Efficient affinity-tagging of M13 phage capsid protein IX for immobilization of protein III-displayed oligopeptide probes on abiotic platforms.

We developed a genetic approach to efficiently add an affinity tag to every copy of protein IX (pIX) of M13 filamentous bacteriophage in a population. Affinity-tagged phages can be immobilized on a surface in a uniform monolayer in order to position the pIII-displayed peptides or proteins for optimal interaction with ligands. The tagging consists of two major steps. First, gene IX (gIX) of M13 phage is mutated in Escherichia coli via genetic recombineering with the gIX::aacCI insertion allele. Second, a plasmid that co-produces the affinity-tagged pIX and native pVIII is transformed into the strain carrying the defective M13 gIX. This genetic complementation allows the formation of infective phage particles that carry a full complement (five copies per virion) of the affinity-tagged pIX. To demonstrate the efficacy of our method, we tagged a M13 derivative phage, M13KE, with Strep-tag II. In order to tag pIX with Strep-tag II, the phage genes for pIX and pVIII were cloned and expressed from pASG-IBA4 which contains the E. coli OmpA signal sequence and Strep-Tag II under control of the tetracycline promoter/operator system. We achieved the maximum phage production of 3 × 1011 pfu/ml when Strep-Tag II-pIX-pVIII fusion was induced with 10 ng/ml of anhydrotetracycline. The complete process of affinity tagging a phage probe takes less than 5 days and can be utilized to tag any M13 or fd pIII-displayed oligopeptide probes to improve their performance.

Complete Genome Sequence of Pseudomonas aeruginosa Mucoid Strain FRD1, Isolated from a Cystic Fibrosis Patient.

We announce here the complete genome sequence of the Pseudomonas aeruginosa mucoid strain FRD1, isolated from the sputum of a cystic fibrosis patient. The complete genome of P. aeruginosa FRD1 is 6,712,339 bp. This genome will allow comparative genomics to be used to identify genes associated with virulence, especially those involved in chronic pulmonary infections.

Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon.

In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSF(ts1). The mutations that resulted in a temperature-sensitive phenotype in mSF(ts1) were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42 degrees C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 h of growth. In order to facilitate its utilization, the mSF(ts1) was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSF(ts1) for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.

Virulence determinants from a cystic fibrosis isolate of Pseudomonas aeruginosa include isocitrate lyase.

Chronic lung infections caused by Pseudomonas aeruginosa are the leading cause of morbidity and mortality for cystic fibrosis (CF) patients. Adaptation of P. aeruginosa to the CF lung results in the loss of acute virulence determinants and appears to activate chronic virulence strategies in this pathogen. In order to identify such strategies, a random transposon mutagenesis was performed and 18 genes that were required for optimal infection of alfalfa seedlings by FRD1, a CF isolate of P. aeruginosa, were recognized. The largest subset of genes (seven of the 18), were associated with central carbon metabolism, including the gene that encodes isocitrate lyase (ICL), aceA. Because FRD1 is avirulent in animal infection models, we constructed an ICL mutant in P. aeruginosa strain PAO1 in order to assess the requirement of ICL in mammalian infection. The PAO1 ICL mutant was less virulent in the rat lung infection model, indicating that ICL is required for the pathogenesis of P. aeruginosa in mammals. Furthermore, FRD1 showed increased ICL activity and expression of an aceA : : lacZ fusion compared to PAO1. We suggest that upregulation of ICL occurred during adaptation of FRD1 to the CF lung and that some of the novel virulence mechanisms employed by FRD1 to infect alfalfa seedlings may be the same mechanisms P. aeruginosa relies upon to persist within human niches.

Development of tools for the genetic manipulation of Pseudomonas aeruginosa.

To facilitate study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, several genetic tools were developed. These tools include a series of cassettes carrying (a) the minimal sequence for the origin of transfer (oriT) of RP4 plasmid for introducing plasmid into P. aeruginosa via conjugation, (b) a minimal sequence for P. aeruginosa replicon (stabilizing fragment or SF) for maintenance of plasmids in P. aeruginosa, and (c) the transcriptionally non-polar tetracycline resistance gene (TcR) for insertional mutagenesis. Additional genetic constructs include (d) two conjugative and suicide lacZ reporter fusion plasmids for studying gene expression at the transcriptional or translational level, (e) a gentamicin resistant promoter-probing mini-Tn5 lacZ, and (f) a tightly regulated T7 promoter/repressor system to control gene expression in P. aeruginosa.