Pilin Promoter Research Articles

A temperature dependent pilin promoter for production of thermostable enzymes in Thermus thermophilus

BackgroundEnzymes from thermophiles are of great interest for research and bioengineering due to their stability and efficiency. Thermophilic expression hosts such as Thermus thermophilus [T. thermophilus] can overcome specific challenges experienced with protein production in mesophilic expression hosts, such as leading to better folding, increased protein stability, solubility, and enzymatic activity. However, available inducible promoters for efficient protein production in T. thermophilus HB27 are limited.ResultsIn this study, we characterized the pilA4 promoter region and evaluated its potential as a tool for production of thermostable enzymes in T. thermophilus HB27. Reporter gene analysis using a promoterless β-glucosidase gene revealed that the pilA4 promoter is highly active under optimal growth conditions at 68 °C and downregulated during growth at 80 °C. Furthermore, growth in minimal medium led to significantly increased promoter activity in comparison to growth in complex medium. Finally, we proved the suitability of the pilA4 promoter for heterologous production of thermostable enzymes in T. thermophilus by producing a fully active soluble mannitol-1-phosphate dehydrogenase from Thermoanaerobacter kivui [T. kivui], which is used in degradation of brown algae that are rich in mannitol.ConclusionsOur results show that the pilA4 promoter is an efficient tool for gene expression in T. thermophilus with a high potential for use in biotechnology and synthetic biology applications.

The Virulence Regulator Rns Activates the Expression of CS14 Pili.

Although many viral and bacterial pathogens cause diarrhea, enterotoxigenic E. coli (ETEC) is one of the most frequently encountered in impoverished regions where it is estimated to kill between 300,000 and 700,000 children and infants annually. Critical ETEC virulence factors include pili which mediate the attachment of the pathogen to receptors in the intestinal lumen. In this study we show that the ETEC virulence regulator Rns positively regulates the expression of CS14 pili. Three Rns binding sites were identified upstream of the CS14 pilus promoter centered at −34.5, −80.5, and −155.5 relative to the Rns-dependent transcription start site. Mutagenesis of the promoter proximal site significantly decreased expression from the CS14 promoter. In contrast, the contribution of Rns bound at the promoter distal site was negligible and largely masked by occupancy of the promoter proximal site. Unexpectedly, Rns bound at the site centered at −80.5 had a slight but statistically significant inhibitory effect upon the pilin promoter. Nevertheless, this weak inhibitory effect was not sufficient to overcome the substantial promoter activation from Rns bound to the promoter proximal site. Thus, CS14 pili belong to a group of pili that depend upon Rns for their expression.

Regulation of the Pap Epigenetic Switch by CpxAR: Phosphorylated CpxR Inhibits Transition to the Phase ON State by Competition with Lrp

Pap pili gene expression is controlled by a reversible OFF/ON phase switch that is orchestrated by binding of Lrp to pap pilin promoter proximal sites 1, 2, and 3 (OFF) or pap promoter distal sites 4, 5, and 6 (ON). Movement of Lrp between proximal and distal sites controls pap pilin transcription and is modulated by PapI and DNA adenine methylase. Here we show that activation of the environmentally responsive CpxAR two-component regulatory system inhibits Pap phase variation by generation of phosphorylated CpxR (CpxR-P). CpxR-P competes with Lrp for binding to both promoter proximal and distal pap DNA binding sites, inhibiting pap transcription in vitro and pili expression in vivo. In contrast to Lrp, CpxR-P is methylation insensitive and does not form DNA methylation patterns in vivo. CpxAR-dependent repression of pap transcription is also observed in response to alkaline growth conditions. These results provide insight into a mechanism for environmental control of epigenetically regulated gene expression.

The intricate workings of a bacterial epigenetic switch.

Bacteria have developed epigenetic mechanisms to control the reversible Off-to-On switching of cell surface structures such as pyelonephritis-associated pili (PAP). The pap pili switch is primarily controlled by the global regulator leucine-responsive regulatory protein (Lrp), the local regulator PapI, and DNA adenine methylase (Dam). There are two sets of binding sites for Lrp in the pap regulatory region: promoter proximal sites 1,2,3 and promoter distal sites 4,5,6. The pilin promoter proximal (GATCprox) and distal (GATCdist) targets for Dam are located within Lrp binding sites 2 and 5, respectively. In the Off state, Lrp binds cooperatively to sites 1,2,3 overlapping the papBA pilin promoter, shutting off pilin transcription, and blocking methylation of GATCprox. Binding of Lrp at sites 1,2,3, together with methylation of GATCdist, reduces the affinity of Lrp for sites 4,5,6, preventing simultaneous binding of Lrp at sites 4,5,6 upstream. Switching to the phase. On state requires the environmentally regulated PapI co-regulator, which increases the affinity of Lrp for sites 5 and 2. PapI binds specifically to Lrp-pap DNA complexes via binding with Lrp as well as contact with DNA sequences within pap sites 5 and 2. Directionality in switching from Off to On appears to be due to methylation of GATCprox, which prevents formation of the PapI-Lrp-pap site 2 ternary complex. A switch model is presented in which DNA replication is proposed to play a critical role by generating a hemimethylated GATCdist site and displacing Lrp from sites 1,2,3. This facilitates methylation of GATCprox and binding of PapI-Lrp to sites 4,5,6, with subsequent activation of pap transcription. The first gene product of the pap operon, PapB, positively regulates papI transcription, resulting in a positive feedback loop that helps maintain the On state. The pap switch is environmentally regulated by a number of factors including the CpxAR two-component regulatory system, the Histone-like nucleoid structuring protein H-NS, and cAMP-Catabolite Gene Activator Protein (CAP), which all involve binding of regulatory binding proteins to pap DNA sequences with subsequent alteration of PapI and Lrp binding. The Pap switch mechanism, with interesting variations, is conserved among a number of enteric bacteria, controlling expression of many unrelated pili-adhesin complexes.

The Mechanism by which DNA Adenine Methylase and PapI Activate the Pap Epigenetic Switch

The expression of pyelonephritis-associated pili (Pap) in uropathogenic Escherichia coli is epigenetically controlled by a reversible OFF to ON switch. In phase OFF cells, the global regulator Lrp is bound to pap sites proximal to the pilin promoter, whereas in phase ON cells, Lrp is bound to promoter distal sites. We have found that the local regulator PapI increases the affinity of Lrp for the sequence “ACGATC,” which contains the target “GATC” site for DNA adenine methylase (Dam) and is present in both promoter proximal and distal sites. Mutational analyses show that methylation of the promoter proximal GATCprox site by Dam is required for transition to the phase ON state by specifically blocking PapI-dependent binding of Lrp to promoter proximal sites. Furthermore, our data support the hypothesis that PapI-dependent binding of Lrp to a hemimethylated GATCdist site generated by DNA replication is a critical component of the switch mechanism.

The size and position of heterologous insertions in a silent locus differentially affect pilin recombination in Neisseria gonorrhoeae.

Gonococcal pilus antigenic and phase variation result from unidirectional, RecA-dependent recombination of DNA sequences from a silent pilin copy (pilS) into the expressed pilin gene (pilE). To develop a quantitative assay for pilin gene recombination that is independent of phase variation, a promoterless cat gene was inserted into pilS, and recombination of "cat into pilE was detected by selection of chloramphenicol-resistant (CmR) variants expressing "cat from the pilin promoter. Although RecA-dependent CmR variants occurred, none were generated by the simple transfer of "cat into pilE. Instead, each CmR variant contained a new pilin locus that was a hybrid of sequences from the pilE and the pilS1::cat loci in addition to the two starting loci. Therefore, this system could not be used to quantify antigenic variation. However, combined studies of these hybrid loci and of recombination products generated during additional pilS mutational analyses demonstrated that both the size and position of an insertion in pilS differentially affect pilin recombination. Also, the hybrid loci appear to be intermediates of antigenic variation. This enabled the creation of molecular models for the recombination reactions that result in pilin antigenic variation.

Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli

We have examined the roles of pap DNA methylation patterns in the regulation of the switch between phase ON and OFF pyelonephritis-associated pili (Pap) expression states in E. coli. Two Dam methyltransferase sites, GATC1028 and GATC1130, were shown previously to be differentially methylated in phase ON versus phase OFF cells. In work presented here, these sites were mutated so that they could not be methylated, and the effects of these mutations on Pap phase variation were examined. Our results show that methylation of GATC1028 blocks formation of the ON state by inhibiting the binding of Lrp and PapI regulatory proteins to this site. Conversely, methylation of GATC1130 is required for the ON state. Evidence indicates that this occurs by the inhibition of binding of Lrp to sites overlapping the pilin promoter. A model describing how the transition between the phase ON and OFF methylation states might occur is presented.

Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli

We have examined the roles of pap DNA methylation patterns in the regulation of the switch between phase ON and OFF pyelonephritis-associated pili (Pap) expression states in E. coli. Two Dam methyltransferase sites, GATC 1028 and GATC 1130, were shown previously to be differentially methylated in phase ON versus phase OFF cells. In work presented here, these sites were mutated so that they could not be methylated, and the effects of these mutations on Pap phase variation were examined. Our results show that methylation of GATC 1028 blocks formation of the ON state by inhibiting the binding of Lrp and Papl regulatory proteins to this site. Conversely, methylation of GATC 1130 is required for the ON state. Evidence indicates that this occurs by the inhibition of binding of Lrp to sites overlapping the pilin promoter. A model describing how the transition between the phase ON and OFF methylation states might occur is presented.

Leucine-responsive regulatory protein controls the expression of both the pap and fan pili operons in Escherichia coli.

The methylation blocking factor gene (mbf) in Escherichia coli is required for specific methylation inhibition of two DNA GATC sites upstream of the papBA pilin promoter and transcriptional activation of pap. Complementation and mutational analysis using pap-lac and ilvIH-lac operon fusions indicates that the mbf gene is identical to a recently described global regulatory gene lrp (leucine-responsive regulatory protein) that acts as a positive regulator of some genes and a negative regulator of others in E. coli. DNA sequence analysis of an mbf::mTn10 insertion showed that the mbfDNA sequence was identical to lrp. Thus Lrp inhibits DNA methylation at specific GATC sites. We also show that Lrp positively regulates transcription of the fan operon, which encodes K99 pili of diarrheagenic E. coli. Purified Lrp was found to bind to DNA fragments encompassing the pap and fan promoters, which is consistent with previous results indicating that Lrp controls gene expression by binding to regulatory DNA sites. Exogenous leucine significantly reduced fan transcription and K99 pili expression, similar to results obtained with the ilvIH operon. However, pap gene expression was unresponsive to leucine, which distinguishes pap from other lrp-regulated genes whose expression is modulated by leucine.

Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle.

A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.

The activity of the Pseudomonas aeruginosa pilin promoter is enhanced by an upstream regulatory site

Recently, a comparison of the nucleotide sequences upstream of the structural pilin genes ( pil) from five different isolates of Pseudomonas aeruginosa identified a sequence homologous to a NifA-binding site, a positive regulatory sequence in Klebsiella pneumoniae which controls the expression of the genes which fix nitrogen [Pasloske et al., J. Bacteriol. 170 (1988) 3738–3741]. This sequence was located between −98 and −114 bp relative to the transcription start point. To establish whether this putative recognition sequence exerted any regulatory effect on pil gene expression, we assayed for the expression of pilin and pili, and assessed the strength of the pilin promoter with and without this upstream sequence. A 48-bp deletion, which removed the putative recognition sequence, decreased the levels of pilin and pilus expression. Also, the equivalent deletion reduced pilin promoter activity by 90% when it was measured using a promoter probe vector. These results provide evidence that a nucleotide sequence upstream of the pilin promoter positively regulates the transcription of the P. aeruginosa pil gene.

Pilin expression in Neisseria gonorrhoeae is under both positive and negative transcriptional control.

We have identified two closely linked genes, pilA and pilB, which act in trans on the pilin promoter. pilA-pilB map downstream of expression loci pilE1 and opaE1 in the gonococcal chromosome. Subcloning data indicate that pilB acts negatively on the pilin promoter, and insertional inactivation of pilB results in hyperpiliated gonococci. A pilA clone activates the pilin promoter in Escherichia coli, and a pilA-/pilA+ heterodiploid gonococcus exhibits a P- phenotype. Our inability to obtain simple pilA- mutants strongly suggests that pilA is an essential gene in the gonococcus. In an in vitro coupled transcription/translation system, inserts spanning the pilA and pilB region direct the synthesis of two proteins of 40 and 58 kd. DNA sequence analysis shows that the pilA and pilB loci encode proteins of 38.6 kd (with a putative DNA binding domain) and 57.9 kd respectively. The pilA and pilB genes are in opposite orientation relative to each other, and the 5' ends of the two genes overlap. We discuss how these two loci may interact to control pilin expression in the gonococcus.