Umu Response

Overview

The umu test, first developed and published by Oda et al., is based on the ability of DNA-damaging agents to induce the expression of the umu operon. In connection with the damage inducible genes (din genes) recA, lexA and umuD, the umuC gene is essentially involved in bacterial mutagenesis through the SOS response.

LexA protein is the repressor of all cellular din genes. Lesions such as single-stranded DNA, depurinic and depytimidinic sites or even free deoxynucletoides seem to activate RecA to LexA processing form that facilitates cleavage of LexA repressor, thus leading to derepression of all din genes. RecA protein appears to have further roles in the mutagenic process: first it facilitates cleavage of UmuD protein, thereby generating a mutagenically active form (UmuD’).

Due to the participation of the umuC in the mutagenic process leading to both point- and frameshift-mutations, only one single bacterial strain is necessary to detect different types of mutagens. Nevertheless, additional strains have been developed in order to increase sensitivity for the detection of genotoxins, belonging to certain classes, such as nitroarenes.

All umu strains carry the plasmid pSK1002 which bears an umuD gene and an umuC gene fused with lacZ, the structural gene for beta-galactosidase. The induction of mutator gene umuC by DNA-damaging agents is detected by measuring intracellular beta-galactosidase levels. Like other tests, the strains have been genetically modified further. A reduction of the lipopolysaccharide structure of the cell wall (rfa) facilitates an increased permeability, epsecailly for hydrophobic chemicals such as polyaromatic hydrocarbons. A further sensitivity enhancing alteration compared with wild type strains is the deficiency in a general pathway for the excision of damaged bases from the DNA: the nucleotide-excision repair. Incision near conformational distortions of the DNA, caused by a variety of bulky adducts, occurs via the formation of multiprotein complex, consisting of 3 proteins, collectively called UvrABC excinuclease. The deletion of this region results in a loss of repair efficiency.

Genotoxicity Testing

The umu operon is useful as it is likely to be more involved in mutageneis than the other known SOS genes (Kato and Shinoura, 1977), and can be taken advantage of in the same way as the SOS Chromotest. Taking advantage of the operon fusion placing the lac operon (responsible for producing beta-galactosidase, a protein which degrades lactose) under the control of an SOS-related protein, a simple colorimetric assay for genotoxicity is possible. If a genotoxin is present, the resulting DNA damage will result in the production of beta-galactosidase instead of DNA repair as a result of the gene fusion. A lactose analog is added to the media which is degraded by the beta-galactosidase, producing a colored compound which can be measured quantitatively through spectrophotometry. The degree of color development is a direct measure of the amount of beta-galactosidase produce, which itself is directly related to the extent of DNA damage.