Abstract
The influence of growth temperature on the ability to ferment d-sorbitol was investigated in Escherichia coli O157:H7. It was found that O157:H7 strains have a temperature-sensitive sorbitol phenotype. d-Sorbitol transport and sorbitol-6-phosphate dehydrogenase activities were expressed in sorbitol-fermenting cells grown at 30°C but only at a low level at 40°C. Sorbitol-positive variants able to transport d-sorbitol were easily selected at 30°C from culture of Sor− E. coli O157:H7 strains.
Enterohemorrhagic Escherichia coli (EHEC), particularly of serotype O157:H7, have emerged in recent years as the predominant cause of hemorrhagic colitis, which can progress into a hemolytic uremic syndrome in humans (11, 14, 16). Molecular and biochemical studies showed that serotype O157:H7 is a discrete clone (17, 18). Unlike other E. coli, isolates of serotype O157:H7 do not ferment d-sorbitol within 24 h, lack β-glucuronidase activity, and do not grow at 45.5°C (2, 5, 6). These traits, especially the absence of sorbitol fermentation, are used extensively to distinguish isolates of serotype O157:H7 from other E. coli serotypes (4, 12). However, the emergence of phenotypic variants has sporadically been observed. It has been reported that serotype O157:H7 can mutate to a sorbitol-positive phenotype either in food products or occasionally during culture in d-sorbitol-containing media (7).
In E. coli, d-sorbitol and some other hexitols are trapped by vectorial phosphorylation (8, 9). Utilization of d-sorbitol is initiated by a specific enzyme II-enzyme III complex of the phosphotransferase system (PTS), which converts the substrate to d-sorbitol-6-phosphate. The phosphate donor in this reaction is phosphoenolpyruvate (PEP). d-Sorbitol-6-phosphate is then converted to d-fructose-6-phosphate by an NAD-linked dehydrogenase. The transport system acts on d-sorbitol with a high affinity and on d-mannitol with a low affinity.
In the present study, we tested the influence of growth temperature on the ability to ferment d-sorbitol in E. coli O157:H7. All strains were from the Centre National de Référence de Typage Moléculaire Entérique at The Institut Pasteur. Non-sorbitol-fermenting O157:H7 strains will be referred to here as Sor−. In a first experiment, 13 Sor− and one sorbitol-fermenting (Sor+; strain 96336) O157:H7 strain were inoculated with 30 μl of culture in d-sorbitol–peptone water containing 10 mM d-sorbitol and bromothymol blue as an indicator (3) and then incubated at either 30, 37, or 40°C. The culture conditions rendered the incubation medium anaerobic. A Sor+ non-O157:H7 strain (strain 75-88) was included as a control. Although all isolates were able to grow in this medium at 30, 37, and 40°C, they could not produce acid from sorbitol at 37 and 40°C. We observed that for 13 Sor− E. coli O157:H7 strains, acid production from d-sorbitol could be detected at 30°C but not at 37 and 40°C after incubation for 3 to 4 days. For the two Sor+ strains (96336 and 75-88), acid production from d-sorbitol was observed within 24 h at all of the temperatures tested. This temperature sensitivity was restricted to strains grown on d-sorbitol. The thirteen Sor− strains were able to produce acid from d-mannitol, d-dulcitol, and l-sorbose within 24 h at 30, 37, and 40°C (data not shown).
To verify the temperature effect on sorbitol fermentation, the anaerobic growth behavior of Sor− and Sor+ strains at 30 and 40°C was studied (Fig. 1). The time course of cell growth and d-sorbitol utilization by one Sor− O157:H7 strain (Ec40) and two Sor+ strains (96336 and 75-88) grown on peptone-water containing 10 mM d-sorbitol at 30 and 40°C were studied. Enzymatic assay of sorbitol was determined by using a sorbitol test kit (Boehringer GmbH, Mannheim, Germany). At 40°C, strain Ec40 (wild type) was unable to utilize d-sorbitol as a carbon source and grew at the expense of peptones in the growth medium. At 30°C, a biphasic growth curve was obtained. At this temperature, strain Ec40 utilized peptones first and then d-sorbitol with a lag period of 72 h (Fig. 1A). Growth inhibition at 40°C was not observed with Ec40 grown on d-mannitol. Furthermore, anaerobic growth on d-mannitol was faster than on d-sorbitol (data not shown). Whatever growth temperature used, the two Sor+ strains (96336 and 75-88) showed high anaerobic growth yields (Fig. 1B).
FIG. 1.
Anaerobic growth of Sor− and Sor+ strains of E. coli O157:H7 on d-sorbitol at 30 and 40°C. The means of at least three determinations are presented, and the standard deviation was 0.01 to 0.05. (A) Cells of Ec40 (circles) and its sorbitol Sor+ variant (triangles) were grown anaerobically at 30°C (open symbols) and at 40°C (filled symbols) on peptone broth medium containing 10 mM d-sorbitol. (B) Cells of 96336 (circles) and 75-88 strains (triangles) growing anaerobically at 30°C (open symbols) and at 40°C (filled symbols) on the same medium.
A Sor+ variant could be isolated from strain Ec40, after four or six overnight transfers at 30°C, in peptone broth medium containing 10 mM d-sorbitol. Anaerobic growth of this variant reached the stationary phase after 24 h at 30°C and after 48 h at 40°C (Fig. 1A). d-Sorbitol was utilized simultaneously. Similarly, Sor+ variants have been obtained for all Sor− strains studied. These latter variants showed the same growth behavior as the Sor+ variant of strain Ec40.
The inability to grow at higher temperatures on hexitols has already been reported (10, 15). Several works suggested that this growth inhibition is apparently due to a temperature-sensitive enzyme. To investigate this possibility, transport and metabolism of d-sorbitol were studied in Sor− and Sor+ strains of E. coli O157:H7 at 30 and 40°C (Table 1). Since d-sorbitol transport system accumulates both d-sorbitol and d-mannitol, the PTS activities towards both hexitols were investigated as described previously (1). We measured the PEP-dependent phosphorylation of [14C]sorbitol and [14C]mannitol in toluene-treated cells. After growth at 30°C on d-sorbitol, strains 75-88 (Sor+ non-O157:H7 strain) and 96336 (Sor+ O157:H7 strain) exhibited PEP-dependent phosphorylation of [14C]sorbitol and [14C]mannitol. Strain of Ec40 (wild type) showed a very low PTS activity for d-sorbitol and d-mannitol, whereas in the Sor+ variant the transport activities were detected. When the same experiments were done with cells grown at 40°C we observed that, in Sor+ O157:H7 strains (96336 and Ec40 variant), the sorbitol transport was lower than in cells grown at 30°C. However, in strain 75-88, transport activities were not inactivated at 40°C. d-Sorbitol- and d-mannitol–phosphotransferase systems were induced in cells grown on d-mannitol, whereas the sorbitol-PTS activity was induced to a low level. Both activities were present at both temperatures. The detection of an ATP-dependent phosphorylation activity of d-sorbitol and d-mannitol suggested the presence of kinases. It thus appears that O157:H7 strains demonstrate a temperature-sensitive d-sorbitol transport.
TABLE 1.
Activities of the PTS and sorbitol-6-phosphate dehydrogenase in Sor+ and Sor− strains of E. coli growth anaerobically at 30 and 40°C
| Strain | Inducera | PEP- and ATP-dependent phosphorylation activities ofb:
|
Sorbitol-6-phosphate dehydrogenase activity (μmol [mg of protein]−1 ml−1)
|
||||
|---|---|---|---|---|---|---|---|
| [14C]sorbitol
|
[14C]mannitol
|
||||||
| 30°C | 40°C | 30°C | 40°C | 30°C | 40°C | ||
| 75-88 (non-O157:H7) | Sorbitol | 156 ± 15 (8) | 105 ± 10 (39) | 127 ± 9 (57) | 122 ± 12 (48) | 1.81 ± 0.05 | 1.65 ± 0.05 |
| Mannitol | 42 ± 9 (27) | 43 ± 5 (21) | 133 ± 11 (82) | 118 ± 6 (34) | ≤0.02 | ≤0.02 | |
| None | 5 | ND | 8 | ND | |||
| 96336 (Sor+ O157:H7) | Sorbitol | 123 ± 2 (91) | 48 ± 8 (25) | 121 ± 5 (54) | 141 ± 10 (49) | 1.42 ± 0.05 | 0.61 ± 0.10 |
| Mannitol | 37 ± 5 (29) | 66 ± 9 (24) | 141 ± 11 (50) | 124 ± 9 (62) | ≤0.02 | ≤0.02 | |
| None | 6 | ND | 20 | ND | |||
| Ec40 (Sor− wild) | Sorbitol | 7 ± 4 (5) | 17 ± 6 (2) | 27 ± 8 (13) | 33 ± 8 (17) | 0.15 ± 0.04 | ≤0.02 |
| Mannitol | 48 ± 6 (20) | 68 ± 10 (34) | 110 ± 16 (46) | 137 ± 20 (54) | ≤0.02 | ≤0.02 | |
| None | 4 | ND | 12 | ND | |||
| Ec40 (Sor+ variant) | Sorbitol | 101 ± 11 (34) | 34 ± 7 (7) | 80 ± 4 (32) | 135 ± 16 (9) | 0.33 ± 0.04 | ≤0.02 |
| Mannitol | 52 ± 8 (19) | 62 ± 11 (26) | 137 ± 5 (5) | 117 ± 20 (52) | ≤0.02 | ≤0.02 | |
| None | 4 | ND | ND | ND | |||
Inducer substrates were used at 0.2% in the anaerobic culture.
PEP- and ATP-dependent phosphorylation was monitored with 0.15 mg (wet weight) of bacteria per ml. Results are expressed as nmol of 14C-labeled substrate phosphorylated min−1. Values in parentheses correspond to the ATP-dependent phosphorylation activity. The means of at least two experiments are presented, along with their standard deviations. ND, not determined.
The effect of the temperature on the sorbitol-6-phosphate dehydrogenase activity was studied in cell extracts of strains grown at 30 and 40°C. The sorbitol-6-phosphate dehydrogenase activity was assayed as described previously (13). It was found that the dehydrogenase activity was lower in cell extracts of strains grown at 40°C than in cells grown at 30°C (Table 1). This dehydrogenase activity was inducible by d-sorbitol but not by d-mannitol and was stable at a high temperature. No appreciable loss of activity was found in cell extracts incubated 60 min at 40°C (data not shown).
Our results show that Sor+ O157:H7 strains express a temperature-sensitive d-sorbitol phenotype. d-Sorbitol transport and sorbitol-6-phosphate dehydrogenase activities were expressed in cells grown at 30°C but only at a low level at 40°C. Sorbitol-positive variants able to transport d-sorbitol were easily selected at 30°C from a culture of Sor− E. coli O157:H7 in d-sorbitol-containing medium. Genetic analysis will be required to establish the type of mutation undergone by these variants.
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