Multiple Control of Flagellum Biosynthesis in Escherichia coli : Role of H-NS Protein and the Cyclic AMP-Catabolite Activator Protein Complex in Transcription of the flhDC Master Operon

Abstract

Little is known about the molecular mechanism by which histone-like nucleoid-structuring (H-NS) protein and cyclic AMP-catabolite activator protein (CAP) complex control bacterial motility. In the present paper, we show that crp and hns mutants are nonmotile due to a complete lack of flagellin accumulation. This results from a reduced expression in vivo of fliA and fliC, which encode the specific flagellar sigma factor and flagellin, respectively. Overexpression of the flhDC master operon restored, at least in part, motility in crp and hns mutant strains, suggesting that this operon is the main target for both regulators. Binding of H-NS and CAP to the regulatory region of the master operon was demonstrated by gel retardation experiments, and their DNA binding sites were identified by DNase I footprinting assays. In vitro transcription experiments showed that CAP activates flhDC expression while H-NS represses it. In agreement with this observation, the activity of a transcriptional fusion carrying the flhDC promoter was decreased in the crp strain and increased in the hns mutant. In contrast, the activity of a transcriptional fusion encompassing the entire flhDC regulatory region extending to the ATG translational start codon was strongly reduced in both hns and crp mutants. These results suggest that the region downstream of the ؉1 transcriptional start site plays a crucial role in the positive control by H-NS of flagellum biosynthesis in vivo. Finally, the lack of complementation of the nonmotile phenotype in a crp mutant by activation-deficient CAP mutated proteins and characterization of cfs, a mutation resulting in a CAP-independent motility behavior, demonstrate that CAP activates flhDC transcription by binding to its promoter and interacting with RNA polymerase.

Regulatory role of UvrY in adaptation of Photorhabdus luminescens growth inside the insect

“…Data were collected on 10 000 cells in a FACScan analyser (Becton Dickinson), and the per cent of dead cells (propidium iodide positive) was determined using the CellQuest software (Becton Dickinson). csrB primer extension, secondary structure prediction and Northern blot analysis Primer extension was performed as previously described (Soutourina et al, 1999) with the following oligonucleotide: 5′-ATTAATTCATTCGATGATGAAACAAGC-3′. The position of the 5′ end was determined using direct sequencing of chromosomal DNA as a reference (Krin et al, 2001).…”

Description and application of a rapid method for genomic DNA direct sequencing

“…These two boxes are separated by a 19-bp spacer and are located 289 and 264 bp upstream from the ATG translational start codon, respectively. In addition, we identi¢ed a consensus CAP binding site (tttTGTGAgttatgTCACAtag) centered at position 371.5 with respect to a putative +1 transcriptional start site, as in E. coli [11]. This suggests that the regulatory mechanism of the £agellar master operon in P. luminescens could be similar to that in E. coli.…”

“…During this work, a partial sequence coding for a gene homologous to £hDC, the £agellar master operon in E. coli, was identi¢ed (E. Duchaud, personal communication). Recently, we demonstrated that the £hDC promoter in E. coli contains a CAP binding site and a large untranslated region, which seems to play a crucial role in the regulation of its expression by H-NS [11]. Therefore, it was of interest to determine the existence of such a regulatory region in P. luminescens.…”

An H-NS-like protein involved in the negative regulation of hrp genes in Xanthomonas oryzae pv. oryzae

“…The target plasmid, which is a pBluescript II SK1 derivative with the putative promoter region of hrpG ( À 686 to 156) amplified by PCR (Table S2 for primers), was digested with SspI, PvuII and BamHI, and incubated with the purified His(6)-tagged XrvB for 15 min at 37 1C in the reaction buffer described by Soutourina et al (1999). After the reaction, the samples were loaded onto a 1% TBE-agarose gel, followed by staining with ethidium bromide.…”