCI2. 2 mM p-nitrophenyl-I'-D­ substrates. I'-glucosides. and ampicillin were purchased from the Sigma Chem­ l.4-glucopyranoside (PNPG) and 2 mM phosphoenolpyruvate. Reactions were ical Company (St. Louis. Mo.). tenninated by adding an equal volume of IM Na2C03' After centrifugation DNA manipulation. Chromosomal DNA was isolated essentially as described (5.000 x g. 5 min).p-nitrophenol was measured at 410 nm. Protein was estimated by Cutting and Vander Horn (9). Standard procedures were used for the con­ with the Bradford Reagent (Bio-Rad Laboratories. Richmond. Calif.) with bo­ struction. isolation. and analysis of plasmids (46). vine serum albumin as a standard. Genomic libraries. Genomic libraries of C. thennocellum and K planticola Nucleotide sequence accession number. The nucleotide sequence data have were generously provided by Arnold Demain (Massachusetts Institute of Tech­ been submitted to GenBank and assigned accession number U6I727. nology. Cambridge. Mass.) and J. Doran (Central Michigan University. Mt. Pleasant. Mich.). respectively. Genomic libraries for seven other organisms were constructed in DH5CI by ligating Sau3AI partial digestion products (4- to 6-kbp RESULTS