A dual function for a bacterial small RNA: SgrS performs base pairing-dependent regulation and encodes a functional polypeptide

Wadler and Vanderpool. 10.1073/pnas.0708102104.

Supporting Information

Files in this Data Supplement:

SI Table 1
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Figure 11
SI Figure 12
SI Table 2




SI Figure 7

Fig. 7. Effects of sgrT mutation on recovery from glucose-phosphate stress. The DsgrS::kan, lacIq+ host strain (CV104) carrying a vector control or one of two plasmids expressing sgrT alleles, pBRCV7 (SgrT; Fig. 2) or pBRCS7 (SgrTUAA2; SI Table 1), was grown in LB with ampicillin and IPTG. Cells were stressed by addition of 0.5% aMG at early log phase. pBRCS7 was derived from pBRCV7 by PCR mutagenesis by using primers O-CV240 and OCV241 to change the start codon of sgrT (AUG) to a stop codon (UAA).





SI Figure 8

Fig. 8. Effect of sgrT overexpression on growth with different carbon sources. The DsgrS::kan, lacIq+ host strain (CV104) carrying a vector control (Left) or the plasmid expressing sgrT (pBRCV7) (Right) was grown on minimal medium with ampicillin, IPTG, and one of the following carbon sources: 0.2% glucose (A), 0.2% N-acetyl glucosamine (NAG) (B), 0.2% mannose (C), 0.2% fructose (D), 0.2% mannitol (E), or 0.1% casamino acids (CAA) (F).





SI Figure 9

Fig. 9. Western blot analysis to detect the SgrT-3XFLAG protein. The sequence specifying the 3XFLAG epitope was fused to the 3' end of sgrT in the context of Plac-sgrT or Plac-sgrTUAA. The DsgrS::kan, lacIq+ strain CV104 carrying this construct was grown to mid-log phase and induced with IPTG. Total cellular proteins were harvested at the indicated times after induction as described in Methods. Detection was performed by using a monoclonal mouse anti-FLAG antibody from Sigma (1:1,000) and a goat anti-mouse IgG horseradish peroxidase conjugate secondary antibody (used at 1:5,000) from Novagen. The predicted size of the SgrT-3XFLAG protein is ~8 kDa. The position of the most abundant induced band is consistent with this prediction.





SI Figure 10

Fig. 10. Northern blot analysis to detect SgrS and SgrSUAA. The DsgrS::kan, lacIq+ host strain (CV104) carrying a vector control or one of two plasmids expressing sgrS alleles, pLCV1 (wild-type sgrS) or pLCV5 (5th codon of sgrT changed to a UAA stop codon), was grown in LB with ampicillin. Total RNA was extracted at the time points indicated after induction of constructs with IPTG and prepared for electrophoresis on a polyacrylamide gel using 2 mg of total RNA. The blot was probed for SgrS by using the RyaA1-bio probe. The position of the SgrS band is indicated at the left.





SI Figure 11

Fig. 11. Growth of cells in rich MOPS medium with glucose. The strains are as described in Fig. 3 and were grown in MOPS defined medium with glucose and amino acids in the presence of ampicillin and IPTG. The growth curves are representative of at least three independent experiments.





SI Figure 12

Fig. 12. SgrT inhibits inducer exclusion. A lac+DsgrS::kan strain (CS136) carrying the vector control (pHDB3) or pPlac-sgrT (pBRCV7) plasmids was grown in TB medium with glucose and lactose. b-Galactosidase activity was assayed at different growth phases: early log (OD600 0.1), mid-log (OD600 0.5), and stationary (OD600 2.0). The results shown are an average of three independent experiments.