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The CRISPR-based regulation tools enable fine-tuning of gene transcription, showing potential in areas of biomanufacturing and live therapeutics. However, the cell toxicity and PAM specificity of existing CRISPR-based regulation systems limit their broad application. The development of new and less-toxic CRISPR-controlled expression systems remains highly desirable for expanding the application scope of CRISPR-based tools. Here, we reconstituted the type I CRISPR-Cas system from Escherichia coli to finely tune gene expression in Bacillus subtilis. Through engineering the 5' untranslated region (UTR) of mRNAs of cas genes, we remarkably improved the efficacy of the type I CRISPRi system. The improved type I CRISPRi system was applied in engineering the D-pantothenic acid (DPA)-producing B. subtilis, which was generated by strengthening the metabolic flux toward ß-alanine and (R)-pantoate via enhancing expression of key enzymes at both transcriptional and translational levels. Through controlling the expression of pdhA with the CRISPRi system for fine-tuning the metabolic flux toward DPA and the TCA cycle, we elevated the DPA titer to 0.88 g/L in shake flasks and 12.81 g/L in fed-batch fermentations without the addition of the precursor ß-alanine. The type I CRISPRi system and the strategy for fine-tuning metabolic flux reported here not only enrich the CRISPR toolbox in B. subtilis and facilitate DPA production through microbial fermentation but also provide a paradigm for programming important organisms to produce value-added chemicals with cheap raw materials.
Assuntos
Bacillus subtilis , Sistemas CRISPR-Cas , Escherichia coli , Engenharia Metabólica , Ácido Pantotênico , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sistemas CRISPR-Cas/genética , Engenharia Metabólica/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Pantotênico/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Regiões 5' não Traduzidas/genética , Regulação Bacteriana da Expressão GênicaRESUMO
Considered a "Generally Recognized As Safe" (GRAS) bacterium, the plant growth-promoting rhizobacterium Paenibacillus has been widely applied in: agriculture, medicine, industry, and environmental remediation. Paenibacillus species not only accelerate plant growth and degrade toxic substances in wastewater and soil but also produce industrially-relevant enzymes and antimicrobial peptides. Due to a lack of genetic manipulation tools and methods, exploitation of the bioresources of naturally isolated Paenibacillus species has long been limited. Genetic manipulation tools and methods continue to improve in Paenibacillus, such as shuttle plasmids, promoters, and genetic tools of CRISPR. Furthermore, genetic transformation systems develop gradually, including: penicillin-mediated transformation, electroporation, and magnesium amino acid-mediated transformation. As genetic manipulation methods of homologous recombination and CRISPR-mediated editing system have developed gradually, Paenibacillus has come to be regarded as a promising microbial chassis for biomanufacturing, expanding its application scope, such as: industrial enzymes, bioremediation and bioadsorption, surfactants, and antibacterial agents. In this review, we describe the applications of Paenibacillus bioproducts, and then discuss recent advances and future challenges in the development of genetic manipulation systems in this genus. This work highlights the potential of Paenibacillus as a new microbial chassis for mining bioresources.
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Metal-organic frameworks (MOFs) are promising drug-delivering platforms for their intrinsic capability of loading and releasing different cargoes. To further extend their biomedical practices, the development of collaborative MOF systems with good biocompatibility and synergistic efficacy is essential. Herein, the near-infrared and pH dual-response collaborative zeolitic imidazolate framework-8 (ZIF-8) platform SOR@ZIF-8@PDA (SZP) was constructed, in which the chemotherapeutic drug sorafenib (SOR) was encapsulated in ZIF-8 and via polydopamine (PDA) coating on ZIF-8 by hierarchical self-assembly. PDA coating serves as a photothermal agent for PPT while reducing the toxicity of ZIF-8. SZP achieves intelligent release of therapeutic drugs by responding to the lower pH of the tumor microenvironment and thermal stimulation generated by near-infrared light irradiation. In addition, under light irradiation, SZP could effectively realize treatment of cancer cells through synergistic chemo-photothermal therapy, as evidenced by the enhanced cell apoptosis, inhibited tumor cell proliferation and migration. This collaborative MOFs system showed excellent biocompatibility and antitumor ability in vivo on a mouse HepG2 tumor model. Our results demonstrated that PDA-modified MOFs exhibited a fantastic good development prospect in biomedical applications.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Estruturas Metalorgânicas , Nanopartículas , Zeolitas , Animais , Camundongos , Carcinoma Hepatocelular/tratamento farmacológico , Doxorrubicina/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Fototerapia , Imidazóis , Nanopartículas/uso terapêutico , Liberação Controlada de Fármacos , Microambiente TumoralRESUMO
In response to a high concentration of glucose, Bacillus subtilis, a microbial chassis for producing many industrial metabolites, rapidly takes up glucose using the phosphotransferase system (PTS), leading to overflow metabolism, a common phenomenon observed in many bacteria. Although overflow metabolism affects cell growth and reduces the production of many metabolites, effective strategies that reduce overflow metabolism while maintaining normal cell growth remain to be developed. Here, we used a quorum sensing (QS)-mediated circuit to tune the glucose uptake rate and thereby relieve overflow metabolism in an engineered B. subtilis for producing d-pantothenic acid (DPA). A low-efficiency non-PTS system was used for glucose uptake at the early growth stages to avoid a rapid glycolytic flux, while an efficient PTS system, which was activated by a QS circuit, was automatically activated at the late growth stages after surpassing a threshold cell density. This strategy was successfully applied as a modular metabolic engineering process for the high production of DPA. By enhancing the translation levels of key enzymes (3-methyl-2-oxobutanoate hydroxymethytransferase, pantothenate synthetase, aspartate 1-decarboxylase proenzyme, 2-dehydropantoate 2-reductase, dihydroxy-acid dehydratase, and acetolactate synthase) with engineered 5'-untranslated regions (UTRs) of mRNAs, the metabolic flux was promoted in the direction of DPA production, elevating the yield of DPA to 5.11 g/L in shake flasks. Finally, the engineered B. subtilis produced 21.52 g/L of DPA in fed-batch fermentations. Our work not only revealed a new strategy for reducing overflow metabolism by adjusting the glucose uptake rate in combination with promoting the translation of key metabolic enzymes through engineering the 5'-UTR of mRNAs but also showed its power in promoting the bioproduction of DPA in B. subtilis, exhibiting promising application prospects.
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Bacillus subtilis , Ácido Pantotênico , Bacillus subtilis/metabolismo , Ácido Pantotênico/metabolismo , Percepção de Quorum , Metabolismo dos Carboidratos , Glucose/metabolismo , Engenharia MetabólicaRESUMO
Genetically engineered microbes, especially Escherichia coli, have been widely used in the biosynthesis of proteins and metabolites for medical and industrial applications. As a traditional probiotic with a well-established safety record, E. coli Nissle 1917 (EcN) has recently emerged as a microbial chassis for generating living therapeutics, drug delivery vehicles, and microbial platforms for industrial production. Despite the availability of genetic tools for engineering laboratory E. coli K-12 and B strains, new genetic engineering systems are still greatly needed to expand the application range of EcN. In this review, we have summarized the latest progress in the development of genetic engineering systems in EcN, as well as their applications in the biosynthesis and delivery of valuable small molecules and biomacromolecules of medical and/or industrial interest, followed by a glimpse of how this rapidly growing field will evolve in the future.
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Sistemas de Liberação de Medicamentos , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia GenéticaRESUMO
ß-alanine has been used in food and pharmaceutical industries. Although Escherichia coli Nissle 1917 (EcN) is generally considered safe and engineered as living therapeutics, engineering EcN for producing industrial metabolites has rarely been explored. Here, by protein and metabolic engineering, EcN was engineered for producing ß-alanine from glucose. First, an aspartate-α-decarboxylase variant ADCK43Y with improved activity was identified and over-expressed in EcN, promoting the titer of ß-alanine from an undetectable level to 0.46 g/L. Second, directing the metabolic flux towards L-aspartate increased the titer of ß-alanine to 0.92 g/L. Third, the yield of ß-alanine was elevated to 1.19 g/L by blocking conversion of phosphoenolpyruvate to pyruvate, and further increased to 2.14 g/L through optimizing culture medium. Finally, the engineered EcN produced 11.9 g/L ß-alanine in fed-batch fermentation. Our work not only shows the potential of EcN as a valuable industrial platform, but also facilitates production of ß-alanine via fermentation. KEY POINTS: ⢠Escherichia coli Nissle 1917 (EcN) was engineered as a ß-alanine producing cell factory ⢠Identification of a decarboxylase variant ADCK43Y with improved activity ⢠Directing the metabolic flux to L-ASP and expressing ADCK43Y elevated the titer of ß-alanine to 11.9 g/L.
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Engenharia Metabólica , Probióticos , Escherichia coli/metabolismo , beta-Alanina/metabolismo , FermentaçãoRESUMO
New Delhi metallo-ß-lactamase (NDM)-producing clinical strains in Acinetobacter spp. have been recently reported in many countries and have received considerable attention. The vast majority of blaNDM cases occur on conjugative plasmids, which play a vital role in disseminating blaNDM. To characterize the conjugative plasmids bearing blaNDM genes in Acinetobacter spp., we analyzed the variants of blaNDM, conjugative transfer regions, genetic contexts of blaNDM, and the phylogenetic pattern of the 62 predicted blaNDM-positive plasmids, which were selected from 1,191 plasmids of Acinetobacter species from GenBank. We identified 30 conjugative plasmids from the 62 blaNDM-harboring plasmids in Acinetobacter species, with the oriT sites similar to plasmid pNDM-YR7 in our study, genes coding for relaxases of the MOBQ family, genes encoding type IV coupling proteins (T4CPs) of the TrwB/TraD subfamily, and VirB-like type IV secretion system (T4SS) gene clusters. The genome sizes of all 30 pNDM-YR7-like plasmids ranged from 39.36 kb to 49.65 kb, with a median size of 44.56 kb. The most common species of Acinetobacter containing the blaNDM-positive conjugative plasmids was A. baumannii, followed by Acinetobacter lwoffii and Acinetobacter indicus. Notably, pNDM-YR7 is the first report on a blaNDM-positive conjugative plasmid in Acinetobacter junii. Moreover, all 30 blaNDM-positive conjugative plasmids in Acinetobacter species were found to contain genetic contexts with the structure ISAba14-aph(3')-VI-ISAba125-blaNDM-ble. Our findings provide important insights into the phylogeny and evolution of blaNDM-positive plasmids of Acinetobacter species and further address their role in acquiring and spreading blaNDM genes in Acinetobacter species. IMPORTANCE Conjugative plasmids harboring the blaNDM gene play a vital role in disseminating carbapenem resistance. In this study, we first report a conjugative plasmid, pNDM-YR7, in Acinetobacter junii. Based on the genomic characteristics of the blaNDM-positive pNDM-YR7, we performed in silico typing and comparative analysis of blaNDM-positive plasmids using the 1,191 plasmids of Acinetobacter species available in the NCBI RefSeq database. We analyzed the characteristics of blaNDM-positive plasmids, including the variants of blaNDM, genetic features associated with blaNDM, conjugative transfer regions, and the phylogenetic pattern of the blaNDM-positive plasmids. All 30 blaNDM-positive conjugative plasmids were found to contain an ISAba14-aph(3')-VI-ISAba125-blaNDM-ble region. This study provides novel insights into the phylogeny and evolution of blaNDM-harboring conjugative plasmids and contributes to the repertoire of knowledge surrounding blaNDM-positive plasmids in the genus Acinetobacter.
Assuntos
Acinetobacter , Filogenia , Acinetobacter/genética , Plasmídeos/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
Bacterial multi-drug resistance (MDR) is a global challenge in the fields of medicine and health, agriculture and fishery, ecology and environment. The cross-region spread of antibiotic resistance genes (ARGs) among different species is one of the main cause of bacterial MDR. However, there is no effective strategies for addressing the intensifying bacterial MDR. The CRISPR-Cas system, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins, can targetedly degrade exogenous nucleic acids, thus exhibiting high application potential in preventing and controlling bacterial MDR caused by ARGs. This review briefly introduced the working mechanism of CRISPR-Cas systems, followed by discussing recent advances in reducing ARGs by CRISPR-Cas systems delivered through mediators (e.g. plasmids, bacteriophages and nanoparticle). Moreover, the trends of this research field were envisioned, providing a new perspective on preventing and controlling MDR.
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Bacteriófagos , Sistemas CRISPR-Cas , Antibacterianos , Bacteriófagos/genética , Farmacorresistência Bacteriana/genética , Plasmídeos/genéticaRESUMO
OBJECTIVES: The prevalence of tet(X) genes threatens the clinical use of last-line tigecycline. The tet(X6) gene has been reported in Proteus strains, but its genetic context is rarely reported. This study aimed to investigate the prevalence and genetic contexts of tet(X6) gene in Proteus spp. METHODS: A tet(X6) variant-bearing P. terrae subsp. cibarius strain was subjected to susceptibility testing, determination of growth curves, scanning electron microscopy, transmission electron microscopy and whole-genome sequencing (WGS). The genomic contexts of the tet(X6)-positive strain were analysed by sequence comparison and annotation. RESULTS: ZJ19PC, a P. terrae subsp. cibarius strain harbouring the tet(X6) variant, was isolated from 20 cecum samples collected in Zhejiang, China. The chromosome size of ZJ19PC was 3 952 084 bp; the GC content was 38.2%; and hugA, sul2, tet(H), floR, dfra1, aadA1, aac(3)-IV and aph(4)-la were found in addition to the tet(X6) variant. Proteus spp. could be classified into three groups based on the tet(X6) gene contexts. Strain ZJ19PC belongs to group 1 (sra-sul2-ISCR2-floR-ISCR2-floR-ISCR2- tet(X6) variant-tnpA-ISEc59-aph(4)-la-aac(3)-Iva-IS26), and this region of group 1 was inserted between modA and guaA. The common antimicrobial resistance (AMR) genes of the three types of AMR gene islands were sul2, floR, tet(X6) and aac(3). The tet(X6) gene contexts and SNP tree showed that ZJ19PC was homologous to HNCF44W and HNCF43W, which indicated that these strains may be clonally transmitted. CONCLUSION: This study analysed the genetic contexts of the tet(X6) gene in Proteus spp. and highlighted the significance of monitoring tigecycline-resistant P. terrae subsp. cibarius.
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Antibacterianos , Proteus , Animais , Antibacterianos/farmacologia , Ceco , TigeciclinaRESUMO
Considered a "Generally Recognized As Safe" (GRAS) bacterium, the plant growth-promoting rhizobacterium Paenibacillus polymyxa has been widely applied in agriculture and animal husbandry. It also produces valuable compounds that are used in medicine and industry. Our previous work showed the presence of restriction modification (RM) system in P. polymyxa ATCC 842. Here, we further analyzed its genome and methylome by using SMRT sequencing, which revealed the presence of a larger number of genes, as well as a plasmid documented as a genomic region in a previous report. A number of mobile genetic elements (MGEs), including 78 insertion sequences, six genomic islands, and six prophages, were identified in the genome. A putative lysozyme-encoding gene from prophage P6 was shown to express lysin which caused cell lysis. Analysis of the methylome and genome uncovered a pair of reverse-complementary DNA methylation motifs which were widespread in the genome, as well as genes potentially encoding their cognate type I restriction-modification system PpoAI. Further genetic analysis confirmed the function of PpoAI as a RM system in modifying and restricting DNA. The average frequency of the DNA methylation motifs in MGEs was lower than that in the genome, implicating a role of PpoAI in restricting MGEs during genomic evolution of P. polymyxa. Finally, comparative analysis of R, M, and S subunits of PpoAI showed that homologs of the PpoAI system were widely distributed in species belonging to other classes of Firmicute, implicating a role of the ancestor of PpoAI in the genomic evolution of species beyond Paenibacillus.
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Improving the creativity is crucial to postgraduate training nowadays. The course of Modern Microbial Biotechnology is an optional specialized course for microbiology-related postgraduates. To explore a new teaching mode for improving the creativity of postgraduate students, we reformed the teaching content, the teaching method and the evaluation mode of this course. Through case study teaching, seminar-style classroom and implementation of a new assessment method, the students not only mastered professional knowledge and disciplinary frontiers of modern microbiology technology, but also improved their ability of discovering, analyzing and solving problems. The reformed course teaching mode is effective in fostering postgraduates' creativity.
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Biotecnologia , Estudantes , HumanosRESUMO
ß-Alanine (3-aminopropionic acid) holds great potential in industrial application. It can be obtained through a chemical synthesis route, which is hazardous to the environment. It is well known that l-aspartate-α-decarboxylase (ADC) can convert l-aspartate to ß-alanine in bacteria. However, due to the low activity of ADC, industrial production of ß-alanine through the green biological route remains unclear. Thus, improving the activity of ADC is critical to reduce the cost of ß-alanine production. In this study, we established a dual-fluorescence high-throughput system for efficient ADC screening. By measuring the amount of ß-alanine and the expression level of ADC using two different fluorescence markers, we can rapidly quantify the relative activity of ADC variants. From a mutagenesis library containing 2000 ADC variants, we obtained a mutant with 33% increased activity. Further analysis revealed that mutations of K43R and P103Q in ADC significantly improved the yield of ß-alanine produced by the whole-cell biocatalysis. Compared with the previous single-fluorescence method, our system can not only quantify the amount of ß-alanine but also measure the expression level of ADC with different fluorescence, making it able to effectively screen out ADC variants with improved relative activity. The dual-fluorescence high-throughput system for rapid screening of ADC provides a good strategy for industrial production of ß-alanine via the biological conversion route in the future.
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Glutamato Descarboxilase/análise , Glutamato Descarboxilase/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Biocatálise , Estabilidade Enzimática , Escherichia coli/genética , Fluorescência , Genes Reporter , Glutamato Descarboxilase/química , Simulação de Acoplamento Molecular , Mutação , beta-Alanina/análise , beta-Alanina/biossínteseRESUMO
Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (P cas ) and suppresses the type I-E CRISPR-Cas system in Escherichia coli Although the H-NS paralogue StpA also binds P cas , its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of P cas Third, by comparing transcriptional activities of the intact P cas and the P cas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in P cas Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of P cas Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coliIMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.
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Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Chaperonas Moleculares/genética , Transformação Bacteriana , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismoRESUMO
OBJECTIVES: To develop a convenient chemical transformation mediated CRISPR/Cas9 (CT-CRISPR/Cas9) system for genome editing in Escherichia coli. RESULTS: Here, we have constructed a CT-CRISPR/Cas9 system, which can precisely edit bacterial genome (replacing, deleting, inserting or point mutating a target gene) through chemical transformation. Compared with the traditional electroporation mediated CRISPR/Cas9 (ET-CRISPR/Cas9) system, genome editing with the CT-CRISPR/Cas9 system is much cheaper and simpler. In the CT-CRISPR/Cas9 system, we observed efficient genome editing on LB-agar plates. The CT-CRISPR/Cas9 system has successfully modified the target gene with the editing template flanked by short homologous DNA fragments (~ 50 bp) which were designed in primers. We used the lab-made CaCl2 solution to perform the CT-CRISPR/Cas9 experiment and successfully edited the genome of E. coli. Potential application of the CT-CRISPR/Cas9 system in high-throughput genome editing was evaluated in two E. coli strains by using a multiwell plate. CONCLUSIONS: Our work provides a simple and cheap genome-editing method, that is expected to be widely applied as a routine genetic engineering method.
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Escherichia coli/genética , Edição de Genes/economia , Edição de Genes/métodos , Sistemas CRISPR-Cas , Fenômenos Químicos , Eletroporação , Genoma Bacteriano , Transformação BacterianaRESUMO
Accompanied by benefits from horizontally transferred genes, bacteria have to face the risk of the invasion of dangerous genes. Bacteria often use the restriction-modification (R-M) system, which is consisted of methyl transferase (MEase) and restrictase (REase), to protect self-DNA and defend against foreign DNA. Paenibacillus polymyxa, widely used as growth promoting rhizobacteria in agriculture, can also produce compounds of medical and industrial interests. It is unclear whether R-M systems exist in P. polymyxa. In this study, we used a shuttle plasmid with epigenetic modification from different bacteria to explore R-M systems in P. polymyxa. We found that DNA which is methylated by DNA adenine methyltransferase (Dam) in E. coli was strongly restricted, indicating the presence of a Dam-methylation-dependent R-M system in P. polymyxa. Whereas, DNA from a dam-E. coli strain was also moderately restricted, indicating the presence of a Dam-methylation-independent R-M system. Degradation of plasmid DNA with Dam methylation by cell-free protein extract of P. polymyxa provides additional evidence for the presence of Dam-methylation-dependent R-M system. Taken together, our work showed that there are two different types of R-M system in P. polymyxa, providing a foundation for the study of innate immunity in P. polymyxa and for the development of genetic engineering tools in P. polymyxa.
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Enzimas de Restrição-Modificação do DNA/genética , DNA/metabolismo , Engenharia Genética/métodos , Paenibacillus polymyxa/genética , Bacillus subtilis/genética , Sistema Livre de Células , Metilação de DNA , Enzimas de Restrição-Modificação do DNA/metabolismo , Epigênese Genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Plasmídeos/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismoRESUMO
Horizontal gene transfer (HGT) plays an important role in bacterial evolution. It is well accepted that DNA is pulled/pushed into recipient cells by conserved membrane-associated DNA transport systems, which allow the entry of only single-stranded DNA (ssDNA). However, recent studies have uncovered a new type of natural bacterial transformation in which double-stranded DNA (dsDNA) is taken up into the cytoplasm, thus complementing the existing methods of DNA transfer among bacteria. Regulated by the stationary-phase regulators RpoS and cAMP receptor protein (CRP), Escherichia coli establishes competence for natural transformation with dsDNA, which occurs in agar plates. To pass across the outer membrane, a putative channel, which may compete for the substrate with the porin OmpA, may mediate the transfer of exogenous dsDNA into the cell. To pass across the inner membrane, dsDNA may be bound to the periplasmic protein YdcS, which delivers it into the inner membrane channel formed by YdcV. The discovery of cell-to-cell contact-dependent plasmid transformation implies the presence of additional mechanism(s) of transformation. This review will summarize the current knowledge about mechanisms of HGT with an emphasis on recent progresses regarding non-canonical mechanisms of natural transformation. Fully understanding the mechanisms of HGT will provide a foundation for monitoring and controlling multidrug resistance.
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Intracellular products (e.g., insulin), which are obtained through cell lysis, take up a big share of the biotech industry. It is often time-consuming, laborious, and environment-unfriendly to disrupt bacterial cells with traditional methods. In this study, we developed a molecular device for controlling cell lysis with light. We showed that intracellular expression of a single lysin protein was sufficient for efficient bacterial cell lysis. By placing the lysin-encoding gene under the control of an improved light-controlled system, we successfully controlled cell lysis by switching on/off light: OD600 of the Escherichia coli cell culture was decreased by twofold when the light-controlled system was activated under dark condition. We anticipate that our work would not only pave the way for cell lysis through a convenient biological way in fermentation industry, but also provide a paradigm for applying the light-controlled system in other fields of biotech industry.
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Bacteriólise , Escherichia coli/citologia , Fermentação , Microbiologia Industrial/métodos , Luz , Biotecnologia , Escherichia coli/efeitos da radiação , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Fosfatos/química , FosforilaçãoRESUMO
To better adapt to the environment, prokaryocyte can take up exogenous genes (from bacteriophages, plasmids or genomes of other species) through horizontal gene transfer. Accompanied by the acquisition of exogenous genes, prokaryocyte is challenged by the invasion of 'selfish genes'. Therefore, to protect against the risk of gene transfer, prokaryocyte needs to establish mechanisms for selectively taking up or degrading exogenous DNA. In recent years, researchers discovered an adaptive immunity, which is mediated by the small RNA guided DNA degradation, prevents the invasion of exogenous genes in prokaryocyte. During the immune process, partial DNA fragments are firstly integrated.to the clustered regularly interspaced short palindromic repeats (CRISPR) located within the genome DNA, and then the mature CRISPR RNA transcript and the CRISPR associated proteins (Cas) form a complex CRISPR/Cas for degrading exogenous DNA. In this review, we will first briefly describe the CRISPR/Cas systems and then mainly focus on the recent advances of the function mechanism and the regulation mechanism of the type I-E CRISPR/Cas system in Escherichia coli.