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Repression of catabolic genes by Hfq.

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posted on 19.06.2014, 15:18 by Elisabeth Sonnleitner, Udo Bläsi

(A–C) The strains were grown to an OD600 of 2.0 in BSM medium supplemented with (A) 40 mM succinate and 40 mM acetamide (to establish CCR and to induce amiE::lacZ transcription) or (B, C) only with 40 mM succinate (CCR). Then, the cells were harvested and the β-galactosidase activities were determined. The bars depict β-galactosidase values conferred by the translational amiE::lacZ fusion encoded by plasmid pME9655 (A), the estA::lacZ fusion encoded by plasmid pTLestA (B) and the phzM::lacZ fusion encoded by plasmid pME10011 (C) in strains PAO1 (blue bar), PAO1Δcrc (green bar), PAO1hfq- (red bar) and PAO1hfqcrc (purple bar), respectively. The error bars represent standard deviations from three independent experiments. (D, E) The strains were grown in BSM medium supplemented with succinate and acetamide. (D) At an OD600 of 1.0 IPTG (1 mM final concentration) was added to strains PAO1Δcrc(pMMBcrcFlag; pME9655) (blue bar), PAO1hfqcrc(pMMBcrcFlag; pME9655) (red bar) and PAO1hfqcrc(pMMB67HE; pME9655) (purple bar), respectively. The bars represent the β-galactosidase values conferred by the plasmid pME9655 encoded translational amiE::lacZ fusion 3 h after induction of the plasmid borne crcFlag gene. The error bars represent standard deviations from three independent experiments. The corresponding CrcFlag levels were determined by western-blot analysis using anti-Flag antibodies in strains PAO1Δcrc(pMMBcrcFlag; pME9655), PAO1hfqcrc(pMMBcrcFlag; pME9655) and PAO1hfqcrc(pMMB67HE; pME9655). Immunodetection of ribosomal protein S2 (loading control) was performed as described in Materials and Methods. (E) At an OD600 of 1.0 IPTG (1 mM final concentration) was added to strains PAO1hfq-(pMMBhfqFlag; pME9655) (blue bar), PAO1hfqcrc(pMMBhfqFlag; pME9655) (green bar) and PAO1hfqcrc(pMMB67HE; pME9655) (purple bar), respectively. The bars represent the β-galactosidase values conferred by the plasmid pME9655 encoded translational amiE::lacZ fusion 3 h after hfqFlag induction. The error bars represent standard deviations from three independent experiments. The corresponding HfqFlag levels were determined by western-blot analysis using anti-Flag antibodies in strains PAO1hfq-(pMMBhfqFlag; pME9655), PAO1hfqcrc(pMMBhfqFlag; pME9655) and PAO1hfqcrc(pMMB67HE; pME9655) (top panel). Immunodetection of ribosomal protein S2 (loading control) was performed as described in Materials and Methods.

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