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. 2009 Mar 6;191(9):3183–3188. doi: 10.1128/JB.00147-09

FIG. 2.

FIG. 2.

RT-QPCR analyses. TaqMan primer sequences were designed to target the middle third of the bacB open reading frame, the second (downstream) gene of the bacAB operon (BACB-F, GCAAGTCTATAAAAAAATAGATAGCGAAAAATATCCA, and BACB-R, CTCCCAATTCAATTAATAACTTTTCATCATTTCCT), and the 16S rRNA gene (EF16S-F, GAAACAGGTGCTAATACCGCATAAC, and EF16S-R, CAGCGACACCCGAAAGC). Probe sequences were as follows: BACB FAM, CAATGTAGATATAGTTCACCAATTTA, and EF16S FAM, CATGCGGCATAAACT. Forty-five cycles were performed using an ABI Prism 7500 sequence detection system, bacAB expression was normalized to 16S rRNA gene expression, and changes (n-fold) in transcript levels relative to those at early exponential phase (A) and baseline (B) were determined using the threshold cycle (ΔΔCT) method with software from Applied Biosystems. Experiments were performed in quadruplicate on two separate occasions. (A) Comparisons of different stages of growth. Results from experiments with hosts that are gelatinase negative (SFJ1 and SFTX3) and gelatinase positive (MC4, SFOS2, and SFOR2) are shown. The data are expressed as changes (n-fold; means ± standard errors of the means) in bacAB transcript levels at late exponential and stationary stages of growth relative to those at early exponential stage (set at 1). Strains were grown in 50 ml of Todd-Hewitt broth with gentle shaking, and aliquots were obtained at early exponential phase (optical density at 650 nm, 0.1), late exponential phase (optical density at 650 nm, 0.8), and 2 h into stationary phase and at 24 h. Transcript levels at late exponential phase were significantly higher than those at other stages in SFJ1, SFTX3, SFOS2, and SFOR2 (P < 0.001) and MC4 (P < 0.01) (values from repeated measures were subjected to two-way analyses of variance with Bonferroni posttests). (B) Results of a test for autoinduction. Changes (n-fold) in bacAB transcript levels following 10- or 30-min exposure of early-exponential-phase SFJ1 cultures to 20% (vol/vol) filter-sterilized culture supernatant (CS) from late-exponential-phase cultures of SFJ1 (JH2-2/pAMS1) or JH2-2 relative to the baseline level (set at 1).