The Pseudomonas aeruginosa nirE gene encodes the S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase required for heme d(1) biosynthesis.

Biosynthesis of heme d(1), the essential prosthetic group of the dissimilatory nitrite reductase cytochrome cd(1), requires the methylation of the tetrapyrrole precursor uroporphyrinogen III at positions C-2 and C-7. We produced Pseudomonas aeruginosa NirE, a putative S-adenosyl-L-methionine (SAM)-dependent uroporphyrinogen III methyltransferase, as a recombinant protein in Escherichia coli and purified it to apparent homogeneity by metal chelate and gel filtration chromatography. Analytical gel filtration of purified NirE indicated that the recombinant protein is a homodimer. NirE was shown to be a SAM-dependent uroporphyrinogen III methyltransferase that catalyzes the conversion of uroporphyrinogen III into precorrin-2 in vivo and in vitro. A specific activity of 316.8 nmol of precorrin-2 h(-1) x mg(-1) of NirE was found for the conversion of uroporphyrinogen III to precorrin-2. At high enzyme concentrations NirE catalyzed an overmethylation of uroporphyrinogen III, resulting in the formation of trimethylpyrrocorphin. Substrate inhibition was observed at uroporphyrinogen III concentrations above 17 microM. The protein did bind SAM, although not with the same avidity as reported for other SAM-dependent uroporphyrinogen III methyltransferases involved in siroheme and cobalamin biosynthesis. A P. aeruginosa nirE transposon mutant was not complemented by native cobA encoding the SAM-dependent uroporphyrinogen III methyltransferase involved in cobalamin formation. However, bacterial growth of the nirE mutant was observed when cobA was constitutively expressed by a complementing plasmid, underscoring the special requirement of NirE for heme d(1) biosynthesis.

Protochlorophyllide: a new photosensitizer for the photodynamic inactivation of Gram-positive and Gram-negative bacteria.

The growing resistance against antibiotics demands the search for alternative treatment strategies. Photodynamic therapy is a promising candidate. The natural intermediate of chlorophyll biosynthesis, protochlorophyllide, was produced, purified and tested as a novel photosensitizer for the inactivation of five model organisms including Staphylococcus aureus, Listeria monocytogenes and Yersinia pseudotuberculosis, all responsible for serious clinical infections. When microorganisms were exposed to white light from a tungsten filament lamp (0.1 mW cm(-2)), Gram-positive S. aureus, L. monocytogenes and Bacillus subtilis were photochemically inactivated at concentrations of 0.5 mg L(-1) protochlorophyllide. Transmission electron microscopy revealed a disordered septum formation during cell division and the partial loss of the cytoplasmic cell contents. Gram-negative Y. pseudotuberculosis and Escherichia coli were found to be insensitive to protochlorophyllide treatment due to the permeability barrier of the outer membrane. However, the two bacteria were rendered susceptible to eradication by protochlorophyllide (10 mg L(-1)) upon addition of polymyxin B nonapeptide at 50 and 20 mg L(-1), respectively. The release of DNA and a detrimental rearrangement of the cytoplasm were observed.

Adaptation of Pseudomonas aeruginosa to various conditions includes tRNA-dependent formation of alanyl-phosphatidylglycerol.

The opportunistic bacterium Pseudomonas aeruginosa synthesizes significant amounts of an additional phospholipid, identified as 2' alanyl-phosphatidylglycerol (A-PG), when exposed to acidic growth conditions. At pH 5.3 A-PG contributed up to 6% to the overall lipid content of the bacterium. Sequence analysis of P. aeruginosa revealed open reading frame PA0920 showing 34% sequence identity to a protein from Staphylococcus aureus involved in tRNA-dependent formation of lysyl-phosphatidylglycerol. The P. aeruginosa deletion mutant DeltaPA0920 failed to synthesize A-PG. Heterologous overproduction of PA0920 in Escherichia coli resulted in the formation of significant amounts of A-PG, otherwise not synthesized by E. coli. Consequently, the protein encoded by PA0920 was named A-PG synthase. The enzyme was identified as an integral component of the inner membrane. The protein was partially purified by detergent solubilization and subjected to an in vitro activity assay. tRNA(Ala)-dependent catalysis was demonstrated. Transcriptional analysis of the corresponding gene in P. aeruginosa using lacZ reporter gene fusion under various pH conditions indicated a 4.4-fold acid-activated transcription. A phenotype microarray analysis was used to identify further conditions for A-PG function.