6-phospho-alpha-D-glucosidase from Fusobacterium mortiferum: cloning, expression, and assignment to family 4 of the glycosylhydrolases

Abstract

The Fusobacterium mortiferum malH gene, encoding 6-phospho-␣-glucosidase (maltose 6-phosphate hydrolase; EC 3.2.1.122), has been isolated, characterized, and expressed in Escherichia coli. The relative molecular weight of the polypeptide encoded by malH (441 residues; M r of 49,718) was in agreement with the estimated value (ϳ49,000) obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the enzyme purified from F. mortiferum. The N-terminal sequence of the MalH protein obtained by Edman degradation corresponded to the first 32 amino acids deduced from the malH sequence. The enzyme produced by the strain carrying the cloned malH gene cleaved [U- Members of the genus Fusobacterium are gram-negative obligate anaerobes that colonize the oral and gastrointestinal tracts of humans and animals (5,24,27,31). The frequent isolation of these species from abscesses and necrotic tissues indicates their causative or contributory roles in a variety of infections (6,7,15,16,25). The prevalence and increased numbers of certain species (e.g., Fusobacterium nucleatum) in patients with gingivitis and periodontitis suggest that this organism may play a role in the etiology of these oral diseases (6,32,50). Although the pathogenic potential and medical importance of fusobacteria are widely recognized, surprisingly little is known about the biochemistry, genetics, or regulation of energy generation in these species.Most species of fusobacteria derive energy for growth via fermentation of amino acids such as glutamate, lysine, and serine (4, 10, 11, 17). Until recently, members of the genus were usually described as asaccharolytic or weakly fermentative (24,31,42). In this context, it was of considerable interest to discover that Fusobacterium mortiferum metabolized a wide variety of carbohydrates, including monosaccharides and both ␣-and ␤-glycosides, as energy sources (40,41,54,55). These compounds are transported by the phosphoenolpyruvate-dependent sugar:phosphotransferase system (PEP:PTS) (29,35,43). This multicomponent group translocation system comprises both membrane-associated enzymes and cytoplasmic proteins. In concert, the PEP:PTS facilitates the simultaneous translocation, phosphorylation, and internalization of sugars by the cell. A functional PEP:PTS requires two general proteins, designated enzyme I and HPr, that are allied with sugarspecific complexes comprising enzymes IIA, IIB, and IIC (for discussions, see references 35 and 44) to effect the sequential transfer of the high-energy phosphoryl moiety from PEP to the incoming sugar.PEP-dependent PTS have been reported for all major groups of carbohydrates in both gram-negative (29, 35) and gram-positive (19, 39, 51, 52) bacteria. However, the existence of a maltose-specific PTS has been a source of controversy, and evidence has been presented both for and against this postulate (for discussions, see references 40 and 53). In our laboratory, studies of maltose utilization by F. mortiferum resulted in the serendipitous isolation of maltose 6-phospha...

Metabolism of sucrose and its five isomers by Fusobacterium mortiferum

“…In contrast to other species, F. mortiferum ferments an extraordinarily wide variety of carbohydrates (Robrish et al, 1991). Previously, in studies of maltose metabolism in F. mortiferum, we cloned and expressed a gene (malH) whose deduced sequence exhibits " 75 % residue identity with the phospho-α-glucosidase of K. pneumoniae (Thompson et al, 1995 ;Bouma et al, 1997). In the present report, we show that the gene adjacent to malH (designated malB) also encodes a putative EII(CB) protein that is " 60 % identical with AglA of K. pneumoniae.…”

“…The amino acid sequence deduced from motif that assigns the two proteins to the GntR family of transcriptional regulators. The complete sequence of the phospho-α-glucosidase gene (malH) of F. mortiferum, together with a partial sequence for the gene (malB), were described in an earlier report (Bouma et al, 1997). The entire sequences for malB, and the upstream gene (malR), have now been obtained by chromosome ' walking ' (GenBank accession no.…”

“…Sau3AI chromosomal DNA fragment of F. mortiferum that includes the malH gene and its promoter has previously been cloned and the enzyme has been expressed from plasmid pCB4.11 (Bouma et al, 1997). This plasmid was transferred by electroporation to E. coli PEP43 ∆cel ∆(bgl-pho) leu metA or B his rpsL lacZ∆4680 lacY + arbT + Tn10 ::bglA dTn10 cam :: ebgA5100 ebgR + L532 (B. G. Hall, Biology Department, University of Rochester, NY, USA, laboratory collection).…”

“…Automated DNA sequencing incorporating Big Dye terminators was used to sequence malB, and an adjacent upstream gene (malR), directly from genomic DNA of F. mortiferum. From data previously reported by Bouma et al (1997), a reverse primer 1R1 (5h-AACTCT-CTCTAACTTGTGGTACTGAAAGTC-3h) was designed to obtain initial sequence information. Subsequent data were obtained by the primer synthesis and the chromosomal ' walking ' technique.…”

Genetic diversity of oral Fusobacterium nucleatum isolated from patients with different clinical conditions

“…Despite the large knowledge about the pathogenic potential and the medical aspects of fusobacteria, their metabolic and genetic aspects are little known 4,16 . In the last years, the use of tools as cloning, sequencing and PCR have given more information about the genetic aspects of F. nucleatum.…”