At each time point, an aliquot of each culture was taken to determine growth and culture medium pH. Data shown in A and B are representative of five and two independent experiments, respectively. To survive in the highly acidic host environment, Hp contains the enzyme urease, which converts urea to ammonia and CO2 [34–38]. Urea supports Hp growth in the absence of CO2 only at acidic pH levels; the CO2 generated from urea plays GDC-0068 in vitro a role in periplasmic and cytoplasmic
buffering [39, 40]. We tested the possibility that CO2 generated from urea was sufficient to support the growth of Hp. We buffered culture medium (pH 6.3) to prevent high pH from inhibiting Hp growth. In the absence of CO2, urea markedly shortened the lag phase of growth, but combining urea with CO2 did not yield additive effects on growth (Figure 2B). We also cultured Hp in the medium supplemented with NH4Cl in the absence or presence of CO2. NH4Cl supply did not support Hp growth in the absence of CO2 nor shortened the lag period in the presence of CO2, excluding the possibility that ammonium produced from urea supports Hp growth. Supplementation of the culture medium with oxaloacetate, which is rapidly converted into pyruvate and CO2, also supported Hp growth in the absence of CO2, but addition Olaparib molecular weight of oxaloacetate to cultures
incubated under 10% CO2 did not increase Hp growth (data not shown). In contrast, pyruvate supplementation could not substitute for CO2 (data not shown). Taken
together, these data demonstrate the CO2 requirement of Hp for optimal growth and its ability to utilize bicarbonate in place of CO2. Lack of CO2 but not high O2 tension transforms Hp into the coccoid form Hp has long been known to transform into the coccoid form under unfavorable conditions, including exposure to atmospheric O2 levels. We examined the morphology of Hp grown under various levels of O2 and CO2 by field emission-scanning electron microscopy (FE-SEM) (Figure 3). The spiral form MRIP of Hp cells was observed at 12 h after inoculation, regardless of gas conditions. However, cultures grown under 8% O2 in the absence of CO2 also contained a significant number of coccoid Hp cells; at 36 h, most of the cells had transformed into U-shaped or coccoid cells. Under 20% O2 without CO2, most cells had very long spiral forms (mean length, 4.5 μm) at 12 h, but more than 60% of the cells were U-shaped, rounded, or coccoid at 36 h. These results indicate that high O2 levels delay Hp transformation into coccoid forms. Under CO2, most cells were spiral-shaped regardless of O2 tension at 12 h; however, at 36 h cells grown under 2% O2 began to convert to coccoid forms, whereas those cultured under 8% or 20% O2 remained in the unstressed spiral form.