Anaerobically induced expression of the nitrite reductase cytochrome c-551 operon from Pseudomonas aeruginosa.

The nitrite reductase gene (denA) and the cytochrome c-551 gene (denB) are located only 50 bp apart from each other in the Pseudomonas aeruginosa chromosome. We report evidence that these two genes are co-transcribed as an operon only under anaerobic (denitrifying) conditions. The nucleotide sequence of the promoter (regulatory) region of the operon is highly AT-rich and contains a sequence closely resembling the consensus FNR binding site in E. coli.     

The sequence of the 6S RNA gene of Pseudomonas aeruginosa.

From the gram-negative eubacterium Pseudomonas aeruginosa we have isolated a stable 6S RNA, approximately 180 nucleotides in length. The RNA was partially sequenced and identified by comparison with the known Escherichia coli 6S RNA sequence. Southern hybridizations revealed a single copy gene coding for the 6S RNA. DNA from other prokaryotes, i.e. E. coli, Thermus thermophilus, Bacillus subtilis, Bacillus stearothermophilus and Halobacterium maris mortui, did not give detectable hybridization signals. The 6S RNA gene was cloned in E. coli and its complete primary structure was determined. Although the 6S RNA sequences from P. aeruginosa and E. coli share only a 60.4% homology, we are able to propose a common secondary structural model.     

Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa.

Type 4 fimbriae are found in a range of pathogenic bacteria, including Bacteroides nodosus, Moraxella bovis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The structural subunits of these fimbriae all contain a highly conserved hydrophobic amino-terminal sequence preceding a variable hydrophilic carboxy-terminal region. We show here that recombinant P. aeruginosa cells containing the B. nodosus fimbrial subunit gene under the control of a strong promoter (pL, from bacteriophage lambda) produced large amounts of fimbriae that were structurally and antigenically indistinguishable from those produced by B. nodosus. This was demonstrated by fimbrial isolation and purification, electrophoretic and Western transfer analyses, and immunogold labeling and electron microscopy. These results suggest that type 4 fimbriated bacteria use a common mechanism for fimbrial assembly and that the structural subunits are interchangeable, thereby providing a basis for the development of multivalent vaccines.     

Synthesis, processing, and transport of Pseudomonas aeruginosa elastase.

Three cell-associated elastase precursors with approximate molecular weights of 60,000 (P), 56,000 (Pro I), and 36,000 (Pro II) were identified in Pseudomonas aeruginosa cells by pulse-labeling with [35S]methionine and immunoprecipitation. In the absence of inhibitors, cells of a wild-type strain as well as those of the secretion-defective mutant PAKS 18 accumulated Pro II as the only elastase-related radioactive protein. EDTA but not EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] inhibited the formation of Pro II, and this inhibition was accompanied by the accumulation of Pro I. P accumulated in cells labeled in the presence of ethanol (with or without EDTA), dinitrophenol plus EDTA, or carbonyl cyanide m-chlorophenyl hydrazone plus EDTA. Pro I and Pro II were localized to the periplasm, and as evident from pulse-chase experiments, Pro I was converted to the mature extracellular enzyme with Pro II as an intermediate of the reaction. P was located to the membrane fraction. Pro I but not Pro II was immunoprecipitated by antibodies specific to a protein of about 20,000 molecular weight (P20), which, as we showed before (Kessler and Safrin, J. Bacteriol. 170:1215-1219, 1988), forms a complex with an inactive periplasmic elastase precursor of about 36,000 molecular weight. Our results suggest that the elastase is made by the cells as a preproenzyme (P), containing a signal sequence of about 4,000 molecular weight and a "pro" sequence of about 20,000 molecular weight. Processing and export of the preproenzyme involve the formation of two periplasmic proenzyme species: proelastase I (56 kilodaltons [kDa]) and proelastase II (36 kDa). The former is short-lived, whereas proelastase II accumulates temporarily in the periplasm, most likely as a complex with the 20-kDa propeptide released from proelastase I upon conversion to proelastase II. The final step in elastase secretion seems to required both the proteolytic removal of a small peptide from proelastase II and dissociation of the latter from P20.