Construction of a genomic map of Moraxella (Branhamella) catarrhalis ATCC 25238 and physical mapping of virulence-associated genes.

A physical genome map of the Moraxella catarrhalis type strain (ATCC 25238) has been constructed using pulsed field gel electrophoresis. Macrorestriction analyses of the genome of M. catarrhalis were performed by digestion with the restriction enzymes SmaI, NotI, and RsrII, which cleave the single circular chromosome into 9, 10, and 6 fragments, respectively. The chromosomal fragments generated by pulsed field gel electrophoresis were converted to a linkage map utilizing a combination of partial digestions, and cross-hybridizations. Moraxella catarrhalis, like a number of other respiratory pathogens, has a relatively small genome estimated at 1750 kilobase pairs or about 40% of the size of the Escherichia coli genome. The locations of the four ribosomal RNA operons (rrnLS) were determined by Southern hybridization and by digestion with I-CeuI endonuclease. A number of genes involved in virulence have been placed onto the physical map by Southern hybridization including those encoding the predominant outer-membrane proteins and the chromosomal gene encoding beta-lactamase.

Overexpression, purification, and analysis of the c1 repressor protein of Pseudomonas aeruginosa bacteriophage D3.

A 3.1-kb region of the bacteriophage D3 genome which contains the immunity functions has recently been sequenced (GenBank accession No. L22692). Sequence analysis indicated the presence of a putative repressor gene (c1) whose protein product functions to maintain the bacteriophage genome as a stably integrated prophage in the chromosome of Pseudomonas aeruginosa. A plasmid was constructed that overexpresses repressor C1 protein under control of P(tac) in Escherichia coli. C1 protein was subsequently purified and characterized as a 223 amino acid protein with specific binding affinity for 14-base imperfect palindromic operator sequences located on the genome of bacteriophage D3. N-terminal protein sequence data obtained from automated Edman degradation (16 cycles) of purified repressor protein were identical to the predicted sequence based on DNA sequence analysis of the c1 open reading frame.

Physical mapping of several heat-shock genes in Pseudomonas aeruginosa and the cloning of the mopA (GroEL) gene.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide or amino acid motifs in heat-shock genes/proteins, we have physically mapped the dnaK, lon, and hptG genes of Pseudomonas aeruginosa. Hybridization data suggest that there is a single copy of the mopBA (GroES/GroEL) operon but several additional copies of mopA. In addition, the map coordinates for the rpoD, rpoS, and rpoH genes were determined. The mopA gene from the mopBA operon was cloned and sequenced. The protein product of this gene showed 79% amino acid identity to the Escherichia coli GroEL and 98% identity to the GroEL sequence from P. aeruginosa ATCC 27853. A number of discrepancies were found with the latter sequence.

Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice.

The disulfide loop domain of Pseudomonas aeruginosa PAO pilin was altered by insertion of a chloramphenicol acetyltransferase gene into the pilin gene so that the C-terminal nine amino acids were replaced with 11 new amino acids. The altered pilin gene was transferred into wild-type PAO by recombination, where it did not affect normal piliation as observed by transmission electron microscopy or change of sensitivity to f116, PO4, B9, and Pf1 pilus-specific bacteriophages. However, the binding to human pneumocyte A549 cells was markedly reduced when tested in an in vitro binding assay (2 to 6 bacteria bound per A549 cell for the mutant bacteria compared with 50 bacteria per A549 cell for the wild-type bacteria). Additionally, when susceptible A.BY/SnJ mice were challenged with wild-type P. aeruginosa PAO and with P. aeruginosa PAO-MP (altered pilin gene), a 50% lethal dose of 3 x 10(6) bacteria per mouse was observed for PAO-MP compared with 7 x 10(4) bacteria per mouse for PAO. Approximately 90 of the adherence capability of P. aeruginosa PAO is seemingly attributable to the C-terminal disulfide loop adherence domain of pili. The pilus adherence function contributes significantly to the virulence of P. aeruginosa PAO in the A.BY/SnJ mouse infection model.

Cloning of the early promoters of Pseudomonas aeruginosa bacteriophage D3: sequence of the immunity region of D3.

The early promoters of bacteriophage D3 of Pseudomonas aeruginosa were cloned and physically mapped to the right 25% of the phage genome. The promoters were cloned into promoter selection vector pQF26, and their relative strengths, the direction of transcription, and whether they were directly regulated by repressor were determined. A 3.3-kb fragment of the genome containing the immunity region was sequenced and analyzed (GenBank accession number: L22692). The promoter activity associated with this region was determined to be bidirectional and repressible, indicating that this region contains operator-promoter complexes. Sequence and functional analyses suggest that this region is analogous to the immunity region of coliphage lambda. Two strong promoters, one of which was repressible, were found to be located adjacent to the immunity region. Clear-plaque mutant phage D3c contains insertion element IS222, which causes it to behave as a repressor-negative (c1) variant. The site of insertion of IS222 was sequenced and determined to lie within the c1 gene open reading frame. This phage shows remarkable similarity in genomic organization to coliphage lambda and its relatives.

Deletion and transposon mutagenesis and sequence analysis of the pRO1600 OriR region found in the broad-host-range plasmids of the pQF series.

The nucleotide sequence of the replicative origin (OriR) region of the small cryptic broad-host-range plasmid, pRO1600, which forms the basis of a number of useful cloning vectors has been determined. In addition it has been subjected to Tn5 mutagenesis, deletion analysis, and subcloning in order to define the regions essential for replication in Pseudomonas aeruginosa. The sequence (1894 bp) contains a fragment derived from transposon Tn1. The OriR region is structurally related to other replication (Rep) protein-dependent origins in that it has an A-T-rich region upstream of four 17-bp direct repeats (iterons) which presumably function in initiator protein binding. The sequence also contains a DNA-A-binding site and an open reading frame which could encode a basic (pI 10.6) 25,343-Da Rep protein with homology to RepA from the Neisseria gonorrhoeae beta-lactamase plasmid pFA3. The possible evolutionary origin of this plasmid in P. aeruginosa (RP1) is discussed.

Nucleotide sequence of the Pseudomonas aeruginosa insertion sequence IS222: another member of the IS3 family.

Sequence analysis of the Pseudomonas aeruginosa insertion sequence element IS222 revealed it to be 1234 bp in size with 23 bp imperfect terminal inverted repeats. Insertion caused a 5-bp duplication of the insertion site. Two ORFs were identified, one of which, ORFA, could encode a basic (pI 10.5) polypeptide with a mass of 11,709. This sequence bears strong homology to the putative ORFA product from the Shigella dysenteriae insertion sequence element IS911, which is a member of the IS3 family of insertion elements. As with other members of this group the nucleotide sequence contains a "frameshift window" (AAAAAAG; M. Chandler and O. Fayet (1993). Mol. Microbiol. 7, 497-503) at which ribosome slippage can result in a fusion protein (ORFAB).

Localization of the virulence-associated genes pilA, pilR, rpoN, fliA, fliC, ent, and fbp on the physical map of Pseudomonas aeruginosa PAO1 by pulsed-field electrophoresis.

Seven virulence-associated genes have been placed on a genomic map of Pseudomonas aeruginosa PAO1, using pulsed-field electrophoresis, on the basis of the previous physical maps of Romling et al. (U. Romling, M. Duchene, D. Essar, D. Galloway, C. Guidi-Rontani, D. Hill, A. Lazdunski, R. Miller, K. Schleifer, D. Smith, H. Toschka, and B. Tummler, J. Bacteriol. 174:327-330, 1992; U. Romling, D. Grothues, W. Bautsch, and B. Tummler, EMBO J. 8:4081-4089, 1989) and Ratnaningsih et al. (E. Ratnaningsih, S. Dharmsthiti, V. Krishnapillai, A. Morgan, M. Sinclair, and B. W. Holloway, J. Gen. Microbiol. 136:2351-2357, 1990). The new locations for the outer membrane enterobactin iron-siderophore receptor ent gene (41 to 42 min) and the fliA gene (59 to 61 min), which encodes a minor sigma factor of RNA polymerase, are given. The pilA (the pilin structural gene), pilR (a pilin regulatory gene), and rpoN (encoding another minor sigma factor of RNA polymerase) genes map together at 71 to 75 min, locations correcting the previously reported values (V. Shortridge, M. Pato, A. Vasil, and M. Vasil, Infect. Immun. 59:3596-3603, 1990). The fbp gene (28 to 29 min), which encodes an outer membrane ferripyochelin-binding protein of low molecular weight, and the fliC gene (64 to 66 min), the flagellin structural gene, were determined to lie in the previously reported locations.

High efficiency electroporation of Pseudomonas aeruginosa using frozen cell suspensions.

High efficiency transformation of Pseudomonas aeruginosa was achieved using frozen cell suspensions and high voltage electroporation. We have obtained frequencies as high as 5.8 x 10(8) transformants/micrograms of plasmid DNA using PA01 strain OT684 and a buffer of 15% glycerol-1 mM MOPS. The method allows for easy and reproducible production of frozen cell suspensions for rapid transformation of P. aeruginosa.

Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters.

We have constructed a series of broad-host-range plasmids which use "visual screens" to detect promoter activity. These plasmids contain the pMB1 and pRO1600 origins of replication and are capable of replicating in a wide range of gram-negative bacteria. The genes encoding beta-galactosidase and alkaline phosphatase from Escherichia coli and bacterial luciferase from Vibrio harveyi supply the promoterless indicator genes. The constructs were tested in E. coli and Pseudomonas aeruginosa.

Transduction of a plasmid containing the bacteriophage D3 cos site in Pseudomonas aeruginosa.

Plasmids harboring the cos sequences of bacteriophage D3 can be transferred, by bacteriophage D3, into Pseudomonas aeruginosa by a mechanism which is insensitive to DNase. Transducing activity was separated from the plaque-forming particles by CsCl equilibrium gradient centrifugation. Restriction endonuclease digestion patterns suggest that the transducing particles contain plasmid concatemers.

Construction of broad-host-range vectors for the selection of divergent promoters.

A series of promoter-probe plasmid vectors has been constructed which allows for the selection of DNA sequences containing divergent control elements. Each vector contains a pair of promoterless genes [encoding beta-galactosidase (lacZ), alkaline phosphatase (phoA), and bacterial luciferase (luxAB)] arranged in an antiparallel fashion and separated by a large intervening multiple cloning site. The vectors permit direct detection of promoter activity on indicator plates after transformation. Cloned promoters are selected based on production of coloured products in the case of lacZ and phoA, and by the emission of light in the case of luxAB. These vectors have been tested using known divergent promoter elements from pBR322 and Pseudomonas phage D3.

Multiple promoters control the regulation of the Pseudomonas aeruginosa regA gene.

Expression studies utilizing the regA promoters, fused in tandem or separately to promoterless reporter genes, indicated that regA is transcribed from two promoters (P1 and P2). Both promoters can act independently. Expression from the P1 promoter is not affected by the iron content of the medium. Expression from the P2 promoter is tightly regulated by iron.

Construction of broad-host-range vectors for general cloning and promoter selection in Pseudomonas and Escherichia coli.

We have constructed two promoter-selection vectors based upon the broad-host-range plasmid pRO1614. pQF40 (6 kb) contains a promoterless tetA gene downstream from a large multiple cloning site while pQF26 (5.4 kb) possesses a promoterless cat cartridge. The latter vector displayed a copy number of 13 in Pseudomonas aeruginosa and 39 in Escherichia coli. When promoter sequences derived from the Pseudomonas phage phi PLS27 were cloned into pQF26, high levels of chloramphenicol-acetyltransferase were detected in P. aeruginosa. In E. coli the activity was approximately one-third that in P. aeruginosa when corrections were made for the plasmid copy number.