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A novel genome-wide accessible chromatin visualization, quantitation, and sequencing method is described, which allows in situ fluorescence visualization and sequencing of the accessible chromatin in the mammalian cell. The cells are fixed by formaldehyde crosslinking, and processed using a modified nick translation method, where a nicking enzyme nicks one strand of DNA, and DNA polymerase incorporates biotin-conjugated dCTP, 5-methyl-dCTP, Fluorescein-12-dATP or Texas Red-5-dATP, dGTP, and dTTP. This allows accessible chromatin DNA to be labeled for visualization and on bead NGS library preparation. This technology allows cellular level chromatin accessibility quantification and genomic analysis of the epigenetic information in the chromatin, particularly accessible promoter, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Genome-wide accessible chromatin sequencing and identification has enabled deciphering the epigenetic information encoded in chromatin, revealing accessible promoters, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding. The starting biological materials are often fixed using formaldehyde crosslinking. Here, we describe accessible chromatin library preparation from low numbers of formaldehyde-crosslinked cells using a modified nick translation method, where a nicking enzyme nicks one strand of DNA and DNA polymerase incorporates biotin-conjugated dATP, dCTP, and methyl-dCTP. Once the DNA is labeled, it can be isolated for NGS library preparation. We termed this method as universal NicE-seq (nicking enzyme-assisted sequencing). We also demonstrate a single tube method that enables direct NGS library preparation from low cell numbers without DNA purification. Furthermore, we demonstrated universal NicE-seq on FFPE tissue section sample., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In mammalian cells, SET8 mediated Histone H4 Lys 20 monomethylation (H4K20me1) has been implicated in regulating mitotic condensation, DNA replication, DNA damage response, and gene expression. Here we show SET8, the only known enzyme for H4K20me1 is post-translationally poly ADP-ribosylated by PARP1 on lysine residues. PARP1 interacts with SET8 in a cell cycle-dependent manner. Poly ADP-ribosylation on SET8 renders it catalytically compromised, and degradation via ubiquitylation pathway. Knockdown of PARP1 led to an increase of SET8 protein levels, leading to aberrant H4K20me1 and H4K20me3 domains in the genome. H4K20me1 is associated with higher gene transcription levels while the increase of H4K20me3 levels was predominant in DNA repeat elements. Hence, SET8 mediated chromatin remodeling in mammalian cells are modulated by poly ADP-ribosylation by PARP1., (© 2022. The Author(s).)

Background: Accessible chromatin landscape allows binding of transcription factors, and remodeling of promoter and enhancer elements during development. Chromatin accessibility along with integrated multiomics approaches have been used for determining molecular subtypes of cancer in patient samples., Results: One-pot Universal NicE-seq (One-pot UniNicE-seq) is an improved accessible chromatin profiling method that negate DNA purification and incorporate sonication free enzymatic fragmentation before library preparation and is suited to a variety of mammalian cells. One-pot UniNicE-seq is versatile, capable of profiling 4% formaldehyde fixed chromatin in as low as 25 fixed cells. Accessible chromatin profile is more efficient on formaldehyde-fixed cells using one-pot UniNicE-seq compared to Tn5 transposon mediated methods, demonstrating its versatility., Conclusion: One-pot UniNicE-seq allows the entire process of accessible chromatin labeling and enrichment in one pot at 4% formaldehyde cross-linking conditions. It doesn't require enzyme titration, compared to other technologies, since accessible chromatin is labelled with 5mC incorporation and deter degradation by nicking enzyme, thus opening the possibility for automation., (© 2021. The Author(s).)

Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negates the optimization step and is suited to a variety of mammalian cells and tissues. Addition of 5-methyldeoxycytidine triphosphate during accessible chromatin labeling and an on-bead library making step substantially improved the signal to noise ratio while protecting the accessible regions from repeated nicking in cell lines, mouse T cells, mouse kidney, and human frozen tissue sections. We also demonstrate one tube UniNicE-seq for the FFPE tissue section for direct NGS library preparation without sonication and DNA purification steps. These refinements allowed reliable mapping of accessible chromatin for high-resolution genomic feature studies.

Chromatin accessibility is a predictor of gene expression, cell division, and cell type specificity. NicE-viewSeq (Nicking Enzyme-assisted viewing and Sequencing) allows accessible chromatin visualization and sequencing with overall lower mitochondrial DNA and duplicated sequences interference relative to ATAC-see. Using NicE-viewSeq, we interrogated the accessibility of chromatin in a cell cycle (G1, S, and G2/M)-specific manner using mammalian cells. Despite DNA replication and subsequent condensation of chromatin to chromosomes, chromatin accessibility remained generally preserved with minimal subtle alterations. Genome-wide alteration of chromatin accessibility within TSS and enhancer elements gradually decreased as cells progressed from G1 to G2M, with distinct differential accessibility near consensus transcription factors sites. Inhibition of histone deacetylases promoted accessible chromatin within gene bodies, correlating with apoptotic gene expression. In addition, reduced chromatin accessibility for the MYC oncogene pathway correlated with downregulation of pertinent genes. Surprisingly, repetitive RNA loci expression remained unaltered following histone acetylation-mediated increased accessibility. Therefore, we suggest that subtle changes in chromatin accessibility are a prerequisite during the cell cycle and histone deacetylase inhibitor-mediated therapeutics., Competing Interests: Conflict of Interest The authors declare no potential conflicts of interest, (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The proportion of people suffering from cardiovascular diseases has risen by 34% in the last 15 years in India. Cardiomyopathy is among the many forms of CVD s present. Infection of heart muscles is the suspected etiological agent for the same. Oral pathogens gaining entry into the bloodstream are responsible for such infections. Streptococcus mutans is an oral pathogen with implications in cardiovascular diseases. Previous studies have shown certain strains of S. mutans are found predominantly within atherosclerotic plaques and extirpated valves. To decipher the genetic differences responsible for endothelial cell invasion, we have sequenced the genome of Streptococcus mutans B14. Pan-genome analysis, search for adhesion proteins through a special algorithm, and protein-protein interactions search through HPIDB have been done. Pan-genome analysis of 187 whole genomes, assemblies revealed 6965 genes in total and 918 genes forming the core gene cluster. Adhesion to the endothelial cell is a critical virulence factor distinguishing virulent and non-virulent strains. Overall, 4% of the total proteins in S. mutans B14 were categorized as adhesion proteins. Protein-protein interaction between putative adhesion proteins and Human extracellular matrix components was predicted, revealing novel interactions. A conserved gene catalyzing the synthesis of branched-chain amino acids in S. mutans B14 shows possible interaction with isoforms of cathepsin protein of the ECM. This genome sequence analysis indicates towards other proteins in the S. mutans genome, which might have a specific role to play in host cell interaction., Competing Interests: Conflict of interestThe authors declare that they do not have any conflict of interests in the publication of this manuscript., (© Association of Microbiologists of India 2019.)

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the INSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5- to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P aeruginosa PGPR2 for root colonization., (Copyright © 2019 Sivakumar et al.)

Photorhabdus bacteria exhibit contrasting lifestyles; they are virulent insect pathogens but symbionts of the entomopathogenic Heterorhabditis nematodes. Photorhabdus genomes encode several secondary metabolites and insecticidal protein toxins. Here, we present the draft genome sequences for five Photorhabdus strains isolated from Heterorhabditis nematodes collected from various geographical regions of India.

Brucellosis is a zoonotic disease caused by Brucella spp. Brucella spp. can be sub-typed by multilocus sequence typing (MLST) method, which targets a set of housekeeping genes. We have developed a core genome MLST (cgMLST) typing scheme to distinguish and differentiate species of Brucella up to biovar level. A total of 407 whole (complete and draft) genome sequences of different Brucella strains were used in this study. Genome sequences were filtered using the BLAST score ratio (BSR)-based allele calling algorithm, and we found that 164 cgMLST target loci are shared in all the 407 genome sequences. These 164 loci were used to develop the cgMLST scheme and further evaluated to sub-type different species of Brucella. Based on our cgMLST scheme, Brucella spp. were classified into 287 sequence types (STs). A phylogenetic tree was constructed based on the STs derived from the cgMLST analysis. The phylogenetic tree differentiated all the 11 Brucella spp. and five biovars of B. suis. B. vulpis formed the outmost clade followed by B. inopinata and B. microti. Among the four subgroups of B. abortus, group A and B were differentiated based on their geographic origins. Similarly, three subgroups of B. melitensis were separated based on their geographical origins with few exceptions. B. neotomae that infect rodents were distinguished from other Brucella spp. B. canis showed the closest relationship with B. suis bv. 4, followed by B. suis bv. 3 and bv. 1. Brucella spp. associated with the marine mammals, such as B. ceti and B. pinnipedialis were closely related. Of these, B. ceti strains isolated from dolphins and porpoise were differentiated into two groups. We incorporated our cgMLST tool in BrucellaBase (http://www.dbtbrucellosis.in/brucella_cgmlst.html), which will be helpful to predict the cgMLST allelic profile and the ST of a newly sequenced genome., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Brucellosis is an important zoonotic disease caused by Brucella spp. We present a phylogeny of 552 strains based on genome-wide single nucleotide polymorphisms (SNPs) determined by an alignment-free k-mer approach. A total of 138,029 SNPs were identified from 552 Brucella genomes. Of these, 31,152 and 106,877 were core and non-core SNPs, respectively. Based on pan-genome analysis 11,937 and 972 genes were identified as pan and core genome, respectively. The pan-genome-wide analysis studies (Pan-GWAS) could not identify the group-specific variants in Brucella spp. Therefore, we focused on SNP based genome-wide association studies (SNP-GWAS) to identify the species-specific genetic determinants in Brucella spp. Phylogenetic tree representing eleven recognized Brucella spp. showed 16 major lineages. We identified 143 species-specific SNPs in Brucella abortus that are conserved in 311 B. abortus genomes. Of these, 141 species-specific SNPs were confined in the positively significant SNPs of B. abortus using SNP-GWAS. Since conserved in all the B. abortus genomes studied, these SNPs might have originated very early during the evolution of B. abortus and might be responsible for the evolution of B. abortus with cattle as the preferred host. Similarly, we identified 383 species-specific SNPs conserved in 132 Brucella melitensis genomes. Of these 379 species-specific SNPs were identified as positively associated using GWAS. Interestingly, >98% of the SNPs that are significantly, positively associated with the traits showed 100% sensitivity and 100% specificity. These identified species-specific core-SNPs identified in Brucella genomes could be responsible for the speciation and their respective host adaptation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Brucella melitensis is an intracellular pathogen resides in the professional and non-professional phagocytes of the host, causing zoonotic disease brucellosis. The stealthy nature of the Brucella makes it's highly pathogenic, and it is hard to eliminate the bacteria completely from the infected host. Hitherto, no licensed vaccines are available for human brucellosis. In this study, we identified potential antigens for vaccine development from non-classically secreted proteins through reverse vaccinology approach. Based on the systemic screening of non-classically secreted proteins of B. melitensis 16M, we identified nine proteins as potential vaccine candidates. Among these, Omp31 and Omp22 are known immunogens, and its role in the virulence of Brucella is known. Roles of other proteins in the pathogenesis are yet to be studied. From the nine proteins, we identified six novel antigenic epitopes that can elicit both B-cell and T-cell immune responses. Among the nine proteins, the epitopes were predicted from Omp31 immunogenic protein precursor, Omp22 protein precursor, extracellular serine protease, hypothetical membrane-associated protein, iron-regulated outer membrane protein FrpB. Further, we designed a multitope vaccine using Omp31 immunogenic protein precursor, Omp22 protein precursor, extra cellular serine protease, iron-regulated outer membrane protein FrpB, hypothetical membrane-associated protein, and LPS-assembly protein LptD and polysaccharide export protein identified in the previous study. Epitopes were joined using amino acid linkers such as EAAAK and GPGPG. Cholera toxin subunit B, the nontoxic part of cholera toxin, was used as an adjuvant and it was linked to the N-terminal of the multitope vaccine candidate. The designed vaccine candidate was modeled, validated and the physicochemical properties were analyzed. Results revealed that the vaccine candidate is soluble, stable, non-allergenic, antigenic and 87% of residues of the designed vaccine candidate is located in the favored region. In conclusion, the computational analysis showed that the newly designed multitope protein could be used to develop a promising vaccine for human brucellosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Small RNAs (sRNAs) are the small regulatory molecules that regulate various biological processes in bacteria. Though the functions of sRNAs are well documented, very little information is available on the sRNAs of Brucella spp. The otpR is the response regulator of a two-component regulatory system, which plays a significant role in Brucella virulence. In this study, we identified the sRNAs expressed in B. melitensis 16M and its otpR mutant under acidic stress from the RNAseq dataset. We identified 94 trans-encoded and 948 cis-encoded sRNAs in B. melitensis 16M. In B. melitensis 16M ΔotpR under acidic stress 99 trans-encoded and 877 cis-encoded sRNAs were identified. Among these, 12 trans-encoded and 43 cis-encoded sRNAs were upregulated in B. melitensis 16M ΔotpR, with an adjusted P-value≤0.05. The mRNA targets of these sRNAs were predicted. Further, the levels of mRNA targets were examined, and the sRNA-mediated regulatory network was predicted. Functional classification and pathway analysis of mRNA targets provided evidence that sRNAs are involved in different metabolic pathways including carbohydrates, amino acids, lipids, nucleotides transport and metabolism, cell membrane biogenesis and intracellular trafficking of Brucella. We also found that eight transcriptional regulators including a quorum sensing regulator, vjbR are down-regulated by sRNAs. These transcriptional regulators might be responsible for the regulation of several other genes in the otpR mutant. The trans-encoded BsnR88 and cis-encoded BsnR980, BsnR998, BsnR881, BsnR1001, BsnR891, BsnR883, BsnR905 are regulating virB operon genes coding for type IV secretion system (T4SS), which is the major virulence factor of Brucella., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Brucella sp. causes a major zoonotic disease, brucellosis. Brucella belongs to the family Brucellaceae under the order Rhizobiales of Alphaproteobacteria. We present BrucellaBase, a web-based platform, providing features of a genome database together with unique analysis tools. We have developed a web version of the multilocus sequence typing (MLST) (Whatmore et al., 2007) and phylogenetic analysis of Brucella spp. BrucellaBase currently contains genome data of 510 Brucella strains along with the user interfaces for BLAST, VFDB, CARD, pairwise genome alignment and MLST typing. Availability of these tools will enable the researchers interested in Brucella to get meaningful information from Brucella genome sequences. BrucellaBase will regularly be updated with new genome sequences, new features along with improvements in genome annotations. BrucellaBase is available online at http://www.dbtbrucellosis.in/brucellabase.html or http://59.99.226.203/brucellabase/homepage.html., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Brucellosis is an important zoonotic disease caused by Brucella spp. Brucella suis is the etiological agent of porcine brucellosis. B. suis is the most genetically diverged species within the genus Brucella. We present the first large-scale B. suis phylogenetic analysis based on an alignment-free k-mer approach of gathering polymorphic sites from whole genome sequences. Genome-wide core-SNP based phylogenetic tree clearly differentiated and discriminated the B. suis biovars and the vaccine strain into different clades. A total of 16,756 SNPs were identified from the genome sequences of 54 B. suis strains. Also, biovar-specific SNPs were identified. The vaccine strain B. suis S2-30 is extensively used in China, which was discriminated from all biovars with the accumulation of the highest number of SNPs. We have also identified the SNPs between B. suis vaccine strain S2-30 and its closest homolog, B. suis biovar 513UK. The highest number of mutations (22) was observed in the phosphomannomutase (pmm) gene essential for the synthesis of O-antigen. Also, mutations were identified in several virulent genes including genes coding for type IV secretion system and the effector proteins, which could be responsible for the attenuated virulence of B. suis S2-30., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Brucella is an intracellular bacterium that causes the zoonotic infectious disease, brucellosis. Brucella species are currently intensively studied with a view to developing novel global health diagnostics and therapeutics. In this context, small RNAs (sRNAs) are one of the emerging topical areas; they play significant roles in regulating gene expression and cellular processes in bacteria. In the present study, we forecast sRNAs in three Brucella species that infect humans, namely Brucella melitensis, Brucella abortus, and Brucella suis, using a computational biology analysis. We combined two bioinformatic algorithms, SIPHT and sRNAscanner. In B. melitensis 16M, 21 sRNA candidates were identified, of which 14 were novel. Similarly, 14 sRNAs were identified in B. abortus, of which four were novel. In B. suis, 16 sRNAs were identified, and five of them were novel. TargetRNA2 software predicted the putative target genes that could be regulated by the identified sRNAs. The identified mRNA targets are involved in carbohydrate, amino acid, lipid, nucleotide, and coenzyme metabolism and transport, energy production and conversion, replication, recombination, repair, and transcription. Additionally, the Gene Ontology (GO) network analysis revealed the species-specific, sRNA-based regulatory networks in B. melitensis, B. abortus, and B. suis. Taken together, although sRNAs are veritable modulators of gene expression in prokaryotes, there are few reports on the significance of sRNAs in Brucella. This report begins to address this literature gap by offering a series of initial observations based on computational biology to pave the way for future experimental analysis of sRNAs and their targets to explain the complex pathogenesis of Brucella.

Brucella spp. are facultative intracellular pathogens that cause brucellosis in various mammals including humans. Brucella survive inside the host cells by forming vacuoles and subverting host defence systems. This study was aimed to predict the secretion systems and the secretomes of Brucella spp. from 39 complete genome sequences available in the databases. Furthermore, an attempt was made to identify the type IV secretion effectors and their interactions with host proteins. We predicted the secretion systems of Brucella by the KEGG pathway and SecReT4. Brucella secretomes and type IV effectors (T4SEs) were predicted through genome-wide screening using JVirGel and S4TE, respectively. Protein-protein interactions of Brucella T4SEs with their hosts were analyzed by HPIDB 2.0. Genes coding for Sec and Tat pathways of secretion and type I (T1SS), type IV (T4SS) and type V (T5SS) secretion systems were identified and they are conserved in all the species of Brucella. In addition to the well-known VirB operon coding for the type IV secretion system (T4SS), we have identified the presence of additional genes showing homology with T4SS of other organisms. On the whole, 10.26 to 14.94% of total proteomes were found to be either secreted (secretome) or membrane associated (membrane proteome). Approximately, 1.7 to 3.0% of total proteomes were identified as type IV secretion effectors (T4SEs). Prediction of protein-protein interactions showed 29 and 36 host-pathogen specific interactions between Bos taurus (cattle)-B. abortus and Ovis aries (sheep)-B. melitensis, respectively. Functional characterization of the predicted T4SEs and their interactions with their respective hosts may reveal the secrets of host specificity of Brucella.

Brucella is a facultative intracellular bacterium belongs to the class alpha proteobacteria. It causes zoonotic disease brucellosis to wide range of animals. Brucella species are highly conserved in nucleotide level. Here, we employed a comparative genomics approach to examine the role of homologous recombination and positive selection in the evolution of Brucella. For the analysis, we have selected 19 complete genomes from 8 species of Brucella. Among the 1599 core genome predicted, 24 genes were showing signals of recombination but no significant breakpoint was found. The analysis revealed that recombination events are less frequent and the impact of recombination occurred is negligible on the evolution of Brucella. This leads to the view that Brucella is clonally evolved. On other hand, 56 genes (3.5 % of core genome) were showing signals of positive selection. Results suggest that natural selection plays an important role in the evolution of Brucella. Some of the genes that are responsible for the pathogenesis of Brucella were found positively selected, presumably due to their role in avoidance of the host immune system.

Here, we report the genome sequence of the intermediate rough vaccine strain mutant, Brucella abortus S19Δper. The length of the draft genome was 3,271,238 bp, with 57.2% G+C content. A total of 3,204 protein-coding genes and 56 RNA genes were predicted., (Copyright © 2015 Chaudhuri et al.)

Global health therapeutics is a rapidly emerging facet of postgenomics medicine. In this connection, Brucella melitensis is an intracellular bacterium that causes the zoonotic infectious disease, brucellosis. Presently, no licensed vaccines are available for human brucellosis. Here, we report the identification of potential vaccine candidates against B. melitensis using a reverse vaccinology approach. Based on a systematic screening of exoproteome and secretome of B. melitensis 16 M, we identified eight proteins as potential vaccine candidates, including LPS-assembly protein LptD, a polysaccharide export protein, a cell surface protein, heme transporter BhuA, flagellin FliC, 7-alpha-hydroxysteroid dehydrogenase, immunoglobulin-binding protein EIBE, and hemagglutinin. Among these, the roles of BhuA and hemagglutinin in the virulence of Brucella are essential to establish infection. Roles of other proteins in the virulence are yet to be studied. Prediction of protein-protein interactions revealed that these proteins can interact with other proteins involved in virulence, secretion system, metabolism, and transport. From these eight potential vaccine candidates, we predicted three surface exposed novel antigenic epitopes that can induce both B-cell and T-cell immune responses. These peptides can be used for the development of either exclusive peptide vaccines or multi-component vaccines against human brucellosis. Reverse vaccinology is an important strategy for discovery of novel global health therapeutics.

We report the draft genome sequences of two Brucella abortus strains LMN1 and LMN2 isolated from cattle and pig. The LMN1 and LMN2 have the genome size of 3,395,952 bp and 3,334,792 bp, respectively. In addition to the conserved genes of Brucella, few novel regions showing similarity to the phages were identified in both strains.

Here, we present the draft genome sequence and annotation of Brucella abortus virulent strain 544. The genome of this strain is 3,289,405 bp long, with 57.2% G+C content. A total of 3,259 protein-coding genes and 60 RNA genes were predicted., (Copyright © 2015 Singh et al.)

Brucella abortus strain S99 is widely used for the preparation of colored, plain, recombinant and smooth lipopolysaccharide antigens for the preparation of Brucella diagnostic kits. The genome of this strain was sequenced and the length of the genome was 3,253,175 bp, with 57.2% G+C content. A total of 3,365 protein coding genes and 53 RNA genes were predicted., (Copyright © 2014 Shome et al.)