Maurice Hofnung

The SOS Chromotest, a colorimetric bacterial assay for genotoxins: procedures

The SOS Chromotest is a quantitative bacterial colorimetric assay for genotoxins. Substantial validation is now available (Quillardet et al., 1985). We describe here in detail the tester strain as well as the effects of the variation of some parameters on the assay. We report a simple spot-test procedure as well as a new standard procedure which incorporate recent technical improvements aimed at simplifying the assay further.

Getting In or Out: Early Segregation Between Importers and Exporters in the Evolution of ATP-Binding Cassette (ABC) Transporters

Abstract. ATP-binding cassette (ABC) systems, also called traffic ATPases, are found in eukaryotes and prokaryotes and almost all participate in the transport of a wide variety of molecules. ABC systems are characterized by a highly conserved ATPase module called here the ABC module, involved in coupling transport to ATP hydrolysis. We have used the sequence of one of the first representatives of bacterial ABC transporters, the MalK protein, to collect 250 closely related sequences from a nonredundant protein sequence database. The sequences collected by this objective method are all known or putative ABC transporters. After having eliminated short protein sequences and duplicates, the 197 remaining sequences were subjected to a phylogenetic analysis based on a mutational similarity matrix. An unrooted tree for these modules was found to display two major branches, one grouping all collected uptake systems and the other all collected export systems. This remarkable disposition strongly suggests that the divergence between these two functionally different types of ABC systems occurred once in the history of these systems and probably before the differentiation of prokaryotes and eukaryotes. We discuss the implications of this finding and we propose a model accounting for the generation and the diversification of ABC systems.

The SOS chromotest: a review☆☆☆

The SOS chromotest is reviewed through over 100 publications corresponding to the testing of 751 chemicals. 404 (54%) of these chemicals present a genotoxic activity detectable in the SOS chromotest. Their SOS inducing potencies span more than 8 orders of magnitude. For 452 compounds, the results obtained in the SOS chromotest could be compared to those obtained in the Ames test. It was found that 373 (82%) of these compounds give similar responses in both tests (236 positive and 137 negative responses). Thus the discrepancies between both tests concern 79 compounds (18%). A case by case analysis shows that many of these compounds are at the same time very weak SOS inducers and very weak mutagens. Thus we think that, most of the time, the discrepancies between the two tests may be accounted for by differences in the interpretation of the results rather than by the experimental results themselves. However, there are some compounds which are clearly SOS inducers but devoid of mutagenic activity in the Ames test (such as quinoline-1-oxide) and to a larger extent, clearly mutagenic compounds which do not induce the SOS response in the SOS chromotest (such as benzidine, cyclophosphamide, acridines, ethidium bromide). We also analyzed the correlation between SOS induction, mutagenesis and carcinogenesis according to the classification of Lewis. For 65 confirmed carcinogens (class 1), the sensitivity, i.e., the capacity to identify carcinogens, was 62% with the SOS chromotest and 77% with the Ames test. For 44 suspected carcinogens (class 2), the sensitivity was 66% with the SOS chromotest and 68% with the Ames test. Thus, we confirmed previous observations made on 83 compounds that there is a close correlation between the results given by both bacterial tests. The capacity of the Ames test to identify carcinogens is higher than that of the SOS chromotest. However, because the number of false positive compounds was lower in the SOS chromotest, the specificity, i.e., the capacity to discriminate between carcinogens and non-carcinogens of the SOS chromotest, appeared higher than that of the Ames test. Thus, the results of the SOS chromotest and of the Ames test can complement each other. The SOS chromotest is one of the most rapid and simple short-term test for genotoxins and is easily adaptable to various conditions, so that it could be used as an early--perhaps the earliest--test in a battery.

The SOS Chromotest, a colorimetric bacterial assay for genotoxins: validation study with 83 compounds

The SOS Chromotest is a simple bacterial colorimetric assay for genotoxicity. It is based on the measure of the induction of sfiA, a gene controlled by the general repressor of the SOS system in E. coli. Expression of sfiA is monitored by means of a gene fusion with lacZ, the structural gene for beta-galactosidase. We have examined 83 compounds of various chemical classes with the SOS Chromotest using a standard procedure. Comparison of the results with those obtained in the Mutatest (the Ames test) showed that most (90%) of the mutagenic compounds were also SOS inducers. For these compounds a quantitative correlation was observed between the mutagenic potency and the SOS-inducing potency (SOSIP). The case of the 10% remaining compounds giving conflicting results in the two tests is discussed. Sensitivity, specificity and accuracy for carcinogenicity prediction have been evaluated for the SOS Chromotest and the Mutatest using 73 chemicals for which carcinogenicity data were available. In spite of some differences, similar results were obtained in the two tests. The present data indicate that the SOS Chromotest has many practical advantages and may be used as a primary screening tool or as part of a battery of short-term tests for carcinogens.

Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface.

The LamB protein is a trimeric integral outer membrane protein from Escherichia coli K12 which functions as a pore for maltodextrins and a receptor for bacteriophages. When inserted into two selected sites of LamB, a foreign antigen, the C3 epitope from poliovirus, was exposed at the cell surface with its normal antigenic properties. Since these genetic insertions did not affect in any essential way the routing, activity and folding of the LamB protein, we conclude that the two corresponding LamB sites are at the cell surface as predicted by our recent model. We discuss the implications of our results for the study of protein topology with a single epitope and the direct cloning and cell surface expression of epitopes of interest as well as the development of live vaccines or diagnostic tests.

Subunit interactions in ABC transporters: a conserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits

The cytoplasmic membrane proteins of bacterial binding protein‐dependent transporters belong to the superfamily of ABC transporters. The hydrophobic proteins display a conserved, at least 20 amino acid EAA‐‐‐G‐‐‐‐‐‐‐‐‐I‐LP region exposed in the cytosol, the EAA region. We mutagenized the EAA regions of MalF and MalG proteins of the Escherichia coli maltose transport system. Substitutions at the same positions in MalF and MalG have different phenotypes, indicating that EAA regions do not act symmetrically. Mutations in malG or malF that slightly affect or do not affect transport, determine a completely defective phenotype when present together. This suggests that EAA regions of MalF and MalG may interact during transport. Maltose‐negative mutants fall into two categories with respect to the cellular localization of the MalK ATPase: in the first, MalK is membrane‐bound, as in wild‐type strains, while in the second, it is cytosolic, as in strains deleted in the malF and malG genes. From maltose‐negative mutants of the two categories, we isolated suppressor mutations within malK that restore transport. They map mainly in the putative helical domain of MalK, suggesting that EAA regions may constitute a recognition site for the ABC ATPase helical domain.

A family of dispersed repetitive extragenic palindromic DNA sequences in E. coli.

We report the properties of 67 members of a family of dispersed repetitive palindromic extragenic bacterial DNA sequences. These sequences, called palindromic units, appear to be present at least several hundred times outside structural genes on the Escherichia coli chromosome. They are found either in clusters ‐ as in a previously described intercistronic element ‐ or in single occurrences. They are not only found within an operon but also between different operons, including between convergent ones. The palindromic units could yield a stem and loop structure at the level of DNA or RNA. The base of the stem is made of eight remarkably conserved base pairs while the rest varies somewhat in length and sequence. We analyse the data available on the palindromic units and we speculate on their possible roles with emphasis on transcription and mRNA stability or processing, as well as on their possible relation to transposition elements and the modular evolution of the genome.

Gene sequence of the λ receptor, an outer membrane protein of E. coli K12

Abstract The Escherichia coli K12 λ receptor is a multifunctional outer membrane protein whose precursor, encoded in gene lamB , is cleaved during export. We present the DNA sequence of lamB and of the distal region that contains repetitive and palindromic sequences and could give rise to highly stable mRNA structures. The calculated molecular weight of the λ receptor is 47,400. Of the 421 amino acids, 89 are charged, mostly negatively. No region devoid of charged amino acids and long enough to serve as a transmembrane portion is detected. The distribution of charges presents special features that we comment upon in relation to the structure, functions and localization of the λ receptor. Gene lamB is followed by molA , an unidentified reading frame corresponding to a 131-amino-acid peptide with the characteristics of an exported protein.

Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein

The highly conserved amino acid sequence ELDKWA of HIV-1 gp41 has been inserted into Escherichia coli MalE protein which had been shown to be an adequate carrier to present foreign epitopes to the immune system. We first investigated whether eight different permissive sites of MalE are able to tolerate an insertion of 7-50 residues encoding this epitope. Secondly, antigenicity of the epitope inserted in MalE protein was estimated from monoclonal antibody 2F5 binding analysis using the BIAcore(R) technology and its immunogenicity in mice was measured as the ability of hybrid proteins to elicit antibodies against a synthetic peptide containing this epitope. This study revealed a good correlation between the antigenicity of the inserted epitope and its immunogenicity. Increasing the length of the inserted epitope, as well as inserting multicopies of this epitope increased both its antigenicity and immunogenicity. However, none of the MalE hybrid proteins tested induced anti-HIV-1 neutralizing antibodies. This study strongly suggests that the capacity of the 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented.

Short palindromic repetitive DNA elements in enterobacteria: a survey

We present a survey of short palindromic repetitive elements in enterobacteria. Seven families are presented. Five were already known (RSA, IRU, 29-bp repeats, BIMEs and boxC), and their properties are updated; in particular, a new composite element is shown to include the formerly identified boxC repeats. Two repetitions, YPAL1 and YPAL2, found primarily in Yersinia, are described here for the first time.