Sequence Alignment Research Articles

Sequence Flow: interactive web application for visualizing partial order alignments

BackgroundMultiple sequence alignment (MSA) has proven extremely useful in computational biology, especially in inferring evolutionary relationships via phylogenetic analysis and providing insight into protein structure and function. An alternative to the standard MSA model is partial order alignment (POA), in which aligned sequences are represented as paths in a graph rather than rows in a matrix. While the POA model has proven useful in several applications (e.g. sequencing reads assembly and pangenome structure exploration), we lack efficient visualization tools that could highlight its advantages.ResultsWe propose Sequence Flow – a web application designed to address the above problem. Sequence Flow presents the POA as a Sankey diagram, a kind of graph visualisation typically used for graphs representing flowcharts. Sequence Flow enables interactive alignment exploration, including fragment selection, highlighting a selected group of sequences, modification of the position of graph nodes, structure simplification etc. After adjustment, the visualization can be saved as a high-quality graphic file. Thanks to the use of SanKEY.js – a JavaScript library for creating Sankey diagrams, designed specifically to visualize POAs, Sequence Flow provides satisfactory performance even with large alignments.ConclusionsWe provide Sankey diagram-based POA visualization tools for both end users (Sequence Flow) and bioinformatic software developers (SanKEY.js). Sequence Flow webservice is available at https://sequenceflow.mimuw.edu.pl/. The source code for SanKEY.js is available at https://github.com/Krzysiekzd/SanKEY.js and for Sequence Flow at https://github.com/Krzysiekzd/SequenceFlow.

First report of Botryosphaeria dothidea causing root rot of sugar beet in Serbia.

Botryosphaeria dothidea (Moug.:Fr.) Ces. & De Not. is predominantly recognized as a pathogen of various woody plants, inducing symptoms of stem canker, dieback, and fruit rot worldwide. However, sporadic reports suggest its impact on field crops, including B. dothidea associated with stem canker in soybean and tobacco (Bian et al. 2015; Chen et al. 2021), as well as B. quercuum on sugar beet (Alfieri et al. 1984). In September 2023, during a survey of root rot pathogens of sugar beet (Beta vulgaris L.) in Rimski Šančevi, Serbia (N 45°19´57″; E 19°49'58″), 3% of collected samples exhibited root rot symptoms. Externally, the lesions exhibited a dark brown coloration. On cross-section, the tissue displayed a gradient of discoloration ranging from light to dark brown throughout the roots. The roots were completely rotted. From these samples, two fungal isolates (SR28/II and SR4/III) were obtained from rotted internal root fragments, after washing, surface disinfection (70% ethanol), and plating on potato dextrose agar (PDA). Colonies on PDA were fluffy with abundant aerial mycelium, surface light to dark grey-brown, reverse black in the centre and grey-brown towards the irregular margin, after 7 days at 25°C in the dark. To induce sporulation, isolates were cultivated on 2% water agar with pine needles and incubated under continuous near ultraviolet (NUV) light at room temperature for 30 days. Conidia were hyaline, aseptate, fusiform, subtruncate at the base, subobtuse at the apex, and measured (19.56-) 24.12 - 26.31 (-28.99) x (5.13-) 5.94 - 6.54 (-7.44) µm (mean 25.06 x 6.26 µm, n=100), consistent with description of B. dothidea (Phillips et al. 2013). For molecular identification, DNA was extracted from mycelium of 7-day-old cultures, and internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha gene (TEF) and partial β-tubulin gene (TUB) were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Multiple sequence alignment and BLAST analyses showed that our isolates had 100% sequence similarity with reference isolates of B. dothidea, including ex-type isolate CBS 115476 (= CMW 8000) in ITS (AY236949), TEF (AY236898), and TUB (AY236927). Maximum likelihood phylogeny of concatenated sequences confirmed the identity of the isolates as B. dothidea. Sequences of ITS, TEF and TUB of isolate SR4/III were submitted to GenBank under accession numbers PP908658, PP911334, PP911333, respectively. The pathogenicity of obtained isolates was assessed on 3-month-old sugar beet plants grown in sterile substrate in the greenhouse. For inoculation, the upper parts of the roots were wounded (10x3 mm) 1 cm above the substrate, using a sterilized nail, and 2x2 mm mycelial plugs of 7-day-old culture grown on PDA were inserted and sealed with parafilm. Control plants were inoculated with sterile PDA plugs. Six plants were used per isolate and for control. After 3 weeks of incubation, the inoculated plants were removed from the substrate and brown rot symptoms were observed upon cross-sectional examination, whereas control roots remained asymptomatic. The pathogen was successfully reisolated and morphologically identified as B. dothidea, fulfilling Koch's postulates. The production of sugar beet, an important industrial crop in Serbia valued for its sugar content, is imperiled by various pathogens. To our knowledge, this is the first report of B. dothidea causing root rot of sugar beet in Serbia and the world. Given its widespread distribution on other hosts and potential aggressiveness, the presence of B. dothidea in sugar beet should not be neglected.

Detection of replication-competent circular single stranded DNA from Deoni cattle

Bovine meat and milk factors (BMMF) having structural similarity to plasmids of Acinetobacter baumanni and some single stranded (ss) DNA viruses are free circulating replication-competent circular ssDNA molecules which were first isolated from serum of Bos taurus animals. Isolation of BMMF from tissues of multiple sclerosis as well as some cancers led researchers to conclude that the consumption of these agents through milk and meat have a positive correlation with these diseases and that the absence of such molecules in B. indicus animals could account to the low occurrence of cancer in those areas consuming products of indigenous animals. Hence, the present study was designed to investigate its presence in Deoni, one of the indigenous cattle breeds of India. The DNA isolated from milk samples were subjected to rolling circle amplification (RCA) using exo-resistant random primers. The products of RCA were then used as template for PCR for amplification of circular DNA. The amplicons obtained of size around 2000bp were cloned in E. coli and sequenced. The BLASTn, multiple sequence alignment and phylogenetic analysis of the obtained sequences were carried out to identify the similarities of these isolates. The sequence showed maximum homology with either BMMF2 or plasmids of Acinetobactor spp. Replication initiation protein (Rep) which showed similarity to bacterial plasmids was present in the sequence. This is the pioneer report on the presence of BMMF in milk of indigenous cattle of India. Keywords: BMMF, replication-competent, circular ssDNA, Deoni

Prediction of conformational states in a coronavirus channel using Alphafold-2 and DeepMSA2: Strengths and limitations

The envelope (E) protein is present in all coronavirus genera. This protein can form pentameric oligomers with ion channel activity which have been proposed as a possible therapeutic target. However, high resolution structures of E channels are limited to those of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for the recent COVID-19 pandemic. In the present work, we used Alphafold-2 (AF2), in ColabFold without templates, to predict the transmembrane domain (TMD) structure of six E-channels representative of genera alpha-, beta- and gamma-coronaviruses in the Coronaviridae family. High-confidence models were produced in all cases when combining multiple sequence alignments (MSAs) obtained from DeepMSA2. Overall, AF2 predicted at least two possible orientations of the α-helices in E-TMD channels: one where a conserved polar residue (Asn-15 in the SARS sequence) is oriented towards the center of the channel, ‘polar-in’, and one where this residue is in an interhelical orientation ‘polar-inter’. For the SARS models, the comparison with the two experimental models ‘closed’ (PDB: 7K3G) and ‘open’ (PDB: 8SUZ) is described, and suggests a ∼60˚ α-helix rotation mechanism involving either the full TMD or only its N-terminal half, to allow the passage of ions. While the results obtained are not identical to the two high resolution models available, they suggest various conformational states with striking similarities to those models. We believe these results can be further optimized by means of MSA subsampling, and guide future high resolution structural studies in these and other viral channels.

Identification of Shemin pathway genes for tetrapyrrole biosynthesis in bacteriophage sequences from aquatic environments

Tetrapyrroles such as heme, chlorophyll, and vitamin B12 are essential for various metabolic pathways. They derive from 5-aminolevulinic acid (5-ALA), which can be synthesized by a single enzyme (5-ALA synthase or AlaS, Shemin pathway) or by a two-enzyme pathway. The genomes of some bacteriophages from aquatic environments carry various tetrapyrrole biosynthesis genes. Here, we analyze available metagenomic datasets and identify alaS homologs (viral alaS, or valaS) in sequences corresponding to marine and freshwater phages. The genes are found individually or as part of complete or truncated three-gene loci encoding heme-catabolizing enzymes. Amino-acid sequence alignments and three-dimensional structure prediction support that the valaS sequences likely encode functional enzymes. Indeed, we demonstrate that is the case for a freshwater phage valaS sequence, as it can complement an Escherichia coli 5-ALA auxotroph, and an E. coli strain overexpressing the gene converts the typical AlaS substrates glycine and succinyl-CoA into 5-ALA. Thus, our work identifies valaS as an auxiliary metabolic gene in phage sequences from aquatic environments, further supporting the importance of tetrapyrrole metabolism in bacteriophage biology.

Genome-Wide Identification of the RALF Gene Family and Expression Pattern Analysis in Zea mays (L.) under Abiotic Stresses.

Rapid Alkalization Factor (RALF) is a signaling molecule in plants that plays a crucial role in growth and development, reproductive processes, and responses to both biotic and abiotic stresses. Although RALF peptides have been characterized in Arabidopsis and rice, a comprehensive bioinformatics analysis of the ZmRALF gene family in maize is still lacking. In this study, we identified 20 RALF genes in the maize genome. Sequence alignment revealed significant structural variation among the ZmRALF family genes. Phylogenetic analysis indicates that RALF proteins from Arabidopsis, rice, and maize can be classified into four distinct clades. Duplication events suggest that the expansion of the RALF gene family in maize primarily relies on whole-genome duplication. ZmRALF genes are widely expressed across various tissues; ZmRALF1/15/18/19 are highly expressed in roots, while ZmRALF6/11/14/16 are predominantly expressed in anthers. RNA-seq and RT-qPCR demonstrated that the expression levels of ZmRALF7, ZmRALF9, and ZmRALF13 were significantly up-regulated and down-regulated in response to PEG and NaCl stresses, respectively. Overall, our study provides new insights into the role of the RALF gene family in abiotic stress.

Genome Survey of Male Rana dybowskii to Further Understand the Sex Determination Mechanism.

Rana dybowskii is one of the important aquaculture species in Northeast China. The fallopian tubes of female R. dybowskii are used to prepare oviductus ranae (an important traditional Chinese medicine). Therefore, R. dybowskii females have higher economical value than males. An increasing female R. dybowskii population can increase the benefits from R. dybowskii culture. However, the genome of amphibians is complex, making it difficult to investigate their sex determination mechanism. In this study, we analyzed the genome of male R. dybowskii using next-generation sequencing technology. A total of 200,046,452,400 bp of clean data were obtained, and the K-mer analysis indicated that the depth was 50×. The genome size of R. dybowskii was approximately 3585.05 M, with a heterozygosity rate, repeat sequence ratio, and genome GC content of 1.15%, 68.96%, and approximately 43.0%, respectively. In total, 270,785 contigs and 498 scaffolds were generated. The size of the contigs and scaffolds was 3,748,543,415 and 3,765,862,278 bp, respectively, with the N50 length of 31,988 and 336,385,783. The longest contig and scaffold were of the size 137,967,485 and 1,808,367,828 bp, respectively. The number of contigs and scaffolds > 10K nt was 99,620 and 451, respectively. Through annotation, 40,913 genes were obtained, including 156,609 CDS (i.e., 3.83 CDS per gene). Sequence alignment was performed with the assembled scaffolding genome in this study. Two and one fragment had high homology with two male-specific DNA molecular markers of R. dybowskii discovered previously (namely, MSM-222 and MSM-261, respectively). In addition, the Dmrt1 gene of R. dybowskii was obtained with a length of 18,893 bp by comparison and splicing. The forward primers amplifying MSM-222 and MSM-261 were located at 322-343 and 14,501-14,526 bp of Dmrt1, respectively. However, sequence alignment revealed that MSM-222 and MSM-261 were not located on Dmrt1, and only some homologous parts were observed. This indicated that in addition to Dmrt1, other important genes may play a crucial role in the sex determination mechanism of R. dybowskii. Our study provided a foundation for the subsequent high-quality genome construction and provided important genomic resources for future studies on R. dybowskii.

Development of Tertiary Structure of Laccase from Geobacillus uzenensis using Homology Modelling

Laccases are a versatile enzyme with immense potential in various biotechnological applications. In this study, the protein structure prediction of laccase from Geobacillus uzenensis, a bacteria known for its robust enzymatic activity is focused. The protein structural elucidation of this laccase is crucial to understand its substrate binding and overall functionality. Protein structure can be determined by computational prediction, which is commonly known as homology modelling. The three-dimensional (3D) model was built based on the available crystal structures of related laccase enzymes and aligned with the primary sequence of the target protein. The resulting model exhibited a high degree of structural similarity and conservation of key catalytic residues, supporting its reliability for further analyses. Firstly, the suitable template was identified with 85.61% of sequence identity determined by sequence alignment. Then, MODELLER program was used to predict the model using the method of satisfaction of spatial restraints. The model was then analyzed for its quality by computational analysis tools such as Ramachandran's Plot. Finally, the binding site of the protein was identified using the GRaSP computational tool. These findings provide significant insight on comprehensive analysis of the laccase from G. uzenensis, by providing a structural framework to unravel its functional properties and substrate specificity.

Serotype independent protection induced by a vaccine based on the IgM protease of Streptococcus suis and proposal for a new immunity-based classification system

The IgM protease (IdeSsuis gene; Gene ID 8153996) of Streptococcus suis is a putative virulence factor that has been shown to be a protective vaccine antigen for pigs (Seele et al. Vaccine 33:2207–12, 2015). To assess its potential as a cross-protective antigen, the amino acid variability among prevalent clinical isolates in various regions and among various serotypes was investigated. Multi-sequence alignment of full-length amino acid sequences of S. suis IgM protease, available in the public domain (status Jan-2022) supplemented with in-house sequences, i.e. a total of 1999 sequences, revealed that the IgM protease of S. suis clusters into three main evolutionary distinct branches: groups A, B and C. Group A, 82% of the sequences in the database, was associated with clinical isolates of various serotypes. Group B, 6% of the strains in the database, was associated with clinical isolates mainly in the EU and mainly belonging to serotype (st) 9. Group C, 12% of the strains in the database, was largely associated with healthy carrier isolates, i.e. nose or tonsil isolates of various serotypes but in particular with st9 and un-typable strains. Within the groups A, B and C, high levels of amino acid identity were observed (> 75%), whereas between groups A and B, the percentage amino acid identity was approximately 30% and between groups A and C approximately 55%. Experimental Escherichia coli expressed recombinant subunit vaccines based on the IgM protease group A sequence of st1 strain B10-99, st2 strain 10 or st7 strain 14009-1, induced serotype independent protection in pigs against challenge with all group A strains tested, i.e. strains of different parts of the phylogenetic tree and of different serotypes including st1, 2, 9 and 14. Protection was observed after vaccination of piglets at 3 and 5 weeks of age and subsequent challenge at 7 weeks but also after vaccination of gilts at 6 and 2 weeks before anticipated parturition and challenge of the offspring up to at least 8 weeks of age. No protection was observed against challenge with st9 strain SZ2000-6264 having group B IgM protease. A recombinant subunit vaccine based on the group B IgM protease sequence, also did not protect against challenge with the homologous group B st9 challenge strain. The results indicate that a vaccine based on a group A IgM protease induces protection against all S. suis strains that express the group A IgM protease. Depending on the geographical region such a vaccine is expected to protect against 60–100% of the virulent S. suis strains. Since the novel proposed IgM protease classification is highly relevant, a PCR was developed and validated, to be able to classify clinical isolates into IgM protease groups A, B and C and predict the cross-protection that can be expected from a group A based IgM protease vaccine.

NovoLign: metaproteomics by sequence alignment

Abstract Tremendous advances in mass spectrometric and bioinformatic approaches have expanded proteomics into the field of microbial ecology. The commonly used spectral annotation method for metaproteomics data relies on database searching, which requires sample-specific databases obtained from whole metagenome sequencing experiments. However, creating these databases is complex, time-consuming, and prone to errors, potentially biasing experimental outcomes and conclusions. This asks for alternative approaches that can provide rapid and orthogonal insights into metaproteomics data. Here we present NovoLign, a de novo metaproteomics pipeline that performs sequence alignment of de novo sequences from complete metaproteomics experiments. The pipeline enables rapid taxonomic profiling of complex communities and evaluates the taxonomic coverage of metaproteomics outcomes obtained from database searches. Furthermore, the NovoLign pipeline supports the creation of reference sequence databases for database searching to ensure comprehensive coverage. We assessed the NovoLign pipeline for taxonomic coverage and false positive annotations using a wide range of in silico and experimental data, including pure reference strains, laboratory enrichment cultures, synthetic communities, and environmental microbial communities. In summary, we present NovoLign, a de novo metaproteomics pipeline that employs large-scale sequence alignment to enable rapid taxonomic profiling, evaluation of database searching outcomes, and the creation of reference sequence databases. The NovoLign pipeline is publicly available via: https://github.com/hbckleikamp/NovoLign.

Graph-based self-supervised learning for repeat detection in metagenomic assembly.

Repetitive DNA (repeats) poses significant challenges for accurate and efficient genome assembly and sequence alignment. This is particularly true for metagenomic data, in which genome dynamics such as horizontal gene transfer, gene duplication, and gene loss/gain complicate accurate genome assembly from metagenomic communities. Detecting repeats is a crucial first step in overcoming these challenges. To address this issue, we propose GraSSRep, a novel approach that leverages the assembly graph's structure through graph neural networks (GNNs) within a self-supervised learning framework to classify DNA sequences into repetitive and nonrepetitive categories. Specifically, we frame this problem as a node classification task within a metagenomic assembly graph. In a self-supervised fashion, we rely on a high-precision (but low-recall) heuristic to generate pseudolabels for a small proportion of the nodes. We then use those pseudolabels to train a GNN embedding and a random forest classifier to propagate the labels to the remaining nodes. In this way, GraSSRep combines sequencing features with predefined and learned graph features to achieve state-of-the-art performance in repeat detection. We evaluate our method using simulated and synthetic metagenomic data sets. The results on the simulated data highlight GraSSRep's robustness to repeat attributes, demonstrating its effectiveness in handling the complexity of repeated sequences. Additionally, experiments with synthetic metagenomic data sets reveal that incorporating the graph structure and the GNN enhances the detection performance. Finally, in comparative analyses, GraSSRep outperforms existing repeat detection tools with respect to precision and recall.

Sequence-Similar Protein Domain Pairs With Structural or Topological Dissimilarity.

For a variety of applications, protein structures are clustered by sequence similarity, and sequence-redundant structures are disregarded. Sequence-similar chains are likely to have similar structures, but significant structural variation, as measured with RMSD, has been documented for sequence-similar chains and found usually to have a functional explanation. Moving two neighboring stretches of backbone through each other may change the chain topology and alter possible folding paths. The size of this motion is compatible to a variation in a flexible loop. We search and find domains with alternate chain topology in CATH4.2 sequence families relatively independent of sequence identity and of structural similarity as measured by RMSD. Structural, topological, and functional representative sets should therefore keep sequence-similar domains not just with structural variation but also with topological variation. We present BCAlign that finds Alignment and superposition of protein Backbone Curves by optimizing a user chosen convex combination of structural derivation and derivation between the structure-based sequence alignment and an input sequence alignment. Steric and topological obstructions from deforming a curve into an aligned curve are then found by a previously developed algorithm. For highly sequence-similar domains, sequence-based structural alignment better represents the chains motion and generally reveals larger structural and topological variation than structure-based does. Fold-switching protein pairs have been reported to be most frequent between X-ray and NMR structures and estimated to be underrepresented in the PDB as the alternate configuration is harder to resolve. Here we similarly find chain topology most frequently altered between X-ray and NMR structures.

Characterization of the myostatin gene in the neotropical species Piaractus mesopotamicus and the possibility of its use in genetic improvement programs.

The myostatin gene has played an important role in the genetic improvement of the main species of economic importance; however, it has not yet been described for some Neotropical fish essential for aquaculture. This study aimed to characterize the myostatin gene of pacu, Piaractus mesopotamicus, and investigate the association of a microsatellite marker in this gene with the weight of fish. The myostatin gene sequence was obtained after following a RACE-PCR strategy based on a partial mRNA sequence available in the GenBank database and the alignment of myostatin sequences from other fish species. The obtained sequence for the P. mesopotamicus gene was analyzed for short tandem repeats, and one dinucleotide was observed at the 3´untranslated region. A short tandem repeat polymorphism was verified in a wild population. Subsequently, the STR was evaluated in a test population of 232 animals in two 220m² concrete tanks at the Aquaculture Center of Unesp. Eight alleles and 22 genotype combinations were identified. A significant association was observed between microsatellite marker polymorphisms and the weight traits (WEIGHT1 and WEIGHT2). Alleles 210, 222, 226, and 230 were found to favor weight gain. In summary, this study contributes to the characterization of the myostatin gene in pacu fish and identifies an association between a STR and weight traits. Thus, this gene could be used as a target for genetic breeding using molecular strategies such as CRISPR and quantitative strategies such as marker-assisted selection, which would contribute to improving the production of the species.

Research on Sequence Clustering and Alignment in DNA Storage Based on the K-Means Model

Research on Sequence Clustering and Alignment in DNA Storage Based on the K-Means Model

Campycins are novel broad-spectrum antibacterials killing Campylobacter jejuni

Pyocins are high molecular weight bacteriocins produced by Pseudomonas aeruginosa that can be retargeted to new bacterial species by exchanging the pyocin tail fibers with bacteriophage receptor binding proteins (RBPs). Here, we develop retargeted pyocins called campycins as new antibacterials to precisely and effectively kill the major foodborne pathogen Campylobacter jejuni. We used two diverse RBPs (H-fibers) encoded by CJIE1 prophages found in the genomes of C. jejuni strains CAMSA2147 and RM1221 to construct campycin 1 and campycin 2, respectively. Campycins 1 and 2 could target all C. jejuni strains tested due to complementary antibacterial spectra. In addition, both campycins led to more than 3 log reductions in C. jejuni counts under microaerobic conditions at 42 °C, whereas the killing efficiency was less efficient under anaerobic conditions at 5 °C. Furthermore, we discovered that both H-fibers used to construct the campycins bind to the essential major outer membrane protein (MOMP) present in all C. jejuni in a strain-specific manner. Protein sequence alignment and structural modeling suggest that the highly variable extracellular loops of MOMP form the binding sites of the diverse H-fibers. Further in silico analyses of 5000 MOMP sequences indicated that the protein falls into three major clades predicted to be targeted by either campycin 1 or campycin 2. Thus, campycins are promising antibacterials against C. jejuni and are expected to broadly target numerous strains of this human pathogen in nature and agriculture.Key points• Campycins are engineered R-type pyocins containing H-fibers from C. jejuni prophages• Campycins reduce C. jejuni counts by >3 logs at conditions promoting growth• Campycins bind to the essential outer membrane protein MOMP in a strain-dependent way

The first report of prion protein gene sequences in Dybowski’s frog and the American bullfrog: high amyloid propensity of the frog prion protein

Prion diseases are fatal infectious neurodegenerative diseases caused by the proteinase K-sensitive form of prion protein (PrPSc). The exact origin of prion seeding and the transition factor of PrPSc has not been elucidated. The main hosts of prion diseases are herbivores, so the feces and corpses of Amphibians can seed PrPSc through ecosystems. The frog is an excellent candidate for transmission studies for this reason, but genetic analyses of the prion protein gene (PRNP) in the context of prion-related characteristics of frog species are lacking. We amplified frog PRNP gene sequences in Dybowski’s frog and the American bullfrog by polymerase chain reaction (PCR) and amplicon sequencing. In addition, we carried out multiple sequencing alignments and annotated major PrP components including signal peptide, tandem repeat domain, and PrPC-PrPSc interaction region of frog PrPs by bioinformatics tools. We predicted secondary and tertiary structures and amyloid propensities of frog PrPs using AlphaFold2 and AMYCO, respectively. We obtained DNA sequences of the PRNP gene in Dybowski’s frog and the American bullfrog, as well as a partially conserved palindromic sequence (PrPC-PrPSc interaction region) and absence of tandem repeat region of PrP in seven frog species. We analyzed protein structure of among these frog species and found that the high Himalaya frog has high aggregation propensity and the western clawed frog does not have the N-terminal signal peptide. To the best of our knowledge, this was the first comparative genetic study regarding prion-related features of frog species.

Characterizing and Engineering Rhamnose-Inducible Regulatory Systems for Dynamic Control of Metabolic Pathways in Streptomyces.

Fine-tuning gene expression is of great interest for synthetic biotechnological applications. This is particularly true for the genus Streptomyces, which is well-known as a prolific producer of diverse natural products. Currently, there is an increasing demand to develop effective gene induction systems. In this study, bioinformatic analysis revealed a putative rhamnose catabolic pathway in multiple Streptomyces species, and the removal of the pathway in the model organism Streptomyces coelicolor impaired its growth on minimal media with rhamnose as the sole carbon source. To unravel the regulatory mechanism of RhaR, a LacI family transcriptional regulator of the catabolic pathway, electrophoretic mobility shift assays (EMSAs) were performed to identify potential target promoters. Multiple sequence alignments retrieved a consensus sequence of the RhaR operator (rhaO). A synthetic biology-based strategy was then deployed to build rhamnose-inducible regulatory systems, referred to as rhaRS1 and rhaRS2, by assembling the repressor/operator pair RhaR/rhaO with the well-defined constitutive kasO* promoter. Both rhaRS1 and rhaRS2 exhibited a high level of induced reporter activity, with no leaky expression. rhaRS2 has been proven successful for the programmable production of actinorhodin and violacein in Streptomyces. Our study expanded the toolkit of inducible regulatory systems that will be broadly applicable to many other Streptomyces species.

Evolution of the lipoxygenase gene family in Pyropia haitanensis and its response to biotic and abiotic stresses

Macroalgae contain a chemically diverse and unique set of oxylipins that derived from lipid peroxidation play pivotal roles in response to various environmental stresses in the intertidal zone. Specific enzymes involved in biosynthesizing these oxylipins remain largely unknown. Lipoxygenase (LOX) is a key enzyme in the oxylipin biosynthetic pathway. Here, six homologous LOX genes were identified in Pyropia haitanensis. To investigate the evolutionary status of the LOX gene family in red algae and its role in the adaptation of P. haitanensis to the intertidal environment, this study analyzed the evolution, structure, and enzymatic activities of these genes. Phylogenetic analysis and homologous sequence alignment indicated that the P. haitanensis LOX genes had two evolutionary origins. PhLOX and PhLOX2 possess an SRPBCC domain at the N-terminus that may have been acquired from marine bacteria through horizontal gene transfer, while PhLOX3–6 may have been acquired from cyanobacteria. Cis-Regulatory analysis revealed numerous elements upstream of the PhLOX genes associated with abiotic stress and hormone regulation, as well as binding sites for defense-related transcription factors. The in-vitro enzymatic activity of PhLOXs was detected, revealing that PhLOX and PhLOX2 exhibited the highest activities. PhLOX3–6 showed a substrate preference for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), but it had lower activity. The transcriptional expression levels of the six PhLOX genes varied under different biotic and abiotic stresses. For example, all PhLOX genes were significantly upregulated after mechanical damage, while PhLOX5 responded to low temperatures. In summary, P. haitanensis acquired a series of LOX genes from marine bacteria and cyanobacteria during its evolutionary history, among which PhLOX and PhLOX2 were selected as the likely primary functional genes. Its functional diversity to cope with various environmental stresses is achieved through the complex transcriptional regulation of PhLOX genes.

The conserved cysteines at position 18, 36, and 49 of Autographa californica multiple nucleopolyhedrovirus VP39 are essential for virus replication.

Autographa californica nucleopolyhedrovirus orf89 (vp39) encodes the major capsid protein VP39. Multiple alignments of protein sequences showed that VP39 has 8 conserved cysteine (Cys) residues. Cysteine residues play an important role in proper function of a protein. To determine the importance of these conserved cysteine residues for virus proliferation, a series of recombinant viruses harboring VP39-Cys mutants were constructed. Viral growth curves and transmission electron microscopy showed that mutation of Cys29, Cys132, Cys169, Cys229, or Cys232 of VP39 to alanine did not affect budded virion production; however, the mutation of Cys18, Cys36, or Cys49 to alanine resulted in interruption of capsid assembly. Co-immunoprecipitation assays showed that mutations of these 8 cysteines individually or simultaneously had no effect on self-association of VP39. Immunofluorescence analysis by confocal microscopy revealed that the subcellular localization of VP39 with mutations in Cys18, Cys36 or Cys49 was exclusively distributed in the cytoplasm of a cell regardless of virus infection or not, while the wild-type VP39 or the VP39 carrying mutations in Cys29, Cys132, Cys169, Cys229, or Cys232 was distributed throughout the cytoplasm and the nucleus. Our results demonstrated that Cys18, Cys36, and Cys49 are essential for the proper localization of VP39, which is a prerequisite for successful nucleocapsid assembly of the virus.

Isolation and functional determination of inward-rectifier K+ channel PbrAKT1 in pear

ABSTRACT Potassium (K+) is essential to plant growth and development. The absorption and transport of K+ by plant roots occurs mainly through K+ channels or transporters. In this study, an AKT1-type channel named PbrAKT1 was successfully cloned and characterised by a series of detailed procedures, including protein sequence alignment, expression and subcellular localisation analysis, as well as functional verification by heterologous expression function validation in yeast mutant R5421, Arabidopsis akt1 mutant, and Xenopus oocytes. The results showed that PbrAKT1 is preferentially expressed in pear roots and exhibits plasma membrane localisation, but its expression is unaffected by low K+ treatment. PbrAKT1 exhibits the function of an inwardly-rectifying K+ channel, and its activity is autonomously regulated when expressed in Xenopus oocytes. In addition, a series of heterologous expression experiments confirmed that PbrAKT1 plays an indispensable role in the processes of K+ transport, as well as alleviating the low-K+ -sensitive phenotype of yeast mutant R5421 and Arabidopsis akt1 mutant. In summary, PbrAKT1 plays an indispensable role in the processes of K+ transport and provide new information on the molecular mechanism of K+ absorption in pear roots.