Transcriptional repressor Gal80 recruits corepressor complex Cyc8–Tup1 to structural genes of the Saccharomyces cerevisiae GAL regulon

Under non-inducing conditions (absence of galactose), yeast structural genes of the GAL regulon are repressed by Gal80, preventing interaction of Gal4 bound to UASGAL promoter motifs with general factors of the transcriptional machinery. In this work, we show that Gal80 is also able to interact with histone deacetylase-recruiting corepressor proteins Cyc8 and Tup1, indicating an additional mechanism of gene repression. This is supported by our demonstration that a lexA–Gal80 fusion efficiently mediates repression of a reporter gene with an upstream lexA operator sequence. Corepressor interaction and in vivo gene repression could be mapped to a Gal80 minimal domain of 65 amino acids (aa 81-145). Site-directed mutagenesis of selected residues within this domain showed that a cluster of aromatic-hydrophobic amino acids (YLFV, aa 118-121) is important, although not solely responsible, for gene repression. Using chromatin immunoprecipitation, Cyc8 and Tup1 were shown to be present at the GAL1 promoter in a wild-type strain but not in a gal80 mutant strain under non-inducing (derepressing) growth conditions. Expression of a GAL1–lacZ fusion was elevated in a tup1 mutant (but not in a cyc8 mutant) grown in derepressing medium, indicating that Tup1 may be mainly responsible for this second mechanism of Gal80-dependent gene repression.

Transcriptional Repressor Gal80 Recruits Corepressor Complex Cyc8-Tup1 to Structural Genes of the Saccharomyces Cerevisiae GAL Regulon

Under non-inducing conditions (absence of galactose), yeast structural genes of the GAL regulon are repressed by Gal80, preventing interaction of Gal4 bound to UASGAL promoter motifs with general factors of the transcriptional machinery. In this work we show that Gal80 is also able to interact with histone deacetylase-recruiting corepressor proteins Cyc8 and Tup1, indicating an additional mechanism of gene repression. This is supported by our demonstration that a lexA-Gal80 fusion efficiently mediates repression of a reporter gene with an upstream lexA operator sequence. Corepressor interaction and in vivo gene repression could be mapped to a Gal80 minimal domain of 65 amino acids (aa 81-145). Site-directed mutagenesis of selected residues within this domain showed that a cluster of aromatic-hydrophobic amino acids (YLFV, aa 118-121) is important, although not solely responsible, for gene repression. Using chromatin immunoprecipitation, Cyc8 and Tup1 were shown to be present at the GAL1 promoter in a wild-type strain but not in a gal80 mig1 mutant under non-inducing (derepressing) growth conditions. Expression of a GAL1-lacZ fusion was elevated in a tup1 mutant (but not in a cyc8 mutant) grown in derepressing medium, indicating that Tup1 may be mainly responsible for this second mechanism of Gal80-dependent gene repression.

Elements in the LftR repressor operator interface contributing to regulation of aurantimycin resistance in Listeria monocytogenes

The bacterium Listeria monocyctogenes ubiquitously occurs in the environment, but can cause severe invasive disease in susceptible individuals when ingested. We recently identified the L. monocytogenes genes lieAB and lftRS, encoding a multi drug resistance ABC transporter and a regulatory module, respectively. These genes jointly mediate resistance against aurantimycin, an antibiotic produced by the soil-dwelling species Streptomyces aurantiacus, and thus contribute to survival of L. monocytogenes in its natural habitat, the soil. Repression of lieAB and lftRS is exceptionally tight, but strongly induced in the presence of aurantimycin. Repression depends on LftR which belongs to subfamily-2 of the PadR-like transcriptional repressors. To better understand this interesting class of transcriptional repressors, we here deduce the LftR operator sequence from a systematic truncation and mutation analysis of the PlieAB promoter. The sequence identified is also present in the PlftRS promoter, but not found elsewhere in the chromosome. Mutational analysis of the putative operator in the PlftRS promoter confirmed its relevance for LftR-dependent repression. The proposed operator sequence was sufficient for DNA binding by LftR in vitro and a mutation in this sequence affected aurantimycin resistance. Our results provide further insights into transcriptional adaptation of an important human pathogen to survive the conditions in its natural reservoir. Importance Listeria monocytogenes is an environmental bacterium that lives in the soil but can infect humans upon ingestion and this can lead to severe invasive disease. Adaptation to these entirely different habitats involves massive reprogramming of transcription. Among the differentially expressed genes is the lieAB operon that encodes a transporter for detoxification of aurantimycin, an antimicrobial compound produced by soil-dwelling competitors. While lieAB is important for survival in the environment, its expression is detrimental during infection. We here identify critical elements in the lieAB promoter and its transcriptional regulator LftR that contribute to habitat-specific expression of the lieAB genes. These results further clarify the molecular mechanisms underlying aurantimycin resistance of L. monocytogenes.

Eliciting the silent lucensomycin biosynthetic pathway in Streptomyces cyanogenus S136 via manipulation of the global regulatory gene adpA

Actinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis (lcm), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA-mediated activation.

The iron-dependent repressor YtgR is a tryptophan-dependent attenuator of the trpRBA operon in Chlamydia trachomatis

The trp operon of Chlamydia trachomatis is organized differently from other model bacteria. It contains trpR, an intergenic region (IGR), and the biosynthetic trpB and trpA open-reading frames. TrpR is a tryptophan-dependent repressor that regulates the major promoter (PtrpR), while the IGR harbors an alternative promoter (PtrpBA) and an operator sequence for the iron-dependent repressor YtgR to regulate trpBA expression. Here, we report that YtgR repression at PtrpBA is also dependent on tryptophan by regulating YtgR levels through a rare triple-tryptophan motif (WWW) in the YtgCR precursor. Inhibiting translation during tryptophan limitation at the WWW motif subsequently promotes Rho-independent transcription termination of ytgR, thereby de-repressing PtrpBA. Thus, YtgR represents an alternative strategy to attenuate trpBA expression, expanding the repertoire for trp operon attenuation beyond TrpL- and TRAP-mediated mechanisms described in other bacteria. Furthermore, repurposing the iron-dependent repressor YtgR underscores the fundamental importance of maintaining tryptophan-dependent attenuation of the trpRBA operon.

Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors

ABSTRACT Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the cop box. Using this motif, we sought to determine whether genes outside the cop operon are also regulated by the CopY repressor, which was previously shown in Lactococcus lactis. We found that S. pneumoniae CopY did not bind to cop operators upstream of these candidate genes in vitro. During this process, we found that the cop box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the S. pneumoniae cop operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant (Kd) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs. IMPORTANCE Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the cop operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.

Regulation of an iron-dependent repressor by tryptophan availability attenuates transcription of the tryptophan salvage genes in Chlamydia trachomatis

The trp operon of Chlamydia trachomatis has a genetic organization that is distinct from other model bacteria. The operon contains the trpR open-reading frame (ORF), an intergenic region (IGR), and the trpB and trpA ORFs. TrpR mediates tryptophan-dependent regulation of the operon from the major promoter upstream of trpR(PtrpR). We recently reported that trpBA is additionally regulated by the iron-dependent repressor YtgR via an operator sequence within the IGR upstream of an alternative promoter for TrpR-independent trpBA expression (PtrpBA). Here we report that YtgR repression of PtrpBA is also dependent on tryptophan levels via a rare triple-tryptophan motif (WWW) in the N-terminal permease domain of the YtgCR precursor. Tryptophan limitation inhibits translation at the WWW motif and subsequently promotes transcription termination of ytgCR in a Rho-independent manner. This regulatory schematic resembles mechanisms of transcriptional attenuation for trp operons described in model bacteria, such as cis-attenuation by TrpL in E. coli or trans-attenuation by TRAP in B. subtilis. YtgR performs an analogous function by sensing both iron and tryptophan levels, with the latter highlighting the unique strategy of Chlamydia to retain a trans-attenuator mechanism for regulating expression of the trpRBA operon.

Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

ABSTRACTCopper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repression. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, these homologs have been characterized to bind a 10-base consensus sequence T/GACAnnTGTA. Here, we bioinformatically and empirically characterize these operator sites across species using S. pneumoniae CopY as a guide for binding. By examining the 21-base repeat operators for the pneumococcal cop operon and comparing binding of recombinant CopY to this, and the operator sites found in Enterococcus hirae, we show using biolayer interferometry that the T/GACAnnTGTA sequence is essential to binding, but it is not sufficient. We determine a more comprehensive S. pneumoniae CopY operator sequence to be RnYKACAAATGTARnY (where “R” is purine, “Y” is pyrimidine, and “K” is either G or T) binding with an affinity of 28 nM. We further propose that the cop operon operator consensus site of pneumococcal homologs be RnYKACAnnYGTARnY. This study illustrates the necessity to explore bacterial operator sites further to better understand bacterial gene regulation.

Approximation by Sequence of Operators Involving Analytic Functions

In this contribution, we define a new operator sequence which contains analytic functions. Using approximation techniques found by Korovkin, some results are derived. Moreover, a generalization of this operator sequence called Kantorovich type generalization is introduced.