scholarly journals Efficient long fragment editing technique enables rapid construction of genetically stable bacterial strains

2020 ◽  
Author(s):  
Chaoyong Huang ◽  
Liwei Guo ◽  
Jingge Wang ◽  
Ning Wang ◽  
Yi-Xin Huo
Keyword(s):  
Open Reading Frames ◽  
Deletion Mutants ◽  
Dna Fragments ◽  
Bacterial Strains ◽  
Chromosome Engineering ◽  
Knock Out ◽  
Rapid Construction ◽  
Stable Plasmid ◽  
Chromosomal Engineering ◽  
Genomic Editing

Abstract Background Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable molecules. Long fragment editing techniques are of great importance for accelerating bacterial chromosome engineering to obtain desirable and genetically stable strains. However, the existing genomic editing methods cannot meet the needs of researchers. Results We herein report an efficient long fragment editing technique for complex chromosomal engineering in Escherichia coli. The technique enabled us to integrate DNA fragments up to 12 kb into the chromosome, and to knock out DNA fragments up to 187 kb from the chromosome, with over 95% positive rates. We applied this technique for E. coli chromosomal simplification, resulting in twelve individual deletion mutants and four cumulative deletion mutants. The simplest chromosome lost a 370.6 kb DNA sequence containing 364 open reading frames. In addition, we applied the technique to metabolic engineering and constructed a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask micro-aerobic fermentation. Conclusions These results suggested that the technique is a powerful chromosomal engineering tool, highlighting its potential to be applied in different fields of synthetic biology.

Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering

2020 ◽  
Author(s):  
Chaoyong Huang ◽  
Liwei Guo ◽  
Jingge Wang ◽  
Ning Wang ◽  
Yi-Xin Huo
Keyword(s):  
Metabolic Engineering ◽  
Large Scale ◽  
Genome Engineering ◽  
Bacterial Genome ◽  
Open Reading Frames ◽  
Deletion Mutants ◽  
Dna Fragments ◽  
E Coli ◽  
Stable Plasmid ◽  
Reading Frames

ABSTRACTBacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering.

The strategy of editing and modification of the genomes of bacterial viruses

Bacteriology ◽  
2020 ◽  
Vol 5 (1) ◽  
pp. 48-59
Author(s):  
A.S. Schurova ◽  
◽  
V.A. Bannov ◽  
A.V. Popova ◽  
◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Phage Genome ◽  
Genetically Engineered ◽  
Bacterial Cells ◽  
Practical Application ◽  
Bacterial Strains ◽  
Recombinant Phage ◽  
Research Problems ◽  
Genomic Editing

In recent decades, a major problem for health systems around the world is the wide spread of bacterial pathogens that are resistant to various antimicrobial agents. A possible approach to solving this problem is the use of bacteriophages, viruses that specifically infect bacterial cells, as well as enzymes and proteins encoded in their genomes. The development of genomic editing technologies, including those based on CRISPR-Cas editing, makes it possible to create genetically engineered or recombinant phage particles with desired properties that are important for further practical application. In this review, we consider issues related to the characterization of bacteriophages as biological objects and as promising candidates for controlling the spread of antibioticresistant bacterial strains. We discuss modern approaches and strategies for modifying the phage genomes using various methods of genetic engineering and molecular biology to solve a variety of practical and research problems. Keywords: bacteriophages, phage genome editing, CRISPR-Cas system

scholarly journals M1CR0B1AL1Z3R—a user-friendly web server for the analysis of large-scale microbial genomics data

2019 ◽  
Vol 47 (W1) ◽  
pp. W88-W92 ◽  
Author(s):  
Oren Avram ◽  
Dana Rapoport ◽  
Shir Portugez ◽  
Tal Pupko
Keyword(s):  
Large Scale ◽  
Ad Hoc ◽  
Evolutionary Dynamics ◽  
Gene Annotation ◽  
Gc Content ◽  
Web Server ◽  
Disease Outbreaks ◽  
Open Reading Frames ◽  
Microbial Genomics ◽  
Bacterial Strains

Abstract Large-scale mining and analysis of bacterial datasets contribute to the comprehensive characterization of complex microbial dynamics within a microbiome and among different bacterial strains, e.g., during disease outbreaks. The study of large-scale bacterial evolutionary dynamics poses many challenges. These include data-mining steps, such as gene annotation, ortholog detection, sequence alignment and phylogeny reconstruction. These steps require the use of multiple bioinformatics tools and ad-hoc programming scripts, making the entire process cumbersome, tedious and error-prone due to manual handling. This motivated us to develop the M1CR0B1AL1Z3R web server, a ‘one-stop shop’ for conducting microbial genomics data analyses via a simple graphical user interface. Some of the features implemented in M1CR0B1AL1Z3R are: (i) extracting putative open reading frames and comparative genomics analysis of gene content; (ii) extracting orthologous sets and analyzing their size distribution; (iii) analyzing gene presence–absence patterns; (iv) reconstructing a phylogenetic tree based on the extracted orthologous set; (v) inferring GC-content variation among lineages. M1CR0B1AL1Z3R facilitates the mining and analysis of dozens of bacterial genomes using advanced techniques, with the click of a button. M1CR0B1AL1Z3R is freely available at https://microbializer.tau.ac.il/.

scholarly journals Induction of Human β-Defensin 2 by the Probiotic Escherichia coli Nissle 1917 Is Mediated through Flagellin

2007 ◽  
Vol 75 (5) ◽  
pp. 2399-2407 ◽  
Author(s):  
Miriam Schlee ◽  
Jan Wehkamp ◽  
Artur Altenhoefer ◽  
Tobias A. Oelschlaeger ◽  
Eduard F. Stange ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Culture Supernatant ◽  
Deletion Mutants ◽  
Strain Atcc ◽  
Bacterial Strains ◽  
Flagellin Gene ◽  
E Coli ◽  
Escherichia Coli Nissle 1917 ◽  
Stimulatory Factor ◽  
Escherichia Coli Nissle

ABSTRACT Human β-defensin 2 (hBD-2) is an inducible antimicrobial peptide synthesized by the epithelium to counteract bacterial adherence and invasion. Proinflammatory cytokines, as well as certain bacterial strains, have been identified as potent endogenous inducers. Recently, we have found that hBD-2 induction by probiotic Escherichia coli Nissle 1917 was mediated through NF-κB- and AP-1-dependent pathways. The aim of the present study was to identify the responsible bacterial factor. E. coli Nissle 1917 culture supernatant was found to be more potent than the pellet, indicating a soluble or shed factor. Chemical analysis demonstrated the factor to be heat resistant and proteinase digestible. Several E. coli Nissle 1917 deletion mutants were constructed and tested for their ability to induce hBD-2 expression in Caco-2 cells. Deletion mutants for flagellin specifically exhibited an impaired immunostimulatory capacity. Reinsertion of the flagellin gene restored the induction capacity to normal levels. Isolated flagellin from E. coli Nissle 1917 and from Salmonella enterica serovar Enteritidis induced hBD-2 mRNA significantly in contrast to the flagellin of the apathogenic E. coli strain ATCC 25922. H1 flagellin antiserum abrogated hBD-2 expression induced by flagellin as well as E. coli Nissle 1917 supernatant, confirming that flagellin is the major stimulatory factor of E. coli Nissle 1917.

scholarly journals A nucleosome positioned in the distal promoter region activates transcription of the human U6 gene.

1997 ◽  
Vol 17 (8) ◽  
pp. 4397-4405 ◽  
Author(s):  
W Stünkel ◽  
I Kober ◽  
K H Seifart
Keyword(s):  
Micrococcal Nuclease ◽  
Deletion Mutants ◽  
Dna Fragments ◽  
Wild Type ◽  
Sequence Element ◽  
Proximal Sequence Element ◽  
Experimental Approaches ◽  
Type Gene ◽  
Distal Promoter ◽  
U6 Gene

To investigate the consequences of chromatin reconstitution for transcription of the human U6 gene, we assembled nucleosomes on both plasmids and linear DNA fragments containing the U6 gene. Initial experiments with DNA fragments revealed that U6 sequences located between the distal sequence element (DSE) and the proximal sequence element (PSE) lead to the positioning of a nucleosome partially encompassing these promoter elements. Furthermore, indirect end-labelling analyses of the reconstituted U6 wild-type plasmids showed strong micrococcal nuclease cuts near the DSE and PSE, indicating that a nucleosome is located between these elements. To investigate the influence that nucleosomes exert on U6 transcription, we used two different experimental approaches for chromatin reconstitution, both of which resulted in the observation that transcription of the U6 wild-type gene was enhanced after chromatin assembly. To ensure that the facilitated transcription of the nucleosomal templates is in fact due to a positioned nucleosome, we constructed mutants of the U6 gene in which the sequences between the DSE and PSE were progressively deleted. In contrast to what was observed with the wild-type genes, transcription of these deletion mutants was significantly inhibited when they were packaged into nucleosomes.

Identification of an 8-vinyl reductase involved in bacteriochlorophyll biosynthesis in Rhodobacter sphaeroides and evidence for the existence of a third distinct class of the enzyme

2013 ◽  
Vol 450 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Daniel P. Canniffe ◽  
Philip J. Jackson ◽  
Sarah Hollingshead ◽  
Mark J. Dickman ◽  
C. Neil Hunter
Keyword(s):  
Chlorophyll A ◽  
Rhodobacter Sphaeroides ◽  
Rhodobacter Capsulatus ◽  
Active Form ◽  
Green Sulfur Bacteria ◽  
Open Reading Frames ◽  
Knock Out ◽  
Sulfur Bacteria ◽  
Vinyl Group ◽  
Vinyl Reductase

The purple phototrophic bacterium Rhodobacter sphaeroides utilises bacteriochlorophyll a for light harvesting and photochemistry. The synthesis of this photopigment includes the reduction of a vinyl group at the C8 position to an ethyl group, catalysed by a C8-vinyl reductase. An active form of this enzyme has not been identified in R. sphaeroides, but its genome contains two candidate ORFs (open reading frames) similar to those reported to encode C8-vinyl reductases in the closely related Rhodobacter capsulatus (bchJ), and in plants and green sulfur bacteria (rsp_3070). To determine which gene encodes the active enzyme, knock-out mutants in both genes were constructed. Surprisingly, mutants in which one or both genes were deleted still retained the ability to synthesize C8-ethyl bacteriochlorophyll. These genes were subsequently expressed in a cyanobacterial mutant that cannot synthesize C8-ethyl chlorophyll a. R. sphaeroides rsp_3070 was able to restore synthesis of the WT (wild-type) C8-ethyl chlorophyll a in the mutant, whereas bchJ did not. The results of the present study demonstrate that Rsp_3070 is a functional C8-vinyl reductase and that R. sphaeroides utilises at least two enzymes to catalyse this reaction, indicating the existence of a third class, while there remains no direct evidence for the activity of BchJ as a C8-vinyl reductase.

scholarly journals A plasmid-encoded papB paralogue modulates autoaggregation of Escherichia coli transconjugants

2020 ◽  
Author(s):  
Ruben Monarrez ◽  
Iruka Okeke
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Genetic Basis ◽  
Mobile Element ◽  
Enteric Bacteria ◽  
Selective Advantage ◽  
Open Reading Frames ◽  
Bacterial Strains ◽  
Element Bearing ◽  
Reading Frames

Abstract Objective: Plasmids are key to antimicrobial resistance transmission among enteric bacteria. It is becoming increasingly clear that resistance genes alone do not account for the selective advantage of plasmids and bacterial strains that harbor them. Deletion of a 32 Kb fitness-conferring region of pMB2, a conjugative resistance plasmid, produced a hyper-autoaggregation phenotype in laboratory Escherichia coli. This study sought to determine the genetic basis for hyper-autoaggregation conferred by the pMB2-derived mini-plasmid. Results: The 32 Kb fragment deleted from pMB2 included previously characterized nutrient acquisition genes as well as putative transposase and integrase genes, a 272 bp papB/ pefB-like gene, and several open-reading frames of unknown function. We cloned the papB/ pefB paralogue and found it sufficient to temper the hyper-autoaggregation phenotype. Hyper-autoaggregation conferred by the mini-plasmid did not occur in a fim-negative background. This study has identified and characterized a gene capable of down-regulating host adhesins and has shown that trans-acting papB/pefB paralogues can occur outside the context of an adhesin cluster. This plasmid-mediated modification of a bacterial host’s colonization program may optimize horizontal transfer of the mobile element bearing the genes.

scholarly journals Biofilm Interaction Mapping and Analysis (BIMA): A tool for deconstructing interspecific interactions in co-culture biofilms

2021 ◽  
Author(s):  
Suzanne M Kosina ◽  
Peter Rademacher ◽  
Kelly M Wetmore ◽  
Markus de Raad ◽  
Marcin Zemla ◽  
...  
Keyword(s):  
Contaminated Soil ◽  
Interspecific Interactions ◽  
Pseudomonas Stutzeri ◽  
Interspecific Interaction ◽  
Deletion Mutants ◽  
Bacterial Strains ◽  
Chromium Vi ◽  
Pseudomonas Species ◽  
Aquaculture Management ◽  
Culture Growth

Pseudomonas species are ubiquitous in nature and include numerous medically, agriculturally and technologically beneficial strains of which the interspecific interactions are of great interest for biotechnologies. Specifically, co-cultures containing Pseudomonas stutzeri have been used for bioremediation, biocontrol, aquaculture management and wastewater denitrification. Furthermore, the use of P. stutzeri biofilms, in combination with consortia based approaches, may offer advantages for these processes. Understanding the interspecific interaction within biofilm co-cultures or consortia provides a means for improvement of current technologies. However, the investigation of biofilm based consortia has been limited. We present an adaptable and scalable method for the analysis of macroscopic interactions (colony morphology, inhibition and invasion) between colony forming bacterial strains using an automated printing method followed by analysis of the genes and metabolites involved in the interactions. Using Biofilm Interaction Mapping and Analysis (BIMA), these interactions were investigated between P. stutzeri strain RCH2, a denitrifier isolated from chromium (VI) contaminated soil, and thirteen other species of pseudomonas isolated from non-contaminated soil. The metabolites and genes associated with both active co-culture growth and inhibitory growth were investigated using mass spectrometry based metabolomics and mutant fitness profiling of a DNA-barcoded mutant library. One interaction partner, Pseudomonas fluorescens N1B4 was selected for mutant fitness profiling; with this approach four genes of importance were identified and the effects on interactions were evaluated with deletion mutants and metabolomics.

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