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J Bacteriol. Nov 1999; 181(22): 7126–7130.
PMCID: PMC94189
Note

d-Allose Catabolism of Escherichia coli: Involvement of alsI and Regulation of als Regulon Expression by Allose and Ribose

Abstract

Genes involved in allose utilization of Escherichia coli K-12 are organized in at least two operons, alsRBACE and alsI, located next to each other on the chromosome but divergently transcribed. Mutants defective in alsI (allose 6-phosphate isomerase gene) and alsE (allulose 6-phosphate epimerase gene) were Als. Transcription of the two allose operons, measured as β-galactosidase activity specified by alsI-lacZ+ or alsE-lacZ+ operon fusions, was induced by allose. Ribose also caused derepression of expression of the regulon under conditions in which ribose phosphate catabolism was impaired.

Conversion of the all cis-hexose allose to fructose 6-phosphate in Aerobacter aerogenes requires the activity of three enzymes—allokinase, allose 6-phosphate isomerase, and allulose 6-phosphate epimerase (7). This pathway appears to operate also in Escherichia coli. Thus, five contiguous genes (alsRBACE), expressed as one operon, encode a periplasmic binding protein-mediated transport system (alsB, alsA, and alsC), a putative hexose phosphate epimerase (alsE), and a regulatory protein for allose utilization (alsR). A potential sixth member of the operon (alsK) has been postulated to encode allokinase (9). Ribose utilization requires among other enzymes ribose 5-phosphate isomerase. In E. coli, two ribose phosphate isomerases, A and B, have been identified biochemically (5, 6) and genetically (18, 20). Ribose phosphate isomerase A, encoded by rpiA, is synthesized constitutively, whereas the synthesis of ribose phosphate isomerase B, encoded by rpiB, appears to be increased following growth of cells in ribose-containing medium. A repressor protein, encoded by the rpiR gene, is involved in regulation of rpiB gene expression. Thus, rpiR strains contain elevated activity of ribose phosphate isomerase B (6, 20). The rpiB and rpiR loci are located next to each other at 92.8 min on the linkage map, but are divergently transcribed, with rpiB transcribed clockwise (20).

alsR and rpiR are the same gene (9). In the present work, we show that the rpiB gene product is also involved in allose catabolism, and presumably rpiB encodes allose 6-phosphate isomerase. Hence rpiB will be redesignated alsI. Although nucleotide sequence analysis implies alsK is the distal cistron of an alsRBACEK operon, our results showed that the alsK cistron was neither necessary for allose utilization nor coordinately expressed with the remaining alsRBACE cistrons. Consequently, we have designated alsK as yjcT (15).

Methods.

The E. coli K-12 strains used in this study are listed in Table Table1.1. Growth media (NZY broth or phosphate-buffered AB minimal medium) were described before (8). The carbon sources used were glucose, ribose, xylose, and glycerol at 0.2% each or allose at 0.05 or 0.1%. The growth of cell cultures was monitored in an Eppendorf PCP6121 photometer as optical density at 436 nm. Bacteriophage P1-mediated transduction (13), transformation with plasmid DNA (10), techniques for the growth of bacteriophage λ (16), and lysogenization by recombinant phage (17) have been previously described, as well as methods for the isolation of plasmid DNA (2) and chromosomal DNA (16). Restriction and ligation of DNA were performed as described by the suppliers of restriction endonucleases (Amersham, Promega, and New England Biolabs) and T4 DNA ligase (Amersham). PCR was performed with chromosomal or plasmid DNA as a template by standard procedures with DynaZyme II DNA polymerase (Finzymes, Oy, Finland). For enzyme assays, exponentially growing cells were harvested by centrifugation and disrupted by sonication for 60 s at 0°C and then centrifuged to remove cell debris. The assay of β-galactosidase activity at 30°C (13), or allokinase activity at 37°C (7) and determination of protein content (19) were performed as previously described.

TABLE 1
Bacterial strains

Isolation and characterization of als and yjcT mutants.

Transposon technology was used to generate one plasmid-harbored alsR::TnphoA′-1 mutation, four alsE::TnphoA′-1 mutations, and four yjcT::TnphoA′-1 mutations (Fig. (Fig.1).1). To avoid effects of a high copy number in an analysis of the regulation of als and yjcT gene expression, allele replacement by homologous recombination was conducted with each of the plasmid-borne als or yjcT mutations. This recombination resulted in the production of strains harboring chromosomally located als or yjcT mutations. The TnphoA′-1 insertions generated polar mutations. For each mutation, a nonpolar version was constructed (Fig. (Fig.1).1). We also constructed an operon fusion allele to the alsI gene [Φ(alsI-lacZ+)139] (Fig. (Fig.2).2). A map of the 10 insertions is shown in Fig. Fig.1A.1A. The nucleotide sequences of the fusion points of the transposon-generated fusions are shown in Fig. Fig.1B.1B. The growth of the als strains, polar as well as nonpolar, on allose was analyzed. The alsR and alsE strains containing polar mutations were Als, whereas the yjcT strains were Als+. The strains containing nonpolar alsE mutations were also Als. In contrast, the strains containing nonpolar alsR21 or yjcT8 mutations were Als+. Furthermore, a strain harboring a mutation in the alsI gene (HO1973) was Als. These results indicated that the alsI and alsE gene products are essential for allose utilization, whereas the repressor, encoded by alsR, is dispensable for allose utilization.

FIG. 1
Structure of the als operon and location of als-lacZ+ and yjcT-lacZ+ insertions. Mutagenization of the alsRBACE operon and yjcT was performed as follows. Strain CC118 (Δlac) harboring pTP680 (alsR+B+A+C ...
FIG. 2
Construction of an alsI-lacZ+ gene fusion. Open reading frames are indicated by open double lines, vector sequences are indicated by thin lines, and flanking DNA sequences or intercistronic regions are shown as black or shaded double lines. Relevant ...

The addition of allose (0.05%) appeared to potently inhibit the growth with glycerol as the carbon source (0.2%) of strains harboring mutations in alsI (HO1973) or alsE (TP2086), encoding allose 6-phosphate isomerase and allulose 6-phosphate epimerase, respectively. In contrast, the growth of the remaining strains, i.e., those defective in the regulatory protein (TP2115 [alsR]) or YjcT (TP2083) were not inhibited by allose. The lack of growth of the alsE and alsI strains in the presence of allose indicated that a compound, which accumulated in these strains, caused inhibition. It is likely that this compound is allose 6-phosphate in the alsI strain and allose 6-phosphate, allulose 6-phosphate, or both in the alsE strain.

We previously showed that the alsI (rpiB)-encoded enzyme is able to isomerase ribose 5-phosphate and ribulose 5-phosphate. Thus, this enzyme appears to have substrate specificity toward both pentose phosphates and hexose phosphates. A similar situation exists for the Streptococcus mutans galactose 6-phosphate isomerase (encoded by lacAB), which is also able to isomerize ribose 5-phosphate (20).

Regulation of alsI operon expression.

The recombinant Φ(alsI-lacZ+)139 fusion-harboring λ phage was used to lysogenize various E. coli strains. A 58-fold increase in β-galactosidase activity was observed when cells of strain YYC1060 were grown in the presence of allose and compared to the activity of cells grown in the absence of allose (Table (Table2).2).

TABLE 2
Regulation of als regulon expression by allose

The β-galactosidase activity specified by the Φ(alsI-lacZ+)139 fusion contained in host strains, which harbored various genetic lesions of ribose catabolism, and grown on different carbon sources is shown in Table Table3.3. In a wild-type strain (YYC1060), only a modest increase (twofold or less) in alsI gene expression was observed when cells were grown with pentose as a carbon source (xylose, ribose, or both) compared to growth in the presence of glucose. In contrast, alsI gene expression was greatly increased in ribose auxotrophic strains (rpiA or rpiA alsI), when grown on ribose. Thus, with growth in the presence of ribose, the β-galactosidase activity of an rpiA strain (HO1686) increased approximately 25-fold compared to growth in the presence of both ribose and glucose. The increase was less pronounced by growth in the presence of both ribose and xylose: approximately fivefold compared to growth in the presence of both ribose and glucose. Furthermore, alsI gene expression increased 25-fold or more in an rpiA alsI strain (HO1693) grown in the presence of ribose and xylose, compared to growth in the presence of ribose and glucose. A mutation in the alsI gene alone was essentially without effect on alsI gene expression, because the alsI strain HO1868 responded like the wild-type strain YY1060. The β-galactosidase activity in an alsR strain harboring the operon fusion (YYC1062) was increased 20- to 100-fold compared to the activity of the otherwise isogenic alsR+ strain (YYC1060).

TABLE 3
Regulation of als regulon expression by pentoses

Regulation of alsRBACE operon expression.

Strains harboring a phoA′-1 (lacZ+) gene fusion to the alsR (TP2115) or alsE (TP2086) cistrons were assayed for β-galactosidase activity in extracts of cells grown in the presence or absence of allose (Table (Table2).2). In the presence of allose, β-galactosidase activity increased 43-fold compared to the activity in the absence of allose in cells harboring a lacZ fusion to alsE (TP2086). Thus, expression of the alsE cistron appeared to be induced by the presence of allose. Cells harboring an alsR-lacZ+ gene fusion (TP2115) contained a high, constitutive level of β-galactosidase activity. The β-galactosidase activity of a nonfusion strain (BW18524) was negligible. In addition, the expression of the alsRBACE operon was regulated by ribose similarly to that described for the alsI operon. Thus, β-galactosidase activity specified by the alsE11::TnphoA′-9 fusion increased approximately 15-fold in cells grown with ribose or 4-fold in cells grown with ribose and xylose, compared to that in cells grown with ribose and glucose (Table (Table33).

Lack of involvement of yjcT (alsK) in allose utilization.

The open reading frames of the distal cistron alsE and the following cistron yjcT overlapped by five codons, which may suggest translational coupling of the two cistrons (3). We constructed four independent insertions in yjcT, all of which had similar properties. Most importantly, expression of yjcT apparently was unaffected by allose (Table (Table2,2, strain TP2083). Strains with transposon insertions in yjcT were Als+. Furthermore, a yjcT mutation had no effect on expression of the alsRBACE and alsI operons: the introduction of yjcT8 into an alsI139-lacZ+ strain had little effect on the fold of induction of β-galactosidase synthesis (Table (Table2,2, strains YYC1060 and HO2190). Supplying yjcT in trans had no effect, as shown by the lack of regulation of the yjcT-lacZ+ fusion strain transformed with pHO390, which contains a wild-type yjcT allele (Table (Table2,2, strains TP2083, TP2083/pHO390 and TP2083/pBR322). The allokinase activity in extracts of cells harboring pHO390, (i.e., with yjcT in multicopy) was identical to the activity in extracts of cells harboring pBR322 (0.5 nmol min−1 mg of protein−1). Finally, allokinase activities were similar in cells of wild-type and yjcT strains grown in glycerol (0.3 nmol min−1 mg of protein−1). Allose did not cause induction of allokinase synthesis, and alsR and alsR+ strains contained identical activities of allokinase. These results suggest that the kinase responsible for phosphorylation of allose either has a broad substrate specificity, which may not be subject to induction by allose, or it utilizes a phosphoryl donor different from ATP.

Conclusion.

We have shown that alsI is essential for allose catabolism and that expression of both of the operons, alsI and alsRABCE, is induced by the presence of allose or ribose. In both cases, regulation is dependent on the alsR gene product. Thus, the alsI and alsRBACE operons constitute the als regulon. Apparently the yjcT gene is not a member of the als regulon.

Acknowledgments

Barry Wanner, Bob Simons, and Bente Mygind are acknowledged for generously providing plasmids, bacteriophages, and bacterial strains. Charlotte Hansen is acknowledged for running the automated DNA sequencing. Tonny D. Hansen and Anne L. Møller are acknowledged for expert technical assistance. We thank Jan Neuhard for carefully reading the manuscript.

Financial support was obtained from the Danish Center of Microbiology and the Center for Enzyme Research.

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