Involvement of the adenylyl cyclase encoded by MSMEG_4279 in the oleic acid-induced resuscitation of NC cells.

<p>NC cells were obtained and resuscitated in batch mode. In part A, the OD₆₀₀ was measured after resuscitation for 5 d for the wild type and the complemented strain, ΔAC(pMindAc), and after 11 d for the ΔAC knock-out strain. Oleic acid and cAMP were added at concentrations of 3.5 µM and 3 mM, respectively. In part B, samples were taken at intervals over the first four days from cultures of the wild type and the mutant, both with and without oleic acid, for the estimation of cellular metabolic activity (uracil incorporation). Asterisks in Fig. 6A indicate that the results are significantly different from the control for each strain by Student’s t-test. Asterisks in Fig. 6B indicate significant difference between wt cells and wt+oleic acid by Student’s t-test.</p

cAMP stimulates the resuscitation of <i>M. smegmatis</i> NC cells.

<p>NC cells were obtained and resuscitated in batch format. The OD₆₀₀ was measured after 5 d of resuscitation. The concentrations of oleic acid, linoleic acid, cAMP and 8-bromo-cAMP were 2 µM, 1.7 µM, 3 mM and 2 mM, respectively. This experiment was repeated three times with similar results; the error bars represent the standard error of the mean.</p

Oleic acid-mediated resuscitation of <i>M. smegmatis</i> NC cells is Rpf-dependent.

<p>NC cells were resuscitated in batch mode in the presence and the absence of both oleic acid (3.5 µM) (A) or cAMP (3.0 mM) (B) and the Rpf inhibitor, BNPT (1 µg/ml) in all four possible combinations. BNPT was added at zero time and again after 48 h of incubation. Samples were withdrawn periodically for OD₆₀₀ determination (A) and estimation of metabolic activity using ³H-uracil incorporation (B) The insert to part A shows the level of uracil incorporation in the four cultures measured after 92 h of incubation. The error bars represent the standard error of the mean. Asterisks indicate significance between ³H-uracil incorporation by cells incubated in the presence of oleate vs both oleate and BNTP (A, insert) or in the presence of cAMP vs both cAMP and BNTP (B) by Student’s t-test.</p

Fatty acid-induced resuscitation <i>of M. smegmatis</i> NC cells.

<p>NC cells were resuspended in reactivation medium to an initial OD₆₀₀ ∼ 0.3 and resuscitated in batch format. The OD₆₀₀ was measured after 5 d of resuscitation. Palmitic, stearic, oleic, linoleic and arachidonic acids were added at their optimum concentrations (4 µM, 4 µM 3.5 µM, 1.7 µM and 1.6 µM, respectively). The insert shows the concentration-dependence of oleic acid-mediated resuscitation. Each point represents the OD₆₀₀ measurement after 5 d of resuscitation. This experiment was repeated three times with similar results; the error bars represent the standard error of the mean. Asterisks indicate that the results are significantly different from the control by Student’s t-test.</p

Fatty acid- and dibutyryl-cAMP-induced resuscitation <i>of M. tuberculosis</i> NC cells.

<p>NC cells were obtained inoculated to an initial OD₆₀₀ = 0.2 and resuscitated in batch format. The OD₆₀₀ was measured after resuscitation for 20 d (A) or 25 d (B). This experiment was repeated two times with similar results; the error bars represent the standard error of the mean. Asterisks indicate that the results are significantly different from the control by Student’s t-test.</p

Expression profiles of three <i>M. smegmatis rpf</i> genes during oleic acid-mediated resuscitation of NC cells.

<p>NC cells were resuscitated in batch mode. Oleic acid was added at a concentration of 3.5 µM and the initial culture density was adjusted to give an OD₆₀₀ = 0.3. RNA was isolated from cells withdrawn from the culture at different time points. Quantitative RT-PCR was performed using equal amounts of RNA (50 ng) as described in Materials and Methods. Each point is the mean of nine measurements (three technical replicates of the three biological replicates). The average OD₆₀₀ values of the three replicate cultures are also shown. Error bars represent the standard error of the mean. Significance between expression level of MSMEG_5700 after 67 h of incubation was demonstrated by ANOVA (P<0,05).</p

Intracellular cAMP levels and ³H-uracil incorporation during oleic acid-induced resuscitation of <i>M. smegmatis</i> NC cells.

<p>NC cells were obtained and resuscitated in batch mode. Oleic acid was added at a concentration of 3.5 µM. The intracellular level of cAMP was estimated after cells had been harvested and disrupted as described in the Materials and Methods. For samples taken during the first 48 h of resuscitation, metabolic activity was determined using ³H-uracil incorporation (denoted CPM on the Figure axis) as detailed in Materials and Methods. Dotted lines divide the overall process into three phases: 0 - true lag, I - metabolic activation, II - cell multiplication. This experiment was repeated three times with similar results. Error bars represent the standard error of the mean. Asterisks indicate that the results are significantly different from the values at zero time by Student’s t-test.</p