Use of a Tn5 derivative that creates lacZ translational fusions to obtain a transposition mutant

We constructed a derivative of Tn5, Tn5 ORFlac, that is capable of creating lacZ translational fusions upon transposition. Lac- strains carrying this construct formed red papillae when plated on MacConkey-lactose media. Lac+ cells isolated from independent papillae expressed distinct beta-galactosidase fusion proteins, suggesting that the Lac+ phenotype resulted from transposition. In support of this, analysis of plasmids carrying Tn5 ORFlac prepared from these cells indicated that the Lac+ phenotypes arose as a result of intermolecular rearrangements. Furthermore, a derivative of Tn5 ORFlac that contains an ochre mutation in the transposase gene formed papillae only in a supB strain. Tn5 ORFlac is useful for obtaining mutants that affect Tn5 transposition and for creating lacZ fusions. We used the papillation phenotype to isolate a spontaneous revertant of IS50L that promotes transposition at a 3.6-fold higher rate than IS50R. The mutation altered the amino acid sequence of both transposase and inhibitor.     

Metastable regulation of type 1 piliation in Escherichia coli and isolation and characterization of a phenotypically stable mutant.

Type 1 piliation in Escherichia coli exhibits phase variation due to the inversion of a small, ca. 300-base-pair, element that regulates pilA (fimA), the gene that encodes the structural subunit of pili (Abraham et al., Proc. Natl. Acad. Sci. USA 82:5724-5727, 1985). We have used the inversion as an assay to characterize a stably piliated mutant. The mutant strain did not exhibit the pilA ON and pilA OFF colonial variants characteristic of the wild type; rather, every clone produced a level of pilA expression intermediate between ON and OFF wild-type populations. The mutant phenotype was conferred by a lesion at a previously undescribed locus between hemA and trpA, which we have termed pilG. Examination of the pilA promoter region in four pilG mutant populations indicated that the phenotypic stability conferred by the pilG mutation was not due to an inability to carry out the inversion. Rather, all pilG mutant populations consisted of approximately equal mixtures of ON and OFF individuals. We suggest that pilG mutants may undergo such rapid switching of the pilA promoter that populations exhibit an intermediate level of pilA expression and phenotypic stability.     

Genetic and molecular study of inability of the yeast Kluyveromyces lactis var drosophilarum to ferment lactose

The fermentation of lactose (Lac+) in the dairy yeast Kluyveromyces lactis var. lactis is controlled by the LAC4 (beta-galactosidase) and LAC12 (lactose permease) genes. The complementation analysis of twelve Kl. lactis var. drosophilarum natural homothallic Lac- strains of different origin was carried out using the genetic heterothallic lines of Kl. lactis var. lactis of the lac4LAC12 and LAC4lac12 genotypes. It was shown that the natural Lac- strains did not possess the LAC4LAC12 gene cluster. Southern hybridization of chromosomal DNA with LAC4 and LAC12 probes, as well as recombination analysis, showed that Kl. lactis var. drosophilarum yeasts do not have even silent copies of these genes. As distinct from this yeast, natural Lac- strains of the yeast Kl. marxianus are mutants impaired in the lactose permease gene (lac12 analogue), but possess an active beta-galactosidase gene (LAC4 analogue). The origin of the LAC4LAC12 gene cluster of the dairy yeasts Kl. lactis is discussed.     

Nonadaptive mutations occur on the F' episome during adaptive mutation conditions in Escherichia coli.

One of the most studied examples of adaptive mutation is a strain of Escherichia coli, FC40, that cannot utilize lactose (Lac-) but that readily reverts to lactose utilization (Lac+) when lactose is its sole carbon source. Adaptive reversion to Lac+ occurs at a high rate when the Lac- allele is on an F' episome and conjugal functions are expressed. It was previously shown that nonselected mutations on the chromosome did not appear in the Lac- population while episomal Lac+ mutations accumulated, but it remained possible that nonselected mutations might occur on the episome. To investigate this possibility, a second mutational target was created on the Lac- episome by mutation of a Tn1O element, which encodes tetracycline resistance (Tetr), to tetracycline sensitivity (Tets). Reversion rates to Tetr during normal growth and during lactose selection were measured. The results show that nonselected Tetr mutations do accumulate in Lac- cells when those cells are under selection to become Lac+. Thus, reversion to Lac+ in FC40 does not appear to be adaptive in the narrow sense of the word. In addition, the results suggest that during lactose selection, both Lac+ and Tetr mutations are created or preserved by the same recombination-dependent mechanism.     

Lactose metabolism in Erwinia chrysanthemi.

Wild-type strains of the phytopathogenic enterobacterium Erwinia chrysanthemi are unable to use lactose as a carbon source for growth although they possess a beta-galactosidase activity. Lactose-fermenting derivatives from some wild types, however, can be obtained spontaneously at a frequency of about 5 X 10(-7). All Lac+ derivatives isolated had acquired a constitutive lactose transport system and most contained an inducible beta-galactosidase. The transport system, product of the lmrT gene, mediates uptake of lactose in the Lac+ derivatives and also appears to be able to mediate uptake of melibiose, raffinose, and galactose. Two genes encoding beta-galactosidase enzymes were detected in E. chrysanthemi strains. That mainly expressed in the wild-type strains was the lacZ product. The other, the lacB product, is very weakly expressed in these strains. These enzymes showed different affinities for the substrates o-nitrophenyl-beta-D-galactopyranoside and lactose and for the inhibitors isopropyl-beta-D-thiogalactopyranoside and galactose. The lmrT and lacZ genes of E. chrysanthemi, together with the lacI gene coding for the regulatory protein controlling lacZ expression, were cloned by using an RP4::miniMu vector. When these plasmids were transferred into Lac- Escherichia coli strains, their expression was similar to that in E. chrysanthemi. The cloning of the lmrT gene alone suggested that the lacZ or lacB gene is not linked to the lmrT gene on the E. chrysanthemi chromosome. One Lac+ E. chrysanthemi derivative showed a constitutive synthesis of the beta-galactosidase encoded by the lacB gene. This mutation was dominant toward the lacI lacZ cloned genes. Besides these mutations affecting the regulation of the lmrT or lacB gene, the isolation of structural mutants unable to grow on lactose was achieved by mutagenic treatment. These mutants showed no expression of the lactose transport system, the lmrT mutants, or the mainly expressed beta-galactosidase, lacZ mutants. The lacZ mutants retained a very low beta-galactosidase level, due to the lacB product, but this level was low enough to permit use of the lacZ mutants for the construction of gene fusions with the Escherichia coli lac genes.     

The two-component regulatory system ompR-envZ controls the virulence of Shigella flexneri.

In Shigella flexneri, the ompB locus (containing the ompR and envZ genes) was found to modulate expression of the vir genes, which are responsible for invasion of epithelial cells. vir gene expression was markedly enhanced under conditions of high osmolarity (300 mosM), similar to that encountered in tissues both extra- and intracellularly. Two ompB mutants were constructed and tested for virulence and for osmotic regulation of vir genes. An envZ::Tn10 mutant remained invasive, although its virulence was significantly decreased as a result of its inability to survive intracellularly. By using a vir::lac operon fusion, this mutation was shown to decrease beta-galactosidase expression both in low- and high-osmolarity conditions but did not affect vir expression in response to changes in osmolarity. A delta ompB deletion mutant was also constructed via allelic exchange with an in vitro-mutagenized ompB locus of Escherichia coli. This mutation severely impaired virulence and abolished expression of the vir::lac fusion in both low- and high-osmolarity conditions. Therefore, a two-component regulatory system modulates virulence according to environmental conditions. In addition, the mutation affecting a spontaneous avirulent variant of S. flexneri serotype 5, M90T, has been mapped at the ompB locus and was complemented by the cloned E. coli ompB locus. Introduction of the vir::lac fusion into this mutant did not result in the expression of beta-galactosidase (Lac-).     

Genetic analysis of the phase variation control of expression of type 1 fimbriae in Escherichia coli.

Expression of type 1 fimbriae in Escherichia coli exhibits phase variation, whereby individual cells can alternate between states of organelle expression (Fim+) and nonexpression (Fim-). Strains with a fimD-lac operon fusion, in which lac, rather than fimD, expression is under the control of the fimD promoter, undergo Lac+ in equilibrium Lac- phase variation, instead. After positioning a lambda prophage adjacent to the operon fusion, we were able to isolate specialized lambda phage carrying both the fimD-lac fusion and the phase variation control region. Introduction of such phage into an Fim+ strain resulted in construction of a strain with a double, independently switching phenotype (Fim+ in equilibrium Fim- and Lac+ in equilibrium Lac-), demonstrating that the region controlling phase variation is contiguous with the fimD-lac operon fusion and is cis acting. When the specialized lambda phage was propagated on a delta lac delta fim strain, phase variation occurred within the plaques, confirming that the phase variation control region is carried on the specialized transducing phage. All lysogens acquired the Lac+ in equilibrium Lac- phenotype, except for two nonswitching Lac+ recombinants, which acquired Lac+ in equilibrium Lac- phase variation only by trans complementation with fim. Phase variation of type 1 fimbriae, therefore, appears to involve both a cis-active element, which is cloned on a specialized lambda phage, and a trans-active permissive factor, which is not present on the phage, but rather must be supplied by the recipient strain in the transduction.     

Different characteristics distinguish early versus late arising adaptive mutations in Escherichia coli FC40

The Escherichia coli strain FC40 has frequently been employed to investigate the mechanism of adaptive mutations. The strain cannot utilize lactose due to a +1 frameshift mutation that reduces beta-galactosidase to about 1% of normal levels. Cells undergo a high rate of mutation from Lac- to Lac+ when cells are grown with lactose as the sole energy source. Almost all Lac+ colonies arising 3-6 days after plating result from a base pair deletion in runs of iterated base pairs within a 130-bp target region. In this study we characterized Lac+ colonies arising 3-10 days after plating. Temperature gradient gel electrophoresis (TGGE) was used to detect mutations in the target region as a function of the day a colony appears. TGGE results confirmed the occurrence of mutations within the target region in 36 of 37 FC40 Lac+ colonies arising on days 3-7. However, mutations in this region were not detected in 23 of 37 Lac+ colonies arising from days 8-10. Sequencing data verified the TGGE results. Half of the Lac+ mutants arising on days 8-10 with no base pair change in the target region were unstable and exhibited a Lac- phenotype after successive growth cycles in rich medium. The results suggest that amplification of the lac operon region is a common factor in late arising colonies, and that different characteristics distinguish early and late arising Lac+ colonies.