Transposon Tn5supF-based reverse genetic method for mutational analysis of Escherichia coli with DNAs cloned in lambda phage

Abstract

An efficient method for systematic mutational analysis of the Escherichia coli genome was developed. It entails TnSsupF transposition to X-E. coli hybrid phage clones (Kohara library) and then transduction of recipient cells to Sup'. Essential and nonessential genes are distinguished by the ability of insertion mutant phage to form haploid versus only heterozygous partial diploid bacterial recombinants.We present here an efficient reverse genetic method for mutational analysis of Escherichia coli K-12, developed to help complete the genetic map of this model organism. We anticipate that new mutational methods will be useful for E. coli because (i) although -1,400 of its genes have been identified by mutation and mapped, one can estimate that more than half remain to be characterized (2, 16); (ii) the DNA sequence by itself does not show which open reading frames and other features contribute to the phenotype and which do not; (iii) traditional searches for mutations with specific phenotypes are not an efficient way of completing the genetic map because mutations in already known genes tend to be isolated repeatedly; and (iv) existing reverse genetic methods are not suitable for large-scale screening. Most of these methods entail insertion of resistance markers into target genes by in vitro mutagenesis of plasmid clones, blockage of plasmid replication, and then screening for plasmid loss (8,10,12,22,26). Obtaining haploid mutant bacterial recombinants can be difficult, especially when the mutated fragment is small or the mutant allele is close to one fragment end, because the vector is not selected against directly. Another method, transformation by linear DNA fragments, generates only haploid recombinants (25,28,31), but it is too inefficient for rapid large-scale screening. A third method entails subcloning specific mutant alleles to K cI857 phage vectors, generating phage-chromosome cointegrates by one crossover, and then selecting nonlysogenic haploid mutant recombinants (6, 13).The method described here ( Fig. 1) (4), and then simple transduction tests to distinguish essential and nonessential genes.Allelic replacement following phage infection. The recovery of bacterial recombinants carrying an allele from a Kohara phage was tested using the Lac-strain V355 and Lac' phage X138 (Table 1). Lac' transductants were obtained at a frequency of _1O-4 per phage, in agreement with a previous report (19). Most Lac+ transductants were nonlysogenic and did not segregate Lac-derivatives.Exchange between phage-borne and chromosomal alleles was studied further by isolating a lacZ: :Tn5supF derivative of X138 (Table 1, footnote b) and using it for transduction. * Corresponding author. Strain DB5508, which contains a plasmid with amber mutations in the tet and amp genes, was infected at a multiplicity of about 1 phage per cell and plated on L medium with tetracycline (to select Sup') containing 10 mM sodium citrate and 0.2% glucose (to inhibit further phage growth). Sup+ transductant colonies were found at a frequency of a...

cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth

“…The parental cysQ mutant alleles were recovered from several revertants by transduction and selection for the appropriate resistance trait (Kanr or Tetr). Two spontaneous reversion mutations that allowed relativEiy good growth on cysteine-free medium were mapped in 14fr x F-crosses and then by transduction with several candidate X phage clones (marked by insertion of a TnScam transposon [44,50]). These reversion mutations were found to be in the segment carried by X419, a phage clone that also carries the cysPTWA (sulfate binding and uptake) operon.…”

“…To generate a cysQ::TnSsupF insertion mutant, TnSsupF was transposed from the donor plasmid in strain DB4496 (43) to cysQ+ phage A656 (23,24), and insertion-containing phage were selected by plaque formation on the dnaB amber strain DK21 (29,43). Haploid Tn5supF-containing bacterial recombinants were obtained by infecting strain DB5508 (which contains amber mutant alleles of amp and tet genes) and selecting Sup' transductants by their resistance to ampicillin or tetracycline (44). Sup' transductants were screened for auxotrophy.…”

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome

“…The A phage containing cloned E. coli DNA (20,21) are cI mutants (repressor deficient) and tend to kill nonlysogens. The A clone corresponding to a particular plasmid clone was identified by restriction mapping, DNA sequencing, genetic complementation (20,26,31), or recombination tests (present work) when there were relatively few candidates to screen. Medium E (40) was the defined minimal medium.…”

“…Standard molecular genetic methods (30,31,35) were employed. The bacterial strains, phage, and plasmids used in this study are listed in Table 1.…”

“…Consequently, a failure to isolate the desired recombinant could reflect either essentiality of the mutated gene or simply a low frequency of the second crossover. Alternatively, a recD or recBC sbcB strain can be transformed with genetically marked linear DNA fragments (18,33,38,41 Recent studies have shown that the X cI mutant phage of the Kohara library of E. coli clones (20, 21) can be used effectively as a specialized transducing phage: a X clone carrying a particular wild-type allele can typically transduce the corresponding mutant bacterial strain at a frequency of about 10-' without need for a lysogenization step (26,31). This formed the basis for a reverse genetic method of random insertion mutagenesis of individual phage clones with transposon Tn5supF followed by recombination of insertion mutant alleles into the E. coli chromosome (30, 31).…”