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B W Holloway

Publications and source records attributed to B W Holloway.

At least 19 recordsLinked to original sources
PubMed1995

Clustering of the trp genes in Burkholderia (formerly Pseudomonas) cepacia.

Chromosomal location of trp genes of a strain Burkholderia cepacia (formerly Pseudomonas cepacia) has been determined by transduction using a generalized transducing phage CP75 and by molecular analysis for a cosmid plasmid clone with trp genes isolated from a genomic gene library of the strain. The trp genes were classified into three linkage groups and they all were closely linked on a short chromosomal region probably in the order (trpA, trpB, trpF)-(trpC, trpD)-trpE.

Bacteria↗
PubMed1993

Mapping of ben genes of Pseudomonas aeruginosa.

Four ben genes responsible for the conversion of benzoate to catechol in Pseudomonas aeruginosa PAO have been mapped to a 4.6 kb KpnI fragment, ben-1 and ben-4 were known to be separate genes but now ben-1508 has been found to be different from ben-2. The two genes were distinguished by Tn5 mutagenesis of a cosmid clone and deletion mapping. It is likely that the four genes mapped (ben-4, ben-2, ben-1508 and ben-1) correspond to the previously characterized benR (regulatory gene) and benABC (benzoate dioxygenase) respectively.

Benzoates↗
PubMed1993

Mechanism of conversion to mucoidy in Pseudomonas aeruginosa infecting cystic fibrosis patients.

Chronic respiratory infections with mucoid Pseudomonas aeruginosa are the leading cause of high mortality and morbidity in cystic fibrosis (CF). The initially colonizing strains are nonmucoid, but in the CF lung they invariably convert into the mucoid, exopolysaccharide alginate-overproducing form causing further deterioration and poor prognosis. Here we report the molecular basis of conversion to mucoidy. The algU gene is required for expression of the key alginate biosynthetic gene algD and encodes a protein homologous to sigma H, an alternative sigma factor regulating sporulation and other post-exponential-phase processes in Bacillus. The algU gene and the negative regulators mucA and mucB constitute the gene cluster controlling conversion to mucoidy. We demonstrate a critical role of mucA in this process based on (i) the presence of frameshift mutations disrupting the mucA coding region in mucoid cells that were absent in nonmucoid parental strains, (ii) genetic complementation of mucA mutations with the mucA+ gene, (iii) allelic replacements with specific mutant mucA genes causing conversion to mucoidy in previously nonmucoid cells, and (iv) detection of identical and additional mucA mutations in clinical mucoid strains isolated from the lungs of CF patients. These results suggest that the switch from the nonmucoid to mucoid state can be caused by inactivation of mucA, resulting in constitutive expression of alginate biosynthetic genes dependent on algU for transcription and that such mutants may be selected in vivo during chronic infections in CF.

Alginates↗
PubMed1993

Mapping of the ben, ant and cat genes of Pseudomonas aeruginosa and evolutionary relationship of the ben region of P. aeruginosa and P. putida.

Genes responsible for the utilization of benzoate, anthranilate or catechol (ben, ant, cat) of Pseudomonas aeruginosa PAO were mapped precisely using a cosmid clone carrying all these genes. Genes were localized either by subcloning and complementation or by Tn5 mutagenesis and mapping of the Tn5 insertions. To achieve this, a novel Tn5 mutagenesis procedure was developed by constructing a Tn5 insertion derivative of the Escherichia coli strain S17-1. Preliminary mapping of the ben cat genes of P. putida PPN was accomplished by complementation using a PPN cosmid bank. Sequence homology was demonstrated by Southern hybridization between the ben regions of both P. aeruginosa and P. putida, implying an evolutionary relationship of this chromosomal region of these two pseudomonads.

Chromosome Mapping↗
PubMed1993

Characterization of a locus determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence similarities with a Bacillus sigma factor.

Overproduction of the exopolysaccharide alginate by Pseudomonas aeruginosa results in mucoid colony morphology and is an important virulence determinant expressed by this organism in cystic fibrosis. Mucoidy is transcriptionally regulated by signal transduction systems and histone-like elements. One point of convergence of regulatory elements controlling mucoidy is the algD promoter. A newly described genetic locus required for algD transcription was characterized in this study. This DNA region, cloned from a nonmucoid PAO strain, was initially isolated on the basis of its ability to suppress mucoidy when present on a plasmid. The suppressing activity was observed in several mucoid PAO derivatives, including strain PAO568, in which the mapped muc-2 mutation is responsible for its mucoid phenotype, and in close to 40% of cystic fibrosis strains tested. Protein expression studies detected two polypeptides with apparent molecular masses of 27.5 and 20 kDa encoded by the region required for the suppression activity. The gene encoding the polypeptide with an apparent molecular mass of 27.5 kDa, termed algU, was further characterized. A functional chromosomal copy of algU was found to be necessary for the expression of mucoidy. Insertional inactivation of algU on the chromosome of the mucoid strain PAO568 abrogated alginate production and algD transcription. DNA sequence analysis revealed sequence similarity of the predicted algU gene product with sigma H (Spo0H), a sigma factor involved in the control of sporulation and competence in Bacillus spp. Physical mapping revealed that algU resided on the same SpeI fragment (F) as did the pruAB locus, known to be tightly linked with genetic determinants (muc) which can confer mucoidy in genetic crosses. When the chromosomal algU copy was tagged with a Tcr cassette (algU::Tcr), a tight genetic linkage of algU with pruAB was demonstrated by F116L-mediated generalized transduction. Moreover, algU::Tcr derivatives of PAO568 (originally carrying the muc-2 marker) lost the ability to transfer mucoidy in genetic crosses. These results suggest that algU, a regulator of algD transcription showing sequence similarity to an alternative sigma factor, and the genes immediately downstream of algU may be associated with a locus participating in the differentiation into the mucoid phenotype.

Alginates↗
PubMed1993

Genetics for all bacteria.

The availability of genetic analysis has now been extended to a wide variety of bacteria. While the traditional methods of conjugation, transduction, and transformation have made major contributions to microbiology and genetics, new recombinant DNA techniques and the development of new equipment for characterization and isolation of DNA fragments have enabled genome analysis of many bacteria for which no genetic information was previously available. These new procedures have enabled the construction of detailed physical/genetic maps as well as precise measurements of genome size, and provided new data on functional arrangements of genes in the bacterial genome. Such information is proving increasingly valuable for many aspects of microbiology as well as for the genetic manipulation of bacteria important in human disease, agriculture, and biotechnology.

Bacteria↗
PubMed1992

The new approaches to whole genome analysis of bacteria.

A range of recombinant DNA techniques now enables whole genome analysis of any bacterium to be carried out without recourse to the classical means of bacterial genetic exchange. Using enzymes which cut infrequently, such as SpeI, combined with pulsed field gel electrophoresis, a physical map of ordered fragments can be constructed. By means of cloned fragments of known genes or oligonucleotides synthesized using data from DNA or protein sequence banks, the location of individual genes on this map can be determined. We have used these techniques to study whole genome structure in three species of Pseudomonas: P. aeruginosa, P. putida and P. solanacearum.

Chromosome Mapping↗
PubMed1992

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.

Alcohols↗
PubMed1991

Chromosomal insertion of TOL transposons in Pseudomonas aeruginosa PAO.

Insertions of the TOL plasmid transposons Tn4651 and Tn4653 into the Pseudomonas aeruginosa PAO chromosome were isolated by a temperature selection technique. The locations and orientations of 16 insertions were determined by pulsed field gel electrophoresis and Southern hybridization with genomic and TOL DNA probes. All insertions occurred within a 334 kb region of the chromosome (representing less than 6% of the genome) with nine of the inserts clustered within a 10 kb area. Each transposon was able to insert in either orientation. An internal duplication of the 39 kb excisable region of pWW0 was seen in two independent insertions.

Chromosome Mapping↗
PubMed1990

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.

Blotting, Southern↗
PubMed1990

Chromosome mapping in Pseudomonas syringae pv. syringae strain PS224.

A conjugation system for mapping the chromosome of Pseudomonas syringae pv. syringae PS224 has been developed using the IncP-10 plasmid R91-5; pMO22, a Tn501-loaded derivative of R91-5; and pMO75, R91-5 loaded with Tn5. Nine different donor origins were identified with R91-5 and pMO22. By insertion of Tn5 into various sites of the chromosome, an additional six donor origins were available using pMO75 as the donor plasmid. In all, 36 markers were located on three linkage groups. Many donor strains were unstable and the limited availability of stable donor strains has limited the extent to which markers have been located. This instability of donor strains is in marked contrast to the highly stable donor strains found in P. putida using the same plasmids. As in P. aeruginosa and P. putida, auxotrophic markers in P. syringae do not show the clustering of related markers found in enterobacteria.

Chromosome Mapping↗
PubMed1987

The molecular genetics of C1 utilizing microorganisms. An overview.

The availability of recombinant DNA techniques has enabled the successful genetic analysis and manipulation of a range of C1 utilizing microorganisms. It has resulted in the identification of genes of interest on both plasmids and the chromosome; enabled the linkage of chromosomal genes to be determined; established the function and regulatory patterns of genes essential for utilization of C1 compounds and provided information on the evolution of methanogenic bacteria.

Bacteria↗
PubMed1987

Interactions between the transposable element IS21 on R68.45 and TN7 in Pseudomonas aeruginosa PAO.

Tn7 transposes from the chromosome of Pseudomonas aeruginosa into the plasmid R68.45 with tandem IS21, at up to 400 times the frequency that it transposes into R68, which has only one copy of IS21. While R68::TN7 derivatives are stable, R68.45::Tn7 isolates undergo frequent deletions. Instability of R68.45 occurs whether Tn7 is inserted into the plasmid (cis configuration) or into the bacterial chromosome (trans configuration). The deletions of R68.45 start at the junction between the tandem IS21 copies and proceed clockwise, ending in the region of oriT. It appears that Tn7 and IS21 can mutually stimulate transposition of each other.

Chromosome Deletion↗
PubMed1986

Genetic analysis of promoters on the insertion sequence IS21 of plasmid R68.45.

Tandem duplication of a 2.1-kb DNA sequence on R68 leads to the active insertion element IS21 on the enhanced chromosome mobilizing plasmid R68.45. The HindIII/SalI fragment which carries the single copy or the tandem duplication of IS21 was cloned from R68 and R68.45, respectively, into the multicopy plasmid pED815. Promoters on the two HindIII/SalI fragments were subsequently identified by cloning Sau3A fragments into the BglII site of the promoter cloning vector pGA46. Three promoters were identified on the HindIII/SalI fragment derived from R68 or R68.45, two of them mapped on Sau3A fragments of 214 bp and 82 bp, respectively, on IS21. The promoter on the 82 bp Sau3A fragment which maps at the SmaI site close to the left end of IS21 reads inward. The Sau3A fragment of 214 bp contains the left end of IS21 and transcription from its promoter proceeds outward. In R68.45, readthrough from this preexisting promoter located near the junction of the tandem copies of IS21 proceeds from the right-hand copy into the left, opposing the reading direction of the promoter mapped at the SmaI site of IS21. The expression of genes on one copy of IS21 by readthrough from a promoter on the other one is a possible explanation for the transpositional activity of the tandem configuration of IS21. The similarity of IS21 to other insertion sequences and especially to "mobile promoters" is discussed.

Cloning, Molecular↗
PubMed1986

Transfer of a chromosomal locus responsible for mucoid colony morphology in Pseudomonas aeruginosa isolated from cystic fibrosis patients to P. aeruginosa PAO.

The locus responsible for mucoid colony morphology in five independent clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients have been transferred by means of pM060-mediated conjugation to the genetically characterised strain P. aeruginosa PAO. Genetic mapping has shown that in all five strains the locus is on the chromosome between 89' and 94', although it is not possible to say that the same locus is involved in each case. The way is now open for a more detailed genetic analysis of the loci responsible for mucoid colony morphology.

Chromosome Mapping↗
PubMed1986

Chromosomal location of TOL plasmid DNA in Pseudomonas putida.

The soil isolate Pseudomonas putida MW1000 can grow on toluene and other hydrocarbons; in this respect it is similar to strains of Pseudomonas which carry the TOL plasmid. By conjugation experiments, the genes conferring these growth abilities have been shown to be located on the bacterial chromosome, linked to vil and catB. A 56-kilobase segment of the bacterial chromosome of MW strains carrying the TOL genes can transpose to the IncP-1 plasmid R18-18. Physical analysis of these TOL R18-18 hybrids has shown that the TOL segment is almost identical to the same region found in the TOL plasmid pWW0.

Chromosome Mapping↗