Localization of alg, opr, phn, pho, 4.5S RNA, 6S RNA, tox, trp, and xcp genes, rrn operons, and the chromosomal origin on the physical genome map of Pseudomonas aeruginosa PAO.

The genes encoding the rrn operons, the 4.5S and 6S RNAs, elements of protein secretion, and outer membrane proteins F and I, and regulatory as well as structural genes for exotoxin A, alkaline phosphatase, and alginate and tryptophan biosynthesis, were assigned on the SpeI/DpnI macrorestriction map of the Pseudomonas aeruginosa PAO chromosome. The zero point of the map was relocated to the chromosomal origin of replication.     

A physical genome map of Pseudomonas aeruginosa PAO.

A complete macrorestriction map of the 5.9 Mb genome of Pseudomonas aeruginosa PAO (DSM 1707) was constructed by the combination of various one- and two-dimensional pulsed field gel electrophoresis techniques. A total of 51 restriction sites (36 SpeI sites, 15 DpnI sites) were placed on the physical map yielding an average resolution of 110 kb. Several genes encoding virulence factors and enzymes of metabolic pathways were located on the anonymous map by Southern hybridization. Distances between the gene loci were similar on the genetic and physical maps, suggesting an even distribution of genome mobility throughout the bacterial chromosome. The four rRNA operons were organized in pairs of inverted repeats. The two-dimensional macro-restriction techniques described herein are generally applicable for the genome mapping of any prokaryote and lower eukaryote which yields resolvable fragment patterns on two-dimensional pulsed field gels.     

A combined physical and genetic map of Pseudomonas aeruginosa PAO

A combined physical and genetic map of Pseudomonas aeruginosa PAO was constructed by pulsed-field gel electrophoresis and Southern hybridization using cosmid clones from a genomic library carrying known genes. A total of 37 SpeI restriction fragments have been mapped on the 5862 kb genome, and fragment contiguity demonstrated by hybridization with clones from a SpeI junction fragment library and fragments obtained by partial SpeI digestion, both derived from the P. aeruginosa PAO chromosome.     

Circularity of the Caulobacter crescentus chromosome determined by pulsed-field gel electrophoresis.

Previous genetic analyses of the Caulobacter crescentus chromosome have resulted in the construction of a linear genetic map. To establish the circularity of the C. crescentus chromosome, restriction fragments generated by digestion with AseI and SpeI were analyzed by pulsed-field gel electrophoresis and Southern hybridization. The size of each fragment was calculated and used to demonstrate that C. crescentus has a genome size of approximately 4,000 kilobases. In addition, both enzymes gave rise to large DNA fragments which contained genes from both ends of the genetic map. Thus, there is physical linkage between the genes at the ends of the genetic map and the chromosome is circular. Since this region of the chromosome appears to contain the replication terminus, we propose that recombination occurs at a high frequency in the vicinity of the terminus. This high frequency of recombination would prevent genetic linkage from being observed between genes on opposite sides of the terminus. Additional experiments using insertions which introduced new AseI and DraI restriction sites into the genome allowed us to calculate the physical distance between genes located in the vicinity of the replication terminus.     

Gradient of genomic diversity in the Pseudomonas aeruginosa chromosome

In 545 Pseudomonas aeruginosa strains, mainly collected from patients with cystic fibrosis, Spel-Dral macrorestriction fragment lenght diversity was scanned for using probes of known map position on th P.earuginosa PAO chromosome. Southern analysis of the 60 unrelated clones uncovered a gradient of macrorestriction fragment lenght polymorphisms (RFLPs) from the origin of replication towards the auxotroh-poor region of the P. aeruginosa population in the region encompassed by the rrn operons. The oriC -reactive Spel fragment was conserved in nearly all isolates examined. Few fragment lenght classes were seen for the alga60-, algR- and toxA -encoding Spel fragments. Fragment siz varied within one class by up to 20 kb. Two probes from the auxotroph-poor region detected a broad size range for the Spel fragment, suggestiong extensive genomic deversity in these reions. Subclonalvariation of fragment size was detected at all investigated loci in at least one of the analysed clones, but within one particlular clone, Spel -RFLPs were found at only few loci.     

High-frequency flp recombinase-mediated inversions of the oriC-containing region of the Pseudomonas aeruginosa genome

The genomes of the two clonally derived Pseudomonas aeruginosa prototypic strains PAO1 and DSM-1707 differ by the presence of a 2. 19-Mb inversion including oriC. Integration of two Flp recombinase target sites near the rrn operons containing the inversion endpoints in PAO1 led to Flp-catalyzed inversion of the intervening 1.59-Mb fragment, including oriC, at high frequencies (83%), favoring the chromosome configuration found in DSM-1707. The results indicate that the oriC-containing region of the P. aeruginosa chromosome can readily undergo and tolerate large inversions.     

Physical and genetic mapping of the catA region of Pseudomonas aeruginosa.

A prime plasmid has been used as the basis for the construction of a physical and genetic map of a 125 kb segment of the Pseudomonas aeruginosa PAO chromosome. Using pMO1811, a prime plasmid selected for the catA region, a series of Tn5 insertions were obtained which identified two new markers gcu (glycine utilization) and oap (organic acids and alcohols permeability) in the 125 kb region and located them in relation to other known markers of this region. A cosmid bank was constructed from the prime plasmid and an ordered array of cosmid clones for this region identified by restriction endonuclease mapping with EcoRI, HindIII and KpnI, as well as complementation mapping and chromosome walking. By Southern hybridization analyses, it was confirmed that the chromosomal insert carried by pMO1811 was flanked by single, tandemly arranged copies of IS21 and the orientation of the insert on this prime was determined. This cosmid bank provides a resource for the further analysis of this region of the P. aeruginosa genome.     

A global response to sulfur starvation in Pseudomonas putida and its relationship to the expression of low-sulfur-content proteins

Staphylococcus xylosus is a ubiquitous bacterium frequently isolated from mammalian skin and occurring naturally on meat and dairy products. A physical and genetic map of the S. xylosus C2a chromosome was constructed by pulsed-field gel electrophoresis analysis after digestion with AscI, ApaI, I-CeuI, SfiI and SmaI enzymes and hybridization analysis. The chromosome size was estimated to be 2868+/-10 kb. Thirty-three genetic markers were mapped. The probable origin of replication (oriC) was positioned. Six rrn loci were located, and their orientation was determined. The chromosomes of six additional S. xylosus strains were also analysed by I-CeuI digestion, and an intraspecies diversity of the chromosome size and the number of rrn operons was shown.     

Structure of Pseudomonas aeruginosa populations analyzed by single nucleotide polymorphism and pulsed-field gel electrophoresis genotyping

Pseudomonas aeruginosa has a wide ecological distribution that includes natural habitats and clinical settings. To analyze the population structure and distribution of P. aeruginosa, a collection of 111 isolates of diverse habitats and geographical origin, most of which contained a genome with a different SpeI macrorestriction profile, was typed by restriction fragment length polymorphism based on 14 single nucleotide polymorphisms (SNPs) located at seven conserved loci of the core genome (oriC, oprL, fliC, alkB2, citS, oprI, and ampC). The combination of these SNPs plus the type of fliC present (a or b) allowed the assignment of a genetic fingerprint to each strain, thus providing a simple tool for the discrimination of P. aeruginosa strains. Thirteen of the 91 identified SNP genotypes were found in two or more strains. In several cases, strains sharing their SNP genotype had different SpeI macrorestriction profiles. The highly virulent CHA strain shared its SNP genotype with other strains that had different SpeI genotypes and which had been isolated from nonclinical habitats. The reference strain PAO1 also shared its SNP genotype with other strains that had different SpeI genotypes. The P. aeruginosa chromosome contains a conserved core genome and variable amounts of accessory DNA segments (genomic islands and islets) that can be horizontally transferred among strains. The fact that some SNP genotypes were overrepresented in the P. aeruginosa population studied and that several strains sharing an SNP genotype had different SpeI macrorestriction profiles supports the idea that changes occur at a higher rate in the accessory DNA segments than in the conserved core genome.     

Replication origin region of Bacillus subtilis chromosome contains two rRNA operons.

The first replicating DNA fragment (BamHI-7) of the Bacillus subtilis chromosome contains two promoters for a rRNA operon. A map of restriction enzyme cleavage sites of the region of replication origin suggests the presence of a second rRNA operon in this region. Hybridization of rRNA genes (rDNA) with DNA fragments derived from the origin region by treatment with various enzymes clearly revealed two rRNA operons in this region, one at the B7-B3 junction and the other at the B5-B6 junction. The restriction enzyme cleavage sites surrounding the rRNA operons show that the operon at the B5-B6 junction corresponds to the rrnA operon. A novel operon at the B7-B3 junction was termed rrnO. Transformation by density-labeled fragments of the origin region showed that the first replicating marker, guaA, is located in the B3 fragment. From these results, a map was constructed for the first time to correlate the genetic markers with the physical structure of the replication origin region of the B. subtilis chromosome. The role of the rrnO operon in regulating the initiation of chromosomal replication is discussed, based on the fact that the promoter of the rrnO operon suppresses the replication of the plasmid carrying the promoter.     

The chromosome of Salmonella paratyphi A is inverted by recombination between rrnH and rrnG.

Salmonella paratyphi A, a human-adapted bacterial pathogen, causes paratyphoid enteric fever. We established the genome map of strain ATCC 9150 by the use of four endonucleases, XbaI, I-CeuI, AvrII (= BlnI), and SpeI, which generated 27, 7, 19, and 38 fragments, respectively; the sum of the fragments in each case indicates a genome size of ca. 4,600 kb. With phage P22, we transduced Tn10 insertions in known genes from Salmonella typhimurium LT2 to S. paratyphi A ATCC 9150 and located these insertions on the S. paratyphi A chromosome through the XbaI and AvrII sites in Tn10 and through the increased size of the SpeI fragment bearing a Tn10. Compared with the maps of other Salmonella species, the S. paratyphi A genomic map showed two major differences: (i) an insertion of about 100 kb of DNA between rrnH/G and proB and (ii) an inversion of half the genome between rrnH and rrnG, postulated to be due to homologous recombination between the rrn genes. We propose that during the evolution of S. paratyphi A, the first rearrangement event was the 100-kb insertion, which disrupted the chromosomal balance between oriC and the termination of replication, forcing the rrnH/G inversion to restore the balance. The insertion and the inversion are both present in all 10 independent wild-type S. paratyphi A strains tested.     

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.     

Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1

Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from Pseudomonas fluorescens and Pseudomonas aureofaciens. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from P. aeruginosa is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes, phzM and phzS, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of phzS alone in Escherichia coli or in enzymes, pyocyanin-nonproducing P. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine. P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene, phzH, which has a homologue in Pseudomonas chlororaphis, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in P. aeruginosa consisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide.     

Clustering of mutations affecting alginic acid biosynthesis in mucoid Pseudomonas aeruginosa.

A 10-kilobase DNA fragment previously shown to contain the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa was used to construct a pBR325-based hybrid that can be propagated in P. aeruginosa only by the formation of a chromosomal-plasmid cointegrate. This plasmid, designated pAD4008, was inserted into the P. aeruginosa chromosome by recombination at a site of homology between the cloned P. aeruginosa DNA and the chromosome. Mobilization of pAD4008 into P. aeruginosa PAO and 8830 and selection for the stable acquisition of tetracycline resistance resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the phosphomannose isomerase gene region of the chromosomes. Chromosomal DNA from the tetracycline-resistant transformants was used to clone the drug resistance determinant with Bg/II or XbaI, thereby allowing the "walking" of the P. aeruginosa chromosome in the vicinity of the pmi gene. Analysis of overlapping tetracycline-resistant clones indicated the presence of sequences homologous to the DNA insert of plasmid pAD2, a recombinant clone of P. aeruginosa origin previously shown to complement several alginate-negative mutants. Restriction mapping, subcloning, and complementation analysis of a 30-kilobase DNA region demonstrated the tight clustering of several genetic loci involved in alginate biosynthesis. Furthermore, the tetracycline resistance determinant in PAO strain transformed by pAD4008 was mapped on the chromosome by plasmid FP2-mediated conjugation and was found to be located near 45 min.     

Physical and genetic map of the Lactococcus lactis subsp. cremoris MG1363 chromosome: comparison with that of Lactococcus lactis subsp. lactis IL 1403 reveals a large genome inversion.

A physical and genetic map of the chromosome of the Lactococcus lactis subsp. cremoris reference strain MG1363 was established. The physical map was constructed for NotI, ApaI, and SmaI enzymes by using a strategy that combines creation of new rare restriction sites by the random-integration vector pRL1 and ordering of restriction fragments by indirect end-labeling experiments. The MG1363 chromosome appeared to be circular and 2,560 kb long. Seventy-seven chromosomal markers were located on the physical map by hybridization experiments. Integration via homologous recombination of pRC1-derived plasmids allowed a more precise location of some lactococcal genes and determination of their orientation on the chromosome. The MG1363 chromosome contains six rRNA operons; five are clustered within 15% of the chromosome and transcribed in the same direction. Comparison of the L. lactis subsp. cremoris MG1363 physical map with those of the two L. lactis subsp. lactis strains IL1403 and DL11 revealed a high degree of restriction polymorphism. At the genetic organization level, despite an overall conservation of gene organization, strain MG1363 presents a large inversion of half of the genome in the region containing the rRNA operons.     

Mapping of genes involved in macromolecular synthesis on the chromosome of Streptomyces coelicolor A3(2).

The genes for the beta, beta', and seven sigma factor subunits of RNA polymerase, for elongation factors EF-Tu1 and EF-Tu3, and for six rRNA operons were mapped on the combined genetic and physical map of the Streptomyces coelicolor chromosome. Like the previously mapped tRNA genes, the RNA polymerase and rRNA genes map to scattered positions. The lack of rRNA operons in the immediate vicinity of the origin of replication (oriC) and the absence of tRNA genes in any of the rRNA operons are novel features of the Streptomyces chromosome.     

Isolation and characterization of Pseudomonas aeruginosa mutants blocked in the synthesis of pyoverdin.

We have isolated and characterized by chemical and enzymatic analyses three distinct types of pyoverdin-defective (pvd) mutants of Pseudomonas aeruginosa PAO1. The pvd-1 mutant is an L-N5-hydroxyornithine (L-N5-OH-Orn) auxotroph unable to hydroxylate L-ornithine (L-Orn) in a cell-free system and requiring L-N5-OH-Orn for pyoverdin production. The other two types of mutants appear to be blocked in further steps of the biosynthetic pathway leading to pyoverdin, namely, the acylation of L-N5-OH-Orn (pvd-2) and chromophore synthesis (pvd-3). The different pvd mutations were all found to be located in the catA1 region at 47 min of the genetic map of P. aeruginosa PAO1.     

Genetic circularity of the Pseudomonas aeruginosa PAO chromosome.

Genetic circularity of the Pseudomonas aeruginosa PAO chromosome was demonstrated by a series of two- and three-factor crosses and double-selection experiments with Cma plasmids FP2, FP5, FP110, and R68.45. A range of additional markers, including catabolic markers, were located on the chromosome map. Plasmid FP2, known to have a major origin of chromosome transfer (0 min) was shown to have at least one other minor origin from which it can transfer the chromosome in the direction opposite to that found for the major origin.     

Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase.

We have cloned a lipopolysaccharide (LPS) biosynthetic gene from Pseudomonas aeruginosa PAO1 that complements the defect in the production and incorporation of LPS O side chains in the LPS-rough strain AK1012. This gene was characterized by pulsed-field gel electrophoresis, deletion and restriction mapping of the cloned DNA, and biochemical analysis of the protein product. The cloned DNA was found to map to the 7-to-11-min region of the P. aeruginosa chromosome, and the gene needed for complementation of the LPS-rough phenotype was contained on a 2.6-kb HindIII-SacI fragment. This same size restriction fragment contains the alginate gene algC, which encodes the enzyme phosphomannomutase (PMM) and also maps to this region of the P. aeruginosa chromosome. The LPS-rough strain AK1012 was deficient in PMM activity, and this activity was restored to parental levels when the cloned gene was transferred to strain AK1012. In addition, the cloned gene could complement the PMM deficiency in the algC mutant strain 8858, and the cloned algC gene could restore the LPS-smooth phenotype to strain AK1012. These results indicate that the gene we have cloned is equivalent to the alginate gene algC. We designate this gene pmm to emphasize that it encodes the enzyme PMM, which has been shown to be essential for alginate production, and we demonstrate that PMM activity is required for the LPS-smooth phenotype in P. aeruginosa PAO1.     

The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY.

The Pseudomonas aeruginosa pilG gene, encoding a protein which is involved in pilus production, was cloned by phenotypic complementation of a unique, pilus-defective mutant of strain PAO1. This mutant, designated FA2, although resistant to the pilus-specific phage D3112 was sensitive to the pilus-specific phages B3 and F116L. In spite of the unusual phage sensitivity pattern, FA2 lacked the ability to produce functional polar pili (pil) and was incapable of twitching motility (twt). Genetic analysis revealed that the FA2 pil mutation, designated pilG1, mapped near the met-28 marker located at 20 min and was distinct from the previously described pilT mutation. This map location was confirmed by localization of a 6.2-kb EcoRI fragment that complemented FA2 on the SpeI and DpnI physical map of the P. aeruginosa PAO1 chromosome. A 700-bp region encompassing the pilG gene was sequenced, and a 405-bp open reading frame, with characteristic P. aeruginosa codon bias, was identified. The molecular weight of the protein predicted from the amino acid sequence of PilG, which was determined to be 14,717, corresponded very closely to that of a polypeptide with the apparent molecular weight of 15,000 detected after expression of pilG from the T7 promoter in Escherichia coli. Moreover, the predicted amino acid sequence of PilG showed significant homology to that of the enteric CheY protein, a single-domain response regulator. A chromosomal pilG insertion mutant, constructed by allele replacement of the wild-type gene, was not capable of pilus production or twitching motility but displayed normal flagellum-mediated motility. These results, therefore, suggest that PilG may be an important part of the signal transduction system involved in the elaboration of P. aeruginosa pili.