Hierarchic regulation of a metabolic pathway: H-NS, CRP, and SsrB control myo-inositol utilization by Salmonella enterica.

IMPORTANCE: The capacity to utilize myo-inositol (MI) as sole carbon and energy source is widespread among bacteria, among them the intestinal pathogen S. Typhimurium. This study elucidates the complex and hierarchical regulation that underlies the utilization of MI by S. Typhimurium under substrate limitation. A total of seven regulatory factors have been identified so far, allowing the pathogen an environment-dependent, efficient, and fine-tuned regulation of a metabolic property that provides growth advantages in different environments.

A signaling complex of adenylate cyclase CyaC of Sinorhizobium meliloti with cAMP and the transcriptional regulators Clr and CycR.

BACKGROUND: Adenylate cyclases (ACs) generate the second messenger cyclic AMP (cAMP), which is found in all domains of life and is involved in the regulation of various cell physiological and metabolic processes. In the plant symbiotic bacterium Sinorhizobium meliloti, synthesis of cAMP by the membrane-bound AC CyaC responds to the redox state of the respiratory chain and the respiratory quinones. However, nothing is known about the signaling cascade that is initiated by cAMP produced by CyaC. RESULTS: Here, the CRP-like transcriptional regulator Clr and the TetR-like regulator CycR (TR01819 protein) were identified to interact with CyaC using the bacterial two-hybrid system (BACTH), co-sedimentation assays, and surface plasmon resonance spectroscopy. Interaction of CycR with Clr, and of CyaC with Clr requires the presence of cAMP and of ATP, respectively, whereas that of CyaC with CycR was independent of the nucleotides. CONCLUSION: The data implicate a ternary CyaC×CycR×cAMP-Clr complex, functioning as a specific signaling cascade which is formed after activation of CyaC and synthesis of cAMP. cAMP-Clr is thought to work in complex with CycR to regulate a subset of genes of the cAMP-Clr regulon in S. meliloti.

Interkingdom Signaling of the Insect Pathogen Photorhabdus luminescens with Plants Via the LuxR solo SdiA.

In bacteria, group-coordinated behavior such as biofilm formation or virulence are often mediated via cell-cell communication, a process referred to as quorum sensing (QS). The canonical QS system of Gram-negative bacteria uses N-acyl homoserine lactones (AHLs) as communication molecules, which are produced by LuxI-type synthases and sensed by cognate LuxR-type receptors. These receptors act as transcriptional regulators controlling the expression of specific genes. Some bacteria harbor LuxR-type receptors lacking a cognate LuxI-type synthases, designated as LuxR solos. Among many other LuxR solos, the entomopathogenic enteric bacterium Photorhabdus luminescens harbors a SdiA-like LuxR solo containing an AHL signal-binding domain, for which a respective signal molecule and target genes have not been identified yet. Here we performed SPR analysis to demonstrate that SdiA acts as a bidirectional regulator of transcription, tightly controlling its own expression and the adjacent PluDJC_01670 (aidA) gene in P. luminescens, a gene supposed to be involved in the colonization of eukaryotes. Via qPCR we could further determine that in sdiA deletion mutant strains, aidA is upregulated, indicating that SdiA negatively affects expression of aidA. Furthermore, the ΔsdiA deletion mutant exhibited differences in biofilm formation and motility compared with the wild-type. Finally, using nanoDSF analysis we could identify putative binding ability of SdiA towards diverse AHLs, but also to plant-derived signals, modulating the DNA-binding capacity of SdiA, suggesting that this LuxR solo acts as an important player in interkingdom signaling between P. luminescens and plants.

Communication Breakdown: Into the Molecular Mechanism of Biofilm Inhibition by CeO2 Nanocrystal Enzyme Mimics and How It Can Be Exploited.

Bacterial biofilm formation is a huge problem in industry and medicine. Therefore, the discovery of anti-biofilm agents may hold great promise. Biofilm formation is usually a consequence of bacterial cell-cell communication, a process called quorum sensing (QS). CeO2 nanocrystals (NCs) have been established as haloperoxidase (HPO) mimics and ecologically beneficial biofilm inhibitors. They were suggested to interfere with QS, a mechanism termed quorum quenching (QQ), but their molecular mechanism remained elusive. We show that CeO2 NCs are effective QQ agents, inactivating QS signals by bromination. Catalytic bromination of 3-oxo-C12-AHL a QS signaling compound used by Pseudomonas aeruginosa, was detected in the presence of CeO2 NCs, bromide ions, and hydrogen peroxide. Brominated acyl-homoserine lactones (AHLs) no longer act as QS signals but were not detected in the bacterial cultures. Externally added brominated AHLs also disappeared in P. aeruginosa cultures within minutes of their addition, indicating that they are rapidly degraded by the bacteria. Moreover, we detected the catalytic bromination of 2-heptyl-1-hydroxyquinolin-4(1H)-one (HQNO), a multifunctional non-AHL QS signal from P. aeruginosa with antibacterial and algicidal properties controlling the expression of many virulence genes. Brominated HQNO was not degraded by the bacteria in vivo. The repression of the Pseudomonas quinolone signal (PQS) production and biofilm formation in P. aeruginosa through the catalytic formation of Br-HQNO on surfaces with coatings containing CeO2 enzyme mimics validates the non-toxic strategy for the development of anti-infectives.

The Insect Pathogen Photorhabdus luminescens Protects Plants from Phytopathogenic Fusarium graminearum via Chitin Degradation.

Phytopathogens represent a large agricultural challenge. The use of chemical pesticides is harmful to the environment, animals, and humans. Therefore, new sustainable and biological alternatives are urgently needed. The insect-pathogenic bacterium Photorhabdus luminescens, already used in combination with entomopathogenic nematodes (EPNs) as a biocontrol agent, is characterized by two different phenotypic cell forms, called primary (1°) and secondary (2°). The 1° cells are symbiotic with EPNs and are used for biocontrol, and the 2° cells are unable to undergo symbiosis with EPNs, remain in the soil after insect infection, and specifically interact with plant roots. A previous RNA sequencing (RNAseq) analysis showed that genes encoding the exochitinase Chi2A and chitin binding protein (CBP) are highly upregulated in 2° cells exposed to plant root exudates. Here, we investigate Chi2A and CBP functions and demonstrate that both are necessary for P. luminescens 2° cells to inhibit the growth of the phytopathogenic fungus Fusarium graminearum. We provide evidence that Chi2A digests chitin and thereby inhibits fungal growth. Furthermore, we show that 2° cells specifically colonize fungal hyphae as one of the first mechanisms to protect plants from fungal phytopathogens. Finally, soil pot bioassays proved plant protection from F. graminearum by 2° cells, where Chi2A and CPB were essential for this process. This work gives molecular insights into the new applicability of P. luminescens as a plant-growth-promoting and plant-protecting organism in agriculture. IMPORTANCE The enteric enterobacterium Photorhabdus luminescens is already being used as a bioinsecticide since it is highly pathogenic toward a broad range of insects. However, the bacteria exist in two phenotypically different cell types, called 1° and 2° cells. Whereas only 1° cells are symbiotic with their nematode partner to infect insects, 2° cells were shown to remain in the soil after an insect infection cycle. It was demonstrated that 2° cells specifically interact with plant roots. Here, we show that the bacteria are beneficial for the plants by protecting them from phytopathogenic fungi. Specific colonization of the fungus mycelium as well as chitin-degrading activity mediated by the chitin binding protein (CBP) and the chitinase Chi2A are essential for this process. Our data give evidence for the novel future applicability of P. luminescens as a plant-growth-promoting organism and biopesticide.

Identification of Pseudomonas asiatica subsp. bavariensis str. JM1 as the first Nε -carboxy(m)ethyllysine-degrading soil bacterium.

Thermal food processing leads to the formation of advanced glycation end products (AGE) such as Nε -carboxymethyllysine (CML). Accordingly, these non-canonical amino acids are an important part of the human diet. However, CML is only partially decomposed by our gut microbiota and up to 30% are excreted via faeces and, hence, enter the environment. In frame of this study, we isolated a soil bacterium that can grow on CML as well as its higher homologue Nε -carboxyethyllysine (CEL) as sole source of carbon. Bioinformatic analyses upon whole-genome sequencing revealed a subspecies of Pseudomonas asiatica, which we named 'bavariensis'. We performed a metabolite screening of P. asiatica subsp. bavariensis str. JM1 grown either on CML or CEL and identified N-carboxymethylaminopentanoic acid and N-carboxyethylaminopentanoic acid respectively. We further detected α-aminoadipate as intermediate in the metabolism of CML. These reaction products suggest two routes of degradation: While CEL seems to be predominantly processed from the α-C-atom, decomposition of CML can also be initiated with cleavage of the carboxymethyl group and under the release of acetate. Thus, our study provides novel insights into the metabolism of two important AGEs and how these are processed by environmental bacteria.

Defect-controlled halogenating properties of lanthanide-doped ceria nanozymes.

Marine organisms combat bacterial colonization by biohalogenation of signaling compounds that interfere with bacterial communication. These reactions are catalyzed by haloperoxidase enzymes, whose activity can be emulated by nanoceria using milli- and micromolar concentrations of Br- and H2O2. We show that the haloperoxidase-like activity of nanoceria can greatly be enhanced by Ln substitution in Ce1-xLnxO2-x/2. Non-agglomerated nanosized Ce1-xLnxO2-x/2 (Ln = Pr, Tb, particle size < 10 nm) was prepared mechanochemically from CeCl3 and Na2CO3 followed by short calcination. Lanthanide metals could be incorporated into the CeO2 host without solubility limit, as shown for Tb. The distribution of the Ln3+ defect sites in the CeO2 host structure was analyzed by electron spin resonance spectroscopy. Ce3+ and superoxide O2- species are present at surface sites. Their formation is promoted by increasing dopant concentration. Ce1-xLnxO2-x/2 was prepared in copious amounts by ball-milling. This energy-saving and residue-free method can be upscaled to industrial scale. The surface defect chemistry of Ce1-xLnxO2-x/2 was unravelled by vibrational spectroscopy. It is associated with the mechanochemical preparation and leads to enhanced catalytic activity. Although Ce0.9Pr0.1O1.95 had a lower BET surface area than pure CeO2, its catalytic activity, calibrated by oxidative bromination of phenol red, was much higher because the ζ-potential increased from 15 mV (for CeO2) to 30 mV (for Ce0.9Pr0.1O1.95). This facilitates adsorption of Br- in aqueous conditions and explains the high catalytic activity of the Ln-substituted CeO2. Ce1-xLnxO2-x/2 is an effective and "green" nanoparticle haloperoxidase mimic for antifouling applications, as no chemicals other than the ubiquitous Br- and H2O2 (generated in daylight) are required, and only natural metabolites are released into the environment.

High-throughput synthesis of CeO2 nanoparticles for transparent nanocomposites repelling Pseudomonas aeruginosa biofilms.

Preventing bacteria from adhering to material surfaces is an important technical problem and a major cause of infection. One of nature's defense strategies against bacterial colonization is based on the biohalogenation of signal substances that interfere with bacterial communication. Biohalogenation is catalyzed by haloperoxidases, a class of metal-dependent enzymes whose activity can be mimicked by ceria nanoparticles. Transparent CeO2/polycarbonate surfaces that prevent adhesion, proliferation, and spread of Pseudomonas aeruginosa PA14 were manufactured. Large amounts of monodisperse CeO2 nanoparticles were synthesized in segmented flow using a high-throughput microfluidic benchtop system using water/benzyl alcohol mixtures and oleylamine as capping agent. This reduced the reaction time for nanoceria by more than one order of magnitude compared to conventional batch methods. Ceria nanoparticles prepared by segmented flow showed high catalytic activity in halogenation reactions, which makes them highly efficient functional mimics of haloperoxidase enzymes. Haloperoxidases are used in nature by macroalgae to prevent formation of biofilms via halogenation of signaling compounds that interfere with bacterial cell-cell communication ("quorum sensing"). CeO2/polycarbonate nanocomposites were prepared by dip-coating plasma-treated polycarbonate panels in CeO2 dispersions. These showed a reduction in bacterial biofilm formation of up to 85% using P. aeruginosa PA14 as model organism. Besides biofilm formation, also the production of the virulence factor pyocyanin in is under control of the entire quorum sensing systems P. aeruginosa. CeO2/PC showed a decrease of up to 55% in pyocyanin production, whereas no effect on bacterial growth in liquid culture was observed. This indicates that CeO2 nanoparticles affect quorum sensing and inhibit biofilm formation in a non-biocidal manner.

Transparent polycarbonate coated with CeO2 nanozymes repel Pseudomonas aeruginosa PA14 biofilms.

Highly transparent CeO2/polycarbonate surfaces were fabricated that prevent adhesion, proliferation, and the spread of bacteria. CeO2 nanoparticles with diameters of 10-15 nm and lengths of 100-200 nm for this application were prepared by oxidizing aqueous dispersions of Ce(OH)3 with H2O2 in the presence of nitrilotriacetic acid (NTA) as the capping agent. The surface-functionalized water-dispersible CeO2 nanorods showed high catalytic activity in the halogenation reactions, which makes them highly efficient functional mimics of haloperoxidases. These enzymes are used in nature to prevent the formation of biofilms through the halogenation of signaling compounds that interfere with bacterial cell-cell communication ("quorum sensing"). Bacteria-repellent CeO2/polycarbonate plates were prepared by dip-coating plasma-treated polycarbonate plates in aqueous CeO2 particle dispersions. The quasi-enzymatic activity of the CeO2 coating was demonstrated using phenol red enzyme assays. The monolayer coating of CeO2 nanorods (1.6 µg cm-2) and the bacteria repellent properties were demonstrated by atomic force microscopy, biofilm assays, and fluorescence measurements. The engineered polymer surfaces have the ability to repel biofilms as green antimicrobials on plastics, where H2O2 is present in humid environments such as automotive parts, greenhouses, or plastic containers for rainwater.

Identification of Gip as a novel phage-encoded gyrase inhibitor protein of Corynebacterium glutamicum.

By targeting key regulatory hubs of their host, bacteriophages represent a powerful source for the identification of novel antimicrobial proteins. Here, a screening of small cytoplasmic proteins encoded by the CGP3 prophage of Corynebacterium glutamicum resulted in the identification of the gyrase-inhibiting protein Cg1978, termed Gip. Pull-down assays and surface plasmon resonance revealed a direct interaction of Gip with the gyrase subunit A (GyrA). The inhibitory activity of Gip was shown to be specific to the DNA gyrase of its bacterial host C. glutamicum. Overproduction of Gip in C. glutamicum resulted in a severe growth defect as well as an induction of the SOS response. Furthermore, reporter assays revealed an RecA-independent induction of the cryptic CGP3 prophage, most likely caused by topological alterations. Overexpression of gip was counteracted by an increased expression of gyrAB and a reduction of topA expression at the same time, reflecting the homeostatic control of DNA topology. We postulate that the prophage-encoded Gip protein plays a role in modulating gyrase activity to enable efficient phage DNA replication. A detailed elucidation of the mechanism of action will provide novel directions for the design of drugs targeting DNA gyrase.

Two novel XRE-like transcriptional regulators control phenotypic heterogeneity in Photorhabdus luminescens cell populations.

BACKGROUND: The insect pathogenic bacterium Photorhabdus luminescens exists in two phenotypically different forms, designated as primary (1°) and secondary (2°) cells. Upon yet unknown environmental stimuli up to 50% of the 1° cells convert to 2° cells. Among others, one important difference between the phenotypic forms is that 2° cells are unable to live in symbiosis with their partner nematodes, and therefore are not able to re-associate with them. As 100% switching of 1° to 2° cells of the population would lead to a break-down of the bacteria's life cycle the switching process must be tightly controlled. However, the regulation mechanism of phenotypic switching is still puzzling. RESULTS: Here we describe two novel XRE family transcriptional regulators, XreR1 and XreR2, that play a major role in the phenotypic switching process of P. luminescens. Deletion of xreR1 in 1° or xreR2 in 2° cells as well as insertion of extra copies of xreR1 into 2° or xreR2 into 1° cells, respectively, induced the opposite phenotype in either 1° or 2° cells. Furthermore, both regulators specifically bind to different promoter regions putatively fulfilling a positive autoregulation. We found initial evidence that XreR1 and XreR2 constitute an epigenetic switch, whereby XreR1 represses xreR2 expression and XreR2 self-reinforces its own gene by binding to XreR1. CONCLUSION: Regulation of gene expression by the two novel XRE-type regulators XreR1 and XreR2 as well as their interplay represents a major regulatory process in phenotypic switching of P. luminescens. A fine-tuning balance between both regulators might therefore define the fate of single cells to convert from the 1° to the 2° phenotype.

Transcriptional regulation of the Nε -fructoselysine metabolism in Escherichia coli by global and substrate-specific cues.

Thermally processed food is an important part of the human diet. Heat-treatment, however, promotes the formation of so-called Amadori rearrangement products, such as fructoselysine. The gut microbiota including Escherichia coli can utilize these compounds as a nutrient source. While the degradation route for fructoselysine is well described, regulation of the corresponding pathway genes frlABCD remained poorly understood. Here, we used bioinformatics combined with molecular and biochemical analyses and show that fructoselysine metabolism in E. coli is tightly controlled at the transcriptional level. The global regulator CRP (CAP) as well as the alternative sigma factor σ32 (RpoH) contribute to promoter activation at high cAMP-levels and inside warm-blooded hosts, respectively. In addition, we identified and characterized a transcriptional regulator FrlR, encoded adjacent to frlABCD, as fructoselysine-6-phosphate specific repressor. Our study provides profound evidence that the interplay of global and substrate-specific regulation is a perfect adaptation strategy to efficiently utilize unusual substrates within the human gut environment.

Nanocomposite antimicrobials prevent bacterial growth through the enzyme-like activity of Bi-doped cerium dioxide (Ce1-xBixO2-δ).

Preventing bacterial adhesion on materials surfaces is an important problem in marine, industrial, medical and environmental fields and a topic of major medical and societal importance. A defense strategy of marine organisms against bacterial colonization relies on the biohalogenation of signaling compounds that interfere with bacterial communication. These reactions are catalyzed by haloperoxidases, a class of metal-dependent enzymes, whose activity can be emulated by ceria nanoparticles. The enzyme-like activity of ceria was enhanced by a factor of 3 through bismuth substitution (Ce1-xBixO2-δ). The solubility of Bi3+ in CeO2 is confined to the range 0 < x < 0.25 under quasi-hydrothermal conditions. The Bi3+ cations are located close to the nanoparticle surface because their ionic radii are larger than those of the tetravalent Ce4+ ions. The synthesis of Ce1-xBixO2-δ (0 < x < 0.25) nanoparticles was upscaled to yields of ∼50 g. The halogenation activity of Ce1-xBixO2-δ was demonstrated with phenol red assays. The maximum activity for x ≈ 0.2 is related to the interplay of the ζ-potential of surface-engineered Ce1-xBixO2-δ nanoparticles and their BET surface area. Ce0.80Bi0.20O1.9 nanoparticles with optimized activity were incorporated in polyethersulfone beads, which are typical constituents of water filter membrane supports. Although Ce1-xBixO2-δ nanoparticles are not bactericidal on their own, naked Ce1-xBixO2-δ nanoparticles and polyethersulfone/Ce1-xBixO2-δ nanocomposites showed a strongly reduced bacterial coverage. We attribute the decreased adhesion of the Gram-negative soil bacterium Pseudomonas aeruginosa and of Phaeobacter gallaeciensis, a primary bacterial colonizer in marine biofilms, to the formation of halogenated signaling compounds. No biocides are needed, H2O2 (formed in daylight) and halide are the only substrates required. The haloperoxidase-like activity of Ce1-xBixO2-δ may be a promising starting point for the development of environmentally friendly, "green" nanocomposites, when the use of conventional biocides is prohibited.

The great potential of entomopathogenic bacteria Xenorhabdus and Photorhabdus for mosquito control: a review.

The control of insects of medical importance, such as Aedes aegypti and Aedes albopictus are still the only effective way to prevent the transmission of diseases, such as dengue, chikungunya and Zika. Their control is performed mainly using chemical products; however, they often have low specificity to non-target organisms, including humans. Also, studies have reported resistance to the most commonly used insecticides, such as the organophosphate and pyrethroids. Biological control is an ecological and sustainable method since it has a slow rate of insect resistance development. Bacterial species of the genera Xenorhabdus and Photorhabdus have been the target of several research groups worldwide, aiming at their use in agricultural, pharmaceutical and industrial products. This review highlights articles referring to the use of Xenorhabdus and Photorhabdus for insects and especially for mosquito control proposing future ways for their biotechnological applicability. Approximately 24 species of Xenorhabdus and five species of Photorhabdus have been described to have insecticidal properties. These studies have shown genes that are capable of encoding low molecular weight proteins, secondary toxin complexes and metabolites with insecticide activities, as well as antibiotic, fungicidal and antiparasitic molecules. In addition, several species of Xenorhabdus and Photorhabdus showed insecticidal properties against mosquitoes. Therefore, these biological agents can be used in new control methods, and must be, urgently considered in short term, in studies and applications, especially in mosquito control.

The Biocontrol Agent and Insect Pathogen Photorhabdus luminescens Interacts with Plant Roots.

The number of sustainable agriculture techniques to improve pest management and environmental safety is rising, as biological control agents are used to enhance disease resistance and abiotic stress tolerance in crops. Here, we investigated the capacity of the Photorhabdus luminescens secondary variant to react to plant root exudates and their behavior toward microorganisms in the rhizosphere. P. luminescens is known to live in symbiosis with entomopathogenic nematodes (EPNs) and to be highly pathogenic toward insects. The P. luminescens-EPN relationship has been widely studied, and this combination has been used as a biological control agent; however, not much attention has been paid to the putative lifestyle of P. luminescens in the rhizosphere. We performed transcriptome analysis to show how P. luminescens responds to plant root exudates. The analysis highlighted genes involved in chitin degradation, biofilm regulation, formation of flagella, and type VI secretion system. Furthermore, we provide evidence that P. luminescens can inhibit growth of phytopathogenic fungi. Finally, we demonstrated a specific interaction of P. luminescens with plant roots. Understanding the role and the function of this bacterium in the rhizosphere might accelerate the progress in biocontrol manipulation and elucidate the peculiar mechanisms adopted by plant growth-promoting rhizobacteria in plant root interactions.IMPORTANCE Insect-pathogenic Photorhabdus luminescens bacteria are widely used in biocontrol strategies against pests. Very little is known about the life of these bacteria in the rhizosphere. Here, we show that P. luminescens can specifically react to and interact with plant roots. Understanding the adaptation of P. luminescens in the rhizosphere is highly important for the biotechnological application of entomopathogenic bacteria and could improve future sustainable pest management in agriculture.

Small RNA-binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli.

The RNA-binding protein RapZ cooperates with small RNAs (sRNAs) GlmY and GlmZ to regulate the glmS mRNA in Escherichia coli. Enzyme GlmS synthesizes glucosamine-6-phosphate (GlcN6P), initiating cell envelope biosynthesis. GlmZ activates glmS expression by base-pairing. When GlcN6P is ample, GlmZ is bound by RapZ and degraded through ribonuclease recruitment. Upon GlcN6P depletion, the decoy sRNA GlmY accumulates through a previously unknown mechanism and sequesters RapZ, suppressing GlmZ decay. This circuit ensures GlcN6P homeostasis and thereby envelope integrity. In this work, we identify RapZ as GlcN6P receptor. GlcN6P-free RapZ stimulates phosphorylation of the two-component system QseE/QseF by interaction, which in turn activates glmY expression. Elevated GlmY levels sequester RapZ into stable complexes, which prevents GlmZ decay, promoting glmS expression. Binding of GlmY also prevents RapZ from activating QseE/QseF, generating a negative feedback loop limiting the response. When GlcN6P is replenished, GlmY is released from RapZ and rapidly degraded. We reveal a multifunctional sRNA-binding protein that dynamically engages into higher-order complexes for metabolite signaling.

Deciphering the Rules Underlying Xenogeneic Silencing and Counter-Silencing of Lsr2-like Proteins Using CgpS of Corynebacterium glutamicum as a Model.

Lsr2-like nucleoid-associated proteins play an important role as xenogeneic silencers (XS) of horizontally acquired genomic regions in actinobacteria. In this study, we systematically analyzed the in vivo constraints underlying silencing and counter-silencing of the Lsr2-like protein CgpS in Corynebacterium glutamicum Genome-wide analysis revealed binding of CgpS to regions featuring a distinct drop in GC profile close to the transcription start site (TSS) but also identified an overrepresented motif with multiple A/T steps at the nucleation site of the nucleoprotein complex. Binding of specific transcription factors (TFs) may oppose XS activity, leading to counter-silencing. Following a synthetic counter-silencing approach, target gene activation was realized by inserting operator sites of an effector-responsive TF within various CgpS target promoters, resulting in increased promoter activity upon TF binding. Analysis of reporter constructs revealed maximal counter-silencing when the TF operator site was inserted at the position of maximal CgpS coverage. This principle was implemented in a synthetic toggle switch, which features a robust and reversible response to effector availability, highlighting the potential for biotechnological applications. Together, our results provide comprehensive insights into how Lsr2 silencing and counter-silencing shape evolutionary network expansion in this medically and biotechnologically relevant bacterial phylum.IMPORTANCE In actinobacteria, Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XS) of horizontally acquired genomic regions, including viral elements, virulence gene clusters in Mycobacterium tuberculosis, and genes involved in cryptic specialized metabolism in Streptomyces species. Consequently, a detailed mechanistic understanding of Lsr2 binding in vivo is relevant as a potential drug target and for the identification of novel bioactive compounds. Here, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and counter-silencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum Our results demonstrated that CgpS distinguishes between self and foreign by recognizing a distinct drop in GC profile in combination with a short, sequence-specific motif at the nucleation site. Following a synthetic counter-silencer approach, we studied the potential and constraints of transcription factors to counteract CgpS silencing, thereby facilitating the integration of new genetic traits into host regulatory networks.

New Vocabulary for Bacterial Communication.

Quorum sensing (QS) is widely accepted as a procedure that bacteria use to converse. However, prevailing thinking places acyl homoserine lactones (AHLs) at the forefront of this communication pathway in Gram-negative bacteria. With the advent of high-throughput genomics and the subsequent influx of bacterial genomes, bioinformatics analysis has determined that the genes encoding AHL biosynthesis, originally discovered to be indispensable for QS (LuxI-like proteins and homologues), are often absent in QS-capable bacteria. Instead, the sensing protein (LuxR-like proteins) is present with an apparent inability to produce any outgoing AHL signal. Recently, several signals for these LuxR solos have been identified. Herein, advances in the field of QS are discussed, with a particular focus on recent research in the field of bacterial cell-cell communication.

Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing.

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.