Regulation of the fimB promoter: a case of differential regulation by ppGpp and DksA in vivo

Abstract

SummaryThe expression of type 1 fimbriae is dependent on the intracellular levels of ppGpp through stimulation of fimB transcription. Here we show that in contrast to the previously described decreased fimbriation observed in a ppGpp-deficient strain, DksA deficiency results in a hyperfimbriated state. In vivo assays show that the effect of DksA deficiency on the type 1 fimbriae occurs at the phase variation level because of elevated transcription from the fimB P2 promoter. In contrast, our in vitro transcription studies demonstrate that ppGpp and DksA can stimulate transcription from the fimB P2 promoter both independently and codependently. We provide evidences that the apparently contradictory results from the in vivo and in vitro transcriptional studies are at least in part a consequence of the increased association of the antipausing factors (GreA and GreB) to the RNA polymerase in the absence of DksA in vivo.

A Proximal Promoter Element Required for Positive Transcriptional Control by Guanosine Tetraphosphate and DksA Protein during the Stringent Response

“…However, it has not been determined whether they are saturated at relatively low concentrations of RNAP and whether ppGpp/DksA affects such saturation kinetics. In the case of the fimB P2 promoter, DksA enhances the ability of RNAP to bind the promoter in vitro while being dispensable for induction in vivo (36), suggesting that several mechanisms of DksA-dependent control may act in parallel. In addition, the GreA and GreB proteins, which are structurally similar to DksA and occupy the secondary channel of RNAP (17,60), can stimulate expression from fimB P2 in the absence of DksA (36), indicating that several factors besides DksA and ppGpp can modulate the transcriptional output of this class of promoters.…”

“…This class of promoters, including those of amino acid synthesis genes (6) and fimB P2 (36), has in common that they form very stable RNAP-promoter open complexes that are destabilized by DksA and/or ppGpp (6,36). These promoters also harbor AT-rich, rather than GC-rich, regions downstream from their Ϫ10 element.…”

“…The destabilization of RNAP-rRNA promoter complexes by ppGpp/DksA observed in vitro might explain why ppGpp/DksA negatively affects transcriptional output from rrn promoters also in vivo (18,34). However, all RNAP-promoter complexes studied in vitro, including promoters positively regulated by ppGpp/DksA, are destabilized by the addition of ppGpp/DksA (5,6,35,36). Thus, it has been difficult to explain positive control of transcription by ppGpp/DksA through changes in RNAP-promoter complex stability.…”

“…The pBG103 plasmid was constructed by GenScript USA Inc. pRLG597 was a kind gift from the Gourse laboratory (47). All plasmids are listed in supplemental Table SII. ␤-Galactosidase Activity Assays-Relative ␤-galactosidase levels were determined as described previously (48) DksA Purification-His-tagged DksA was overexpressed and purified according to Åberg et al (36), with the exception that the cells were disrupted using a French press. RNA Extraction and Quantitative PCR (qPCR)-Wild type, ⌬dksA, and the ⌬relA ⌬spoT strains containing promoter-lacZ fusions were grown at 37°C in M9 defined medium (see above).…”

ppGpp Conjures Bacterial Virulence

“…In this manner, ppGpp and DksA contribute indirectly to bacterial adaptation. (3,113). Accordingly, other factors likely influence regulation by ppGpp 0 and dksA mutant cells.…”