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.     

Transductional analysis of chloramphenicol biosynthesis genes in Streptomyces venezuelae.

Auxotrophs isolated from two chloramphenicol-nonproducing mutants of Streptomyces venezuelae included three requiring pyridoxal (Pxl-), VS248 (cml-11 pdx-2), VS253 (cml-11 pdx-3), and VS258 (cml-12 pdx-4), and one requiring thiosulfate, VS263 (cml-12 cys-28). Results of SV1-mediated transductions were consistent with the relative marker order cys-28-cml-12-cml-11-pdx-2,3,4,5, all of which were cotransducible and must therefore span less than 45 kilobases of DNA, the approximate length of DNA packaged by SV1. cys-28 was also cotransducible with arg-4 and arg-6, but arg and pdx were not cotransducible. Results of crosses with donors carrying any one of 11 cml mutations were consistent with the location of all cml mutations between cys-28 and pdx markers. Also, a new Pxl- auxotroph (pdx-6) and two new Cml- mutants were recovered after localized hydroxylamine mutagenesis of a cys-28 cml+ strain derived from VS263 by transduction.     

Construction of recombination-deficient strains of Pseudomonas aeruginosa

Summary The rec-102 mutation had pleiotropic effects in Pseudomonas aeruginosa: low recombination proficiency in conjugation and transduction; high UV sensitivity; inability to induce pyocin R2 by mitomycin C; and increased susceptibility to mitomycin C and nalidixic acid. The rec-102 locus was mapped by R68.45-mediated conjugation in the 45 min region of the PAO chromosome, between argF and thr-9001. By selection for a marker in this region, rec-102 can be introduced into a P. aeruginosa strain of interest using an R68.45 rec-102 donor. The recombination-deficient strains constructed in this way were phenotypically similar to Escherichia coli recA mutants.     

Genetic mapping of the structural gene for phospholipase C of Pseudomonas aeruginosa PAO.

An insertion mutation constructed by gene replacement methods was used to map the gene corresponding to the hemolytic phospholipase C (plcS gene) in Pseudomonas aeruginosa PAO1 by R68.45-mediated conjugation. plcS mapped approximately at 67 min on the 75-min chromosomal map (B. W. Holloway, K. O'Hoy, and H. Matsumoto, p. 213-221, in S. J. O'Brien, ed., Genetic Maps 1987, vol. 4, 1987), between the markers pur-67 and pru-375 and considerably distal to the regulatory genes plcA and plcB, which are located at approximately 12 min.     

Locus of the Pseudomonas aeruginosa toxin A gene.

The gene for Pseudomonas aeruginosa toxin A has been mapped in the late region of the chromosome of strain PAO. Strain PAO-PR1, which produces parental levels of toxin A antigen that is enzymatically inactive and nontoxic, was used as the donor for R68.45 plasmid-mediated genetic exchange. Strain PAO-PR1 (toxA1) was mated with toxin A-producing strains, and exconjugates for selected prototrophic markers were tested for the transfer of toxA1. The toxA1 gene was located between cnu-9001 and pur-67 at approximately 85 min on the PAO chromosome.     

Characterization and Genetic Mapping of Phosphoglucoisomerase Mutations in Mannitol-Negative Mutants of Pseudomonas aeruginosa

Phosphoglucoisomerase (pgi) mutations in a number of independently isolated mannitol-negative mutants of Pseudomonas aeruginosa PAO1 were mapped on the chromosome by plasmid FP5-mediated conjugation and by cotransduction with the generalized transducing phages G101 and F116L. Mutant allele pgi-9001 exhibited linkage to ilvB, C-9059 (46–85%), car-9003 (93–100%), and pur-9047 (70%), but not with met-9011, in FP5-mediated conjugational crosses. All known pgi mutations and several previously uncharacterized mannitol-negative mutations exhibited transductional linkage to two independent car mutations at frequencies ranging from 13% to 42% and 53% to 99%, in transductional crosses mediated by phages G101 and F116L respectively. These pgi and mannitol-negative mutations also were cotransducible at very low frequencies (<1%) with two independent ilv mutations. Cotransduction of the car and ilv loci could not be detected. These data suggest the location of pgi within the first minute of the P. aeruginosa chromosome closely linked to the car marker and probably between the ilv and car loci. All of the mannitol-negative mutations that exhibited linkage to the car and ilv loci were characterized as pgi mutations by enzyme assays. A phenotypically similar, mannitol-negative mutatant was shown to contain a mutation in glucose-6-phosphate dehydrogenase (zwf-9012) that maps to a different region on the chromosome. Received: 26 April 1996 / Accepted: 10 June 1996     

Constitutive mutation of cysJIH operon in a cysB deletion strain of Salmonella typhimurium.

Summary In a cysB deletion strain a new mutation, denoted cys-2332 was isolated, which causes the constitutive expression of the cysJIH operon. cys-2332 is closely linked to cysJIH and presumably is located in the initiator region of this operon, rendering its expression independent of the cysB gene product and the internal inducer O-acetyl-L-serine. The presence of salfite reductase (encoded by cysI and cysJ) activity in a cysB ^- cys-2332 double mutant indicates that cysG, which is not linked to cysJIH but is required for the synthesis of the sulfite reductase co-factor siroheme, is not controlled by cysB.     

Generalized transduction in Rhizobium meliloti

Summary The phage 11 of R. meliloti performs generalized transduction. This was confirmed by the variety of single markers transferred and by separating transducing particles containing BUdR-labelled bacterial DNA. The transduction frequencies depended on the marker. Linked alleles were mapped by cotransduction on fragments of bacterial DNA equal in size to the phage DNA. With crosses between antibiotic resistancy and auxotrophic markers a partial map was constructed with str, cml, pur-19, and leu-44 sites. With a few multi-auxotrophic mutants linkage data of conjugation were compared with the linkage by cotransduction.     

Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors

The expression of sacB, the Bacillus subtilis gene encoding levansucrase, is lethal to mycobacteria in the presence of 10% sucrose. In this study, we describe the use of sacB as a marker for positive selection of gene-replacement events into Mycobacterium smegmatis. A sucrose counter-selectable suicide plasmid was used to deliver an inactivated copy of the pyrF gene (pyrFKm) into the M. smegmatis genome. Only uracil auxotroph clones, resulting from replacement of the endogenous pyrF allele, survived in a one-step selection on plates containing kanamycin and 10% sucrose. This demonstrated that selection on sucrose against the maintenance of the vector bearing the sacB gene is 100% efficient, enabling the positive selection of allelic-exchange mutants. Two-step selection is also feasible; it was used to construct unmarked pyrF mutants in which the gene was inactivated by a frameshift mutation. This method of generating unmarked, directed mutations is rapid and simple, making it a powerful tool for the genetic characterization of mycobacteria.     

Chromosome mobilization by the R plasmid R68.45: a tool in Pseudomonas genetics.

Summary The conjugative plasmid R68.45 mobilizes the chromosome of Pseudomonas aeruginosa strain PAO from multiple sites located in different chromosome regions. In interrupted matings on the plate, selection for any single marker tested resulted in entry times of 3–5 min. When selection was imposed for two markers linked in R68.45-mediated conjugation, double recombinants appeared after a delay which corresponded approximately to the map distance between the two markers as measured by the sex factor FP2. Thus, R68.45 and FP2 appear to promote chromosome transfer at similar rates, but R68.45, unlike FP2, seems to give non-polarized transfer. R68.45 may be used to estimate map distances between linked markers located in those chromosome regions where other sex factors do not produce enough recombinants to permit accurate measurement of entry times.In R68.45 matings on the plate, most recombinants inherited short donor chromosome fragments (usually less than 10 min long) and lost the R plasmid during purification. Used like a large generalized transducing phage, R68.45 has proved valuable in construction of PAO strains with desired genotypes.     

Mapping of a gene controlling the production of phospholipase C and alkaline phosphatase in Pseudomonas aeruginosa

Summary Using plasmid R68.45 mediated conjugation and transduction with bacteriophage F116L, a genetic locus controlling at least two orthophosphate repressible proteins in Pseudomonas aeruginosa has been mapped at about 22–23 min on the PAO chromosome.     

Tryptophan genes in Rhizobium--their organization and their transfer to other bacterial genera.

Summary R. leguminosarum trp alleles mapped by R68.45-mediated recombination were located in three distinct chromosomal regions. We isolated three derivatives of R68.45 that carried different trp genes of R. meliloti. Each of the plasmids suppressed all of the R. leguminosarum trp alleles in a particular region. The R-primes were transferred to strains of P. aeruginosa carrying mutations in different trp genes. The plasmid pAJ24JI suppressed trpA, B and F mutants, pAJ73JI suppressed trpC and D and pAJ88JI suppressed a trpE mutant. When the R-primes were transferred to E. coli trp strains they failed to suppress any trp mutants. A derivative of pAJ24JI was isolated which was able to suppress trpA and F mutants of E. coli.     

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.     

Analysis of flagellar genes in Pseudomonas aeruginosa by use of Rfla plasmids and conjugations.

Over 300 flagellar mutants were isolated in Pseudomonas aeruginosa PAO. R-prime plasmids carrying segments of bacterial chromosome which can complement the mutant phenotypes were isolated by means of plasmid R68.45. Among the R-prime plasmids, pMT6 complemented 167 out of 307 mutants examined, and pMT19 complemented the remaining 140 mutants. We found no mutant which was complemented by both of these plasmids. Hence, the flagellar genes were divided into two clusters by these two plasmids, namely, region I on pMT19 and region II on pMT6. By FP5- and R68.45-mediated conjugation, these two regions were located on the P. aeruginosa PAO chromosome with an order of puuF--region I--region II--oru-325.     

Localization of symbiotic mutations in Rhizobium meliloti

A total of 5 Nod- and 57 Fix- symbiotic mutants of Rhizobium meliloti strain 41 have been isolated after either nitrosoguanidine or Tn5 transposition mutagenesis. Chromosomal locations of mutations in 1 Nod- and 11 Fix- derivatives were ascertained by transferring the chromosome (mobilized by plasmid R68.45), in eight fragments, into symbiotically effective recipients and testing the recombinants for symbiotic phenotype. Alternatively, the kanamycin resistance marker of Tn5 was mapped. In five mutants the fix alleles were localized on different chromosomal regions, but six other fix mutations and one nod mutation tested did not map onto the chromosome. It was shown that the chromosome-mobilizing ability (Cma+) of R68.45 was not involved in the mobilization of genes located extrachromosomally. Moreover, Cma- derivatives of R68.45 could mobilize regions of the indigenous plasmid pRme41b but not chromosomal genes. Thus, mobilization of a marker by Cma- R68.45 indicates its extrachromosomal location. With a 32P-labeled DNA fragment carrying Tn5 as a hybridization probe, it was shown that in five extrachromosomally located Tn5-induced fix mutants and one nod mutant Tn5 was localized on plasmid pRme41b. This is in agreement with the genetic mapping data.     

Mapping of mutations affecting pyoverdine production in Pseudomonas aeruginosa

Abstract Pyoverdine, the yellow-green fluorescent pigment produced by Pseudomonas aeruginosa , is a highly efficient siderophore. Pyoverdine-deficient ( pvd ) mutants of P. aeruginosa PAO isolated after mutagenesis were non-fluorescent and unable to grow in the presence of 2.8 mM ethylenediamine-di-( o -hydroxyphenylacetate) (EDDHA). Addition of purified pyoverdine to media containing EDDHA restored growth of pvd mutants. 6 pvd mutations were mapped between catA and mtu -9002 (at 65–70 min on the chromosome map) by R68.45-mediated conjugation. 2 slightly leaky pvd mutations were localised between argC and strA (at 35 min) by transduction. Thus, we have identified at least 2 genes or gene clusters required for pyoverdine production in P. aeruginosa .     

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.     

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.     

pyrF as a Counterselectable Marker for Unmarked Genetic Manipulations in Treponema denticola

The pathophysiology of Treponema denticola, an oral pathogen associated with both periodontal and endodontic infections, is poorly understood due to its fastidious growth and recalcitrance to genetic manipulations. Counterselectable markers are instrumental in constructing clean and unmarked mutations in bacteria. Here, we demonstrate that pyrF, a gene encoding orotidine-5′-monophosphate decarboxylase, can be used as a counterselectable marker in T. denticola to construct marker-free mutants. T. denticola is susceptible to 5-fluoroorotic acid (5-FOA). To establish a pyrF-based counterselectable knockout system in T. denticola, the pyrF gene was deleted. The deletion conferred resistance to 5-FOA in T. denticola. Next, a single-crossover mutant was constructed by reintroducing pyrF along with a gentamicin resistance gene (aacC1) back into the chromosome of the pyrF mutant at the locus of choice. In this study, we chose flgE, a flagellar hook gene that is located within a large polycistronic motility gene operon, as our target gene. The obtained single-crossover mutant (named FlgEⁱⁿ) regained the susceptibility to 5-FOA. Finally, FlgEⁱⁿ was plated on solid agar containing 5-FOA. Numerous colonies of the 5-FOA-resistant mutant (named FlgE^out) were obtained and characterized by PCR and Southern blotting analyses. The results showed that the flgE gene was deleted and FlgE^out was free of selection markers (i.e., pyrF and aacC1). Compared to previously constructed flgE mutants that contain an antibiotic selection marker, the deletion of flgE in FlgE^out has no polar effect on its downstream gene expression. The system developed here will provide us with a new tool for investigating the genetics and pathogenicity of T. denticola.     

Mapping and characterization of two mutations to antibiotic supersusceptibility in Pseudomonas aeruginosa

Two mutations associated with antibiotic supersusceptibility in Pseudomonas aeruginosa strain Z61 were transferred separately into strain PAO222, using R68.45-mediated conjugation and phage F116L transduction. One mutation (absA) was 40% contransducible with pro-82 at 26 min on the P. aeruginosa chromosome and was associated with increased susceptibility to beta-lactams, gentamicin and hydrophobic agents. Strains carrying the absA mutation also displayed enhanced uptake of a hydrophobic fluorescent probe, 1-N-phenylnaphthylamine, and were found, by SDS-PAGE, to be altered in the pattern of lipopolysaccharide O-antigen distribution. The other mutation (absB), associated with increased susceptibility to beta-lactams and gentamicin but not to hydrophobic agents, was cotransducible with met-28 and proC at 20 min on the chromosome. The absB mutation caused a structurally undefined alteration in the physical interaction of EDTA and gentamicin with the outer membrane.