The growth of Lactobacillus casei strain Cl-16 at the expense of ribitol was inhibited if the non-metabolizable substrate xylitol was included in the medium at concentrations of 6 mM or greater. At these concentrations, xylitol did not competitively inhibit ribitol transport. The cessation of growth was caused by the intracellular accumulation of xylitol-5-phosphate, which occurred because growth on ribitol had gratuitously induced a functional xylitol-specific phosphotransferase system but not the enzymes necessary for the further metabolism of xylitol-5phosphate. Eventually, the cells overcame the xylitol-mediated inhibition by repressing the synthesis of enzyme II of the xylitol phosphotransferase system so that xylitol-5-phosphate would no longer be accumulated within the cell.

A futile xylitol cycle appears to be responsible for xylitol-mediated inhibition of growth of Lactobacillus casei CI-16 at the expense of ribitol. The gratuitously induced xylitol-specific phosphoenolpyruvate-dependent phosphotransferase accumulates the pentitol as xylitol-5-phosphate, a phosphatase cleaves the latter, and an export system expels the xylitol. Operation of the cycle rapidly dissipates the ribitol-5-phosphate pool (and ultimately the energy supply of the cell), thereby producing bacteriostasis.Those strains of Lactobacillus casei capable of growing at the expense of ribitol (rtl) are sensitive to xylitol (xtl) when the former is used as a source of energy (7). Previous studies have shown that growth at the expense of rtl results in the coincidental induction of an xtl-specific phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). The xtl PTS transports xtl into the cell to form xylitol-5phosphate (xtl-5-P); however, the absence of an xtl-5-P dehydrogenase prevents the further metabolism of this intermediate, and it accumulates in the cell (7). Depending upon its external concentration, xtl can either completely inhibit growth (at >5 mM) or merely increase the generation time of the organism (at <2 mM). Although much is known about the metabolic events that occur during the xtl-mediated inhibition of L. casei, the actual mechanism responsible for producing the bacteriostatic effects is unknown. Thompson and Chassy (15) demonstrated that the inhibition of growth by 2-deoxy-D-glucose (2-DG) in cultures of Streptococcus lactis 133 growing on lactose or sucrose was due to the initiation and maintenance of a futile cycle. The 2-DG cycle catalyzed the import and export of the nonmetabolizable hexose at the expense of the energy reserves (PEP) of the cells, thereby inhibiting growth.In this report, we demonstrate that the bacteriostatic effects of xtl are probably due to the futile cycling of the nonmetabolizable pentitol which (like the 2-DG-mediated inhibition) dissipates the energy reserves of L. casei Cl-16. MATERIALS AND METHODSCultivation and maintenance of L. casei CI-16. L. casei Cl-16 was obtained from the private collection of M. Rogosa, National Institute of Dental Research, Bethesda, Md. The organism was maintained and stored in fortified litmus milk medium (9). Lactobacillus-carrying medium (5) was used for all growth experiments and to prepare washed suspensions for resting-cell studies. All substrates were filter sterilized, and the microorganism was growth at 37°C in static culture.Growth experiments. The fate of the metabolic intermediate products of rtl before and after the addition of xtl was monitored by periodic sampling of an exponential-phase culture. Replicate sterile screw-capped test tubes were filled with 15 ml of filter-sterilized lactobacillus-carrying medium containing 30 mM [1-'4C]rtl (specific activity, 1 pCi/p.mol) * Corresponding author. and was adjusted to a density of 100 to 110 Klett units (660nm filter) with a washed-cell suspension harvested from a mi...

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The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [3H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP greater than ATP greater than GTP greater than CTP greater than TTP for pertussis toxin and ATP greater than GTP greater than TTP greater than CTP for the B oligomer. Phosphate ions inhibited the binding of [3H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [3H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

A simple three-step procedpre is described which yields electrophoretically homogeneous preparations of ribitol-5-phosphate dehydrogenase and xylitol-5-phosphate dehydrogenase. The former enzyme is a 115,000-molecular-weight protein composed of two subunits of identical size and is specific for its substrate, ribitol. The xylitol-5-phosphate dehydrogenase exists as a tetrameric protein with a molecular weight of 180,000; this enzyme oxidizes the phosphate esters of both xylitol and D-arabitol. Characterization of the physical, kinetic, and immunological properties of the two enzymes suggests that the functionally similar enzymes may not be structurally related.Only two species of lactic-acid bacteria, Streptococcus avium and Lactobacillus casei, have strains that are capable of growing at the expense of one or more five-carbon polyalcohols (9, 16). These bacteria utilize pentitols via a pathway not found in eucaryotes or other procaryotes (9, 10). The sugar alcohol is transported into the cell as pentitol-5-phosphate by a phosphoenolpyruvate-dependent phosphotransferase system in which a NAD-linked dehydrogenase oxidizes the phosphate ester to xylulose-5-phosphate, an intermediate of the pentose phosphate pathway. The transport systems and dehydrogenases are substrate specific, recognizing either the threo-(ribitol pathway) or erythro-(xylitol and D-arabitol pathway) configuration of the sugar phosphates and their corresponding phosphate esters. The xylitol pathway of L. casei C183 consists of only three novel components, a membrane-bound EIIXti, a soluble 111xt1, and a soluble xylitol-5-phosphate dehydrogenase; the ribitol pathway appears to be similarly constituted (10). II"xt1 of L. casei C183 has been purified, and its physical and biochemical properties have been described (12). In this report, we describe the purification and properties of the xylitol-5-phosphate and ribitol-5-phosphate dehydrogenases from L. casei C183 and C116, respectively. Purification of xylitol-5-phosphate and ribitol-5-phosphate dehydrogenases. Between 18 and 20 g (wet weight) of L. casei C183 or C116 cells was suspended in 60 ml of 0.02 M potassium phosphate buffer (pH 7.2) containing 10 mM 2-mercaptoethanol (PBME). Cells were disrupted by two 6-min treatments in an ice-water-cooled chamber of a sonifier (model 350; Branson Sonic Power Co., Danbury, Conn.) operating at 70% of maximum power. Intact cells were removed from the extract by centrifugation at 13,200 x g for 30 min. Comminuted membranes were removed from the cell-free preparation by centrifugation at 104,000 x g for 120 min in an ultracentrifuge (model L2-65B; Ivan Sorvall, Inc., Norwalk, Conn.). MATERIALS AND METHODSThe supernatant fluid was decanted from the membrane pellet and applied to a DE52 column (15 by 2.5 cm; DEAEcellulose [Whatman, Inc., Clifton, N.J.]) equilibrated with PBME. After the column was washed with 100 ml of PBME, dehydrogenase activity was eluted with a 0 to 0.4 M KCI gradient in PBME, and 10-ml fractions were collected. Xylitol-5-phosphate dehydrog...