Ute Römling

Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa CMG103 was isolated from a metal-polluted river in Pakistan and displayed a high level of Zn and Cd resistance. An omega-Km transposon mutant of strain CMG103, which showed a substantial decrease in resistance to Zn and Cd, was obtained. A 12.8 kb region determining Zn and Cd resistance in strain CM103 was cloned by complementing the mutant strain, and its nt sequence was determined. Five genes, czrSRCBA, involved in Zn and Cd resistance, were identified. The predicted gene products of czrCBA show a significant similarity with the proteins encoded by the plasmid borne metal resistant determinants czc, cnr and ncc of Ralstonia strains, which determine a chemiosmotic cation-antiporter efflux system. The predicted CzrS and CzrR proteins show a significant similarity to the sensor and regulatory protein, respectively, of two component regulatory systems, such as CopS/CopR and PcoS/PcoR involved in the regulation of plasmid-borne Cu-resistant determinants, and CzcS/CzcR involved in the regulation of czc. The cloned czr region contained downstream of czrCBA additional ORFs whose predicted gene products are similar to proteins involved in catabolism of aromatic compounds. DNA-DNA hybridization indicated strong conservation of czr in other environmental P. aeruginosa isolates and in the P. aeruginosa type strain PAO1, a clinical isolate. This was confirmed by a comparison of the sequence of the CMG103 czr region with the currently available genome sequence of strain PAO1. A high sequence identity (till 99% at the nt level) and organizatory conservation of the czr region of CMG103 was found in PAO1 as well regarding coding sequences as intervening sequences between ORFs. The czr locus was localized between coordinates 2400 and 2550 kb on the physical map of the chromosome of PAO1.

Genomic mapping of Pseudomonas aeruginosa PAO

The extent of knowledge of genome arrangement in any bacterium is directly proportional to the degree of interest in the organism, its importance to medicine, agriculture, the environment and industry and the availability of means of genetic analysis. Until the introduction of recombinant DNA techniques, genome mapping was dependent on the availability and user-friendliness of systems of genetic exchange conjugation, transduction and transformation being the most common. New DNA technologies have enabled the genetic mapping of those organisms for which in vivo systems of analysis are not available, and this topic has been recently reviewed (Smith & Condemine, 1990; Holloway, 1993). For Psewdomonas aerzlginosa, a fortunate situation has been created by the availability of all three major systems of in vivo gene exchange, conjugation, transduction and transformation (Holloway & Morgan, 1986), as well as the newer technologies (Holloway e t al., 1992). Combined with an increasing research interest in this organism, mainly stimulated by its medical importance, the extent of genome mapping has increased markedly in the last few years.

A physical genome map of the Burkholderia cepacia type strain

Burkholderia cepacia (basonym Pseudomonas cepacia), the type speciesof the new genus Burkholderia, is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil‐borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis. A physical map of the 8.1 Mb genome of the B. cepacia type‐strain ATCC 25416 was constructed by applying two‐dimensional pulsed‐field gel electrophoresis techniques. Placed onto the macrorestriction map were 38 Spel, 11 Swal, 11 Pacl, 11 Pmel and six l‐Ceul sites, resulting in an average resolution of 1O5 kbp. Random single‐hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size. The largest replicon harbours four rrn operons while the other two Megabase‐size replicons each contain a single rrn operon, suggesting that the genome has three chromosomes and a large plasmid. Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined to B. cepacia. The phylogenetically related species Burkholderia glumae, Burkholderia pickettii, Burkholderia solanacearum, Alcaligenes eutrophus and the so far unassigned Pseudomonas glathei were also found to harbour more than one Megabase‐size replicon.

Genome Organization of Pseudomonas stutzeri and Resulting Taxonomic and Evolutionary Considerations

In order to determine the genome variability within Pseudomonas stutzeri, 20 strains representing the seven described genomovars and strain JM300 were analyzed by using various resolution levels of rare cutting enzymes. XbaI and SpeI fingerprints revealed a high degree of heterogeneity of restriction patterns that did not correlate with the division into genomovars. However, a fragment pattern comparison led to the establishment of several groups of clonal variants within genomovars. One circular chromosome ranging in size from 3.75 to 4.64 Mb constitutes the genome of P. stutzeri strains. The I-CeuI, PacI, and SwaI low-resolution map of P. stutzeri type strain CCUG 11256 shows the locations of 12 genes, including rrn operons and the origin of replication. I-CeuI digests of the 20 strains studied plus the positions of six genes allowed a comparison of the rrn backbone organization within genomovars; the four rrn operons seemed to be at similar locations with respect to the origin of replication, as did the rest of the genes. However, a comparison of I-CeuI cleavage maps of the genomovar reference strains revealed a diverse genome organization in the genomovars relative to rrn operons and gene locations. In most genomovars, rrn operons are not arranged around the origin of replication but are equally distributed on the chromosome. Strain JM300 does not belong to any described genomovar, as determined from the organization of its genome. Large chromosomal rearrangements seem to be responsible for the differences in superordinate genome structure and must have played an important role in P. stutzeri diversification and niche colonization. An ancestral chromosome is suggested, and some plausible pathways for the generation of the various genome structures are proposed.

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.

Comparative hygienic surveillance of contamination with pseudomonads in a cystic fibrosis ward over a 4-year period

In order to study the long-term distribution and population dynamics of Pseudomonas aeruginosa strains in a highly contaminated hospital environment, two 4-week epidemiological studies, with an interval of 4 years, were carried out in the cystic fibrosis (CF) ward of the Paediatric Clinic of the Medical School of Hannover. Out of the 1948 specimens taken, P. aeruginosa was mainly identified in those from moist, inanimate sources (200 isolates) and hospitalized CF patients (168 isolates). A correlation was established between the frequency with which P. aeruginosa-positive patients came into contact with hospital facilities and the rate of contamination of these facilities. Rooms reserved for colonized patients were more frequently contaminated with P. aeruginosa in contrast to function rooms in the same ward and the outpatient clinic. However, no direct exchange between patients strains and the inanimate hospital environment was detected. Out of the 11 genotypes of P. aeruginosa found in 1989 and the 13 genotypes found in 1993, four genotypes were present on both occasions. The most predominant clone was found in tap-water, sinks, wash-basins and creams with an incidence of 34 and 68% in the environmental isolates. The strains seemed to have spread into the adjacent control ward during the 4-year interval. Thus, the separation of colonized and non-colonized patients was undermined through the transfer of strains from a highly contaminated environment without additional hygiene precautions.

Direct sputum analysis of Pseudomonas aeruginosa macrorestriction fragment genotypes in patients with cystic fibrosis

Abstract The distribution of bacterial populations in the airways of 13 patients with cystic fibrosis who were colonized for 6 – 23 years with Pseudomonas aeruginosa was investigated by genotyping of bacterial chromosomes directly isolated from 21 sputa. After removal of host material from sputum by hypotonic cell lysis and repetitive washing and centrifugation steps, agarose-embedded bacterial cells were lysed, residual eukaryotic DNA separated by field inversion gel electrophoresis, and the purified bacterial chromosomes subjected to macrorestriction fragment pattern and Southern analyses. Bacterial populations consisted of a single P. aeruginosa clone in 17 sputa, of which more than one clonal variant was apparent in two SpeI fragment fingerprints. Two clones of P. aeruginosa and another species co-existed in four samples. Genomically homogeneous populations of P. aeruginosa are characteristic for chronically colonized lungs in most cases of cystic fibrosis.

Cloning, mapping and characterization of thePseudomonas aeruginosa hemL gene

The rate-limiting step in the biosynthesis of tetrapyrroles is the formation of 5-aminolevulinic acid (ALA). InPseudomonas aeruginosa ALA is synthesized via a two-step reaction from aminoacylated tRNAGlu by the action of glutamyl-tRNA reductase and glutamate-1-semialdehyde-2,1-amino mutase. To initiate an investigation of the regulation of the second step in ALA formation, thehemL gene was cloned fromP. aeruginosa by complementation of anEscherichia coli hemL mutant. An open reading frame of 1284 by encoding a protein of 427 amino acids with a calculated molecular mass of 45 404 Da was identified. ThehemL gene was mapped to theSpeI fragment Z and theDpnI fragment J1 of theP. aeruginosa chromosome corresponding approximately to min 0.3–0.9. One transcription start site was located 280 by upstream of the translational start site of thehemL gene. No classicalσ70-dependent promoter was detected. Oxygen stress induced by the addition of H2O2 to the growth medium led to an approximately 3.5-fold increase inhemL expression as determined by mRNA dot blot assays. Anaerobic denitrifying growth led to a 2-fold stimulation ofhemL transcription. Two additional open reading frames were detected downstream of thehemL gene. One open reading frame (orf1) of 549 by encodes a protein of 182 amino acids with a calculated molecular mass of 19 638 Da. The second open reading frame (orf2) of 1341 by encodes a protein of 446 amino acids with a calculated molecular mass of 49 967 Da and showed similarity to a protein of unknown function fromMycobacterium leprae and to a P-methylase fromStreptomyces hygroscopicus.

Bacterial genome mapping

Abstract This review provides a survey of the rationales and techniques of ‘top-down’ and ‘bottom-up’ approaches for physical mapping of bacterial genomes and reports on the application of comparative intra- and interspecies chromosome mapping in medical microbiology, biotechnology and molecular evolution.

6 – Macrorestriction Mapping and Analysis of Bacterial Genomes

This chapter focuses on the construction of macrorestriction maps of bacterial genomes. The relatively small genome of 0.5–10 Mbp makes bacteria an appropriate target for the comprehensive study of genome organization by pulsed-field gel electrophoresis (PFGE) techniques. The number, size, and topology of genetic entities are the fundamental characteristics of a genome. PFGE permits visualization of chromosomes and plasmids. Unsheared bacterial DNA is prepared by the inclusion of intact bacteria into agarose blocks prior to cell lysis. It is found that determining the size of circular chromosomes requires the additional step of linearizing the DNA prior to PFGE, since large circular DNA remains trapped in the plug instead of migrating through the gel. It is observed that after PFGE of embedded samples, circular chromosomes will produce an intense fluorescent signal from the agarose plug, which reflects the trapped circles, and faint bands in the megabase range. For the construction of a macrorestriction map, the genomic DNA is digested with restriction endonucleases that cut only rarely and the fragments are subsequently separated by PFGE. It is suggested that the mapping strategy requires an optimal quality of the agarose-embedded DNA and optimal separation conditions.