Minicells from Highly Genome Reduced Escherichia coli: Cytoplasmic and Surface Expression of Recombinant Proteins and Incorporation in the Minicells

Minicells, small cells lacking a chromosome, produced by bacteria with mutated min genes, which control cell division septum placement, have many potential uses. Minicells have contributed to basic bacterial physiology studies and can enable new biotechnological applications, including drug delivery and vaccines. Genome-reduced (GR) bacteria are another informative area of investigation. Investigators identified that with even almost 30% of the E. coli genome deleted, the bacteria still live. In biotechnology and synthetic biology, GR bacteria offer certain advantages. With GR bacteria, more recombinant genes can be placed into GR chromosomes and fewer cell resources are devoted to purposes apart from biotechnological goals. Here, we show that these two technologies can be combined: min mutants can be made in GR E. coli. The minCminD mutant GR E. coli produce minicells that concentrate engineered recombinant proteins within these spherical delivery systems. We expressed recombinant GFP protein in the cytoplasm of GR bacteria and showed that it is concentrated within the minicells. We also expressed proteins on the surfaces of minicells made from GR bacteria using a recombinant Gram-negative AIDA-I autotransporter expression cassette. As some autotransporters, like AIDA-I, are concentrated at the bacterial poles, where minicells bud, and because the surface-to volume ratio of the small minicells is higher than bacteria, recombinant proteins expressed on surfaces of the GR bacteria are concentrated on the minicells. Minicells made from GR bacteria can enable useful biotechnological innovations, such as drug delivery vehicles and vaccine immunogens.

CDK control pathways integrate cell size and ploidy information to control cell division

Maintenance of cell size homeostasis is a property that is conserved throughout eukaryotes. Cell size homeostasis is brought about by the co-ordination of cell division with cell growth, and requires restriction of smaller cells from undergoing mitosis and cell division, whilst allowing larger cells to do so. Cyclin-CDK is the fundamental driver of mitosis and therefore ultimately ensures size homeostasis. Here we dissect determinants of CDK activity in vivo to investigate how cell size information is processed by the cell cycle network in fission yeast. We develop a high-throughput single-cell assay system of CDK activity in vivo and show that inhibitory tyrosine phosphorylation of CDK encodes cell size information, with the phosphatase PP2A aiding to set a size threshold for division. CDK inhibitory phosphorylation works synergistically with PP2A to prevent mitosis in smaller cells. Finally, we find that diploid cells of equivalent size to haploid cells exhibit lower CDK activity in response to equal cyclin-CDK enzyme concentrations, suggesting that CDK activity is reduced by increased DNA levels. Therefore, scaling of cyclin-CDK levels with cell size, CDK inhibitory phosphorylation, PP2A, and DNA-dependent inhibition of CDK activity, all inform the cell cycle network of cell size, thus contributing to cell-size homeostasis.

CdrS is a global transcriptional regulator influencing cell division in Haloferax volcanii

Transcriptional regulators that integrate cellular and environmental signals to control cell division are well known in bacteria and eukaryotes, but their existence is poorly understood in archaea. We identified a conserved gene (cdrS) that encodes a small protein and is highly transcribed in the model archaeon Haloferax volcanii. The cdrS gene could not be deleted, but CRISPRi-mediated repression of the cdrS gene caused slow growth, cell division defects, and changed the expression of multiple genes and their products associated with cell division, protein degradation and metabolism. Consistent with this complex regulatory network, overexpression of cdrS inhibited cell division, whereas overexpression of the operon encoding both CdrS and a tubulin-like cell division protein (FtsZ2) stimulated division. ChIP-Seq identified 18 DNA-binding sites of the CdrS protein including one upstream of the promoter for diadenylate cyclase, which is an essential gene involved in c-di-AMP signalling implicated in the regulation of cell division. These findings suggest that CdrS is a transcription factor that plays a central role in a regulatory network coordinating metabolism and cell division.

Ser/Thr kinase-dependent phosphorylation of the peptidoglycan hydrolase CwlA controls its export and modulates cell division in Clostridioides difficile

Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The serine/threonine kinases (STKs) of the Hanks family control cell division and envelope homeostasis, but only a few kinase substrates and associated molecular mechanisms have been identified. In this work, we identified CwlA as the first STK-PrkC substrate in the human pathogen Clostridiodes difficile and showed that CwlA is an endopeptidase involved in daughter cell separation. We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, therefore controlling the hydrolytic activity in the cell wall. High level of CwlA at the cell surface led to cell elongation, whereas low level caused cell separation defects. We thus provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division.

Noc corrals migration of FtsZ protofilaments during cytokinesis in Bacillus subtilis

ABSTRACTBacteria that divide by binary fission form FtsZ rings at the geometric midpoint of the cell between the bulk of the replicated nucleoids. In B. subtilis, the DNA- and membrane-binding Noc protein is thought to mediate nucleoid occlusion to prevent FtsZ rings from forming over the chromosome. To explore the role of Noc, we used time-lapse fluorescence microscopy to monitor FtsZ and the nucleoid of cells growing in microfluidic channels. Our data show that Noc does not prevent FtsZ formation over the chromosome or control cell division site selection. Instead, Noc inhibits migration of FtsZ protofilaments from one FtsZ structure to the next. Moreover, we show that FtsZ protofilaments travel due to a local reduction in ZapA association, and the Noc mutant phenotype can be suppressed by ZapA overexpression. Thus, Noc maintains a high local concentration of FtsZ to stabilize FtsZ rings during cytokinesis.IMPORTANCEIn bacteria, a condensed structure of FtsZ (Z-ring) recruits cell division machinery, and Z-ring formation is inhibited over the chromosome by a poorly understood phenomenon called nucleoid occlusion. In B. subtilis, nucleoid occlusion has been reported to be mediated by the DNA-membrane bridging protein, Noc. Using time-lapse fluorescence microscopy of cells growing in microchannels, we show that Noc neither protects the chromosome from proximal Z-ring formation nor determines the future site of cell division. Rather, Noc plays a corralling role by preventing protofilaments from leaving a Z-ring undergoing cytokinesis and traveling over the nucleoid.

EXPRESSION AND CHARACTERISATION OF CUCUMBER FRUIT FLESH THICKNESS-RELATED GENE CSA2M058670.1

Bulked segregant analysis combined with specific length amplified fragment sequencing techniques have been applied to determine the fine genetic mapping of fruit flesh thickness-related genes in cucumber. Herein, the Csa2M058670.1 gene was subjected to real time fluorescence quantitative PCR (qRT-PCR) and sequence analysis, indicating a strong correlation with cucumber fruit flesh thickness. Expression and characterisation of the Csa2M058670.1 gene were performed based on previous studies. The results of the fluorescence-based quantitative PCR showed that Csa2M058670.1 was expressed in all organs, but levels were highest in fruit peel, fruit flesh, and female flowers. Furthermore, Csa2M058670.1 expression was induced by abiotic stresses including drought, low temperature, and high salt. Domain analysis revealed that the protein encoded by Csa2M058670.1 possesses an SET (Su(var), Enhancern of zeste CE(z), and Trithorax) domain that may control cell division and differentiation. Therefore, we speculated that Csa2M058670.1 might affect fruit flesh thickness in cucumbers by influencing cell division.

IDENTIFIKASI PERUBAHAN STRUKTUR DNA TERHADAP PEMBENTUKAN SEL KANKER MENGGUNAKAN DEKOMPOSISI GRAF

IDENTIFIKASI PERUBAHAN STRUKTUR DNA TERHADAP PEMBENTUKAN SEL KANKER MENGGUNAKAN DEKOMPOSISI GRAFABSTRAKKerusakan DNA adalah salah satu penyebab yang dapat mebuat sel normal bertumbuh menjadi sel kanker. Hal ini dikarenakan DNA yang rusak dapat menyebabkan mutasi di gen vital yang mengontrol pembelahan sel sampai terjadi pembelahan sel yang tidak terkendali dan memicu pertumbuhan sel kanker. Beberapa mutasi dibutuhkan untuk mengubah sel normal menjadi sel kanker. Dalam hal ini, teori dekomposisi graf digunakan untuk menganalisa proses terjadinya pertumbuhan sel kanker yang dimulai dari kerusakan DNA yang menyebabkan terjadinya mutasi gen. Dengan teori dekomposisi graf, sebuah graf bisa difaktorkan ke dalam beberapa subgraf. Pemfaktoran ini dapat digunakan untuk melihat pola perubahan hubungan antar objek. Tujuan dari penelitian ini untuk mengidentifikasi struktur DNA terhadap pembentukan sek kanker dengan menggunakan dekomposisi graf. Yang diidenfikasi adalah mutasi delesi, addisi, dan substitusi dimana dari mutasi-mutasi ini dilihat hasil dekomposisi graf dan apakah dari ketiga mutasi ini dapat membentuk sel kanker.Kata Kunci : Struktur DNA, Sel Kanker, Dekomposisi Graf, Perfect Matching, Hamilton cycle IDENTIFICATION OF CHANGES OF DNA STRUCTURES ON CANCER CELL FORM USING GRAPH DECOMPOSITIONABSTRACTDNA damage is one of the causes that can make normal cells grow into cancer cells. This is because damaged DNA can cause mutations in vital genes that control cell division until uncontrolled cell division and trigger the growth of cancer cells. Some mutations are needed to convert normal cells into cancer cells. In this case theory of graph decomposition will be used to analyze the process of cancer cell growth that starts from the DNA damage that causes gene mutation. With the graph decomposition theory, a graph can be factored into several subgraphs. This factoring can be used to see patterns of relationship changes between objects. The purpose of this study was to identify the structure of DNA against the formation of cancer cells by using decomposition graph. What will be identified are the deletion mutations, additions, and substitutions from which these mutations are seen in the decomposition of the graph and whether these three mutations can form cancer cells.Keywords : Structure of DNA, Cancer Sel, Dekomposition Graph, Perfect Maching, Hamilton cycle

Nuclear survivin expression correlates with endoglin-assessed microvascularisation in laryngeal carcinoma

AimsSurvivin—a member of the family of inhibitor of apoptosis proteins that control cell division, apoptosis and metastasis—is overexpressed in virtually all human cancers, including laryngeal squamous cell carcinoma (LSCC). Recent findings also correlate survivin expression with the regulation of angiogenesis. The novel main aim of this study was a preliminary investigation into the potential role of survivin expression in LSCC neoangiogenesis, as determined by endoglin-assessed microvascular density (MVD).MethodsImmunohistochemical expression of nuclear survivin and endoglin-assessed MVD were ascertained by image analysis in 75 consecutive LSCCs.ResultsStatistical analysis disclosed a strong direct correlation between nuclear survivin expression and MVD. Patients whose nuclear survivin expression was ≥6.0% had a significantly higher LSCC recurrence rate, and a significantly shorter disease-free survival (DFS) than those with a nuclear survivin expression <6.0%. The LSCC recurrence rate was also higher and the DFS shorter in patients with endoglin-assessed MVD ≥6.89%. The OR for recurrence was 2.79 in patients with LSCC with a nuclear survivin expression ≥6.0%, and 12.31 in those with an MVD≥6.89%.ConclusionsSurvivin-targeting strategies to enhance tumour cell response to apoptosis and inhibit tumour growth should receive more attention with a view to developing agents for use in multimodality advanced LSCC treatment, or combined with conventional chemotherapy. Given the present preliminary evidence in LSCC, survivin targeting should also be further investigated for anti-angiogenic purposes, to reduce tumour blood flow and induce cancer necrosis.