Fine-structure mapping and complementation analysis of the Escherichia coli cysB gene.

Sixty-two point mutations were isolated in Escherichia coli by means of transduction with mutagenized phage P1. Twenty-two deletions extending into cysB but able to recombine with at least some of the point mutations were isolated on a transmissible E. coli plasmid. Mapping of the point mutations against the deletions divided the former into 16 deletion groups. Nine merodiploids were constructed in which the chromosome carried one of the three point mutations most distal to the trp operon and in which a plasmid carried one of the three point mutations most proximal to the trp operon. All of these showed a Cys-phenotype. It follows that mutations at the two extreme ends of the region belong to the same complementation group.     

Distribution of Tn551 insertion sites responsible for auxotrophy on the Staphylococcus aureus chromosome

A method was devised to efficiently select isolates of Staphylococcus aureus 8325 in which Tn551, a transposon originating on the pI258 plasmid responsible for erythromycin resistance (Emr), had translocated to the host chromosome. This method consisted of selecting for Emr at 43 degrees C with a strain in which the pI258 plasmid was unable to replicate at 43 degrees C because of a temperature-sensitive plasmid mutation. By selecting isolates that were Emr at 43 degrees C and auxotrophic for nutrients not required by the parent strain. Tn551-induced auxotrophic mutants were readily isolated. The incidence of auxotrophic classes was not random; 80% of the isolates in one experiment were Trp-, whereas only a single example of each of some of the other classes was isolated. Among the Trp- mutants, the distribution of trp genes affected and the frequency of precise excision of Tn551 from individual sites varied. When analyzed by transformation, the Tn551-induced ala, his, ilv, lys, rib, thrA, thrB, and trp mutations were shown to occupy sites previously defined by nitrosoguanidine-induced mutations. Tn551-induced mutagenesis provided three previously unrecognized classes of auxotrophs (tyr, met, and thrC), and the Tn551 integration sites resulting in these mutations have been identified. In addition, a chromosomal region (uraB) was identified by Tn551 mutagenesis that is distinct from uraA (previously defined by chemical mutagenesis). Some Tn551-induced mutations (most notably pur) could not be linked to the known linkage groups of the chromosome by transformation. With the exception of two pur mutations, all of the Tn551-induced auxotrophic mutational sites cotransformed at unity with Tn551 and, in cases in which they were selected, prototrophic transformants were always Ems. Thus, the Tn551 and auxotrophic sites are identical.     

Genetic and Segregation Analysis of Escherichia coli Strains Containing a Tandem Duplication of the trpD-purB Region of the Chromosome

Genetic and segregation analysis of Escherichia coli strains containing a partial duplication of the trp operon reveal that the 2.5-min-long region trpD-purB is duplicated in tandem in the chromosome. The adjacent loci cysB and fabD are not duplicated. Although one copy of the duplicated region is longer than the maximum size of bacteriophage P1kc transducing fragments, the frequency at which the duplicated segment trpDCBA is transferred by transduction to tonB-trp deletion strains is equal to that observed for transfer of the normal trp operon. This suggests that three-point recombination events believed to account for transduction of long duplications occur as frequently as two-point recombination events believed to account for normal transduction. Cotransduction frequencies of trpDCBA with the duplicated loci tonB, galU, tyrT, and hemA are very similar to those for the trp operon with the same loci. This indicates that normal genetic linkage is maintained during the three-point recombination event. However, purB, which is normally unlinked to trp by transduction, is closely linked to trpDCBA and thus must be near the repeat point of the duplication. Transduction tests with point mutations in the trp operon indicated that the repeat point occurs near the normal boundary between trpE and trpD. Segregation analysis of heterogenotes constructed from tonB-trp deletion strains shows that the frequency at which a marker is lost is approximately proportional to its distance from the repeat point. This finding is consistent with a random, singlesite crossover event during segregation. Several observations indicate that non-reciprocal genetic exchange also occurs between copies of the duplication. Analysis of heterogenotes containing dadR1 and dadR(+) demonstrate that the mutant allele is transdominant.     

Genetic mapping of the bacitracin synthetase gene(s) in Bacillus licheniformis

The map position of a mutation in the bacitracin synthetase gene(s) in Bacillus licheniformis ATCC 10716 was determined by transduction with phage SP-15. Results indicate that it is linked to the lys and trp loci and is distinct from the known sporulation loci on the chromosome of Bacillus licheniformis. The defect(s) of the enzyme complex were analysed in terms of its ability to bind covalently 14C-labelled amino acid precursors of the bacitracin molecule.     

Location of trpR Mutations in the serB-thr Region of Salmonella typhimurium

Tryptophan biosynthesis in Salmonella is controlled by at least one regulatory gene, trpR, which is cotransducible with thr genes and not with the trp operon. Mutations in trpR cause derepression of tryptophan enzyme synthesis and confer resistance to growth inhibition by 5-methyltryptophan. Nineteen trpR mutations were mapped with respect to thrA and serB markers by two-point (ratio) and three-point transduction tests. The results are all consistent with the site order serB80-trpR-thrA59 on the Salmonella chromosome. Very low or undetectable levels of recombination between different trpR mutations have so far prevented the determination of fine structure in the trpR gene. Thirteen other 5-methyltryptophan-resistant mutants previously found not to be cotransducible with either the trp operon or thrA, and designated trpT, were also used in these experiments. Lack of cotransducibility with thrA was confirmed, and no linkage with serB was detected. The nature and location of trpT mutations remain obscure.     

Isolation and Characterization of Specialized φ80 Transducing Phages Carrying Regions of the Salmonella typhimurium trp Operon

We have isolated a series of nondefective phi80 specialized transducing phage which carry segments of the Salmonella typhimurium trp operon. These phage were obtained from a lysogenic derivative of a merozygote constructed by transferring an S. typhimurium trp episome into an Escherichia coli strain which lacks the normal phi80 attachment site. The deoxyribonucleic acid (DNA) from one such phage was purified and employed in DNA-ribonucleic acid (RNA) hybridization studies. The results obtained show that, under our hybridization conditions, heterologous hybridization is less efficient than homologous hybridization. It was also observed that not all S. typhimurium trp messenger RNA can readily anneal to E. coli trp operon DNA. Heterologous hybrids consisting of S. typhimurium trp messenger RNA and E. coli trp operon DNA were estimated to have a dissociation constant 10-fold larger than that of homologous hybrids.     

Translocation of transposons Tn10 and Tn5 in the Yersinia pestis chromosome

The possibility of translocation of the transposons Tn5 and Tn10 into the genome of Yersinia pestis, with the subsequent mutagenic effect was demonstrated. We revealed transposon harbouring clones at frequency 10(-4) to 10(-2). Derivatives of P1cml clr100ts phage served as vectors. Insertion of Tn10 transposon induced mutations in ilv, ser, arg, pur, pro, leu, nic, tyr, gua genes. The number of the insertion sites on the chromosome obtained for Tn5 was the same, these being arg, ade, pyr, leu, gua, trp, his, pan, ilv. The majority of auxotrophs did not revert. Occasionally, revertants were observed at frequencies 10(-8) to 10(-6). Unlike Escherichia coli, reversion was not accompanied by the loss of transposons. The rearrangements induced by transposons, presumably, near the insertion site, as well as duplications of transposons followed by incorporation of copies into novel sites, led to the appearance of additional defective genes, which made it possible to select various types of polyauxotrophs. Based on reiteration of coinciding double and triple mutant markers, we proposed a linkage group of genes within a segment of Y. pestis chromosome: lys ... tyr - ser - arg - ilv - leu - gua - ade(pur) - pro ... his ... pyr ... trp. The reasons for peculiarities of the behaviour of transposons in Y. pestis bacteria are discussed.     

Control of phenylalanyl-tRNA synthetase genetic expression. Site-directed mutagenesis of the pheS, T operon regulatory region in vitro

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli strongly suggested that the pheS, T operon was regulated by a phenylalanine-mediated attenuation mechanism. To investigate the functions of the different segments composing the pheS, T attenuator site, a series of insertion, deletion and point mutations in the pheS, T leader region have been constructed in vitro on a recombinant M13 phage. The effects of these alterations on the regulation of the operon were measured after transferring each mutation onto a lambda phage carrying a pheS, T-lacZ fusion. The behaviours of the various mutants agree with the predictions of the attenuation model. The role of the antiterminator (2-3 pairing) as competitor of the terminator (3-4 pairing) is demonstrated by several mutations affecting the stability of the 2-3 base-pairing. The existence of deletions and point mutations in the 3-4 base-pairing shows that the terminator is essential for both expression level and regulation of the operon. Mutations in the translation initiation site of the leader peptide show that the expression of the leader peptide is essential for attenuation control. However, alteration of the translation initiation rate of the leader peptide derepresses the pheS, T operon, which is the opposite of what is observed with the trp operon. This difference is explained in terms of different translation initiation efficiencies of the leader peptides. Finally, insertion mutations, increasing gradually the distance between the leader peptide stop codon and the first strand of the antiterminator, derepress the pheS, T operon and show that formation of the antiterminator structure is under the control of the translation of the leader peptide.     

Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage.

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.     

Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.     

A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.

This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.     

色氨酸操纵子调控机理详析

色氨酸操纵子是最早被研究的细菌合成代谢调控、基因表达调控的模型之一。其中阻遏蛋白对转录起始的抑制作用、色氨酸作为辅阻遏物的作用以及通过定点突变揭示的弱化作用的分子机制已基本被阐明。此外,色氨酸操纵子RNA结合弱化蛋白、NusA、NusG、TrpY等调节蛋白对细菌色氨酸操纵子弱化作用的调节机制也在近年来得到进一步揭示。特别是在枯草芽孢杆菌中,色氨酸操纵子主要依赖于转录衰减机制调控,包括由色氨酸激活的色氨酸操纵子RNA结合弱化蛋白与新生转录产物结合形成内部终止子,导致5′非翻译区（5′UTR）转录终止。NusA、NusG通过刺激RNA聚合酶在5′UTR的U107和U144位点暂停,释放出RNA聚合酶,最终造成转录终止。不同的是,在U144位点NusA参与的转录弱化机制依赖其发夹结构,且NusA与RNA聚合酶作用促进了RNA结合弱化蛋白与新生转录产物的结合,使转录终止。而NusG是通过与非模板DNA链中的一段富含T碱基序列和RNA聚合酶同时互作,阻止了RNA聚合酶向下游移动,从而引起RNA聚合酶高效停滞。但在细菌操纵子中,绝大多数调节因子参与的弱化机制最终依赖于ρ因子,从而导致多达一半的转录终止事件发生。近年来,随着学科的发展,越来越多关于色氨酸操纵子调节机制新概念被挖掘报道,这也使人类对色氨酸操纵子的表达调控机制的认知愈加详尽。     

Construction of a chromosome map for the phage group II Staphylococcus aureus Ps55.

The genome size and a partial physical and genetic map have been defined for the phage group II Staphylococcus aureus Ps55. The genome size was estimated to be 2,771 kb by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes SmaI, CspI, and SgrAI. The Ps55 chromosome map was constructed by transduction of auxotrophic and cryptic transposon insertions, with known genetic and physical locations in S. aureus NCTC 8325, into the Ps55 background. PFGE and DNA hybridization analysis were used to detect the location of the transposon in Ps55. Ps55 restriction fragments were then ordered on the basis of genetic conservation between the two strains. Cloned DNA probes containing the lactose operon (lac) and genes encoding staphylococcal protein A (spa), gamma hemolysin (hlg), and coagulase (coa) were also located on the map by PFGE and hybridization analysis. This methodology enabled a direct comparison of chromosomal organization between NCTC 8325 and Ps55 strains. The chromosome size, gene order, and some of the restriction sites are conserved between the two phage group strains.