Structural basis and synergism of ATP and Na+ activation in bacterial K+ uptake system KtrAB.

The K+ uptake system KtrAB is essential for bacterial survival in low K+ environments. The activity of KtrAB is regulated by nucleotides and Na+. Previous studies proposed a putative gating mechanism of KtrB regulated by KtrA upon binding to ATP or ADP. However, how Na+ activates KtrAB and the Na+ binding site remain unknown. Here we present the cryo-EM structures of ATP- and ADP-bound KtrAB from Bacillus subtilis (BsKtrAB) both solved at 2.8 Å. A cryo-EM density at the intra-dimer interface of ATP-KtrA was identified as Na+, as supported by X-ray crystallography and ICP-MS. Thermostability assays and functional studies demonstrated that Na+ binding stabilizes the ATP-bound BsKtrAB complex and enhances its K+ flux activity. Comparing ATP- and ADP-BsKtrAB structures suggests that BsKtrB Arg417 and Phe91 serve as a channel gate. The synergism of ATP and Na+ in activating BsKtrAB is likely applicable to Na+-activated K+ channels in central nervous system.

Combining hybrid nanoflowers with hybridization chain reaction for highly sensitive detection of SARS-CoV-2 nucleocapsid protein.

BACKGROUND: COVID-19 (coronavirus disease 2019) pandemic has had enormous social and economic impacts so far. The nucleocapsid protein (N protein) is highly conserved and is a key antigenic marker for the diagnosis of early SARS-CoV-2 infection. RESULTS: In this study, the N protein was first captured by an aptamer (Aptamer 58) coupled to magnetic beads (MBs), which in turn were bound to another DNA sequence containing the aptamer (Aptamer 48-Initiator). After adding 5'-biotinylated hairpin DNA Amplifier 1 and Amplifier 2 with cohesive ends for complementary hybridization, the Initiator in the Aptamer 48-Initiator began to trigger the hybridization chain reaction (HCR), generating multiple biotin-labeled DNA concatamers. When incubated with synthetic streptavidin-invertase-Ca3(PO4)2 hybrid nanoflower (SICa), DNA concatamers could specifically bind to SICa through biotin-streptavidin interaction with high affinity. After adding sucrose, invertase in SICa hydrolyzed sucrose to glucose, whose concentration could be directly read with a portable glucometer, and its concentration was positively correlated with the amount of captured N protein. The method is highly sensitive with a detection limit as low as 1 pg/mL. SIGNIFICANCE: We believe this study provided a practical solution for the early detection of SARS-CoV-2 infection, and offered a new method for detecting other viruses through different target proteins.

Gas and light: triggers of c-di-GMP-mediated regulation.

The widespread bacterial second messenger c-di-GMP is responsible for regulating many important physiological functions such as biofilm formation, motility, cell differentiation, and virulence. The synthesis and degradation of c-di-GMP in bacterial cells depend, respectively, on diguanylate cyclases and c-di-GMP-specific phosphodiesterases. Since c-di-GMP metabolic enzymes (CMEs) are often fused to sensory domains, their activities are likely controlled by environmental signals, thereby altering cellular c-di-GMP levels and regulating bacterial adaptive behaviors. Previous studies on c-di-GMP-mediated regulation mainly focused on downstream signaling pathways, including the identification of CMEs, cellular c-di-GMP receptors, and c-di-GMP-regulated processes. The mechanisms of CME regulation by upstream signaling modules received less attention, resulting in a limited understanding of the c-di-GMP regulatory networks. We review here the diversity of sensory domains related to bacterial CME regulation. We specifically discuss those domains that are capable of sensing gaseous or light signals and the mechanisms they use for regulating cellular c-di-GMP levels. It is hoped that this review would help refine the complete c-di-GMP regulatory networks and improve our understanding of bacterial behaviors in changing environments. In practical terms, this may eventually provide a way to control c-di-GMP-mediated bacterial biofilm formation and pathogenesis in general.

In silico design of a promiscuous chimeric multi-epitope vaccine against Mycobacterium tuberculosis.

Tuberculosis (TB) is a global health threat, killing approximately 1.5 million people each year. The eradication of Mycobacterium tuberculosis, the main causative agent of TB, is increasingly challenging due to the emergence of extensive drug-resistant strains. Vaccination is considered an effective way to protect the host from pathogens, but the only clinically approved TB vaccine, Bacillus Calmette-Guérin (BCG), has limited protection in adults. Multi-epitope vaccines have been found to enhance immunity to diseases by selectively combining epitopes from several candidate proteins. This study aimed to design a multi-epitope vaccine against TB using an immuno-informatics approach. Through functional enrichment, we identified eight proteins secreted by M. tuberculosis that are either required for pathogenesis, secreted into extracellular space, or both. We then analyzed the epitopes of these proteins and selected 16 helper T lymphocyte epitopes with interferon-γ inducing activity, 15 cytotoxic T lymphocyte epitopes, and 10 linear B-cell epitopes, and conjugated them with adjuvant and Pan HLA DR-binding epitope (PADRE) using appropriate linkers. Moreover, we predicted the tertiary structure of this vaccine, its potential interaction with Toll-Like Receptor-4 (TLR4), and the immune response it might elicit. The results showed that this vaccine had a strong affinity for TLR4, which could significantly stimulate CD4+ and CD8+ cells to secrete immune factors and B lymphocytes to secrete immunoglobulins, so as to obtain good humoral and cellular immunity. Overall, this multi-epitope protein was predicted to be stable, safe, highly antigenic, and highly immunogenic, which has the potential to serve as a global vaccine against TB.

Influence of neo-adjuvant radiotherapy on the intestinal microbiota of rectal cancer patients.

PURPOSE: Neo-adjuvant radiotherapy (NART) is a widely used pre-surgery radiotherapy for rectal cancer patients. Although NART is effective in reducing tumor burden before surgery, it may cause dysbiosis of intestinal microbiota. The intestinal microbiota shapes tumor inflammatory environment and influences cancer progression. However, how NART remodels the microbiota and how the microbiota affects therapeutic efficacy has been largely elusive. This study aimed to reveal the details of how NART affects the intestinal microbiota in patients with rectal cancer. METHODS: Rectal cancer patients who received NART were recruited into the study, and their healthy family members on the same diet served as controls. Stool samples from five rectal cancer patients (28 in total) and five healthy individuals (16 in total) were collected for intestinal microbiota analysis by 16S rRNA gene amplicon sequencing. Samples from patients were divided into earlier- and later-NART according to the number of NART. RESULTS: NART did not significantly affect the α diversity of intestinal microbiota. However, the abundance of bacterial genera associated with cancer progression tended to decrease in later-NART patients. More importantly, a variety of oral pathogenic bacteria were enriched in the intestine of later-NART patients. NART also affected functional pathways associated with the microbiota in DNA repair, metabolism, and bacterial infection. CONCLUSION: NART significantly altered the microbiota composition and function in rectal cancer patients, and some oral pathogens were found to translocate to the intestine. This is the first report to study the effect of NART on intestinal microbiota in patients with rectal cancer, exploring the importance of intestinal microbiota during the process of NART.

Systematic Comparison and Rational Design of Theophylline Riboswitches for Effective Gene Repression.

Riboswitches are promising regulatory tools in synthetic biology. To date, 25 theophylline riboswitches have been developed for regulation of gene expression in bacteria. However, no one has systematically evaluated their regulatory effects. To promote efficient selection and application of theophylline riboswitches, we examined 25 theophylline riboswitches in Escherichia coli MG1655 and found that they varied widely in terms of activation/repression ratios and expression levels in the absence of theophylline. Of the 20 riboswitches that activate gene expression, only one exhibited a high activation ratio (63.6-fold) and low expression level without theophylline. Furthermore, none of the five riboswitches that repress gene expression were more than 2.0-fold efficient. To obtain an effective repression system, we rationally designed a novel theophylline riboswitch to control a downstream gene or genes by premature transcription termination. This riboswitch allowed theophylline-dependent downregulation of the TurboRFP reporter in a dose- and time-dependent manner. Its performance profile exceeded those of previously described repressive theophylline riboswitches. We then introduced as the second part a RepA tag (protein degradation tag) coding sequence fused at the 5'-terminal end of the turborfp gene, which further reduced protein level, while not reducing the repressive effect of the riboswitch. By combining two tandem theophylline riboswitches with a RepA tag, we constructed a regulatory cassette that represses the expression of the gene(s) of interest at both the transcriptional and posttranslational levels. This regulatory cassette can be used to repress the expression of any gene of interest and represents a crucial step toward harnessing theophylline riboswitches and expanding the synthetic biology toolbox. IMPORTANCE A variety of gene expression regulation tools with significant regulatory effects are essential for the construction of complex gene circuits in synthetic biology. Riboswitches have received wide attention due to their unique biochemical, structural, and genetic properties. Here, we have not only systematically and precisely characterized the regulatory properties of previously developed theophylline riboswitches but also engineered a novel repressive theophylline riboswitch acting at the transcriptional level. By introducing coding sequences of a tandem riboswitch and a RepA protein degradation tag at the 5' end of the reporter gene, we successfully constructed a simple and effective regulatory cassette for gene regulation. Our work provides useful biological components for the construction of synthetic biology gene circuits.

Staggered intercalation of DNA duplexes with base-pair modulation by two distinct drug molecules induces asymmetric backbone twisting and structure polymorphism.

The use of multiple drugs simultaneously targeting DNA is a promising strategy in cancer therapy for potentially overcoming single drug resistance. In support of this concept, we report that a combination of actinomycin D (ActD) and echinomycin (Echi), can interact in novel ways with native and mismatched DNA sequences, distinct from the structural effects produced by either drug alone. Changes in the former with GpC and CpG steps separated by a A:G or G:A mismatch or in a native DNA with canonical G:C and C:G base pairs, result in significant asymmetric backbone twists through staggered intercalation and base pair modulations. A wobble or Watson-Crick base pair at the two drug-binding interfaces can result in a single-stranded 'chair-shaped' DNA duplex with a straight helical axis. However, a novel sugar-edged hydrogen bonding geometry in the G:A mismatch leads to a 'curved-shaped' duplex. Two non-canonical G:C Hoogsteen base pairings produce a sharply kinked duplex in different forms and a four-way junction-like superstructure, respectively. Therefore, single base pair modulations on the two drug-binding interfaces could significantly affect global DNA structure. These structures thus provide a rationale for atypical DNA recognition via multiple DNA intercalators and a structural basis for the drugs' potential synergetic use.

The RNA Chaperone Protein Hfq Regulates the Characteristic Sporulation and Insecticidal Activity of Bacillus thuringiensis.

Bacillus thuringiensis (Bt) is one of the most widely used bio-insecticides at present. It can produce many virulence factors and insecticidal crystal proteins during growth and sporulation. Hfq, on the other hand, is a bacterial RNA chaperone that can regulate the function of different kinds of RNAs, thereby affecting various bacterial phenotypes. To further explore the physiological functions of Hfq in Bt, we took BMB171 as the starting strain, knocked out one, two, or three hfq genes in its genome in different combinations, and compared the phenotypic differences between the deletion mutant strains and the starting strain. We did observe significant changes in several phenotypes, including motility, biofilm formation, sporulation, and insecticidal activity against cotton bollworm, among others. Afterward, we found through transcriptome studies that when all hfq genes were deleted, 32.5% of the genes in Bt were differentially transcribed, with particular changes in the sporulation-related and virulence-related genes. The above data demonstrated that Hfq plays a pivotal role in Bt and can regulate its various physiological functions. Our study on the regulatory mechanism of Hfq in Bt, especially the mining of the regulatory network of its sporulation and insecticidal activity, could lay a theoretical foundation for the better utilization of Bt as an effective insecticide.

Clp protease and antisense RNA jointly regulate the global regulator CarD to mediate mycobacterial starvation response.

Under starvation conditions, bacteria tend to slow down their translation rate by reducing rRNA synthesis, but the way they accomplish that may vary in different bacteria. In Mycobacterium species, transcription of rRNA is activated by the RNA polymerase (RNAP) accessory transcription factor CarD, which interacts directly with RNAP to stabilize the RNAP-promoter open complex formed on rRNA genes. The functions of CarD have been extensively studied, but the mechanisms that control its expression remain obscure. Here, we report that the level of CarD was tightly regulated when mycobacterial cells switched from nutrient-rich to nutrient-deprived conditions. At the translational level, an antisense RNA of carD (AscarD) was induced in a SigF-dependent manner to bind with carD mRNA and inhibit CarD translation, while at the post-translational level, the residual intracellular CarD was quickly degraded by the Clp protease. AscarD thus worked synergistically with Clp protease to decrease the CarD level to help mycobacterial cells cope with the nutritional stress. Altogether, our work elucidates the regulation mode of CarD and delineates a new mechanism for the mycobacterial starvation response, which is important for the adaptation and persistence of mycobacterial pathogens in the host environment.

A 'time bomb' in the human intestine-the multiple emergence and spread of antibiotic-resistant bacteria.

Antibiotics have a strong killing effect on bacteria and are the first choice for the prevention and treatment of bacterial infectious diseases. Therefore, they have been widely used in the medical field, animal husbandry and planting industry. However, with the massive use of antibiotics, more and more antibiotic-resistant bacteria (ARB) have emerged. Because human intestines are rich in nutrients, have suitable temperature, and are high in bacterial abundance, they can easily become a hotbed for the spread of ARB and antibiotic-resistant genes (ARGs). When opportunistic pathogenic bacteria in the intestine acquire ARGs, the infectious diseases caused by such opportunistic pathogens will become more difficult to treat, or even impossible to cure. Therefore, ARB in the human intestine are like a 'time bomb'. In this review, we discuss the sources of intestinal ARB and the transmission routes of ARGs in the human intestine from the perspective of One Health. Further, we describe various methods to prevent the emergence of ARB and inhibit the spread of ARGs in the human intestine. Finally, we may be able to overcome ARB in the human intestine using an interdisciplinary 'One Health' approach.

The spatial position effect: synthetic biology enters the era of 3D genomics.

Microbial cell factories are critical to achieving green biomanufacturing. A position effect occurs when a synthetic gene circuit is expressed from different positions in the chassis strain genome. Here, we propose the concept of the 'spatial position effect,' which uses technologies in 3D genomics to reveal the spatial structure characteristics of the 3D genome of the chassis. On this basis, we propose to rationally design the integration sites of synthetic gene circuits, use reporter genes for preliminary screening, and integrate synthetic gene circuits into promising sites for further experiments. This approach can produce stable and efficient chassis strains for green biomanufacturing. The proposed spatial position effect brings synthetic biology into the era of 3D genomics.

Sequence Conservation, Domain Architectures, and Phylogenetic Distribution of the HD-GYP Type c-di-GMP Phosphodiesterases.

The HD-GYP domain, named after two of its conserved sequence motifs, was first described in 1999 as a specialized version of the widespread HD phosphohydrolase domain that had additional highly conserved amino acid residues. Domain associations of HD-GYP indicated its involvement in bacterial signal transduction and distribution patterns of this domain suggested that it could serve as a hydrolase of the bacterial second messenger c-di-GMP, in addition to or instead of the EAL domain. Subsequent studies confirmed the ability of various HD-GYP domains to hydrolyze c-di-GMP to linear pGpG and/or GMP. Certain HD-GYP-containing proteins hydrolyze another second messenger, cGAMP, and some HD-GYP domains participate in regulatory protein-protein interactions. The recently solved structures of HD-GYP domains from four distinct organisms clarified the mechanisms of c-di-GMP binding and metal-assisted hydrolysis. However, the HD-GYP domain is poorly represented in public domain databases, which causes certain confusion about its phylogenetic distribution, functions, and domain architectures. Here, we present a refined sequence model for the HD-GYP domain and describe the roles of its most conserved residues in metal and/or substrate binding. We also calculate the numbers of HD-GYPs encoded in various genomes and list the most common domain combinations involving HD-GYP, such as the RpfG (REC-HD-GYP), Bd1817 (DUF3391-HD-GYP), and PmGH (GAF-HD-GYP) protein families. We also provide the descriptions of six HD-GYP-associated domains, including four novel integral membrane sensor domains. This work is expected to stimulate studies of diverse HD-GYP-containing proteins, their N-terminal sensor domains and the signals to which they respond.

Bacterial quorum sensing quenching activity of Lysobacter leucyl aminopeptidase acts by interacting with autoinducer synthase.

Acyl-homoserine lactone (AHL) is the most studied autoinducer in gram-negative bacteria controlling infections of various pathogens. Quenching of AHL signaling by inhibiting AHL synthesis or AHL-receptor binding via small molecular chemicals or enzymatically degrading AHL is commonly used to block bacterial infections. Here, we describe a new quorum-quenching strategy that directly "acquires" bacterial genes/proteins through a defined platform. We artificially expressed a typical AHL synthase gene pcoI from the biocontrol Pseudomonas fluorescens 2P24 in the antifungal bacterium Lysobacter enzymogenes OH11 lacking AHL production. This step led to the discovery of multiple PcoI interacting protein candidates from L. enzymogenes. The individual expression of these candidate genes in 2P24 led to the identification of Le0959, which encodes leucyl aminopeptidase, an effective protein that inhibits AHL synthesis in 2P24. Therefore, we define Le0959 as LqqP (Lysobacterquorum-quenching protein). The expression of pcoI in E. coli could produce AHL, and the introduction of lqqP into E. coli expressing pcoI could prevent the production of AHL. LqqP directly binds to PcoI, and this protein-protein binding reduced the abundance of free PcoI (capable of AHL synthesis) in vivo, thereby blocking PcoI-dependent AHL production. Overall, this study highlights the discovery of LqqP in quenching AHL quorum sensing by binding to AHL synthase via developing a previously-uncharacterized screening technique for bacterial quorum quenching.

The Multiple Regulatory Relationship Between RNA-Chaperone Hfq and the Second Messenger c-di-GMP.

RNA chaperone protein Hfq is an important post-transcriptional regulator in bacteria, while c-di-GMP is a second messenger signaling molecule widely distributed in bacteria. Both factors have been found to play key roles in post-transcriptional regulation and signal transduction pathways, respectively. Intriguingly, the two factors show some common aspects in the regulation of certain physiological functions such as bacterial motility, biofilm formation, pathogenicity and so on. Therefore, there may be regulatory relationship between Hfq and c-di-GMP. For example, Hfq can directly regulate the activity of c-di-GMP metabolic enzymes or alter the c-di-GMP level through other systems, while c-di-GMP can indirectly enhance or inhibit the hfq gene expression through intermediate factors. In this article, after briefly introducing the Hfq and c-di-GMP regulatory systems, we will focus on the direct and indirect regulation reported between Hfq and c-di-GMP, aiming to compare and link the two regulatory systems to further study the complicated physiological and metabolic systems of bacteria, and to lay a solid foundation for drawing a more complete global regulatory network.

Lysobacter enzymogenes antagonizes soilborne bacteria using the type IV secretion system.

Soil microbiome comprises numerous microbial species that continuously interact with each other. Among the modes of diverse interactions, cell-cell killing may play a key role in shaping the microbiome composition. Bacteria deploy various secretion systems to fend off other microorganisms and Type IV Secretion System (T4SS) in pathogenic bacteria was shown to function as a contact-dependent, inter-bacterial killing system only recently. The present study investigated the role played by T4SS in the killing behaviour of the soilborne biocontrol bacterium Lysobacter enzymogenes OH11. Results showed that L. enzymogenes OH11 genome encompasses genes encoding all the components of T4SS and effectors potentially involved in inter-bacterial killing system. Generation of knock-out mutants revealed that L. enzymogenes OH11 uses T4SS as the main contact-dependent weapon against other soilborne bacteria. The T4SS-mediated killing behaviour of L. enzymogenes OH11 decreased the antibacterial and antifungal activity of two Pseudomonas spp. but at the same time, protected carrot from infection by Pectobacterium carotovorum. Overall, this study showed for the first time the involvement of T4SS in the killing behaviour of L. enzymogenes and its impact on the multiple interactions occurring in the soil microbiome.

Clp is a "busy" transcription factor in the bacterial warrior, Lysobacter enzymogenes.

Cyclic AMP receptor protein (CRP) is a well-characterized group of global transcription factors in bacteria. They are known to regulate numerous cellular processes by binding DNA and/or cAMP (a ligand called bacterial second messenger) to control target gene expression. Gram-negative Lysobacter enzymogenes is a soilborne, plant-beneficial bacterium without flagella that can fight against filamentous fungi and oomycete. Driven by the type IV pilus (T4P) system, this bacterium moves to nearby pathogens and uses a "mobile-attack" antifungal strategy to kill them via heat-stable antifungal factor (HSAF) and abundant lyases. This strategy is controlled by a unique "busy" transcription factor Clp, which is a CRP-like protein that is inactivated by binding of c-di-GMP, another ubiquitous second messenger of bacteria. In this review, we summarize the current progress in how Clp initiates a "mobile-attack" strategy through a series of previously uncharacterized mechanisms, including binding to DNA in a unique pattern, directly interacting with or responding to various small molecules, and interacting specifically with proteins adopting distinct structure. Together, these characteristics highlight the multifunctional roles of Clp in L. enzymogenes, a powerful bacterial warrior against fungal pathogens.

Antifungal weapons of Lysobacter, a mighty biocontrol agent.

Bacteria interact with fungi in a variety of ways to inhibit fungal growth, while the underlying mechanisms remain only partially characterized. The plant-beneficial Bacillus and Pseudomonas species are well-known antifungal biocontrol agents, whereas Lysobacter are far less studied. Members of Lysobacter are easy to grow in fermenters and are safe to humans, animals and plants. These environmentally ubiquitous bacteria use a diverse arsenal of weapons to prey on other microorganisms, including fungi and oomycetes. The small molecular toxins secreted by Lysobacter represent long-range weapons effective against filamentous fungi. The secreted hydrolytic enzymes act as intermediate-range weapons against non-filamentous fungi. The contact-dependent killing devices are proposed to work as short-range weapons. We describe here the structure, biosynthetic pathway, action mode and applications of one of the best-characterized long-range weapons, the heat-stable antifungal factor (HSAF) produced by Lysobacter enzymogenes. We discuss how the flagellar type III secretion system has evolved into an enzyme secretion machine for the intermediate-range antifungal weapons. We highlight an intricate mechanism coordinating the production of the long-range weapon, HSAF and the proposed contact-dependent killing device, type VI secretion system. We also overview the regulatory mechanisms of HSAF production involving specific transcription factors and the bacterial second messenger c-di-GMP.

Which Is Stronger? A Continuing Battle Between Cry Toxins and Insects.

In this article, we review the latest works on the insecticidal mechanisms of Bacillus thuringiensis Cry toxins and the resistance mechanisms of insects against Cry toxins. Currently, there are two models of insecticidal mechanisms for Cry toxins, namely, the sequential binding model and the signaling pathway model. In the sequential binding model, Cry toxins are activated to bind to their cognate receptors in the mid-intestinal epithelial cell membrane, such as the glycophosphatidylinositol (GPI)-anchored aminopeptidases-N (APNs), alkaline phosphatases (ALPs), cadherins, and ABC transporters, to form pores that elicit cell lysis, while in the signaling pathway model, the activated Cry toxins first bind to the cadherin receptor, triggering an extensive cell signaling cascade to induce cell apoptosis. However, these two models cannot seem to fully describe the complexity of the insecticidal process of Cry toxins, and new models are required. Regarding the resistance mechanism against Cry toxins, the main method insects employed is to reduce the effective binding of Cry toxins to their cognate cell membrane receptors by gene mutations, or to reduce the expression levels of the corresponding receptors by trans-regulation. Moreover, the epigenetic mechanisms, host intestinal microbiota, and detoxification enzymes also play significant roles in the insects' resistance against Cry toxins. Today, high-throughput sequencing technologies like transcriptomics, proteomics, and metagenomics are powerful weapons for studying the insecticidal mechanisms of Cry toxins and the resistance mechanisms of insects. We believe that this review shall shed some light on the interactions between Cry toxins and insects, which can further facilitate the development and utilization of Cry toxins.

Coordinated control of the type IV pili and c-di-GMP-dependent antifungal antibiotic production in Lysobacter by the response regulator PilR.

In the soil gammaproteobacterium Lysobacter enzymogenes, a natural fungal predator, the response regulator PilR controls type IV pili (T4P)-mediated twitching motility as well as synthesis of the heat-stable antifungal factor (HSAF). Earlier we showed that PilR acts via the second messenger, c-di-GMP; however, the mechanism remained unknown. Here, we describe how PilR, c-di-GMP signalling, and HSAF synthesis are connected. We screened genes for putative diguanylate cyclases (c-di-GMP synthases) and found that PilR binds to the promoter region of lchD and down-regulates its transcription. The DNA-binding affinity of PilR, and therefore its repressor function, are enhanced by phosphorylation by its cognate histidine kinase, PilS. The lchD gene product is a diguanylate cyclase, and the decrease in LchD levels shifts the ratio of c-di-GMP-bound and c-di-GMP-free transcription factor Clp, a key activator of the HSAF biosynthesis operon expression. Furthermore, Clp directly interacts with LchD and enhances its diguanylate cyclase activity. Therefore, the PilS-PilR two-component system activates T4P-motility while simultaneously decreasing c-di-GMP levels and promoting HSAF production via the highly specific LchD-c-di-GMP-Clp pathway. Coordinated increase in motility and secretion of the "long-distance" antifungal weapon HSAF is expected to ensure safer grazing of L. enzymogenes on soil or plant surfaces, unimpeded by fungal competitors, or to facilitate bacterial preying on killed fungal cells. This study uncovered the mechanism of coregulated pili-based motility and production of an antifungal antibiotic in L. enzymogenes, showcased the expanded range of functions of the PilS-PilR system, and highlighted exquisite specificity in c-di-GMP-mediated circuits.

A non-flagellated, predatory soil bacterium reprograms a chemosensory system to control antifungal antibiotic production via cyclic di-GMP signalling.

Lysobacter enzymogenes is a non-flagellated, soil proteobacterium that secretes a diffusible antibiotic known as heat-stable antifungal factor (HSAF) to kill nearby fungi for food. The genome of the model strain OH11 encodes a homologous Wsp system, which is generally deployed by flagellated bacteria to achieve flagella-dependent outputs via a c-di-GMP-FleQ complex, in which c-di-GMP is a ubiquitous dinucleotide second messenger and FleQ is a transcription factor (TF). Here, we show that the Wsp system in the non-flagellated OH11 participates in a unique c-di-GMP-dependent signalling pathway and forms a WspR-CdgL binary complex to alter HSAF production, in which WspR and CdgL act as a c-di-GMP diguanylate cyclase (DGC) and a non-TF binding protein respectively. We found that the phosphorylation of WspR activates its DGC activity and enhances c-di-GMP production while inhibiting HSAF biosynthesis. The phosphorylation of WspR also plays a key role in weakening WspR-CdgL binding and HSAF generation. Interestingly, c-di-GMP binding to CdgL did not seem to induce the disassociation of the WspR-CdgL complex. These observations, along with our earlier findings, lead us to propose a model in which L. enzymogenes re-programs the Wsp system via c-di-GMP signalling to regulate HSAF biosynthesis for the benefit of ecological adaptation.