Identification of the DNA-binding sites for two response regulators involved in control of bacteriocin synthesis in Lactobacillus plantarum C11

In Lactobacillus plantarum C11, bacteriocin production has previously been shown to be an inducible process, in which a secreted peptide, produced by the host itself, is involved. The inducing factor, designated plantaricin A (PlnA), is a bacteriocin-like peptide encoded by a gene (plnA) located on the same operon as the genes for a two-component regulatory system (plnBCD). This system consists of a histidine kinase (PlnB) and two response regulators (PlnC,D), and belongs to a recently defined subfamily of two-component regulatory systems, which are activated by secreted peptide pheromones through a quorum-sensing mechanism. We show here that the two response regulators PlnC and PlnD bind specifically to imperfect direct repeats found within the adjacent promoter of the plnABCD operon, and to similar sequences found within the promoter regions of two nearby operons containing bacteriocin structural genes (plnEFI and plnJKLR). Binding of PlnC and PlnD was increased two to three fold in the presence of acetyl phosphate. The results suggest that bacteriocin synthesis in L. plantarum C11 is regulated by the DNA-binding activity of the two response regulators PlnC and PlnD.

     

Identification of the DNA-binding sites for two response regulators involved in control of bacteriocin synthesis in Lactobacillus plantarum C11

In Lactobacillus plantarum C11, bacteriocin production has previously been shown to be an inducible process, in which a secreted peptide, produced by the host itself, is involved. The inducing factor, designated plantaricin A (PlnA), is a bacteriocin-like peptide encoded by a gene (plnA) located on the same operon as the genes for a two-component regulatory system (plnBCD). This system consists of a histidine kinase (PlnB) and two response regulators (PlnC,D), and belongs to a recently defined subfamily of two-component regulatory systems, which are activated by secreted peptide pheromones through a quorum-sensing mechanism. We show here that the two response regulators PlnC and PlnD bind specifically to imperfect direct repeats found within the adjacent promoter of the plnABCD operon, and to similar sequences found within the promoter regions of two nearby operons containing bacteriocin structural genes (plnEFI and plnJKLR). Binding of PlnC and PlnD was increased two to three fold in the presence of acetyl phosphate. The results suggest that bacteriocin synthesis in L. plantarum C11 is regulated by the DNA-binding activity of the two response regulators PlnC and PlnD. Received: 21 January 1998 / Accepted: 28 April 1998     

Inducible bacteriocin production in Lactobacillus is regulated by differential expression of the pln operons and by two antagonizing response regulators,the activity of which is enhanced upon phosphorylation

Expression of the five (pln) operons involved in the bacteriocin production of Lactobacillus plantarum C11 is regulated by a so-called pheromone-based signal-transducing network, in which the peptide pheromone (PlnA) induces bacteriocin production through the action of a histidine protein kinase (PlnB) and two antagonizing response regulators (PlnC as an activator and PlnD as a negative regulator). All pln-regulated promoters contain a conserved pair of direct repeats that serve as binding sites for PlnC and PlnD. In the present work, we show that the five PlnA-responsive operons are differentially expressed with regard to both timing and strength, and that the pheromone triggers a strong autoactivating loop of the regulatory unit (plnABCD) during an early stage of induction that gradually leads to enhanced activation of the other operons. The transport operon (plnGHSTUV), which is involved in the secretion of the pheromone and bacteriocins, is also expressed relatively early upon induction, but is quickly turned off soon after peak expression. Further investigation of the various promoters revealed that, although subtle differences within the promoter regions could account for the observed differential regulation, the presence of a downstream promoter-proximal sequence in one promoter was found to cause delayed peak activity. How phosphorylation regulates the activity of the pln response regulators was also accessed by direct mutagenesis at their phosphorylation sites. It was found that the two response regulators exert activity at two different levels: a low level when they are not phosphorylated and an elevated level when they are phosphorylated. The present data demonstrate that bacteriocin production in L. plantarum C11 is a highly regulated process, in which different regulatory mechanisms are applied to fine tune the timing and strength of expression of the five pln operons.     

Protein factors specifically binding to the regulatory elements of non-transcribed spacer of rat ribosomal genes

Mouse, rat and human protein factors recognizing regulatory elements of nontranscribed spacer of rat ribosomal genes were studied by gel retardation assay. Protein factors bind specifically to the DNA fragments containing the core promoter sequence of RNA-polymerase I, to "spacer" promoter and to a putative enhancer sequence. Factors of mouse, rat and human nuclear extracts that recognize the region containing the core promoter sequence have similar molecular masses and are not identical to the previously described protein factor TIF-1B. Two factors that bind the "spacer" promoter region differ from the factors of the core promoter. "Spacer" promoter factors of mouse and rat nuclear extracts are probably identical, but differ from those of human extract. Protein factors, recognizing the putative enhancer region of rat and human extracts are alike but were not detected in mouse extract. Regions of nontranscribed spacer containing dispersed and tandem repeats do not bind any specific protein factors.     

Regulation of dev, an operon that includes genes essential for Myxococcus xanthus development and CRISPR-associated genes and repeats

Expression of dev genes is important for triggering spore differentiation inside Myxococcus xanthus fruiting bodies. DNA sequence analysis suggested that dev and cas (CRISPR-associated) genes are cotranscribed at the dev locus, which is adjacent to CRISPR (clustered regularly interspaced short palindromic repeats). Analysis of RNA from developing M. xanthus confirmed that dev and cas genes are cotranscribed with a short upstream gene and at least two repeats of the downstream CRISPR, forming the dev operon. The operon is subject to strong, negative autoregulation during development by DevS. The dev promoter was identified. Its -35 and -10 regions resemble those recognized by M. xanthus sigma(A) RNA polymerase, the homolog of Escherichia coli sigma(70), but the spacer may be too long (20 bp); there is very little expression during growth. Induction during development relies on at least two positive regulatory elements located in the coding region of the next gene upstream. At least two positive regulatory elements and one negative element lie downstream of the dev promoter, such that the region controlling dev expression spans more than 1 kb. The results of testing different fragments for dev promoter activity in wild-type and devS mutant backgrounds strongly suggest that upstream and downstream regulatory elements interact functionally. Strikingly, the 37-bp sequence between the two CRISPR repeats that, minimally, are cotranscribed with dev and cas genes exactly matches a sequence in the bacteriophage Mx8 intP gene, which encodes a form of the integrase needed for lysogenization of M. xanthus.     

Characterization of DNA binding sites of the ComE response regulator from Streptococcus mutans

In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.     

Insulation and wiring specificity of BceR‐like response regulators and their target promoters in Bacillus subtilis

BceRS and PsdRS are paralogous two‐component systems in Bacillus subtilis controlling the response to antimicrobial peptides. In the presence of extracellular bacitracin and nisin, respectively, the two response regulators (RRs) bind their target promoters, P_bceA or P_psdA, resulting in a strong up‐regulation of target gene expression and ultimately antibiotic resistance. Despite high sequence similarity between the RRs BceR and PsdR and their known binding sites, no cross‐regulation has been observed between them. We therefore investigated the specificity determinants of P_bceA and P_psdA that ensure the insulation of these two paralogous pathways at the RR–promoter interface. In vivo and in vitro analyses demonstrate that the regulatory regions within these two promoters contain three important elements: in addition to the known (main) binding site, we identified a linker region and a secondary binding site that are crucial for functionality. Initial binding to the high‐affinity, low‐specificity main binding site is a prerequisite for the subsequent highly specific binding of a second RR dimer to the low‐affinity secondary binding site. In addition to this hierarchical cooperative binding, discrimination requires a competition of the two RRs for their respective binding site mediated by only slight differences in binding affinities.     

Mouse integrin alphav promoter is regulated by transcriptional factors Ets and Sp1 in melanoma cells

A 17-bp region between the -31 and -15 bp region of the mouse integrin alphav gene is known to be one of the cis-acting elements for promoter activity. Experimental binding of nuclear proteins to the -31/-15 region reveals that the -27/-16 region mediates the binding. The -27/-16 region, GGCTCCTCCTCC, has a TCCTCC motif, one of the Sp1 binding motifs. An anti-Sp1 IgG and an Sp1-binding oligonucleotide interfered with the binding of nuclear proteins to the -27/-16 oligonucleotide, demonstrating that Sp1 binds to the -27/-16 region. In addition to the -27/-16 region, two other regions, -108/-89 and -64/-44, were found to bind to nuclear proteins within the -108/+1 alphav promoter region. An oligonucleotide containing the Ets-binding consensus sequence of CAGGAAGT interfered with their binding, indicating that both regions have a functional Ets-binding site; which is ACGGAAGT from -106 to -99 bp and ACTTCCTC from -61 to -54 bp, as deduced from the sequence. Mutations in or deletions from any one of three cis-acting elements, the two Ets-binding sites or one Sp1-binding site, remarkably decreased the promoter activity detected in the -108/+1 region. Cotransfection of both Sp1 and Ets-1 cDNAs with the -108/+1 region into B16F10 cells increased the promoter activity 2.9-fold. These results demonstrate that Sp1 and Ets cooperate to activate the -108/+1-alphav promoter region.