Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD, was identified by its ability to complement the pilD mutation in P. aeruginosa. Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes (tapABCD) that are homologous to P. aeruginosa type IV pilus biogenesis genes (pilABCD). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N-methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila.

RNA-OUT, the 69-nucleotide antisense RNA that regulates Tn10/IS10 transposition folds into a simple stem-loop structure. The unusually high metabolic stability of RNA-OUT is dependent, in part, on the integrity of its stem-domain: mutations that disrupt stem-domain structure (Class II mutations) render RNA-OUT unstable, and restoration of structure restores stability. Indeed, there is a strong correlation between the thermodynamic and metabolic stabilities of RNA-OUT. We show here that stem-domain integrity determines RNA-OUT's resistance to 3' exoribonucleolytic attack: Class II mutations are almost completely suppressed in Escherichia coli cells lacking its principal 3' exoribonucleases, ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase). RNase II and PNPase are individually able to degrade various RNA-OUT species, albeit with different efficiencies: RNA-OUT secondary structure provides greater resistance to RNase II than to PNPase. Surprisingly, RNA-OUT is threefold more stable in wild-type cells than in cells deficient for RNase II activity, suggesting that RNase II somehow lessens PNPase attack on RNA-OUT. We discuss how this might occur. We also show that wild-type RNA-OUT stability changes only two-fold across the normal range of physiological growth temperatures (30-44 degrees C) in wild-type cells, which has important implications for IS10 biology.

Although there is substantial evidence that type IV pili purified from diarrhea-associated Aeromonas species (designated Bfp for bundle-forming pilus) are intestinal colonization factors (S. M. Kirov, L. A. O'Donovan, and K. Sanderson, Infect. Immun. 67:5447-5454, 1999), nothing is known regarding the function of a second family of Aeromonas type IV pili (designated Tap for type IV Aeromonas pilus), identified following the cloning of a pilus biogenesis gene cluster tapABCD. Related pilus gene clusters are widely conserved among gramnegative bacteria, but their significance for virulence has been controversial. To investigate the role of Tap pili in Aeromonas pathogenesis, mutants of Aeromonas strains (a fish isolate of A. hydrophila and a human dysenteric isolate of A. veronii bv. sobria) were prepared by insertional inactivation of the tapA gene which encodes the type IV pilus subunit protein, TapA. Exotoxic activities were unaffected by the mutation in tapA. Inactivation of tapA had no effect on the bacterial adherence of these two isolates to HEp-2 cells. For the A. veronii bv. sobria isolate, adhesion to Henle 407 intestinal cells and to human intestinal tissue was also unaffected. There was no significant effect on the duration of colonization or incidence of diarrhea when the A. veronii bv. sobria strain was tested in the removable intestinal tie adult rabbit diarrhea model or on its ability to colonize infant mice. Evidence was obtained that demonstrated that TapA was expressed by both Aeromonas species and was present on the cell surface, although if assembled into pili this pilus type appears to be an uncommon one under standard bacterial growth conditions. Further studies into factors which may influence Tap expression are required, but the present study suggests that Tap pili may not be as significant as Bfp pili for Aeromonas intestinal colonization.