Brittan L. Pasloske

Cloning and sequencing of the Pseudomonas aeruginosa PAK pilin gene

A 1.2‐kilobase (kb) HindIII restriction fragment containing the pilin gene from Pseudomonas aeruginosa PAK has been cloned and sequenced. The pilin protein is 144 amino acids in length with a positively charged leader sequence of 6 amino acids. There is probably only one copy of the gene per chromosome.

PfEMP3 and HRP1: co-expressed genes localized to chromosome 2 of Plasmodium falciparum

A malarial protein, Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3), has been recently characterized as a high-molecular-mass component (approx. 315 kDa) localized to the erythrocyte membrane of knob-bearing (K+), cytoadherent (C+) mature stages of P. falciparum-parasitized erythrocytes (PE) [Pasloske et al., Mol. Biochem. Parasitol. 59 (1993) 59-72]. Knobless (K-), non-cytoadherent (C-) parasites of the same strain were shown to lack the PfEMP3 gene. In view of the biological importance of the knobby and cytoadherent phenotypes with regard to parasite virulence, we extended the analysis of PfEMP3 and its gene product to other K+/K- and C+/C- parasites. Previously, other studies have shown that the malarial protein, knob-associated histidine-rich protein 1 (HRP1), is also strongly correlated with knob expression. Here, we show that PfEMP3 and HRP1 were absent from all the K- parasites tested, including the Palo Alto (PA) K-C+ strain. This result demonstrates that PfEMP3 and HRP1 are not essential for cytoadherence. PfEMP3 was localized to chromosome 2 of the K+ parasites, within no more than 130 kb of HRP1, between the telomere and HRP1. Stage-specific analysis of the mRNA for HRP1 and PfEMP3 indicated maximal transcription of the genes in ring-stage parasites, with little or no mRNA present during the mature parasite stages. Analysis of PfEMP3 and HRP1 by immunofluorescence assay (IFA) revealed identical staining patterns of fixed PE at all stages of the asexual life cycle. Hence, PfEMP3 and HRP1 are adjacent to each other in chromosome 2, co-expressed temporally and their gene products co-localized to the PE membrane.

The expression of Pseudomonas aeruginosa PAK pilin gene mutants in Escherichia coli

Previous work has demonstrated the expression of the cloned pilin gene of Pseudomonas aeruginosa PAK within Escherichia coli and has pinpointed this proteins localization exclusively to the cytoplasmic membrane (Finlay et al., 1986). To define regions of the pilin subunit necessary for its stability and transport within E. coli, we constructed six mutants of the pilin gene and studied their expression and localization using a T7 promoter system. Two of the mutants have either a 4‐ or 8‐amino‐acid deletion at the N‐terminus and both were stably expressed and transported primarily to the cytoplasmic membrane of E. coli. The other four mutants are C‐terminal truncations having between 36 and 56 amino acids of the C‐terminal region of the unprocessed pilin. Studies with these truncated mutants revealed that only the first 36 residues of the unprocessed pilin subunit were required for insertion into the E. coli membrane.

The activity of the Pseudomonas aeruginosa pilin promoter is enhanced by an upstream regulatory site

Recently, a comparison of the nucleotide sequences upstream of the structural pilin genes (pil) from five different isolates of Pseudomonas aeruginosa identified a sequence homologous to a NifA-binding site, a positive regulatory sequence in Klebsiella pneumoniae which controls the expression of the genes which fix nitrogen [Pasloske et al., J. Bacteriol. 170 (1988) 3738-3741]. This sequence was located between -98 and -114 bp relative to the transcription start point. To establish whether this putative recognition sequence exerted any regulatory effect on pil gene expression, we assayed for the expression of pilin and pili, and assessed the strength of the pilin promoter with and without this upstream sequence. A 48-bp deletion, which removed the putative recognition sequence, decreased the levels of pilin and pilus expression. Also, the equivalent deletion reduced pilin promoter activity by 90% when it was measured using a promoter probe vector. These results provide evidence that a nucleotide sequence upstream of the pilin promoter positively regulates the transcription of the P. aeruginosa pil gene.