This suggests that B. burgdorferi has already adapted its growth rate to that permitted by its reduced number of rRNA genes. It thus appears that ascertainment of the biological role of differences in rRNA gene copy number in various bacterial species will require an extensive comparative analysis of the adaptability of bacteria with high and low numbers of rRNA genes to different growth conditions before any clear cut conclusions can be drawn. Two major mechanisms regulating rRNA synthesis in E. coli are growth BI 2536 rate and the stringent response [9, 11]. Our efforts to determine if B. burgdorferi rRNA synthesis was controlled by growth rate at a single temperature have been repeatedly frustrated by the still undefined

nutritional requirements

of B. burgdorferi and the lack of simple culture media for this organism [38, 39]. We previously reported that (p)ppGpp levels in B. burgdorferi grown in BSK did not vary despite 10-fold reductions of yeastolate, neopeptone or rabbit serum EX 527 [18]. We have now found that complete omission of rabbit serum from BSK-H did not affect growth of B. burgdorferi B31 at 34°C (Figure 3) or (p)ppGpp levels at 34°C or 23°C (Figure 4). It was thus not possible to alter B. burgdorferi growth rate at a given temperature by changing the composition of its culture medium [11, 40]. The slower growth of B. burgdorferi B31 at 23°C compared to 34°C correlated well with slower accumulation of total DNA, RNA and protein. Although there was a lag in cell number, total DNA and total protein in cells grown at the lower temperature, the amounts of DNA and protein per cell were similar at both temperatures. As expected, the amount of DNA per rapidly dividing exponential phase cells was higher than in more slowly dividing stationary phase cells. The slower rate of increase in total RNA in stationary phase cultures at the lower temperature also resulted in a significant Interleukin-2 receptor difference

in RNA per cell under these two conditions. Although these results were in agreement with the hypothesis that growth rate regulates rRNA synthesis in B. burgdorferi, further investigation determined that growth phase and not growth rate regulated rRNA levels under these conditions (Figure 5). Importantly, even though B. burgdorferi was grown for up to 11 days in 34°C culture and for 28 days in 23°C culture in our experiments, about 80% of all cells at this stationary phase stage are viable (R. Iyer and I. Schwartz, unpublished results), and non-viability cannot therefore account for the large decrease in rRNA levels in stationary phase in these cultures. Amounts of 16S and 23S rRNA that were first normalized to mRNA amounts for constitutively expressed flaB and then additionally normalized to levels at 23°C and 106 cells/ml were similar in rapidly growing (34°C) and slowly growing (23°C) cultures when compared at the same growth phase; both RNA species decreased as the cultures progressed toward stationary phase (Figure 5).