fliA Promoter Research Articles

The bacterial division protein MinDE has an independent function in flagellation

Before preparing for division, bacteria stop their motility. During the exponential growth phase in Escherichia coli, when the rate of bacterial division is highest, the expression of flagellar genes is repressed and bacterial adhesion is enhanced. Hence, it is evident that cell division and motility in bacteria are linked; however, the specific molecular mechanism by which these two processes are linked is not known. While observing E.coli, we found that compared to the WT, the E.coli (Δmin) cells show higher motility and flagellation. We demonstrated that the higher motility was due to the absence of the Min system and can be restored to normal in the presence of Min proteins, where Min system negatively regulates flagella formation. The Min system in E.coli is widely studied for its role in the inhibition of polar Z-ring formation through its pole-to-pole oscillation. However, its role in bacterial motility is not explored. MinD homologs, FlhG and FleN, are known to control flagellar expression through their interaction with FlrA and FleQ, respectively. AtoC, a part of the two-component system AtoSC complex, is homologous to FlrA/FleQ, and the complex is involved in E.coli flagellation via its interaction with the fliA promoter. We have shown that MinD interacts directly with the AtoS of AtoSC complex and controls the fliA expression. Our findings suggest that the Min system acts as a link between cell division and motility in E.coli.

The evolutionary impact of intragenic FliA promoters in proteobacteria.

In Escherichia coli, one sigma factor recognizes the majority of promoters, and six 'alternative' sigma factors recognize specific subsets of promoters. The alternative sigma factor FliA (σ28 ) recognizes promoters upstream of many flagellar genes. We previously showed that most E. coli FliA binding sites are located inside genes. However, it was unclear whether these intragenic binding sites represent active promoters. Here, we construct and assay transcriptional promoter-lacZ fusions for all 52 putative FliA promoters previously identified by ChIP-seq. These experiments, coupled with integrative analysis of published genome-scale transcriptional datasets, strongly suggest that most intragenic FliA binding sites are active promoters that transcribe highly unstable RNAs. Additionally, we show that widespread intragenic FliA-dependent transcription may be a conserved phenomenon, but that specific promoters are not themselves conserved. We conclude that intragenic FliA-dependent promoters and the resulting RNAs are unlikely to have important regulatory functions. Nonetheless, one intragenic FliA promoter is broadly conserved and constrains evolution of the overlapping protein-coding gene. Thus, our data indicate that intragenic regulatory elements can influence bacterial protein evolution and suggest that the impact of intragenic regulatory sequences on genome evolution should be considered more broadly.

Post-Transcriptional and Post-Translational Control of the Flagellar Regulon Rescues Motility of a Salmonella Enterica Type III Export Flio Mutant

The bacterial flagellum is made using a type III secretion pathway. For Salmonella enterica, this requires 6 transmembrane proteins FlhA, FlhB, FliO, FliP, FliQ, and FliR. FliO is not absolutely required, because bypass mutations in the fliP gene can improve motility for a fliO mutant. In this study, an extended screen was made for randomly selected mutations that improved motility of cells bearing a ΔfliO null mutation. Using whole genome sequencing two novel mutations were identified, a “silent” mutation localized to the fliA gene, which encoded a U36C substitution in the fliAZ mRNA. A missense mutation was found in the clpP gene, and encoded a V20F substitution in the ClpXP protease. Transcriptional and translational fusions of the lacZ gene to the fliA promoter demonstrated that the silent mutation improved translation of the highly structured fliAZ mRNA. Real-time quantitative RT-PCR revealed that late flagellar gene expression was blocked in a ΔfliO mutant, but the clpP(V20F) or fliA(mRNA U36C) mutations improved expression. Examination of flagellar biogenesis by immunoblotting and transmission electron microscopy found that the fliO mutant synthesized rare flagella, but introducing the fliA(mRNA U36C) mutation together with previously identified mutations in fliP cooperatively restored flagellar biogenesis to near wild-type levels.

The σ‐factor FliA, ppGpp and DksA coordinate transcriptional control of the aer2 gene of Pseudomonas putida

Here the sigma-factor requirement for transcription of three similar, but differentially regulated, aer genes of Pseudomonas putida KT2440 is investigated. Previous work has shown that the three Aer proteins, like chemoreceptors, colocalize to a single pole in a CheA-dependent manner. Lack of Aer2 - the most abundant of these three proteins - mediates defects in metabolism-dependent taxis and aerotaxis, while lack of Aer1 or Aer3 has no apparent phenotype. We show, using wild-type and mutant P. putida derivatives combined with P. putida reconstituted FliA- (sigma(28)) and sigma(70)-dependent in vitro transcription assays, that transcription of aer2 is coupled to motility through the flagella sigma-factor FliA, while sigma(70) is responsible for transcription of aer1 and aer3. By comparing activities of the wild-type and mutant forms of the aer2 promoter, we present evidence (i) that transcription from FliA-dependent Paer2 is enhanced by changes towards the Escherichia coli consensus for FliA promoters rather than towards that of P. putida, (ii) that the nature of the AT-rich upstream region is important for both output and sigma(70) discrimination of this promoter, and (iii) that Paer2 output is directly stimulated by the bacterial alarmone ppGpp and its cofactor DksA.

DksA and ppGpp directly regulate transcription of the Escherichia coli flagellar cascade

The components of the Escherichia coli flagella apparatus are synthesized in a three-level transcriptional cascade activated by the master regulator FlhDC. The cascade co-ordinates the synthesis rates of a large number of gene products with each other and with nutritional conditions. Recent genome-wide studies have reported that flagellar transcription is altered in cells lacking the transcription regulators DksA or ppGpp, but some or all reported effects could be indirect, and some are contradictory. We report here that the activities of promoters at all three levels of the cascade are much higher in strains lacking dksA, resulting in overproduction of flagellin and hyperflagellated cells. In vitro, DksA/ppGpp inhibits the flhDC promoter and the sigma(70)-dependent fliA promoter transcribing the gene for sigma(28). However, DksA and ppGpp do not affect the sigma(28)-dependent fliA promoter or the sigma(28)-dependent fliC promoter in vitro, suggesting that the dramatic effects on expression of those genes in vivo are mediated indirectly through direct effects of DksA/ppGpp on FlhDC and sigma(28) expression. We conclude that DksA/ppGpp inhibits expression of the flagellar cascade during stationary phase and following starvation, thereby co-ordinating flagella and ribosome assembly and preventing expenditure of scarce energy resources on synthesis of two of the cell's largest macromolecular complexes.

NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility

The Escherichia coli NsrR protein is a nitric oxide-sensitive repressor of transcription. The NsrR-binding site is predicted to comprise two copies of an 11 bp motif arranged as an inverted repeat with 1 bp spacing. By mutagenesis we confirmed that both 11 bp motifs are required for maximal NsrR repression of the ytfE promoter. We used chromatin immunoprecipitation and microarray analysis (ChIP-chip) to show that NsrR binds to 62 sites close to the 5' ends of genes. Analysis of the ChIP-chip data suggested that a single 11 bp motif (with the consensus sequence AANATGCATTT) can function as an NsrR-binding site in vivo. NsrR binds to sites in the promoter regions of the fliAZY, fliLMNOPQR and mqsR-ygiT transcription units, which encode proteins involved in motility and biofilm development. Reporter fusion assays confirmed that NsrR negatively regulates the fliA and fliL promoters. A mutation in the predicted 11 bp NsrR-binding site in the fliA promoter impaired repression by NsrR and prevented detectable binding in vivo. Assays on soft-agar confirmed that NsrR is a negative regulator of motility in E. coli K12 and in a uropathogenic strain; surface attachment assays revealed decreased levels of attached growth in the absence of NsrR.

Indole cell signaling occurs primarily at low temperatures in Escherichia coli

We have shown that the quorum-sensing signals acylhomoserine lactones, autoinducer-2 (AI-2) and indole influence the biofilm formation of Escherichia coli. Here, we investigate how the environment, that is, temperature, affects indole and AI-2 signaling in E. coli. We show in biofilms that indole addition leads to more extensive differential gene expression at 30 degrees C (186 genes) than at 37 degrees C (59 genes), that indole reduces biofilm formation (without affecting growth) more significantly at 25 and 30 degrees C than at 37 degrees C and that the effect is associated with the quorum-sensing protein SdiA. The addition of indole at 30 degrees C compared to 37 degrees C most significantly repressed genes involved in uridine monophosphate (UMP) biosynthesis (carAB, pyrLBI, pyrC, pyrD, pyrF and upp) and uracil transport (uraA). These uracil-related genes are also repressed at 30 degrees C by SdiA, which confirms SdiA is involved in indole signaling. Also, compared to 37 degrees C, indole more significantly decreased flagella-related qseB, flhD and fliA promoter activity, enhanced antibiotic resistance and inhibited cell division at 30 degrees C. In contrast to indole and SdiA, the addition of (S)-4,5-dihydroxy-2,3-pentanedione (the AI-2 precursor) leads to more extensive differential gene expression at 37 degrees C (63 genes) than at 30 degrees C (11 genes), and, rather than repressing UMP synthesis genes, AI-2 induces them at 37 degrees C (but not at 30 degrees C). Also, the addition of AI-2 induces the transcription of virulence genes in enterohemorrhagic E. coli O157:H7 at 37 degrees C but not at 30 degrees C. Hence, cell signals cause diverse responses at different temperatures, and indole- and AI-2-based signaling are intertwined.

Completion of the hook-basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription.

The flhDC operon of Salmonella typhimurium is the master control operon required for the expression of the entire flagellar regulon. The flagellar master operon was placed under the tetracycline-inducible promoter PtetA using the T-POP transposon. Cells containing this construct are motile in the presence of tetracycline and non-motile without inducer present. No flagella were visible under the electron microscope when cells were grown without inducer. The class 1, class 2 and class 3 promoters of the flagellar regulon are temporally regulated. After addition of tetracycline, the class 1 flhDC operon was transcribed immediately. Transcription of flgM (which is transcribed from both class 2 and class 3 promoters) began 15 min after induction. At 20 min after induction, the class 2 fliA promoter became active and intracellular FliA protein levels increased; at 30 min after induction, the class 3 fliC promoter was activated. Induction of fliC gene expression coincides with the appearance of FlgM anti-sigma factor in the growth medium. This also coincides with the completion of hook-basal body structures. Rolling cells first appeared 35 min after induction, and excess hook protein (FlgE) was also found in the growth medium at this time. At 45 min after induction, nascent flagellar filaments became visible in electron micrographs and over 40% of the cells exhibited some swimming behaviour. Multiple flagella assemble and grow on individual cells after induction of the master operon. These results confirm that the flagellar regulatory hierarchy of S. typhimurium is temporally regulated after induction. Both FlgM secretion and class 3 gene expression occur upon completion of the hook-basal body structure.

Structure and expression of the fliA operon of Salmonella typhimurium

The fliA gene encodes the flagellum-specific sigma factor sigma28 In Salmonella typhimurium. The transcription in vivo and in vitro of this gene was analysed and it was found that there are two promoters for the expression of this gene. One is a class 2 promoter which is recognized by sigma70-RNA polymerase in the presence of the FlhD and FlhC activator proteins. The other is a class 3 promoter which is recognized by sigma28-RNA polymerase. Therefore, the fliA operon is under dual positive control from FlhD/FlhC and from FliA itself. The nucleotide sequence downstream of the fliA gene was determined. The sequence contains two ORFs following the fliA gene. On the basis of their sequence homology, it is concluded that these two correspond to the fliZ and fliY genes of Escherichia coil. Northern blot analysis revealed that the fliZ gene is transcribed from the fliA promoters, whereas the fliY gene is transcribed from both the fliA promoters and its own FlhD/FlhC-independent promoter. A fliZ-disruption mutant was constructed by inserting a kanamycin-resistance gene cassette into the fliZ gene on the chromosome. The mutant showed poor motility, and introduction of a fliZ+ plasmid into this mutant restored the wildtype level of motility. These results suggest that the fliZ gene may be required for expression of maximal motility.