Nonlethal deleterious mutation-induced stress accelerates bacterial aging.

Random mutagenesis, including when it leads to loss of gene function, is a key mechanism enabling microorganisms' long-term adaptation to new environments. However, loss-of-function mutations are often deleterious, triggering, in turn, cellular stress and complex homeostatic stress responses, called "allostasis," to promote cell survival. Here, we characterize the differential impacts of 65 nonlethal, deleterious single-gene deletions on Escherichia coli growth in three different growth environments. Further assessments of select mutants, namely, those bearing single adenosine triphosphate (ATP) synthase subunit deletions, reveal that mutants display reorganized transcriptome profiles that reflect both the environment and the specific gene deletion. We also find that ATP synthase α-subunit deleted (ΔatpA) cells exhibit elevated metabolic rates while having slower growth compared to wild-type (wt) E. coli cells. At the single-cell level, compared to wt cells, individual ΔatpA cells display near normal proliferation profiles but enter a postreplicative state earlier and exhibit a distinct senescence phenotype. These results highlight the complex interplay between genomic diversity, adaptation, and stress response and uncover an "aging cost" to individual bacterial cells for maintaining population-level resilience to environmental and genetic stress; they also suggest potential bacteriostatic antibiotic targets and -as select human genetic diseases display highly similar phenotypes, - a bacterial origin of some human diseases.

The Catalysis Mechanism of E. coli Nitroreductase A, a Candidate for Gene-Directed Prodrug Therapy: Potentiometric and Substrate Specificity Studies.

E. coli nitroreductase A (NfsA) is a candidate for gene-directed prodrug cancer therapy using bioreductively activated nitroaromatic compounds (ArNO2). In this work, we determined the standard redox potential of FMN of NfsA to be -215 ± 5 mV at pH 7.0. FMN semiquinone was not formed during 5-deazaflavin-sensitized NfsA photoreduction. This determines the two-electron character of the reduction of ArNO2 and quinones (Q). In parallel, we characterized the oxidant specificity of NfsA with an emphasis on its structure. Except for negative outliers nitracrine and SN-36506, the reactivity of ArNO2 increases with their electron affinity (single-electron reduction potential, E17) and is unaffected by their lipophilicity and Van der Waals volume up to 386 Å. The reactivity of quinoidal oxidants is not clearly dependent on E17, but 2-hydroxy-1,4-naphthoquinones were identified as positive outliers and a number of compounds with diverse structures as negative outliers. 2-Hydroxy-1,4-naphthoquinones are characterized by the most positive reaction activation entropy and the negative outlier tetramethyl-1,4-benzoquinone by the most negative. Computer modelling data showed that the formation of H bonds with Arg15, Arg133, and Ser40, plays a major role in the binding of oxidants to reduced NfsA, while the role of the π-π interaction of their aromatic structures is less significant. Typically, the calculated hydride-transfer distances during ArNO2 reduction are smallwer than for Q. This explains the lower reactivity of quinones. Another factor that slows down the reduction is the presence of positively charged aliphatic substituents.

Regulation of Cysteine Homeostasis and Its Effect on Escherichia coli Sensitivity to Ciprofloxacin in LB Medium.

Cysteine and its derivatives, including H2S, can influence bacterial virulence and sensitivity to antibiotics. In minimal sulfate media, H2S is generated under stress to prevent excess cysteine and, together with incorporation into glutathione and export into the medium, is a mechanism of cysteine homeostasis. Here, we studied the features of cysteine homeostasis in LB medium, where the main source of sulfur is cystine, whose import can create excess cysteine inside cells. We used mutants in the mechanisms of cysteine homeostasis and a set of microbiological and biochemical methods, including the real-time monitoring of sulfide and oxygen, the determination of cysteine and glutathione (GSH), and the expression of the Fur, OxyR, and SOS regulons genes. During normal growth, the parental strain generated H2S when switching respiration to another substrate. The mutations affected the onset time, the intensity and duration of H2S production, cysteine and glutathione levels, bacterial growth and respiration rates, and the induction of defense systems. Exposure to chloramphenicol and high doses of ciprofloxacin increased cysteine content and GSH synthesis. A high inverse relationship between log CFU/mL and bacterial growth rate before ciprofloxacin addition was revealed. The study points to the important role of maintaining cysteine homeostasis during normal growth and antibiotic exposure in LB medium.

Plasmid partitioning driven by collective migration of ParA between nucleoid lobes.

The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy-number plasmids. However, despite extensive research, the spatiotemporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo have remained unclear. In this study, we utilize high-throughput imaging, quantitative data analysis, and computational modeling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid. As previously observed, we find that F-plasmid ParA undergoes collective migrations ("flips") between cell halves multiple times per cell cycle. We reveal that a constricting nucleoid is required for these migrations and that they are triggered by a plasmid crossing into the cell half with greater ParA. Using simulations, we show that these dynamics can be explained by the combination of nucleoid constriction and cooperative ParA binding to the DNA, in line with the behavior of other ParA proteins. We further show that these ParA flips act to equally partition plasmids between the two lobes of the constricted nucleoid and are therefore important for plasmid stability, especially in fast growth conditions for which the nucleoid constricts early in the cell cycle. Overall, our work identifies a second mode of action of the ParABS system and deepens our understanding of how this important segregation system functions.

Expression, purification and preliminary crystallographic analysis of bacterial transmembrane protein EfeU for iron import.

The bacterial Efe system functions as an importer of free Fe2+ into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from Escherichia coli, selected E. coli Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system.

Patchy and widespread distribution of bacterial translation arrest peptides associated with the protein localization machinery.

Regulatory arrest peptides interact with specific residues on bacterial ribosomes and arrest their own translation. Here, we analyse over 30,000 bacterial genome sequences to identify additional Sec/YidC-related arrest peptides, followed by in vivo and in vitro analyses. We find that Sec/YidC-related arrest peptides show patchy, but widespread, phylogenetic distribution throughout the bacterial domain. Several of the identified peptides contain distinct conserved sequences near the C-termini, but are still able to efficiently stall bacterial ribosomes in vitro and in vivo. In addition, we identify many arrest peptides that share an R-A-P-P-like sequence, suggesting that this sequence might serve as a common evolutionary seed to overcome ribosomal structural differences across species.

Nucleoside-diphosphate kinase of uropathogenic Escherichia coli inhibits caspase-1-dependent pyroptosis facilitating urinary tract infection.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infection (UTI). UPEC invades bladder epithelial cells (BECs) via fusiform vesicles, escapes into the cytosol, and establishes biofilm-like intracellular bacterial communities (IBCs). Nucleoside-diphosphate kinase (NDK) is secreted by pathogenic bacteria to enhance virulence. However, whether NDK is involved in UPEC pathogenesis remains unclear. Here, we find that the lack of ndk impairs the colonization of UPEC CFT073 in mouse bladders and kidneys owing to the impaired ability of UPEC to form IBCs. Furthermore, we demonstrate that NDK inhibits caspase-1-dependent pyroptosis by consuming extracellular ATP, preventing superficial BEC exfoliation, and promoting IBC formation. UPEC utilizes the reactive oxygen species (ROS) sensor OxyR to indirectly activate the regulator integration host factor, which then directly activates ndk expression in response to intracellular ROS. Here, we reveal a signaling transduction pathway that UPEC employs to inhibit superficial BEC exfoliation, thus facilitating acute UTI.

YeeD is an essential partner for YeeE-mediated thiosulfate uptake in bacteria and regulates thiosulfate ion decomposition.

Uptake of thiosulfate ions as an inorganic sulfur source from the environment is important for bacterial sulfur assimilation. Recently, a selective thiosulfate uptake pathway involving a membrane protein YeeE (TsuA) in Escherichia coli was characterized. YeeE-like proteins are conserved in some bacteria, archaea, and eukaryotes. However, the precise function of YeeE, along with its potential partner protein in the thiosulfate ion uptake pathway, remained unclear. Here, we assessed selective thiosulfate transport via Spirochaeta thermophila YeeE in vitro and characterized E. coli YeeD (TsuB) as an adjacent and essential protein for YeeE-mediated thiosulfate uptake in vivo. We further showed that S. thermophila YeeD possesses thiosulfate decomposition activity and that a conserved cysteine in YeeD was modified to several forms in the presence of thiosulfate. Finally, the crystal structures of S. thermophila YeeE-YeeD fusion proteins at 3.34-Å and 2.60-Å resolutions revealed their interactions. The association was evaluated by a binding assay using purified S. thermophila YeeE and YeeD. Based on these results, a model of the sophisticated uptake of thiosulfate ions by YeeE and YeeD is proposed.

Protein engineering a PhotoRNR chimera based on a unifying evolutionary apparatus among the natural classes of ribonucleotide reductases.

Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the de novo transformation of nucleoside 5'-di(tri)phosphates [ND(T)Ps, where N is A, U, C, or G] to their corresponding deoxynucleotides. Despite the diversity of factors required for function and the low sequence conservation across RNRs, a unifying apparatus consolidating RNR activity is explored. We combine aspects of the protein subunit simplicity of class II RNR with a modified version of Escherichia coli class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme. The design of this RNR involves fusing a truncated form of the active site containing α subunit with the functionally important C-terminal tail of the radical-generating ß subunit to render a chimeric RNR. Inspired by a recent cryo-EM structure, a [Re] photooxidant is located adjacent to Y356[ß], which is an essential component of the radical transport pathway in class I RNRs. Combination of this RNR photochimera with cytidine diphosphate (CDP), adenosine triphosphate (ATP), and light resulted in the generation of Y356• along with production of deoxycytidine diphosphate (dCDP) and cytosine. The photoproducts reflect an active site chemistry consistent with both the consensus mechanism of RNR and chemistry observed when RNR is inactivated by mechanism-based inhibitors in the active site. The enzymatic activity of the RNR photochimera in the absence of any ß metallocofactor highlights the adaptability of the 10-stranded αß barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs.

Molecular insights into capsular polysaccharide secretion.

Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.

Iron-sulfur cluster assembly scaffold protein IscU is required for activation of ferric uptake regulator (Fur) in Escherichiacoli.

It was generally postulated that when intracellular free iron content is elevated in bacteria, the ferric uptake regulator (Fur) binds its corepressor a mononuclear ferrous iron to regulate intracellular iron homeostasis. However, the proposed iron-bound Fur had not been identified in any bacteria. In previous studies, we have demonstrated that Escherichia coli Fur binds a [2Fe-2S] cluster in response to elevation of intracellular free iron content and that binding of the [2Fe-2S] cluster turns on Fur as an active repressor to bind a specific DNA sequence known as the Fur-box. Here we find that the iron-sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur, as deletion of IscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content. Additional studies reveal that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur and restores its Fur-box binding activity in vitro. While IscU is also required for the [2Fe-2S] cluster assembly in the Haemophilus influenzae Fur in E. coli cells, deletion of IscU does not significantly affect the [2Fe-2S] cluster assembly in the E. coli ferredoxin and siderophore-reductase FhuF. Our results suggest that IscU may have a unique role for the [2Fe-2S] cluster assembly in Fur and that regulation of intracellular iron homeostasis is closely coupled with iron-sulfur cluster biogenesis in E. coli.

Identification of a novel DNA repair inhibitor using an in silico driven approach shows effective combinatorial activity with genotoxic agents against multidrug-resistant Escherichia coli.

Increasing antimicrobial drug resistance represents a global existential threat. Infection is a particular problem in immunocompromised individuals, such as patients undergoing cancer chemotherapy, due to the targeting of rapidly dividing cells by antineoplastic agents. We recently developed a strategy that targets bacterial nucleotide excision DNA repair (NER) to identify compounds that act as antimicrobial sensitizers specific for patients undergoing cancer chemotherapy. Building on this, we performed a virtual drug screening of a ~120,000 compound library against the key NER protein UvrA. From this, numerous target compounds were identified and of those a candidate compound, Bemcentinib (R428), showed a strong affinity toward UvrA. This NER protein possesses four ATPase sites in its dimeric state, and we found that Bemcentinib could inhibit UvrA's ATPase activity by ~90% and also impair its ability to bind DNA. As a result, Bemcentinib strongly diminishes NER's ability to repair DNA in vitro. To provide a measure of in vivo activity we discovered that the growth of Escherichia coli MG1655 was significantly inhibited when Bemcentinib was combined with the DNA damaging agent 4-NQO, which is analogous to UV. Using the clinically relevant DNA-damaging antineoplastic cisplatin in combination with Bemcentinib against the urological sepsis-causing E. coli strain EC958 caused complete growth inhibition. This study offers a novel approach for the potential development of new compounds for use as adjuvants in antineoplastic therapy.

The SecM arrest peptide traps a pre-peptide bond formation state of the ribosome.

Nascent polypeptide chains can induce translational stalling to regulate gene expression. This is exemplified by the E. coli secretion monitor (SecM) arrest peptide that induces translational stalling to regulate expression of the downstream encoded SecA, an ATPase that co-operates with the SecYEG translocon to facilitate insertion of proteins into or through the cytoplasmic membrane. Here we present the structure of a ribosome stalled during translation of the full-length E. coli SecM arrest peptide at 2.0 Å resolution. The structure reveals that SecM arrests translation by stabilizing the Pro-tRNA in the A-site, but in a manner that prevents peptide bond formation with the SecM-peptidyl-tRNA in the P-site. By employing molecular dynamic simulations, we also provide insight into how a pulling force on the SecM nascent chain can relieve the SecM-mediated translation arrest. Collectively, the mechanisms determined here for SecM arrest and relief are also likely to be applicable for a variety of other arrest peptides that regulate components of the protein localization machinery identified across a wide range of bacteria lineages.

The colonization factor CS6 of enterotoxigenic Escherichia coli contributes to host cell invasion.

Enterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in children and travelers in low-income regions. The virulence of ETEC is attributed to its heat-labile and heat-stable enterotoxins, as well as its colonization factors (CFs). CFs are essential for ETEC adherence to the intestinal epithelium. However, its invasive capability remains unelucidated. In this study, we demonstrated that the CS6-positive ETEC strain 4266 can invade mammalian epithelial cells. The invasive capability was reduced in the 4266 ΔCS6 mutant but reintroduction of CS6 into this mutant restored the invasiveness. Additionally, the laboratory E. coli strain Top 10, which lacks the invasive capability, was able to invade Caco-2 cells after gaining the CS6-expressing plasmid pCS6. Cytochalasin D inhibited cell invasion in both 4266 and Top10 pCS6 cells, and F-actin accumulation was observed near the bacteria on the cell membrane, indicating that CS6-positive bacteria were internalized via actin polymerization. Other cell signal transduction inhibitors, such as genistein, wortmannin, LY294002, PP1, and Ro 32-0432, inhibited the CS6-mediated invasion of Caco-2 cells. The internalized bacteria of both 4266 and Top10 pCS6 strains were able to survive for up to 48 h, and 4266 cells were able to replicate within Caco-2 cells. Immunofluorescence microscopy revealed that the internalized 4266 cells were present in bacteria-containing vacuoles, which underwent a maturation process indicated by the recruitment of the early endosomal marker EEA-1 and late endosomal marker LAMP-1 throughout the infection process. The autophagy marker LC3 was also observed near these vacuoles, indicating the initiation of LC-3-associated phagocytosis (LAP). However, intracellular bacteria continued to replicate, even after the initiation of LAP. Moreover, intracellular filamentation was observed in 4266 cells at 24 h after infection. Overall, this study shows that CS6, in addition to being a major CF, mediates cell invasion. This demonstrates that once internalized, CS6-positive ETEC is capable of surviving and replicating within host cells. This capability may be a key factor in the extended and recurrent nature of ETEC infections in humans, thus highlighting the critical role of CS6.

Analysis of Transmembrane ß-Barrel Proteins by Native and Semi-native Polyacrylamide Gel Electrophoresis.

Membrane-embedded ß-barrel proteins are important regulators of the outer membrane permeability barrier of Gram-negative bacteria. ß-barrels are highly structured domains formed by a series of antiparallel ß-strands. Each ß-strand is locked in position by hydrogen bonds between its polypeptide backbone and those of the two neighbouring strands in the barrel structure. Some transmembrane ß-barrel proteins form larger homo- or hetero-multimeric complexes that accomplish specific functions. In this chapter, we describe native and semi-native polyacrylamide gel electrophoresis (PAGE) methods to characterize the organization of transmembrane ß-barrel proteins. We illustrate blue native (BN)-PAGE as an analytical method to assess the formation of protein complexes. Furthermore, we describe a heat-modifiability assay via semi-native PAGE as a rapid method to investigate the folding of transmembrane ß-barrels.

Regulation of Aerobic Succinate Transporter dctA of E. coli by cAMP-CRP, DcuS-DcuR, and EIIAGlc: Succinate as a Carbon Substrate and Signaling Molecule.

INTRODUCTION: C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae during their colonization of mammalian hosts. Particularly noteworthy is the essential role of fumarate respiration during colonization of pathogenic bacteria. To investigate the regulation of aerobic C4-DC metabolism, the study explored the transcriptional control of the main aerobic C4-DC transporter, dctA, under different carbohydrate conditions. In addition, mutants related to carbon catabolite repression (CCR) and C4-DC regulation (DcuS-DcuR) were examined to better understand the regulatory integration of aerobic C4-DC metabolism into CCR. For initial insight into posttranslational regulation, the interaction between the aerobic C4-DC transporter DctA and EIIAGlc from the glucose-specific phosphotransferase system was investigated. METHODS: The expression of dctA was characterized in the presence of various carbohydrates and regulatory mutants affecting CCR. This was accomplished by fusing the dctA promoter (PdctA) to the lacZ reporter gene. Additionally, the interaction between DctA and EIIAGlc of the glucose-specific phosphotransferase system was examined in vivo using a bacterial two-hybrid system. RESULTS: The dctA promoter region contains a class I cAMP-CRP-binding site at position -81.5 and a DcuR-binding site at position -105.5. DcuR, the response regulator of the C4-DC-activated DcuS-DcuR two-component system, and cAMP-CRP stimulate dctA expression. The expression of dctA is subject to the influence of various carbohydrates via cAMP-CRP, which differently modulate cAMP levels. Here we show that EIIAGlc of the glucose-specific phosphotransferase system strongly interacts with DctA, potentially resulting in the exclusion of C4-DCs when preferred carbon substrates, such as sugars, are present. In contrast to the classical inducer exclusion known for lactose permease LacY, inhibition of C4-DC uptake into the cytoplasm affects only its role as a substrate, but not as an inducer since DcuS detects C4-DCs in the periplasmic space ("substrate exclusion"). The work shows an interplay between cAMP-CRP and the DcuS-DcuR regulatory system for the regulation of dctA at both transcriptional and posttranslational levels. CONCLUSION: The study highlights a hierarchical interplay between global (cAMP-CRP) and specific (DcuS-DcuR) regulation of dctA at the transcriptional and posttranslational levels. The integration of global and specific transcriptional regulation of dctA, along with the influence of EIIAGlc on DctA, fine-tunes C4-DC catabolism in response to the availability of other preferred carbon sources. It attributes DctA a central role in the control of aerobic C4-DC catabolism and suggests a new role to EIIAGlc on transporters (control of substrate uptake by substrate exclusion).

Communication between DNA and nucleotide binding sites facilitates stepping by the RecBCD helicase.

Double-strand DNA breaks are the severest type of genomic damage, requiring rapid response to ensure survival. RecBCD helicase in prokaryotes initiates processive and rapid DNA unzipping, essential for break repair. The energetics of RecBCD during translocation along the DNA track are quantitatively not defined. Specifically, it's essential to understand the mechanism by which RecBCD switches between its binding states to enable its translocation. Here, we determine, by systematic affinity measurements, the degree of coupling between DNA and nucleotide binding to RecBCD. In the presence of ADP, RecBCD binds weakly to DNA that harbors a double overhang mimicking an unwinding intermediate. Consistently, RecBCD binds weakly to ADP in the presence of the same DNA. We did not observe coupling between DNA and nucleotide binding for DNA molecules having only a single overhang, suggesting that RecBCD subunits must both bind DNA to 'sense' the nucleotide state. On the contrary, AMPpNp shows weak coupling as RecBCD remains strongly bound to DNA in its presence. Detailed thermodynamic analysis of the RecBCD reaction mechanism suggests an 'energetic compensation' between RecB and RecD, which may be essential for rapid unwinding. Our findings provide the basis for a plausible stepping mechanism' during the processive translocation of RecBCD.

Chi hotspot Control of RecBCD Helicase-nuclease: Enzymatic Tests Support the Intramolecular Signal-transduction Model.

Repair of broken DNA is essential for life; the reactions involved can also promote genetic recombination to aid evolution. In Escherichia coli, RecBCD enzyme is required for the major pathway of these events. RecBCD is a complex ATP-dependent DNA helicase with nuclease activity controlled by Chi recombination hotspots (5'-GCTGGTGG-3'). During rapid DNA unwinding, when Chi is in a RecC tunnel, RecB nuclease nicks DNA at Chi. Here, we test our signal transduction model - upon binding Chi (step 1), RecC signals RecD helicase to stop unwinding (step 2); RecD then signals RecB (step 3) to nick at Chi (step 4) and to begin loading RecA DNA strand-exchange protein (step 5). We discovered that ATP-γ-S, like the small molecule RecBCD inhibitor NSAC1003, causes RecBCD to nick DNA, independent of Chi, at novel positions determined by the DNA substrate length. Two RecB ATPase-site mutants nick at novel positions determined by their RecB:RecD helicase rate ratios. In each case, we find that nicking at the novel position requires steps 3 and 4 but not step 1 or 2, as shown by mutants altered at the intersubunit contacts specific for each step; nicking also requires RecD helicase and RecB nuclease activities. Thus, altering the RecB ATPase site, by small molecules or mutation, sensitizes RecD to signal RecB to nick DNA (steps 4 and 3, respecitvely) without the signal from RecC or Chi (steps 1 and 2). These new, enzymatic results strongly support the signal transduction model and provide a paradigm for studying other complex enzymes.

Current understandings of colibactin regulation.

The biosynthetic machinery for the production of colibactin is encoded by 19 genes (clbA - S) within the pks pathogenicity island harboured by many E. coli of the B2-phylogroup. Colibactin is a potent genotoxic metabolite which causes DNA-damage and which has potential roles in microbial competition and fitness of pks+ bacteria. Colibactin has also been strongly implicated in the development of colorectal cancer. Given the genotoxicity of colibactin and the metabolic cost of its synthesis, the regulatory system governing the clb cluster is accordingly highly complex, and many of the mechanisms remain to be elucidated. In this review we summarise the current understanding of regulation of colibactin biosynthesis by internal molecular components and how these factors are modulated by signals from the external environment.

A c-di-GMP signaling module controls responses to iron in Pseudomonas aeruginosa.

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a bacterial second messenger that modulates various processes including biofilm formation, motility, and host-microbe symbiosis. Numerous studies have conducted comprehensive analysis of c-di-GMP. However, the mechanisms by which certain environmental signals such as iron control intracellular c-di-GMP levels are unclear. Here, we show that iron regulates c-di-GMP levels in Pseudomonas aeruginosa by modulating the interaction between an iron-sensing protein, IsmP, and a diguanylate cyclase, ImcA. Binding of iron to the CHASE4 domain of IsmP inhibits the IsmP-ImcA interaction, which leads to increased c-di-GMP synthesis by ImcA, thus promoting biofilm formation and reducing bacterial motility. Structural characterization of the apo-CHASE4 domain and its binding to iron allows us to pinpoint residues defining its specificity. In addition, the cryo-electron microscopy structure of ImcA in complex with a c-di-GMP analog (GMPCPP) suggests a unique conformation in which the compound binds to the catalytic pockets and to the membrane-proximal side located at the cytoplasm. Thus, our results indicate that a CHASE4 domain directly senses iron and modulates the crosstalk between c-di-GMP metabolic enzymes.