Functional characterization of a serine/threonine protein kinase of Pseudomonas aeruginosa.

Protein kinases play a key role in signal transduction pathways in both eukaryotic and prokaryotic cells. Using in vivo expression technology, we have identified several promoters in Pseudomonas aeruginosa which are preferentially activated during infection of neutropenic mice. One of these promoters directs the transcription of a gene encoding a putative protein kinase similar to the enzymes found in eukaryotic cells. The full characterization of this protein, termed PpkA, is presented in this communication. The ppkA gene encodes a 1,032-amino-acid polypeptide with an N-terminal catalytic domain showing all of the conserved residues of protein kinases with the substrate phosphorylation specificities for serine and threonine residues. The catalytic domain is linked to the rest of the protein by a short proline-rich segment. The enzymes showed anomalous migration behavior when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be attributed to autophosphorylation activity. The full-length enzyme was expressed as an oligohistidine fusion protein and was shown to phosphorylate several artificial protein substrates. Both autophosphorylation and phosphorylation of added substrates were strongly reduced by a single-amino-acid substitution in the catalytic domain of PpkA. Although PpkA appears to be differentially phosphorylated by autocatalysis, the levels of phosphorylation have minimal effect on its overall enzymatic activity. Our results, therefore, indicate the operation of a novel protein phosphorylation mechanism during transduction of signals in P. aeruginosa, and this pathway may be important in regulating the expression of virulence factors by this pathogen during certain phases of infection.

Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion.

The general secretion pathway (GSP), found in a wide range of bacteria, is responsible for extracellular targeting of a subset of proteins from the periplasm. In Pseudomonas aeruginosa, the GSP requires the participation of 12 proteins, of which XcpT, XcpU, XcpV, XcpW are homologues of PilA, the major subunit of type IV pili. The interaction between the pilin-like Xcp proteins was investigated using bifunctional crosslinking reagents. Cross-linking analysis of whole cells of wild-type P. aeruginosa, followed by immunoblot analysis, revealed a 34-kDa XcpT-containing complex. This complex was shown to consist of XcpT/PilA heterodimers. The role of PilA in the GSP was examined, using P. aeruginosa mutants in the pilA gene, or in rpoN, a gene regulating pilA expression. Each mutant showed a significant reduction in the efficiency of extracellular protein secretion, and this defect could be restored by expression of the cloned pilA gene in the mutant cells. The formation of the PilA/XcpT complex did not require XcpR or XcpQ, two other components of the secretion machinery, nor did it require the pilus biogenesis factors PilB and PIlC. The dimeric XcpT/PilA complex was also formed in a pilD mutant, which lacks the leader peptidase enzyme, demonstrating that the leader peptide at the N-terminus or PilA or XcpT did not have to be removed for the dimerization to occur. XcpW and XcpU can also be crosslinked to form dimeric complexes with PilA. When expression of XcpT is increased, its homodimers, as well as XcpT/XcpW heterodimers, can be detected. Finally, an oligohistidine-tagged XcpT was shown to form stoichiometric complexes with PilA, and with XcpT, U, V and W. These dimers were co-purified by nickel-affinity chromatography. The results of this study suggest that XcpT can form heterodimers with PilA, and Xcp U, V and W, which may be assembly intermediates of the secretion apparatus. Alternatively, these may represent dynamic intermediates that facilitate protein secretion by continuous association and dissociation. The requirement for PilA for efficient protein secretion argues for a critical role played by PilA in two related processes during P. aeruginosa infections: formation of an adhesive pilus organelle and secretion of exoenzymes.

Platelet-derived growth factor receptor alpha subunit deleted Patch mouse exhibits severe cardiovascular dysmorphogenesis.

Patch (Ph) mice, whose platelet-derived growth factor receptor alpha subunit (alpha PDGFR) gene has been deleted, have been used to elucidate requirements for alpha PDGFR for normal murine development. In this report we evaluate the role of alpha PDGFR in cardiovascular development by using in situ hybridization to follow the changing pattern of alpha PDGFR expression in cardiovascular tissues after embryonic day 13, and comparing this pattern with the pattern of cardiovascular defects observed in homozygous Ph mutants. Both mesodermally derived and neural crest-derived components of the cardiovascular system are severely dysmorphic in Ph/Ph embryos and those structures most severely affected are those that normally express alpha PDGFR mRNA at the highest levels and for the longest duration. Ph/Ph vessels appear to be lined with a normal endothelium, but contain a reduced number of smooth muscle cells and are fragile during processing for histology. The myocardium is thin, the heart is small and dysmorphic, the valves are malformed, and the interventricular and interatrial septa of the heart are defective. In the outflow tract, the spectrum of defects includes both persistent truncus arteriosus and double outlet right ventricle. This pattern of abnormalities is consistent with the hypothesis that deletion of alpha PDGFR results in a functional ablation of cranial neural crest cells, and that mesodermally derived components of the vascular system also require alpha PDGFR.

Osteoporosis induced in mice by overproduction of interleukin 4.

Osteoporosis is a common disease in which loss of bone mass results in skeletal fragility. The development of therapies for this disorder has been hampered by the lack of a convenient animal model. Here we describe a disorder in bone homeostasis in transgenic mice that inappropriately express the cytokine interleukin 4 (IL-4) under the direction of the lymphocyte-specific proximal promoter for the lck gene. Bone disease in lck-IL-4 mice appeared to result from markedly decreased bone formation by osteoblasts, features strikingly similar to those observed in cases of severe low-turnover human involutional osteoporosis. By 2 months of age, female and male lck-IL-4 mice invariably developed severe osteoporosis of both cortical and trabecular bone. Osteoporosis was observed in two independently derived founder animals, indicating that this phenotype was directly mediated by the IL-4 transgene.

Use of synthetic oligonucleotide DNA probes for identification and direct detection of Bacteroides forsythus in plaque samples.

Oligonucleotide DNA probes complementary to the hypervariable region of the 16S rRNA of Bacteroides forsythus were tested for their specificity and sensitivity against reference and clinical isolates of 73 different species of bacteria. The probes were used to detect the organism directly from nucleic acid extracts of subgingival plaque samples, and the results were compared with results of detection by culture methods. The data demonstrate that the probes are very specific (100%) and sensitive (100% when they are compared with those obtained by the culture method) and could reliably detect the organism in plaque samples. When a probe to a conserved region of the 16S rRNA (UP9A) was used to estimate the quantity of bacteria present in plaque samples, it gave results comparable to those of culture methods. The data suggest that total microbial load may be a parameter in periodontal disease.

Atypical structure of the 23S ribosomal RNA molecule in certain oral bacteria.

Ribosomal RNA (rRNA) isolated from Wolinella recta and seven related bacteria was examined by agarose gel electrophoresis. The 23S rRNA molecule could not be detected in W. recta, Wolinella curva, Bacteroides gracilis, or Bacteroides ureolyticus. In place of the 23S molecule, there were three smaller molecules of approximately 1700, 650, and 600 bases designated 23S alpha, 23S beta, and 23S delta, respectively. An intact 23S rRNA molecule could be isolated from Wolinella succinogenes, Campylobacter concisus, and Campylobacter sputorum. The cleavage sites of the W. recta 23S rRNA molecule were located by direct RNA sequence analysis and were found to be in similar locations, nucleotides 546 and 1180, as cleavage sites described in other prokaryotes. The presence or absence of the 23S rRNA molecule may be a useful marker for these micro-organisms.