Plasmid selection in Escherichia coli using an endogenous essential gene marker

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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YhdJ, a Nonessential CcrM-Like DNA Methyltransferase of Escherichia coli and Salmonella enterica

Journal of Bacteriology ◽

10.1128/jb.01854-06 ◽

2007 ◽

Vol 189 (11) ◽

pp. 4325-4327 ◽

Cited By ~ 20

Author(s):

Sarah E. Broadbent ◽

Roberto Balbontin ◽

Josep Casadesus ◽

Martin G. Marinus ◽

Marjan van der Woude

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Dna Methyltransferase ◽

Caulobacter Crescentus ◽

Essential Gene ◽

Gene Products ◽

E Coli ◽

Dna Adenine Methyltransferase

ABSTRACT The Caulobacter crescentus DNA adenine methyltransferase CcrM and its homologs in the α-Proteobacteria are essential for viability. CcrM is 34% identical to the yhdJ gene products of Escherichia coli and Salmonella enterica. This study provides evidence that the E. coli yhdJ gene encodes a DNA adenine methyltransferase. In contrast to an earlier report, however, we show that yhdJ is not an essential gene in either E. coli or S. enterica.

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Titration and Conditional Knockdown of the prfB Gene in Escherichia coli: Effects on Growth and Overproduction of the Recombinant Mammalian Selenoprotein Thioredoxin Reductase

Applied and Environmental Microbiology ◽

10.1128/aem.02019-06 ◽

2006 ◽

Vol 73 (2) ◽

pp. 432-441 ◽

Cited By ~ 14

Author(s):

Olle Rengby ◽

Elias S. J. Arnér

Keyword(s):

Escherichia Coli ◽

Growth Rate ◽

Essential Gene ◽

Thioredoxin Reductase ◽

Lower Growth ◽

E Coli ◽

Native Promoter ◽

Lower Growth Rate ◽

The Cost ◽

Translational Termination

ABSTRACT Release factor 2 (RF2), encoded by the prfB gene in Escherichia coli, catalyzes translational termination at UGA and UAA codons. Termination at UGA competes with selenocysteine (Sec) incorporation at Sec-dedicated UGA codons, and RF2 thereby counteracts expression of selenoproteins. prfB is an essential gene in E. coli and can therefore not be removed in order to increase yield of recombinant selenoproteins. We therefore constructed an E. coli strain with the endogenous chromosomal promoter of prfB replaced with the titratable PBAD promoter. Knockdown of prfB expression gave a bacteriostatic effect, while two- to sevenfold overexpression of RF2 resulted in a slightly lowered growth rate in late exponential phase. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and recombinant selenoprotein expression was subsequently studied, using production of mammalian thioredoxin reductase as model system. This showed that lowering the levels of RF2 correlated directly with increasing Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a lower growth rate. This study thus demonstrates that expression of prfB can be titrated through targeted exchange of the native promoter with a PBAD-promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01444-15 ◽

2015 ◽

Vol 81 (16) ◽

pp. 5650-5659 ◽

Cited By ~ 4

Author(s):

Shan Goh ◽

Angela Hohmeier ◽

Timothy C. Stone ◽

Victoria Offord ◽

Francisco Sarabia ◽

...

Keyword(s):

Escherichia Coli ◽

Target Gene ◽

Essential Gene ◽

Transcript Level ◽

Essential Genes ◽

Content Type ◽

Reading Frame ◽

Knockout Mutants ◽

Bacterial Genes ◽

Relationship Of

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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