Tn917-lac mutagenesis of Streptococcus mutans to identify environmentally regulated genes.

Previously, we demonstrated successful Tn917 mutagenesis of the oral pathogen Streptococcus mutans using pTV1-OK (Km(r), repATs), a temperature conditional replicative delivery vector carrying a lactococcal pWVO1Ts backbone. In this report we describe the construction and utilization of pTV32-OK, a plasmid harboring Tn917-lac (em(r), beta-gal(+)) that was employed to isolate transcriptional fusions of the Escherichia coli lacZ reporter gene with streptococcal promoters in S. mutans strain NG8. Tn917-lac transposition occurred at a frequency of ca. 10(-6) with 20% of the resultant em(r) clones displaying varying levels of lacZ expression. Tn917-lac mutants that expressed beta-galactosidase activity under growth conditions of glucose limitation, acidic pH, 35 mM NaCl, and elevated (42 degrees C) temperature were isolated. Further characterization of one of the mutants with increased beta-gal activity under glucose limitation, strain AS42, revealed maximal activity in batch culture in stationary phase after glucose depletion. The beta-gal activity of AS42 also was found to be repressed 3-fold in medium containing 2% glucose relative to measured activity from cells suspended in the same medium containing no glucose. Further phenotypic analysis revealed that AS42 had a 30% lower growth yield than the parent strain NG8 when grown in pH 5 medium. Sequence analysis of the region harboring the transposon revealed that the lacZ fusion occurred near the 3'-end of a gene encoding a homolog of an ATP binding protein from a family of Gram-positive ABC transporters. These findings demonstrate that Tn917-lac mutagenesis can be used to identify environmentally regulated genes in S. mutans and possibly in other medically relevant streptococcal species.

Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus.

We describe a general method for random mutagenesis of cloned genes by error-prone PCR or DNA shuffling that eliminates the need for post-amplification subcloning following each cycle of mutagenesis. This method exploits the highly efficient and recombinogenic nature of DNA uptake during natural transformation in the Gram-positive bacterium Bacillus subtilis and the Gram-negative bacterium Acinetobacter calcoaceticus. Plasmid systems were designed that allow capture of PCR-amplified DNA fragments by marker-replacement recombination with a structurally similar helper plasmid resident in the transformation recipient. This recombination event simultaneously transfers the amplified sequences into the helper plasmid and restores the integrity of a drug resistance gene, thereby affording a direct selection for fragment capture. Although this strategy was sufficiently effective to permit recovery in B. subtilis of up to 10(3) transformants/microgram of PCR product, equivalent plasmid systems were approximately 100 times more efficient in A.calcoaceticus. Acinetobacter calcoaceticus also offers the advantage of essentially constitutive transformation competence in ordinary complex broth, such as LB, in contrast to two-step growth in semi-synthetic media required for optimal transformation of B.subtilis.

Construction and properties of Tn917-lac, a transposon derivative that mediates transcriptional gene fusions in Bacillus subtilis.

A derivative of Tn917 was constructed, referred to as Tn917-lac, which is capable of generating fusions that connect the transcripts of Bacillus subtilis chromosomal genes to the coding sequence of the lacZ gene of Escherichia coli. Two independent insertions of Tn917-lac into the gltA gene and one insertion into the trpE gene (in the trpEDCFBA operon) of B. subtilis were studied in detail, and the results confirmed that Tn917-lac-mediated transcriptional fusions produce levels of beta-galactosidase that reflect accurately the regulated expression of interrupted genes. To facilitate these studies, a procedure was developed that permits the analysis of Tn917-lac-mediated fusions in partial diploids where insertional mutations are complemented by an intact copy of the interrupted genes. Tn917 is known to function efficiently in bacteria representing three quite different Gram-positive genera (Streptococcus, Bacillus, and Staphylococcus) and is known to display a relatively high degree of randomness in its insertions into bacterial genomes, making it likely that Tn917-lac will be useful for the identification and study of many kinds of regulated genes in a wide range of Gram-positive species.

A physical and functional analysis of Tn917, a Streptococcus transposon in the Tn3 family that functions in Bacillus.

The erythromycin-resistance (Emr)-conferring transposon Tn917, first isolated in the genus Streptococcus, has in previous work been shown to function efficiently in the spore-forming species Bacillus subtilis, where it has been developed as a tool for identifying and studying sporulation genes. In the present work, a physical analysis of Tn917 was undertaken, including detailed restriction mapping, chemical DNA sequencing, heteroduplex studies, and Southern hybridization analysis, as a first step in understanding the genetic organization of this useful insertion element. The location and transcriptional orientation of the transposon-borne erm gene (the gene responsible for the Emr phenotype) have been determined, and a partial sequence of DNA 5' to the coding sequence of this gene indicates that its inducibility is probably the result of "translational attenuation," a mechanism known to be responsible for the regulation of at least two other gram-positive erm genes. Restriction mapping and heteroduplex analysis have revealed extensive homology between Tn917 and the Staphylococcus transposon Tn551, throughout virtually their entire lengths, and DNA sequencing studies have revealed a remarkably high degree of sequence correspondence within the terminal inverted repeats of Tn917, Tn551 and the gram-negative transposon Tn3. Tn917 was also shown to generate a 5-bp duplication upon insertion, as do Tn3 and Tn551 (and all of the other Tn3-related elements studied thus far), strengthening the conclusion that these three transposons are members of a highly dispersed family of related insertion elements which populate both gram-positive and gram-negative genera.

Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917.

The Streptococcus faecalis transposon Tn917 was introduced into Bacillus subtilis by transformation of competent cells with the plasmid pAM alpha 1::Tn917 and was tested for transposition activity by selection for insertions into the temperate phage SP beta. Insertions were obtained at a frequency indicating relatively efficient movement of the element, and Southern hybridization analysis of a particular insertion confirmed it to be the result of a genuine transposition event. A restriction fragment from pAM alpha 1::Tn917 containing the transposon sequences was ligated into a temperature-sensitive plasmid (pBD95), and transpositions into the B. subtilis chromosome were selected by requiring the transposon drug resistance to be maintained at temperatures nonpermissive for plasmid replication. Insertions have been recovered at many chromosomal sites, including ones that produced auxotrophy of different kinds and ones that produced various different sporulation-defective phenotypes, indicating good prospects for the use of Tn917 as a tool for insertional mutagenesis in B. subtilis.

Two Multiallelic Mating Compatibility Loci Separately Regulate Zygote Formation and Zygote Differentiation in the Myxomycete PHYSARUM POLYCEPHALUM.

The mating of Physarum polycephalum amoebae, the ultimate consequence of which is a "plasmodium," was recently shown to be governed by two compatibility loci, matA (or mt) and matB (Dee 1978; Youngmanet al. 1979). We present evidence that matA and matB separately regulate two discrete stages of mating: in the first stage, amoebae (which are normally haploid) fuse in pairs, with a specificity determined by matB genotype, to form diploid zygotes; subsequent differentiation of the zygotes into plasmodia is regulated by matA and is unaffected by matB. Mixtures of amoebae carrying unlike matA and matB alleles formed diploids to the extent of 10 to 15% of the cells present, and the diploids differentiated into plasmodia. When only the matB alleles differed, diploid cells still formed to a comparable (5 to 10%) extent, but rather than differentiating, these diploids remained amoebae. When strains carried the same alleles of matB, formation of diploid cells was greatly reduced: in like-matB, like-matA mixtures, none of 320 cells examined was diploid; in like-matB, unlike mat-A mixtures, differentiating diploids could be detected, but at only 10(-3) to 10(-2) the frequency of unlike-matB, unlike-matA mixtures. The nondifferentiating diploid amoebae recovered from unlike-matB, like-matA mixtures were genetically stable through extensive growth, even though they grew more slowly than haploids (10-hr vs. 8-hr doubling period), and could be crossed with both haploids and diploids. The results of such higher ploidy and mixed ploidy crosses indicate that karyogamy does not invariably accompany zygote formation and differentiation.

A New Mating Compatibility Locus in PHYSARUM POLYCEPHALUM.

The rate and extent of plasmodium formation were studied in mating tests involving pairs of largely isogenic amoebal strains compatible for mating-type (mt) alleles. A systematic variability was observed: plasmodia formed either rapidly and extensively or slowly and inefficiently. Plasmodium formation was found to be 10(3)- to 10(4)-fold more extensive in "rapid" crosses than in "slow" crosses. A genetic analysis revealed that the variability reflects the influence of a multiallelic compatibility locus that determines mating efficiency. This compatibility locus (designated matB), together with the original mating type locus, mt (in this work designated matA), constitute a tetrapolar mating specificity system in Physarum polycephalum.

A gene,imz, affecting the pH sensitivity of zygote formation inPhysarum polycephalum.

Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore "dominant". We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.

An extracellular inducer of asexual plasmodium formation in Physarum polycephalum.

Asexual conversion of amoebae to plasmodia was studied in the Colonia isolate of the myxomycete, Physarum polycephalum. When a culture of Colonia amoebae is grown on a bacterial lawn, a period of amoebic growth precedes the appearance of cells committed to the plasmodial state. The onset of plasmodium production appears to be related to amoebic nutrition since cultures supplied with fewer bacteria display earlier differentiation. For a period of time after differentiation is initiated, conversion of amoebae to plasmodia is rapid and proceeds as an exponential function of time. A filter-transmissible substance, apparently released by differentiating cells, is implicated in the control of this rapid conversion.