Pseudomonas aeruginosa is an opportunistic human pathogen that causes a variety of infections in immunocompromised hosts and individuals with cystic fibrosis. Expression of elastase, one of the virulence factors produced by this organism, requires the transcriptional activator LasR. Experiments with gene fusions show that gene lasl is essential for high expression of elastase. The lasl gene is involved in the synthesis of a diffusible molecule termed Pseudomonas autoinducer (PAI). PAI provides P. aeruginosa with a means of cell-to-cell communication that is required for the expression of virulence genes and may provide a target for therapeutic approaches.

Calf 5 to 3 exo/endonuclease, the counterpart of the human FEN-1 and yeast RTH-1 nucleases, performs structure-specific cleavage of both RNA and DNA and is implicated in Okazaki fragment processing and DNA repair. The substrate for endonuclease activity is a primer annealed to a template but with a 5 unannealed tail. The results presented here demonstrate that the nuclease must enter the 5 end of the unannealed tail and then slide to the region of hybridization where the cleavage occurs. The presence of bound protein or a primer at any point on the single-stranded tail prevents cleavage. However, biotinylation of a nucleotide at the 5 end or internal to the tail does not prevent cleavage. The sliding process is bidirectional. If the nuclease slides onto the tail, later binding of a primer to the tail traps the nuclease between the primer binding site and the cleavage site, preventing the nuclease from departing from the 5 end. A model for 5 entry, sliding, and cleavage is presented. The possible role of this unusual mechanism in Okazaki fragment processing, DNA repair, and protection of the replication fork from inappropriate endonucleolytic cleavage is presented.A 5Ј to 3Ј exonuclease purified from calf has been shown to cooperate functionally with calf DNA polymerase ⑀ to perform nick translation (Siegal et al., 1992). In this reaction, exonucleolytic activity on a downstream primer required synthesis from an upstream primer. The exonuclease was later found to be much more active on a nicked compared with a gapped double-stranded DNA substrate . This suggests that stimulation of nuclease activity by DNA synthesis resulted from continuous generation of the favored substrate.The calf 5Ј to 3Ј exonuclease has been implicated in processing of Okazaki fragments . A DNA template, primed with an oligoribonucleotide that had been extended at the 3Ј end with DNA, was used as a model Okazaki fragment. Calf RNase H1 removed the initiator RNA with a single cut at the 5Ј side of the ribonucleotide at the RNA-DNA junction. The 5Ј to 3Ј exonuclease then could remove the remaining ribonucleotide, creating a 5Ј DNA. Extension of an upstream primer by polymerization in the presence of DNA ligase I allowed the two primers to be joined, as expected on the lagging replication fork strand in vivo.In addition to the above activities, the 5Ј to 3Ј exonuclease could also cleave endonucleolytically . The substrate for this reaction was a DNA primer-template, having a downstream primer with a noncomplementary, unannealed region at the 5Ј end. Additionally, an upstream primer was annealed directly adjacent to the first annealed nucleotide of the downstream primer. Endonucleolytic cleavage removed the unannealed region as an intact segment. Cleavage occurred either at the point of annealing to make a nicked substrate or one nucleotide downstream to make a 1-nucleotide gap. The 5Ј to 3Ј exonuclease domains of Escherichia coli DNA polymerase I and Thermus aquaticus (Taq) DNA polymerase (Lyamichev et al., 1993) are functionally homologous ...

The enzyme elastase is an important virulence factor of the opportunistic human pathogen Pseudomonas aeruginosa. Previous studies have shown that expression of the P. aeruginosa elastase gene (lasB) requires both an activator protein, LasR, and an N-acylhomoserine lactone compound termed Pseudomonas autoinducer (PAI). In this study, we analyzed the lasB promoter region to learn more about lasB activation by LasR and PAI. We report that the lasB transcriptional start is located 141 nucleotides upstream from the lasB translational start. It was also discovered that the lasB promoter region contains two putative operator sequences (OP1 and OP2) that are similar to each other and the Vibrio fischeri lux operator. OP1 is located directly upstream from, and may overlap with, the lasB promoter region, and OP2 is centered 102 nucleotides upstream from the lasB transcriptional start site. To study the effects of these putative operators and other sequences upstream from the lasB transcriptional start site on lasB activation, a series of transcriptional lasBp-lacZ gene fusions was constructed. Data from these fusions indicate that both putative operators are involved in LasR-and PAImediated lasB activation, with OP1 being more important than OP2.Pseudomonas aeruginosa produces a variety of extracellular enzymes contributing to its pathogenesis (22,32,33,36). One of these enzymes, elastase, is well established as a P. aeruginosa virulence factor. P. aeruginosa elastase is produced during the course of clinical infections, and it contributes to the development of infections in animal models (5,8,14,18,19,22,23,25,26). This enzyme is capable of degrading or inactivating important biologic tissues and immune system components, including immunoglobulin (9, 15), serum complement factors (21, 43), ␣ 1 -proteinase inhibitor (34, 35), collagen (16), fibrin (32), and elastin (32, 52). Elastase cleaves host proteases MMP9 and MMP2 in corneal culture (50) and acts synergistically with alkaline protease to inactivate the human cytokines gamma interferon and tumor necrosis factor alpha (38).The elastase structural gene, lasB, has been cloned and sequenced (3), and its expression was shown to require an intact lasR gene (12) and P. aeruginosa autoinducer (PAI) (39). PAI has been identified as N-(3-oxododecanoyl) homoserine lactone and is one of a family of N-acylhomoserine lactone compounds that, together with a regulatory protein (e.g., LasR), are involved in cell density signalling and gene activation (41). The best-characterized member of this family of quorum-sensing systems is the LuxR and Vibrio autoinducer (VAI) autoinduction system of Vibrio fischeri (11). The properties of this family of autoinducers have been recently reviewed by Fuqua et al. (11) and Swift et al. (48).Two recent reports indicate a complexity of lasB gene regulation beyond the requirement of LasR and PAI (37,42). A second activator with homology to LasR (RhlR) is required for rhamnolipid synthesis in P. aeruginosa (37). An rhlR mutant strain was shown to be de...

No abstract

The role of the exonucleolytic activity of the calf 5' to 3' exo/endonuclease, a RAD2 homolog 1 (RTH-1) class nuclease, in lagging-strand DNA replication has been examined using model Okazaki fragment substrates. These substrates exemplify the situation in Okazaki fragment processing which occurs after the initiator RNA primer is cleaved off, and released intact, by calf RNase HI, leaving a single ribonucleotide at the 5' end of the RNA-DNA junction. This final RNA is then removed by the calf RTH-1 nuclease [Turchi et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9803-9807]. The cleavage specificity of calf RTH-1 nuclease for different junction ribonucleotides was compared. These were removed without the usual requirement of calf RTH-1 for an immediately adjacent upstream primer. In most cases, the presence of an upstream DNA or RNA primer, separated from the monoribonucleotide-DNA segment by either a nick or a gap, reduced the efficiency of removal of the monoribonucleotide compared to the removal seen with no upstream primer. Substrates in which the monoribonucleotide-DNA segment had been replaced by an oligomer of the same sequence but consisting entirely of DNA also exhibited upstream primer inhibition. Results with various sequences indicated that the upstream primer is generally inhibitory for ribonucleotide removal but is sometimes neutral. For deoxynucleotide removal it could be stimulatory, neutral, or inhibitory. Possible reasons for the unexpected lack of upstream primer dependence have been explored. The ratio of RNase HI to RTH-1 was also shown to be critical for both enzymes to work together efficiently. These results suggest that regions of upstream primer inhibition within the genome may play a role in determining the mechanism by which mammalian Okazaki fragments are processed.