This paper reports the isolation and characterization of the first Lactobacillus (L. reuteri) gene (inu) encoding an FTF (Inu), producing in E. coli a high-molecular-weight inulin with β-(2→1) glycosidic bonds only and inulin FOS (mainly consisting of 1-kestose). FOS production was also observed with L. reuteri strain 121 cells, but no inulin formation was detected. In another work, we have raised antibodies in rabbits against the purified recombinant Inu protein (unpublished results). Unfortunately, no specific immunostaining was observed with L. reuteri cells, culture supernatants, or cell wall material. In these studies, we observed a high background response. Very likely, the rabbits used to raise antibodies contained endogenous lactobacilli. Northern blot hybridization experiments with a probe comprising the region in inu corresponding to the family 68 core region did not reveal the presence of inu mRNA. In summary, FOS synthesis was observed in L. reuteri culture supernatants but the inulin type of fructan produced by the recombinant Inu was not observed in L. reuteri strain 121. Possible explanations for this clear discrepancy are that (i) the inu gene is silent in L. reuteri or not expressed under the growth conditions tested, (ii) the Inu enzyme synthesizes FOS only under the conditions tested in its natural host, (iii) the inulin polymer already has been degraded at the time of harvesting of the cultures (no evidence, however, has been found for the presence of inulin-degrading activities in L. reuteri strain 121 supernatants), or (iv) Inu has activities in E. coli CEs other than those in L. reuteri strain 121. InuHis protein produced in E. coli showed smearing on SDS-PAGE gels, and the His tag could not be detected. These observations may suggest that in E. coli, the InuHis protein is in fact truncated at its C terminus. At present, we cannot exclude the possibility that the products synthesized by the L. reuteri Inu protein are different from the products synthesized by the recombinant InuHis protein.
In the process of cloning the inu
gene from L. reuteri
strain 121, a PCR step was performed with a specific primer based on the (incomplete) inu
sequence (20FTFi) and a degenerate primer based on the N-terminal amino acid sequence of a previously purified levansucrase protein from L. reuteri
strain 121. Misannealing of the degenerate primer (19FTF) yielded an amplicon overlapping with the inu
DNA sequence. This PCR was done at 50°C with a proofreading DNA polymerase. The calculated melting temperature of the DNA sequence to which the primer 19FTF annealed (5′-TAAACGTTTAGCAAAAAGGTAAA-3′) was 36°C (based on the following formula: melting temperature = 2×AT + 4×CG + 4). This result might be explained by a misannealing of the primer at the start of the PCR. A “hot start” involving separation of the DNA polymerase from the PCR mixture at temperatures lower than 90°C ensures that no misannealing takes place (5
). Conceivably, no PCR product had been obtained when such a hot start had been used in the PCR involving primers 19FTF and 20FTFi.
A typical secretion signal peptide (26
) is present in the N terminus of the Inu protein, with a possible initiation from either translation start codons at positions 1432 and 1459. The N-terminal amino acid sequence of the purified recombinant InuΔ699His protein corresponded to the amino acid sequence following the predicted signal peptidase cleavage site in the deduced Inu sequence. The lack of FTF activity (glucose release from sucrose) in arabinose-induced E. coli
(harboring the inu
gene) culture supernatants (results not shown) indicates that the recombinant InuΔ699His protein is not secreted by E. coli
. N-terminal amino acid sequence analysis of the E. coli
purified InuΔ699His protein shows that E. coli
does cleave the signal sequence from the Inu protein, and thus that the E. coli
protein export machinery recognizes the signal sequence. A similar observation was done for the S. salivarius
). Most likely the Inu protein is present either in the cell membrane, or in the periplasmic space of the E. coli
A cell wall-anchoring motif reported for various gram-positive cell wall-associated proteins (24
) was present at the C terminus of the deduced Inu amino acid sequence. It consisted of a 20 times repeat of the amino acids PXX, an LPXTG motif, a hydrophobic domain, and was ended by three positively charged amino acids (Fig. ). This is the first report of this motif for an FTF enzyme. A model for proteins bearing this cell wall-anchoring motif has been proposed (23
). In this model, the stretch of hydrophobic residues acts as a membrane spanning region with the positively charged amino acid residues directed towards the cytosol and the N-terminal part of the protein directed outwards of the cell spaced by a proline-glycine and/or threonine-serine rich region (in the case of Inu the PXX repeats). The LPXTG motif is proteolytically cleaved between the threonine and the glycine residues by a sortase enzyme resulting in a protein covalently linked to the peptidoglycan layer. Major problems arose when attempting to introduce and express the full-length Inu (no transformants) and InuHis (protein smears on gel) constructs in E. coli
. Introduction and expression in E. coli
of the InuΔ699 and InuΔ699His contructs were straightforward. Apparently the C-terminal region of Inu is problematic for the E. coli
protein expression machinery.
The products of Inu incubated with its substrate sucrose were mainly 1-kestose and an inulin of high molecular weight. High-molecular-weight inulin production was reported before only in S. mutans
). The inulin polymers produced by these streptococci, however, contain β-(2→6) branches (5%). Exclusive production of 1-kestose has been reported for Aspergillus niger
). Plant FTFs are known to synthesize 1-kestose as primer for the production of inulin polymers (46
). The combined production of 1-kestose and a levan polymer has been reported for the G. diazotrophicus
levansucrase enzyme (17
). The combination of the production of 1-kestose and levan is remarkable, because levan polymers consist of β-(2→6)-linked fructosyl units, while 1-kestose consists of a β-(2→1) fructosyl unit coupled to sucrose. The production of 1-kestose by Inu is the first elongation step in the polymerization reaction. The large amounts of FOS formed under the incubation conditions used thus may represent aborted polymerization attempts of the Inu enzyme.
The deduced amino acid sequence of the Inu protein of the generally regarded as safe bacterium L. reuteri
shows highest homology to FTFs from streptococcal origin. Streptococci are well-studied inhabitants of the oral cavity, with fructan synthesized from sucrose most likely contributing to the cariogenicity of dental plaque formation (33
). L. reuteri
strains, in contrast, are residents of the mammalian gut system. It will be interesting to study the in situ functional properties of L. reuteri
strain 121 and the fructans produced and their possible roles in the probiotic properties attributed to L. reuteri
Previously, we reported the presence of a levansucrase in L. reuteri
strain 121 (43
). Here we report the isolation and characterization of a novel inulosucrase-encoding gene from L. reuteri
strain 121. Southern hybridization studies under non-stringent conditions with two probes against the inulosucrase gene and strain 121 chromosomal DNA revealed one hybridizing band. The N-terminal sequence as well as the internal amino acid sequences determined for the purified levansucrase from L. reuteri
strain 121 (44
) could not be identified in the deduced strain 121 inulosucrase sequence. L. reuteri
strain 121 thus contains both a levansucrase gene and an inulosucrase gene. Apparently, these two ftf
genes are significantly different not only in products formed, but also in amino acid sequence.
Future work will involve (i) a detailed biochemical characterization of the recombinant Inu enzyme and (ii) inu gene expression studies in L. reuteri strain 121. This will enable a more detailed investigation of the catalytic mechanism of FTF enzymes producing inulin polymers, and analysis of the Inu activities and products in L. reuteri itself. The biological relevance and potential health benefits of an inulosucrase associated with a L. reuteri strain remain to be established.