Molecular analysis on the lipid A (endotoxin) biosynthesis of aerosolized Pseudomonas spp. (y-Proteobacteria) isolated from different workplaces

Nevertheless, several lipid A effects on the human health remain to be elucidated. Moreover, lipid As from Gram-negative bacteria like Pseudomonas spp. exhibit endotoxic activity but which specific steps in the lipid A biosynthesis are essential for the manifestation of the endotoxic activity still remain to be resolved. In this study we show molecular results obtained from isolates mainly belonging to airborne Pseudomonas spp. collected from bioaerosol at different workplaces. The morphology of the microbial strains was examined via phase-contrast and confocal scanning laser microscopy. Also, we used direct microscopic cell counting after DNA-staining. PCR mediated reactions and cycle-sequencing were performed using primers for the genes 16S rDNA, RpoD , Rho and for specific genes of interest, mainly the lipid A biosynthesis genes. The nucleotides and the inferred amino acid residues were compared with accessible data in GenBank databases (NCBI). For the phylogenetic analyses, evolutionary distances were computed both for the DNA sequences and for the deduced amino acid residues of the protein coding genes, calculating 1,000 bootstrap replicates. Finally, all the molecular data from our own sequencing studies and from genomes which have been completely sequenced were elaborated to design oligonucleotide probes for DNA microarray screening to simultaneously perform gene expression assays for lipid A biosynthesis genes. All the obtained sequence data, through an integrated approach, can give information on the effects of bacterial endotoxin on the human health and can offer insight for antibacterial agents that target the lipid A biosynthesis.