Core Gene Cluster Research Articles

A two-step regulatory circuit involving Spo0A-AbrB activates mersacidin biosynthesis in Bacillus subtilis

Due to intramolecular ring structures, the ribosomally produced and post-translationally modified peptide mersacidin shows antimicrobial properties comparable to those of vancomycin without exhibiting cross-resistance. Although the principles of mersacidin biosynthesis are known, there is no information on the molecular control processes for the initial stimulation of mersacidin bioproduction. By using Bacillus subtilis for heterologous biosynthesis, a considerable amount of mersacidin could be produced without the mersacidin-specific immune system and the mersacidin-activating secretory protease. By using the established laboratory strain Bacillus subtilis 168 and strain 3NA, which is used for high cell density fermentation processes, in combination with the construction of reporter strains to determine the promoter strengths within the mersacidin core gene cluster, the molecular regulatory circuit of Spo0A, a master regulator of cell differentiation including sporulation initiation, and the global transcriptional regulator AbrB, which is involved in cell adaptation processes in the transient growth phase, was identified to control the initial stimulation of the mersacidin core gene cluster. In a second downstream regulatory step, the activator MrsR1, encoded in the core gene cluster, acts as a stimulatory element for mersacidin biosynthesis. These findings are important to understand the mechanisms linking environmental conditions and microbial responses with respect to the bioproduction of bioactive metabolites including antimicrobials such as mersacidin. This information will also support the construction of production strains for bioactive metabolites with antimicrobial properties.

GBP2 acts as a member of the interferon signalling pathway in lupus nephritis

Lupus nephritis (LN) is a common and serious clinical manifestation of systemic lupus erythematosus. However, the pathogenesis of LN is not fully understood. The currently available treatments do not cure the disease and appear to have a variety of side effects in the long term. The purpose of this study was to search for key molecules involved in the LN immune response through bioinformatics techniques to provide a reference for LN-specific targeted therapy. The GSE112943 dataset was downloaded from the Gene Expression Omnibus database, and 20 of the samples were selected for analysis. In total, 2330 differentially expressed genes were screened. These genes were intersected with a list of immune genes obtained from the IMMPORT immune database to obtain 128 differentially expressed immune-related genes. Enrichment analysis showed that most of these genes were enriched in the interferon signalling pathway. Gene set enrichment analysis revealed that the sample was significantly enriched for expression of the interferon signalling pathway. Further analysis of the core gene cluster showed that nine genes, GBP2, VCAM1, ADAR, IFITM1, BST2, MX2, IRF5, OAS1 and TRIM22, were involved in the interferon signalling pathway. According to our analysis, the guanylate binding protein 2 (GBP2), interferon regulatory factor 5 and 2′-5′-oligoadenylate synthetase 1 (OAS1) genes are involved in three interferon signalling pathways. At present, we do not know whether GBP2 is associated with LN. Therefore, this study focused on the relationship between GBP2 and LN pathogenesis. We speculate that GBP2 may play a role in the pathogenesis of LN as a member of the interferon signalling pathway. Further immunohistochemical results showed that the expression of GBP2 was increased in the renal tissues of LN patients compared with the control group, confirming this conjecture. In conclusion, GBP2 is a member of the interferon signalling pathway that may have implications for the pathogenesis of LN and serves as a potential biomarker for LN.

Transcriptome Analysis of Peripheral Blood Mononuclear Cells Response in Patients with Severe COVID-19 Reveals Crucial Genes Regulating Protein Ubiquitination

BackgroundWe sought to further our understanding of the biological characteristics underlying severe COVID-19.Material/MethodsRNA sequencing (RNA-Seq) analysis was used to evaluate peripheral blood mononuclear cells from 4 patients with severe COVID-19 and 4 healthy controls. We performed gene expression analyses to detect differentially expressed genes (DEGs). Enrichment analyses were performed to identify their molecular processes and signaling pathways, and the protein–protein interaction network was constructed to extract the core gene cluster. The investigation of protein-chemical interactions and regulatory signatures for specific regulatory checkpoints and powerful chemical agents was then conducted for these essential genes. Finally, we used single-cell RNA-Seq analysis from an online platform to show how these genes were expressed differently, depending on the kind of cell.ResultsA total of 268 DEGs were found. The biological process of protein ubiquitination was later discovered to be highly influenced by the core gene cluster (ITCH, TRIM21, RNF130, FBXO11, UBE2J1, and ASB16) at the transcriptome level. Six transcription factors, FNIC, FOXA1, YY1, GATA2, MET2A, and FOXC1, as well as miRNAs hsa-miR-1-3p and hsa-miR-27a-3p were identified. We found a potent chemical agent, copper sulfate, may regulate protein ubiquitination genes cooperatively, and the genes regulating protein ubiquitination could be expressed highly on the macrophages.ConclusionsTaken together, we suggest that protein ubiquitination is a crucial functional process in patients with severe COVID-19. This study will give a deeper insight into biological characteristics and progression of COVID-19 and facilitate development of novel therapeutics, leading to significant advancements in the COVID-19 pandemic.

Genome Investigation of a Cariogenic Pathogen with Implications in Cardiovascular Diseases.

The proportion of people suffering from cardiovascular diseases has risen by 34% in the last 15years 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.

Genetic engineering and heterologous expression of the disorazol biosynthetic gene cluster via Red/ET recombineering

Disorazol, a macrocyclic polykitide produced by the myxobacterium Sorangium cellulosum So ce12 and it is reported to have potential cytotoxic activity towards several cancer cell lines, including multi-drug resistant cells. The disorazol biosynthetic gene cluster (dis) from Sorangium cellulosum (So ce12) was identified by transposon mutagenesis and cloned in a bacterial artificial chromosome (BAC) library. The 58-kb dis core gene cluster was reconstituted from BACs via Red/ET recombineering and expressed in Myxococcus xanthus DK1622. For the first time ever, a myxobacterial trans-AT polyketide synthase has been expressed heterologously in this study. Expression in M. xanthus allowed us to optimize the yield of several biosynthetic products using promoter engineering. The insertion of an artificial synthetic promoter upstream of the disD gene encoding a discrete acyl transferase (AT), together with an oxidoreductase (Or), resulted in 7-fold increase in disorazol production. The successful reconstitution and expression of the genetic sequences encoding for these promising cytotoxic compounds will allow combinatorial biosynthesis to generate novel disorazol derivatives for further bioactivity evaluation.

A Deeply Branching Thermophilic Bacterium with an Ancient Acetyl-CoA Pathway Dominates a Subsurface Ecosystem

A nearly complete genome sequence of Candidatus ‘Acetothermum autotrophicum’, a presently uncultivated bacterium in candidate division OP1, was revealed by metagenomic analysis of a subsurface thermophilic microbial mat community. Phylogenetic analysis based on the concatenated sequences of proteins common among 367 prokaryotes suggests that Ca. ‘A. autotrophicum’ is one of the earliest diverging bacterial lineages. It possesses a folate-dependent Wood-Ljungdahl (acetyl-CoA) pathway of CO2 fixation, is predicted to have an acetogenic lifestyle, and possesses the newly discovered archaeal-autotrophic type of bifunctional fructose 1,6-bisphosphate aldolase/phosphatase. A phylogenetic analysis of the core gene cluster of the acethyl-CoA pathway, shared by acetogens, methanogens, some sulfur- and iron-reducers and dechlorinators, supports the hypothesis that the core gene cluster of Ca. ‘A. autotrophicum’ is a particularly ancient bacterial pathway. The habitat, physiology and phylogenetic position of Ca. ‘A. autotrophicum’ support the view that the first bacterial and archaeal lineages were H2-dependent acetogens and methanogenes living in hydrothermal environments.

Differing Evolutionary Histories of WFDC8 (Short-Term Balancing) in Europeans and SPINT4 (Incomplete Selective Sweep) in Africans

The whey acidic protein four-disulfide core (WFDC) gene cluster on human chromosome 20q13, harbors 15 small serine protease inhibitor genes with roles in innate immunity, reproduction, and regulation of endogenous proteases kallikreins. The WFDC cluster has emerged as a prime example of rapid diversification and adaptive evolution in primates. This study sought a better understanding of the evolutionary history of WFDC genes in humans and focused on exploring the adaptive selection signatures found in populations of European (Utah residents with ancestry from northern and western Europe [CEU]) and African (Yoruba from Ibadan, in Nigeria [YRI]) ancestry in a genome-wide scan for putative targets of recent adaptive selection. Our approach included resequencing coding and noncoding regions of WFDC6, EPPIN, and WFDC8 in 20 CEU and of SPINT4 in 20 YRI individuals. We generated 302 kb and 60 kb of high-quality sequence data from CEU and of YRI populations, respectively, enabling the identification of 72 single nucleotide polymorphisms. Using classic neutrality tests, empirical and haplotype-based analysis, we pinpointed WFDC8 and SPINT4 as the likely targets of short-term balancing selection in the CEU population, and recent positive selection (incomplete selective sweep) in the YRI population. Putative candidate variants targeted by selection include 44A (rs7273669A) for WFDC8, which may downregulate gene expression by abolishing the binding site of two transcription factors; and a haplotype configuration [Ser73+98A] (rs6017667A-rs6032474A) for SPINT4, which may simultaneously affect protein function and gene regulation. We propose that the evolution of WFDC8 and SPINT4 has been shaped by complex selective scenarios due to the interdependence of variant fitness and ecological variables.

Completing the puzzle of aurachin biosynthesis in Stigmatella aurantiaca Sg a15

The aurachins are a family of secondary metabolites, with the main members aurachin A, B, C, and D, produced by the myxobacterium Stigmatella aurantiaca Sg a15. These isoprenoid quinoline alkaloids are classified as A-type or C-type aurachins according to the position of the farnesyl residue either at C4 or C3 of the quinoline core, respectively. Previous feeding studies revealed that the C-type aurachins are converted to A-type aurachins by late stage tailoring reactions. While the core gene cluster coding for the functionalities required for the biosynthesis of the basic structure aurachin D is known, neither of the genes encoding for the successively acting tailoring enzymes was known up to date, which was assumed to be due to a split cluster organisation. Here we describe the identification of a total of five genes, located upstream of the aurachin core cluster and at additional two loci elsewhere in the genome, encoding for the aforementioned functionalities. The generation and evaluation of respective inactivation mutants of S. aurantiaca Sg a15 allowed for the first time to propose an exhaustive model for aurachin biosynthesis. One of the deduced biosynthetic transformations corresponds to a pinacol rearrangement, an unprecedented tailoring reaction in secondary metabolite biosynthesis.

AcMNPV ac143 ( odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 ( odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100–114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC ac142 REP -ac143KO ). Fluorescence and light microscopy showed that infection by AcBAC ac142 REP- ac143KO is limited to a single cell and titration assays confirmed that AcBAC ac142 REP- ac143KO was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC ac142 REP- ac143KO transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription.

Molecular analysis of the cercosporin biosynthetic gene cluster in Cercospora nicotianae

We describe a core gene cluster, comprised of eight genes (designated CTB1-8), and associated with cercosporin toxin production in Cercospora nicotianae. Sequence analysis identified 10 putative open reading frames (ORFs) flanking the previously characterized CTB1 and CTB3 genes that encode, respectively, the polyketide synthase and a dual methyltransferase/monooxygenase required for cercosporin production. Expression of eight of the genes was co-ordinately induced under cercosporin-producing conditions and was regulated by the Zn(II)Cys(6) transcriptional activator, CTB8. Expression of the genes, affected by nitrogen and carbon sources and pH, was also controlled by another transcription activator, CRG1, previously shown to regulate cercosporin production and resistance. Disruption of the CTB2 gene encoding a methyltransferase or the CTB8 gene yielded mutants that were completely defective in cercosporin production and inhibitory expression of the other CTB cluster genes. Similar 'feedback' transcriptional inhibition was observed when the CTB1, or CTB3 but not CTB4 gene was inactivated. Expression of four ORFs located on the two distal ends of the cluster did not correlate with cercosporin biosynthesis and did not show regulation by CTB8, suggesting that the biosynthetic cluster was limited to CTB1-8. A biosynthetic pathway and a regulatory network leading to cercosporin formation are proposed.