coli K12 strains HB101, DH5α and TG1α (obtained from Cedarlane Laboratories). To test the effect of a cya mutation, we used the K12 strains C600 and TP610, of genotype C600 cya610 (Hedegaard & Danchin, 1985). To test the effect of a relA mutation, we used the K12 strains BW25113 and the corresponding relA mutant obtained from the Keio collection (Baba et al., 2006).
Only 2787 possess the aah and aidA genes. The plasmids used in this study were pIB264 (Benz & Schmidt, 1989), pMC1871 (Shapira et al., 1983) and pFB01. The pIB264 plasmid harbors a fragment of 2787 DNA encompassing the native aah-aidA region of 2787. The sequence of the insert was obtained using Sotrastaurin molecular weight primers extending upstream of aah and aidA. The plasmids pMC1871, and its derivative pFB01, are reporter vectors for measuring gene expression based on the β-galactosidase gene, lacZ. pFB01 was constructed by PCR amplification from pIB264 ABT-263 solubility dmso of a 426 bp fragment of upstream region of aah using the primers promo-F (5′-TATATCCCGGGATTAATACCACGTTTATACCGGTGAG-3′) and promo-R (5′-TAATACCCGGGCATAATCCCTCCTATATAATGTAATATCC-3′). The fragment was then digested with AseI and SmaI and cloned at the same sites in pMC1871, directly upstream of the promoterless lacZ gene. The construction was verified by restriction analysis and sequencing. Bacteria containing pMC1871 or pFB01 were grown overnight in Luria–Bertani (LB) broth containing 12.5 μg mL−1
tetracycline at 37 °C with agitation and then diluted 150-fold in a fresh medium under the conditions to be investigated. When the cultures reached the specific OD at 600 nm (OD600 nm), the β-galactosidase activity was assessed as described previously (Mourez et al., 1997). In some experiments, the cultures were grown to an OD600 nm of 0.7, the bacteria from 1 mL samples were pelleted, resuspended with 4 mL of conditioned supernatants and grown for an additional 30 min at 37 °C before assessing β-galactosidase activity. Conditioned supernatants came from cultures grown at 37 °C with agitation until an OD600 nm of approximately 0.1, 0.7 and selleck chemicals 2.5 and were filtered using 0.22-μm syringe filters. In some experiments, the bacterial supernatants were
diluted 1 : 2 in water or a fresh LB medium. The results were expressed in Miller units or in percentage of activity compared with a control growth condition. Statistical comparisons were performed by an anova and Dunnet’s post-tests using prism 4.0 software (Graphpad Software). Overnight cultures of 2787 in LB were diluted 150-fold in a fresh medium and grown at 37 °C with agitation. At various times, RNA was extracted using the RiboPure-Bacteria kit (Ambion) according to the manufacturer’s instructions. qRT-PCR reactions were performed using the QuantiTech SYBR green RT-PCR kit (Qiagen) with 500 ng of RNA. Thermal cycling conditions were an initial step at 50 °C for 30 min and 95 °C for 15 min, followed by 40 cycles of denaturation (94 °C, 15 s), annealing (55 °C, 20 s) and extension (72 °C, 30 s).