Abstract
Previous studies suggested that the first step in utilization of starch by Bacteroides thetaiotaomicron was binding of the polysaccharide to the cell surface, followed by translocation of the polysaccharide across the outer membrane into the periplasm. In this study, we report the molecular characterization of a gene that encodes an outer membrane protein that is essential for utilization of both maltooligosaccharides and starch. The gene, susC, encoded a protein of 115.3 kDa. Antibodies were raised against SusC, and the outer membrane location of SusC could be confirmed by Western blot (immunoblot) analysis. SusC had a possible signal sequence of between 20 and 39 amino acids, depending on which N-terminal methionine initiates the start of the protein. It also had some features typical of well-characterized outer membrane proteins from members of the family Enterobacteriaceae, such as a terminal phenylalanine residue and a region in the amino portion of the protein thought to be involved in stabilizing the protein in the outer membrane. The amino acid sequence, together with results of gene disruption experiments, suggested that SusC was not an amylolytic enzyme. Transcriptional fusion experiments, using beta-glucuronidase as a reporter group, showed that expression of susC was maltose regulated at the transcriptional level. This is the first molecular characterization of a B. thetaiotaomicron outer membrane protein involved in maltooligosaccharide and starch utilization.
References
52
Referenced
138
-
Altschul , S. F. , W. Gish , W. Miller , E. W. Myers , and D. J. Lipman . 1990 . Basic local alignment search tool . J. Mol. Biol. 215 : 403 - 410 .
(
10.1016/S0022-2836(05)80360-2
) / J. Mol. Biol. / Basic local alignment search tool by Altschul S. F. (1990) -
Anderson , K. A. , and A. A. Salyers . 1989 . Genetic evidence that outer membrane binding of starch is required for starch utilization by Bacteroides thetaiotaomicron . J. Bacteriol. 171 : 3199 - 3204 .
(
10.1128/jb.171.6.3199-3204.1989
) / J. Bacteriol. / Genetic evidence that outer membrane binding of starch is required for starch utilization by Bacteroides thetaiotaomicron by Anderson K. A. (1989) -
Anderson , K. A. , and A. A. Salyers . 1989 . Biochemical evidence that starch breakdown by Bacteroides thetaiotaomicron involves outer membrane starchbinding sites and periplasmic starch-degrading enzymes . J. Bacteriol. 171 : 3192 - 3198 .
(
10.1128/jb.171.6.3192-3198.1989
) / J. Bacteriol. / Biochemical evidence that starch breakdown by Bacteroides thetaiotaomicron involves outer membrane starchbinding sites and periplasmic starch-degrading enzymes by Anderson K. A. (1989) -
Cheng , Q. , V. Hwa , and A. A. Salyers . 1992 . A locus that contributes to colonization of the intestinal tract by Bacteroides thetaiotaomicron contains a single regulatory gene (chuR) that links two polysaccharide utilization pathways . J. Bacteriol. 174 : 7185 - 7193 .
(
10.1128/jb.174.22.7185-7193.1992
) / J. Bacteriol. / A locus that contributes to colonization of the intestinal tract by Bacteroides thetaiotaomicron contains a single regulatory gene (chuR) that links two polysaccharide utilization pathways by Cheng Q. (1992) -
Cheng , Q. , M. C. Yu , A. R. Reeves , and A. A. Salyers . 1995 . Identification and characterization of a Bacteroides gene, csuF, which encodes an outer membrane protein that is essential for growth on chondroitin sulfate . J. Bacteriol. 177 : 3721 - 3727 .
(
10.1128/JB.177.13.3721-3727.1995
) / J. Bacteriol. / Identification and characterization of a Bacteroides gene, csuF, which encodes an outer membrane protein that is essential for growth on chondroitin sulfate by Cheng Q. (1995) -
Chou , P. Y. , and G. D. Fasman . 1978 . Empirical predictions of protein conformation. Annu . Rev. Biochem. 47 : 251 - 276 .
(
10.1146/annurev.bi.47.070178.001343
) / Rev. Biochem. / Empirical predictions of protein conformation. Annu by Chou P. Y. (1978) -
Coulton , J. W. , G. K. Reid , and A. Campana . 1988 . Export of hybrid proteins FhuA~-~LacZ and FhuA~-~PhoA to the cell envelope of Escherichia coli K-12 . J. Bacteriol. 170 : 2267 - 2275 .
(
10.1128/JB.170.5.2267-2275.1988
) / J. Bacteriol. / Export of hybrid proteins FhuA~-~LacZ and FhuA~-~PhoA to the cell envelope of Escherichia coli K-12 by Coulton J. W. (1988) -
Dean , C. R. , and K. Poole . 1993 . Cloning and characterization of the ferric enterobactin receptor gene (pfeA) of Pseudomonas aeruginosa . J. Bacteriol. 175 : 317 - 324 .
(
10.1128/jb.175.2.317-324.1993
) / J. Bacteriol. / Cloning and characterization of the ferric enterobactin receptor gene (pfeA) of Pseudomonas aeruginosa by Dean C. R. (1993) -
Emr , S. D. , J. Hedgpeth , J. -M. Clement , T. J. Silhavy , and M. Hofnung . 1980 . Sequence analysis of mutations that prevent export ofreceptor, an Escherichia coli outer membrane protein . Nature (London) 285 : 82 - 85 .
(
10.1038/285082a0
) / Nature (London) / Sequence analysis of mutations that prevent export ofreceptor, an Escherichia coli outer membrane protein by Emr S. D. (1980) -
Feldhaus , M. J. , V. Hwa , Q. Cheng , and A. A. Salyers . 1991 . Use of an Escherichia coli ~-glucuronidase gene as a reporter gene for investigation of Bacteroides promoters . J. Bacteriol. 173 : 4540 - 4543 .
(
10.1128/JB.173.14.4540-4543.1991
) / J. Bacteriol. / Use of an Escherichia coli ~-glucuronidase gene as a reporter gene for investigation of Bacteroides promoters by Feldhaus M. J. (1991) -
Fujino , T. , P. Beguin , and J. P. Aubert . 1993 . Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface . J. Bacteriol. 175 : 1891 - 1899 .
(
10.1128/jb.175.7.1891-1899.1993
) / J. Bacteriol. / Organization of a Clostridium thermocellum gene cluster encoding the cellulosomal scaffolding protein CipA and a protein possibly involved in attachment of the cellulosome to the cell surface by Fujino T. (1993) -
Gherardini , F. C. , and A. A. Salyers . 1987 . Characterization of an outer membrane mannanase from Bacteroides ovatus . J. Bacteriol. 169 : 2031 - 2037 .
(
10.1128/JB.169.5.2031-2037.1987
) / J. Bacteriol. / Characterization of an outer membrane mannanase from Bacteroides ovatus by Gherardini F. C. (1987) - Glazer A. N. and H. Nikaido. 1994. Microbial biotechnology: fundamentals of applied microbiology. W. H. Freeman and Co. New York.
-
Goldberg , M. B. , S. A. Boyko , J. R. Butterton , J. A. Stoebner , S. M. Payne , and S. B. Calderwood . 1992 . Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins . Mol. Microbiol. 6 : 2407 - 2418 .
(
10.1111/j.1365-2958.1992.tb01415.x
) / Mol. Microbiol. / Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB-dependent proteins by Goldberg M. B. (1992) -
Guiney , D. G. , and E. Yacobson . 1983 . Location and nucleotide sequence of the transfer origin of the broad host range plasmid RK2 . Proc. Natl. Acad. Sci. USA 80 : 3593 - 3598 .
(
10.1073/pnas.80.12.3595
) / Proc. Natl. Acad. Sci. USA / Location and nucleotide sequence of the transfer origin of the broad host range plasmid RK2 by Guiney D. G. (1983) -
Hanahan , D. 1983 . Studies on the transformation of Escherichia coli with plasmids . J. Mol. Biol. 166 : 557 - 580 .
(
10.1016/S0022-2836(83)80284-8
) / J. Mol. Biol. / Studies on the transformation of Escherichia coli with plasmids by Hanahan D. (1983) -
Heller , K. , and R. J. Kadner . 1985 . Nucleotide sequence of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli . J. Bacteriol. 161 : 904 - 908 .
(
10.1128/JB.161.3.904-908.1985
) / J. Bacteriol. / Nucleotide sequence of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli by Heller K. (1985) -
Jeanteur , D. , J. H. Lakey , and F. Pattus . 1991 . The bacterial porin superfamily: sequence alignment and structure prediction . Mol. Microbiol. 5 : 2153 - 2164 .
(
10.1111/j.1365-2958.1991.tb02145.x
) / Mol. Microbiol. / The bacterial porin superfamily: sequence alignment and structure prediction by Jeanteur D. (1991) -
Johnson , J. L. , and B. Harich . 1986 . Ribosomal ribonucleic acid homology among species of the genus Bacteroides. Int. J. Syst. Bacteriol. 36 : 71 - 79 .
(
10.1099/00207713-36-1-71
) / Bacteroides. Int. J. Syst. Bacteriol. / Ribosomal ribonucleic acid homology among species of the genus by Johnson J. L. (1986) - Koebnik R. Personal communication.
-
Kotarski , S. F. , and A. A. Salyers . 1984 . Isolation and characterization of outer membranes of Bacteroides thetaiotaomicron grown on different carbohydrates . J. Bacteriol. 158 : 102 - 109 .
(
10.1128/jb.158.1.102-109.1984
) / J. Bacteriol. / Isolation and characterization of outer membranes of Bacteroides thetaiotaomicron grown on different carbohydrates by Kotarski S. F. (1984) -
Krone , J. A. , F. Stegehuis , G. Koningstein , C. van Doorn , B. Roosendaal , F. K. de Graaf , and B. Oudega . 1985 . Characterization of the pColV-K30 encoded cloacin DF13/aerobactin outer membrane receptor protein of Escherichia coli: isolation and purification of the protein and analysis of its nucleotide sequence and primary structure . FEMS Microbiol. Lett. 26 : 153 - 161 .
(
10.1111/j.1574-6968.1985.tb01583.x
) / FEMS Microbiol. Lett. / Characterization of the pColV-K30 encoded cloacin DF13/aerobactin outer membrane receptor protein of Escherichia coli: isolation and purification of the protein and analysis of its nucleotide sequence and primary structure by Krone J. A. (1985) -
Kyte , J. , and R. F. Doolittle . 1982 . A simple method for displaying the hydropathic character of a protein . J. Mol. Biol. 157 : 105 - 132 .
(
10.1016/0022-2836(82)90515-0
) / J. Mol. Biol. / A simple method for displaying the hydropathic character of a protein by Kyte J. (1982) -
Lamed , R. , J. Naimark , E. Morgenstern , and E. A. Bayer . 1987 . Specialized cell surface structures in cellulolytic bacteria . J. Bacteriol. 169 : 3792 - 3800 .
(
10.1128/JB.169.8.3792-3800.1987
) / J. Bacteriol. / Specialized cell surface structures in cellulolytic bacteria by Lamed R. (1987) -
Lederberg , E. M. , and S. M. Cohen . 1974 . Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid . J. Bacteriol. 119 : 1072 - 1074 .
(
10.1128/jb.119.3.1072-1074.1974
) / J. Bacteriol. / Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid by Lederberg E. M. (1974) - Litz , J. S. 1990 . Direct immunization with antigen in ProSieveTm agarose gel slices . FMC BioProducts Resolutions 6 ( 3 ): 6 . / FMC BioProducts Resolutions / Direct immunization with antigen in ProSieveTm agarose gel slices by Litz J. S. (1990)
-
Lowry , O. H. , N. J. Rosebrough , A. L. Farr , and R. J. Randall . 1951 . Protein measurement with the Folin phenol reagent . J. Biol. Chem. 193 : 265 - 275 .
(
10.1016/S0021-9258(19)52451-6
) / J. Biol. Chem. / Protein measurement with the Folin phenol reagent by Lowry O. H. (1951) - Maniatis T. E. F. Fritsch and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Cold Spring Harbor N.Y.
- Marston , F. A. O. , P. A. Lowe , M. T. Doel , J. M. Schoemaker , S. White , and S. Angal . 1984 . Purification of calf prochymosin (prorennin) synthesized in Escherichia coli . Bio/Technology 2 : 800 . / Bio/Technology / Purification of calf prochymosin (prorennin) synthesized in Escherichia coli by Marston F. A. O. (1984)
-
McCarty , R. E. , S. F. Kotarski , and A. A. Salyers . 1985 . Location and characteristics of enzymes involved in the breakdown of polygalacturonic acid by Bacteroides thetaiotaomicron . J. Bacteriol. 161 : 493 - 499 .
(
10.1128/JB.161.2.493-499.1985
) / J. Bacteriol. / Location and characteristics of enzymes involved in the breakdown of polygalacturonic acid by Bacteroides thetaiotaomicron by McCarty R. E. (1985) -
Morag , E. , A. Lapidot , D. Govorko , R. Lamed , M. Wilchek , E. A. Bayer , and Y. Shoham . 1995 . Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum . Appl. Environ. Microbiol. 61 : 1980 - 1986 .
(
10.1128/AEM.61.5.1980-1986.1995
) / Appl. Environ. Microbiol. / Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum by Morag E. (1995) -
Murphy , C. K. , V. I. Kalve , and P. E. Klebba . 1990 . Surface topology of the Escherichia coli K-12 ferric enterobactin receptor . J. Bacteriol. 172 : 2736 - 2746 .
(
10.1128/JB.172.5.2736-2746.1990
) / J. Bacteriol. / Surface topology of the Escherichia coli K-12 ferric enterobactin receptor by Murphy C. K. (1990) -
Nakajima , R. , T. Imanaka , and S. Aiba . 1986 . Comparison of amino acid sequences of eleven different ~-amylases . Appl. Microbiol. Biotechnol. 23 : 355 - 360 .
(
10.1007/BF00257032
) / Appl. Microbiol. Biotechnol. / Comparison of amino acid sequences of eleven different ~-amylases by Nakajima R. (1986) -
Nau , C. D. , and J. Konisky . 1989 . Evolutionary relationship between the TonB-dependent outer membrane transport proteins: nucleotide and amino acid sequences of the Escherichia coli colicin I receptor gene . J. Bacteriol. 171 : 1041 - 1047 .
(
10.1128/jb.171.2.1041-1047.1989
) / J. Bacteriol. / Evolutionary relationship between the TonB-dependent outer membrane transport proteins: nucleotide and amino acid sequences of the Escherichia coli colicin I receptor gene by Nau C. D. (1989) -
Needleman , S. B. , and C. D. Wunsch . 1970 . A general method applicable to the search for similarities in the amino acid sequences of two proteins . J. Mol. Biol. 48 : 443 - 453 .
(
10.1016/0022-2836(70)90057-4
) / J. Mol. Biol. / A general method applicable to the search for similarities in the amino acid sequences of two proteins by Needleman S. B. (1970) -
Pugsley , A. P. 1993 . The complete general secretory pathway in gram-negative bacteria . Microbiol. Rev. 57 : 50 - 108 .
(
10.1128/MR.57.1.50-108.1993
) / Microbiol. Rev. / The complete general secretory pathway in gram-negative bacteria by Pugsley A. P. (1993) - Reeves A. R. Unpublished results.
-
Rutz , J. M. , J. Liu , J. A. Lyons , J. Goranson , S. K. Armstrong , M. A. McIntosh , J. B. Feix , and P. Klebba . 1992 . Formation of a gated channel by a ligand-specific transport protein in the bacterial outer membrane . Science 258 : 471 - 475 .
(
10.1126/science.1411544
) / Science / Formation of a gated channel by a ligand-specific transport protein in the bacterial outer membrane by Rutz J. M. (1992) -
Saito , H. , and K. I. Miura . 1963 . Preparation of transforming deoxyribonucleic acid by phenol treatment . Biochim. Biophys. Acta 72 : 619 - 629 .
(
10.1016/0926-6550(63)90386-4
) / Biochim. Biophys. Acta / Preparation of transforming deoxyribonucleic acid by phenol treatment by Saito H. (1963) -
Sanger , F. , S. Nicklen , and A. R. Coulson . 1977 . DNA sequencing with chain-terminating inhibitors . Proc. Natl. Acad. Sci. USA 74 : 5463 - 5467 .
(
10.1073/pnas.74.12.5463
) / Proc. Natl. Acad. Sci. USA / DNA sequencing with chain-terminating inhibitors by Sanger F. (1977) -
Shoemaker , N. B. , C. Getty , E. P. Guthrie , and A. A. Salyers . 1986 . Regions in Bacteroides plasmids pBFTM10 and pB8-51 that allow Escherichia coli-Bacteroides shuttle vectors to be mobilized by IncP plasmids and by a conjugative Bacteroides tetracycline resistance element . J. Bacteriol. 166 : 959 - 965 .
(
10.1128/JB.166.3.959-965.1986
) / J. Bacteriol. / Regions in Bacteroides plasmids pBFTM10 and pB8-51 that allow Escherichia coli-Bacteroides shuttle vectors to be mobilized by IncP plasmids and by a conjugative Bacteroides tetracycline resistance element by Shoemaker N. B. (1986) -
Smith , C. J. 1985 . Development and use of cloning systems for Bacteroides fragilis: cloning of a plasmid-encoded clindamycin resistance determinant . J. Bacteriol. 164 : 294 - 301 .
(
10.1128/JB.164.1.294-301.1985
) / J. Bacteriol. / Development and use of cloning systems for Bacteroides fragilis: cloning of a plasmid-encoded clindamycin resistance determinant by Smith C. J. (1985) -
Struyve , M. , M. Moons , and J. Tommassen . 1991 . Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein . J. Mol. Biol. 218 : 141 - 148 .
(
10.1016/0022-2836(91)90880-F
) / J. Mol. Biol. / Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein by Struyve M. (1991) -
Studier , F. W. , and B. A. Moffett . 1986 . Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes . J. Mol. Biol. 189 : 113 - 130 .
(
10.1016/0022-2836(86)90385-2
) / J. Mol. Biol. / Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes by Studier F. W. (1986) -
Studier , F. W. , A. H. Rosenberg , J. J. Dunn , and J. W. Dubendorff . 1990 . Use of T7 RNA polymerase to direct expression of cloned genes . Methods Enzymol. 185 : 60 - 89 .
(
10.1016/0076-6879(90)85008-C
) / Methods Enzymol. / Use of T7 RNA polymerase to direct expression of cloned genes by Studier F. W. (1990) -
Tancula , E. , M. J. Feldhaus , L. A. Bedzyk , and A. A. Salyers . 1992 . Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron . J. Bacteriol. 174 : 5609 - 5616 .
(
10.1128/jb.174.17.5609-5616.1992
) / J. Bacteriol. / Location and characterization of genes involved in binding of starch to the surface of Bacteroides thetaiotaomicron by Tancula E. (1992) -
Valentine , P. J. , and A. A. Salyers . 1992 . Analysis of proteins associated with growth of Bacteroides ovatus on the branched galactomannan guar gum . Appl. Environ. Microbiol. 58 : 1534 - 1540 .
(
10.1128/AEM.58.5.1534-1540.1992
) / Appl. Environ. Microbiol. / Analysis of proteins associated with growth of Bacteroides ovatus on the branched galactomannan guar gum by Valentine P. J. (1992) -
Vercellotti , J. R. , A. A. Salyers , W. S. Bullard , and T. D. Wilkins . 1977 . Breakdown of mucin and plant polysaccharides in the human colon . Can. J. Biochem. 55 : 1190 - 1196 .
(
10.1139/o77-178
) / Can. J. Biochem. / Breakdown of mucin and plant polysaccharides in the human colon by Vercellotti J. R. (1977) -
Von Heijne , G. 1985 . Signal sequences: the limits of variation . J. Mol. Biol. 184 : 99 - 105 .
(
10.1016/0022-2836(85)90046-4
) / J. Mol. Biol. / Signal sequences: the limits of variation by Von Heijne G. (1985) -
Woese , C. R. 1987 . Bacterial evolution . Microbiol. Rev. 51 : 221 - 271 .
(
10.1128/MR.51.2.221-271.1987
) / Microbiol. Rev. / Bacterial evolution by Woese C. R. (1987) -
Wren , B. W. 1991 . A family of clostridial and streptococcal ligand-binding proteins with conserved C-terminal repeat sequences . Mol. Microbiol. 5 : 797 - 803 .
(
10.1111/j.1365-2958.1991.tb00752.x
) / Mol. Microbiol. / A family of clostridial and streptococcal ligand-binding proteins with conserved C-terminal repeat sequences by Wren B. W. (1991) -
Yoshihisa , T. , Y. Ohsumi , and Y. Anraku . 1988 . Solubilization and purification of ~-mannosidase, a marker enzyme of vacuolar membranes in Saccharomyces cerevisiae . J. Biol. Chem. 263 : 5158 - 5163 .
(
10.1016/S0021-9258(18)60693-3
) / J. Biol. Chem. / Solubilization and purification of ~-mannosidase, a marker enzyme of vacuolar membranes in Saccharomyces cerevisiae by Yoshihisa T. (1988)
Dates
Type | When |
---|---|
Created | 8 years, 9 months ago (Nov. 10, 2016, 9:07 a.m.) |
Deposited | 4 years, 1 month ago (July 29, 2021, 2 p.m.) |
Indexed | 4 weeks, 1 day ago (Aug. 6, 2025, 9:47 a.m.) |
Issued | 29 years, 7 months ago (Feb. 1, 1996) |
Published | 29 years, 7 months ago (Feb. 1, 1996) |
Published Print | 29 years, 7 months ago (Feb. 1, 1996) |
@article{Reeves_1996, title={A Bacteroides thetaiotaomicron outer membrane protein that is essential for utilization of maltooligosaccharides and starch}, volume={178}, ISSN={1098-5530}, url={http://dx.doi.org/10.1128/jb.178.3.823-830.1996}, DOI={10.1128/jb.178.3.823-830.1996}, number={3}, journal={Journal of Bacteriology}, publisher={American Society for Microbiology}, author={Reeves, A R and D’Elia, J N and Frias, J and Salyers, A A}, year={1996}, month=feb, pages={823–830} }