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Microbial genetics

The art and design of genetic screens: Escherichia coli

Nature Reviews Genetics volume 4, pages 419–431 (2003)Cite this article

Key Points

  • Bacterial genetics is based on the ability to select for rare mutants among large populations (1 in >1010) on the basis of the ability of the bacteria to survive lethal challenges, grow in the absence of supplements or take advantage of different growth substrates.

  • Bacterial genetics also uses a wide range of screens that are based on visual readouts of metabolic activity, including indicator agars (such as MacConkey and tetrazolium) and agars that contain chromogenic substrates (such as Xgal).

  • One of the most powerful genetic systems is the lactose operon of Escherichia coli. The lac genes encode an enzyme for breaking down lactose — lacZ (β-galactosidase) — and the membrane transporter for lactose — lacY (lac permease).

  • The lacZ gene can be fused to most other genes in many organisms. Transcriptional lacZ gene fusions produce wild-type β-galactosidase at levels that are determined by the promoter activity of the gene to which lacZ is fused. Translational lacZ gene fusions result in the production of hybrid β-galactosidase molecules that are encoded by both the gene to which lacZ is fused, and lacZ. The levels of production of the hybrid can depend on many post-transcriptional factors.

  • LacZ fusions allow the powerful tools that have been developed for the lac system to be applied to almost any bacterial system.

  • The properties of different LacZ fusions can provide important information about transcriptional and translational regulation as well as other cell functions, which include protein stability, protein folding, protein secretion and electron transport.

  • Modern 'high-tech' approaches that involve genomics and microarrays can be combined with genetic analysis to yield new insights into complex biological problems such as host–pathogen interactions.

Abstract

This article summarizes the general principles of selections and screens in Escherichia coli. The focus is on the lac operon, owing to its inherent simplicity and versatility. Examples of different strategies for mutagenesis and mutant discovery are described. In particular, the usefulness and effectiveness of simple colour-based screens are illustrated. The power of lac genetics can be applied to almost any bacterial system with gene fusions that hook any gene of interest to lacZ, which is the structural gene that encodes β-galactosidase. The diversity of biological processes that can be studied with lac genetics is remarkable and includes DNA metabolism, gene regulation and signal transduction, protein localization and folding, and even electron transport.

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Figure 1: The lactose operon of Escherichia coli.
Figure 2: MacConkey and tetrazolium agars.
Figure 3: α-complementation.
Figure 4: Development of Mal+ papillae on MacConkey maltose agar.
Figure 5: Two types of LacZ fusion.
Figure 6: The use of diploid analysis to distinguish negative control by a repressor from positive control by an activator.
Figure 7: The unexpected secretion-related phenotypes that are caused by lacZ translational fusions.

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Acknowledgements

Work in our laboratories has been supported by the National Institutes of Allergy and Infectious Disease and the National Science Foundation (H.A.S.), and by the National Institute of General Medical Sciences (T.J.S.). Thanks to R. Freudenberg for help with the photographs of the agar plates and N. Ruiz for help with figures.

Author information

Authors and Affiliations

  1. Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, 10032, USA

    Howard A. Shuman

  2. Department of Molecular Biology, Princeton University, Princeton, 08544, New Jersey, USA

    Thomas J. Silhavy

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  1. Howard A. Shuman
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Correspondence to Thomas J. Silhavy.

Related links

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DATABASES

BioCyc

CRP

CpxA

CpxR

DegP

degP

DsbA

galE

lacA

LacA

lacI

lacY

LacY

lacZ

LacZ

lamB

malE

MalF

MalK

malT

MalT

mutD

mutY

polA

rpsL

rssB

RpoS

rpoS

secA

Glossary

BACTERIOPHAGE

A virus that infects and replicates in bacteria. These are either lytic viruses, which always kill the host, or temperate viruses, which can either lyse the host cell or establish a stable relationship in which the bacteriophage genome is stably maintained in the host genome (see also lysogen).

LYSOGEN

A bacterial strain that harbours the genome of a temperate bacteriophage.

PLAQUE

A zone of lysed bacterial lawn that is formed by the growth of a bacteriophage.

REVERSION ANALYSIS

The isolation and characterization of revertants, or secondary mutants, that regain a wild-type-like phenotype. This analysis can provide clues to the nature of the original mutation.

PERMEABILIZED CELLS

Cells that have disrupted membranes.

CHROMOGENIC

A colourless substrate that produces a coloured product following the action of one or more enzymes.

INDUCER

A substance that causes a gene, or set of genes, to be expressed at a higher level.

INSERTION SEQUENCES

(IS elements). Sequences that can insert themselves into target sites on DNA.

PSEUDOREVERTANT

A revertant that does not re-establish a wild-type genotype.

ENTERIC BACTERIA

Bacteria that normally inhabit the intestines.

LEAKY DOWN PHENOTYPE

A phenotype in which there is a moderate decrease in gene function.

CLONING VECTORS

Engineered plasmids with convenient cloning sites and markers.

COMPLEMENTATION

The use of partial diploids to determine whether two mutations affect the same or different genes. If the mutations are in the same gene, they generally fail to complement each other and the diploid retains the mutant phenotype. By contrast, mutations in different genes usually complement one another and restore a wild-type phenotype to the diploid. However, exceptions to both cases abound.

POLAR

A phenomenon in bacterial transcriptional units in which the transcription of downstream genes is decreased by a translation stop in an upstream gene.

PAPILLATION

The formation of secondary colonies that grow on top of a colony that has already formed on a nutrient agar surface.

HETERODUPLEX

A DNAmolecule that is formed by base pairing between strands that are derived from two DNA molecules that are not identical in sequence.

REGULON

A group of transcriptional units or operons that are coordinately controlled by a regulator.

SMALL RNAS

Small untranslated RNAs that function in regulation.

STATIONARY PHASE

The phase of growth of a batch culture in which nutrients become limiting and growth ceases.

PULSE-CHASE EXPERIMENT

An experiment in which a radioactive form of a precursor is added for a brief period ('pulse') so that it is incorporated into a macromolecule, then a non-radioactive identical chemical is added to dilute the incorporation of the radioactive species ('chase').

SIGNAL SEQUENCE

An amino-acid sequence at the amino terminus of a protein that directs the protein to the secretion system for translocation across the cell membrane.

MALTOPORIN

The Escherichia coli outer-membrane protein that functions as a diffusion channel for maltooligosaccharides.

POLYTOPIC

A membrane-spanning protein with more than one membrane-spanning segment.

FLUOROPHORE

A small molecule, or a part of a larger molecule, that can be excited by light to emit fluorescence.

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Shuman, H., Silhavy, T. The art and design of genetic screens: Escherichia coli. Nat Rev Genet 4, 419–431 (2003). https://doi.org/10.1038/nrg1087

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