Eukaryotic Acquisition of a Bacterial Operon.

Operons are a hallmark of bacterial genomes, where they allow concerted expression of functionally related genes as single polycistronic transcripts. They are rare in eukaryotes, where each gene usually drives expression of its own independent messenger RNAs. Here, we report the horizontal operon transfer of a siderophore biosynthesis pathway from relatives of Escherichia coli into a group of budding yeast taxa. We further show that the co-linearly arranged secondary metabolism genes are expressed, exhibit eukaryotic transcriptional features, and enable the sequestration and uptake of iron. After transfer, several genetic changes occurred during subsequent evolution, including the gain of new transcription start sites that were sometimes within protein-coding sequences, acquisition of polyadenylation sites, structural rearrangements, and integration of eukaryotic genes into the cluster. We conclude that the genes were likely acquired as a unit, modified for eukaryotic gene expression, and maintained by selection to adapt to the highly competitive, iron-limited environment.

A Host-Produced Quorum-Sensing Autoinducer Controls a Phage Lysis-Lysogeny Decision.

Vibrio cholerae uses a quorum-sensing (QS) system composed of the autoinducer 3,5-dimethylpyrazin-2-ol (DPO) and receptor VqmA (VqmAVc), which together repress genes for virulence and biofilm formation. vqmA genes exist in Vibrio and in one vibriophage, VP882. Phage-encoded VqmA (VqmAPhage) binds to host-produced DPO, launching the phage lysis program via an antirepressor that inactivates the phage repressor by sequestration. The antirepressor interferes with repressors from related phages. Like phage VP882, these phages encode DNA-binding proteins and partner antirepressors, suggesting that they, too, integrate host-derived information into their lysis-lysogeny decisions. VqmAPhage activates the host VqmAVc regulon, whereas VqmAVc cannot induce phage-mediated lysis, suggesting an asymmetry whereby the phage influences host QS while enacting its own lytic-lysogeny program without interference. We reprogram phages to activate lysis in response to user-defined cues. Our work shows that a phage, causing bacterial infections, and V. cholerae, causing human infections, rely on the same signal molecule for pathogenesis.

Long-Distance Cooperative and Antagonistic RNA Polymerase Dynamics via DNA Supercoiling.

Genes are often transcribed by multiple RNA polymerases (RNAPs) at densities that can vary widely across genes and environmental conditions. Here, we provide in vitro and in vivo evidence for a built-in mechanism by which co-transcribing RNAPs display either collaborative or antagonistic dynamics over long distances (>2 kb) through transcription-induced DNA supercoiling. In Escherichia coli, when the promoter is active, co-transcribing RNAPs translocate faster than a single RNAP, but their average speed is not altered by large variations in promoter strength and thus RNAP density. Environmentally induced promoter repression reduces the elongation efficiency of already-loaded RNAPs, causing premature termination and quick synthesis arrest of no-longer-needed proteins. This negative effect appears independent of RNAP convoy formation and is abrogated by topoisomerase I activity. Antagonistic dynamics can also occur between RNAPs from divergently transcribed gene pairs. Our findings may be broadly applicable given that transcription on topologically constrained DNA is the norm across organisms.

A Bacterial Chromosome Structuring Protein Binds Overtwisted DNA to Stimulate Type II Topoisomerases and Enable DNA Replication.

When DNA is unwound during replication, it becomes overtwisted and forms positive supercoils in front of the translocating DNA polymerase. Unless removed or dissipated, this superhelical tension can impede replication elongation. Topoisomerases, including gyrase and topoisomerase IV in bacteria, are required to relax positive supercoils ahead of DNA polymerase but may not be sufficient for replication. Here, we find that GapR, a chromosome structuring protein in Caulobacter crescentus, is required to complete DNA replication. GapR associates in vivo with positively supercoiled chromosomal DNA, and our biochemical and structural studies demonstrate that GapR forms a dimer-of-dimers that fully encircles overtwisted DNA. Further, we show that GapR stimulates gyrase and topo IV to relax positive supercoils, thereby enabling DNA replication. Analogous chromosome structuring proteins that locate to the overtwisted DNA in front of replication forks may be present in other organisms, similarly helping to recruit and stimulate topoisomerases during DNA replication.

A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation.

Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes.

Structural basis of transcriptional activation by the Mycobacterium tuberculosis intrinsic antibiotic-resistance transcription factor WhiB7.

In pathogenic mycobacteria, transcriptional responses to antibiotics result in induced antibiotic resistance. WhiB7 belongs to the Actinobacteria-specific family of Fe-S-containing transcription factors and plays a crucial role in inducible antibiotic resistance in mycobacteria. Here, we present cryoelectron microscopy structures of Mycobacterium tuberculosis transcriptional regulatory complexes comprising RNA polymerase σA-holoenzyme, global regulators CarD and RbpA, and WhiB7, bound to a WhiB7-regulated promoter. The structures reveal how WhiB7 interacts with σA-holoenzyme while simultaneously interacting with an AT-rich sequence element via its AT-hook. Evidently, AT-hooks, rare elements in bacteria yet prevalent in eukaryotes, bind to target AT-rich DNA sequences similarly to the nuclear chromosome binding proteins. Unexpectedly, a subset of particles contained a WhiB7-stabilized closed promoter complex, revealing this intermediate's structure, and we apply kinetic modeling and biochemical assays to rationalize how WhiB7 activates transcription. Altogether, our work presents a comprehensive view of how WhiB7 serves to activate gene expression leading to antibiotic resistance.

Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis.

WhiB7 represents a distinct subclass of transcription factors in the WhiB-Like (Wbl) family, a unique group of iron-sulfur (4Fe-4S] cluster-containing proteins exclusive to the phylum of Actinobacteria. In Mycobacterium tuberculosis (Mtb), WhiB7 interacts with domain 4 of the primary sigma factor (σA4) in the RNA polymerase holoenzyme and activates genes involved in multiple drug resistance and redox homeostasis. Here, we report crystal structures of the WhiB7:σA4 complex alone and bound to its target promoter DNA at 1.55-Å and 2.6-Å resolution, respectively. These structures show how WhiB7 regulates gene expression by interacting with both σA4 and the AT-rich sequence upstream of the -35 promoter DNA via its C-terminal DNA-binding motif, the AT-hook. By combining comparative structural analysis of the two high-resolution σA4-bound Wbl structures with molecular and biochemical approaches, we identify the structural basis of the functional divergence between the two distinct subclasses of Wbl proteins in Mtb.

c-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

Streptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.

RNA-RNA Interactomes of ProQ and Hfq Reveal Overlapping and Competing Roles.

Base pairing RNAs modulate gene expression in all studied organisms. In many bacteria, the base pairing between most small regulatory RNAs (sRNAs) and their targets is mediated by the Hfq RNA chaperone. However, recent studies have shown FinO-domain proteins also bind sRNAs. To examine the global contribution of the FinO-domain ProQ protein in Escherichia coli, we carried out RIL-seq to identify RNA pairs bound to this protein. The RNA-RNA interactome for ProQ contains hundreds of pairs. Intriguingly, a significant fraction of the ProQ-bound RNA pairs are also found associated with Hfq, indicating overlapping, complementary, or competing roles for the two proteins. Characterization of one novel RNA pair bound by both chaperones revealed that while Hfq is required for RNA sponge-mediated downregulation of the sRNA, ProQ can inhibit this regulation. Overall, our results uncover increased complexity in RNA regulatory networks involving RNA chaperone proteins, RNases, sRNAs, and mRNAs.

Catalytically Active Cas9 Mediates Transcriptional Interference to Facilitate Bacterial Virulence.

In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.

Obstacles to Scanning by RNase E Govern Bacterial mRNA Lifetimes by Hindering Access to Distal Cleavage Sites.

The diversity of mRNA lifetimes in bacterial cells is difficult to reconcile with the relaxed cleavage site specificity of RNase E, the endonuclease most important for governing mRNA degradation. This enzyme has generally been thought to locate cleavage sites by searching freely in three dimensions. However, our results now show that its access to such sites in 5'-monophosphorylated RNA is hindered by obstacles-such as bound proteins or ribosomes or coaxial small RNA (sRNA) base pairing-that disrupt the path from the 5' end to those sites and prolong mRNA lifetimes. These findings suggest that RNase E searches for cleavage sites by scanning linearly from the 5'-terminal monophosphate along single-stranded regions of RNA and that its progress is impeded by structural discontinuities encountered along the way. This discovery has major implications for gene regulation in bacteria and suggests a general mechanism by which other prokaryotic and eukaryotic regulatory proteins can be controlled.

Substrate Recognition and Autoinhibition in the Central Ribonuclease RNase E.

The endoribonuclease RNase E is a principal factor in RNA turnover and processing that helps to exercise fine control of gene expression in bacteria. While its catalytic activity can be strongly influenced by the chemical identity of the 5' end of RNA substrates, the enzyme can also cleave numerous substrates irrespective of the chemistry of their 5' ends through a mechanism that has remained largely unexplained. We report structural and functional data illuminating details of both operational modes. Our crystal structure of RNase E in complex with the sRNA RprA reveals a duplex recognition site that saddles an inter-protomer surface to help present substrates for cleavage. Our data also reveal an autoinhibitory pocket that modulates the overall activity of the ribonuclease. Taking these findings together, we propose how RNase E uses versatile modes of RNA recognition to achieve optimal activity and specificity.

Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation.

Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome may also play an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutation and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and significantly change the chromosome organization at domain scales. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulator (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (>200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.

Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase.

Transcription termination factor Rho is known for its ubiquitous role in suppression of pervasive, mostly antisense, transcription. In the model Gram-positive bacterium Bacillus subtilis, de-repression of pervasive transcription by inactivation of rho revealed the role of Rho in the regulation of post-exponential differentiation programs. To identify other aspects of the regulatory role of Rho during adaptation to starvation, we have constructed a B. subtilis strain (Rho+) that expresses rho at a relatively stable high level in order to compensate for its decrease in the wild-type cells entering stationary phase. The RNAseq analysis of Rho+, WT and Δrho strains (expression profiles can be visualized at http://genoscapist.migale.inrae.fr/seb_rho/) shows that Rho over-production enhances the termination efficiency of Rho-sensitive terminators, thus reducing transcriptional read-through and antisense transcription genome-wide. Moreover, the Rho+ strain exhibits global alterations of sense transcription with the most significant changes observed for the AbrB, CodY, and stringent response regulons, forming the pathways governing the transition to stationary phase. Subsequent physiological analyses demonstrated that maintaining rho expression at a stable elevated level modifies stationary phase-specific physiology of B. subtilis cells, weakens stringent response, and thereby negatively affects the cellular adaptation to nutrient limitations and other stresses, and blocks the development of genetic competence and sporulation. These results highlight the Rho-specific termination of transcription as a novel element controlling stationary phase. The release of this control by decreasing Rho levels during the transition to stationary phase appears crucial for the functionality of complex gene networks ensuring B. subtilis survival in stationary phase.

A natural single nucleotide mutation in the small regulatory RNA ArcZ of Dickeya solani switches off the antimicrobial activities against yeast and bacteria.

The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster named sol was recently shown to produce an antifungal molecule. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters sol, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The cluster sol, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters sol and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the D. solani type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form.

The divalent metal ion exporter IhpABC is required to maintain iron homeostasis under low to moderate environmental iron conditions in the bacterium Bradyrhizobium japonicum.

Bacterial iron export mitigates high iron stress, but a role for it under lower iron conditions has not been established. MbfA is the high iron stress exporter in Bradyrhizobium japonicum. Here, we identify the ihpABC genes in a selection for secondary site mutations that suppress the poor growth phenotype of feoAB mutants defective in iron acquisition. IhpABC belongs to the RND tripartite efflux pump family. High iron conditions that derepress the mbfA gene partially rescued the growth of an ihpC mutant but reverted the feoB ihpC mutant to the feoB growth phenotype. The ihpA mutant grown under low iron conditions accumulated higher levels of iron compared to the wild type, and it displayed aberrant iron-responsive gene expression. The mbfA mutant was more sensitive than the wild type to H2 O2 , but the ihpA mutant was not sensitive. The ihpA mutant accumulated more Zn, Co and Cd than was found in the wild type, and growth of the mutant was more sensitive to inhibition by ZnCl2 , CoCl2 and CdCl2 . The findings suggest that IhpABC is a divalent metal ion exporter that helps maintain iron homeostasis under low to moderate environmental iron levels. Thus, iron export is not limited to managing high iron stress.

The multifarious MerR family of transcriptional regulators.

The MerR family of transcriptional regulators includes a variety of bacterial cytoplasmic proteins that respond to a wide range of signals, including toxins, metal ions, and endogenous metabolites. Its best-characterized members share similar structural and functional features with the family founder, the mercury sensor MerR, although most of them do not respond to metal ions. The group of "canonical" MerR homologs displays common molecular mechanisms for controlling the transcriptional activation of their target genes in response to inducer signals. This includes the recognition of distinctive operator sequences located at suboptimal σ70 -dependent promoters. Interestingly, an increasing number of proteins assigned to the MerR family based on their DNA-binding domain do not match in structure, sequence, or mode of action with any of the canonical MerR-like regulators. Here, we analyzed several members of the family, including this last group. Based on a phylogenetic analysis, and similarities in structural/functional features and position of their target operators relative to the promoter elements, we propose to assign these "atypical/divergent" MerR regulators to a phylogenetically separated group. These atypical/divergent homologs represent a new class of transcriptional regulators with novel regulatory mechanisms.

Redox homeostasis in Mycobacterium tuberculosis is modulated by a novel actinomycete-specific transcription factor.

Mycobacterium tuberculosis (Mtb) has evolved diverse cellular processes in response to the multiple stresses it encounters within the infected host. We explored available TnSeq datasets to identify transcription factors (TFs) that are essential for Mtb survival inside the host. The analysis identified a single TF, Rv1332 (AosR), conserved across actinomycetes with a so-far uncharacterized function. AosR mitigates phagocyte-derived oxidative and nitrosative stress, thus promoting mycobacterial growth in the murine lungs and spleen. Oxidative stress induces formation of a single intrasubunit disulphide bond in AosR, which in turn facilitates AosR interaction with an extracytoplasmic-function sigma factor, SigH. This leads to the specific upregulation of the CysM-dependent non-canonical cysteine biosynthesis pathway through an auxiliary intragenic stress-responsive promoter, an axis critical in detoxifying host-derived oxidative and nitrosative radicals. Failure to upregulate AosR-dependent cysteine biosynthesis during the redox stress causes differential expression of 6% of Mtb genes. Our study shows that the AosR-SigH pathway is critical for detoxifying host-derived oxidative and nitrosative radicals to enhance Mtb survival in the hostile intracellular environment.

The noncoding RNA CcnA modulates the master cell cycle regulators CtrA and GcrA in Caulobacter crescentus.

Bacteria are powerful models for understanding how cells divide and accomplish global regulatory programs. In Caulobacter crescentus, a cascade of essential master regulators supervises the correct and sequential activation of DNA replication, cell division, and development of different cell types. Among them, the response regulator CtrA plays a crucial role coordinating all those functions. Here, for the first time, we describe the role of a novel factor named CcnA (cell cycle noncoding RNA A), a cell cycle-regulated noncoding RNA (ncRNA) located at the origin of replication, presumably activated by CtrA, and responsible for the accumulation of CtrA itself. In addition, CcnA may be also involved in the inhibition of translation of the S-phase regulator, GcrA, by interacting with its 5' untranslated region (5' UTR). Performing in vitro experiments and mutagenesis, we propose a mechanism of action of CcnA based on liberation (ctrA) or sequestration (gcrA) of their ribosome-binding site (RBS). Finally, its role may be conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, representing indeed a potentially conserved process modulating cell cycle in Caulobacterales and Rhizobiales.

Inferred regulons are consistent with regulator binding sequences in E. coli.

The transcriptional regulatory network (TRN) of E. coli consists of thousands of interactions between regulators and DNA sequences. Regulons are typically determined either from resource-intensive experimental measurement of functional binding sites, or inferred from analysis of high-throughput gene expression datasets. Recently, independent component analysis (ICA) of RNA-seq compendia has shown to be a powerful method for inferring bacterial regulons. However, it remains unclear to what extent regulons predicted by ICA structure have a biochemical basis in promoter sequences. Here, we address this question by developing machine learning models that predict inferred regulon structures in E. coli based on promoter sequence features. Models were constructed successfully (cross-validation AUROC > = 0.8) for 85% (40/47) of ICA-inferred E. coli regulons. We found that: 1) The presence of a high scoring regulator motif in the promoter region was sufficient to specify regulatory activity in 40% (19/47) of the regulons, 2) Additional features, such as DNA shape and extended motifs that can account for regulator multimeric binding, helped to specify regulon structure for the remaining 60% of regulons (28/47); 3) investigating regulons where initial machine learning models failed revealed new regulator-specific sequence features that improved model accuracy. Finally, we found that strong regulatory binding sequences underlie both the genes shared between ICA-inferred and experimental regulons as well as genes in the E. coli core pan-regulon of Fur. This work demonstrates that the structure of ICA-inferred regulons largely can be understood through the strength of regulator binding sites in promoter regions, reinforcing the utility of top-down inference for regulon discovery.