Fig. 3.

PutR is a transcriptional activator of putA in B. abortus. (a) Genetic organization of putR and putA on chromosome II in B. abortus 2308. putR (bab2_0517) is divergently oriented to putA (bab2_0518). (b) β-Galactosidase activity of a putA-lacZ transcriptional fusion. The indicated Brucella strains carrying a plasmid containing the putA-lacZ transcriptional fusion were grown in Brucella broth to the late exponential phase, and the β-galactosidase activity from each strain was determined. The bar graph shows the average number of Miller units from each strain, and a significant difference between the parental strain 2308 and the ΔputR strain was determined using a t-test (P<0.05) and is indicated by an asterisk (*). (c) Quantitative RT-PCR (qRT-PCR) analysis of putA mRNA levels. The relative levels of putA mRNA in B. abortus 2308, ΔputR and ΔputR-RC were determined by qRT-PCR using RNA isolated from the Brucella strains following cultivation of the bacteria in Brucella broth to the late exponential phase. Oligonucleotide primers specific for either putA or 16S rRNA were employed to amplify the target genes by PCR, and quantification of the amplified DNA fragments was performed using SYBR green incorporation. The values represent the abundance of specific mRNA relative to the level of mRNA from the parental strain 2308 designated as 1.000. (d) β-Galactosidase activity of a putR-lacZ transcriptional fusion. Brucella strains carrying a plasmid containing the putR-lacZ transcriptional fusion were cultured in Brucella broth to the late exponential phase, and the β-galactosidase activity from each strain was measured. The bar graph shows the average number of Miller units from each strain, and a significant difference between the parental strain 2308 and the ΔputR strain was determined using a t-test (P<0.05) and is indicated by an asterisk (*).