Regulation In Bacteria Research Articles

Programmable synthetic biomolecular condensates for cellular control.

The formation of biomolecular condensates mediated by a coupling of associative and segregative phase transitions plays a critical role in controlling diverse cellular functions in nature. This has inspired the use of phase transitions to design synthetic systems. While design rules of phase transitions have been established for many synthetic intrinsically disordered proteins, most efforts have focused on investigating their phase behaviors in a test tube. Here, we present a rational engineering approach to program the formation and physical properties of synthetic condensates to achieve intended cellular functions. We demonstrate this approach through targeted plasmid sequestration and transcription regulation in bacteria and modulation of a protein circuit in mammalian cells. Our approach lays the foundation for engineering designer condensates for synthetic biology applications.

A Brief Overview of LacI-Family Transcriptional Regulators in Bacteria

A Brief Overview of LacI-Family Transcriptional Regulators in Bacteria

Plant associated protists-Untapped promising candidates for agrifood tools.

The importance of host-associated microorganisms and their biotic interactions for plant health and performance has been increasingly acknowledged. Protists, main predators and regulators of bacteria and fungi, are abundant and ubiquitous eukaryotes in terrestrial ecosystems. Protists are considered to benefit plant health and performance, but the community structure and functions of plant-associated protists remain surprisingly underexplored. Harnessing plant-associated protists and other microbes can potentially enhance plant health and productivity and sustain healthy food and agriculture systems. In this review, we summarize the knowledge of multifunctionality of protists and their interactions with other microbes in plant hosts, and propose a future framework to study plant-associated protists and utilize protists as agrifood tools for benefiting agricultural production.

The LeuO regulator and quiescence: About transcriptional roadblocks, multiple promoters, and crispr-cas.

LeuO is a LysR-type transcriptional regulator in bacteria. It determines the regulation of numerous genes related to stress response and virulence. Thus, four exciting areas of research are discussed herein. One pertains the leuO gene, which in S. Typhi and in E. coli contains multiple forward promoters as well as reverse promoters, even though it is expressed at very low levels, that is, it is quiescent. Such multiplicity might allow for a greater plasticity in regulation, or even aid in maintaining the quiescence, in processes that appear to involve many nucleoid-associated proteins in a second area of opportunity. A third one relates to the effector-binding domain of the LeuO regulator, which is highly conserved in S. enterica and in E. coli and determines its activity as a regulator of transcription. A fourth area regards the role of the CRISPR-Cas system in gene regulation in S. Typhi; a system that is regulated by LeuO.

Structural insights into RNA-mediated transcription regulation in bacteria

RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination.

Quantitative phosphoproteome analysis of Streptomyces coelicolor by immobilized zirconium (IV) affinity chromatography and mass spectrometry reveals novel regulated protein phosphorylation sites and sequence motifs

Streptomycetes are multicellular gram-positive bacteria that produce many bioactive compounds, including antibiotics, antitumorals and immunosuppressors. The Streptomyces phosphoproteome remains largely uncharted even though protein phosphorylation at Ser/Thr/Tyr is known to modulate morphological differentiation and specialized metabolic processes. We here expand the S. coelicolor phosphoproteome by optimised immobilized zirconium (IV) affinity chromatography and mass spectrometry to identify phosphoproteins at the vegetative and sporulating stages. We mapped 361 phosphorylation sites (41% pSer, 56.2% pThr, 2.8% pTyr) and discovered four novel Thr phosphorylation motifs (“Kxxxx(pT)xxxxK”, “DxE(pT)”, “D(pT)” and “Exxxxx(pT)”) in 351 phosphopeptides derived from 187 phosphoproteins. We identified 154 novel phosphoproteins, thereby almost doubling the number of experimentally verified Streptomyces phosphoproteins. Novel phosphoproteins included cell division proteins (FtsK, CrgA) and specialized metabolism regulators (ArgR, AfsR, CutR and HrcA) that were differentially phosphorylated in the vegetative and in the antibiotic producing sporulating stages. Phosphoproteins involved in primary metabolism included 27 novel ribosomal proteins that were phosphorylated during the vegetative stage. Phosphorylation of these proteins likely participate in the intricate and incompletely understood regulation of Streptomyces development and secondary metabolism. We conclude that Zr(IV)-IMAC is an efficient and sensitive method to study protein phosphorylation and regulation in bacteria and enhance our understanding of bacterial signalling. SignificanceTwo thirds of the secondary metabolites used in clinic, especially antibiotics, were discovered in Streptomyces strains. Antibiotic resistance became one of the major challenges in clinic, and new antibiotics are urgently required in clinic. Next-generation sequencing analyses revealed that streptomycetes harbour many cryptic secondary metabolite pathways, i.e. pathways not expressed in the laboratory. Secondary metabolism is tightly connected with hypha differentiation and sporulation, and understanding Streptomyces differentiation is one of the main challenges in industrial microbiology, in order to activate the expression of cryptic pathways in the laboratory.Protein phosphorylation at Ser/Thr/Tyr modulates development and secondary metabolism, but the Streptomyces phosphoproteome is still largely uncharted. Previous S. coelicolor phosphoproteomic studies used TiO2 affinity enrichment and LC-MS/MS identifying a total of 184 Streptomyces phosphoproteins. Here, we used by first time zirconium (IV) affinity chromatography and mass spectrometry, identifying 186 S. coelicolor phosphoproteins. Most of these phosphoproteins (154) were not identified in previous phosphoproteomic studies using TiO2 affinity enrichment. Thereby we almost doubling the number of experimentally verified Streptomyces phosphoproteins. Zr(IV)-IMAC affinity chromatography also worked in E. coli, allowing the identification of phosphoproteins that were not identified by TiO2 affinity chromatography.We conclude that Zr(IV)-IMAC is an efficient and sensitive method for studies of protein phosphorylation and regulation in bacteria to enhance our understanding of bacterial signalling networks. Moreover, the new Streptomyces phosphoproteins identified will contribute to design further works to understand and modulate Streptomyces secondary metabolism activation.

Uncovering lipopolysaccharide regulation in bacteria via the critical lipid binding tunnel of YciS/YciM

SummaryGram-negative bacteria contain an asymmetric outer membrane, in which the outer leaflet is composed of lipopolysaccharide (LPS). LPS, a drug target of polymyxin, plays an essential role in drug resistance, biofilm formation, and pathogenesis. An important inner membrane protein, YciM, may be responsible for the regulation of LPS biosynthesis and transport. Here, we report the crystal structure of YciM from Salmonella typhimurium in a complex with a non-specifically bond molecule, an ethylene glycol, which identified a tunnel that could bind lipids. Our in vitro assays showed that YciM could bind lipid molecules with affinity in the micromolar range, while mutagenic and functional studies confirmed that lipid-binding residues are critical for the function of YciM. Additionally, our data also showed that YciM accurately regulates LPS biosynthesis and transport with YciS, which could help to better understand the regulation mechanism of LPS.

Genome-wide transcription start site mapping in the facultative intracellular pathogen Brucella melitensis by Capping-seq

Brucella melitensis (B. melitensis) is an important facultative intracellular bacterium that causes global zoonotic diseases. Continuous intracellular survival and replication are the main obstruction responsible for the accessibility of prevention and treatment of brucellosis. Bacteria respond to complex environment by regulating gene expression. Many regulatory factors function at loci where RNA polymerase initiates messenger RNA synthesis. However, limited gene annotation is a current obstacle for the research on expression regulation in bacteria. To improve annotation and explore potential functional sites, we proposed a novel genome-wide method called Capping-seq for transcription start site (TSS) mapping in B. melitensis. This technique combines capture of capped primary transcripts with Single Molecule Real-Time (SMRT) sequencing technology. We identified 2,369 TSSs at single nucleotide resolution by Capping-seq. TSSs analysis of Brucella transcripts showed a preference of purine on the TSS positions. Our results revealed that −35 and −10 elements of promoter contained consensus sequences of TTGNNN and TATNNN, respectively. The 5′ ends analysis showed that 57% genes are associated with more than one TSS and 47% genes contain long leader regions, suggested potential complex regulation at the 5′ ends of genes in B. melitensis. Moreover, we identified 52 leaderless genes that are mainly involved in the metabolic processes. Overall, Capping-seq technology provides a unique solution for TSS determination in prokaryotes. Our findings develop a systematic insight into the primary transcriptome characterization of B. melitensis. This study represents a critical basis for investigating gene regulation and pathogenesis of Brucella.

Quantitative contribution of the spacer length in the supercoiling-sensitivity of bacterial promoters.

DNA supercoiling acts as a global transcriptional regulator in bacteria, but the promoter sequence or structural determinants controlling its effect remain unclear. It was previously proposed to modulate the torsional angle between the −10 and −35 hexamers, and thereby regulate the formation of the closed-complex depending on the length of the ‘spacer’ between them. Here, we develop a thermodynamic model of this notion based on DNA elasticity, providing quantitative and parameter-free predictions of the relative activation of promoters containing a short versus long spacer when the DNA supercoiling level is varied. The model is tested through an analysis of in vitro and in vivo expression assays of mutant promoters with variable spacer lengths, confirming its accuracy for spacers ranging from 15 to 19 nucleotides, except those of 16 nucleotides where other regulatory mechanisms likely overcome the effect of this specific step. An analysis at the whole-genome scale in Escherichia coli then demonstrates a significant effect of the spacer length on the genomic expression after transient or inheritable superhelical variations, validating the model’s predictions. Altogether, this study shows an example of mechanical constraints associated to promoter binding by RNA Polymerase underpinning a basal and global regulatory mechanism.

Staphylococcal saoABC Operon Codes for a DNA-Binding Protein SaoC Implicated in the Response to Nutrient Deficit.

Whilst a large number of regulatory mechanisms for gene expression have been characterised to date, transcription regulation in bacteria still remains an open subject. In clinically relevant and opportunistic pathogens, such as Staphylococcus aureus, transcription regulation is of great importance for host-pathogen interactions. In our study we investigated an operon, exclusive to staphylococci, that we name saoABC. We showed that SaoC binds to a conserved sequence motif present upstream of the saoC gene, which likely provides a negative feedback loop. We have also demonstrated that S. aureus ΔsaoB and ΔsaoC mutants display altered growth dynamics in non-optimal media; ΔsaoC exhibits decreased intracellular survival in human dermal fibroblasts, whereas ΔsaoB produces an elevated number of persisters, which is also elicited by inducible production of SaoC in ΔsaoBΔsaoC double mutant. Moreover, we have observed changes in the expression of saoABC operon genes during either depletion of the preferential carbon or the amino acid source as well as during acidification. Comparative RNA-Seq of the wild type and ΔsaoC mutant demonstrated that SaoC influences transcription of genes involved in amino acid transport and metabolism, and notably of those coding for virulence factors. Our results suggest compellingly that saoABC operon codes for a DNA-binding protein SaoC, a novel staphylococcal transcription factor, and its antagonist SaoB. We linked SaoC to the response to nutrient deficiency, a stress that has a great impact on host-pathogen interactions. That impact manifests in SaoC influence on persister formation and survival during internalisation to host cells, as well as on the expression of genes of virulence factors that may potentially result in profound alternations in the pathogenic phenotype. Investigation of such novel regulatory mechanisms is crucial for our understanding of the dynamics of interactions between pathogenic bacteria and host cells, particularly in the case of clinically relevant, opportunistic pathogens such as Staphylococcus aureus.

Role of Hfq in glucose utilization, biofilm formation and quorum sensing system in Bacillus subtilis.

Hfq is an RNA-binding protein, its main function is to participate in post-transcriptional regulation of bacteria and regulate small regulatory RNA (sRNA) and messenger RNA (mRNA) stability, but the Hfq function of Bacillus subtilis (B. subtilis) has not been fully explained. In this study, we used the strains of B. subtilis168 (BS168), BS168Δhfq and BS168Δhfq-C to explore the effects of Hfq on the glucose utilization, biofilm formation and quorum sensing (QS) system of B. subtilis. The results showed that the knockout of hfq resulted in growth defects when bacteria were cultured in the Luria-Bertani (LB) medium, but we did not observe the same effects in Nitrogen medium (NM) and Inorganic Salt-free medium (ISM). We further found that the growth of strains under different glucose concentrations was also different, which was related to the expression of CcpA. Interestingly, the hfq mutant showed increased resistance to a high-glucose environment. Furthermore, the biofilm and extracellular poly saccharides (EPS) formation of BS168Δhfq decreased significantly. At the same time, changes were observed in the morphology of the biofilm, such as larger intercellular space of the biofilm and thinner edge. The qRT-PCR results confirmed that the hfq knockout caused significant up-regulation or down-regulation of gene expression in QS system, and down-regulated genes were involved in the positive regulation of biofilm formation. Taken together, we demonstrated that Hfq plays a vital role in glucose utilization, biofilm formation and QS of B. subtilis, which provides a new perspective for subsequent related research.

An overview of gene regulation in bacteria by small RNAs derived from mRNA 3' ends.

Over the past two decades, small noncoding RNAs (sRNAs) that regulate mRNAs by short base pairing have gone from a curiosity to a major class of post-transcriptional regulators in bacteria. They are integral to many stress responses and regulatory circuits, affecting almost all aspects of bacterial life. Following pioneering sRNA searches in the early 2000s, the field quickly focused on conserved sRNA genes in the intergenic regions of bacterial chromosomes. Yet, it soon emerged that there might be another rich source of bacterial sRNAs—processed 3′ end fragments of mRNAs. Several such 3′ end-derived sRNAs have now been characterized, often revealing unexpected, conserved functions in diverse cellular processes. Here, we review our current knowledge of these 3′ end-derived sRNAs—their biogenesis through ribonucleases, their molecular mechanisms, their interactions with RNA-binding proteins such as Hfq or ProQ and their functional scope, which ranges from acting as specialized regulators of single metabolic genes to constituting entire noncoding arms in global stress responses. Recent global RNA interactome studies suggest that the importance of functional 3′ end-derived sRNAs has been vastly underestimated and that this type of cross-regulation between genes at the mRNA level is more pervasive in bacteria than currently appreciated.

Genome-Wide Identification of Novel sRNAs in Streptococcus mutans.

Streptococcus mutans is a major pathobiont involved in the development of dental caries. Its ability to utilize numerous sugars and to effectively respond to environmental stress promotes S. mutans proliferation in oral biofilms. Because of their quick action and low energetic cost, noncoding small RNAs (sRNAs) represent an ideal mode of gene regulation in stress response networks, yet their roles in oral pathogens have remained largely unexplored. We identified 15 novel sRNAs in S. mutans and show that they respond to four stress-inducing conditions commonly encountered by the pathogen in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. To better understand the role of sRNAs in S. mutans, we further explored the function of the novel sRNA SmsR4. Our data demonstrate that SmsR4 regulates the enzyme IIA (EIIA) component of the sorbitol phosphotransferase system, which transports and phosphorylates the sugar alcohol sorbitol. The fine-tuning of EIIA availability by SmsR4 likely promotes S. mutans growth while using sorbitol as the main carbon source. Our work lays a foundation for understanding the role of sRNAs in regulating gene expression in stress response networks in S. mutans and highlights the importance of the underexplored phenomenon of posttranscriptional gene regulation in oral bacteria. IMPORTANCE Small RNAs (sRNAs) are important gene regulators in bacteria, but the identities and functions of sRNAs in Streptococcus mutans, the principal bacterium involved in the formation of dental caries, are unknown. In this study, we identified 15 putative sRNAs in S. mutans and show that they respond to four common stress-inducing conditions present in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. We further show that the novel sRNA SmsR4 likely modulates sorbitol transport into the cell by regulating SMU_313 mRNA, which encodes the EIIA subunit of the sorbitol phosphotransferase system. Gaining a better understanding of sRNA-based gene regulation may provide new opportunities to develop specific inhibitors of S. mutans growth, thereby improving oral health.

Epigenetic Mechanisms of Gene Expression Regulation in Bacteria of the Genus Bacillus

Epigenetic Mechanisms of Gene Expression Regulation in Bacteria of the Genus Bacillus

A Peroxide-Responding sRNA Evolved from a Peroxidase mRNA.

Small RNAs (sRNAs) are important gene regulators in bacteria, but it is unclear how new sRNAs originate and become part of regulatory networks that coordinate bacterial response to environmental stimuli. Using a covariance modeling-based approach, we analyzed the presence of hundreds of sRNAs in more than a thousand genomes across Enterobacterales, a bacterial order with a confluence of factors that allows robust genome-scale sRNA analyses: several well-studied organisms with fairly conserved genome structures, an established phylogeny, and substantial nucleotide diversity within a narrow evolutionary space. We discovered that a majority of sRNAs arose recently, and uncovered protein-coding genes as a potential source from which new sRNAs arise. A detailed investigation of the emergence of OxyS, a peroxide-responding sRNA, revealed that it evolved from a fragment of a peroxidase messenger RNA. Importantly, although it replaced the ancestral peroxidase, OxyS continues to be part of the ancestral peroxide-response regulon, indicating that an sRNA that arises from a protein-coding gene would inherently be part of the parental protein’s regulatory network. This new insight provides a fresh framework for understanding sRNA origin and regulatory integration in bacteria.

Growth-phase specific regulation of cviI/R based quorum sensing associated virulence factors in Chromobacterium violaceum by linalool, a monoterpenoid.

Quorum sensing (QS)-dependent gene regulation in bacteria performs a vital role in synchronization of cell-density-dependent functions. In Chromobacterium violaceum QS-dependent cviI/R regulatory genes are activated during the mid- or late-exponential phase of growth. However, sufficient evidence is lacking on the role of QS inhibitors on gene regulation at different phases of growth. Hence, we report the role of linalool, a natural monoterpenoid on QS mediated gene regulation at different stages of growth in C. violaceum by performing biosensor, growth kinetic and gene expression studies. In vitro and in vivo studies were performed for establishing role of linalool in reducing the virulence and infection by using HEK-293T cell lines and Caenorhabditis elegans models respectively. C. violaceum CV026 with C6-HSL was used as control. The results showed linalool to be a QS inhibitor with an estimated IC50 of 63µg/mL for violacein inhibition. At this concentration the cell density difference (delta OD600) of 0.14 from the compound was observed indicating the quorum concentration. The expression of cviI/R was initiated at mid-log phase (~ 18h) and reached the maximum at 36h in control whereas in treatment it remained significantly downregulated at all time points. The expression of violacein biosynthetic genes vioA, vioC, vioD and vioE was also downregulated by linalool. Infection studies with linalool showed higher survival rates in HEK-293T cell lines and C. elegans compared to the infection control. Taken together, this study proves linalool to be a QS inhibitor capable of attenuation of QS by controlling the cell density through cviI/R downregulation at the early phase of growth and hence offering scope for its application for controlling infections.

Localized Peptidoglycan Biosynthesis in Chlamydia trachomatis Conforms to the Polarized Division and Cell Size Reduction Developmental Models.

Cell size regulation in bacteria is a function of two basic cellular processes: the expansion of the cell envelope and its constriction at spatially defined points at what will eventually become the division plane. In most bacterial species, both cell wall expansion and restriction are dependent on peptidoglycan (PG), a structural polymer comprised of sugars and amino acids that imparts strength and rigidity to bacterial membranes. Pathogenic Chlamydia species are unique in that their cell walls contain very little PG, which is restricted almost entirely to the apparent division plane of the microbe’s replicative forms. Very little is known about the degree to which PG affects the size and shape of C. trachomatis during its division process, and recent studies suggest the process is initiated via a polarized mechanism. We conducted an imaging study to ascertain the dimensions, orientation, and relative density of chlamydial PG throughout the organism’s developmental cycle. Our analysis indicates that PG in replicating C. trachomatis can be associated with four, broad structural forms; polar/septal disks, small/thick rings, large rings, and small/thin rings. We found that PG density appeared to be highest in septal disks and small/thick rings, indicating that these structures likely have high PG synthesis to degradation ratios. We also discovered that as C. trachomatis progresses through its developmental cycle PG structures, on average, decrease in total volume, indicating that the average cell volume of chlamydial RBs likely decreases over time. When cells infected with C. trachomatis are treated with inhibitors of critical components of the microbe’s two distinct PG synthases, we observed drastic differences in the ratio of PG synthesis to degradation, as well as the volume and shape of PG-containing structures. Overall, our results suggest that C. trachomatis PG synthases differentially regulate the expansion and contraction of the PG ring during both the expansion and constriction of the microbe’s cell membrane during cell growth and division, respectively.

Using cryo-EM to uncover mechanisms of bacterial transcriptional regulation.

Transcription is the principal control point for bacterial gene expression, and it enables a global cellular response to an intracellular or environmental trigger. Transcriptional regulation is orchestrated by transcription factors, which activate or repress transcription of target genes by modulating the activity of RNA polymerase. Dissecting the nature and precise choreography of these interactions is essential for developing a molecular understanding of transcriptional regulation. While the contribution of X-ray crystallography has been invaluable, the ‘resolution revolution’ of cryo-electron microscopy has transformed our structural investigations, enabling large, dynamic and often transient transcription complexes to be resolved that in many cases had resisted crystallisation. In this review, we highlight the impact cryo-electron microscopy has had in gaining a deeper understanding of transcriptional regulation in bacteria. We also provide readers working within the field with an overview of the recent innovations available for cryo-electron microscopy sample preparation and image reconstruction of transcription complexes.

Status quo of tet regulation in bacteria.

SummaryThe tetracycline repressor (TetR) belongs to the most popular, versatile and efficient transcriptional regulators used in bacterial genetics. In the tetracycline (Tc) resistance determinant tet(B) of transposon Tn10, tetR regulates the expression of a divergently oriented tetA gene that encodes a Tc antiporter. These components of Tn10 and of other natural or synthetic origins have been used for tetracycline‐dependent gene regulation (tet regulation) in at least 40 bacterial genera. Tet regulation serves several purposes such as conditional complementation, depletion of essential genes, modulation of artificial genetic networks, protein overexpression or the control of gene expression within cell culture or animal infection models. Adaptations of the promoters employed have increased tet regulation efficiency and have made this system accessible to taxonomically distant bacteria. Variations of TetR, different effector molecules and mutated DNA binding sites have enabled new modes of gene expression control. This article provides a current overview of tet regulation in bacteria.

Structural basis for the inhibition of the Bacillus subtilis c-di-AMP cyclase CdaA by the phosphoglucomutase GlmM

Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes.