The genetic organization of arginine biosynthesis in Pseudomonas aeruginosa

Summary Six loci coding for arginine biosynthetic enzymes in Pseudomonas aeruginosa strain PAO were identified by enzyme assay: argA (N-acetylglutamate synthase), argB (N-acetylglutamate 5-phosphotransferase), argC (N-acetylglutamate 5-semialdehyde dehydrogenase), argF (anabolic ornithine carbamoyltransferase), argG (argininosuccinate synthetase), and argH (argininosuccinase). One-step mutants which had a requirement for arginine and uracil were defective in carbamoylphosphate synthase, specified by a locus designated car. To map these mutations we used the sex factor FP2 in an improved interrupted mating technique as well as the generalized transducing phages F116L and G101. We confirmed earlier studies, and found no clustering of arg and car loci. However, argA, argH, and argB were mapped on a short chromosome segment (approx. 3 min long), and argF and argG were cotransducible, but not contiguous. N-Acetylglutamate synthase, the enzyme which replenishes the cycle of acetylated intermediates in ornithine synthesis of Pseudomonas, appears to be essential for arginine synthesis since argA mutants showed no growth on unsupplemented minimal medium.     

Characterization and genetic mapping of fructose phosphotransferase mutations in Pseudomonas aeruginosa

Pseudomonas aeruginosa transports and phosphorylates fructose via a phosphoenolpyruvate-dependent fructose phosphotransferase system (PTS). Mutant strains deficient in both PTS activity and glucose-6-phosphate dehydrogenase activity were isolated and were used to select mannitol-utilizing revertant strains singly deficient in PTS activity. These mutants were unable to utilize fructose as a carbon source and failed to accumulate exogenously provided [14C]fructose, and crude cell extracts lacked phosphoenolpyruvate-dependent fructose PTS activity. Thus, the PTS was essential for the uptake and utilization of exogenously provided fructose by P. aeruginosa. Mutations at a locus designated pts, which resulted in a loss of PTS activity, exhibited 57% linkage to argF at 55 min on the chromosome in plasmid R68.45-mediated conjugational crosses. The pts mutations in four independently isolated mutant strains exhibited from 11 to 20% linkage to argF, and one of these mutations exhibited 3% linkage to lys-9015 in phage F116L-mediated transductional crosses.     

R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa

Summary The R factor R68 readily promotes chromosome transfer in Pseudomonas aeruginosa strain PAT, but shows little such sex factor activity in strain PAO. A variant of this plasmid, R68.45, has been isolated which produces recombinants in PAO plate matings at frequencies of 10^-3–10^-5 per donor cell for markers in the 0–60 min region of the chromosome. Little or no chromosome transfer was shown in liquid media. The kinetics of chromosome transfer were studied by interrupting matings on solid media with nalidixic acid. Five chromosomal markers, mapping in widely spaced regions of the chromosome all entered 3–5 min after initiation of mating. These results, combined with linkage studies, indicate that R68.45, unlike the Pseudomonas sex factors FP2 and FP39, promotes chromosome transfer from a range of origin sites and can thus be used for mapping the region of the P. aeruginosa chromosome later than 40 min.R68.45 and other similar variants were isolated from rare chromosomal recombinants appearing in crosses between PAO(R68) donors and PAO recipients in which selection for argB ⁺ was made. Selection for other chromosomal markers did not result in such variants suggesting that plasmids of the R68.45 type arise by recombination of genetic material between the R68 plasmid and certain regions of the bacterial chromosome.     

Snr,New Genetic Loci Common to the Nitrate Reduction Systems of Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is able to both assimilate and dissimilate nitrate. On the basis of the characteristics of mutants unable to dissimilate or assimilate nitrate to nitrite, it was revealed that two different sets of genes (represented by Class I and Class II mutants) were shared between the nitrate-to-nitrite reduction steps of both pathways. The genes represented by Class I and II mutants have been separated into distinct genetic loci using two cosmids, pAD1695/96. The two different genetic loci have been designated snr (shared nitrate reduction) and mol (MoCo processing genes) based on the phenotypic characteristics of the mutants complemented. Restriction analyses of pAD1695/96 followed by subcloning confirmed the complementation results. The snr loci, which represent a unique and hitherto uninvestigated set of genes for nitrate reduction, were mapped on the P. aeruginosa chromosome by linkage analysis with sex factor FP2. Received: 22 November 1996 / Accepted: 3 December 1996     

Identification of the protein products of the rrnC, ilv, rho region of the Escherichia coli K-12 chromosome

Summary Two methods have been used to identify the protein products of the Escherichia coli K-12 ilv region at 84 min and the flanking rrnC (counterclockwise) and rho (clockwise) loci. First, a set of dilv specialized transducing phages, including some phages that carry rho and others that carry part of rrnC, was used to infect UV irradiated cells. The proteins produced by the infecting dilv phage were selectively labelled with radioactive amino acids and identified by SDS gel electrophoresis and autoradiography. Second, restriction enzyme fragments were cloned from the dilv phage into pBR322 and the plasmid specific gene products produced in maxicells were similarly identified by SDS gel electrophoresis and autoradiography. The proteins produced were correlated with specific genes and restriction enzyme fragments present in the dilv phage and the pBR322 derivatives. Several ilv gene products that have previously been refractory to protein purification attempts have been identified for the first time by this technique. The presence of mutations at the ilvO site is shown to activate the cryptic ilvG gene and to result in the production of a 62,000 dalton protein. A 15,000 dalton protein of unknown function is synthesized from a DNA segment between ilv and rrnC. The rho gene was cloned from dilv phage into pBR322 and shown to be dominant to a rho mutation on the host cromosome. The rho gene product and four additional proteins coded by genes near or between rho and ilv have been detected.     

Revised locations of the hisI and pru (proline utilization) genes on the Pseudomonas aeruginosa chromosome map

Summary The location of genes in the vicinity of the major FP2 origin on the chromosome of Pseudomonas aeruginosa PAO has been revised. The markers hisI (a transduction group of histidine biosynthetic genes) and pru (a gene cluster encoding proline utilization functions) were located in the 90 to 95/0 min chromosome region by a series of plate matings mediated by R68.45. Three-factor-crosses using this plasmid established the following marker order: pur-67 pru hisI/cys-59 proB ilvB/C. Genetic evidence is presented to confirm the previous observations that FP2 can mobilize the chromosome from at least two origins near proB and in both directions. Thus, when markers in this chromosome region are analyzed by FP2 crosses only, the mapping data may be difficult to interpret. This complication can be overcome by the use of R68.45 and Tfr (transposon-facilitated recombination) or Hfr donors.     

Rough and fine linkage mapping of the Rhizobium meliloti chromosome.

Summary A circular linkage map of the Rhizobium meliloti chromosome, obtained from R68.45-mediated crosses, has been revised by cotransductional analysis, after general transduction by DF2 phage.Three short chromosomal regions have been mapped by cotransduction. Comparison between conjugal and cotransductional data suggests that R68.45-mediated linkage measures are indeed rough. Cotransduction seems to be a useful tool for improving the linkage map of R. meliloti.     

Mapping of the locus involved in the catabolic oxidation of D-amino acids in Pseudomonas aeruginosa PAO.

Summary Mutants of Pseudomonas aeruginosa PAO deficient in their utilization of DL-valine have been found to have lost their capacity to utilize DL-alanine and L-proline. Use of conjugal and transductional mediated gene transfers have established the chromosomal location of this gene and also its pleotropic function in the induction of the D-amino acid oxidase, involved in the oxidative utilization of DL-valine, DL-alanine and L-proline. These point mutations are clustered in a single locus designated as Val D and mapped around the 19th minute on the chromosome.     

Only one gene is required for the glpT-dependent transport of sn-glycerol-3-phosphate in Escherichia coli

Summary Deletion and point mutants defective in the glpT-dependent sn-glycerol-3-phosphate transport system were isolated and located on the Escherichia coli chromosome. They mapped in glpT in the clockwise order gyrA, glpA, glpT at around 48 min on the Escherichia coli linkage map. The mutations within glpT were ordered by deletion mapping, three factor crosses, and by crosses involving transducing bacteriophages carrying glpT-lac operon fusions. Results obtained using these fusion phages indicated that glpT is transcribed in the counterclockwise direction on the E. coli linkage map.Complementation analysis using these mutants revealed only one complementation group. Thus, one gene is necessary and sufficient for the proton motive force-dependent sn-glycerol-3-phosphate transport system.     

Genetic characterization of the Escherichia coli cyclopropane fatty acid (cfa) locus and neighboring loci

Summary The phenotypically silent cyclopropane fatty acid synthesis (cfa) gene of Escherichia coli K-12 has been located on the genetic linkage map. This was accomplished by integrating (via homologous recombination) the selectable marker of a recombinant plasmid into the host chromosome near the cfa locus. This integration allowed the subsequent isolation of a cfa-linked transposon Tn10 insertion. Genetic mapping of the Tn10 insertion, using conventional techniques, placed the cfa locus at min 36.5 on the linkage map in the vicinity of several other non-selectable markers. We ordered cfa and these other loci by three-factor transductional analyses. Selection for excision of the Tn10 element resulted in several types of mutants which harbor mutations of cfa and of neighboring genes, presumably as a consequence of Tn10-catalyzed chromosomal rearrangements.     

Genetic and physical mapping of recF in Escherichia coli K-12

Summary Two factor transductional crosses place recF at approximately 82 min on the E. coli chromosome; recF is highly cotransducible with dnaA and gyrB (cou). Transductional analysis with a series of tna specialized transducing phages carrying chromosomal DNA from the tnaA region place recF between dnaA and gyrB. This analysis also indicates that a gene lying in the same region and producing an easily detectable protein (estimated MW of 45 kD) is dnaN and not recF.     

Glyceraldehyde 3-P dehydrogenase, glycerate 3-P kinase and enolase mutants of Escherichia coli: Genetic studies

Summary The genetic loci for two enzymes of glycolysis have been established by transductional crosses. The eno locus, likely to be the structural gene for enolase maps in the 52-minute region of the Escherichia coli chromosome. A structural determinant for glycerate 3-P kinase (pgk) is located near serA. The map order is speB-pgk–serA-lysA-argA-eno-cysC. In the 35-minute region maps a locus affecting the structure of glyceraldehyde 3-P dehydrogenase.     

Genetic analysis and molecular cloning of the Escherichia coli ruv gene

Summary The genetic organisation of the ruv gene, a component of the SOS system for DNA repair and recombination in Escherichia coli, was investigated. New point mutations as well as insertions and deletions were generated using transposon Tn10 inserted in eda as a linked marker for site specific mutagenesis, or directly as a mutagen. The mutations were ordered with respect to one another and previously isolated ruv alleles by means of transductional crosses. The direction of chromosome mobilization from ruv:: Mud(Ap^R lac)strains carrying F42lac ⁺ established that ruv is transcribed in a counterclockwise direction. Recombinant phages able to restore UV resistance to ruv mutants were identified, and the ruv ⁺ region was subcloned into a low copy number plasmid. The ruv ⁺ plasmid was able to correct the extreme radiation sensitivity and recombination deficiency of ruv recBC sbcB strains.     

A novel transducing phage

Summary Two newly isolated generalized transducing phages, F126 and F130, are in many respects similar to the previously described phage F116 of P. aeruginosa strain PAO. They also share with F116 the property of not responding to host-specific restriction in strain PAO. However, while the transduction ability of phages F116 and F130 is also inert to PAO-specific restriction, transduction mediated by phage F126 is 8–120 fold reduced in restrictive conditions. In experiments designed to explore the conditions necessary to obtain restriction in F126 transduction it was found that it differed from those previously known to specify host-controlled restriction in P. aeruginosa PAO (Rolfe and Holloway, 1968). These observations suggest that more than one independent restriction system operates in strain PAO.     

Chromosomal location of a gene (idg) that specifies production of the blue pigment indigoidine inErwinia chrysanthemi

Production of a blue pigment, indigoidine, is a variable trait among wild-type strains ofErwinia chrysanthemi; it is also influenced by the composition of the growth medium. Starting with a nonpigmented wild-type strain (ICPB EC183) ofE. chrysanthemi, we obtained by nitrosoguanidine mutagenesis a pigmented (idg ⁺) mutant strain (AC6055), which simultaneously was Arg^–. Linkage betweenarg andidg was established in two-factor transductional and conjugational crosses. Coinheritance ofidg withhis ⁺,ilv ⁺,leu ⁺,ser ⁺,thr ⁺, orura ⁺ transductants was not observed. Spontaneous Arg⁺ revertants of AC6055 were invariably Idg⁺. The pigments produced by AC6055, Idg⁺ recombinants, and wild-type Idg⁺ strains were identical, judged by absorption spectra (_max = 615 nm) of the dimethylsulfoxide extracts of whole cells. We concluded that nitrosoguanidine caused comutation in thearg andidg loci that are linked on theE. chrysanthemi chromosome.     

Linkage relationships of mutations endowing Streptococcus pyogenes with resistance to antibiotics that affect the ribosome

Summary Several mutations conferring resistance to streptomycin, kanamycin, spectinomycin, erythromycin, and lincomycin on the group A streptococcal strain 56188 have been mapped by two- and three-point crosses using transduction with bacteriophage A25. The markers are located in two linkage regions too distant to be cotransduced. One harbors the streptomycin and kanamycin loci which are transduced jointly at 78% and the other bears loci for spectinomycin (spc), erythromycin (eryA), and lincomycin (linA) resistance, in this order. spc and linA are cotransduced at a frequency of about 27%. Analysis of three-point crosses involving spc-4, eryA300, and linA12 according to the Wu model for random general transduction shows consistency of the theoretical predictions with the experimental data and indicates that the intervals of the above sequence are about 22% and 6% of the average length of the transduced piece.     

Convenient construction of strains useful for transducing recA mutations with bacteriophage P1

Abstract The generalized transducing phage P1 grew well on heterozygous Escherichia coli carrying recA srlC 300::Tn 10 on the chromosome and recA ⁺ on a pBR322-derived plasmid. Because of the close linkage of Tn 10 to recA mutations, including recA 1, recA 13, recA 56, recA deletion and recA allele of E. coli BNG30, the latter can be moved to other strains in transductional crosses for selective resistance to tetracycline.     

RAD‐seq linkage mapping and patterns of segregation distortion in sedges: meiosis as a driver of karyotypic evolution in organisms with holocentric chromosomes

Meiotic drive, the class of meiotic mechanisms that drive unequal segregation of alleles among gametes, may be an important force in karyotype evolution. Its role in holocentric organisms, whose chromosomes lack localized centromeres, is poorly understood. We crossed two individuals of Carex scoparia (Cyperaceae) with different chromosome numbers (2n = 33^II = 66 × 2n = 32^II = 64) to obtain F1 individuals, which we then self‐pollinated to obtain second‐generation (F2) crosses. RAD‐seq was performed for 191 individuals (including the parents, five F1 individuals and 184 F2 individuals). Our F2 linkage map based on stringent editing of the RAD‐seq data set yielded 32 linkage groups. In the final map, 865 loci were located on a linkage map of 3966.99 cM (linkage groups ranged from 24.39 to 193.31 cM in length and contained 5–51 loci each). Three linkage groups exhibit more loci under segregation distortion than expected by chance; within linkage groups, loci exhibiting segregation distortion are clustered. This finding implicates meiotic drive in the segregation of chromosome variants, suggesting that selection of chromosome variants in meiosis may contribute to the establishment and fixation of chromosome variants in Carex, which is renowned for high chromosomal and species diversity. This is an important finding as previous studies demonstrate that chromosome divergence may play a key role in differentiation and speciation in Carex.     

Mapping of the loci involved in the catabolic oxidation of L-hydroxyproline in Pseudomonas aeruginosa PAO

Summary Genes specifying the oxidative utilization of hydroxyproline (Hyp) in P. aeruginosa PAO were located on the chromosome, around 19th minute by conjugation experiments. A map order of his-68-his-07-Hyp was assigned. Confirmation of this gene order was also demonstrated by transductional mapping studies. All the genes determining the enzymes of Hyp dissimiliatory pathway were closely linked.