Genetic mapping of the Salmonella typhimurium pncB locus.

The nicotinic acid phosphoribosyltransferase locus pncB was located on the Salmonella typhimurium linkage map counterclockwise relative to pyrC. P22 and P1 transductional analyses revealed linkage of pncB with aroA and pyrD, indicating a pncB map position of approximately 20 map units. The results of these cotransduction experiments also indicated that the genetic map distance between gal and pyrD is greater than the published 2.2 map units.     

Mapping of the fabD locus for fatty acid biosynthesis in Escherichia coli.

fabD mutants of Escherichia coli contain a thermolabile malonyl-coenzyme A-acyl carrier protein transacylase which causes defective fatty acid synthesis and temperature-sensitive growth. By conjugation and P1 transduction the fabD locus has now been mapped at min 24, between pyrC and purB and close to cat. The order of sites is tentatively given as pyrC, cat, fabD, and purB, though the orientation of cat and fabD could be reversed. The possible relationship of fabD with another mutation lying in this region and also affecting acid synthesis is discussed. In the course of these studies we also confirmed the location of the fabA gene, determined that poaA lies between fabA and pyrC, and inadvertently found that the pyr mutation in strain AT3143 is probably pyrF and not pyrC.     

Generation of auxotrophic mutants of Enterococcus faecalis.

A 22-kb segment of chromosomal DNA from Enterococcus faecalis OG1RF containing the pyrimidine biosynthesis genes pyrC and pyrD was previously detected as complementing Escherichia coli pyrC and pyrD mutations. In the present study, it was found that the E. faecalis pyrimidine biosynthetic genes in this clone (designated pKV48) are part of a larger cluster resembling that seen in Bacillus spp. Transposon insertions were isolated at a number of sites throughout the cluster and resulted in loss of the ability to complement E. coli auxotrophs. The DNA sequences of the entire pyrD gene of E. faecalis and selected parts of the rest of the cluster were determined, and computer analyses found these to be similar to genes from Bacillus subtilis and Bacillus caldolyticus pyrimidine biosynthesis operons. Five of the transposon insertions were introduced back into the E. faecalis chromosome, and all except insertions in pyrD resulted in pyrimidine auxotrophy. The prototrophy of pyrD knockouts was observed for two different insertions and suggests that E. faecalis is similar to Lactococcus lactis, which has been shown to possess two pyrD genes. A similar analysis was performed with the purL gene from E. faecalis, contained in another cosmid clone, and purine auxotrophs were isolated. In addition, a pool of random transposon insertions in pKV48, isolated in E. coli, was introduced into the E. faecalis chromosome en masse, and an auxotroph was obtained. These results demonstrate a new methodology for constructing defined knockout mutations in E. faecalis.     

Structure of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster.

A 10.5-kilobase PstI endonuclease fragment encoding the entire Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster was cloned in Escherichia coli by transformation of a carB strain to uracil-independent growth. The cloned fragment also complemented E. coli pyrB, pyrC, pyrD, pyrE, and pyrF mutants. From the ability of subclones to complement E. coli pyr mutants, the gene order was deduced to be pyrBCADFE. The B. subtilis pyrB gene was shown to be expressed in E. coli, but synthesis of the enzyme was not repressible by the addition of uracil to the growth medium. The approximate molecular weights of the polypeptides encoded by B. subtilis pyrA, pyrB, pyrC, pyrD, pyrE, and pyrF were found to be 110,000, 36,000, 46,000, 34,000, 25,000, and 27,000, respectively.     

TolF locus in Escherichia coli: chromosomal location and relationship to loci cmlB and tolD.

The tentative map position on the Escherichia coli chromosome of the tolF locus, determining tolerance to colicins A, E2, E3, K, and L, has been confirmed by three-point transduction. It lies between the aroA and pyrD loci at about 21 min on the linkage map of Bachmann et al. (1976). The cmlB locus, determining increased resistance to the antibiotics chloramphenicol and tetracycline, also lies in this region (Reeve, 1966). Phenotypic and genetic comparison of isogenic strains that carry a mutation in either the tolF or cmlB locus makes it likely that these loci are closely related or identical. The tolD locus determining tolerance to colicins E2 and E3 as well as increased resistance to antibiotics has been reported to be located close to the aroA locus as a result of conjugation experiments (Eriksson-Grennberg et al. 1965). However, tolD did not cotransduce with any of several loci in this region, indicating that the mutation is not located within the region of the genetic map corresponding to approximately 19 to 22.5 min.     

Mapping of sul, the suppressor of lon in Escherichia coli.

The suppressor sul, which is allele specific for the ultraviolet sensitivity gene lon, has been mapped by conjugation and transductional crosses in Escherichia coli K-12 and B/r. Previously, sul was reported to lie in the azi region of the Escherichia coli chromosome. Evidence is presented which positions sul close to and clockwise of fabA on the Escherichia coli map. Cotransductional frequencies of 31.3% were obtained between sul and pyrD, and frequencies of 82% were obtained between sul and fabA. Also, the mucoid phenotype of K-12 lon strains grown on minimal glucose agar plates at 37 C was not significantly effected in sul derivatives. No differences between the sul of Escherichia coli B/r and that of K-12 derivatives with regard to map location or effect on mucoid production were observed.     

pryB mutations as suppressors of arginine auxotrophy in Salmonella typhimurium.

Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation were found to grow more slowly in minimal medium than pyrH+ controls. The addition of arginine or citrulline but not ornithine restored normal growth rates. This requirement for arginine was completely suppressed by pyrB mutations and partially suppressed by pyrC and pyrD mutations. No suppression was observed with mutants at the pyrF locus. Introduction of leaky mutation argI2002 resulted in a more extreme arginine requirement and accentuated suppression by pyrB mutations. Suppression by the pyrC and pyrD mutations was reduced as a result of the incorporation of the leaky argI2002 allele. These results indicate that in pyrH700 strains carbamoyl phosphate is preferentially directed toward the formation of intermediates in the pyrimidine biosynthetic pathway. Arginine auxotrophy results from the reduced availability of carbamoyl phosphate for the biosynthesis of arginine. Suppression of this arginine dependence for growth is used as a convenient positive selection technique for pyrB mutations.     

Toxicity of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate in Salmonella typhimurium

Growth of Salmonella typhimurium pyrC or pyrD auxotrophs was severely inhibited in media that caused derepressed pyr gene expression. No such inhibition was observed with derepressed pyrA and pyrB auxotrophs. Growth inhibition was not due to the depletion of essential pyrimidine biosynthetic pathway intermediates or substrates. This result and the pattern of inhibition indicated that the accumulation of the pyrimidine biosynthetic pathway intermediate carbamyl aspartate was toxic. This intermediate is synthesized by the sequential action of the first two enzymes of the pathway encoded by pyrA and pyrB and is a substrate for the pyrC gene product. It should accumulate to high levels in pyrC or pyrD mutants when expression of the pyrA and pyrB genes is elevated. The introduction of either a pyrA or pyrB mutation into a pyrC strain eliminated the observed growth inhibition. Additionally, a direct correlation was shown between the severity of growth inhibition of a pyrC auxotroph and the levels of the enzymes that synthesize carbamyl aspartate. The mechanism of carbamyl aspartate toxicity was not identified, but many potential sites of growth inhibition were excluded. Carbamyl aspartate toxicity was shown to be useful as a phenotypic trait for classifying pyrimidine auxotrophs and may also be useful for positive selection of pyrA or pyrB mutants. Finally, we discuss ways of overcoming growth inhibition of pyrC and pyrD mutants under derepressing conditions.     

Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis.

The aroA locus of Bordetella pertussis, encoding 5-enolpyruvylshikimate 3-phosphate synthase, has been cloned into Escherichia coli by using a cosmid vector. The gene is expressed in E. coli and complemented an E. coli aroA mutant. The nucleotide sequence of the B. pertussis aroA gene was determined and contains an open reading frame encoding 442 amino acids, with a calculated molecular weight for 5-enolpyruvylshikimate 3-phosphate synthase of 46,688. The amino acid sequence derived from the nucleotide sequence shows homology with the published amino acid sequences of aroA gene products of other microorganisms.     

A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19), encoded by the aroA locus, is a target site of glyphosate inhibition in bacteria. A glyphosate-resistant aroA allele has been cloned in Escherichia coli from a mutagenized strain of Salmonella typhimurium. Subcloning of this mutant aroA allele shows the gene to reside on a 1.3-kilobase segment of S. typhimurium DNA. Nucleotide sequence analysis of this mutant gene indicates a protein-coding region 427 amino acids in length. Comparison of the mutant and wild type aroA gene sequences reveals a single base pair change resulting in a Pro to Ser amino acid substitution at the 101st codon of the protein. A hybrid gene fusion between mutant and wild type aroA gene sequences was constructed. 5-Enolpyruvylshikimate-3-phosphate synthase was prepared from E. coli cells harboring this construct. The glyphosate-resistant phenotype is shown to be associated with the single amino acid substitution described above.     

Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function.

A defined synthetic medium was used to determine the amino acid requirements of Enterococcus faecalis OG1 and to demonstrate the absence of a requirement for exogenous purines or pyrimidines. Genomic libraries prepared from strain OG1 were transduced into Escherichia coli auxotrophic mutants, and cloned DNAs which complemented pyrC, pyrD, purF, purL, and guaAB mutations were identified. These and other cloned DNAs with known functions were localized on a restriction map of OG1 which was generated with SfiI (5 fragments), AscI (9 fragments), and NotI (15 fragments); the size of the OG1 chromosome was revised from a previous estimate of approximately 2,750 kb to 2,825 kb. The synthetic medium and the restriction map should be useful for studying enterococcal metabolic functions and the relationships between chromosomally encoded genes.     

Structural and regulatory mutations allowing utilization of citrulline or carbamoylaspartate as a source of carbamoylphosphate in Escherichia coli K-12.

Escherichia coli mutants lacking carbamoylphosphate synthase require arginine and uracil for growth. It is, however, possible to obtain mutants in which carbamoylphosphate is obtained by phosphorolysis of citrulline or carbamyolaspartate. Citrulline utilizers are argG bradytrophs or strains in which the synthesis of ornithine carbamoyltransferase (either of the F or I type) is specifically depressed by unstable chromosomal rearrangements or stable mutations that presumably affect the operators of those genes. Carbamoylaspartate utilization as a source of carbamoylphosphate appears to require more than one mutation; the best-understood strains are pyrD pyrH or pyrC pyrH mutants in which aspartate carbamoyltransferase activity is high and the pool of cytidine triphosphate (feedback inhibitor of aspartate carbamoyl-transferase) is presumably low and in which channeling of carbamoylaspartate towards pyrimidine biosynthesis is considerably reduced. Selection of enzyme overproducers based on a metabolic dependency for a reversed enzymatic reaction can be regarded as a means for isolating regulatory mutants.     

Dihydroorotase from Escherichia coli. Cloning the pyrC gene and production of tryptic peptide maps

We have inserted a 1.7-kilobase pair Escherichia coli DNA fragment containing the 1-kilobase pair pyrC gene into the high copy number plasmid pKC16. Dihydroorotase expressed by the pyrC plasmid in E. coli constituted 6.3% of the soluble protein in frozen cell paste. Pure dihydroorotase derived from this frozen cell paste was compared with pure enzyme derived from an E. coli strain lacking the pyrC plasmid: tryptic peptide maps from the two dihydroorotase preparations, produced using reverse-phase high performance liquid chromatography, were indistinguishable. We conclude that the entire pyrC gene is present on the hybrid plasmid and that the dihydroorotase produced from this plasmid is identical to the wild type.     

副猪嗜血杆菌aroA基因鉴定及遗传进化分析

[目的]细菌aroA基因参与芳香族氨基酸的生物合成,被成功应用于细菌分类和基因失活致弱突变菌株的构建.副猪嗜血杆菌(Hps)是感染猪出现多发性浆膜炎和关节炎的一种病原细菌,鉴定该菌aroA全基因序列将有助于鉴定遗传进化关系和突变分析.[方法]利用PCR和细菌基因组步移技术鉴定Hps的aroA基因序列,进而对不同血清型菌株该基因序列进行鉴定,并与其它革兰氏阴性细菌进行比对和遗传进化分析.[结果]自Hps血清5型基因组DNA中获得包含完整aroA基因的3.7 kb基因片段,其中aroA基因全长1314 bp,编码产物长度437 aa,分子量大小47.9 kDa,该基因上游为磷酸烯醇式丙酮酸羧化酶基因.自本试验选择的Hps不同血清型菌株中均可扩增出包含完整aroA基因的1476 bp片段,且这些不同血清型菌株间核酸序列同源性在97.7%以上.Hps血清5型aroA基因序列与巴氏杆菌科其它成员核酸序列同源性为70.6%-78.9%,与E.coli和S.typhi-murium的同源性分别为66.4%和67.2%.[结论]本试验首次对Hps的15个血清型国际参考菌株及地方分离株aroA全基因序列进行了鉴定,序列比较结果显示aroA基因在革兰氏阴性细菌中具有较高的同源性.aroA基因鉴定对构建基因失活突变菌株以研究Hps生物学特性奠定了基础.     

Mapping of a locus (mdoA) that affects the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

Mutants of Escherichia coli defective in the newly discovered mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides. The mutation has now been mapped and found to be located near 23 min on the E. coli chromosome between putA and pyrC. The mdoA mutants are defective in the membrane-localized component of the glucosyl transferase system described by Weissborn and Kennedy (A. C. Weissborn and E. P. Kennedy, Fed. Proc. 42:2122, 1983).