All-in-one platform: Versatile, Easy, and User-friendly System (VEUS) based on automated and expert-independent antibody immobilization and immunoassay by utilizing customized movement of magnetic particles.

The ELISA is the most worldwide method for immunoassay. However, the ELISA is losing ground due to low reproducibility of manual experimental processes in both R&D and IVD areas. An automated platform is a good solution, but there are still limitations owning to extremely high cost and requiring large space to set up especially for a small size laboratory. Here, we present a novel all-in-one platform called "VEUS" settable on the laboratory table that offers comprehensive automation of the entire multiplex immunoassay process by exploiting antibody conjugated magnetic particles, quality control and then immunoanalytical reaction, thereby enhancing detection sensitivity and high reproducibility. As a proof of concept, the system exhibits a sensitive LOD of 0.6 and 3.1 pg mL-1 within 1 h run, comparable precision that of molecular diagnostic systems based on PCR method, enabling rapid multiplex diagnosis of Influenza A, Influenza B, and COVID-19 viruses with similar symptoms. Through automation by the all-in-one system, it can be used by novice users, something innovative for immunoassays, relying heavily on user experience. Furthermore, it can contribute to streamline entire immunoassay processes of diverse biomarkers with high reproducibility and convenience in laboratories.

Zinc-dependent regulation of zinc import and export genes by Zur.

In most bacteria, zinc depletion is sensed by Zur, whereas the surplus is sensed by different regulators to achieve zinc homeostasis. Here we present evidence that zinc-bound Zur not only represses genes for zinc acquisition but also induces the zitB gene encoding a zinc exporter in Streptomyces coelicolor, a model actinobacteria. Zinc-dependent gene regulation by Zur occurs in two phases. At sub-femtomolar zinc concentrations (phase I), dimeric Zur binds to the Zur-box motif immediately upstream of the zitB promoter, resulting in low zitB expression. At the same time, Zur represses genes for zinc uptake. At micromolar zinc concentrations (phase II), oligomeric Zur binding with footprint expansion upward from the Zur box results in high zitB induction. Our findings reveal a mode of zinc-dependent gene activation that uses a single metalloregulator to control genes for both uptake and export over a wide range of zinc concentrations.

Functional elucidation of the non-coding RNAs of Kluyveromyces marxianus in the exponential growth phase.

BACKGROUND: Non-coding RNAs (ncRNAs), which perform diverse regulatory roles, have been found in organisms from all superkingdoms of life. However, there have been limited numbers of studies on the functions of ncRNAs, especially in nonmodel organisms such as Kluyveromyces marxianus that is widely used in the field of industrial biotechnology. RESULTS: In this study, we measured changes in transcriptome at three time points during the exponential growth phase of K. marxianus by using strand-specific RNA-seq. We found that approximately 60% of the transcriptome consists of ncRNAs transcribed from antisense and intergenic regions of the genome that were transcribed at lower levels than mRNA. In the transcriptome, a substantial number of long antisense ncRNAs (lancRNAs) are differentially expressed and enriched in carbohydrate and energy metabolism pathways. Furthermore, this enrichment is evolutionarily conserved, at least in yeast. Particularly, the mode of regulation of mRNA/lancRNA pairs is associated with mRNA transcription levels; the correlation between the pairs is positive at high mRNA transcriptional levels and negative at low levels. In addition, significant induction of mRNA and coverage of more than half of the mRNA sequence by a lancRNA strengthens the positive correlation between mRNA/lancRNA pairs. CONCLUSIONS: Transcriptome sequencing of K. marxianus in the exponential growth phase reveals pervasive transcription of ncRNAs with evolutionarily conserved functions. Studies of the mode of regulation of mRNA/lancRNA pairs suggest that induction of lancRNA may be associated with switch-like behavior of mRNA/lancRNA pairs and efficient regulation of the carbohydrate and energy metabolism pathways in the exponential growth phase of K. marxianus being used in industrial applications.

Determination of single nucleotide variants in Escherichia coli DH5α by using short-read sequencing.

Escherichia coli DH5α is a common laboratory strain that provides an important platform for routine use in cloning and synthetic biology applications. Many synthetic circuits have been constructed and successfully expressed in E. coli DH5α; however, its genome sequence has not been determined yet. Here, we determined E. coli DH5α genome sequence and identified genetic mutations that affect its phenotypic functions by using short-read sequencing. The sequencing results clearly described the genotypes of E. coli DH5α, which aid in further studies using the strain. Additionally, we observed 105 single nucleotide variants (SNVs), 83% of which were detected in protein-coding regions compared to the parental strain E. coli DH1. Interestingly, 23% of the protein-coding regions have mutations in their amino acid residues, whose biological functions were categorized into two-component systems, peptidoglycan biosynthesis and lipopolysaccharide biosynthesis. These results underscore the advantages of E. coli DH5α, which tolerates the components of transformation buffer and expresses foreign plasmids efficiently. Moreover, these SNVs were also observed in the commercially available strain. These data provide the genetic information of E. coli DH5α for its future application in metabolic engineering and synthetic biology.

The architecture of ArgR-DNA complexes at the genome-scale in Escherichia coli.

DNA-binding motifs that are recognized by transcription factors (TFs) have been well studied; however, challenges remain in determining the in vivo architecture of TF-DNA complexes on a genome-scale. Here, we determined the in vivo architecture of Escherichia coli arginine repressor (ArgR)-DNA complexes using high-throughput sequencing of exonuclease-treated chromatin-immunoprecipitated DNA (ChIP-exo). The ChIP-exo has a unique peak-pair pattern indicating 5' and 3' ends of ArgR-binding region. We identified 62 ArgR-binding loci, which were classified into three groups, comprising single, double and triple peak-pairs. Each peak-pair has a unique 93 base pair (bp)-long (±2 bp) ArgR-binding sequence containing two ARG boxes (39 bp) and residual sequences. Moreover, the three ArgR-binding modes defined by the position of the two ARG boxes indicate that DNA bends centered between the pair of ARG boxes facilitate the non-specific contacts between ArgR subunits and the residual sequences. Additionally, our approach may also reveal other fundamental structural features of TF-DNA interactions that have implications for studying genome-scale transcriptional regulatory networks.

Exploring the functional residues in a flavin-binding fluorescent protein using deep mutational scanning.

Flavin mononucleotide (FMN)-based fluorescent proteins are versatile reporters that can monitor various cellular processes in both aerobic and anaerobic conditions. However, the understanding of the role of individual amino acid residues on the protein function has been limited and has restricted the development of better functional variants. Here we examine the functional amino acid residues of Escherichia coli flavin mononucleotide binding fluorescent protein (EcFbFP) using the application of high-throughput sequencing of functional variants, termed deep mutational scanning. The variants were classified into 329 function-retained (FR) and 259 function-loss (FL) mutations, and further the mutational enrichment in each amino acid residues was weighed to find the functionally important residues of EcFbFP. We show that the crucial amino acid residues of EcFbFP lie among the FMN-binding pocket, turns and loops of the protein where conformation changes occur, and spatially clustered residues near the E56-K97 salt bridges. In addition, the mutational sensitivity of the critical residues was confirmed by site-directed mutagenesis. The deep mutational scanning of EcFbFP has demonstrated important implications for constructing better functioning protein variants.

Comprehensive analysis to improve the validation rate for single nucleotide variants detected by next-generation sequencing.

Next-generation sequencing (NGS) has enabled the high-throughput discovery of germline and somatic mutations. However, NGS-based variant detection is still prone to errors, resulting in inaccurate variant calls. Here, we categorized the variants detected by NGS according to total read depth (TD) and SNP quality (SNPQ), and performed Sanger sequencing with 348 selected non-synonymous single nucleotide variants (SNVs) for validation. Using the SAMtools and GATK algorithms, the validation rate was positively correlated with SNPQ but showed no correlation with TD. In addition, common variants called by both programs had a higher validation rate than caller-specific variants. We further examined several parameters to improve the validation rate, and found that strand bias (SB) was a key parameter. SB in NGS data showed a strong difference between the variants passing validation and those that failed validation, showing a validation rate of more than 92% (filtering cutoff value: alternate allele forward [AF] ≥ 20 and AF<80 in SAMtools, SB<-10 in GATK). Moreover, the validation rate increased significantly (up to 97-99%) when the variant was filtered together with the suggested values of mapping quality (MQ), SNPQ and SB. This detailed and systematic study provides comprehensive recommendations for improving validation rates, saving time and lowering cost in NGS analyses.

Inverse regulation of Fe- and Ni-containing SOD genes by a Fur family regulator Nur through small RNA processed from 3'UTR of the sodF mRNA.

Superoxide dismutases (SODs) are widely distributed enzymes that convert superoxides to hydrogen peroxide and molecular oxygen, using various metals as cofactors. Many actinobacteria contain genes for both Ni-containing (sodN) and Fe-containing (sodF) SODs. In Streptomyces coelicolor, expression of the sodF and sodN genes is inversely regulated by nickel-specific Nur, a Fur-family regulator. With sufficient nickel, Nur directly represses sodF transcription, while inducing sodN indirectly. Bioinformatic search revealed that a conserved 19-nt stretch upstream of sodN matches perfectly with the sodF downstream sequence. We found that the sodF gene produced a stable small-sized RNA species (s-SodF) of ∼ 90 nt that harbors the anti-sodN sequence complementary to sodN mRNA from the 5'-end up to the ribosome binding site. Absence of nearby promoters and sensitivity to 5'-phosphate-specific exonuclease indicated that the s-SodF RNA is a likely processed product of sodF mRNA. The s-SodF RNA caused a significant decrease in the half-life of the sodN mRNA. Therefore, Nur activates sodN expression through inhibiting the synthesis of sodF mRNA, from which inhibitory s-SodF RNA is generated. This reveals a novel mechanism by which antagonistic regulation of one gene is achieved by small RNA processed from the 3'UTR of another gene's mRNA.

Current challenges in bacterial transcriptomics.

Over the past decade or so, dramatic developments in our ability to experimentally determine the content and function of genomes have taken place. In particular, next-generation sequencing technologies are now inspiring a new understanding of bacterial transcriptomes on a global scale. In bacterial cells, whole-transcriptome studies have not received attention, owing to the general view that bacterial genomes are simple. However, several recent RNA sequencing results are revealing unexpected levels of complexity in bacterial transcriptomes, indicating that the transcribed regions of genomes are much larger and complex than previously anticipated. In particular, these data show a wide array of small RNAs, antisense RNAs, and alternative transcripts. Here, we review how current transcriptomics are now revolutionizing our understanding of the complexity and regulation of bacterial transcriptomes.

Alternative zinc-binding sites explain the redox sensitivity of zinc-containing anti-sigma factors.

Certain bacterial zinc-containing anti-sigma (ZAS) factors respond sensitively to thiol-induced oxidative stress by undergoing conformational changes, which in turn reduce binding affinities for their cognate sigma factors. This redox sensitivity provides a mechanism for coping with oxidative stress by activating the transcription of antioxidant genes. Not all ZAS proteins are redox-sensitive, but the mechanism of redox sensitivity is not fully understood. Here we propose that alternative zinc-binding sites determine redox sensitivity. To support this proposal, we performed protein modeling and zinc docking on redox-sensitive and redox-insensitive ZAS proteins complexed with their cognate sigma factors. At least one strong alternative zinc-binding pocket was detected for all known redox-sensitive ZAS factors in actinomycetes, while no strong alternative zinc-binding pocket was identified in redox-insensitive ZAS factors, except for one controversial case. This hypothesis of alternative zinc-binding sites can also explain residue-specific contributions to the redox sensitivity of RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor, for which alanine mutagenesis experiments are available. Our results suggest a mechanistic model for redox sensitivity as follows: zinc ion can probabilistically occupy multiple sites in redox-sensitive ZAS proteins, increasing the susceptibility of zinc-coordinating cysteine residues to oxidation. This picture of probabilistic zinc occupation agrees with a previous structure and energy analysis on zinc finger proteins, and thus it may be more widely applicable to other classes of reactive zinc-binding proteins.

Conservation of thiol-oxidative stress responses regulated by SigR orthologues in actinomycetes.

Numerous thiol-reactive compounds cause oxidative stress where cells counteract by activation of survival strategies regulated by thiol-based sensors. In Streptomyces coelicolor, a model actinomycete, a sigma/antisigma pair SigR/RsrA controls the response to thiol-oxidative stress. To unravel its full physiological functions, chromatin immuno-precipitation combined with sequence and transcript analyses were employed to identify 108 SigR target genes in S. coelicolor and to predict orthologous regulons across actinomycetes. In addition to reported genes for thiol homeostasis, protein degradation and ribosome modulation, 64 additional operons were identified suggesting new functions of this global regulator. We demonstrate that SigR maintains the level and activity of the housekeeping sigma factor HrdB during thiol-oxidative stress, a novel strategy for stress responses. We also found that SigR defends cells against UV and thiol-reactive damages, in which repair UvrA takes a part. Using a refined SigR-binding sequence model, SigR orthologues and their targets were predicted in 42 actinomycetes. This revealed a conserved core set of SigR targets to function for thiol homeostasis, protein quality control, possible modulation of transcription and translation, flavin-mediated redox reactions, and Fe-S delivery. The composition of the SigR regulon reveals a robust conserved physiological mechanism to deal with thiol-oxidative stress from bacteria to human.

Determinants of redox sensitivity in RsrA, a zinc-containing anti-sigma factor for regulating thiol oxidative stress response.

Various environmental oxidative stresses are sensed by redox-sensitive regulators through cysteine thiol oxidation or modification. A few zinc-containing anti-sigma (ZAS) factors in actinomycetes have been reported to respond sensitively to thiol oxidation, among which RsrA from Streptomyces coelicolor is best characterized. It forms disulfide bonds upon oxidation and releases bound SigR to activate thiol oxidative stress response genes. Even though numerous ZAS proteins exist in bacteria, features that confer redox sensitivity to a subset of these have been uncharacterized. In this study, we identified seven additional redox-sensitive ZAS factors from actinomycetes. Comparison with redox-insensitive ZAS revealed characteristic sequence patterns. Domain swapping demonstrated the significance of the region K(33)FEHH(37)FEEC(41)SPC(44)LEK(47) that encompass the conserved HX(3)CX(2)C (HCC) motif. Mutational effect of each residue on diamide responsive induction of SigR target genes in vivo demonstrated that several residues, especially those that flank two cysteines (E39, E40, L45, E46), contribute to redox sensitivity. These residues are well conserved among redox-sensitive ZAS factors, and hence are proposed as redox-determinants in sensitive ZAS. H37A, C41A, C44A and F38A mutations, in contrast, compromised SigR-binding activity significantly, apparently affecting structural integrity of RsrA. The residue pattern around HCC motif could therefore serve as an indicator to predict redox-sensitive ZAS factors from sequence information.

Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur.

Zinc is one of the essential transition metals in cells. Excess or lack of zinc is detrimental, and cells exploit highly sensitive zinc-binding regulators to achieve homeostasis. In this article, we present a crystal structure of active Zur from Streptomyces coelicolor with three zinc-binding sites (C-, M-, and D-sites). Mutations of the three sites differentially affected sporulation and transcription of target genes, such that C- and M-site mutations inhibited sporulation and derepressed all target genes examined, whereas D-site mutations did not affect sporulation and derepressed only a sensitive gene. Biochemical and spectroscopic analyses of representative metal site mutants revealed that the C-site serves a structural role, whereas the M- and D-sites regulate DNA-binding activity as an on-off switch and a fine-tuner, respectively. Consistent with differential effect of mutations on target genes, zinc chelation by TPEN derepressed some genes (znuA, rpmF2) more sensitively than others (rpmG2, SCO7682) in vivo. Similar pattern of TPEN-sensitivity was observed for Zur-DNA complexes formed on different promoters in vitro. The sensitive promoters bound Zur with lower affinity than the less sensitive ones. EDTA-treated apo-Zur gained its DNA binding activity at different concentrations of added zinc for the two promoter groups, corresponding to free zinc concentrations of 4.5×10(-16) M and 7.9×10(-16) M for the less sensitive and sensitive promoters, respectively. The graded expression of target genes is a clever outcome of subtly modulating Zur-DNA binding affinities in response to zinc availability. It enables bacteria to detect metal depletion with improved sensitivity and optimize gene-expression pattern.

Positive and negative feedback regulatory loops of thiol-oxidative stress response mediated by an unstable isoform of sigmaR in actinomycetes.

Alternate sigma factors provide an effective way of diversifying bacterial gene expression in response to environmental changes. In Streptomyces coelicolor where more than 65 sigma factors are predicted, sigma(R) is the major regulator for response to thiol-oxidative stresses. sigma(R) becomes available when its bound anti-sigma factor RsrA is oxidized at sensitive cysteine thiols to form disulphide bonds. sigma(R) regulon includes genes for itself and multiple thiol-reducing systems, which constitute positive and negative feedback loops respectively. We found that the positive amplification loop involves an isoform of sigma(R) (sigma(R')) with an N-terminal extension of 55 amino acids, produced from an upstream start codon. A major difference between constitutive sigma(R) and inducible sigma(R') is that the latter is markedly unstable (t(1/2) approximately 10 min) compared with the former (> 70 min). The rapid turnover of sigma(R') is partly due to induced ClpP1/P2 proteases from the sigma(R) regulon. This represents a novel way of elaborating positive and negative feedback loops in a control circuit. Similar phenomenon may occur in other actinomycetes that harbour multiple start codons in the sigR homologous gene. We observed that sigH gene, the sigR orthologue in Mycobacterium smegmatis, produces an unstable larger isoform of sigma(H) upon induction by thiol-oxidative stress.

Structural basis for the specialization of Nur, a nickel-specific Fur homolog, in metal sensing and DNA recognition.

Nur, a member of the Fur family, is a nickel-responsive transcription factor that controls nickel homeostasis and anti-oxidative response in Streptomyces coelicolor. Here we report the 2.4-A resolution crystal structure of Nur. It contains a unique nickel-specific metal site in addition to a nonspecific common metal site. The identification of the 6-5-6 motif of the Nur recognition box and a Nur/DNA complex model reveals that Nur mainly interacts with terminal bases of the palindrome on complex formation. This contrasts with more distributed contacts between Fur and the n-1-n type of the Fur-binding motif. The disparity between Nur and Fur in the conformation of the S1-S2 sheet in the DNA-binding domain can explain their different DNA-recognition patterns. Furthermore, the fact that the specificity of Nur in metal sensing and DNA recognition is conferred by the specific metal site suggests that its introduction drives the evolution of Nur orthologs in the Fur family.

Synthesis of gamma-glutamylcysteine as a major low-molecular-weight thiol in lactic acid bacteria Leuconostoc spp.

Some members of lactic acid bacteria are known to synthesize glutathione (GSH) or to import it from growth medium, whereas others are not. Analysis of the genome sequences of several Leuconostoc spp. indicate the presence of the gene gshA that encodes gamma-glutamylcysteine synthetase, but not the gene gshB encoding glutathione synthetase. We report here that, in cells of Leuconostoc kimchii and Leuconostoc mesenteroides, gamma-glutamylcysteine (gamma-GC) is present in large amount, whereas GSH is not detectable. The level of gamma-GC was higher at the stationary phase than at the exponential phase. Expression of the gshA gene in Leuconostoc spp. analyzed by S1 mapping showed the increased mRNA level upon hydrogen peroxide treatment. From high-resolution S1 mapping, the transcriptional start site was mapped and the putative promoter elements were suggested. This work suggests that gamma-GC has a significant role in Leuconostoc spp. as the major low-molecular-weight thiol.

SNP@Domain: a web resource of single nucleotide polymorphisms (SNPs) within protein domain structures and sequences.

The single nucleotide polymorphisms (SNPs) in conserved protein regions have been thought to be strong candidates that alter protein functions. Thus, we have developed SNP@Domain, a web resource, to identify SNPs within human protein domains. We annotated SNPs from dbSNP with protein structure-based as well as sequence-based domains: (i) structure-based using SCOP and (ii) sequence-based using Pfam to avoid conflicts from two domain assignment methodologies. Users can investigate SNPs within protein domains with 2D and 3D maps. We expect this visual annotation of SNPs within protein domains will help scientists select and interpret SNPs associated with diseases. A web interface for the SNP@Domain is freely available at http://snpnavigator.net/ and from http://bioportal.net/.

PSIbase: a database of Protein Structural Interactome map (PSIMAP).

UNLABELLED: Protein Structural Interactome map (PSIMAP) is a global interaction map that describes domain-domain and protein-protein interaction information for known Protein Data Bank structures. It calculates the Euclidean distance to determine interactions between possible pairs of structural domains in proteins. PSIbase is a database and file server for protein structural interaction information calculated by the PSIMAP algorithm. PSIbase also provides an easy-to-use protein domain assignment module, interaction navigation and visual tools. Users can retrieve possible interaction partners of their proteins of interests if a significant homology assignment is made with their query sequences. AVAILABILITY: http://psimap.org and http://psibase.kaist.ac.kr/