Table 2.

Analysis by qRT-PCR of the relative transcriptional levels of selected genes in parental B. ovis PA and the ΔvjbR mutant cultured in the presence or absence of C₁₂-HSL to the exponential- or stationary-growth phase^a

B. ovis strain	Culture medium supplement	Growth phase of RNA extraction	Relative transcription level of indicated target gene (2−ΔΔCT)
			babR (blxR)	bvrR	cgs	fliF	omp25c	virB2	vjbR
PA	ACN	Exponential	1.000	1.000	1.000	1.000	1.000	1.000	1.000
PA	C12-HSL	Exponential	2.570	ND	1.170	1.330	1.006	0.086	0.893
ΔvjbR	ACN	Exponential	2.093	1.078	0.963	1.219	1.601	0.009	0.000
ΔvjbR	C12-HSL	Exponential	2.013	ND	ND	1.086	1.583	0.008	0.000
PA	ACN	Stationary	1.000	1.000	1.000	1.000	1.000	1.000	1.000
PA	C12-HSL	Stationary	1.049	ND	1.215	0.721	0.482	0.328	0.931
ΔvjbR	ACN	Stationary	0.906	0.871	1.188	0.889	0.482	0.220	ND
ΔvjbR	C12-HSL	Stationary	0.925	ND	ND	0.842	0.619	0.138	ND

^aRelative transcription quantification was performed with the 2^−ΔΔCT method (32), using the IF-1 gene as an endogenous reference gene and B. ovis PA cultured in the presence of ACN (solvent for C₁₂-HSL) up to the exponential-growth phase (DO₆₀₀ ∼ 0.7) or stationary-growth phase (DO₆₀₀ ∼ 2.3) as the benchmark. Two cDNA samples—and three PCRs per gene and cDNA—were used to calculate relative transcription levels under each set of strain and culture conditions. Increased or reduced transcription of each gene in comparison with that seen with B. ovis PA cultured with ACN (2^−ΔΔCT = 1) is indicated by values > 1 or < 1, respectively. Gene expression levels were considered different when the value was >1.5 or <0.5. ND, not determined.