Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli

ABSTRACTEssential bacterial genes located within operons are particularly challenging to study independently because of coordinated gene expression and the nonviability of knockout mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene disruption of genes may help establish essentiality but can lead to polar effects on genes downstream or upstream of the target gene. Here, theEscherichia coliribF-ileS-lspA-fkpB-ispHoperon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. The gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition as the minimum transcript level required for 50% growth (MTL50). Mupirocin and globomycin, the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and open reading frame-expressing plasmids to examine deregulation of the overlappingileS-lspAgenes. We found upstream and downstream polar silencing effects when eitherileSorlspAwas silenced, indicating coupled expression. Weighted MTL50values (means and standard deviations) ofribF,ileS, andlspAwere 0.65 ± 0.18, 0.64 ± 0.06, and 0.76 ± 0.10, respectively. However, they were not significantly different (P= 0.71 by weighted one-way analysis of variance). The gene requirement forispHcould not be determined due to insufficient growth reduction. Mupirocin and globomycin sensitization experiments indicated thatileS-lspAexpression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA-silencing antimicrobials.

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A novel way to purify recombinant baculoviruses by using bacmid

Bioscience Reports ◽

10.1042/bsr20080074 ◽

2009 ◽

Vol 29 (2) ◽

pp. 71-75 ◽

Cited By ~ 1

Author(s):

Wu-Jie Su ◽

Wei-De Shen ◽

Bing Li ◽

Yan Wu ◽

Guang Gao ◽

...

Keyword(s):

Insect Cells ◽

Nuclear Polyhedrosis Virus ◽

Essential Gene ◽

Essential Genes ◽

Reading Frame ◽

Recombinant Viruses ◽

Recombinant Bacmid ◽

Bmnpv Bacmid ◽

Nuclear Polyhedrosis ◽

Recombinant Bacmids

In the present study, we studied the feasibility of deleting essential genes in insect cells by using bacmid and purifying recombinant bacmid in Escherichia coli DH10B cells. To disrupt the orf4 (open reading frame 4) gene of BmNPV [Bm (Bombyx mori) nuclear polyhedrosis virus], a transfer vector was constructed and co-transfected with BmNPV bacmid into Bm cells. Three passages of viruses were carried out in Bm cells, followed by one round of purification. Subsequently, bacmid DNA was extracted and transformed into competent DH10B cells. A colony harbouring only orf4-disrupted bacmid DNA was identified by PCR. A mixture of recombinant (white colonies) and non-recombinant (blue colonies) bacmids were also transformed into DH10B cells. PCR with M13 primers showed that the recombinant and non-recombinant bacmids were separated after transformation. The result confirmed that purification of recombinant viruses could be carried out simply by transformation and indicated that this method could be used to delete essential genes. Orf4-disrupted bacmid DNA was extracted and transfected into Bm cells. Viable viruses were produced, showing that orf4 was not an essential gene.

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Elucidation of Regulatory Modes for Five Two-Component Systems in Escherichia coli Reveals Novel Relationships

mSystems ◽

10.1128/msystems.00980-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Kumari Sonal Choudhary ◽

Julia A. Kleinmanns ◽

Katherine Decker ◽

Anand V. Sastry ◽

Ye Gao ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Target Gene ◽

Target Genes ◽

Rna Seq ◽

Content Type ◽

E Coli ◽

Component Systems ◽

Two Component ◽

Two Component Systems

ABSTRACT Escherichia coli uses two-component systems (TCSs) to respond to environmental signals. TCSs affect gene expression and are parts of E. coli’s global transcriptional regulatory network (TRN). Here, we identified the regulons of five TCSs in E. coli MG1655: BaeSR and CpxAR, which were stimulated by ethanol stress; KdpDE and PhoRB, induced by limiting potassium and phosphate, respectively; and ZraSR, stimulated by zinc. We analyzed RNA-seq data using independent component analysis (ICA). ChIP-exo data were used to validate condition-specific target gene binding sites. Based on these data, we do the following: (i) identify the target genes for each TCS; (ii) show how the target genes are transcribed in response to stimulus; and (iii) reveal novel relationships between TCSs, which indicate noncognate inducers for various response regulators, such as BaeR to iron starvation, CpxR to phosphate limitation, and PhoB and ZraR to cell envelope stress. Our understanding of the TRN in E. coli is thus notably expanded. IMPORTANCE E. coli is a common commensal microbe found in the human gut microenvironment; however, some strains cause diseases like diarrhea, urinary tract infections, and meningitis. E. coli’s two-component systems (TCSs) modulate target gene expression, especially related to virulence, pathogenesis, and antimicrobial peptides, in response to environmental stimuli. Thus, it is of utmost importance to understand the transcriptional regulation of TCSs to infer bacterial environmental adaptation and disease pathogenicity. Utilizing a combinatorial approach integrating RNA sequencing (RNA-seq), independent component analysis, chromatin immunoprecipitation coupled with exonuclease treatment (ChIP-exo), and data mining, we suggest five different modes of TCS transcriptional regulation. Our data further highlight noncognate inducers of TCSs, which emphasizes the cross-regulatory nature of TCSs in E. coli and suggests that TCSs may have a role beyond their cognate functionalities. In summary, these results can lead to an understanding of the metabolic capabilities of bacteria and correctly predict complex phenotype under diverse conditions, especially when further incorporated with genome-scale metabolic models.

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High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

Applied and Environmental Microbiology ◽

10.1128/aem.00313-14 ◽

2014 ◽

Vol 80 (13) ◽

pp. 3826-3834 ◽

Cited By ~ 44

Author(s):

Junjie Yang ◽

Bingbing Sun ◽

He Huang ◽

Yu Jiang ◽

Liuyang Diao ◽

...

Keyword(s):

Escherichia Coli ◽

Genetic Modification ◽

Target Gene ◽

Donor Plasmid ◽

Content Type ◽

Recognition Sites ◽

Genetic Modifications ◽

Pcr Targeting ◽

Λ Red ◽

Red Recombination

ABSTRACTGenetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of theEscherichia colichromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used forcadAknockout,gdhAknock-in, seamless deletion ofpepD, site-directed mutagenesis of the essentialmetKgene, and replacement ofmetKwith theRickettsiaS-adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.

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Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering

Microbiology ◽

10.1099/mic.0.079376-0 ◽

2014 ◽

Vol 160 (11) ◽

pp. 2341-2351 ◽

Cited By ~ 77

Author(s):

Mario Juhas ◽

Daniel R. Reuß ◽

Bingyao Zhu ◽

Fabian M. Commichau

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Essential Gene ◽

Building Blocks ◽

Essential Genes ◽

Minimal Cell ◽

E Coli ◽

Cell Factories ◽

Gene Sets ◽

K 12

Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell ‘chassis’. Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnological applications. Determination of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome reduction analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications.

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The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00066-12 ◽

2012 ◽

Vol 56 (6) ◽

pp. 3216-3223 ◽

Cited By ~ 20

Author(s):

Tina Manzhu Kang ◽

Jessica Yuan ◽

Angelyn Nguyen ◽

Elinne Becket ◽

Hanjing Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Bacillus Anthracis ◽

Gene Knockout ◽

Aminoglycoside Antibiotic ◽

Double Strand Breaks ◽

Repair Capacity ◽

Content Type ◽

Strand Breaks ◽

Knockout Mutants ◽

Increased Sensitivity

ABSTRACTThe distribution of mutants in the Keio collection ofEscherichia coligene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ,holC,holD, andpriAknockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded byrecA,recB, andrecC, among others. Additionally, caffeine partially protects cells of bothEscherichia coliandBacillus anthracisfrom killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

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ThechbGGene of the Chitobiose (chb) Operon of Escherichia coli Encodes a Chitooligosaccharide Deacetylase

Journal of Bacteriology ◽

10.1128/jb.00533-12 ◽

2012 ◽

Vol 194 (18) ◽

pp. 4959-4971 ◽

Cited By ~ 19

Author(s):

Subhash Chandra Verma ◽

Subramony Mahadevan

Keyword(s):

Escherichia Coli ◽

Metal Binding ◽

Inflammatory Diseases ◽

Regulatory Protein ◽

Loss Of Function ◽

Content Type ◽

Reading Frame ◽

E Coli ◽

Evolutionarily Conserved

ABSTRACTThechboperon ofEscherichia coliis involved in the utilization of the β-glucosides chitobiose and cellobiose. The function ofchbG(ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show thatchbGencodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of thechbpromoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations inchbRconferring the ability to grow on both cellobiose and chitobiose are independent ofchbGfunction for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-β-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence ofchbG, suggesting an additional role ofchbGin the hydrolysis of chitooligosaccharides. The homologs ofchbGin metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function ofE. colichbGhas a broader significance.

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Mini-Tn7Insertion in an ArtificialattTn7Site Enables Depletion of the Essential Master Regulator CtrA in the Phytopathogen Agrobacterium tumefaciens

Applied and Environmental Microbiology ◽

10.1128/aem.01392-16 ◽

2016 ◽

Vol 82 (16) ◽

pp. 5015-5025 ◽

Cited By ~ 16

Author(s):

Wanda Figueroa-Cuilan ◽

Jeremy J. Daniel ◽

Matthew Howell ◽

Aliyah Sulaiman ◽

Pamela J. B. Brown

Keyword(s):

Agrobacterium Tumefaciens ◽

Cell Cycle Progression ◽

Essential Gene ◽

Essential Genes ◽

Master Regulator ◽

Cycle Progression ◽

Content Type ◽

Inducible Promoters ◽

Bacterial Plant Pathogen

ABSTRACTMechanistic studies of many processes inAgrobacterium tumefacienshave been hampered by a lack of genetic tools for characterization of essential genes. In this study, we used a Tn7-based method for inducible control of transcription from an engineered site on the chromosome. We demonstrate that this method enables tighter control of inducible promoters than plasmid-based systems and can be used for depletion studies. The method enables the construction of depletion strains to characterize the roles of essential genes inA. tumefaciens. Here, we used the strategy to deplete the alphaproteobacterial master regulator CtrA and found that depletion of this essential gene results in dramatic rounding of cells, which become nonviable.IMPORTANCEAgrobacterium tumefaciensis a bacterial plant pathogen and natural genetic engineer. Thus, studies of essential processes, including cell cycle progression, DNA replication and segregation, cell growth, and division, may provide insights for limiting disease or improving biotechnology applications.

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The essential genome ofEscherichia coliK-12

10.1101/237842 ◽

2017 ◽

Author(s):

Emily C. A. Goodall ◽

Ashley Robinson ◽

Iain G. Johnston ◽

Sara Jabbari ◽

Keith A. Turner ◽

...

Keyword(s):

Escherichia Coli ◽

Statistical Analysis ◽

Essential Gene ◽

Automated Analysis ◽

Transposon Mutagenesis ◽

Essential Genes ◽

High Density ◽

Bacterial Physiology ◽

E Coli ◽

K 12

ABSTRACTTransposon-Directed Insertion-site Sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries and therefore it remains unclear whether the two methodologies are comparable. To address this, a high density transposon library was constructed inEscherichia coliK-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false positive identification of essential gene candidates, statistical data analysis included corrections for both gene length and genome length. Through this analysis new essential genes and genes previously incorrectly designated as essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects and fine resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis datasets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.IMPORTANCEIncentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes inE. coli, we constructed a very high density transposon mutant library. Initial automated analysis of the resulting data revealed many discrepancies when compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high density TraDIS sequencing data for each putative essential gene for the model laboratory organism,Escherichia coli. This paper is important because it provides a better understanding of the essential genes ofE. coli, reveals the limitations of relying on automated analysis alone and a provides new standard for the analysis of TraDIS data.

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Dissemination and Systemic Colonization of Uropathogenic Escherichia coli in a Murine Model of Bacteremia

mBio ◽

10.1128/mbio.00262-10 ◽

2010 ◽

Vol 1 (5) ◽

Cited By ~ 46

Author(s):

Sara N. Smith ◽

Erin C. Hagan ◽

M. Chelsea Lane ◽

Harry L. T. Mobley

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Murine Model ◽

Systemic Disease ◽

Content Type ◽

E Coli ◽

Bacterial Genes ◽

Tract Infections ◽

K 12

ABSTRACTInfection with uropathogenicEscherichia coli(UPEC), the causative agent of most uncomplicated urinary tract infections, proceeds in an ascending manner and, if left untreated, may result in bacteremia and urosepsis. To examine the fate of UPEC after its entry into the bloodstream, we developed a murine model of sublethal bacteremia. CBA/J mice were inoculated intravenously with 1 × 106 CFU of pyelonephritis strainE. coliCFT073 carrying a bioluminescent reporter. Biophotonic imaging, used to monitor the infection over 48 h, demonstrated that the bacteria disseminated systemically and appeared to localize at discrete sites. UPEC was recovered from the spleen, liver, kidneys, lungs, heart, brain, and intestines as early as 20 min postinoculation, peaking at 24 h postinoculation. A nonpathogenicE. coliK-12 strain, however, disseminated at significantly lower levels (P< 0.01) and was cleared from the liver and cecum by 24 h postinoculation. Isogenic mutants lacking type 1 fimbriae, P fimbriae, capsule, TonB, the heme receptors Hma and ChuA, or particularly the sialic acid catabolism enzyme NanA were significantly outcompeted by wild-type CFT073 during bacteremia (P< 0.05), while flagellin and hemolysin mutants were not.IMPORTANCEE. coliis the primary cause of urinary tract infections. In severe cases of kidney infection, bacteria can enter the bloodstream and cause systemic disease. While the ability ofE. colito cause urinary tract infection has been extensively studied, the fate of these bacteria once they enter the bloodstream is largely unknown. Here we used an imaging technique to develop a mouse model ofE. colibloodstream infection and identify bacterial genes that are important for the bacteria to spread to and infect various organs. Understanding how urinary tract pathogens likeE. colicause disease after they enter the bloodstream may aid in the development of protective and therapeutic treatments.

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Redesigned TetR-Aptamer System To Control Gene Expression in Plasmodium falciparum

mSphere ◽

10.1128/msphere.00457-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Krithika Rajaram ◽

Hans B. Liu ◽

Sean T. Prigge

Keyword(s):

Gene Expression ◽

Plasmodium Falciparum ◽

Target Gene ◽

Essential Genes ◽

Untranslated Regions ◽

Dependent Manner ◽

Control Of Gene Expression ◽

Content Type ◽

Target Gene Expression ◽

The Impact

ABSTRACT One of the most powerful approaches to understanding gene function involves turning genes on and off at will and measuring the impact at the cellular or organismal level. This particularly applies to the cohort of essential genes where traditional gene knockouts are inviable. In Plasmodium falciparum, conditional control of gene expression has been achieved by using multicomponent systems in which individual modules interact with each other to regulate DNA recombination, transcription, or posttranscriptional processes. The recently devised TetR-DOZI aptamer system relies on the ligand-regulatable interaction of a protein module with synthetic RNA aptamers to control the translation of a target gene. This technique has been successfully employed to study essential genes in P. falciparum and involves the insertion of several aptamer copies into the 3′ untranslated regions (UTRs), which provide control over mRNA fate. However, aptamer repeats are prone to recombination and one or more copies can be lost from the system, resulting in a loss of control over target gene expression. We rectified this issue by redesigning the aptamer array to minimize recombination while preserving the control elements. As proof of concept, we compared the original and modified arrays for their ability to knock down the levels of a putative essential apicoplast protein (PF3D7_0815700) and demonstrated that the modified array is highly stable and efficient. This redesign will enhance the utility of a tool that is quickly becoming a favored strategy for genetic studies in P. falciparum. IMPORTANCE Malaria elimination efforts have been repeatedly hindered by the evolution and spread of multidrug-resistant strains of Plasmodium falciparum. The absence of a commercially available vaccine emphasizes the need for a better understanding of Plasmodium biology in order to further translational research. This has been partly facilitated by targeted gene deletion strategies for the functional analysis of parasite genes. However, genes that are essential for parasite replication in erythrocytes are refractory to such methods, and require conditional knockdown or knockout approaches to dissect their function. One such approach is the TetR-DOZI system that employs multiple synthetic aptamers in the untranslated regions of target genes to control their expression in a tetracycline-dependent manner. Maintaining modified parasites with intact aptamer copies has been challenging since these repeats can be lost by recombination. By interspacing the aptamers with unique sequences, we created a stable genetic system that remains effective at controlling target gene expression.

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